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The effect of temperature, frequency of stimulation and external calcium on myocardial contractility Longhurst, Penelope Anne 1981

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THE EFFECT OF TEMPERATURE, FREQUENCY OF STIMULATION AND EXTERNAL CALCIUM CONCENTRATION ON MYOCARDIAL CONTRACTILITY by PENELOPE ANNE LONGHURST B.Sc, University of London, England, 1975 M.Sc, University of British Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FUIJILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1981 Penelope Anne Longhurst, 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s un d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f PHARMACOLOGY, FACULTY OF PHAI^CEUTTCAL SCIENCES The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 DE-6 (2/79) - i i -ABSTRACT Changing the temperature, frequency of stimulation and calcium concentration altered the dose-response curves for isoproterenol and histamine on guinea pig and rabbit papillary muscles. Basal developed force, maximal developed force and sensitivity to the agonists were a l l affected. Increasing the temperature stepwise from 25° to 42° resulted i n a progressive decrease i n BDF and sensitivity to the agonists. The response of MDF was different i n the guinea pig and rabbit. In the guinea pig, MDF was not affected by changing the temperature, and the size of the response to isoproterenol and histamine was similar. In the rabbit, the largest MDF response was seen at 37.5° when the calcium concentration was maintained at 2.2 mM. At. this calcium concentration the response to histamine was. less than that to isoproterenol at each temperature, although the difference was not significant. Increasing the frequency of stimulation stepwise from 0.5 to 4 Hz i n the guinea pig, and 0.2.to 3 Hz in the rabbit affected the dose-response curves i n a slightly different manner. In both species, BDF was reduced by low frequency stimulation. At other frequencies BDF was not changed. With isoproterenol, the MDF was only reduced by high frequency stimulation, and the sensitivity increased stepwise with increasing frequency. With histamine, the MDF was reduced by both low and high frequency stimulation. In the rabbit the response to histamine was consistently less than that to isoproterenol. The sensitivity to histamine was not affected by changing the frequency i n the guinea pig, but was increased by high f r e -quency stimulation i n the rabbit. - i i i -Increasing the calcium content stepwise from 1.5 to 8 mM i n the guinea pig, and 0.5 to 6 mM in the rabbit resulted in a progessive increase i n BDF, MDF and sensitivity. In both species, the increase i n MDF ap-peared to reach a maximum between 2.2 and 6 mM calcium. In the rabbit this effect was less noticeable i n situations where the frequency or tem-perature was also reduced. The response to histamine was reduced compared to that of isoproterenol. We postulate that the response to histamine i s reduced in rabbit papillary muscles due to stimulation of H^- receptors i n this tissue. It has been shown that stimulation of 3 and E^- receptors results in an increase i n cyclic AMP, while stimulation of H^- receptors has no effect on cyclic AMP. The increase i n cyclic AMP may enhance calcium binding by the SR resulting i n an augmented response.. In the guinea pig papillary muscle which contains 3- and H^- receptors, the inotropic responses to isoproterenol and histamine are similar, while i n the rabbit papillary muscle which contains 3- and H^- receptors, the response to histamine is reduced compared to that of isoproterenol. This difference may be due to the lack of cyclic AMP involvement in the response to histamine i n this tissue. Use of the calcium antagonist D600 produced a decrease in the sensi-t i v i t y of rabbit papillary muscles to isoproterenol, but did not depress the'.MDF. There was no difference i n the sensitivity to histamine. D600 therefore could not distinguish any difference i n dependence on extracellu-lar calcium between isoproterenol and histamine in this tissue. 45 Isoproterenol stimulated an increase i n Ca content of rabbit right ventricle strips at 2 minutes after administration. No effect could be - i v -detected at any other time, nor when histamine was used. It i s sugges-4 5 ted that at times greater than 2 minutes any increase in Ca content induced by isoproterenol was masked by a "pool saturation" phenomenon, and that this increase which was detected i s consistent with a difference in the mechanism of action of isoproterenol and histamine i n rabbit ventricular muscle. John H. McNeill, Thesis Supervisor. -V-TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES x i v INTRODUCTION 1 1. The effect of changing frequency of stimulation on cardiac contraction 1 2. The role of calcium i n cardiac contraction 5 3. The effect of calcium antagonists on cardiac :.contraction 16 4. The effect of temperature on cardiac contraction 20 5. The. effect of catecholamines on cardiac contraction 28 6. The effect of. histamine on cardiac contraction 34 7. The purpose of the current investigation 42 SUMMARY OF EXPERIMENTAL AIMS 44 METHODS 45 A. CONTRACTILE STUDIES 45 1. Preparation of tissues 45 2. FD,-Q measurements 46 3. Use of antagonists 46 B. RADIOISOTOPE STUDIES 47 1. Measurement of the extracellular space 47 45 2. Removal of extracellular Ca 47 45 3. Measurement of Ca uptake with time 52 4. Radioactive studies in stimulated tissues 57 C. STATISTICAL ANALYSIS 57 - v i -PAGE D. DRUGS AND CHEMICALS 57 Sci n t i l l a t i o n f l u i d 58 RESULTS 59 A. GUINEA PIG 59 1. Basal developed force 59 2. Maximal developed force 59 3. ED 5 Q data 62 B. RABBIT 63 I. The effect of changing frequency of stimulation and calcium concentration 63 1. Basal developed force 63 2. Maximal developed force 64 3. ED™ data 67 oU II. The effect of changing temperature and calcium concentration 68 1. Basal developed force 68 2. Maximal developed force 68 3. ED c n data 70 ou III. Use of D600 71 C. RADIOISOTOPE STUDIES 72 1. Measurement of the extracellular space 72 a. rabbit papillary muscles 72 b. rabbit l e f t a t r i a l and right ventricle strips 72 c. guinea pig right ventricle strips 72 2. Removal of extracellular calcium 73 a. rabbit right ventricle strips 73 b. guinea pig right ventricle strips 74 - v i i -PAGE 45 3. Ca uptake with time 74 a. quiescent tissues 74 b. stimulated tissues 76 DISCUSSION 157 1. The effect of changing temperature on myocardial contractility 157 2. The effect of changing .frequency of stimulation on myocardial contractility 161 3. The effect of changing calcium concentration on myocardial contractility 165 4. Summary 177 CONCLUSIONS 179 BIBLIOGRAPHY 182 APPENDIX I 195 A. Method of calculating ED^Q values 195 B. Use of Newman-Keuls test for multiple comparisons 197 C. Use. of Bonferroni st a t i s t i c s 203 APPENDIX II 206 - v i i i -LIST OF TABLES TABLE PAGE I . Upper significance limits' of the F-distribution (P=0.05) 199 II.. Upper 5% percentage points, Q, i n the studentized range 200 III , The distribution of t (two tailed tests) 205 IV . The -effect of changing temperature on the dose-response curve.of guinea pig papillary muscles to isoproterenol 206 V , The effect of changing temperature on the dose-response curve of guinea pig papillary muscles to histamine 210 VI • The effect of changing frequency of stimulation on the dose-response curve of guinea pig papillary muscles to isoproterenol 208 VII: The effect of changing frequency of stimulation on the dose-response curve of guinea pig papillary muscles to histamine. 211 VIII . The effect of changing calcium concentration on the dose-response curve of guinea pig papillary muscles to isoproterenol 212 IX The effect of changing calcium concentration on the dose-response curve of guinea pig papillary muscles to histamine 214 X . The effect of changing temperature, frequency of stimulation.and calcium concentration on the BDF and MDF to isoproterenol and histamine i n guinea pig papillary muscles 216 -ix-XI The effect of changing temperature, frequency of stimulation and calcium concentration on the % increase.of maximal developed force over basal developed force in guinea pig papillary muscles XII The effect of changing temperature, frequency of stimulation and calcium concentration on the EDJ-Q value for isoproterenol in guinea pig papillary muscles XIII The effect of changing temperature, frequency of stimulation and calcium-concentration on the. E D ^ value for histamine in guinea pig papillary muscles XIV The effect of changing the calcium concentration while stimulating at 0.2 Hz frequency on the dose-response curve of. rabbit papillary muscles to isoproterenol XV The. effect, of changing the calcium concentration while stimulating at 0.5 Hz frequency on the dose-response curve of rabbit papillary muscles to i soproterenol XVI The effect of changing the calcium concentration while stimulating at 1 Hz frequency on the dose-response curve of rabbit papillary muscles to isoproterenol XVII The effect of changing the calcium concentration while stimulating at 3 Hz frequency on the dose-response curve of rabbit papillary muscles to isoproterenol -X-PAGE XVIII The effect of changing the calcium concentration while stimulating at 0.2 Hz frequency on the dose-response curve of rabbit papillary muscles to" histamine 225 XIX The effect of changing the calcium concentration while stimulating at 0.5 Hz frequency on the dose-response curve of rabbit papillary muscles to histamine 226 XX . The effect of changing the calcium concentration while stimulating at 1 Hz frequency on the dose-response curve of rabbit papillary muscles to histamine 227 XXI The effect of changing the calcium concentration while stimulating at 3 Hz frequency on the dose-response, curve of rabbit papillary muscles to histamine 228 XXII The effect of changing frequency of stimulation and calcium concentration on the BDF and MDF to isopro-terenol, and histamine in rabbit papillary muscles 229 XXIII : The effect of changing frequency of stimulation and calcium concentration, on the % increase of maximal developed force over basal developed force in rabbit papillary muscles 230 XXIV The. effect of changing frequency of stimulation and calcium .concentration on the ED^ n, value for isopro-terenol in rabbit papillary muscles 231 -xi-P A G E XXV The effect of changing frequency of stimulation and calcium concentration on the E D 5 Q value for histamine in rabbit papillary muscles 231 XXVI The effect of changing the calcium concentration while maintaining the temperature at 25° on the dose-response, curve for isoproterenol in rabbit papillary muscles 233 XXVII The effect of changing the calcium concentration while maintaining the temperature at 37.. 5° on the dose-response curve for isoproterenol in rabbit papillary muscles 234 XXVIII The effect of changing the calcium concentration while maintaining the temperature at 42° on the ..dose-response, curve for isoproterenol in rabbit papillary muscles 235 XXIX The effect of changing the calcium concentration while maintaining the temperature at 25° on the dose-response, curve for histamine in rabbit papillary muscles 236 XXX The effect of changing the calcium concentration while maintaining the.temperature at 37.5° on the dose-response curve for histamine in rabbit papillary muscles 237 XXXI The effect of changing the calcium concentration while maintaining the temperature at 42° on the dose-response curve for histamine in rabbit papillary muscles 238 - x i i -PAGE XXXII The effect, of changing temperature and calcium concentration .on the BDF and MDF to isoproterenol and histamine in rabbit papillary muscles 239 XXXIII The effect of changing temperature and calcium concentration on the % increase of maximal developed force over.basal developed force in rabbit papillary muscles 240 XXXIV The effect of changing temperature and calcium concentration on.the E D ^ Q value for isoproterenol in rabbit papillary muscles 241 XXXV The effect of changing temperature and calcium concentration on the ED c n value for. histamine in DU rabbit papillary muscles 242 XXXVI The effect of 60 minutes pretreatment with D600 on the dose-response curves of isoproterenol and histamine in rabbit.papillary muscles 243 14 XXXVII C-sorbitol measurement of the extracellular space of rabbit- papillary muscles 245 14 XXXVIII C-sorbitol measurement of the extracellular space of rabbit left atrial and right ventricle strips 246 14 XXXIX- C-sorbitol measurement of the extracellular space of guinea pig right ventricle strips 247 45 XXXX Determination of Ca content of rabbit right ventricle strips with time using different quenching solutions 248 45 XXXXI Compartmental analysis of the release of Ca from rabbit right ventricle strips 250 - x i i i -PAGE 145 XXXXII Determination of Ca content of rabbit right ventricle strips with time using modified Chenoweth-Koelle solutions 252 XXXXIII Determination of Ca content of guinea-pig right ventricle strips with time using modified Chenoweth-Koelle solutions 253 45 XXXXIV Measurements of- Ca uptake into quiescent rabbit right ventricle strips 254 45 XXXXV Measurement of Ca uptake by quiescent tissues over 2-10 minutes 255 45 XXXXVI Measurement of Ca uptake by quiescent tissues after 80 minutes loading followed by 10 minutes treatment 256 45 XXXXVII Measurement of Ca uptake by stimulated tissues over 2-4 minutes 257 45 XXXXVIII Measurement of Ca uptake by.stimulated tissues after 80 minutes loading followed by 10 minutes treatment 258 -xiv-LIST OF FIGURES PAGE  FIGURE 1. Proposed mechanism of action of catecholamines in glycogenolysis and cardiac contraction 32 2. Schematic model for calcium involvement in the inotropic responses to isoproterenol and histamine 39 3. Method for measurement of the extracellular space 49 4. Method for removal of extracellular calcium 51 45 5. Method for measurement of Ca uptake over 2-10 minutes 54 45 6. Method for measurement of uptake of Ca following loading of the tissue 56 7. The effect of changing temperature, frequency of stimulation and calcium concentration on the basal developed force.of guinea pig papillary muscles;. 78 8. The effect of changing temperature on the dose-response curves of isoproterenol and histamine in guinea pig papillary muscles 80 9. The effect of changing temperature on the maximal developed force of guinea pig papillary muscles to isoproterenol and histamine 82 10. The effect of changing frequency of stimulation on the dose-response curves of isoproterenol and hista-mine in guinea pig papillary muscles 84 -XV-P A G E 11. The effect of changing frequency of stimulation on the maximal developed force of guinea pig papillary muscles to.isoproterenol and histamine 86 12. The effect of changing calcium concentration on the dose-response curves of isoproterenol and hista-mine in guinea pig papillary muscles 88 13. The effect of changing calcium concentration on the maximal developed force of guinea pig papillary muscles to isoproterenol and histamine 90 14. The effect of changing temperature,, frequency of stimulation and calcium concentration on the % increase of maximal developed force over basal developed force in guinea pig papillary muscles 92 15. The effect of changing temperature, frequency of stimulation and calcium concentration on the EDJ-Q value.for isoproterenol in guinea pig papillary muscles 94 16. The effect of changing temperature, frequency of stimulation, and calcium concentration on the EL\-Q value for histamine in guinea pig papillary muscles 96 17. The effect of changing frequency of stimulation and calcium concentration on the basal developed force of rabbit papillary muscles 98 18. The effect of changing frequency of stimulation and calcium concentration on the dose-response curve of -xvi-P A G E isoproterenol in rabbit papillary muscles 100 19. The effect of changing frequency of stimulation and calcium concentration on the maximal developed force of rabbit papillary muscles to isoproterenol 102 20. The effect of changing frequency of stimulation on the dose-response curve of histamine in rabbit papillary muscles 104 21. The effect of changing frequency of stimulation and calcium concentration on the maximal developed force of rabbit papillary muscles to histamine 106 22. The effect of changing frequency of stimulation and calcium concentration on the % increase of maximal developed force over basal developed force in rabbit papillary muscles 108 23. The effect of changing frequency of stimulation and calcium concentration on the E D ^ Q value for isoproterenol in rabbit papillary muscles 110 24.. The effect of changing frequency of stimulation and calcium concentration on the ED,--, value for histamine oU in rabbit papillary muscles 112 25. The effect of changing temperature and calcium concen-tration on the basal developed force of rabbit papillary muscles 114 26. The effect of changing temperature and calcium concen-tration on the dose-response curve of isoproterenol in rabbit papillary muscles 116 -xvii-P A G E 2 7 . The effect of changing temperature and calcium concentration on the maximal developed force of rabbit papillary muscles to isoproterenol 1 1 8 2 8 . The effect of changing temperature and calcium concentration on the dose-response curve of histamine in rabbit papillary muscles 1 2 0 2 9 . The effect of changing temperature and calcium concen-tration on the maximal developed force of rabbit papillary muscles to histamine 1 2 2 3 0 . The effect of changing temperature and calcium concen-tration on the % increase of maximal developed force over basal developed force in rabbit papillary muscles 1 2 4 3 1 . The effect.of changing temperature and calcium concen-tration on the E D ^ Q value for isoproterenol in rabbit papillary muscles, 1 2 6 3 2 . The effect of changing temperature and calcium concen-tration on the E D 5 0 value for histamine in rabbit papillary muscles 1 2 8 3 3 . The effect of 6 0 minutes pretreatment with D 6 0 0 on the dose-response curves of isoproterenol and histamine in rabbit, papillary muscles 1 3 0 1 4 3 4 . C-sorbitol measurement of the extracellular space of rabbit papillary muscles 1 3 2 1 4 3 5 . C-sorbitol measurement of the extracellular space of rabbit left atrial and right ventricle strips 1 3 4 - x v i i i -PAGE 14 36. C-sorbitol measurement of the extracellular space of guinea pig right ventricle strips 136 45 37. Residual Ca content of rabbit right ventricle strips as a function of incubation time using different isotope-free quenching solutions 138 45 38. Compartmental analysis of the release of Ca from rabbit right ventricle strips into isotope-free solutions at 0° and 37° 140 45 39. Residual Ca content of rabbit right ventricle strips as a function of incubation time using modified Chenoweth-Koelle solutions 142 45 40. Residual Ca content of guinea pig right ventricle strips as a function of incubation time using modified. Chenoweth-Koelle solutions 144 45 41. Measurement of Ca uptake with quiescent rabbit right ventricle strips 146 45 42. Measurement of Ca uptake by quiescent tissues over 2-10 minutes 148 45 43. Measurement of Ca uptake by quiescent tissues after 80. minutes loading followed by 10 minutes treatment 150 45 44. Measurement of Ca uptake by stimulated tissues over 2-4 minutes 152 45 45. Measurement of Ca uptake by. stimulated tissues after 80 minut s l ading follow d by. 10 minutes treatment 154 -xix-PAGE 46. Representative tracings-of the contractile response of rabbit right ventricle strips to ten minutes exposure to 126 mM K, isoproterenol or histamine 156 ACKNCWIZDGEMENTS I am deeply grateful to Dr. John H. McNeill for his guidance and patience over many years. I would like to thank Dr. Casey van Breemen for allowing me to 45 carry out the Ca studies in his laboratory, and for his financial support during my stay in Miami. Also Dr. Vladimir Palaty for his valuable help in interpreting the data. I owe special.gratitude to Judy Wyne for her expert typing skills. The financial support of the Canadian Heart Foundation is gratefully acknowledged. -1-^ . - INTRODUCTION 1. The Effect of Changing Frequency of Stimulation on Cardiac Contraction The Bowditch staircase phenomenon or Treppe was fi r s t described in 1871. Bowditch showed that following a period of rest, application of repetitive stimuli to frog, cardiac muscle produced progressively greater contractions until a plateau was reached. Several theories were put forward in the early part of the twentieth century to explain the pheno-menon. Mines (1913a) suggested that under normal conditions there was an optimal hydrogen ion concentration (pH) for tissues. His theory was that changing pH of the medium produced effects depending on whether the pH became closer to the optimal value (when the contractions should become larger), or further away from the optimal pH (when the contractions should become smaller). The staircase effect was attributed to lactic acid production by the muscle, resulting in local changes in pH. Mines suggested that as the frequency of stimulation was increased, i t became more difficult for the lactic acid to diffuse away. This would then increase the'contraction i f the medium was previously on the alkaline side of the optimal pH. Dale in 1930 demonstrated the staircase phenomenon in rabbit ventricular muscle. Increasing the frequency of stimulation from 25 to 50 beats per minute produced an i n i t i a l decrease in the tension developed by the beat following the change. With subsequent beats the tension gradually increased to a plateau where the tension developed was larger than that seen at the lower frequency. When the change was reversed, from 50 to 25 beats per minute, the first beat was larger. Subsequent beats became progressively smaller, reaching a plateau which was smaller -2-than that seen before. Dale (1932) found in the rabbit ventricle that changes in pH, and halving or doubling the normal potassium or calcium levels had no effect on the ratio of tension developed at 60 beats per minute to that at 30. In the frog ventricle, the staircase phenomenon was not altered by changing the pH (which disproved Mines' theory), or addition of atropine (with which Bowditch claimed the staircase could be reversed). In addition the pattern of changes seen when the frequency was altered differed from the rabbit. When the frequency was doubled, in the frog the fi r s t beat was larger than its predecessor whereas in the rabbit i t was smaller. With subsequent beats force continued to increase until a plateau was reached. VJhen the frequency was halved, the fi r s t beat was reduced while in the rabbit i t was increased, and a l l sub-sequent beats were progressively reduced until a plateau was reached which was lower than the original tension developed. Dale supported the theory that the staircase phenomenon was due to "a cumulative action of the contractions." She suggested that this was brought by accumulation of some metabolic product which favoured contraction. During the rising phase of the staircase this substance would be formed more rapidly than i t could diffuse away, resulting in increased tension development.. Then as the concentration increased further the rate of diffusion would also increase until a point was reached where the rate of diffusion and formation were in equilibrium. At this point the plateau would form. On the descending phase of the staircase, the substance was formed at a slower rate than the diffusion process, resulting in. decreased tension development. The theory could explain the data obtained for the frog heart, but was unable to explain the increased f i r s t beat in the rabbit following halving of the frequency. -3-In 1961-63, Koch-Weser and Blinks attempted to analyze the interval-strength relationship more closely (Blinks and Koch-Wester, 1961; 1963; Koch-Weser, 1963; Koch-Weser and Blinks, 1963). They showed (Koch-Weser and Blinks, 1963) that the kitten atrial strip and papillary muscle responded to changes in frequency of stimulation in a manner similar to that described for the rabbit by Dale (1932). When the frequency was decreased the next beat was larger, and when the frequency was increased the next beat was smaller. It was apparent however that when more frequencies were examined, the staircase phenomenon was somewhat differ-ent in the kitten atrial and ventricle strips. In the atrial strips the interval-force curve was "triphasic. At very low frequencies between 1 beat every 5 minutes and 1 beat every 90 seconds the .".tens ion developed was large. At these" frequencies the period of rest between contractions is of sufficient length that the strength of the contraction is indepen-dent of previous beats. These contractions were therefore termed rested-state. Between frequencies of 1 beat every 90 seconds and 19 beats per minute the tension developed decreased steadily. As the frequency was further increased, the tension increased again reaching a maximum at about 120 beats per minute which was lower than the rested state contrac-tion, and decreasing again as the frequency was raised further. The terms optimum and pessimum frequency were introduced to describe frequen-cies where the contractions were largest and smallest respectively. A similar interval-force curve was seen in the guinea pig left atrium • (Blinks and Koch-Weser, 1961). In contrast, in the kitten papillary muscle the curve • was monophasic. In addition the rested state contraction was extremely weak. The tension started to increase at a frequency of 3 beats per minute reaching a plateau between 120 and 300 beats per minute. -4-Koch-Weser and Blinks (1963) also confirmed Dales' observations in the frog that when the frequency of stimulation was decreased the next beat was smaller, and when the frequency was increased the next beat was larger. The interval-force curve for the frog ventricle was similar to that described for the kitten papillary muscle. The rested state contraction was again extremely weak. As the frequency was increased up to 24 beats per minute the tension developed increased, but at higher frequencies decreased. Koch-Weser and Blinks (1963) introduced the concept of the positive inotropic effect of activation (PIEA) and. negative inotropic effect of activation (NIEA) to explain the interval-force curves. These 2 inter-val-dependent processes were considered to affect myocardial contractil-ity by acting in opposite directions. The NIEA decreased contractility and became prominent as the interval between beats was lengthened. The PIEA increased contractile strength, becoming prominent as the interval between beats.was shortened. Both changes were postulated to disappear with time (not.necessarily at the same rate) and be capable of cumulation. In addition they were algebraically additive, unless some limit on the extent of contractile ability of the muscle prevented this. Under normal circumstances the contractile force of each beat was equal to the resting state contraction + PIEA - NIEA. For the atrial tissues during the portion of the force-interval curve from 1 beat every 90 seconds to 19 beats per minute where the tension decreased from a large resting state contraction to the pessimum, Koch-Weser and Blinks postulated that NIEA was cumulating to a greater extent than PIEA, due to the long interval between the contractions. As the frequency was increased further up to.120 beats per minute, there -5-was additional cumulation of PIEA, finally reaching a point where i t exceeded NIEA and resulted in increased tension development. The further decline in tension at higher frequencies was suggested to result from renewed predominance of NIEA cumulation. In the kitten ventricular tissues, due to the very small rested state contraction, accumulation of PIEA was more obvious. Koch-Weser and Blinks suggested that more PIEA was produced by each beat than was in the atrium, so that at a l l stages the cumulation of PIEA exceeded that of NIEA. In the frog ventricle, they postulated that there was no NIEA present. Over the range covered by a frequency of 1 beat every 90 sec-onds to 18 beats per minute the increased tension development was suggested to be totally due to PIEA cumulation. The decline in tension at higher frequencies was thought to be due to the decreased duration of the active state. Hollander and Webb (1955); Abbott and Mommaerts-. (1959) ,.arid Koch-Weser (1963) a l l demonstrated that as the frequency of stimulation was increased there was a reduction in the duration of the active state, time to peak tension and relaxation time. More recent work on the Bowditch staircase phenomenon suggests that calcium ions play a major role in its production. These findings will be discussed in the next section. 2. The Role of Calcium in Cardiac Contraction The requirement for calcium to maintain myocardial contractility was f i r s t recognized.by Ringer in. 1882. He showed that removal of calcium from the bathing solution surrounding a frog heart resulted in cardiac arrest. This is in contrast to skeletal muscle, where contractions s t i l l occur for some time in the absence of calcium. Later work by -6-Mines (1913b) showed that although the beating of the heart stopped quite rapidly (within a few beats) following calcium removal, electrical activity continued for some time. By the 1960's i t was apparent that calcium was an absolute require-ment for excitation-contraction coupling in a l l mammalian muscles. (For reviews see Ebashi and Endo, 1968; H.E. Huxley, 1969; A.F. Huxley, 1974). Niedergerke in 1963 carried out experiments in frog heart which demon-45 strated that beating ventricular muscle accumulated Ca, and that the amount taken up was increased when either Ca was increased or Na was r o o decreased. These findings supported the then current hypothesis that calcium entering the cell during an action potential was responsible for the muscle contraction, i.e. that calcium coupled excitation with contraction. The study was extended to mammalian tissues by Winegrad and Shanes (1962), who discovered that contractions of guinea pig atria were accom-45 panied by a .large Ca uptake. Alterations of the strength of contraction (by changing the frequency or calcium concentration) were accompanied by 45 45 proportional changes in Ca uptake, although the total Ca content was unchanged. They postulated that the calcium entry into the cell during excitation resulted in inhibition of a relaxing factor (previously postulated by Ebashi in 1960 to exist in skeletal muscle) and therefore initiated contraction. Similar work by Grossman and Furchgott (1964a, b, c) verified the observations of Winegrad and Shanes that the calcium content of the guinea pig left atrium was divided into a least 3 compartments or stores: i) a rapidly exchangeable fraction which was probably extracellular; i i ) a slowly, exchangeable fraction which was probably intracellular; -7-and i i i ) a fraction which appeared to be non-exchangeable or "unaccounted for" calcium and which fraction Grossman and Furchgott (1964a) suggested included the calcium associated with contraction. 45 Grossman and Furchgott supported the finding that Ca exchange increased at a higher frequency, although the total calcium content was not dissimilar from resting tissues. Similar findings were reported by Langer (1965) and Sands and Winegrad (1970). They also 45 (1964c) examined the effect of various drugs on Ca exchange. Use of acetylcholine, noradrenaline and stropanthin-- K produced changes in contractile strength which were unaccompanied by changes in total calcium content. Acetylcholine depressed both contractile force and calcium exchange. These effects were both antagonized by atropine. Noradrenaline and stropanthin produced significant increases in calcium exchange accompanied by contraction. In 1967, Reuter first produced evidence suggesting that a slow inward current, carried at least in part by calcium occurred in cardiac muscle at a time just after the spike of the action potential. Later work (Beeler and Reuter, 1970), led them to suggest that calcium entered the cell during the plateau phase of the action potential, and was then stored intracellularly. This calcium was released by an unknown mecha-nism to e l i c i t contraction. Wood, Fleppner and Weidmann (1969) formu-lated the hypothesis that the calcium entering the cell during the action potential produced contraction i) directly and/or i i ) due to release of intracellularly bound calcium. This calcium was released from the contractile proteins and stored in the sarcotubular system or extruded from the cell in order for relaxation to occur. In this manner, subsequent depolarizations could build up the amount of calcium -8-bound in the intracellular? stores and available for contraction. In a similar manner, the contracture induced by high potassium concentra-tions is thought to be due to persistent depolarization resulting in an increased calcium influx (Haeusler et al . , 1968; Morad and Orkand, 1971). Wood, Heppner and Weidmann (1969) likened the calcium entry into the cell to the PIEA of Koch-Weser and Blinks (1963). The NIEA was attributed to the relative slowness of calcium redistribution from intracellular surfaces to the sarcotubular network from which i t could be released during the next action potential. The inotropic state of the tissue was considered to be proportional to the amount of calcium present in the internal stores. The site and number of internal calcium stores, and the mechan-ism by which calcium is released from them has provoked much interest during the last decade. In the early 1970's a 2 compartment model was proposed (Bassingthwaite and Reuter 1972; Tritthart et al. , 1973; Kaufmann et al . , 1974; Edman and Johannsson, 1975). During membrane depolarization calcium entered the cell through the slow channel (Reuter, 1967; Beeler and Reuter, 1970). The quantity of calcium entering was considered not to be of sufficient quantity to activate the contractile proteins directly (Winegrad and Shanes, 1962; Grossman and Furchgott, 1964b; Manring and Hollander, 1971; Bassingthwaite and Reuter, 1972; Reuter, 1974a), and was probably distributed.to various internal calcium stores labelled compartment 1. The depolarization induced by the action potential would then spread down the t-tubules to. release calcium from other stores labelled compartment 2, probably located in the lateral cisternae of the sarcoplasmic reticulum (SR). Following release from -9-the contractile proteins and relaxation, calcium was taken up by an active process into compartment 1, probably located in another region of the SR, and subsequently transferred to compartment 2 for release to produce the next contraction. This transfer between the 2 compart-ments, making the calcium available for contraction was postulated to take a certain period of time. This would explain why a premature beat is reduced in size, since.the store might not have sufficient time to r e f i l l . Calcium not stored intracellularly was extruded from the cell by a sodium-calcium exchange (Langer, 1968; Glitsch et a l . , 1970). In addition Manring and Hollander (1971) assumed the presence of an active pump, which extruded calcium from compartment 2 to the outside. The model of Edman and Johannsson (1975) differed slightly in that i t assumed that calcium was extruded from both compartments to the outside. ' Calcium is thought to be transported into the SR from the cytosol by means of a Mg-dependent Ca-ATPase, similar to that described for skeletal muscle (Tada et al . , 1978; Sumida et al., 1980).. This same Ca-ATPase may be present on the sarcolemma (SL) (Tada et al . , 1972; Sulakhe and St. Louis, 1976), although in cardiac cells Na-Ca exchange seems to be more important (Reuter, 1974; Langer, 1976; Chapman and Tunstall, 1980). Inside the SR, calcium is bound to acidic proteins which may include calsequestrin and a "high affinity calcium-binding protein." These proteins produce, a reduction in the gradient of calcium ions across the SR membrane (Tada et al . , 1978). Some calcium is, in addition stored in mitochondria, but i t is not considered to be of importance in the normal regulation of myocardial contractility (Carafoli, 1975). Currently the most popular theory of the mechanism of calcium release from SR stores is that of calcium-induced calcium release or trigger calcium (Fabiato and Fabiato, 1972, 1975a). The theory suggests that the calcium entering the cell during the action potential induces release of additional calcium from the SR which then activates the contractile proteins. They demonstrated that in skinned cardiac fibres where the SL was removed smaller amounts of calcium were required to produce a contraction, than were required for direct activation of the contractile proteins. In a later paper (Fabiato and Fabiato, 1978), they demonstrated variations among species in the threshold of free calcium required to induce calcium release, amount of calcium released and rate of calcium reaccumulation. From their data they constructed models to explain the interactions of 1) the transsarcolemmal calcium current and Na.:Ca exchange, 2) the calcium-induced release of calcium from the SR, and 3) the calcium binding to the SR. They found that skinned cells of rat, cat, dog, rabbit and human atria, and rat ventricle had a similar low threshold for free calcium required to induce calcium —8 —7 release from the SR (range about 3 x 10 to 1 x 10 M). They suggested that this was the reason why the plateau phase of the action potential is of short duration in these tissues. With skinned ventricle cells -of the cat, dog, rabbit and human, the threshold for calcium-induced calcium release increased steadily and at a higher free calcium concen-tration than that seen for the atria. The threshold for the rabbit ventricle occurred at a much higher calcium.concentration (about 3 x _7 10 M) than any other tissue. Fabiato and Fabiato proposed that this could, explain the lower rates of tension development seen in the rabbit ventricle compared to ventricles of other species. -11-In addition., a progressive increase in the threshold for free calcium (such as was seen most extremely for the rabbit ventricle) was accompanied by a progressive decrease in the amount of calcium released from the SR, and progressive decrease in the rate of calcium reaccumulation by the SR. They found that in rabbit ventricle they could alter these characteristics to give values which closely resembled the rat ventricle. This was achieved by enhancing calcium accumulation and by release from the SR by changing the pH from 7.00 to 7.40, or by adding cyclic AMP which has been shown to enhance the rate of calcium accumulation by the SR (Fabiato and Fabiato, 1975b). Conversely, by decreasing the pH from 7.00 to 6.60 or decreasing the temperature, 2 procedures which decrease the rate of calcium accumu-lation by the SR, the rat ventricle could be made to behave more like rabbit ventricle. Data obtained for the frog ventricle led them to suggest that in this tissue contraction was brought about directly by the calcium entering the cell during the action potential.. Additional studies pertinent to this discussion were carried out by Naylor and co-workers (1975) who demonstrated.that guinea pig and rabbit ventricle micro-somal fractions accumulated and released calcium at a relatively slow rate compared to rat ventricle. Rat microsomes bound calcium at a + 2+ mean net rate of 8.82 - 0.66 nmol Ca /mg protein/second during the fi r s t 5 seconds of incubation compared to 3.92 - 0.55 and 4.53 - 0.78 nmol/mg protein/second for the rabbit and guinea pig respectively. The mean net rate of calcium release similarly was 27.70 - 3.60 nmol 2+ + Ca released/mg protein/minute in the rat compared to 3.21 - 0.24 and 12.50 - 1.20 nmol/mg protein/minute in the rabbit and guinea pig. -12-In addition the onset of net calcium release was significantly-shorter in the rat than in the guinea pig or rabbit, the rabbit having the longest time of onset of calcium release of the 3 species. These findings complement those of Fabiato and Fabiato (1978) who, although they did not study the guinea pig did find that the rat and rabbit ventricle represented the 2 extremes of the species studied. Another series of studies showing species differences are those involving cardiac ATPase activity. Yazaki. and Raben in 1974 published data showing that ventricular myosin Ca-ATPase activity increased in dif-ferent species in the ascending order rabbit < dog < rat < mouse. There was no difference in the activities of skeletal muscle ATPase. A similar experiment by Delcayre and Swynghedauw (1975) correlated increasing Ca-ATPase activity in the species rabbit < dog < guinea pig < rat with an increase in the V.max of myocardial contractility reported by Henderson et al . , (1970).. Similarly Hjalmarson et al . , (1970) and Dowell (1976) showed that decreasing the maximum dP/dt by hypophysectomy, sympathectomy and adrenalectomy could.be correlated with decreases in. rat myosin ATPase activities. In general then, an increase in myosin ATPase activity seems to be associated with an * increase in the rate of shortening among species, although Naylor et a l . , (19.75). found l i t t l e difference between myofibrillar Ca-ATPase activities of rat, guinea pig and rabbit. In a more recent study Yazaki (Yazaki et.al., 1979) found, a higher level of myosin Ca-ATPase activity in atria compared to ventricles of rat, rabbit, dog and human hearts. The ascending order of activities in both atria and ventricles was dog = human = rabbit < rat, although the activities were two-fold higher in atria. This greater activity in atrial -13-tissue appears to be correlated with the rate of tension development, which is known to be greater in atria than ventricles. An alternative method of calcium release from the SR which has already been alluded to is that of depolarization-induced release. Ochi and Trautwein (1971) working with the sucrose gap technique on guinea pig papillary muscles came to the conclusion that the ampli-tude and duration of depolarization was more important for triggering contractions than was the slow inward current. They found that the amplitude of the contraction increased with increasing depolarization even when the slow inward current decreased as the membrane approached the calcium equilibrium potential. Morad and Orkand (1971) using a modified sucrose gap technique on frog ventricle strips have also shown that tension increased as the membrane was progressively depolar-ized, and that this was independent of the magnitude of the inward current, which was in fact reversed at +60 mV. The mechanism by which the depolarization induces contraction is not clearly understood. In skeletal muscle, the current hypothesis is that the depolarization spreads down the t-tubules to the SR, resulting in calcium release due to a change in the permeability of the membrane to calcium (Ebashi, 1976). It is possible that at least part of the calcium released from cardiac intracellular stores is by a similar mechanism. Edman and Johannsson (1976) suggested that increasing the duration of depolarization per unit time resulted in an increased inflow of calcium into the cell. This can be seen with increasing rate. This calcium was then taken up by a 2 compartment system and redistributed for release during the next action potential. By increasing the duration -14-of depolarization per unit time, the increased calcium influx would result in an increase in calcium in compartment 2, for release and activation of the contractile proteins providing that the rate was not so high as to interfere with uptake. Langer is the strongest proponent of the theory that the calcium which interacts with the contractile proteins is mainly derived from SL stores rather than SR stores (Langer, 1976, 1980; Langer et al., 1976). They postulate that calcium is bound to negatively charged sites on the outside of the SL, and that these calcium stores are made available for calcium entry into the cell either through the calcium channels or via the sodium-calcium exchange. Langer has shown that the ability of a number of di- and tri-valent cations to displace calcium from SL sites correlates well with their ability to uncouple excitation-contraction coupling (Bers and Langer, 1979). In addition, the sialic acid moities of the glycocalyx appear to play a rate-limiting role in control of calcium flux across the membrane, as well as representing a large potential calcium store (Langer, 1980). In concluding the direct role of calcium in cardiac contraction I would like to.present the model, of Noble (1979) for calcium compart-ments, and movement of calcium ions in cardiac muscle. Noble based his model partially on the data obtained by Edman and Johannsson (1976) for long stimulation intervals on rabbit papillary muscles. He sugges-ted that there must be a gradual loss of calcium from compartment 2 to account for the steady decline in tension of ventricular muscle following an increased interval between stimuli. However, since the rested state contraction never falls to zero tension, some portion of compartment 2 must be leak-proof and contain a certain fraction of calcium which is never lost. He therefore suggested that compartment 2 contained a subcompartment labelled R for residual, resistant or "rested state calcium". In addition he postulated the presence of a second compartment taking up intracellular.calcium during relaxation and extruding i t to the outside only (compartment 3). The mechanism for this extrusion was not discussed but would probably include Na: Ca exchange and the Ca-ATPase. The amount of calcium extruded from compart-ment 3 increases to equal any increase in calcium influx in order to maintain a steady state. The existence of such a mechanism was suggested by Niedergerke (1963). The staircase phenomenon was explained by postulating a time lag between calcium entry and the stimulation of calcium efflux from compartment 3. Therefore there would be an increase in intracellular calcium due to the increased depolarizations, resulting in an increased tension development and finally reaching a steady state plateau as efflux from compartment 3 started. In addition to its direct role in activating the contractile proteins, calcium may also act indirectly, through stimulation of myosin light chain kinase. Stull et a l . , (1980) propose that calcium and calmodulin bind to form a complex. This complex subsequently binds to the kinase to form an active enzyme complex which then phosphorylates the P-light chains of the myosin head. The dephosphorylation of the light chains is brought about by the enzyme myosin light chain phospha-tase. However;in cardiac muscle, the interaction of myosin and actin is not dependent on phosphorylation of the P-light chains, in contrast to the situation thought to be present in smooth muscle. Therefore the role of myosin phosphorylation in cardiac muscle is somewhat obscure. -16-Stull (Stull et al . , 1980) suggests that in skeletal muscle, since the rate of phosphorylation of the light chains is fairly slow, requiring a few contractions to become maximal, i t may play a role in enhancing contractile activity during sustained exercise. A similar situation may be found in the heart where activation of the protein kinase and phosphorylation of the P-light chains is thought to be analogous to that of skeletal muscle. In concluding this section, i t should be pointed out that although the role of calcium is obvious, the various mechanisms by which this role is achieved have s t i l l not been clearly elucidated. At present i t seems that the best choice available to us is to remain open minded and to allow for a l l the various theories of calcium release, interactions, sequestrations etc. to be come incorporated in a general model of calcium's action in cardiac contraction. 3. The Effect of Calcium Antagonists on Cardiac Contraction. Fleckenstein CFleckenstein et al. , 1971) introduced the term calcium antagonists to describe drugs such as verapamil and methoxyverapamil (D600) which inhibited the slow inward current carried by calcium ions; In one of the earliest papers showing an effect of calcium antag-_7 onists on the heart, Endoh and co-workers (1975) showed that 10 M and _7 •- • 3 x 10 M D600 shifted the dose-response curves for phenylephrine and isoproterenol on rabbit papillary muscles to the right. It also markedly depressed the basal tension and the maximal response to phenylephrine, but not to isoproterenol. The similarity between the depression of the force-interval curve produced in the presence of D600 or by increasing the temperature led them to suggest that both increased temperature and -17-D600 might inhibit calcium movements across the SL. The different effects of D600 on phenylephrine and isoproterenol they attributed to the fact that phenylephrine acted mainly by stimulating calcium fluxes, while isoproterenol could in addition cause calcium release from intra-cellular stores. Bayer and his co-workers (1975a, b, c) carried out an extensive investigation of the effects of verapamil and D600 on cat papillary muscles. Both agents drastically affected the staircase phenomenon. In the control situation, increasing the frequency of stimulation stepwise from 6 to 60 beats per minute produced a positive staircase which rapidly reached a steady state. In contrast 2 mg/ml of (.-) vera-pamil or D600 converted the response into a negative staircase, which only slowly reached a steady state. The pattern of response seen when the extracellular calcium was reduced from 2.5 to 0.75 mM, or 0.4 mM lanthanium or 1.0 mM nickel was added to the bath was quite different. The amplitude of the response was reduced compared to the control, but the staircase remained positive. Increasing the calcium concentration to 7.5 mM did not reverse the negative staircase seen in the presence of verapamil, but increased the amplitude of the contractions. One interesting.observation that they made was. that i f 5 mg/ml verapamil was administered to a tissue stimulated at 6 beats per minute in the pres-ence of 2.5 mM calcium, the amplitude was barely changed. If the fre- . quency was increased to 60 beats per minute, the amplitude was almost totally reduced. Reestablishment of a frequency of 6 beats per minute resulted in a potentiated f i r s t beat. .Subsequent beats gradually declined in amplitude to reach a steady state where the tension developed was the same as that seen originally at 6 beats per minute. In addition, -18-i f the muscle was stimulated at 60 beats per minute and a test contrac-tion elicited 2, 4 or 8 seconds after'rhythmic stimulation'was stopped, verapamil and D600 produced quite unusual effects. In the control situation, or in the presence of 0.5 mM calcium, 1.0 mM nickel or 0.2 mM lanthanum the test contractions were only a l i t t l e larger than the previous contractions, and the amplitude of the contractions at 2, 4 and 8 seconds were similar. However, in the presence of 5 mg/ml Verapamil or D600 the test contractions were several times larger than the previous contractions, and also increased greatly with each increase in the resting interval (Bayer et al. , 1975a). These strange findings made i t obvious that verapamil and D600 besides having effects on the slow inward current, also had some other site(s) of action which affected cardiac exitation-contraction. Using computer-generated curves they calculated that verapamil and D600 must in addition to affecting calcium influx, be reducing the translocation of calcium from compartment 1 of the SR to compartment 2, from which i t would be released and produce contraction. This could then explain why the contractions following a pause at high frequency were potentiated, and why at low frequencies the contractions were barely affected by the calcium antagonists. In their second .paper (Bayer, et al., 1975b) they showed that (-) and (-) - verapamil both changed the staircase phenomenon to a negative staircase as described previously. In contrast, the (+) - isomer had no effect on the tension developed, and the staircase remained positive at doses up to 10 mg/ml. The optical isomers of D600 gave similar results to those of verapamil, but were about 8 times more potent. (-) -verapamil and D600 appeared to affect the translocation of calcium in a manner similar to the racemic compounds, but the (+) - isomers did -19-not seem to. have any effect on i t . They therefore suggested that the negative inotropic effects of the racemic compounds were primarily determined by the (-) - isomer. Further studies on (+) - verapamil and D600 showed that at doses greater than 10 mg/ml the threshold of excitation was increased. This was particularly noticeable at frequencies greater than 30 beats per minute. Electrophysiological studies (Bayer et a l . , 1975c) showed that the (-) - isomers depressed the plateau phase of the action potential, while the (+) - isomers decreased the maximum velocity of depolarization. This depression became increasingly prominent as the frequency was increased from 15 to 90 beats per minute. The racemic isomers produced the characteristics of both the (+) - and (-) - isomers. Their data led them to conclude that the (+) - isomers of verapamil and D600 were inhibitors of the sodium channels, with l i t t l e or no calcium antagonistic activity, while the (-) - isomers had l i t t l e or no effect on the sodium channel. Reinhardt et al.., (1977) using guinea pig left atria and (.-) - D600 _7 found that 10 M D600 significantly depressed the interval-force curve in this tissue also. The depression was most noticeable at stimulation frequencies greater than 0.25 Hz. In the presence of D600, the tension _7 developed in response to 10 M isoproterenol was reduced at frequencies greater than 1 Hz, and became progressively more depressed as the fre-quency was increased. Increasing the temperature of the bathing medium from 27° to 42° produced a progressive decrease in the basal tension. In the presence of D600, the basal tension was almost totally'abolished'at a l l tempera-tures studied. At 27°, the dose response curve for isoproterenol in the _7 presence of 5 x 10 M D600 was shifted to the right at 0.5 Hz, and -20-non-existent at 2 Hz. At 37°, the responses at 0.5 Hz were similar to those at 27°, but at 2 Hz, a small positive inotropic effect of isopro-terenol could be detected, this was significantly depressed compared to the control and also shifted to the right. It therefore was apparent that D600 depressed the response to isoproterenol most at the lower temperature and higher frequency, leading them to conclude that this effect must indicate some action on calcium stores. Currently, the mechanism of action of the calcium antagonists in the heart is s t i l l not certain. It seems likely that in addition to their well-known effect on the slow inward current, the (+) - isomers also affect the fast inward current. It also appears that they may affect transfer of calcium between compartments in some fashion, but this will depend on whether they can be shown to enter the cell to produce this effect. 4. The Effect of Temperature on Cardiac Contraction. The influence of temperature on cardiac contraction has attracted less attention than frequency of stimulation or calcium concentraction, although obviously the fact that temperature does affect contraction makes i t of equal importance. Hollander and Webb (1955) carried out a very thorough study where they recorded the action potentials of rat atria stimulated at 200 beats per minute, as the temperature was increased from 23° to 42°. They found that increasing the temperature caused a progressive decrease in tension development. This was accompanied by a decrease in the duration of the action potential. They suggested that this effect on the action potential was responsible for the reduced tension. Their data also showed that the rate of contraction and relaxation decreased as the temperature was -21-decreased. Their conclusion was that a major mechanism behind these changes was the effect of the reduced temperature on the physical prop-erties of the membrane, and which could be reversed by increasing the temperature. Garb and Penna (1956) determined the effect of changing temperature on the response of spontaneously beating cat and guinea pig atria to isoproterenol and adrenaline. They found that the control rate of contraction rose 71% in the cat and 87% in the guinea pig when the temperature was raised from 29° to 37°. At the same time there was a decrease in tension development. At 37° in the guinea pig the response decreased so much that no tension could be recorded. At 37° the response of cat atria to isoproterenol and histamine was reduced compared to 29°. Garb and Penna suggested that the differences in contraction at the 2 temperatures might be due to some decrease in the availability of oxygen, however at a higher temperature of only 37° this seems unlikely. In contrast to these findings with sympathomimetic amines, Saunders and Sanyal (1955) have shown that the positive inotropic effect of ouabain on guinea pig ventricle strips was increased when the temperature was increased when the temperature was raised from 27° to 37°. The suggested that this could be related to a loss of intracellular potassium at low temperatures. This would result in an increase in intracellular calcium. Blinks and Koch-Weser (1963) recognized that i t was important to examine the effects of temperature on tension in a preparation where the rate could be controlled, since the rate was also affected by temperature and this in turn affected the strength of contraction. They pointed out in their review that in mammalian tissues the optimum temperature' for -22-maximal strength of contraction varied with the frequency of stimulation, but that the tension was always greatest at temperatures more than 10° below the norm for that species. As the temperature was lowered below the optimum, the strength of contraction was known to decline slowly. However i t declined much more rapidly as the temperature was increased above the optimum, and especially above the normal temperature. The interaction of temperature and calcium was postulated by Kaufmann and Fleckenstein (1965). They proposed that at 37° and in their normal buffer containing 1.8 mM calcium, calcium entry was a rate-limiting process and when i t was not maximal prevented f u l l activation of the contractile proteins. However, by lowering the temperature to 17° and prolonging the duration of the action potential sufficient calcium could enter the cell to produce maximal contractions. In contrast at 37° they found that by raising the calcium concentration to 14.4 mM, enough calcium could enter the cell during the brief action potential at that temperature to activate the contractile proteins as fully as with normal calcium and 17°. Sumbera and colleagues (1966, 1967) used a rapid step change of temperature to follow the effect of changing temperature on the contrac-tions of guinea pig left atria. They noted that in adapting to a differ-ent temperature the tension developed by the muscle displayed a stair-case phenomenon and- suggested that this pattern, since i t was also seen following changes in rate and input or output pressure represented a general adaption pattern, based on some property specific to cardiac muscle.(Sumbera et al . , 1966). In normal buffer containing 2.5 mM calcium at 32°, they found that following a 10 second pause the next contraction was larger. If during the 10 second, pause.the temperature was rapidly dropped to 22 , the tension developed and duration of tension development was increased compared to 32° throughout. Opposite effects were seen i f the temperature was increased from 22° to 32° during the pause. Both the tension and duration of tension development were decreased compared to 22° throughout. In addition, the tension developed was greater than that seen when the tissue was switched from 32° to 22°, and the duration of tension development was longer than at 32° throughout. It was therefore obvious that the effect of changing temperature produced fairly rapid effects on contraction. They repeated the experiment in the presence of 0.25 mM calcium, and could detect no contraction at 32° following a 10 second pause. Even after decreasing the temperature to 19° during the pause, no contraction could be detected. However, follow-ing exposure to 19°, a contraction was seen after the pause even i f a rapid switch to 32° was instituted during the pause. They concluded that larger contractions could be produced at low temperatures because of the greater influx of calcium which should, occur during the prolonged action potential at these temperatures, and that some cumulative effect was taking place. Langer and Brady (1968) showed that decreasing the temperature from about 28° to 21° produced an average increase in systolic tension of 3.44 - .85 g and no change in calcium exchange. However the diastolic tension development was large (in the only example shown i t was 6 g) and the tissues were paced at different rates, ranging between 19 and 60 beats per minute. Langer and Brady concluded that cooling decreased the capacity of the sodium pump, and increased the concentration of Na±. This would.then participate in Ma:Ca exchange, allowing increased calcium binding to regions of the membrane adjacent to the contractile 45 proteins. Reuter and Seitz (1968) measured Ca efflux from guinea pig -24-left atria and showed that efflux was decreased when the temperature was reduced from 35° to 25°. 2, 4-dinitrophenol at a dose of 2.7 x 10~^ M 45 had no effect on Ca efflux, leading them to propose that i t was controlled by a physical process, rather than an enzymic process analogous to the sodium pump. Mattiazzi and Nilsson (1976) examined the influence of temperature on the time course of mechanical activity in rabbit papillary muscle. They found that at 30 beats per minute and 2 mM calcium reducing the temperature from 32.5 to 26.5° did not affect the rate of development of tension although time to peak tension was delayed, but increased the maximal tension developed and the duration of the tension development. If the frequency was increased to 42-48 beats per minute and/or the calcium concentration increased to 14 mM the peak tension was not differ-ent at the 2 temperatures, but the rate of development of tension was decreased slightly at 26.5°. They suggested that cooling reduced calcium uptake by the SR, resulting in increased activity at low temperatures, due to decreased removal of calcium from the region of the contractile proteins. They concluded that the extracellular calcium concentration and contraction frequency both influenced the maximal tension developed, but that the rate of calcium release from intracellular stores was hardly affected by temperature since the rate of rise of activity was only slightly changed. A slightly different approach which appeared in the literature at about this time was the temperature-induced receptor interconversion theory. Reinhardt et al., (1972) showed increasing the temperature stepwise from 25° to 42° decreased the affinity of sympathomimetic amines for the 8-receptors of guinea pig left atria, and also decreased the maximal tension developed in response to the drugs. They pointed out that a number of different factors might be responsible for the increased contractile response at low temperatures, including a partial inhibition of the sodium pump indirectly resulting in increased intra-cellular calcium and allowing lower agonist concentrations to produce maximal tension development. Conversely, higher temperatures could activate the pump, meaning that higher agonist concentrations would be required to produce an inotropic effect. In a later paper (Endoh et al. , 1975) the same group showed that after increasing the temperature from 37° to' 42° the maximal response of rabbit papillary muscle to isopro-terenol was not different, although in the guinea pig i t was significantly less at 42° (Reinhardt et al., 1972). In the rabbit the maximal response to phenylephrine and calcium was also not different at 37° and 42°. However, with a l l 3 agonists the dose response curve was shifted to the right at 42° as was seen in the guinea pig. Since the maximal response seen at 42° was not different from that at 37° there was obviously no _7 deterioration of the proteins at the higher temperature. D600 (10 and _7 3 x 10 M) shifted the dose-response curves to isoproterenol to the right, resembling the shift seen with increasing temperature. This led them to postulate that both affected calcium movement across the membrane, D600 by inhibiting calcium influx and increased temperature by increasing calcium efflux. The observation that D600 inhibited phenylephrine more than isoproterenol led them to further suggest that while both agonists stimulated calcium influx across the membrane, isoproterenol could in addition evoke calcium release from intracellular stores. Then in 1976, Kunos and Nickerson presented evidence which led them to suggest that the a and 3 -adrenoceptors of the frog heart represented allosteric conformations of a single receptor whose characteristics were governed by the metabolic state of the receptor. They showed that at 14° the tissues retained more - phenoxybenzamine than they did at 23 . Phenoxybenzamine (7--.3 mM for 40 minutes) inhibited the inotropic response to adrenaline at 17°, and potentiated i t at 24°. Conversely propranolol inhibited the response to adrenaline at 23°, but its activity was reduced at the lower temperature. In addition, significantly greater amounts of [ll4C 1 - propranolol were bound at 24° than at 14°. They postulated the presence of a single receptor population which had . 8-characteristics at high temperatures and a -characteristics at low temperatures. They used this theory to explain why phenoxybenzamine shifted the dose-response curve for adrenaline to the right at 14° and to the left at 24°. However, most other workers- have been unable to confirm the existence of adrenoceptor interconversion. Benfey (1977, 1979a, b) has been unable to reproduce the data of Kunos. He suggests that the reduced response to propranolol at low temperatures was because the tissue was equilibrated with the antagonist for only 10 minutes, resulting in incomplete blockade. Benfey further suggests that the concentration of phenoxybenzamine used by Kunos was higher than the concentration required to block a -receptors, probably resulting in binding of the antagonist to a number of other membrane components as well as the a -receptor and making i t unlikely.that the binding sites reported by Kunos and Nlckerson represent physiological a -receptors. Workers in our own laboratory have also been unable to confirm the adrenoceptor conversion hypothesis. Martinez and McNeill (1977) pointed out that the basal tension was so great at 17°, that the positive inotropic effect of the amines studied was greatly reduced. An increase in cyclic AMP in response to noradrenaline was seen at 37°, 22° and 17°, and was blocked by propranolol but not phentolamine. Exposure to 10 ^ M phenoxybenzamine for 45 minutes at 17°, did not affect the _5 positive inotropic effect of 10 M noradrenaline or the concommitant increase in cyclic AMP following warming to 37°. According to Kunos, the incubation with phenoxybenzamine at 17° should have "locked" the receptors into an a -configuration, resulting in a reduced 3 -response after warming to 37°. Interestingly Phan et al . , (1980) have shown that rat atrial muscarinic receptors may change their configuration to that of a high agonist affinity receptor-agonist complex at low temper-atures, and suggest that this explains the greater inhibitory effects of acetylcholine on the heart seen at lower temperatures. A similar situation may occur with other receptors. The mechanism by which temperature affects the rate and force of contraction of the heart then has been suggested to be due to a number of mechanisms, most of them involving calcium, and not a l l of them fully tested. Chiesi (1979) has shown that the Ca-ATPase activity of dog heart microsomal.fraction increased as the temperature was increased from about 10° to 40°. As the temperature was increased further the activity dropped off sharply. She also showed that in conjunction with this, calcium uptake into the SR increased steadily over the temperature range 17.5° to 40°. Yazaki and Raben (1974) also showed that the Ca-ATPase increased with increasing temperature. In addition they found that the activity of rabbit ventricular muscle ATPase was consistently lower than that of rat at each temperature studied. In a later paper (Yazaki et al.,. 1979) they showed that dog atrial myosin had a greater ATPase activity than did ventricle. The temperature dependence of the two tissues differed. For both atria and ventricles a greater activity was seen at higher temperatures, but as the temperature was reduced, activity decreased more rapidly in the ventricle fraction. This sug-gested to them that the structures of the myosin molecule was different. It therefore seems that increasing the temperature both increases enzymic activity, resulting in more efficient calcium removal from the contractile proteins, and reduces the duration of the action potential, limiting calcium entry into the cell and ultimately resulting in a decreased tension development. 5. The Effect of Catecholamines on Cardiac Contraction. Langley in 1901 was among the f i r s t investigators to realize that the physiological effects produced by dog supra-renal gland extract were similar to those produced by stimulation of the sympathetic nerves. In 1905, Elliott showed that adrenaline accelerated the heart rate and increased the force of contraction of amphibian and mammalian hearts in a manner similar to.that seen when the sympathetic nerves were stimulated. The responses of other muscles also showed similar effects to the two methods of stimulation. Langley (1905) postulated that the stimulation by adrenaline was produced by some interaction at the junction of the nerve and muscle since i) direct application of adrenaline to the nerve had no effect, and i i ) degeneration of the nerve sometimes produced a greater effect in the presence of adrenaline. He pointed out that the action of adrenaline depended however on an association of the sympathetic nervous system with the muscle, and this association was postulated to result in the development of a reacting mechanism on the muscle side of the junction. This contrasted with the prior concept that adrenaline acted by some action on sympathetic nerve endings (Brodie and Dixon, -29-1904). By 1910, a whole series of amines structurally related to adrenaline had been tested pharmacologically. Barger and Dale (1910) found that the closer the structural similarity to adrenaline, the greater the sympathetic activity. They found that noradrenaline was usually more potent than adrenaline, and that i t and other amines produced effects which corresponded more closely to those of sympathetic stimulation than did those of adrenaline. Finally, in 1946 von Euler, using a variety of tissues showed that sympathetic nerve extract and noradrenaline produced physiological effects which were identical, and suggested that noradrenaline was the neurotransmitter released following sympathetic nerve stimulation. The concept of a receptive substance which was the recipient of stimuli from chemicals or nervous stimulation, and which transmitted these stimuli to the target tissue was introduced by Langley in 1905 to explain the action of curare on skeletal muscle. The concept of a receptor-chemical agent reaction explains the following characteristics of many drugs: i) they act at micromolar concentrations; i i ) they exhibit structure-activity relationships; i i i ) they can be antagonized selectively by antagonists; and iv) the antagonists also exhibit struc-ture-activity relationships (Clark, 1937; Stephenson, 1956; Paton, 1961; Ariens and Simonis, 1964; Furchgott, 1964; and Waud3 1968). In 1948, Ahlquist published a study where he examined the responses of six physio-logical systems to five different sympathomimetic amines including noradrenaline, adrenaline and isoproterenol. The relative activities of the amines in the systems f e l l into two distinct categories in the first category, the order of activities was adrenaline > noradrenaline » isoproterenol. These responses were mainly excitatory (vasoconstric-tion, and stimulation,-of the uterus, nictitating membrane and ureter), -30-but there was also one inhibitory effect (intestinal relaxation) which had the same order of amine activity. Ahlquist termed the receptor mediating these actions the a -receptor. The second category of orders of activity of the amines was isoproterenol» adrenaline» noradrenaline. These responses were mostly inhibitory (vasodilation and relaxation of the uterus and bronchi), except for excitation of the heart. The receptors mediating these actions were termed 3 -recep-tors. Adrenergic blocking drugs have since also been characterized according to the receptor which they occupy. It is now known that sub-types of the a -and 3 -receptor exist. Lands and co-workers (1967) carried out an extension of Ahlquist's study, examining fifteen amines. They suggested that there were two sub-types of the 8 -receptor. The f i r s t category, termed 3i, mediated lipolysis and cardiac stimulation; and the second, termed 32 mediated the bronchodilator and vasodilator effects of the amines. Similarly, Langer (1974) suggested that a -receptors should be divided into sub-types ai, representing postsynaptic and a2, representing presynaptic receptors. This has been supported by the work of Drew (1977) and Wikberg (1978). Wikburg has shown that a i -receptors, are present when the relative affinity of phenylephrine for a -receptor is greater than that of clonidine, and the a2--receptors are present when the relative affinities are reversed. Sutherland and Robison (1966) showed that the membrane bound enzyme adenylate cyclase, which is responsible for formation of cyclic ++ AMP from ATP m the presence of Mg , appeared to be coupled to the function of the 3 -receptors. Formation of cyclic AMP has since been shown to be stimulated by a number of other substances (Robison, -Butcher --31-and Sutherland, 1971). Cyclic AMP in its turn activates a cyclic AMP-dependent protein kinase. In liver slices following stimulation by catecholamines phosphorylase kinase is then activated, resulting in formation of phosphorylase a from phosphorylase b, and resulting in glycogenolysis (figure 1). In muscle, protein kinase is thought to phosphorylate the Tn-I subunit of troponin (Bailey and Villar-Palasi, 1971; England, 1976; Stull et al., 1980). In cardiac muscle i t has been suggested that phosphorylation of troponin modifies its properties allowing inter-action of actin and myosin and producing contraction (Stull et al., 1972; England et al., 1973). The activation of protein kinase has also been suggested to phos-phorylate the protein phospholamban,- located on the SR membrane. This has been shown to be associated with stimulation, of calcium transport by the SR (Katz et a l . , 1975). It has been suggested that this increase in rate of calcium uptake may be partially responsible for the increased rate of relaxation seen with agents which stimulate cyclic AMP formation (Katz, 1977; Tsien, 1977) (figure 1). In addition, as previously mentioned, the P-light chains of myosin are also phosphorylatable, although by the myosin light chain kinase, which is stimulated by the calcium-calmodulin complex, rather than by cyclic AMP. (Stull et a l . , 1980). Cheung and Williamson (1965) reported that following administration of adrenaline to rat hearts there was a positive inotropic response, preceeded by an increase in cyclic AMP activity, and followed by an increase in phosphorylase a activity. This difference in peak activity of cyclic AMP, contraction and phosphorylase a has since been confirmed -32-Figure 1 Proposed Mechanism of Action of Catecholamines in Glycogenolysis and Cardiac Contraction. (Adapted from Krebs, 1972; and Nimmo and Cohen, 1977). Catecholamines cyclic AMP increased protein kinases activated phosphorylase kinase activated phosphorylation of proteins including Tn-I and phospholamban phosphorylase a glycogenolysis stimulated cardiac contraction -33-many times (Namm and Mayer, 1968; Schumann, Endoh and Brodde, 1975; McNeill and Verma, 1973; McNeill, 1978). Cyclic AMP may also as previously reported affect the calcium capacity of, and release from the SR (Fabiato.. and Fabiato, 1975b). In addition to stimulation of adenylate cyclase, the catecholamines are thought to affect the slow inward current and increase calcium influx (Rasmussen et al . , 1972). Reuter (1974b) provided data showing that the catecholamines and dibutyryl cyclic AMP increased the calcium current of cat papillary muscles. Isoproterenol and noradrenaline applied intracellularly had no effect, indicating that they must inter-act with a receptor located on the outside of the membrane. Previously Grossman and Furchgott (1964c) had demonstrated that noradrenaline 45 increased Ca exchange of the guinea pig left atrium. In 1973, Meinertz (Meinertz et al . , 1973a) published data showing that adrenaline 45 and dibutyryl cyclic AMP both increased contractile force, .and Ca uptake of rat left atria, although a later study could not repeat the results (Meinertz et al . , 1973b). Watanabe and Besch (1974) in their turn suggested that the increase in cyclic AMP seen following adminis-tration of certain inotropic agents to the guinea pig Langendorff heart activated the slow inward current, since incubation with calcium antagonists reduced the tension developed, but did not affect the increase in cyclic AMP in response to the drugs. The mechanism of the contractile action of the catecholamines, then s t i l l remains obscure. Effects on cyclic AMP and calcium are definitive, but the manner in which they are tied together is s t i l l largely specula-tive. -34-6. The Effect of Histamine on Cardiac Contraction. Dale and Laidlaw (1910, 1911) f i r s t showed that histamine produced a variety of effects on mammalian tissues. These included stimulation of a number of smooth muscles and a vasodepressor effect. In addition, i t produced positive inotropic and chronotropic effects in isolated cat and rabbit hearts. It is only recently that the direct effects of histamine on the heart have been extensively studied. Trendelenburg (1960) and Bartlet (1963) showed that in certain species, histamine had positive inotropic and chronotropic effects, and that they were poorly antagonized by large doses of the conventional antihistamines then available. In 1966, Ash and Schild suggested that the lack of antagonism of some histamine effects was due to the presence of two different hista-mine receptors. The effects of histamine which could be easily antag-onized by classical antihistamines (vasodilation, bronchiolar constric-tion and allergic reactions) were termed H^ . Those effects which were not antagonized (stimulation of gastric secretion, relaxation of the rat uterus and stimulation of the heart) were termed non -H-^ . Finally in 1972, Black and his co-workers introduced burimamide the fi r s t antagonist shown to antagonize the non-H^ effects of hista-mine. The histamine receptor mediating these actions was now termed H^. A number of ^-receptor antagonists have since been synthesized (metiamide, cimetidine and tiotidine) and used both clinically and experimentally (Black et al . , 1973; Reinhardt et al . , 1974; McNeill and Verma, 1974; Finkelstein and Isselbacher, 1978; Yellin et al . , 1979; Polanin et al . , 1980). In addition, stimulation of H9-receptors has -35-been shown to be associated with an increase in cyclic AMP, (Klein and Levey, 1971; McNeill and Muschek, 1972; Reinhardt et al . , 1977; Verma and McNeill, 1977). H-^-receptors are not involved with changes in cyclic AMP levels. An interesting discovery has been made that the cardiac effects of histamine may be mediated by different receptors in different species, and even in different parts of the heart within the same species. In the guinea pig i t is now fairly well established that the positive chronotropic effects of histamine on the right atrium are mediated by H^-receptors, - .antagonized by burimamide (Reinhardt et a l . , 1974; Steinberg and Holland, 1975), and associated with an increase in cyclic AMP levels (Verma and McNeill, 1977). In the left atrium, the positive inotropic effects of histamine are mediated through H-^-receptors (Reinhardt et al., 1974; Steinberg and Holland, 1975; Verma and McNeill, 1977), antagonized by promethazine, and not accompanied by changes in cyclic AMP levels. The receptors mediating the positive inotropic effect of histamine in guinea pig ventricular muscle, similar to the right atrium have been shown to be of the H^-type. Reinhardt and co-workers (1977) and Verma and McNeill (1977) showed that an increase in cyclic AMP preceeded the positive inotropic response of ventricular muscle to histamine, and that both effects could be antagonized by burimamide. In addition, Verma and McNeill showed that large doses of H-^-agonists could produce inotropic effects in the ventricle, and these were later shown to be antagonized by propranolol and promethazine (Laher and McNeill, 1980). Recently Shigenobu et ..al., (1980) showed that in ventricular muscle -36-from immature (0-5 day) guinea pigs, chlorpheniramine had no effect on the positive inotropic response to histamine. In mature guinea pigs (300-500 g) the dose-response curve for histamine was shifted to the right by chlorpheniramine, as was that of 2-(2-pyridyl) ethyl-amine (PEA). Doses of PEA greater than 10 were required to produce a positive effect in the presence of propranolol. Klein and Levey (1971) found an increase in adenylate cyclase activity of enzyme prepared from the left ventricle following addition of histamine. -5 The stimulation was blocked by 8 x 10 M diphenhydramine hydro-chloride, a dose which probably interacted with f^-receptors. Johnson et al. (1979) suggest that the depressant effects of H-^-antagonists on adenylate cyclase from guinea pig ventricular tissue are due to non-specific binding to the H^-receptor in high concentrations. It therefore seems fairly well established that in the guinea pig the predominant histamine receptor is En, which is present in the right atrium and ventricular muscle, and stimulation of which is asso-ciated with increased cyclic AMP levels. The left atrium and right ventricle have been shown to contain H^-receptors, those of the right ventricle being either fewer in number, or with a reduced affinity for histamine. Stimulation of these H-^-receptors is not associated with any change in cyclic AMP levels. Tenner and McNeill (1978) further characterized the interaction of histamine with H-^-receptors in the guinea pig left atrium. They showed that conditions which enhanced the ability of the guinea pig left atrium to develop force (hypothermia, high frequency stimulation and high extracellular calcium concentrations) also increased the -37-sensitivity to isoproterenol, although efficacy (maximum change of developed force) was decreased. In contrast these same conditions depressed both the efficacy and sensitivity to histamine. Conversely, conditions which depressed the ability of the tissue to develop force (only low frequency stimulation was used) decreased the sensitivity to both isoproterenol and histamine while increasing the efficacy of iso-proterenol and decreasing that of histamine. It was suggested by Tenner and McNeill (1978) that the differences in the contractile responses to isoproterenol and histamine seen under the differing environmental conditions was due to the inotropic effects of H-^-receptor stimulation occurring through a different process than that of 3-stimulation. Verma and McNeill (1977) had previously proposed that the actions of histamine paralleled those of the adrenergic agents, in that stimulation of one receptor (H^ or 3 ) was associated with an increase in cyclic AMP, while stimulation of the other(H-^ or a) was not. From their data, Tenner and McNeill (1978) concluded that the inotropic effects of H-^ -,;and a-stimulation were brought about by calcium influx into the cel l , producing release of activator calcium from the SR, but that H^ -..and 8-stimulation, in addition stimulated cyclic AMP formation, resulting in augmented calcium release from the SR (Fabiato." and Fabiato, 1975b). The possible mechanisms of action of the agonists are summarized in figure 2 (Longhurst and McNeill, 1980, 1981). Activation of cardiac 3- or H^ - and H^ - receptors results in depolarization of the membrane. This may lead to release of sarcolemmal (SL) calcium (1) and opens the slow calcium channel (2) allowing external -38-Figure 2 Schematic Model for Calcium Involvement in the Inotropic Responses to Isoproterenol and Histamine. (1) Activation of receptor by agonist leads to release of sar-colemmal calcium and opens a channel (2) which allows extracellular calcium to enter the ce l l . This trigger calcium may cause release of activator calcium from the SR (3), resulting in contraction (4). During relaxation calcium is taken up by the Ca-ATPase of the SR (5), to be utilized again for activation of the contractile system. Stim-ulation of 8- or H^ - receptors also results in activation of cyclic AMP (6) which may promote uptake and release of activator calcium by the SR. In addition (7) cyclic AMP may phosphorylate the contrac-t i l e proteins and modify contraction. Calcium not stored in the SR may be extruded from the cell by a Na-Ca exchange mechanism (8) or a sarcolemmal Ca-ATPase (9). This model is not intended to be final, but simply to present some possible calcium interactions in the response of the cardiac muscle to isoproterenol and histamine. For a more detailed account of calcium involvement in muscle contraction see Chapman (1979) and van Breemen et a l . , (1979). -39-Ca++ HIS -40-calcium to enter the cell (Inoue et al . , 1979). This small trans-sarcolemmal influx of calcium, termed "electrogenic" by Langer (1976), has been shown to be insufficient in quantity to activate the contrac-t i l e proteins itself.(Bassingthwaite and Reuter, 1972; Reuter, 1974), but has been suggested (Fabiato and Fabiato, 1975a) to act as trigger calcium, releasing much larger amounts of calcium from the SR (3). This activator calcium binds to the troponin of the cardiac muscle, resulting in modification of the tropomyosin molecules so that the myosin heads can contact the actin molecules, ATP is split and the muscle contracts (4). During relaxation, calcium is actively sequestered into the SR by the Ca-ATPase (5). As well, isoproterenol and histamine through their interactions with 3- and E^- receptors have the potential to activate adenylate cyclase, resulting in cyclic AMP formation (6) (0ye, 1975). - This cyclic AMP may enhance the capacity and rate of calcium binding within the SR (Fabiato and Fabiato, 1975b), resulting in additional calcium release and an augmented contractile response. While H^ - receptor stimulation has been shown to induce an increase in. myocardial cyclic AMP levels, stimulation of H^ - receptors has been shown to produce inotropic effects which are not associated with changes in cyclic AMP (Verma and McNeill, 1977). In addition (7) cyclic AMP has been shown in vitro to phqsphorylate cardiac troponin I and troponin T in the presence of protein kinase (Bailey and Villar-Palasi, 1971). England (1976) has shown that the increase in contrac-t i l e force in rat hearts perfused with isoproterenol was paralleled by an increase in phosphorylation of Troponin I. Calcium not stored may be extruded from the cell by the Na: Ca -41-exchange carrier which couple exchange of 3 or 4 Na per Ca (Pitts, 1979), or by the Ca-ATPase, driven by ATP hydrolysis (Sulakhe and St. Louis, 1976). McNeill and Verma (1978, 1979) investigated the histamine receptors in rabbit atria and ventricle strips and showed that in contrast to the guinea pig, the rabbit heart contained predominantly H^ - receptors. H^ - receptors were found only in the right atrium, but both and H^ -receptors were involved in the positive chronotropic response to hista-mine. H^ - receptor stimulation was associated with an increase in cyclic AMP levels. However, more recent work from our laboratory (Polanin et al., 1980; Polanin and McNeill, 1981) has shown the pres-ence of H^ - receptors in the left atrium as well. Unpublished data from our laboratory indicates that the positive inotropic effect of H£- agonists in left atria is also accompanied by an increase in cyclic AMP. One interesting observation is that the H^ - receptors of the right and left atrium might be different (Polanin et al . , 1980; Longhurst and McNeill, 1981) since the- '"pA^ values for cimetidine and metiamide were higher in the right atrium than in the left. According to Schild (1957), drugs which act on the same receptors are expected to give rise to the same pA^  value, suggesting that there is some difference in the H^ - receptor type between the two tissues. However, the pA^  values for tiotidine were similar in right and left atria. This difference between the three antagonists could be due to the fact that metiamide and cimetidine share a similar structure, while that of tiotidine differs in that the ring structure is a thiazole, rather than an imidazole, and has a second large functional group attached to i t . In addition, a concept of H_- receptor subtypes has been postulated by Chand and Eyre -42-(1977, 1978) to exist in the rabbit trachea and cat bronchus. They suggest that this "atypical" receptor may be an isoreceptor of H^ , analogous to the subgrouping of 8i,- and 6? - adrenoceptor. The mechanism of the contractile response to histamine is even more obscure than that of the catecholamines, since less work has been carried out on the autacoid. It seems likely that the mechanism of Ry- stimulation is similar to that of 8 - stimulation, and H^ - to that of a - stimulation. Once again calcium is obviously playing a major role. 7. The Purpose of the Present Investigation. The present investigation was to determine the effects of changing temperature, frequency of stimulation and calcium concentration on the contractile effects of isoproterenol and histamine on guinea pig. and rabbit papillary muscles. McNeill and Tenner (1978) showed that conditions which enhanced the ability of muscle to develop force (hypothermia, increased frequency and increased extracellular calcium) increased the basal developed'' force and sensitivity to isoproterenol of guinea pig left atria. In contrast, while increased frequency enhanced the sensitivity to histamine, hypothermia and increased extracellular calcium decreased the sensitivity. The guinea pig left atrium has been shown to contain H^ - receptors, stimulation of which is not associated with changes in cyclic AMP levels, while stimulation of 8 - receptors by isoproterenol does increase cyclic AMP. Tenner and McNeill proposed that there was a similarity between 6 - and H_- receptors since stimulation of them resulted in an increase -43-in cyclic AMP; and between a- and H^ - receptors, since their stimu-lation was not associated with a change in cyclic AMP levels. Initially i t was of interest to us to determine whether similar responses to isoproterenol and histamine could be seen in the guinea pig or rabbit papillary muscle. Papillary muscles were chosen because the rabbit papillary muscle contains 8 - and H^ - receptors (similar to the guinea pig left atrium) and the guinea pig papillary muscle contains 8- and K - receptors (Reinhardt et al., 1977; McNeill and Verma, 1978). In addition we expanded the study to investigate the effects of increas-ing and decreasing temperature, frequency of stimulation and calcium concentration compared to the normal values currently used in our labor-atory. In the rabbit, we also investigated the effect of increasing one condition while simultaneously decreasing another, to determine the effects on the inotropic response to isoproterenol and histamine. In the later studies we tried to investigate the effects of the two agonists on calcium entry into the muscle or calcium content of the muscle. At f i r s t this was done indirectly, using the calcium antagonist D600 to see whether H^ - induced responses which should solely depend on calcium entry into the cell and subsequent release from the SR were affected by slow inward current blockade to a greater extent than 8 - induced responses which in addition increase cyclic AMP and have the potential to augment calcium release and storage by the SR. A direct measurement of calcium content following adminis-45 tration of isoproterenol and histamine was also carried out using Ca. -44-Summary of Experimental Aims. 1. Determine how changing experimental conditions affects basal response of guinea pig and rabbit papillary muscles. 2. Determine how changing experimental conditions affects contractile response to isoproterenol and histamine of guinea pig and rabbit papillary muscles. 3. Determine whether response of rabbit papillary muscle to isopro-terenol and histamine has different sensitivity to calcium antag-onists . 4. Determine whether isoproterenol and histamine have different effects on calcium uptake. -45-METHODS A. Contractile Studies . 1. Preparation of Tissues Cardiac preparations were obtained from albino guinea pigs (400-500g) and New Zealand White rabbits (1.5-2.5 kg) of either sex. A l l animals received food and water ad libitum. They were pretreated with heparin sodium (8 mg/kg i.p.), ten minutes prior to sacrifice. Guinea pigs were killed by cervical dislocation and rabbits by a blow to the head, followed by exsanguination. The hearts were rapidly removed and placed in aerated Chenoweth-Koelle (CK) solution at 37.5°. The compo-sition of the solution was as follows (millimolar): NaCl, 120; KCl, 5.6; CaCl 2, 2.2; MgCl2, 2.1; dextrose, 10; NaHCC>3, 19.2. One small papillary muscle was dissected from the right and left ventricle of each guinea pig heart with a small piece of ventricle attached for placement on the electrode (for a description of the elec-trode see Longhurst, 1978). Initial experiments showed that the basal developed force and maximal developed force were similar in muscles taken from the two chambers. Two small papillary muscles were dissected from the right ventricle only of the rabbit. A l l tissues were suspended in 20ml organ baths containing CK solu-tion at 37.5° and aerated with 95% oxygen and 5% carbon dioxide. Cont-ractile force was measured by means of a Palmer clip attached to the chordae tendinea, connected to a Grass force displacement transducer and recorded on a Grass model 79D polygraph. -46-Diastolic tension was adjusted to lg. Muscles were driven at 1Hz with 5 millisecond square pulses at a voltage approximately twice threshold, using a Grass model S6 stimulator. Following equil-ibration for at least 30 minutes under conditions of 37.5°, 1Hz and 2.2mM calcium, the experimental conditions were changed. In experi-ments where two conditions were changed, the calcium concentration was changed fi r s t . Once the tissue had again equilibrated in terms of tension development, the second condition was changed. The tissues were allowed to reach a new steady state tension prior to addition of drugs , dissolved in buffer and in a volume of 0.2ml to the bath. Dose-response curves were produced by the cumulative addition of drugs using the method described by van Rossum and van den Brink (1963). Only one dose-response curve was obtained per tissue. 2. F D ^ Q Measurements Geometric mean ED r n values were determined by the method of oU Fleming et a l . , (1972). The ED 5 0 data are presented as geometric means and their 95% confidence intervals (antilog of mean log, and log 95% confidence intervals). See Appendix I for a description of the method. 3. Use of Antagonists In the experiments where D600 was used, the drug was left in contact with the tissues for 60 minutes before addition of the agonists. -47-B. Radioisotope Studies 1. Measurement of the Extracellular Space The extracellular space was measured as described in figure 3 to determine the length of time required for the cut edges of the tissue to heal and exclude an extracellular marker. Following removal from the animal, papillary muscles and right ventricle strips measuring l-2mm x 10mm were allowed to equilibrate in aerated CK solution at 37.5° for varying periods of time before 14 being transferred to CK solution containing C-sorbitol for 15, 30 or 60 minutes. Following this incubation, tissues were blotted and weighed. They were then placed in vials containing 3 ml of water overnight. In the morning 7 ml of scintillation cocktail was added 14 . . . and the vials were analysed for C m a liquid scintillation counter. 45 2. Removal of Extracellular Ca 45 Extracellular Ca was removed as described in figure 4. Fol-lowing 60 minutes equilibration in physiological solution (PSS), the tissues were transferred into a loading solution of PSS containing 45 Ca for 150 minutes. The tissues were then passed sequentially, every 10 minutes through test tubes containing 3 ml of the PSS to be investigated, maintained at 0°. This was achieved by attaching the tissues to small stainless wires which were then inserted into the large end of a Pasteur pipette and placed in the test tube so that the tissue was in the PSS. The appropriate aerating gas was passed through the -48-Figure 3. Method for Measurement of the Extracellular Space. Tissues were equilibrated in Chenoweth-Koelle solution for varying periods of time before transfer into Chenoweth-Koelle solution 14 . . containing C-sorbitol for 15, 30 or 60 minutes. Tissues were blotted and weighed, and soaked in 3 ml water overnight. 7 ml of scintillation fluid containing Triton X-100 was added to the vials 14 which were then analyzed for C. The diagram at the top. of the page illustrates the. predicted effect of increasing periods of preincubation time on the extracellular space measurement. -49-Measurement of Extracellular Space l 37° Physiol, buffer 1 4OSorbitol X min 20X Count blot and weigh 1 [ 7 ml cocktail containing Triton X-100 Count -50-Figure 4. Method for Removal of Extracellular Calcium. Tissues were allowed to equilibrate for 60 minutes in physiological 45 solution before loading with Ca for 150 minutes. They were then passed sequentially every 10 minutes through a series of test tubes containing 3 ml of the.solution being investigated. Tissues were blotted and weighed, and soaked in 3 ml 5 mM EDTA overnight. 7 ml of scintillation fluid containing Triton X-100 was added to the vials 45 which were then analyzed for Ca. The diagram at the top of the page illustrates the predicted effect of 45 a quenching solution on Ca efflux from the tissues, compared to efflux into a normal solution at 37°. -51-Quenching Quenched Time (t min) 37 Physiol, buffer 45ca 150 min 20X Count Quenching solution 0°, 10 in each L ™ TO ran 3ml Count 7 ml cocktail containing Triton X-100 Count "il f blot and weigh 3 ml 5mM EDTA overnight -52-pipette allowing mixing of the PSS. At the end of 150 minutes (15 tubes) the tissue was removed from the wires, blotted and weighed. Tissues were transferred to vials containing 3 ml 5 mM EDTA and left overnight. In the morning 7 ml of scintillation fluid was added and 45 the vials were analysed for Ca. The 3 ml of PSS in the test tubes 45 was poured into vials and also analysed for Ca after addition of scintillation fluid. A sample'of the loading solution was added to 3 ml 5 mM EDTA and 7 ml of scintillation fluid and similarly analysed. 45 3. Measurement of Ca Uptake with Time . 45 Uptake of Ca into tissues was measured as described in figure 5. Tissues were placed in a loading solution of the appropriate CK buffer 45 + 45 containing Ca - drug for 2-10 minutes. Extracellular Ca was removed by placing the tissues for 60 minutes in a modified CK solution 45 containing 0 Ca and 10 mM Mg. Ca content was then determined as described above. 45 For determination of Ca uptake over longer periods of time a slightly different approach was taken (figure 6 ). Initially the 45 tissues were placed m a loading solution of normal CK buffer plus Ca for up to 120 minutes to determine an optimal loading period for sub-45 sequent experiments. Ca content was determined as described above. A time of 80 minutes loading was determined to be sufficient. Sub-45 sequently, to determine Ca content after exposure to drugs or a 45 depolarizing solution, tissues were fi r s t loaded with Ca for 80 minutes. They were then exposed for a further 10 minutes to the same 45 45 amount of Ca plus the treatment being investigated. Ca content Figure 5. Method for Measurement.of "^Ca Uptake over 2-10 Minutes. Following equilibration in Chenoweth-Koelle solution, tissues were 45 placed in the loading solution for 2-10.minutes. Extracellular Ca 45 was removed and Ca content was determined as described in figure 4. The diagram at the top of the page illustrates the predicted uptake of 45 Ca into the tissues in the control situation, and in the presence of 45 a drug which stimulates Ca influx. -54-Uptake 3 / Physiol, buffer 45ca L i Quench Sol'n 20X Count 7 ml cocktail containing Triton X-100 Count 1 ( blot and weigh 3 ml 5 m M EDTA overnight -55-Figure 6. 45 Method for Measurement of Uptake of Ca Following Loading of the Tissue. Following equilibration in Chenoweth-Koelle-solution, tissues were placed in the loading solution for up to 120 45 minutes. Extracellular Ca was removed by placing the t i s -sues for 60 minutes in a modified Chenoweth-Koelle solution 45 containing 0 Ca and 10 mM Mg. Ca was then determined as 45 described in figure 4. Net Ca uptake was measured by load-45 ing the tissue with Ca for 80 minutes, and then exposing 45 the tissues to a further 10 minutes of Ca plus the treat-45 ment being investigated. Extracellular Ca was removed 45 and the Ca.content determined as described in figure 4. The. diagram at the top of the .page illustrates the predicted effect of loading the tissue for up to 120 minutes. To measure net uptake a time was chosen.when equilibration was reached (marked .with the arrow), after which the treatment being investi-gated was administered.. -56-37^ Physiol, buffer 1 r 4 5 C c 1 r 4 5 C a + drug Quench Sol'n Loading solution 20X Count 7 ml cocktail containing Triton X-100 Count 1 I blot and weigh 3 ml 5mM EDTA overnight -57-was determined as described above. 4. Radioactive Studies in Stimulated Tissues Many of the radioisotope studies described above were carried out in both quiescent and stimulated tissues. The electrodes used for the stimulated preparations were the same as described for the contrac-45 ti l e studies. Ca was removed from the electrodes by soaking them m 5 mM EDTA overnight. Tension recordings were carried out as described in the same section. Tissues were only stimulated during loading and uptake studies. They were never stimulated in the part of the experiment where extra-45 cellular Ca was removed. C. Statistical Analysis Simple statistical analysis was carried out.using the Student's t-test. For multiple comparisons either the Newman-Keuls (Ziven and Bartko, 1978) or Bonferroni (Miller, 1966) test was used. A probabil-ity of P <0.05 was chosen as the criterion of significance. See Appen-dix I for methods. D. Drugs and Chemicals DL-isoproterenol HCI and histamine HCI were obtained from Sigma Biochemical Company. D600 was a gift from Knoll AG, Chemische Fabriken, Ludwigshafen am Rhein, FDR. -58-""^ Ca (specific activity 20.8 m Ci/mg) and ^C-sorbitol (180 m Ci/mmol) were obtained from New England Nuclear. Scintillation Fluid The scintillation fluid contained 15.2 g PPO and 380 mg POPOP in 3.8 1 toluene and 3.256 1 Triton X-100. E. Definitions BDF: Basal developed force - the tension developed by the tissue under 1 g diastolic tension following equilibration and prior to adminis-tration, of agonists. MDF: Maximal developed force - the maximal tension developed by the tissue in the presence of a particular agonist. PSS: Physiological salt solution. 45 Quenchxng Solution: An altered PSS, designed to retard exchange of Ca across the sarcolemmal membrane. -59-RESULTS A. Guinea Pig 1. Basal Developed Force The effect of changing temperature, frequency of stimulation and calcium concentration on the basal developed force (BDF) of guinea pig papillary muscles is illustrated in figure 7. Raising the temperature from 25° to 42° produced a -decrease in the BDF from 0.554 - 0.075 to 0.209 - 0.032 g. There was a significant difference between the BDF seen under conditions of 25 and 30° (0.554 - 0.075 and 0.524 - 0.060 g), compared to that seen at 37.5° (0.288 - 0.036 g). Little difference was seen between the groups when the frequency of stimulation was altered. Only when the muscles were stimulated at 0.5 Hz producing a BDF of 0.105 - 0.020 g was there any significant difference from the BDF of 0.288 - 0.036 g at 1 Hz. Raising the calcium concentration from 1.5 to 8 mM produced very noticeable stepwise increases in the BDF from 0.070 - 0.008 to 0.703 -0.049 g. The BDF seen in the presence of 6 mM (0.6C8 - 0.069 g)-and 8 mM calcium (0.703 - 0.049 g) were significantly larger than the BDF at 2.2 mM calcium (0.288 - 0.036 g), the concentration of calcium in the normal CK solution. 2. Maximal Developed Force Dose-response curves for isoproterenol and histamine were carried -60-out using the various experimental conditions described above. Figure 8 shows the dose-response curves for isoproterenol and histamine at different temperatures. Although the BDF values covered a large range (figure 7 ) from 0.554 - 0.075 to 0.209 - 0.032 g, in the presence of the higher doses of the agonists the tension values were similar at the different temperatures studied. For isoproterenol they ranged from 0.989 - 0.093 g at 42° to 1.1278 -0.139 g at 37.5°. and for histamine from 1.008 - 0.123 g at 25° to 1.392 - 0.215 g at 30°. The maximal developed force (MDF) from these curves was deter-mined and replotted in figure 9. It can be seen that there is l i t t l e difference in the magnitude of the MDF either under the influence of the two agonists, or due to any of the temperature changes. The dose-response curves for isoproterenol and histamine at dif-ferent frequencies of stimulation are shown in figure 10. Here the BDF data were much closer together, as was described before, but a wider range was seen in the presence of the agonists. This is more evident in figure 11'. The response to both agonists was significantly reduced by high frequency stimulation ranging with isoproterenol from a low of. 0.440'i 0.043 g at 4 Hz .to a high of 1.128 - 0.139 g at 1 Hz, and with histamine from a low of 0.388 - 0.040 g at 4 Hz_,to a high of 1.372 - 0.162 g at 1 Hz. In addition, the MDF for histamine was significantly reduced to 0.790 - 0.245 g by stimulation at 0.5 Hz compared to 1.372 - 0.162 g at 1 Hz. The dose-response curves for isoproterenol and histamine at different calcium concentrations are shown in figure 12. The spread of both the BDF data and the response to the agonists is quite evident. -61-The MDF data (figure 13) show that although there were significant differences between the BDF (figure 7), the MDF values are quite similar, ranging with isoproterenol from a low of 1.054 - .145 g at 1.5 mM to a high of 1.568 - 0.096 g at 6 mM calcium, and with hista-mine from a low of 1.214 - 0.165 g at 1.5 mM to a high of 1.800 -0.215 g at 6 mM calcium. These points were not significantly-different from each other. The data was replotted as % increase of MDF over BDF (figure 14). With both agonists, as the temperature was raised, the % increase became larger, ranging from 156 - 30% with isoproterenol at 25° to 591 - 91% at 37.5°, and 567 - 122% at 42°. With histamine the + o + smallest % increase was 148 - 13% at 30 , changing to 546 - 65% at 42 . The magnitude of response seen when the frequency of stimulation or calcium concentration was changed was much larger. As the frequency of stimulation was increased the magnitude of the % increase in MDF decreased. The largest response with isoproterenol of 1231 - 205% at 0.5 Hz, was twice the size of the largest temperature response of 567 - 122%, seen at 42°. The smallest response of 152 - 21% seen at 4 Hz was similar to the smallest temperature response (156 - 30% at 25°). Similarly with histamine, the largest response was 788 - 189% seen at 0.5 Hz, and declining to 175 - 34% at 4 Hz. Raising the calcium concentration from 1.5 to 8 mM produced a decrease in the magnitude of the % increase response to the two agonists. With isoproterenol the change was from 2114 - 296% at 1.5 mM to 131 - 16% at 8 mM calcium, and with histamine from 1853 - 273% at 1.5 mM to 166 - 15% at 8 mM calcium. This % increase seen with isoproterenol at 1.5 mM was twice the size of that seen at 0.5 Hz (1231 - 205%). No difference was noted between the % increase of MDF -62-produced by the two agonists. 3. ED c n Data oU Figures 15 and 16 illustrate the effect of changing the temper-ature, frequency of stimulation and calcium concentration on the E D ^ values for isoproterenol and histamine in guinea pig papillary muscles. The changes in ED R N seen with the altered conditions seemed to ou occur in a stepwise manner. This was most obvious with isoproterenol (figure 15). Raising the temperature to 42° significantly increased —8 the ED C N to 3.72 x 10~ M (with a 95% confidence interval of 2.77 x ou 10 - 8 to 5.01 x 10 - 8 M) compared to the value of 1.37 x 10~8 M (95% C.I. = 8.99 x 10~9 to 2.09 x 10~8 M) obtained at 37.5°. Lowering the frequen-cy of stimulation from 1 Hz to 0.5 Hz significantly increased the ED^Q to 3.57 x 10~8 M (95% C.I. = 2.32 x 10~8 to 5.55 x 10~8 M) from 1.37 x 10"8 M (95% C.I. = 8.99 x 10~9 to 2.09 x 10~8 M) obtained at 1 Hz. In addition lowering the.calcium concentration to 1.5 mM significantly increased the ED c n value to.3.44 x 10~8 M (95% C.I. = 2.44 x 10~8 to oU 4.86 x 10"8 M) compared to 2.2 mM. Raising the.calcium concentration.to 6 and 8 mM significantly decreased the ED R„ value compared to 2.2 mM. At 6 mM the ED R N was o(J oU 6.11 x 10"9 M (95% C.I. = 3.91 x 10~9 to 9.53 x 10~9 M), and at 8 mM, 5.64 x 10"9 M (95% C.I. = 4.11 x 10 _ 9 to 7.72 x 10"9 M). The results with histamine (figure 16) were less clear-cut. •FT, , 50: Raising the temperature to 42° did produce a • .increase in ED--  -63-to 8.97 x 10 7 M (95% C.I. = 6.29 x 10 7 to 1.28 x 10 6 M) compared to the value of 8.46 x 10~7 M (95% C.I. = 3.90 x 10~7 to 1.83 x 10~6 M) obtained at 37.5°, but at no temperature studied was there any signifi-cant difference from 37.5°. No gradation of response could be detected with changing frequency of stimulation, and raising the calcium concen-tration produced only a very slight stepwise decrease in the ED^, from 8.46 x 10-7M (95% C.I. = 3.90 x 10~7 to 1.83 x l O - 6 M) at 2.2 mM, to 3.83 x 10~7 M (95% C.I. = 2.41 x 10~7 to 6.09 x 10 - 7 M) at 4 mM calcium, the lowest EDr_ value obtained. None of the ED c n values obtained with bU oU histamine was significantly different from the response seen at 37.5°, 1 Hz and 2.2 mM calcium. It should be noted that the ED c n values for oU isoproterenol were, in general, at least 2 orders of magnitude more potent than those for histamine. B. Rabbit The same procedure was repeated for the rabbit, but in addition the frequency of stimulation and calcium concentration (part I) and tempera-ture and calcium concentration .(part I-I) were changed together. I. The Effect of Changing Frequency of Stimulation and Calcium Concen- tration 1. Basal Developed Force The effect of changing frequency of stimulation and calcium concen-tration on the BDF of rabbit papillary muscles is illustrated in figure 17. -64-At each frequency studied, as the calcium concentration was raised there was a stepwise increase in BDF. At 0.2 Hz, the BDF increased significantly from 0.0441— .0070 g to 0.1365 - .0257 g at 1 Hz and 0.3119 - .0312 g at 3 Hz. A similar response was seen at 0.5 Hz. At 1 Hz and 6 mM calcium the largest BDF was seen (0.5750 - .0649 g). In addition, at each calcium concentration as the frequency was raised from 0.2 to 3 Hz, the BDF initially f e l l , then increased again to reach a maximum at 1 Hz and then decreased again at 3 Hz. With 0.5 mM calcium the BDF responses at a l l frequencies were extremely low, the maximum occurring at 3 Hz. In comparison with the guinea pig data, (figure 7 ) the response to changing the frequency of stimulation was similar. Only when the tissues were stimulated at 0.2 or 0.5 Hz was the BDF reduced compared to that at 1 Hz. Increasing the calcium concentration at 1 Hz frequency of stimulation also produced a stepwise increase in BDF similar to that seen in the guinea pig. The response at 2.2 mM calcium (0.3093 -.0234 g) was significantly different from the values of 0.0398 - .'0059 g at 0.5 mM and 0.5750 - .0649 g at 6 mM calcium. 2. Maximal Developed Force Dose-response curves for isoproterenol and histamine were carried out using the various experimental ..conditions - described above. Figure 18 shows the dose-response curves for isoproterenol when both frequency of stimulation and calcium concentration were altered. The response to isoproterenol seemed to f a l l into three general groupings. Starting from the top of the graph, the response to isoproterenol was -65-greatest under conditions of 0.2 Hz and 6 mM calcium. The second grouping contained the responses to isoproterenol under conditions of 1 Hz and 6 mM calcium, 1 Hz and 2.2 mM calcium, 0.5 Hz and 6 mM cal-cium, 0.5 Hz and 2.2 mM calcium, and 0.2 Hz and 2.2 mM calcium. The inotropic responses to isoproterenol under these conditions were very similar. The final group contained very poorly developed responses to isoproterenol. The conditions were 0.5 Hz and 0.5 mM calcium, 3 Hz and 2.2 mM calcium, 0.2 Hz and 0.5 mM calcium, 1 Hz and 0.5 mM calcium, 3 Hz and 6 mM calcium, 3 Hz and 0.. 5 mM calcium. The MDF data (figure 19 ) shows that as the calcium concentration was raised at 0.2 Hz frequency there was a stepwise increase in MDF from 0.8000 - .2083 g at 0.5 mM to 1.5429 - v2467 g at 2.2 mM and 2.4091 - .1626 g at 6 mM calcium. These values were significantly greater than the MDF at 0.5 mM. However at the other frequencies, the MDF at 2.2 and 6 mM calcium were similar. As the frequency of stimu-lation was raised at each calcium concentration, the MDF either increased slightly and then f e l l off (0.5 and 2.2 mM), or decreased in a stepwise manner (6 mM). The largest MDF was that at 0.2 Hz and 6 mM calcium. The responses at 3 Hz were very depressed compared to the other frequen-cies. The smallest MDF of 0.5250 - .0785 g was seen at 0.5 Hz. The largest MDF seen at 1 Hz was 0.8900 - .0872 g and was significantly greater than that at 0.5 Hz. At 2.2 mM calcium the effect of changing the frequency of stimulation on MDF of isoproterenol was similar to that seen in the guinea pig (figure R5). There was l i t t l e difference between the MDF at 0.5 and 1 Hz and a significant depression of MDF at 3 Hz. The data for histamine is illustrated in figure 20. In contrast -66-to Isoproterenol, the 3 largest curves were those obtained under conditions of 1 Hz and 6 mM, 1 Hz and 2.2 mM and 0.2 Hz and 6 mM calcium. The curves for the other conditions f e l l into one large grouping, the biggest response being seen with 0.5 Hz and 6 mM calcium. The MDF data (figure 21 ) was similar to that for isopro-terenol , but smaller in magnitude. At both 0.2 and 0.5 Hz as the calcium concentration was raised there was a stepwise increase in MDF. The values obtained at 2.2 and 6 mM calcium were significantly greater than those at 0.5 mM. The MDF with 0.2 Hz and 6 mM calcium (1.2550™ .1947 g) was larger"than that obtained with 0.5 Hz and 6 mM calcium (0.8938 - .1519 g). At 1 Hz frequency there was no significant difference between the MDF at 2.2 mM (1.3100 - .1233 g) and 6 mM calcium (1.3625 •- .1401 g).. They were both significantly greater than the MDF at 0.5 mM calcium. At 3 Hz frequency the MDF was at a maximum at 22 mM. (0.5500 - .1095 g) and declined when the calcium concentration was increased to 6 mM (0.4563 - .0952 g). There was no significant differ-ence between MDF at 3 Hz. At four points (0.2 Hz and 6 mM, .0.5 Hz.and 0.5 mM, 0.5 Hz and 2.2 mM, and 1 Hz and 0.5 mM calcium) the responses to histamine were signi-ficantly less than those.to isoproterenol. The data for isoproterenol and histamine, replotted as % increase of MDF over BDF is illustrated in figure 22.- With both agonists, as the calcium concentration was raised at each frequency of stimulation the % increase f e l l . This was most extreme for isoproterenol at 0.5 Hz where the % increase was 3138 - 454% at 0.5 mM calcium, falling to 844 -163% at 6 mM calcium, and at 1 Hz where i t went from 2400 - 558% to 269 - 46%. The responses to isoproterenol were at least two times larger than the responses to histamine. In addition, as the frequency of stimulation was raised at each calcium concentration the % increase first increased and then f e l l . 3_. EDr-Q Data Figures 23 and 24 illustrate the effect of changing the frequency of stimulation and calcium concentration on the E D J - Q values for isopro-terenol and histamine in rabbit papillary muscles. For isoproterenol, the E D J - Q values changed in a stepwise manner as the conditions were altered. At 0.2 Hz, the ED,-n value decreased from 1.42 x 10_7M (95% C.I. = 4.84 ou x 10~8 to 4.20 x 10"7 M) at 0.5 mM, to 2.35 x 10~8 M (95% C.I. = 1.47 x —8 —8 10 to 3.76 x 10 M) at 6 mM calcium. The difference was significant at P< 0.05. At 0.5 Hz there was no difference between the ED c n value oil obtained at any calcium concentration. At 1 Hz, the E F ^ Q values at 2.2 and 6 mM calcium were again significantly lower than that at 0.5 mM. The lowest EDrr,values were seen under conditions of 3 Hz stimulation. The ol) value of 6.12 x 10 - 9 M (95% C.I. = 3.63 x 10 - 9 to 1.03 x 10~8 M) obtained —8 at 6 mM calcium was significantly lower than that of 2.08 x 10 M (95% C.I. = 1.20 x 10~8 to 3.62 x 10~8 M) seen at 0.5 mM calcium. With histamine, the changes in F D ^ Q values were less noticeable. The FD^ values in the presence of 0.5 and 2.2 mM calcium were similar at a l l frequencies studied. The value of 9.14 x 10~7 M (95% C.I. = 6.18 x 10~7 -6 -7 to 1.35 x 10 M) seen under conditions of 1 Hz and 6 mM and 3.00 x 10 M (95% C.I. = 7.58 x 10"8 to 1.19 x 10~6 M) at 3 Hz and 6 mM calcium were —6 significantly reduced compared to that at 0.5 mM calcium (4.76 x 10 M -68-(95% C.I. = 2.48 x 10" to 9.12 x 10 M) and 1.58 x 10 M (95% C.I. 1 P ..... = 8.89 x 10 to 2.81 x 10 M) respectively). The ED 5 Q values for isoproterenol were an average, of 2 orders of magnitude more potent than those for histamine. II. The Effect of Changing Temperature and Calcium Concentration 1. Basal Developed Force The effect of changing temperature and calcium concentration of the BDF of rabbit papillary muscles is illustrated in figure 25. At each temperature studied, as the calcium concentration was raised the BDF increased. The BDF under conditions of 6 mM calcium were always signif-icantly larger than those at 0.5 mM. This was especially noticeable at 25°, where the BDF at 0.5 mM calcium was 0.4382 - .0499 g, compared to 1.9633 - .1143 g at 6 mM. The increases in BDF were less extreme at the other temperatures, that at 37.5° and 6 mM calcium was 0.5750 -.0649 g and at 42° and 6 mM calcium 0.8333 - .0890 g. In addition at 37.5°, the BDF at 2.2 mM calcium (0.3093 - .0234 g) was significantly larger than that at 0.5 mM calcium (0.0398 - .0059). The values obtained at 37.5° compare favourably with the observations made on guinea pig papillary muscles (figure 7 ). In general as the temperature was raised, at each calcium concentration the BDF decreased and then rose again slightly at 42°. 2. Maximal Developed Force Dose-response curves for isoproterenol and histamine were carried out using the various experimental conditions described above. -69-Figure 26 shows the dose-response curves for isoproterenol when both temperature and calcium concentration were altered. The response to isoproterenol at 25° and 6 mM calcium was much larger than the response at any other condition studied. This was totally due to the huge BDF value of 1.9633 - .1143 g. The efficacy of isoproterenol under these conditions was not very large. Conditions under which isoproterenol produced good inotropic effects were 37.5° and 6 mM, 37.5° and 2.2 mM, 42° and 6 mM, and 42° and 6 mM calcium. The response in the presence of 42° and 2.2 mM calcium was slightly lower. The response to isoproterenol was compromised under conditions of 25° and 0.5 mM, 25° and 2.2 mM, and 37.5° and 0.5 mM calcium. The MDF data (figure 27 ) shows that only 3 conditions produced a significant increase in MDF compared to 0.5 mM calcium. These were 25° and 6 mM calcium where the MDF increased from 0.9167 - .1801 g at 0.5 mM calcium to 2.5000 - .2573 g, 37.5° and 2.2 mM calcium where the MDF increased from 0.7900 - .1352 g to 1.8111 - .2245 g, and 37.5° and 6 mM calcium where the MDF increased from 0.7900 - .1352 g to 1.8375 - .1413 g. At 42°, altering the calcium concentration had l i t t l e effect on the MDF. In general, raising the calcium concentration increased the tension developed. The dose-response curves for histamine are shown in figure 28. Again, an exceptionally large response was seen under conditions of 25° and 6 mM calcium. The other responses were lower than those seen with isoproterenol. The response to 37.5° and 0.5 mM calcium was especially reduced. The MDF data (figure . 29) shows that the same conditions produced a significant increase in the MDF compared to 0.5 mM calcium. At 25° the MDF increased significantly from 0.8550 - .1230 g at 0.5 mM to 2.7688 - .2962 g at 6 mM. At 37.5° the MDF increased significantly from 0.309 - .0803 g at 0.5 mM, to 1.3111 - .1233 g at 2.2 mM and 1.3625 - .1401 g at 6 mM calcium. Only at 37.5° and 0.5 mM was the response to histamine significantly less than that to isoproterenol. The data for isoproterenol and histamine, replotted as % increase of MDF over BDF is illustrated in figure 30 . The % increase for both agonists was greatest with low calcium concentrations, with isoproterenol decreasing from 2400 - 558% at 37.5° and 0.5 mM to 269 - 46% at 1 Hz and 6 mM calcium. An even larger change was seen at 42°, where the % increase decreased from 3025 - 911% at 0.5 mM to 155 - 33% at 6 mM cal-cium. With histamine the values were smaller, at 37.5° decreasing from 945 - 282% at 0.5 mM to 227 - 70% at 6 mM calcium. At 42° the change was larger, from 1310 - 300% at 0.5 mM to 63 - 15% at 6 mM calcium. 3. ED c n Data oU Figures 31 and 32 illustrate the effect of changing the temperature and calcium concentration on the.ED^g values for isoproterenol and hista-mine in rabbit papillary muscles. For isoproterenol, at 25° and 37.5° the ED c n decreased from 1.62 x-10-8 M (95%. C.I. = 5.53 x 10~9 to 4.76 x oU 10~8 M) and 6.41 x 10~8 M (95% C.I. = 4.74 x 10 _ 8 to 8.67 x 10 _ 8 M) at -9 -10 -9 0.5 mM calcium, to 1.60 x 10 M (95% C.I. = 3.63 x 10 to 7.04 x 10 M) and 1.50 x 10~8 M (95% C.I. = 1.28 x 10 - 8 to 1.75 x 10 - 8 M) respec-}50 tively at 6 mM calcium. At 42° the largest ED 5 Q value of 8.13 x 10 " M (95% C.I. = 5.94 x 10~8 to 1.11 x 10~7 M), was seen at 2.2 mM calcium. -71-With 2.2 and 6 mM calcium, as the temperature was raised, an increase in the E D R N was seen. For histamine, the same increase of oU E D J - Q with raising of the temperature was seen, but as the calcium concentration was raised there was l i t t l e effect on the E D ^ . Only when the calcium concentration at 25° was raised from 0.5 mM where the E D 5 Q was 2.73 x 10 M (95% C.I. = 1.11 x 10" to 6.67 x 10" M) t o 2.2 mM where the E D C N was 6.00 x 10 - 7 M (95% C.I. = 3.56 x 10 - 7 bu to 1.01 x 10~6 M), and at 37.5° when the calcium concentration was —6 raised from 0.5 mM where the E D 5 Q was 4.76 x 10 M (95% C I . = 2.48 x 10"6 to 9.12 x 10~6 M) to 6 mM where the E D 5 Q was 9.14 x 10 - 7 M 1 K (95% C.I. = 6.18 x 10" to 1.35 x 10~ M) was any significant differ-ence seen. Again on average, the E D 5 0 values for isoproterenol were 2 orders of magnitude more potent than those for histamine. Ill Use of D600. Dose-response curves for isoproterenol and histamine were carried out in the absence and presence of D600 (figure 33 ). The dose of D600 _7 used was 5 x 10 M. Higher doses almost totally depressed the response to isoproterenol, smaller doses did not shift the curve. The dose-res-ponse curve for isoproterenol was shifted to the. right by D600, but the maximal response was not affected. There was a significant difference between the E D ^ Q values for the 2 curves, they increased from 2.04 x 10"8 M (95% C.I. = 1.07 x 10~8 to 3.90 x 10"8 M) in the control situation, to 5.66 x 10"8 M (95% C.I. = 3.44 x 10~8 to 9.29 x 10 - 8 M) in the presence of D600. The curve for histamine was also shifted to the right, but ~6 the ED 5 0 values were not significantly different, at 5.43 x 10 M (95% C.I. = 3.36 x 10 to 8.78 x 10 M) in the control situation, and 9.95 x 10 6 M (95% 'C.l. = 2.84 x,10~6to 1.04 x 10-5• M). in .the. .presence of D600. C. Radioisotope Studies 1. Measurement of the. Extracellular Space a. Rabbit Papillary Muscles Figure 34 illustrates the determination of the extracellular space (ECS) for rabbit papillary muscle using "^C-sorbitol. Using whole muscles, following 30 minutes incubation in CK solution, 1 4C-sorbitol uptake increased rapidly up to 30 minutes and then more slowly up to 60 minutes, giving a mean ECS value of 0.4787 - 0.052 (n=2). Using split 14 muscles, the C-sorbitol uptake increased steadily up to 60 minutes. At this time the value for ECS was 0.6752 - 0.089 (n=2). b. Rabbit Left Atrial and Right Ventricle Strips With both left atrial and right ventricle strips (figure 35 ) following 60 minutes equilibration in CK solution, there was a rapid 14 uptake of C-sorbitol up. to 30 minutes, followed by .a plateau phase to 60 minutes. ECS values were 0.4665 - 0.015 (n=3) for left atrial strips,-and 0.4261 - 0.025 (n=3) for right ventricle strips. c. Guinea Pig Right Ventricle Strips C-sorbitol uptake was similar in the guinea pig right ventricle strips (figure 36). Following 60 minutes equilibration in CK solution -73-there was a rapid uptake of the labelled compound up to 15 minutes, followed by a plateau phase up to 30 minutes. The ECS value was 0.3509 - 0.033 (n=4). 2. Removal of Extracellular Calcium a. Rabbit. Right Ventricle Strips Seven different modifications of sodium Hepes solution of 0° were used to determine which.was the most efficient at removing extracellular calcium from rabbit right ventricle strips. In addition a control sodium Hepes solution at 37° was used (figure 37). Using the control solution o 45 at 37 , there was a steady decrease of tissue Ca with time, which appeared to slow, down around 100 minutes. The curve seen with 0 Ca and 10 mM La also decreased steadily with time, but a plateau phase was never seen, suggesting a constant leakage of intracellular calcium was taking place. The curves for.0 Ca and 0.5 mM EGTA, 0. Ca and 10 mM Mg, and control solution at 0° were a l l similar. The 0 Ca and 10 mM Mg curve produced a more prominent plateau phase than the .other 2 curves. The curve for the 0 Ca., 0 Mg and 140 mM Na solution was similar to these 3 curves, 45 but gave a larger value for tissue Ca content. The solutions contain-45 ing isotonic sucrose and choline gave much larger values for Ca content, and did not appear to remove a l l the extracellular calcium. 45 The release of Ca into the control solution could be approximated by the sum of two first-order processes (figure 38). The i n i t i a l points were not included in the calculations. At 0° the value for calcium -74-assumed to be intracellular and effluxing with a rate constant of 0.006 min ^  was 839 ymoles calcium/kg. At 37° the values for calcium assumed to be bound in the extra-cellular space and washing out with a rate constant of 0.061 min \ was calculated to be 1260 ymoles calcium/kg. The value for calcium assumed to be intracellular and effluxing with a rate constant of 0.015 min was 535 ymoles calcium/kg. The control curve at 37° and the 0 Ca and 10 mM Mg curve were repeated using a modified CK solution (figure 39). The results were similar to those seen using the Hepes buffers. In a l l subsequent experi-ments tissues were placed in the 0 Ca and 10 mM Mg CK solution at 0° for 60 minutes to remove extracellular calcium. b. Guinea Pig Right Ventricle Strips The 2 control curves and the 0 Ca and 10 mM Mg at 0° curve were repeated using modified CK solution, and guinea pig right ventricle strips (figure. 40). Basically,, the curves were similar to those using rabbit ventricle strips, and a time of 60 minutes was chosen to remove extracellular calcium. 4^  3. Ca Uptake with Time a. Quiescent Tissues When rabbit right ventricle strips were placed in the loading 45 solution of Ca for up to 120 minutes, there was almost linear uptake -75-45 of Ca up to 60 minutes, followed by a plateau phase (figure 41). 45 + The Ca content at 120 minutes was 1336.7 - 22.4 ymoles/kg wet weight, which was not different from the values of 1227.3 - 52.7 ymoles/kg at 60 minutes, or 1283.6 - 70.7 ymoles/kg at 90 minutes. 45 The uptake of Ca into quiescent rabbit right ventricle strips during the fi r s t 10 minutes after placement in a CK solution containing 45 . 45 Ca is shown in figure 42. In the control CK solution, Ca uptake proceeded quite linearly up to 10 minutes, reaching a value of 511.8 -—6 41.6 ymoles/kg wet weight. Addition of 10 M isoproterenol to the 45 solution had no effect on the Ca uptake at any time studied. The value at 10 minutes was 442.1 - 4.5 ymoles/kg wet weight. However, when the tissues were placed in a modified CK solution containing 0 Na and 126 mM K, (subsequently referred to as 126 mM K), -6 45 or 126 mM K plus 10 M isoproterenol the uptake, of Ca was increased compared to the control, reaching a value of 860.8- - 52.1 ymoles/ kg wet weight at 10 minutes with 126 mM K alone, and 834.5 - 16.4 ymoles/ —6 kg wet weight with 126 mM K plus 10 M isoproterenol. 45 If the tissues were f i r s t loaded with Ca for 80 minutes, and then placed in the 126 mM K solution for 10 minutes, a significant increase' in 45 Ca content was seen compared to the control (figure 43 ), rising from 1167.16 - 83.66 ymoles/kg wet weight to 1720.75 ymoles/kg wet weight. —6 This same increase in 126 mM K was seen when either 10 M isoproterenol (1959.80 - 71.45 ymoles/kg wet weight) or 10 M histamine (1611.72 -68.32 ymoles/kg wet weight) was added. However in the normal CK solution containing isoproterenol (1454.30 - 49.14 ymoles/kg wet weight) or 45 histamine (1162.65 ymoles/kg wet weight), the Ca content was not -76-different from the control. b. Stimulated Tissues When the tissues were stimulated at 1 Hz, similar results were 45 seen (figure 44). Up to 4 minutes there was a linear increase of Ca uptake in control tissues, reaching a value of 239.63 - 17.80 ymoles/kg —6 wet weight, as well as those in the presence of 10 M isoproterenol (258.76 - 22.68 ymoles/kg wet weight) or 10~4 M histamine (222.84 -20.84 ymoles/kg wet weight). The values of 160.21 - 11.71 ymoles/kg wet weight obtained in the presence of isoproterenol for 2 minutes was significantly larger than that of 115.95 - 9.02 obtained from control tissues. No significant difference from control was seen for histamine. Following 80 minutes loading, tissues in 126 mM K solutions had 45 ' + a higher Ca content than controls, ranging from 2121.10 - 493.19 ymoles/ kg wet weight for 126 mM K plus 10~ M isoproterenol, to 1316.42 -228.14 ymoles/kg wet weight for isoproterenol alone (figure-45). Controls ranged from 1428.10 - 107.83 ymoles/kg wet weight for 126 mM K, to 1120.39 - 135.12 ymoles/kg wet weight for normal CK, but unlike the quiescent tissues, none of the values were significantly different from each other. Following exposure to 126 mM K, the tissues went into contracture (figure 46). It was not possible to quantitate whether the contracture g was changed by concommitant administration of 10 M isoproterenol (b) -5 or 10 M histamine (d). Use of isoproterenol or histamine alone prod-uced no change in 4 5Ca content (figure 45) compared to controls although a large inotropic effect was seen (figure 46 c,e~). -77-Figure . 7. The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the Basal Developed Force of Guinea Pig Papillary Muscles. a: temperature was altered while the frequency of stimulation and calcium concentration were held constant at 1 Hz and 2.2 mM. b: frequency of stimulation was altered while the temperature and calcium concentration were held constant at 37.5° and 2.2 mM. c: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. The bars represent the mean BDF - SEM. The numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to 37.5°, 1 Hz and 2.2 mM calcium (Newman-Keuls test). 1.0-0.8 J ~ 0 . 6 l Q 00 0.4 0.2 (25) (31) (33) (26) 25 30 37.5 42 + 1Hz,2.2mMCa1 T * (20) 1( 1 (33)|(22)|(25) (18) 0.5 1.0 20 3.0 4.0 Hz + 37.5? 2.2mM C a 4 4 I (33)1 (34]|(24)|(31) 1.5 2.2 4 6 8mM (Ca^) + 37.5° 1 Hz i . 00 I Figure 8. The Effect of Changing Temperature on the. .Dose-Response Curves of Isoproterenol and Histamine in Guinea Pig Papillary Muscles. The plot depicts the tension developed in response to isoproterenol and histamine at 4 different temperatures while frequency of stimu-lation and calcium concentration were held constant a 1 Hz and 2.2 mM. Each point represents the mean tension from 5-18 different experi-ments (see figure 9). SEM were omitted for the sake of clarity. -81-Figure 9. The Effect of Changing Temperature on the Maximal Developed Force of Guinea Pig Papillary Muscles to Isoproterenol and Histamine. •The bars represent the mean MDF - SEM in response to isoproterenol and histamine., Frequency of stimulation and calcium concentration were held constant at..l Hz and 2.2 mM. The numbers in parentheses indicate the number of observations. -82-2.0i 1.61 B 1.21 Q 0.^ ISO 1 HIS l 1 0.41 (9) (18) (18) (9) 25 3 0 37.5 42 (1 2) (6) (9) (5) 25 3 0 37.5 42 T° +1Hz, 2.2mMCa -83-Figure 10. The Effect of Changing Frequency of Stimulation on the Dose-Response Curves of. Isoproterenol and Histamine in Guinea Pig Papillary Muscles. The plot depicts the tension developed in response to isoproterenol and histamine at 5 different frequencies of stimulation. Temperature and calcium concentration were .held constant at 37.5° and 2.2 mM. Each point represents the mean tension from 5-18 different experiments (see figure 11). SEM were omitted for the sake of clarity. Histamine (-log M) -85-Figure 11. The Effect of Changing Frequency of Stimulation on the Maximal Developed Force, of Guinea Pig Papillary Muscles, to Isoproterenol and Histamine. The bars represent the mean MDF - SEM in response to isoproterenol and histamine. Temperature and calcium concentration were held constant at 37.5° and 2.2 mM. The numbers in .parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to 37.5°, 1 Hz and 2.2 mM calcium (Newman-Keuls test). -86-ISO 1 JL (12) I (18) (10) (10) (10) 0.5 1 2 3 4 HIS (5) 1 ( 9 ) (7) T* (13) (8) 0.5 1 2 3 4 Hz + 37.5? 2.2 mMCa -87-Figure 12. The Effect of Changing Calcium Concentration on the Dose-Response Curves of Isoproterenol and Histamine in Guinea Pig Papillary Muscles. The plot depicts the tension developed in response to isoproterenol and histamine at 5 different calcium concentrations. Temperature and frequency of stimulation were held constant at 37.5°'and 1 Hz. Each point represents the mean tension from 7-18 different experiments (see figure 13). SEM were omitted for the sake of clarity. -89-Figure 13. The Effect of Changing Calcium Concentration on the Maximal Developed Force of Guinea Pig Papillary Muscles to Isoproterenol and Histamine. The bars represent the mean MDF - SEM in response to isoproterenol and histamine. Temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. The numbers in parenthese indicate the number of observations. -90-2.0-. ISO HIS 1.6 1.21 I T i I I 1 1 I 0.8 A 0.4 (12) (18) (18) (11) (13) 1.5 2.2 4 6 8mM (7) (9) (14) (8) 1(15) 1.5 2.2 4 6 8mM (Ca^) + 37.5? 1 Hz -91-Figure 14. The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the % Increase of Maximal Developed Force over Basal Developed Force, in Guinea Pig Papillary Muscles. a: temperature was altered while the frequency, of stimulation and calcium concentration were held constant at 1 Hz and 2.2 mM. b: frequency of stimulation was altered while the temperature and calcium concentration were held constant at 37.5° and 2.2 mM. c: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. The bars represent the mean % increase of force - SEM. The number of observations is the same as in figures 9, 11 and 13. -92-75-5-2.5-15-10-cs o T -X 5-0) o o c rease 25-inci 32 20-2 . 1 a 25 30 37.5 42 2 5 30 375 42° +1Hz, 2.2 mM Ca I 05 1 2 3 4 05 1 2 3 4 Hz +37.5;2.2mM Can 15 10H 1 T , Iso SHIS 1.5 2.2 4 6 8 1.5 2.2 4 6 8mM +37.5? 1Hz -93-Figure 15. The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the ED 5 Q Value for Isoproterenol in Guinea Pig Papillary Muscles. The bars represent the. geometric mean ED^ Q values - 95% confidence intervals. The numbers in parentheses indicate the number of observations-. Asterisks indicate a significant difference at P<0.05, compared to 37.5°, 1 Hz and 2.2 mM calcium (Newman-Keuls test). 60 50 A 40 o> 'o £ 30 o Q m 201 101 ( 1 1 ) ( 1 6 ) ( 1 8 ) ( 9 ) 25 30 37.5 42 T° +1Hz,2.2mM Ca4 ( 1 0 ) ( 1 8 ) ( 1 0 X C 1 1 ) ( 1 0 ) 0.5 1 2 3 4 Hz +375° 2.2 mM Ca"1 ( 1 2 ) i I D -P I ( 1 8 ) ( 1 8 ) ( 1 1 ) 4h ( 1 4 ) 1.5 2.2 4 6 (Ca^) +37.5? 1 Hz 8mM - 9 5 -Figure 16. The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the EDgn Value for Histamine in Guinea Pig Papillary Muscles. The bars represent the geometric mean ED^ values - 95% confidence intervals. The numbers in parentheses indicate the number of observations. 20-. o It) Q u 8 41 (10) (9) (9) (15) 25 30 375 42 T° +1Hz,2.2mMCat ( 8 ) (9) (7) (13) ( 8 ) Q5 1 2 3 4 Hz + 37.5?2.2mMCa'!+ (7 ) ( 9 ) (15) (8 ) (16) 1.5 2.2 4 6 8mM (Ca- ) + 375°1Hz. Figure 17 The Effect of Changing Frequency of Stimulation and Calcium Concen-tration on the Basal Developed Force of Rabbit Papillary Muscles. a: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 0.2 Hz. b: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 0.5 Hz. c: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. d: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 3 Hz. The bars represent the mean BDF - SEM. The numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium concentration (Newman-Keuls test). -98-0.7-0.6 A 0.5" 0.4 H £ 0.3 H 0.2 H o.H 2*1 ( 1 7 ) ( 1 3 ) ( 2 1 ) Hz 0.2 — [Catt]0.5 2.2 6 ( 1 9 ) 23 0.5 ( 1 5 ) 21 ( 2 2 ) ( 2 7 ) ( 1 6 ) 0.5 2.2 6 0.5 2.2 6 J t ( 1 6 ) ( 1 2 ) ( 1 6 ) 0.5 2.2 6 M M -99-Figure 18. The Effect of Changing Frequency of Stimulation and Calcium Concen-tration on the Dose-Response Curve of Isoproterenol in Rabbit Papillary Muscles. The plot depicts the tension developed in response to isoproterenol at four different frequencies of stimulation and three different calcium concentrations. The temperature was maintained at 37.5°. Each point represents the mean tension from 5-11 different experiments (see figure 19). SEM were omitted for clarity. -100--101-Figure 19. The Effect of Changing Frequency of Stimulation and Calcium Concentration on the Maximal Developed Force of Rabbit Papillary Muscles to Isoproterenol. The bars represent.the mean MDF - SEM in response to isoproterenol. The temperature was maintained at 37.5°. The numbers, in parentheses indicate the number, of observations. Asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls test). -102-I JL (8) (7) (11) Hz 0.2 (8) (9) 0.5 (7) (10) (9) (8) (8) (5) (8) [Catt]0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 M M -103-Figure 20. The Effect of Changing Frequency of Stimulation and Calcium Concentration on the Dose-Response Curve of Histamine in Rabbit Papillary Muscles. The plot depicts the tension developed in response to histamine at four different frequencies of stimulation and three different calcium concentrations. The temperature was maintained at 37.5°. Each point represents the mean tension from 6-15 different experiments (see figure 21). SEM were omitted for clarity. -104-O 0 . 2 H z + 0 . 5 mM Ca++ 1.6-c o v> c 0) 1.2-0.8-0.4H • 0 . 2 2 . 2 A 0 . 2 6 A 0 . 5 0 . 5 • 0 . 5 2 . 2 • 0 . 5 6 V 1 0 . 5 • 1 2 . 2 O 1 6 • 3 0 . 5 X 3 2 . 2 * 3 6 I I I I 8 7 6 5 Histamine (-log M) C -105-Figure 21. The Effect of Changing Frequency of Stimulation and Calcium Concen-tration on the Maximal Developed Force of Rabbit Papillary Muscles to Histamine. The bars represent the mean MDF - SEM in response to histamine. The temperature was maintained at 37.5°. The numbers in parentheses indicate the number of observations. The black asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls test). The open asterisks indicate a significant difference at P<0.05, compared to the value obtained with isoproterenol (Bonfer-roni test). - 1 0 6 -2.CH 5> 1.6-o 1.2H 0.8-0.4-1 Jt ti (8) ( 6 ) (10)1 Hz 0.2 (12) | 0.5 (8) (1D (15) (8) (8) ( 6 ) J_ (8) [Catt]0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 M M -107-Figure 22. The Effect of Changing Frequency of Stimulation and Calcium Concentration on the % Increase of Maximal Developed Force over Basal Developed Force in Rabbit Papillary Muscles. a: Calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37..5° and 0.2 Hz. b: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 0.5 Hz. c: calcium' concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 3 Hz. The bars represent the mean % increase of force - SEM. The number of observations is the same as in figures 19 and 21. -108--109-Figure 23. The Effect of Changing Frequency of Stimulation and Calcium Concentration on the ED^ Value for Isoproterenol in Rabbit Papillary Muscles. The bars represent.the geometric mean EL\.Q values - 95% confidence intervals. The .numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls test). - 1 1 0 -45-40-1 3 5 H _ 30-» 20H o Q HI 15H 10-1 5H ( 6 ) (8) Hz (7) 0.2 (11) (8) (9) 0.5 (7) (10) (11) "(8)1 (8) .(8) [Catt]0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 M M -111-Figure 2U. The Effect of Changing Frequency of Stimulation and Calcium Concentration on the E D 5 Q Value for Histamine.in Rabbit Papillary Muscles. + The bars represent the geometric mean ED^ Q values - 95% confidence intervals. The numbers in parentheses indicate the number of obser-vations. Asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls). -112-20^ 18H 12H f 10-I o in Q LU 8H 6 H 4H 2H (8) ( 6 ) (10) Hz 0.2 (9) (12) (8) (11) (15) (8) | 0.5 (5) (8) (8) [Catt]0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 0.5 2.2 6 M M -113-Figure 25. The Effect of Changing Temperature and Calcium Concentration on the Basal Developed Force of Rabbit Papillary Muscles. a: calcium concentration was altered while- the temperature and frequency of stimulation were held constant at 25° and 1 Hz. b: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. c: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. The bars represent the mean BDF - SEM. The numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls test). -114-a (17) 1 (16)(15) (22) (27j(16) 25 (Ca + + ) 0 5 2 2 6 — 3 7 . 5 — 0.5 2.2 6 4 (16M(14)(18) 42 0.5 2.2 6 m M -115-Figure 26. The Effect of Changing Temperature and Calcium Concentration on the Dose-Response Curve of Isoproterenol in Rabbit Papillary-Muscles . The plot depicts the tension developed in response to isoproterenol at three different temperatures and three different calcium concentra-tions. The frequency was maintained at 1 Hz- Each point represents the mean tension from 6-10 different experiments (see figure 27). SEM were omitted for clarity. -116-o 25° (X5mMCa++ C 9 8 7 6 5 Isoproterenol (-log M) -117-Figure 27. The Effect of Changing Temperature and Calcium Concentration on the Maximal Developed Force of Rabbit Papillary Muscles to Isopro-terenol . The bars represent the mean MDF - SEM in response to isoproterenol. The frequency was maintained at 1 Hz. The numbers in parentheses indicate the. number .of observations. Asterisks indicate a significant difference at P<0.05, compared.to 0.5 mM calcium (Newman-Keuls"test). 2 B i 2.4--118-2D-I 1.6 i uT 1.2-1 o I 04 (6) (8) 25 (7) (Ca + + ) 0 5 2 2 6 00) ( 9 ) (8) 37.5— 0.5 2.2 6 (8* C7JI 42 (8)1 0.5 2.2 6 m M -119-Figure 28. The Effect of Changing Temperature and Calcium Concentration on the Dose-Response Curve of Histamine in Rabbit Papillary Muscles. The plot depicts the tension developed in response to histamine at three different temperatures and three different calcium concen-trations. The frequency was. maintained at 1 Hz. Each point represents the mean tension from 7-15 different experiments. SEM were omitted for clarity. -120--121-Figure 29. The. Effect of Changing Temperature and ^ Calcium Concentration on the Maximal Developed Force of Rabbit Papillary Muscles to Histamine. The bars represent the mean MDF - SEM in response to histamine. The frequency was maintained at 1 Hz. The numbers in parentheses indicate the number of observations. The black asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls test). The open asterisks indicate a significant difference at P<0.05, com-pared to the value obtained with isoproterenol (Bonferroni test). - 1 2 2 -2Si 24 4 2JO 1.6 O ) iT 1.2 J Q s 0.8^  IJ. 4 1 0.4-1 (10) (8) 25 (8)1 (Ca + + ) 0 5 2 2 6 (11) d5) (8) — 3 7 . 5 — 0.5 2.2 6 \tsi (7)| 42 (10) 0.5 2.2 6mM -123-Figure 30. The Effect of Changing Temperature and Calcium Concentration on the % Increase of Maximal. Developed Force over Basal Developed Force in Rabbit Papillary Muscles. a: calcium concentration was altered, while the temperature and frequency of stimulation were held constant at 25° and 1 Hz. b: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 37.5° and 1 Hz. c: calcium concentration was altered while the temperature and frequency of stimulation were held constant at 42° and 1 Hz. The bars represent the mean % increase of force - SEM. The number of observations is the same as in figures 27 and 29. -125-Figure 31. The Effect of Changing Temperature and Calcium Concentration on the ED^ Q Value for Isoproterenol in Rabbit Papillary Muscle. The bars represent the. geometric mean E D ^ values - 95% confidence intervals. The numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to. 0.5 mM calcium (Newman-Keuls test). - 1 2 6 -14-. i 101 00 I O in (6) i (8) i(6) T 25 (Ca + + ) 0 5 2 2 6 (10) 1(9) (8) — 3 7 . 5 — 0.5 2.2 6 Ic 3 I (7) (8)1 42 0.5 2.2 6mM -127-Figure 32. The Effect of Changing Temperature and Calcium Concentration on the EDgQ Value for Histamine in Rabbit Papillary Muscles. The bars represent the geometric mean ED^ Q values - 95% confidence intervals. The numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to 0.5 mM calcium (Newman-Keuls test). -128-(8) C I O ) 25 (C a++)0.5 2.2 6 (11) (15) ft 37.5— 0.5 2.2 6 ( 8 4 2!l 42 10) 0.5 2.2 6mM -129-Figure 33. The Effect of 60 Minutes Pretreatment with D600 on the Dose-Response Curves of Isoproterenol and Histamine in Rabbit Papillary Muscles. The plots depict the tension developed in response to isoproterenol -7 and histamine m the absence.and presence of 5 x 10 M D600. The experimental conditions were 37.5°, 1 Hz and 2.2 mM calcium. Tissues were incubated with D600 for 60 minutes before the agonists were added to the bath. Each point represents the mean tension developed from 8-10 experiments - SEM. -130-Iso Control lso +5x10~7M D600 His Control His+5x10"7M D600 E D 5 0 2.04 x10"8 M, n = 8 5.66 x10 _ 8M, n = 9 5.43x10"6M, n = 10 9.95x 10 8 M, n=9 3.2 2.81 24 ^20 1 .21-61 (A i2 1.2 0.8 0.41 T 7 6 Dose (-log M) -131-Figure 34. 14 C-Sorbitol Measurement of the Extracellular Space of Rabbit Papillary Muscles. 14 The plot depicts the uptake of C-sorbitol by whole and split papillary muscles at 30 and 60 minutes. Each point represents the mean ECS - SEM. The numbers in parentheses indicate the number of observations. Tissues were initially allowed to equilibrate in aerated CK solution 14 for 30 minutes prior to administration of C-sorbitol for 30 and 60 14 minutes. C was analyzed as described in the methods section. -132-0.8, Rabbit Papillary Muscles 14-C Sorbitol Measurement of Ext race 11 u la r Space 0.6] * 0.* U) CO o LU 0.2 Split muscles 30 Time (min) 60 -133-Figure 35. 14 C-Sorbitol Measurement of the Extracellular Space, of Rabbit Left Atrial and Right Ventricle Strips. 14 The plot depicts the uptake of C-sorbitol by left atrial and right ventricle strips at 30.and 60 minutes. Each point represents the mean ECS - SEM. The numbers in parentheses indicate the number of observations. Tissues were initiall y allowed to equilibrate in aerated CK solution 14 for 60 minutes prior to administration of C-sorbitol for 30 and 60 14 minutes. C was analyzed as described in the methods section. -134-Rabbit Left Atrium & Right Ventricle Strips 0.61 1 4 S o r b i t o l Measurement of Extracellular Space * 0.4 (0 o LU 0.2 0 30 Time (min) 60 -135-Figure 36. C-Sorbitol Measurement of the Extracellular Space of Guinea Pig Pdght Ventricle Strips. The plot depicts the uptake of """^C-sorbitol by guinea pig right ventricle strips at 15 and 30 minutes. Each point represents the mean ECS - SEM. The numbers in parentheses indicate the number of observations. Tissues were initiall y allowed to equilibrate in aerated CK solution 14 for 60 minutes prior to administration of C-sorbitol for 15 and 30 14 minutes. C was analyzed as described in the methods section. -136-Guinea Pig Right Ventricle Strips 14-C Sorbitol Measurement of Extracellular Space 0 15 30 Time (min) -137-Figure 37. Residual Ca Content of Rabbit Right Ventricle Strips as a Function of Incubation Time Using Different Isotope-Free Quenching Solutions. The plot depicts residual content of 't°Ca in rabbit right ventricle strips as a function of incubation time in 7 different isotope-free solutions at 0°, and a control at 37°. Each point represents the tissue 45 Ca content in ymoles/kg wet weight from duplicate samples. 45 o Tissues were loaded with Ca in a sodium Hepes solution at 37 for 150 minutes. They were then ..passed sequentially every 10 minutes through test tubes containing 3 ml of the PSS being investigated, as labelled on 45 the diagram. The tissues and solutions were assayed for Ca as described in the methods section. The points were plotted sequentially by adding the counts present, in the tissue to those present in the PSS from 150 to zero minutes. -138-45, Rabbit RV strips Ca Content as a Function of Time OmM Ca,0Mg,0Na,260 Sucrose 0Mg,0Na,140Choline •o-0Ca,10La Control,37' 20 40 60 80 100 120 140 Time (min) -139-Figure 38. 45 Compartmental Analysis of the Release of Ca from Rabbit Right Ventricle Strips into Isotope-Free Solutions at 0° and 37°. 45 The plot depicts the residual content of Ca in rabbit right ventricle strips as a function of incubation time in isotope-free solutions at 0° ( A , A) and 37° (• , O ) , resolved into 2 exponential 45 components. Each point represents the tissue Ca content in limoles/kg wet weight from duplicate experiments. 45 o Resolution of the efflux of Ca from the control solution at 37 and 0° into 2 exponential components was carried out by 'curve peeling'. The equation used for both exponential components was a^ = Ae -^, where a^ is the quantity of label present in the tissue at time t; A is the y-intercept of the regression line for the efflux; and b is the rate 45 constant for the efflux of Ca from the tissue. -140-500O, -141-Figure 39. Residual Ca Content of Rabbit Right Ventricle Strips as a Function of Incubation Tine using Modified Chenoweth-Koelle Solutions. The plot depicts the residual content of Ca in rabbit right ventricle strips as a function of incubation time in isotope-free solution at 37°, and 2 modified isotope-free CK solutions at 0°. 45 Each point represents the tissue Ca content in ymoles/kg wet weight from duplicate samples. 45 o Tissues-were-loaded.with Ca in CK, solution at- 37 for 150 minutes. They were.then passed sequentially every 10 minutes through test tubes containing 3 ml of the PSS being investigated, as labelled in the diagram. The tissues and solutions were assayed.for Ca as described in the. methods section. The points were plotted, sequentially by adding the.counts present in the tissue to those present in the PSS from 150 to zero minutes. -142-10000-1 Time (min) -143-Figure 40. 45 Residual Ca Content of Guinea Pig Right Ventricle Strips as a Function of Incubation Time using Modified Chenoweth-Koelle Solutions. 45 The plot depicts the residual content of Ca in guinea pig right ventricle strips as a function of incubation time in isotope-free CK solution at 0° and 37°, and isotope-free CK solution at 0° containing 45 10 mM Mg, 0 Ca. Each point represents the tissue Ca content in u moles/kg wet weight from duplicate samples. 45 o Tissues were loaded with Ca in CK solution at 37 for 150 minutes. They were then passed sequentially every 3 minutes through test tubes containing 3 ml of the PSS being investigated, as labelled in the diagram. 45 The tissues and solutions were assayed for Ca as described in the methods section. The points were plotted sequentially by. adding the counts present in the tissue to.those present in the PSS from 150 to. zero minutes. -144-10000 5000 Guinea Pig RV strips ^ C a Content as a Function of Time 20 40 60 80 100 Time (min) 120 140 -145-Figure 41. 45 Measurement of Ca Uptake into Quiescent Rabbit Right Ventricle Strips. 45 The plot depicts the Ca content of quiescent rabbit right ventricle strips as a function of time, over 10-120 minutes after placement in a 45 loading solution. Each point represents the tissue Ca content in ymoles/kg wet weight - SEM. The numbers in parentheses indicate the number of observations. -146--147-Figure 42, 45 Measurement of Ca Uptake by Quiescent Tissues over 2-10 Minutes. 45 The plot depicts the Ca content of quiescent rabbit right ventricle strips as a function of time, over 2-10 minutes following exposure to 126 mM K and/or 10 M isoproterenol. Each point repre-45 sents the tissue Ca content in unoles/kg wet weight from duplicate samples - SEM (except the 2 minute point for 126 mM K, which is a single observation). - 1 4 8 -Quiescent Rabbit RV strips Uptake 0 2 4 6 8 10 Time in Ca(min) -149-Figure 43. Measurement of Ca Uptake by Quiescent Tissues After 80 Minutes Loading Followed by 10 Minutes Treatment. The bars represent the mean L+°Ca content in ymoles/kg wet weight - SEM of quiescent rabbit right ventricle strips after 80 45 minutes loading in Ca, followed by 10 minutes drug or 126 mM K 45 treatment in the presence of Ca. The numbers in parentheses indicate the number of observations. Asterisks indicate a signi-ficant difference at P <0.05, compared to the control value (Newman-Keuls test). -150-Quiescent Rabbit RV strips Net Uptake 2200-1 a >„_ s_4S 80min Ca followed by 10min treatment+45Ca I* * Significantly "> Con at p< 0.05 4 CON 126K 126K 10"6M 126 K 10~6M +10"6M ISO +10_6M HIS ISO HIS -151-Figure 44. Measurement^of Ca Uptake by Stimulated Tissues over 2-4 Minutes. The plot depicts the ^ °Ca content of rabbit right ventricle strips stimulated at 1 Hz, as a function of time over 2-4 minutes -6 -4 following exposure to 10 M isoproterenol or 10 M histamine. 45 Each point represents the tissue Ca content in ymoles/kg wet weight - SEM. The numbers in parentheses indicate the number of observations. Asterisks indicate a significant difference at P<0.05, compared to the control value (Newman-Keuls test). -152-Stimulated Rabbit RV strips Uptake 10"6M lso(8) Control ( 7 ) 10"4M His(8) * Significantly y Con at p<0.05 Timein 4 5Ca (min) -153-Figure 45. 45 Measurement of Ca Uptake by Stimulated Tissues After 80 Minutes Loading Followed by 10 Minutes Treatment. 45 The bars represent the mean Ca content in ymoles/kg wet weight - SEM of stimulated rabbit right ventricle strips after 80 minutes 45 loading in Ca, followed by 10 minutes drug or 126 mM K treatment in 45 the presence of Ca. The numbers in parentheses indicate the number of observations. -154-Stimulated Rabbit RV strips Net Uptake 80min 4 5Ca followed by 10min treatment +45Ca 2600-1 2200-1 1800-1 4 0 0 H 10004 C O N 1 2 6 K 1 2 6 K 1 0 ~ 6 M 1 2 6 K 1 0 ~ 6 M + 1 0 ' 6 M I S O + 1 0 ~ 6 M H I S I S O H I S -155-Figure 46. Representative Tracings of the Contractile Response of Rabbit Right Ventricle Strips to Ten Minutes Exposure to 126 mM K, Isoproterenol or Histamine. The tracings are from individual experiments, and illustrate the effect of 10 minutes exposure to: a: 126 mM K. g b: 126 mM K and 10 M isoproterenol. —6 c: 10 M isoproterenol. d: 126 mM K and 10"5 M histamine. -5 e: 10 M histamine. The tension scale represents 2 cm to 1 g, and the time scale 2 cm to 4 minutes. N.B. In tracing c, the.jump in .tension at the arrow is due to the ex-perimental procedure, not addition of the drug. - 1 5 6 -A 1 0 6 M Iso -157-DISCUSSION 1. The Effect of Changing Temperature on Myocardial Contractility In both the guinea pig and rabbit, increasing the temperature decreased the BDF. In the guinea pig the BDF at 25 and 30° were similar, suggesting that under these conditions calcium utilization was at a maxi-mum (figure 7). In the rabbit, combining the change in temperature with changed calcium concentration produced mixed results (figure 25). A combination of 25° and 6 mM calcium produced an extremely large BDF. It was so large that when isoproterenol or histamine was added (figures 27 and 29) the efficacy was very low. Surprisingly, the BDF seen under conditions of 42° and 6 mM calcium was greater than that at 37.5° and' 6 mM calcium, but both were considerably lower than the BDF at 25° and 6 mM calcium. It seems likely that, at the lowest temperatures, the long duration of the active state must play a major role in calcium metabolism and contraction, due to its effects.on the slow inward current. At 42°, the duration.of the action potential is very brief. Hollander and Webb (1955) showed that at 42° i t was only 1/4 of the duration at 23°. Our tissues were a l l paced at 1 Hz, therefore the duration of the action potential per unit tine-would be less' at'. 42° • than- at1 25°. It seems probable that at 42° the influx of calcium per beat is restricted and that the major portion of calcium available for contraction at this temperature is a reflection of increased SR Ca-ATPase activity (Chiesi, 1979; Yazaki et al., 1979) resulting in more effective recycling of the calcium which does enter the cell during the action potential. At 37.5°, both the duration of the action potential and Ca-ATPase activity must be sufficiently reduced so that the BDF is less than .that at 25° and 42°. -158-In the guinea pig, with both isoproterenol and histamine, decreasing the temperature shifted the dose-response curves to the left, and increas-ing the temperature shifted them to the right (figure 8). Similar responses were seen in guinea pig left atria by Reinhardt and co-workers (1972). These shifts were reflected in the EDcn values which were lowest at 25° oU and highest at 42° for both agonists (figures 31 and 32). In the rabbit at 2.2 mM calcium, increasing the temperature to 42° shifted the dose-response curves for isoproterenol and histamine to the right (figures 26 and 28). At 25°, the dose-response curves for both agonists were strangely flattened. A similar effect was seen by Tenner and McNeill (1978). However, according to the ED 5 Q values (figures 31 and 32), the sensitivity increased stepwise from 42° to 25° despite the shape of the dose-response curve. The MDF (figures 27 and 29) were very low, certainly well below the maximum tension which the tissue was cap-able of developing. Under conditions of 0.5 mM calcium, the dose-response curves at 25° and 42° were both shifted to the right. The dose-response curve at 37.5° reached a similar maximum to that at 25° and 2.2 mM cal-cium, but started at a lower BDF. This was most prominent with isoproter-enol, where the ED^ data shows that the tissues were least sensitive under conditions of 37.5° and.0.5 mM calcium, compared to 25° or 42°. With histamine, the sensitivity increased stepwise as the temperature was decreased from 42° to 25°. At 6 mM calcium, again the dose-response curves at 25° and 42° were shifted to the right. There was l i t t l e varia-tion in sensitivity when using isoproterenol, compared to histamine where the sensitivity at 42° and .6 mM calcium was very low compared to 25° or 37.5°. When the effect of changing temperature on the MDF developed in -159-response to the agonists was examined, the guinea pig and rabbit differed considerably. When the effect of isoproterenol was examined in the guinea pig, the MDF was similar at a l l temperatures (figure 9). In the rabbit at 2.2 mM calcium, the MDF was considerably reduced at 25° as described above (figure 27). This did not appear to be a function of the BDF which was.similar to that seen under several other tempera-tures and calcium concentrations. The MDF in response to isoproterenol in the rabbit at 37.5° and 2.2 mM calcium was much larger than that seen in the guinea pig, and similar to that seen under conditions of 37.5° and 6 mM calcium, and 42° and 0.5 mM and 6 mM calcium. There was an extremely large MDF in response to both agonists at 25° and 6 mM calcium. This was entirely due to the large BDF. The efficacy was very low under these conditions. This probably means that the contractile proteins were almost maximally activated in the absence of the drugs, and there was l i t t l e additional inotropic effect when they were added to the system. The MDF of isoproterenol and .histamine were similar under these conditions. In the rabbit.papillary muscle, histamine produces its effects by binding to H^ - receptors. This interaction has no effect on cyclic AMP levels. In contrast, isoproterenol, by binding to 3-receptors produces an increase in cyclic AMP. Cyclic AMP is thought to,phosphorylate troponin I and the SR membrane (Bailey and Villar-Palasi, 1972; England, 1976), and augment calcium release from SR.stores (Fabiato and Fabiato, 1975b). At 25° and 6 mM calcium, the MDF of isoproterenol and histamine were similar, suggesting that the effects of cyclic AMP on excitation-contraction coupling following administration of isoproterenol produced l i t t l e extra effect under these conditions, where the calcium content of the cell is probably maximal. -160-At 37.5° and 42° and a l l calcium concentrations, the response to histamine was much less than that to isoproterenol. The difference was only significant at 37.5° and 0.5 mM calcium (figure 29). At 25°, the responses to isoproterenol and histamine were similar. This would tend to suggest that at 25° where the duration of the active state is pro- • longed (Hollander and Webb, 1955; Kaufmann and Fleckenstein, 1965; Mattiazzi and Nilsson, 1976), sufficient calcium can enter the cell to produce an inotropic effect in response to histamine that is of a similar magnitude to that of isoproterenol. However, when the duration of the active state is reduced by increasing the temperature, and the external calcium concentration is.reduced, the availability of calcium entering the cell is restricted. Under these conditions, the response to histamine may be reduced compared to isoproterenol,.since.isoproterenol has the ability to increase cyclic AMP levels and augment its inotropic effect as described above (see figure 2). The guinea pig papillary muscle contains 8- and H^-receptors, both of which have the potential to increase cyclic AMP levels. In this tissue, under a l l conditions, the MDF of isoproterenol and histamine were similar, suggesting perhaps that they produced their inotropic effects by a similar mechanism. Tenner and McNeill (1978) showed that at both 25° and 37.5° the MDF of isoproterenol on the guinea pig left atrium was greater than that of histamine. The guinea pig left atrium is a tissue which, like the rabbit papillary muscle contains 8- and H^ - receptors, again suggesting that the inotropic effect of the agonists is brought about by a different means, and that cyclic AMP stimulation augments the positive inotropic effect of agonists in some way. In addition, it.should be pointed out that temperature also has a considerable effect on contractility. -161-Sonnenblick and co-workers (Yeatman et al. , 1969) showed that decreasing temperature Increased the stiffness of cat papillary muscles. The maximum velocity of shortening at zero load (Vmax) and dP/dt decreased, while the maximum tension (Po) increased when the temperature was reduced stepwise from 37 to 24°. These effects probably at least partially account for the prolongation of contraction and increase in tension seen at low temperatures. 2. The Effect of Changing Frequency of Stimulation on Myocardial Contractility In the guinea pig, increasing the frequency of stimulation from 1 to 4 Hz had l i t t l e effect on the BDF (figure 7). Only when the frequency was decreased to 0.5 Hz was there a significant decrease in the BDF. The response in the rabbit was similar (figure 17). At 0.2 and 0.5 Hz, the BDF was significantly lower than at 1 and 3 Hz, under conditions of 2.2 mM calcium. Since, by increasing the frequency, the number of action potentials per unit time is also increased, this may allow a greater amount of calcium to enter the c e l l . With both the guinea pig and rabbit, under basal conditions and stimulation at 3 and 4 Hz, the BDF response was similar to that at 1 Hz, indicating that despite the r e l -ative brevity of the action potential, the amount of calcium entering the cell was sufficient to maintain the BDF.. ..-.In the rabbit, when the calcium concentration was reduced to 0.5 mM the BDF at a l l frequencies was very small, the largest value being seen at 3 Hz. Under conditions of 6 mM calcium, the BDF was largest at 1 Hz and significantly reduced at 3 Hz suggesting that when large.amounts of calcium are available, the duration of the.action potential is important in regulating the amount of calcium made available to the contractile proteins. -162-A number of workers have shown that the duration of tension development is decreased as the frequency is increased (Abbott- and Monmaerts, 1959; Blinks and Koch-Weser, 1961; Koch-Weser, 1963). Hollander and Webb (1955) using rat atria showed no change in duration of tension development, but a significant decrease in action potential duration and area.of the action potential when the frequency was increased from 200 to .420 beats per minute. In general, the duration of the action potential has been shown to be related to the duration of tension devel-opment (Carmeliet, • 1977). Braveny and Sumbera (1970) investigated the relationship between the duration of the action potential and contraction in guinea pig papillary muscles. They found a positive correlation between time to peak tension and the duration of the action potential. Wohlfart (1979) also found a positive correlation between time to peak tension and duration of the action potential in rabbit papillary muscles when the interval between beats was greater than 0.8 sec. At this inter-val (1.25 Hz) the tissues developed maximal tension. At shorter inter-vals the correlation was lost and the peak force decreased. • Braveny and Sumbera could find no constant level of membrane polarization at which relaxation occurred and suggested that i t might be dependent on the intracellular calcium concentration. Wohlfart proposed the exist-ence of a small compartment attached to the inside of the cell membrane (MC).. This compartment contained a variable amount of calcium, the quantity of which reflected the calcium content of compartment 2 from which calcium was released to the myofilaments. His data f i t a model where the duration of the action potential was inversely related to the calcium content of MC, and suggested that this represented a stabilizing negative feedback mechanism. A large amount of calcium stored in com-partment 2 would produce a large inotropic effect and would also f i l l MC. -163-The f i l l i n g of MC would decrease the action potential duration and thus reduce the amount of calcium entering the cell. Both groups of workers found a correlation between the duration of the action potential and the tension developed by the next contraction. Wohlfart calculated that only a quarter of the calcium used to produce a contraction was recirculated via the SR to produce subsequent contractions, indicating that the tiss-ues were relatively dependent on external calcium for contractions. However, most data indicates that the amount of calcium entering the cell during an action potential is inadequate to produce contraction (Reuter, 1974a). In the guinea pig changing the frequency of stimulation from 1 Hz shifted the dose-response curves for isoproterenol and histamine to the right (figure 10). The inotropic effects of isoproterenol and histamine were depressed under 3 and 4 Hz stimulation (see also figure 11). These shifts are reflected in the E D ^ Q values (figures 15 and 16). With iso-proterenol, use of 0.5 Hz significantly reduced the sensitivity of the tissues to isoproterenol. At 2 and 3 Hz, the sensitivity was only slightly decreased. At 4 Hz, the sensitivity was increased slightly compared to 1 Hz, presumably due to the flattened shape of the dose-response curve. The results with histamine were even less clear-cut. It appears that when the frequency is increased to 3 and 4 Hz, although under basal conditions the tissue appears to behave quite normally, when an inotropic agent is added, the availability of calcium through the slow inward channels becomes restricted compared to the lower frequencies. Wohlfart showed that between frequencies of O.'l and 1.25 Hz the basal peak force increased quite gradually to a maximum and then f e l l off sharply at higher frequencies. However, while the duration of the action potential of the next beat increased sharply up to 3 Hz frequency and - 1 6 4 -then decreased, the duration of the action potential of subsequent beats decreased gradually at a l l frequencies between 0 . 1 and 2 Hz, before falling off sharply at higher frequencies. Since the data in this study consists of tension measurements, made over a period of time, rather than instantan-eous measurements after changing conditions, this "subsequent beat" action potential duration is probably the more appropriate parameter to consider here. This being the case, i t is likely that.at frequencies less than 1 Hz there is l i t t l e difference in the duration of the action potential, but at higher frequencies the duration becomes significantly decreased, resulting in an impaired calcium influx, which is only of importance when the agonist is added. In the rabbit at 2 . 2 mM calcium changing the frequency from 1 Hz also shifted the dose-response curves for isoproterenol and histamine to the right (figures.18 and 2 0 ) , although the E D ^ Q values show that there was only a slight or no change in sensitivity (figures 2 3 and 2 4 ) . At 3 Hz, the sensitivity was increased, and the dose-response curves were flattened similar to those in the guinea pigs. Under conditions of 0 . 5 mM calcium, the dose-response curves at a l l frequencies were flattened, resulting in fairly low E D ^ values for both agonists, and presumably due to.reduced calcium influx. At 6 mM calcium, the dose-response curve for isoproterenol was very large at 0 . 2 Hz. Under conditions of 6 mM calcium the amount of calcium entering the cell through the slow channel and by Na:Ca exchange must be maximal, and at 0 . 2 Hz, according to Wohlfart ( 1 9 7 9 ) the action potential duration is close to a maximum, which could explain the augmented dose-response curve. The dose-response curve for histamine was reduced compared to that for isoproterenol, sug-gesting that cyclic AMP may have been involved in producing the larger response. At 3 Hz and 6 mM calcium, the dose-response curves for iso--165-proterenol and histamine were s t i l l flattened, indicating that the depression could not simply be due to a lack of external calcium avail-ability. It seems most likely that at 3 Hz, the depression is due to the relative slowness of transfer of calcium between compartments 1 and 2 of the SR, and therefore the limited amount of calcium released by each action potential. It is quite possible that this transfer becomes incom-plete at frequencies greater than 1.25 Hz resulting in the depression of peak force seen by Wohlfart (1979) and Edman and "Johannsson (1976). In addition, at 3 Hz the MDF to isoproterenol and histamine in both the rabbit and guinea pig were depressed at a l l calcium concentrations. In both species the response to histamine was greatest at 1 Hz, while with isoproterenol there was no difference between the responses at 0.5 and 1 Hz. This suggests that in response to histamine the inotro-pic effect was dependent to a greater extent on internal calcium stores, while with isoproterenol calcium entering the cell, and cyclic AMP stim-ulation could, also produce an effect. Again, in the rabbit the responses to histamine were greatly reduced compared to isoproterenol. These differ-ences were significant under conditions of 0.2 Hz and 6 mM, 0.5 Hz and 0...5 or 2.2 mM, and 1 Hz and 0.5 mM calcium. At 3 Hz, the responses to the two agonists were not different. 3. The Effect of Changing Calcium Concentration on Myocardial Contractility Increasing the calcium concentration increased the BDF of both guinea pig and rabbit papillary muscles. In the guinea pig (figure 7) there was l i t t l e difference between the BDF responses under conditions of 6 and 8- mM calcium. Both were significantly greater than those at 2.2 and 4 mM cal-cium. These in turn were significantly greater than the. BDF under condi--166-tions of 1.5 mM calcium. In contrast, in. the presence of isoproterenol and histamine the MDF responses were similar at a l l calcium concentrations (figure 13), suggesting that in the presence of these agonists, calcium utilization is stimulated to such an extent that i t is able to overcome the limiting effect of the external calcium concentration. Although a concentration of 1.5 mM calcium significantly reduced the BDF in the guinea pig, i t is s t i l l close to the calcium concentration used in most physiological salt solutions. This would suggest that at 1.5 mM calcium i t is not surprising that the MDF in response to isoproterenol and hista-mine was not different from that seen at higher concentrations. In the rabbit at each frequency . (figure 17) and temperature studied (figure 25) there was. a stepwise increase in MDF as the calcium concen-tration was increased. In this species the lowest calcium concentration used was 0.5 mM. Under this condition the MDF in response to isoproterenol and histamine was reduced to a greater extent than was seen in the guinea pig. Under conditions of 3 Hz stimulation (figures 19 and 21), and 25° and 42° (figures 27 and 29) there was less difference between the responses under different calcium concentrations. This suggests that these condi-tions either i) increase calcium utilization to augment the response under conditions of low extracellular calcium (at 42° and 0.5 mM calcium), or i i ) decrease calcium utilization to reduce the response under condi-tions of higher extracellular calcium to that seen under conditions of low extracellular calcium (at 3 Hz and 2.2 or 6 mM calcium, and 25° and 2.2 mM calcium). In the guinea pig, the dose-response curves for isoproterenol and histamine were shifted to the left at 6 and 8. mM calcium, and to the right at 1.5 mM calcium (figure 12). This is reflected by the FD 5 Q data -167-(figures 15 and 16) where the sensitivity to the agonists is increased at higher calcium .concentrations and decreased at lower concentrations. A similar shift of the dose-response curves was seen in the rabbit at each frequency (figures 18 and 20) and temperature (figures 26 and 28), although the shifts.are only reflected in the E D ^ Q data under conditions of changing frequency (figures 23 and 24) and 25 and 37.5° in response to isoproterenol (figure 31). Therefore.it appears that generally the availability of extracellular calcium is important for maintaining the BDF and determining the. potency of the agonists, but except at very low calcium concentrations the MDF seems to depend only partially on the extracellular calcium level, and partially on the temperature and frequency of stimulation. To test the hypothesis that cyclic AMP involvement produced a greater inotropic response to isoproterenol than histamine in rabbit papillary muscles we investigated the effect of D600 on the two agonists. According to our theory, i f isoproterenol has the potential to produce at least part of its inotropic.effect through cyclic AMP involvement, i t should be less affected by administration of calcium antagonists than histamine which in the rabbit papillary muscle has no. effect on cyclic AMP. The -7 data obtained using 5 x 10 M D600 did not appear to substantiate our hypothesis. Following 60 minutes incubation..with.D600, the dose-response curves for both isoproterenol and histamine were shifted to the right (figure 33). There was a significant difference between the EDJ-Q values for isoproterenol in the absence and presence of D600. The EDJ-Q values for histamine were unchanged. The shift of the dose-response curve seen with isoproterenol was similar to that seen following use of a 3-blocking agent. Similar shifts with isoproterenol were seen by Endoh et al . , -168-(1975) and Reinhardt et al . (1977) in rabbit papillary muscles and guinea pig left atria. No suggestion has been made that the calcium antagonists act by binding to the S-receptor. Rather, current evidence suggests that their mechanism of action is at some point beyond the receptor, for example by inhibition of the slow inward current (Fleck-enstein et a l . , 1971) and/or altering internal calcium transfer (Bayer et al., 1975a; Reinhardt et al . , 1977). In contrast, two studies have suggested that calcium antagonists may act as a-blocking agents. Blackmore (Blackmore et a l . , 1979) working on rat liver homogenate found that verapamil inhibited adrenaline binding in the presence of propranolol, in similar concentrations to those inhibiting phosphorylase 45 activation and Ca efflux. Fairhurst (Fairhurst et a l . , 1980) working with rat brain homogenates found that D600 inhibited binding of specific a- and muscarinic agonists in a competitive manner. They postulated that this might occur by an allosteric mechanism, rather, than by direct binding to the receptor. At present, no receptor binding studies in the presence of D600 or other calcium antagonists have been carried out in cardiac tissues. Our findings that D600 did not suppress the response to histamine more than that to isoproterenol do not entirely negate our hypothesis. The response of isoproterenol to D600 treatment was different to that of histamine, and this suggests that there is indeed a difference in the. mechanism of action of these two inotropic agents. Endoh and co-workers (1975) suggested a similar effect with isoproterenol and phenyl-ephrine. In their study D600 significantly depressed the maximal res-ponse, to phenylephrine. We were unable to show.a depression with hista-mine, but the inotropic response was relatively.small, and the standard errors were large. We used slightly more D600 than .Endoh, but both our -169-control and D600-treated BDF were much larger. Our results with iso-proterenol were similar to those of Endoh, except that our BDF values were again larger. According to our theory and those of Verma and McNeill (1977) and Tenner and McNeill (1978), the inotropic responses to stimu-lation by 3- and H^ - receptor agonists should be similar, as should those produced by stimulation of a- and H^ - receptors. Endoh pointed out that the biochemical processes producing the inotropic effect of phenylephrine was obviously different from that produced by isoproterenol, resulting .in an increased sensitivity of the a-r-receptor induced inotropic effect to D600. If the theory of Verma and McNeill (1977) and Tenner and McNeill (1978) holds, then our study with histamine should produce similar results to those of Endoh using phenylephrine. It is possible that the results differed because the large BDF and small efficacy of histamine in this preparation masked the effect of D600. 45 We had hoped that the studies using Ca would be definitive in determining' whether isoproterenol and histamine had had different effects on calcium metabolism. This did not prove to be the case. Ventricle 45 strips were chosen to follow Ca exchange for the following reason. We required a large number of tissues for each - experiment, and splitting the ventricle seemed to be more physiological than splitting the papillary muscles. This was evident from the ECS data. With the split papillary 14 muscles C was s t i l l being accumulated by the tissues at 60 minutes, probably, indicating that part of the label was being taken up intracellu-larly (figure 34). The ECS values obtained for intact rabbit papillary muscles, and left atrial and right ventricle strips were similar (figures 34 and 35). This suggests that splitting the atria and ventricles and allowing 60 minutes equilibration in isotope-free solutions before addi-tion of label, was sufficient to allow healing, of the cut edges and exclu--170-sion of the label from the intracellular space. The values obtained were similar to those reported i n the literature. Poole-Wilson and Lauger 51 (1979) using rabbit intra ventricular septum and Cr-EDTA reported ECS values of 0.412 ml/g wet weight, and Winegrad and Shanes (1962) using 14 + guinea pig l e f t a t r i a and G-sucrose reported ECS values of 0.25 - 0.1 ml/g wet weight. Grossman and Furchgott (1964a) reported values between 0.21 and 0.41 ml/g wet wt i n guinea pig l e f t auricles depending on the method used. The ECS value calculated for the guinea pig right ventricle strip was slightly lower than that found for the rabbit (figure 36). Right ventricle strips were chosen for subsequent experiments because in the rabbit they contain only 3- and H1~ receptors (McNeill and Verma, 1978; 1979), while the l e f t atrium which would also have been suitable to use contains B-, H^- and H^- receptors (McNeill and Verma, .1978, 1979; Polanin et al.., 1980; Polanin and McNeill, 1981). In addition, we wanted to use the same.tissue in the guinea pig, and were cognizant of the finding that guinea pig ventricular muscle contains 8- and H^- receptors (Reinhardt et a l . , 1977; Verma and McNeill, 1977), while the l e f t atrium contains 8- and H^- receptors. Since i t was our intention to compare the effects of 8- and H^- receptor stimulation i n rabbit tissues, and 8- and E^- receptor stimulation i n guinea pig tissues., we therefore chose to use right ventricle strips. 45 The concept of removing extracellular Ca with ice-cold buffers, 4 5 i n order to determine the intracellular Ca content, is a relatively new technique. In smooth muscle, the trivalent cation lanthanum is fre-45 quently used to selectively remove extracellular Ca (van Breemen and McNaughton,. 1970, van Breemen et a l . , 1972). More recently however, Aaronson and co-workers (1979) developed a technique using an ice-cold -171-isotope-free physiological salt solution containing 6.5 mM calcium and 5 mM EGTA. They considered that this method would preserve cellular function and membrane integrity more than the lanthanum method. In a joint experiment we examined the effects of a number of different ice-cold calcium-free solutions for their a b i l i t y to remove extracellular Ca from rabbit right ventricle strips and aortic rings. The results i n the cardiac tissues are presented i n this thesis (figure 37). A solution containing 0 Ca and 10 mM Mg was chosen for subsequent experiments because i t produced the longest plateau phase compared to the other solutions. 45 Slightly higher intracellular Ca levels were obtained using an ice-cold control solution, or 0 Ca and 0.5 mM EGTA solution. In this tissue, the 0 Ca, and 10 mM La solution produced an efflux curve which was similar to that seen with the control solution at 37°. This suggests that i n this preparation, lanthanum entered the c e l l s and displaced intracellular as 45 well as extracellular Ca. Use of the ice-cold 0 Ca and 10 mM Mg solu-tion i n the guinea pig right ventricle strips produced a similar efflux curve (figure 39). It i s possible that omitting calcium from the quench-ing solutions did produce some membrane dysfunction. Aaronson et a l . (1979) used a solution containing 5 mM Ca EGTA plus an extra 1.5 mM calcium in an attempt to overcome this problem. Inclusion of calcium resulted in an increase i n the rate constant of calcium assumed to be washing out of the extracellular space, and a decrease in the rate constant of calcium assumed to be intracellular. This suggests that there i s an increase i n 45 the rate of Ca removal from extracellular sources when calcium i s inclu-ded in the medium, and appears to be associated with a decrease in the 45 rate of loss of intracellular Ca. Karaki and Weiss (1980) working with rabbit aortic strips characterized four different calcium binding sites. -172-In the extracellular space they found two high and low affinity lantha-num-accessible sites, and intracellularly two high and low affinity lan-thanum-inaccessible sites. They found that calcium was released from both low affinity sites in a manner that was independent of the calcium concentration while that bound to high affinity sites was only released i f i t could exchange with extracellular calcium. In our study, we found that when the effect of the control solution 45 o o on residual Ca content was determined at 37 and 0 , decreasing the temperature produced a decrease.in both rate constants (figure 38). The decrease in the rate constant for loss of extracellular calcium was quite small, and the amount of calcium assumed to be bound in the extracellular space was similar at both temperatures, as would be expected, since the 45 o tissues were a l l loaded with Ca at 37 . However the difference in the rate constant for calcium assumed bound in the intracellular space was much larger at the higher temperature, indicating that at this temperature 45 o a large proportion of intracellular Ca was lost compared to 0 . If the findings of Karaki and.Weiss (1980) that high affinity calcium bound both intracellular ly and extracellularly is only released i f calcium is present 45 in the bathing medium also applies to cardiac muscle, our estimate of Ca bound extracellularly is likely to be an overestimate., since we had 0 Ca in our quenching solutions. 45 The rabbit ventricle strips took up Ca rapidly (figure 41). Uptake was complete within 60 minutes therefore in subsequent experiments tissues 45 were loaded with Ca for 80 minutes. The use of 126 mM K produced a contracture of the rabbit right vent-ricle strips (figure 46) which was associated with an augmented uptake 45 of Ca (figures 42, 43 and 45). This increased uptake was not changed by concomitant administration of isoproterenol or histamine. The increase -173-was evident with 6 minutes in quiescent tissues, and continued to increase .fairly linearly until at least 10 minutes (figure 42). The use —6 of 10 M isoproterenol in a control solution containing 5.6 mM K did 45 not change Ca uptake compared ..to the. control solution. In stimulated tissues, fewer, time points were examined (figure 44). Histamine in a -4 45 dose of 10 M did not change Ca uptake compared to the control, but —6 at 2 minutes, 10 M isoproterenol did cause a significant increase. It 45 seems unlikely that this increase in Ca content could account for the larger inotropic effect in the rabbit ventricular muscles compared to 45 histamine, since by four minutes Ca content was not different from that seen in control or histamine-treated tissues,, while the inotropic effect s t i l l remained larger. 45 Following 80 minutes loading in Ca, 10 minutes•exposure to high 45 K produced a significant increase in Ca content of quiescent rabbit right ventricle strips (figure 43). This same significant increase was —6 seen when 10 M isoproterenol or histamine was added to the high K medium. The same dose of isoproterenol or histamine -in the control medium 45 did not affect Ca content compared to control, although in the presence of isoproterenol there was a slight increase. The data for stimulated tissues were similar, but the variation among, samples was greater (figure 45). Although there was a slight 45 . + . increase in Ca content when high K - isoproterenol or histamine was used, the differences were not significant compared to controls. It is possible that increasing the n values could make this difference significant. The stimulated tissues went into contracture when, exposed to the high K solutions (figure 46). The contracture did not appear to be altered by either agonist. Even though use of the agonists in the control solution -174-45 produced a significant inotropic effect, they had no effect on Ca content. Morad (Morad, 1969; Morad and Rolett, 1972) showed that catechola-mines have the potential to relax the contracture produced by KCl in cat ventricular muscle. He proposed that this effect was due to stimulation of the calcium sequestering system by the catecholamines, resulting in an enhanced calcium uptake, and therefore relaxation. In our study, isoproterenol did not- appear to affect the magnitude of the high-K contracture, although i t was not possible to quantitate the data (figure 46). There was a possibility that isoproterenol by stimulating cyclic AMP which is known- to enhance calcium sequestration by the SR, would decrease the contracture in rabbit right ventricle strips to a greater extent than, histamine which has no effect on cyclic AMP in this tissue. We were not able to determine i f this in fact happened. It is possible that by pretreating the animals with reserpine so that a larger contrac-ture could be induced, a prominent .relaxant effect of isoproterenol on the contracture could be seen. This experiment is one which could easily 45 be considered for the future, and would not necessarily require Ca studies to be carried out in conjunction. Unfortunately due to lack of time, i t was not possible to repeat these studies in the guinea pig. However, since we had expected to find differences between the effects of isoproterenol and histamine in the rabbit, yet did not, i t is not likely that we would detect changes in the guinea pig where we expected the response to the two agonists to be similar.. In the rabbit right ventricle strips, we found similar effects 45 of isoproterenol and histamine on Ca content, except in one instance, —6 when 10 M isoproterenol following 2 minutes exposure caused a signifi--175-45 cant, increase in Ca content. It seems unlikely that this increase could be responsible for the increase in tension since i t was not main-tained. However at 37.5°, 1 Hz and 2.2 mM calcium, the conditions used 45 in the Ca studies, the response to isoproterenol although greater than that to histamine was not significant using Bonferroni statistics (figure 21) although i t was using the Student's t-test. In hindsight, i t appears that i t might have been preferable to use conditions of 37.5°, 0.5 Hz and 2.2 mM calcium, where the difference was significant. Alternatively, Bonferroni analysis is a very rigorous test, and fewer points were signi-ficantly different when using i t rather than the t-test. It is possible that we erred on the conservative side by using Bonferroni analysis and in an attempt to avoid a Type II error, (accepting Ho when i t was false) committed instead a Type I error (rejecting Ho when i t was true). The.possibility arises that i t is not possible to measure an increase 45 in total Ca content.in cardiac muscle following use of inotropic agents, as was reported by.Grossman and Furchgott (1964c). They showed that 1.6 g x 10 M noradrenaline had no effect.on total calcium content of guinea 45 pig left atria. However they found an increase in Ca exchange after use of the drug, which was more noticeable as the frequency was increased. Since we did not measure the calcium. content of our tissues we were not able to calculate this parameter, however i t should be emphasized that 45 we did find an increase in Ca content following use of isoproterenol, and that this does not appear to be an artifact. Alternatively, the method used in the current study was not sensitive enough to detect what 45 could be quite subtle changes in Ca content induced by the agonists, compared to high K. This could be due to a number of reasons. The rabbit right ventricle strip, although appearing to be viable from the ECS data, -176-45 may not have been suitable for Ca uptake studies. Dobson et a l . (1974) concluded that the right ventricle strip was prone to protein loss due to dissection, and tissue hypoxia to a greater extent than isolated per-fused hearts., in situ hearts and papillary muscles from guinea pigs. Although we equilibrated the.tissues for 60 minutes before use, and tried to keep the size to a minimum, i t is possible that uptake of the label was not optimal. In addition, the quenching solution which we chose could have been inappropriate for the study. Addition of calcium to the solution might have improved the sensitivity, of the assay by maintain-ing membrane integrity and increasing removal of high affinity extracel-45 lularly bound ' Ca (Aaronson et a l . , 1979; Karaki and Weiss, 1980). The hypothesis proposed in the early part of this thesis could not be substantiated by the data obtained. However, this does not mean that the model must necessarily be discarded, Rather, i t is likely that certain limitations.in the methods used, could have been responsible for the lack of positive data in. support of the hypothesis. 45 Meinertz et al., (1973b) were unable to detect an increase in Ca exchange following exposure to adrenaline in certain cardiac tissues, and concluded that this was due to a "pool saturation phenomenon." It is possible that in our experiment, at exposure times greater than 2 minutes 45 . . . . Ca uptake had reached a" point where influx and efflux were in equilib-45 rium, and the effect of isoproterenol on Ca content at later times could not be. detected. If this is indeed so, i t is possible that the signifi-45 cant difference in Ca content seen with isoproterenol at 2 minutes does represent a difference in the mechanism of action of isoproterenol and histamine which is masked at later times, and supports.our hypothesis. -177-4. Summary 1. Hypothermia and increased calcium concentration increased the BDF of guinea,pig and rabbit papillary muscles. Hyperthermia, low .frequency stimulation and decreased.calcium concentration, decreased the BDF. 2. Hypothermia did not increase the MDF to isoproterenol or histamine unless i t was combined with increased calcium concentration. Increased frequency of stimulation significantly depressed the MDF for isoproter-enol and histamine. Increasing the calcium concentration did not improve the response. Increasing the calcium concentration only increased, the MDF when i t was combined, with low frequency stimulation or low temperature. 3. Hyperthermia had l i t t l e effect on. the MDF. to isoproterenol or hista-mine. Low. frequency stimulation decreased the response slightly com-pared to 1 Hz. This was partially reversed by increasing the calcium concentration. Decreasing the calcium concentration in the rabbit depressed the MDF. This was partially reversed, by both increasing and decreasing the temperature. 4. In the guinea pig the inotropic effect of isoproterenol and histamine was similar. In the rabbit, the inotropic effect of histamine was always less than that of isoproterenol. We proposed that this was due to the histamine receptors, of the rabbit papillary muscle being of the H-^ -type and therefore unable to increase cyclic AMP levels. Use of Isoproterenol in both species and histamine in the guinea pig would in contrast stimulate cyclic AMP and allow increased calcium uptake -178-and. release by the SR, resulting in a greater inotropic effect. 5. Use of D600 shifted the dose-response curve for isoproterenol to the- right, but did not depress the MDF. The curve for histamine was slightly shifted to the right. 45 6. Isoproterenol produced an increase in Ca content following 2 minutes exposure in rabbit right ventricle strips. Histamine did not change 45 45 Ca content. If there is calcium pool saturation with Ca, and this can occur as early as 4 minutes after exposure to the label, i t is possible that this could indicate a difference in mechanism of action of. isoproterenol and histamine in rabbit ventricular muscle and support our hypothesis. -179-CONCLUSIONS Changing the temperature, frequency of stimulation and calcium concentration altered the dose-response curves for isoproterenol and histamine on guinea pig and rabbit papillary muscles. Basal developed force, maximal developed force and sensitivity to the agonists were a l l affected. Increasing the temperature stepwise from 25° to 42° resulted in a progressive decrease in BDF and sensitivity to the agonists. The response of MDF was different in the guinea pig and rabbit. In the guinea pig, MDF was not affected by changing the temperature, and the magnitude of the responses to isoproterenol and histamine were similar. In the rab-bit, the largest MDF response was seen at 37.5° when the calcium con-centration was maintained at 2.2 mM. At this calcium concentration, the response to histamine was less than that to isoproterenol at each temperature, although the difference was not significant. There are several possible interacting mechanisms by which changing the temperature could affect the response of the tissues to isoproterenol and histamine. They include i) increased SR Ca-ATPase activity as the temperature is raised, resulting in augmented calcium storage by the SR and a reduction of calcium levels in the region of the contractile pro-teins, i i ) A decrease in the duration of the action potential as the temperature is raised, resulting in a reduction in the amount of calcium entering the cell per unit time, i i i ) A decrease in the stiffness of the tissue as the temperature is raised, resulting in an increase in Vmax and dP/dt, and a decrease in Po. These effects probably at least partially -180-account for the decreased duration of the action potential described in i i ) . Increasing the frequency of stimulation stepwise from 0.5 to 4 Hz in the guinea pig, and 0.2 to 3 Hz in the rabbit produced the following effects. In both species, BDF was reduced by low frequency stimulation. With isoproterenol, the MDF was only reduced by high frequency stimu-lation, and the sensitivity increased stepwise with increasing frequency. With histamine, the MDF was reduced by both low and high frequency stim-ulation. In the rabbit the response to histamine was consistently less than that to isoproterenol. The sensitivity to histamine was not affected by changing the frequency in the guinea pig, but was increased by high frequency stimulation in the rabbit. Possible mechanisms by which changing the frequency could affect the responses to isoproterenol and histamine include i) the duration of the action potential is decreased as the frequency is raised, resulting in a reduction in the amount of calcium entering the cell per unit time, i i ) A relative slowness of calcium transfer between intracellular compart-ments at higher frequencies could result in reduced calcium availability for interaction with the contractile proteins. Increasing the.calcium content stepwise from 1.5 to 8 mM in the guinea pig, and 0.5 to 6 mM in the rabbit resulted in a progressive increase in BDF, MDF and sensitivity. In both species, the increase in MDF appeared to reach a maximum between 2.2 and 6 mM calcium. In the rabbit, this effect was less noticeable in situations where the frequency or temperature was also reduced. Possible mechanisms by which changing the calcium concentration could affect the responses to isoproterenol and histamine include i) increased -181-extracellular calcium results in an increase in calcium entry through the calcium channels and therefore an increased loading of the SR and avilability for interaction with the contractile proteins, i i ) Increased calcium may interact with calmodulin and stimulate phosphorylation of myosin, thus promoting contraction. Use of the calcium antagonist D600 produced a decrease in the sen-sitivity of rabbit papillary muscles to isoproterenol, but did not depress the MDF. There was no difference in the sensitivity to histamine. The use of D600 therefore, could not distinguish any difference in dependence on extracellular calcium between isoproterenol and histamine in this tissue. 45 Isoproterenol stimulated an increase in Ca content of rabbit right ventricle strips at 2 minutes after administration. 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Cardiac atrial myosin adenosine triphosphatase of animals and humans. Distinctive enzymatic properties compared with cardiac ventricular myosin. Circ. Res. 45: 522-527. Yeatman, L.A. Jr., Parmley, W.W., and Sonnenblick, E.H. 1969. Effect of temperature on series elasticity and contractile element motion in heart muscle. Amer. J. Physiol. 217: 1030-1034. Yellin, T.O., Buck, S.H., Gilman, D.J., Jones, D.F., and Wardleworth, J.M. 1979. ICI 125, 211: A new gastric antisecretory agent acting on hista-mine H2-receptors. Life Sci. 25: 2001-2009. Zivin, J.A., and Bartko, J.J. 1978. Statistics for disinterested scien-tists. Life Sci. 18: 15-26. Appendix I -195-A. Method of Calculating EE>50 Values (Fleming et al. , 1972) 1. Calculate the arithmetic E D , - 0 from each individual dose-response curve. The example used will be the dose-response curve for isopro-terenol on rabbit papillary muscles at 37.5°, 0.5 Hz and 2.2 mM calcium (figure 18).. ' —8 2. Assign to the smallest exponent (10 ) the prefix 0. To the next —7 —6 largest exponent (10 ) assign the prefix 1, to the next (10 ) 2, etc. 3. Ignore the exponent and prefix, and calculate the log of the arith-metic ED Q^. 4. Calculate the mean - SEM of the log EDJ-Q values (Including the prefix). 5. Find t ,^  , at P = 0.05. Calculate (SEM) (t) to give the 95% df = n-1 to confidence interval. 6. Find the antilog of the mean log E D ^ . This value is the geometric mean E D ^ . To determine the exponent, check the prefix against those assigned in step 2. 7. Add (SEM) (t) and the mean log E D 5 Q . Find the antilog. This value is the upper limit of the 95% confidence interval. 8. Subtract (SEM) (t) from the mean log E D ^ . Find the antilog. This value is the lower limit of the 95% confidence interval. - 1 9 6 -Example of Calculation of the ED 5 Q Value for Isoproterenol on Rabbit Papillary Muscles at 3 7 . 5 ° , 0.5 Hz and 2.2 mM Calcium Concentration. Arithmetic E D 5 0 (M) Log E D 5 Q 1.45 X 1 0 " 7 ( 1 ) 1 . 1 6 1 4 ( 2 ) ( 3 ) 1.2 X lo" 7 1.0792 1.5 X lo" 7 1 . 1 7 6 1 7.2 X lo" 8 0.8573 9.4 X I Q " 8 0 . 9 7 3 1 2.4 X lo" 7 1.3802 7.8 X I D " 8 0 . 8 9 2 1 5.4 X I D " 8 0.7324 7.2 X lo" 8 0.8573 n = 9 mean lo 1 . 0 1 2 1 + .0678 V - 1 = 8 2.3060 (SEM) (t) 0.1564 Geometric Mean ED,-Q 1.0283 X 1 0 " 7 M 9 5 % C. I. (lower) 7 . 1 7 3 2 X 1 0 " 8 M 9 5 % C.I. (upper) , 1 . 4 7 4 1 X 1 0 ~ 7 M. Numbers in parentheses indicate the step in the method section--197-B. Use of Newman-Keuls Test for Multiple Comparisons (Zivan and  Bartko, 1978) 1. Carry out a one way AN.OVA classification on the groups being tested. The example used will be the effect of changing the frequency of stimulation on the MDF of rabbit papillary muscles to histamine (figure 21). 2. Tabulate the following statistical data: Degrees of freedom among groups df^ 2 Mean square among groups S ^  Degrees of freedom within groups df 2 Mean square within groups S Check that the F value exceeds the F value given in the P = 0.05, F-distribution tables (Table I). If the calculated F value is less than F^^, none of the treatment groups are significantly different from each other, and there is no need to continue with the test. 3. If the F value shows that there is a difference between the groups calculate If the groups are not of equal size, calculate the harmonic mean to use as n. i) Add the reciprocal of the number of subjects in each group. Divide by the number of groups. i i ) Take the reciprocal of this number. The value obtained is the harmonic mean which may be substituted for n in the above equation. w F value F d -198-Arrange the group means in order from lowest to highest, and calculate the differences between these means. Using a Studentized Range Table (Table II) calculate the Studentized Range Statistic for the df from the ANOVA data, and the number w of comparisons made.. Multiply the Studentized Range Statistic by to obtain a critical value which the difference between each pair of means must exceed to differ significantly at P< ,0.05. -199-Table I. Upper Significance Limits' Qf the F-Dietributlon P»Q.05 P - P (right) - inttgal between F end infinity I.II O-T^ » i w 6.61 5 79 5 41 5.1» S.0S 4.95 4.88 4.82 4.77 4.74 4.70 4.68 4.66 4.64 4.62 toi 5 14 4.53 4 39 4 28 4.21 4.15 4.10 4.06 4.03 4.00 3.98 3.96 3.94 tS A1A 4 j t 412 Ml 3 87 3.79 3.73 3.68 3.64 3.60 3.57 3.55 3.53 3.51 SJ2 AM 4J7 384 169 3.58 3.50 3.44 3J9 3J5 3.31 3.28 3.26 3.24 3.22 tS AM J.86 IS iM 3.37 3.29 3.23 3.18 3.14 3.10 3.07 3.05 3.03 3.01 «• 1 2 3 »• 1 161.44 200 216 22 2 18.51 19.0 19.2 1 3 10.13 9.55 9.28 4 7.71 6.94 6.59 S 6.61 5.79 5.41 6 5.99 5.14 4.76 7 5.59 4.74 4J5 8 5J2 4.46 4.07 9 5.12 4.26 3.86 0 4.96 4.10 3.71 11 4.84 3.98 3.59 12 4.75 3.89 3.49 13 4.67 3.81 3.41 14 4.60 3.74 334 15 4.54 3.68 3.29 16 4.49 3.63 3.2* 17 4.45 3.59 3.20 IB 4.41 3.55 3.16 19 4.38 3.52 3.13 20 4J5 3.49 3.10 21 4.32 3.47 3.07 22 4.30 3.44 3.05 23 4.28 3.42 3.03 24 4.26 3.40 3.01 2S 4.24 J.W 2.99 26 4.23 3.37 2.98 27 AM 3J5 2.96 28 AM SJ4 2.95 29 4.18 3.33 2.93 30 4.17 3.32 2.92 32 4.15 3.29 2.90 34 4.13 3.28 2.88 36 4.11 3.26 2.87 38 4.10 iM 2.85 40 4.08 3.23 2.84 42 4.07 3.22 2.83 44 4.06 3.21 2.82 46 4.05 3.20 2.81 48 4.04 3.19 2.80 80 4.03 3.18 2.79 60 4.00 3.15 176 70 3.98 3.13 2.74 80 3.96 3.11 2.72 90 3.95 3.10 171 too 3.94 3.09 2.70 125 3.92 3.07 2.68 ISO 3.90 3.06 2.66 200 349 3.04 2.65 300 3*7 3.03 243 800 3.86 3.01 2.62 1000 SJto iM 2.61 10 11 12 14 IS 230 234 237 239 241 242 243 244 245 245 246 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 8.70 3.48 3.33 3.36 3.20 3.26 3.11 3.18 3.03 3.11 2.96 3.06 2.90 3.01 2.85 196 2.81 2.93 2.77 2.90 2.74 187 2.71 284 2.68 2.82 166 2.80 2.64 178 2.62 2.76 2.60 2.1 A 159 2.73 2.57 2.71 Z.56 2.70 2.55 2.69 2.53 2.67 2.51 2.65 2.49 163 2.48 2.62 146 2.61 2.45 159 144 158 143 157 142 157 141 156 140 153 137 150 135 149 133 147 132 146 131 144 129 143 127 142 126 140 124 139 123 138 122 3.22 3.09 3.00 192 185 179 174 170 166 2.63 160 157 155 153 151 149 147 146 145 143 142 140 138 136 135 134 132 131 130 129 129 125 123 121 120 119 117 116 114 113 112 111 3.14 3.01 191 183 176 171 166 161 158 154 151 149 146 144 142 140 139 137 136 135 133 131 129 128 126 125 124 123 122 121 3.07 3.02 195 190 185 180 177 171 170 165 164 159 159 154 155 149 151 146 148 142 145 139 142 137 140 134 137 132 136 130 134 128 132 127 131 125 129 124 128 122 127 121 124 119 123 117 2.21 115 119 114 118 112 117 111 116 110 115 109 114 108 198 185 175 167 160 154 149 145 141 138 135 132 130 127 125 124 122 120 119 118 116 114 112 111 109 108 106 105 104 103 2.94 182 172 163 157 151 146 141 2.37 134 131 128 126 123 121 120 118 117 l t 5 114 113 110 108 107 105 2.04 103 2.01 200 1.99 191 189 179 176 169 166 160 238 153 151 148 145 142 140 138 135 134 131 131 128 128 125 125 122 123 120 120 118 118 115 2.16 114 115 112 113 H O 112 109 2.86 285 174 172 164 162 155 153 148 146 142 140 137 135 133 131 129 2.27 126 123 122 120 120 118 117 115 115 113 113 111 111 109 109 107 108 106 106 104 110 108 105 103 109 106 107 104 105 102 103 100 102 1.99 2.00 1.97 1.99 1.96 1.98 1.95 1.97 1.94 1.96 1.93 120 113 107 103 1.99 1.95 1.92 117 110 104 1.99 1.95 1.92 1.89 114 107 102 1.97 1.93 113 106 100 1.95 1.91 111 104 1.99 1.94 1.90 110 103 1.97 1.93 1.89 108 101 1.96 1.91 1.87 107 100 1.94 1.89 1.85 106 1.98 1.93 1.88 1.84 104 1.97 1.91 1.86 1.82 103 1.96 1.90 1.85 1.81 1.77 1.74 102 I.9S 1.89 1.84 l JO 1.76 1.73 104 101 101 1.99 1.99 1.97 1.98 1.95 1.96 1.94 1.95 1.92 1.93 1.91 1.92 1.90 1.91 1.89 1.90 1.88 1.89 1.87 1.86 1.84 1.84 1.81 1.82 1.79 1.89 1.86 1.88 1.84 1.86 1.83 1.80 1.78 1.85 1.82 1.83 1.80 1.82 1.79 1.80 1.77 1.79 1.77 1.77 1.75 1.76 1.73 1.74 1.72 1.78 1.75 1.72 1.70 1.71 1.69 1.70 1.68 Reproduced from Documenta Geigy Scientific Tables. 7th Edition, page M-0. -200-Table II. TABLE A 15 UPPER 5"„ PERCENTAGE POINTS, Q. IN THE STUDENTIZEO RANGE* Degrees of Freedom./ 1 •> 3 4 5 6 7 8 9 IU 11 12 13 14 15 16 17 18 19 20 24 30 40 60 120 ac 18.0 27.0 32.8 6.09 8.33 9.80 4.50 5.91 6.83 3.93 5.04 5.76 3.64 4.60 3.46 4.34 3.34 4.16 3.26 4.04 3.20 3.95 3.15 3.88 3.11 3.82 3.08 3.77 3.06 3.73 3.03 3.70 3.01 3.67 3.00 3.65 2.98 3.62 2.97 3.61 2.96 3.59 2.95 3.58 2.92 3.53 2.89 3.48 2.86 3.44 i 2.83 3.40 i 2.80 3.36 ! 2.77 3.32 5.22 4.90 4.68 4.53 4.42 4.33 4.26 4.20 4.15 4.11 4.08 4.05 4.02 4.00 3.98 3.96 3.90 3.84 3.79 37.2 10.89 7.51 6.29 5.67 5.31 5.06 4.89 4.76 4.66 4.58 4.51 4.46 4.41 4.37 4.34 4.31 4.28 4.26 4.24 4.17 4.11 4.04 Number of Treatments, a 40.5 11.73 8.04 6.71 6.03 5.63 5.35 5.17 5.02 4.91 4.82 4.75 4.69 4.64 4.59 4.56 4.52 4.49 4.47 4.45 4.37 4.30 4.23 3.74 3.98 4.16 3.69 3.92 4.10 3.63 3.86 4.03 43.1 12.43 8.47 7.06 6.33 5.89 5.59 5.40 5.24 5.12 5.03 4.95 4.88 4.83 4.78 4.74 4.70 4.67 4.64 4.62 4.54 4.46 4.39 4 31 4.24 4.17 45.4 13.03 8.85 7.35 6.58 6.12 5.80 5.60 5.43 5.30 5.20 5.12 5.05 4.99 4.94 4.90 4.86 4.83 4.79 4.77 4.68 4.60 4.52 444 4.36 4.29 47.3 13.54 9.18 7.60 10 49.1 13.99 9.46 7.83 6.80 6.99 6.32 6.49 5.99 6.15 5.77 5.92 5.60 5.74 II 50.6 14.39 9.72 8.03 7.17 6.65 6.29 6.05 5.87 12 51.9 14.75 9.95 8.21 7.32 6.79 6.42 6.18 5.98 5.46 5.35 5.27 5.19 5.13 5.08 5.03 4.99 4.96 4.92 5.60 5.49 5.40 5.32 5.25 5.20 5.15 5.11 5.07 5.04 5.72 5.83 5.61 5.71 5.51 5.61 5.43 5.53 5.36 5.46 5.31 5.26 5.21 5.17 5.14 4.90 5.01 5.11 4.81 4.92 5.01 4.72 4.83 4.92 4.63 4.74 4.82 13 14 15 53.2 54.3 55.4 15.08 15.38 15.65 10.16 10.35 10.52 8.37 8.52 8.67 4.55 4.47 4.39 4.65 4.56 4.47 5.40 5.35 5.31 5.27 5.23 5.20 5.10 5.00 4.90 7.47 6.92 6.54 6.29 6.09 5.93 5.81 5.71 5.63 5.56 5.49 5.44 5.39 5.35 5.32 4.73 4.64 4.55 7.60 7.04 6.65 6.39 6.19 7.72 7.14 6.75 6.48 6.28 6.03 6.12 5.90 5.98 5.80 5.88 5.71 5.79 5.64 5.72 5.57 5.52 5.47 5.43 5.39 5.65 5.59 5.55 5.50 5.46 5.28 5.36 5.43 5.18 5.25 5.32 5.08 5.15 5.21 4.98 5.05 5.11 4.81 4.88 4.94 5.00 4.71 4.78 4.84 4.90 4 62 4.68 4.74 4.80 • Reprinted from Biomelrika, 39:192 (1952) by permission of the author. Joyce M. May. and the editor. Reproduced from Snedecor and Cochran's Statistical.Methods, 6th Edition, 1967. Iowa State University Press, Ames, Iowa, page 568. -201-Example of Calculation of Newman-Keuls Test on Data of Effect of Changing Frequency of Stimulation on the MDF of Rabbit Papillary Muscles to Histamine 1 Hz, 2. 2 mM +.- 0.5 Hz 1 Hz 3 Hz 0.8* 2.2 0.65 0.6 1.0 0.5 0.45 1.4 0.4 1.6 1.8 0.7 0.4 1.6 0.55 1.5 2.1 0.5 0.25 . 1.4 0.3 1.4 0.5 1.0 0.35 1.2 0.3 1.4 0.75 1.0 0.9 0.55 0.7 n 12 15 6 mean 0.6500 1.3100 0.5500 - SEM - .1314 .1233 .0447 numbers indicate MDF in g from individual tissues. -202-df A = 2 (2) S 2 A = 1.9865 df = 30 w S2 = 0.1844 w Fcalc ~ 1 0 • 7 7 5 0 ' F tab = 3.3158 Harmonic average = 1/(1/12 + 1/15 +1/6) = 9.47 (3) d = >/0.1844/9.47 1 - 0.1395 (3) Array of Differences (means ordered low to high) (4) Mean 0.5500 0.6500 1.3100 Studentized Frequency 3 Hz 0.5 Hz 1 Hz Range Statistic 3 Hz 0.1000 ^  0.7600* — 3.48 (5) 0.5 Hz 0.6600* — 2.89 Critical values: d x 3.48 = 0.4855 (5) d x 2.89 = 0.4032 Numbers in parentheses indicate the step in the method section. The differences connected by the dotted lines are compared to the critical values. Significant differences, at P<0.05 are indicated by an asterisk. In this example the MDF produced by Histamine at 1 Hz frequency is significantly different from those at 0.5 and 3 Hz. There is no significant difference between the MDF at 0.5 and 3 Hz. -203-C. Use of Bonferroni Statistics (Miller, 1966) 1. Calculate the students t-value in the normal manner for the two samples being tested. The example used will be a comparison of the effects of isoproterenol and histamine on the MDF of rabbit papillary muscles under conditions of 37.5°, 0.5 Hz and 2.2 mM calcium (figures 13 and 15). 2. Instead of using P = 0.05 to check the t value the Bonferroni test at a probability of 95% uses the value for 0.05/the number of groups experimented with. In this case the groups are 0.5, 1 and 3 Hz = 3, P = 0.05/3. (For the effect of changing frequency in the guinea pig (figure R5) the P value would be 0.05/5). 3. Determine at this new value for P and the number of degrees of freedom - 2 for the two groups being tested (Table III). 4. If t ~*"tab' ^ e r e a significant difference between the two groups using Bonferroni analysis at P <-0.05. Example of Calculation of Bonferroni Statistics on the Effects of Isoproterenol and Histamine on the MDF of Rabbit Papillary Muscles Under Conditions of 37.5°, 0.5 Hz and 2.2 mM Calcium. (Step 1.) MDF - SEM (n) Isoproterenol Histamine "^calc 1.7167 - .2646 (9) 0.6500 - .1314 (12) 3.8973 -204-In the particular experiment illustrated in figure 21, three different conditions are investigated (0.5, 1 and 3 Hz). In the t tables the number for t ^ ^ must be found in the column headed by P = 0.05/3 = 0.0167, and bisected by df -2 = 19. At P = 0.01, •t-j-gb = 2.861. Since t ^ c 't-tab' i s a significant difference between the MDF for isoproterenol and histamine at P <0.05. -205-Table III. TABLE A 4 THE DISTRIBUTION OF I* (TWO-TAILED TESTS) Degrees ; Freedom : 0.500 ; 0.400 | 0.200 I 0.100 Probability of a Larger Value, Sign Ignored 1 • 1.000 2 0.816 3 765 4 741 5 727 6 718 7 : 711 8 i 706 9 .703 1 10 .700 11 .697 12 .695 13 .694 14 .692 15 .691 16 .690 17 .689 18 .688 19 .688 20 .687 21 .686 22 .686 23 .685 24 .685 25 .684 2 6 i .684 27 ! .684 28 .683 29 i .683 30 ! .683 35 .682 40 .681 45 .680 50 .680 55 .679 60 .679 70 .678 80 .678 90 .678 100 .677 120 .677 X .6745 1.376 1.061 0.978 .941 .920 .906 .896 .889 .883 .879 .876 .873 .870 .868 .866 .865 .863 • .862 : .861 .860 : .859 ' .858 .858 .857 : .856 '. : .856 ; .855 ; .855 .854 i .854 | ! .852 ! .851 : .850 ' .849 .849 .848 .847 .847 .846 .846 .845 .8416 3.078 1.886 1.638 1.533 1.476 1.440 ! 1.415 1.397 ' 1.383 | 1 3 7 2 : 1.363 1.356 1.350 1.345 1.341 1.337 ' • 1.333 : 1.330 1.328 : 1.325 ' 1.323 1.321 1.319 : 1.318 ' 1.316 : ! ' i 1.315 | ! 1.314 ! j 1.313 i ! 1.311 ! j 1.310 | i 1.306 ! ; 1.303 ; 1301 ; 1.299 ; 1.297 1.296 1.294 . 1.293 j 1.291 I 1.290 i I 1.289 ! 1.2816 6.314 2.920 2.353 2.132 2.015 1.943 1.895 1.860 1.833 1.812 1.796 1.782 1.771 1.761 1.753 0.050 0.025 0.010 12.706 4.303 | 3.182 i 2.776 ! 2.447 2.365 2.306 2.262 2.228 2.201 2.179 2.160 2.145 2.131 ! 25.452 ' 6.205 i 4.176 3.495 3.163 2.969 2.841 2.752 2.685 2.634 2.593 2.560 2.533 2.510 2.490 63.657 9.925 5.841 4.604 4.032 ! 3.707 | 3.499 I 3.355 I 3.250 j 3.169 ! 3.106 ', 3.055 3.012 • 2.977 . 2.947 0.005 14.089 7.453 5.598 4.773 4.317 4.029 3.832 3.690 3.581 0.001 31.598 12.941 8.610 6.859 5.959 5.405 5.041 4.781 4.587 3.497 3.428 4.437 4.318 3.372 4.221 3.326 | 4.140 3.286 ! 4.073 1.746 2.120 2.473 ! 2.921 3.252 4.015 1.740 : 2.110 2.458 ; 2.898 3.222 ; 3.965 1.734 : 2.101 2.445 2.878 3.197 ' 3.922 1.729 2.093 2.433 2.861 3.174 : 3.883 1.725 2.086 2.423 2.845 3.153 3.850 1.721 1 2.080 2.414 2.831 3.135 3.819 1.717 2.074 2.406 2.819 3.119 3.792 1.714 i 2.069 2.398 2.807 3.104 3.767 1.711 1 2.064 . 2.391 2.797 3.090 3.745 1.708 : 2.060 2.385 ; 2.787 3.078 3.725 1.706 i 2.056 1 i 2.379 2.779 3.067 3.707 1.703 i 2.052 2.373 2.771 3.056 3.690 1.701 : 2.048 : 2.368 2.763 3.047 3.674 1.699 ! 2.045 2.364 2.756 3.038 3.659 1.697 j 2.042 ! 2.360 2.750 3.030 3.646 1.690 1.684 1.680 1.676 1.673 1.671 1.667 1.665 1.662 1.661 1.658 1.6448 2.030 : 2.342 : 2.724 2.996 ' 3.591 2 021 • 2.329 ' 2.704 ! 2.971 . 3.551 2.014 j 2.319 I 2.690 ! 2.952 : 3.520 2.008 ! 2.310 i 2.678 '> 2.937 . 3.496 2.004 ! 2.304 ] 2.669 | 2.925 3.476 2.000 ! 2.299 2.660 2.915 3.460 1.994 I 2.290 2.648 2.899 3.435 1.989 : 2.284 2.638 2.887 3.416 1.986 ! 2.279 2.631 2.878 3.402 1.982 j 2.276 2.625 2.871 3.390 1.980 i 2.270 2.617 2.860 3.373 1.9600 1 2.2414 2.5758 2.8070 3.2905 • Parts of this table are reprinted by permission from R. A. Fisher's Statistical Methods _ . ... . . i i i... r\\: c^ mkurftt./107^ —IOVIV fmm M a x i n e for Research Workers, published by Oliver and Boyd. Edinburgh (1925-1950). from Maxine Merrington's "Table of Percentage Points of the f-Distribution," Biometnka, 32:300 (1942); and from Bernard Ostle's Statistics in Research, Iowa State University Press (1954). Reproduced from Snedecor and Cochran's Statistical Methods, 6th Edition, 1967. Iowa State University Press, Ames, Iowa, page 549. -206-Appendix II Table IV The Effect of Changing Temperature on the Dose-Response Curve of Guinea Pig Papillary Muscles to Isoproterenol. (Data for figures 7,8,9 ) 1 Hz, 2.2 mM + 25° 3C ° 37. 5° 42 o Control 0.4636* 0.4306 0.2500 0.2500 ±.1009 (11) ±.0680 (18) ±.0470 (20) ±.0707 (10) -9 10 M 0.5227 0.4583 0.3275 0.2650 ±.1001 (11) ±.0727 (18) ±.0730 ±.0711 (10) _9 5 x 10 M 0.5909 0.7591 0.3000 ± .1181 (11) ±.1232 (11) ±.0876 (10) 10"8 M 0.7000 0.8111 0.5684 0.4050 ±.1304 (11) ±.1118 (18) ±.0869 (19) ±.1341 (10) 5 x 10~8 M 0.9318 1.0833 1.0463 0.6722 ±.1337 (11) ±.1215 (18) ±.1248 (19) ±.1014 (9) 10"7 M 1.0722 1.0250 1.1278 0.8667 ±.1323 (9) ±.1192 (16) ±.1393 (18) ±.1000 (9) 10"5 M 1.0500 0.9000 1.0808 0.9889 ±.1359 (8) ±.1257 (15) ±.1541 (13) ±.0931 (9) 10"5 M 1.0375 0.9625 1.0615 0.8714 ±.1391 (8) ±.1447 (12) ±.1541 (13) ±.1112 (7) -207-Table IV, continued -4 10 M 0.8500 0.8944 1.0423 0.8000 ± .2300 (4) ± .1597 (9) ± .1612 (13) ± .1323 (6) -'numbers indicate mean tension in g, - SEM (n). -208-Table V The Effect of Changing Temperature on the Dose-Response Curve of Guinea Pig Papillary Muscles to Histamine. (Data for figures 7,8,9 ) 1 Hz. 2.2 mM+ 25 30 o 37.5 o 42^  Control -9 10 M 10 M 10 7 M 5 x 10 7 M 10 6 M 5 x 10 M 10"5 M 5 x 10 5 M 0.5654 0.6538 ± .0975 (13) ± .1012 (13) .0.5769 0.6731 ±.0916 (13) ±.0962 (13) 0.5846 0.6692 ±.0903 (13) " ± .0938.(13) 0.6769 0.7423 ± .0928 (13) ±.0935 (13) 0.7692 ±.0985 (13) 0.8577 1.2269 ±.1034 (13) ± .1404 (13) 1.3000 ±.1765 (11) 1.0083 1.3786 ±.1226 (12) ±.2081 (7) 1.3917 ±.2154 (6) 0.3462 0.1929 ± .0532 (13) ± .0275 (17) 0.3654 0.1059 ± .0526 (13) ± .0263 (17) 0.3769 ± .0479 (13) 0.5077 ± .0635 (13) 0.8792 ± .1232 (12) 0.9600 ± .1120 (10) 1.3722 ± .1623 (9) 0.2006 ± .0255 (17) 0.2535 ± .0421 (17) 0.4790 ± .1230 (10) 0.6576 ± .1144 (17) 1.0122 ± .1911 (9) 0.8977 ± .1225 (13) 0.9920 ±.2487 (5) -209-Table V, continued 10 - 4 M 1.0045 1.1375 1.3375 0.9433 ±.1341 (11) ±.2249 (4) ±.1295 (8) ±.1329 (9) "numbers indicate mean tension in g, - SEM (n). Table VI -210-The Effect of Changing Frequency of Stimulation on the Dose-Response Curve of Guinea Pig Papillary Muscles to Isoproterenol. (Data for figures 7, 10, 11 ) 37.5° 2.2 mM 0.5 Hz 1 Hz'"' 2 Hz 3'Hz 4 Hz Control 0.0850- 0.2500 0.2909 0 .2000 0 .2050 ±..0167 (10) ± .0470 (20) ± .0653 (11) ± .0544 (11) + .0203 (10) -9 10 M 0.0950 0.3275 0.3282 0 .2091 0 .2300 ± .0189 (10) ± .0545 (20) ± .0730 (11) ± .0521 (11) + .0367 (10) 10 - 8 M 0.2050 0.5684 0.4918 0 .2818 0 . 3500 ± .0705 (10) ± .0869 (19) ± .1108 (11) + .0746 (11) + .0459 (10) 5 x 10"8 M 0.6250 1.0463 1.0136 0 .4318 0 .4400 ± .1165 (10) ± .1248 (19) ± .1372 (11) + .0772 (11) ± .0433 (10) 10"7 M 0.8550 1.1278 1.0600 0 .4550 0 .4100 ± .1353 (10) ± .1393 (18) ± .1462 (10) ± .0815 (10) + .0400 (10) 5 x 10"7 M 0.9944 1.0767 1.0214 0 .4600 0 .3650 ± .1531 (9) ± .1459 (15) ± .1728 (7) ± .0816 (10) + .0317 (10) 10"6 M 1.0800 1.0808 1.0100 0 .4063 0 .3300 ± .2107 (5) ± .1541 (13) ± .2147 (5) + .0942 (8) ± .0343 (10) 10"5 M 1.2500 1.0615 0.8900 0 .3643 0 .3056 ± .2500 (2) ± .1541 (13) ± .1965 (5) ± .0992 ± .0395 (9) -4 10 M 1.2500 1.0423 0.7750 0 .3429 0 .2813 ± .2500 (2) ± .1612 (13) ± .2175 (4) ± .0862 (7) ± .0365 (8) ''numbers indicate mean tension in g, - SEM (n). -211-Table VII The Effect of Changing Frequency of Stimulation on the Dose-Response Curve of Guinea Pig Papillary Muscles to Histamine. (Data for figures 7, 10, 11 ) 37.5° 2.2 mM 0.5 Hz 1 Hz 2 Hz 3 Hz 4 Hz Control 0.1250* 0.3462 0.2200 0.2964 0.2063 ±.0359 (10) ±.0532 (13) ±.0503 (11) ±.0479 (14) ±.0333 (8) -9 10 M 0.1250 0.3654 0.2291 0.2714 0.1813 ±.0359 (10) ±.0526 (13) ±.0494 (11) ±.0358 (14) ±.0340 (8) 10"8 M 0.1250 0.3769 0.2382 0.2379 0.1813 ±.0359 (10) ±.0479 (13) +.0417 (11) ±.0298 (14) ±.0377 (8) 5 x 10~8 M 0.1350 0.4192 0.2609 0.2486 0.1938 ±.0358 (10) ±0.0505 (13) ±.0485 (11) ±.0298 (14) ±.0486 (8) 10" 7 M 0.2000 0.5077 0.2918 0.2929 .0.2063 ±.0624 (10) ±0.0635 (13) ±.0543 (11) ±.0309 (14) ±.0546 (8) 5 x 10"7 M 0.5456 0.8792 0.5090 0.4143 0.2813 ±.1477 (10) ±.1232 (12) ±.0981 (10) ±.0487 (14) ±.0526 C8) 10"6 M 0.6944 0.9600 0.6338 0.4829 0.3250 ±.1632 (9) ±.1120 (10) ±.1444 (8) ±.0522 (14) ±.0401 (8) 10"5 M 0.7786 1.3722 0.9029 0.6308 0.3875 ±.1861 (7) ±.1623 (9) ±.1582 (7) ±.0609 (13) ±.0398 (8) -4 10 M 0.7900 1.3375 0.7300 0.5182 0.3375 ±.2446 (5) ±.1295 (8) ±.2022 (5) ±.0523 (11) ±.0310 (8) *numbers indicate mean tension in g, - SEM (n). -212-Table VIII The Effect of Changing Calcium Concentration on the Dose-Response Curve of Guinea Pig Papillary Muscles to Isoproterenol. (Data for figures 7, 12, 13) 37.5° 1 Hz 1.5 mM 2.2. mM ' 4 mM 6 mM 8 mM Control 0.0600' 0.2500 0.2816 0.6636 0.7733 ± .0053 (15) ± .0470 (20) ± .0443 (19) ± .0775 (11) ± .0842 (15) -9 10 M 0.0867 0.3275 0.2947 0.7182 0.8100 ± .0186 (15) ± .0545 (20) ± .0487 (19) ± .0733 (11) ± .0924 (15) 5 x 10"9M 1.0545 ± .0870 (11) 1.1308 ± .0957 (13) 10"8 M 0.1767 0.5684 0.4974 1.2636 1.3733 ± .0238 (15) ± .0869 (19) ± .0698 (19) + .0789 (11) + .1026 (15) 5 x 10"8M 0.6571 1.0463 1.0711 1.5682 1.5308 ± .0695 (14) ± .1248 (19) ± .1098 (19) ± .0964 (11) ± .1100 (13) 10"7 M 0.9536 1.1278 1.2306 1.5636 1.4923 ± .1038 (14) ± .1393 (18) ± .1282 (18) ± .0897 (11) ± .1089 (13) 5 x 10"7M 1.0542 1.0767 1.1469 ± .1454 (12) ± .1459 (15) ± .0791 (16) 10"6 M 1.0333 1.0808 1.1000 1.5200 1.4538 ± .1698 (9) ± .1541 (13) ± .0900 (13) ± .0814 (10) ± .1147 (13) 10"5 M 1.0063 1.0615 0.9833 1.4200 1.2917 ± .1848 (8) ± .1541 (13) ± .0538 (12) ± .0757 (10) ± .0996 (12) -213-Table VIII, continued 10 4 M 0.9438 1.0423 0.9625 '1.3600 1.2583 ± .1743 (8) ± .1612 (13) ± .0574 (12) ± .0748 (10) ± .0949 (12) '''numbers indicate mean tension in g, - SEM (n). -214-Table IX The Effect of Changing Calcium Concentration on the Dose-Response Curve of Guinea Pig Papillary Muscles to Histamine. (Data for figures 7, 12, 13) 37 5° n ' + 1.5 mM 2.2 mM 4 mM 6mM 8 mM 1 Hz Control 0.0667* 0.3462 0.3600 0.7150 0.6375 ± .0118 (9) ± .0532 (13) ± .0572 (15) ± .1111 (10) ± .0515 (16) 10"9 M 0.0667 0.3654 0.3667 0.7050 0.6267 ± .0118 (9) ± .0526 (13) ±..0564 (15) ± .1086 (10) ± .0547 (15) 10"8 M 0.0667 0.3769 0.3733 0.7200 0.6333 ± .0118 (9) ± .0479 (13) ± .0583 (15) ± .1114 (10) ± .0645 (15) 5 x 10~8M 0.0667 0.4067 ± .0118 ± .0643 (15) 10"7 M 0.1000 0.5077 0.5200 0.8400 0.7533 ± .0264 (9) ± .0635 (13) ± .0732 (15) ± .1327 (10) ± .0822 (15) 5 x 10"7M 0.3278 0.8792 0.9300 1.2850 1.1000 ± .0693 (9) ± .1232 (12) ± .1396 (15) ± .1955 (10) ± .1284 (15) 10"6 M 0.6500 0.9600 1.1433 1.5889 1.3233 ± .1395 (8) ± .1120 (10) ± .1406 (15) ± .2003 (9) ± .1481 (15) 5 x 10"6M 1.8000 1.5733 ± 2146 (8) ± .1599 (15) 10"5 M 1.2143 1.3722 1.3393 1.7429 1.3423 ± .1654 (7) ± .1623 (9) ± .1234 (14) ± .1784 (7) ± .1361 (13) -215-Table IX, continued 10~4 M 1.1643 1.3375 1.3000 1.6714 1.2167 ±.1285 (7) ±.1295 (8) ±.1120 (12) ±.1658 (7) ±.1107 (12) '''numbers indicate mean tension in g, - SEM (n). -216-Table X 7 The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the BDF, and MDF to Isoproterenol and Histamine in Guinea Pig Papillary Muscles (Data for figures 7,' 9, 11, 13). BDF Isoproterenol MDF Histamine MDF 1 Hz, 2.2 mM+ 25° 0.5540 + .0754 (25)* 1.0722 + .1323 (9) 1.0083 + .1226 (12) 30° 0.5242 + .0604 (31) 1.0833 + .1215 (18 1.3917 + .2154 (6) 37.5° 0.2879 + .0358 (33) 1.1278 + .1393 (18 1.3722 + .1623 (9) 42° 0.2088 + .0318 (26) 0.9889 + .0931 (9) 1.0122 + .1911 (9) 37.5°,2.2 mM+ 0.5 Hz 0.1050 + .0198 (20) 0.9944 + .1531 (9) 0.7886 + .1861 (7) 1 0.2879 + .0358 (33) 1.1278 + .1393 (18) 1.3722 + .1623 (9) 2 0.2555 + .0410 (22) 1.0600 + .1462 (10) 0.9029 + .1582 (7) 3 0.2540 + .0365 (25) 0.4600 + .0816 (10) 0.6308 + .0609 (13) 4 0.2056 + .0180 (18) 0.4400 + .0433 (10) 0.3875 + .0398 (8) 37.5°, 1 Hz+ 1.5 mM 0.0700 + .0076 (20) 1.0542 + .1454 (12) 1.2143 + .1654 (7) 2.2 0.2879 + . 0358 (33) 1.1278 + .1393 (18) 1.3722 + .1623 (9) 4 0.3162 + .0354 (34) 1.2306 + .1282 (18) 1.3393 + .1234 (14) 6 0.6083 + .0686 (24) 1.5682 + .0964 (11) 1.8400 + .2146 (8) 8 0.7032 + .0494. (31) 1.5308 + .1100 (13) 1.5733 + .1599 (15) '''numbers indicate mean tension in g, - SEM (n). -217-Table XI The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the % Increase of Maximal Developed Force over Basal Developed Force in Guinea Pig Papillary Muscles. (Data for figure 14 )  25° 30° 37.5° 42° 1. Isoproterenol 156* 312 591 567 - 30% (11) - 73% (16) - 91% (18) - 122% (9) 2. Histamine 184 148 459 546 - 79% (11) - 13% (9) - 123% (9) - 65% (15) 0.5 Hz 1 Hz 2 Hz 3 Hz 4 Hz 1. Isoproterenol 1231 591 521 259 152 - 205% (10) - 91% (18) - 186% (10) - 75% (11) - 21% (10) 2. Histamine 788 459 637 226 175 - 189% (8) - 123% (9) - 232% (7) - 45% (13) - 34% (8) 1.5 mM 2.2 mM 4 mM 6 mM 8 mM 1. Isoproterenol 2114 591 515 173 131 - 296% (7) - 91% (18) - 104% - 29% (11) - 16% (14) 2. Histamine 1853 459 368 174 166 - 273% (7) - 123% (9) - 51% (15) - 20% (8) - 15 (15) *numbers indicate mean % increase - SEM (n). -218-Table XII The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the ED^Q Value for Isoproterenol in Guinea Pig Papillary Muscles. (Data for figure 15 ) 25° 30° 37.5° 42° a 11 16 18 9 b 1.12 x 10"8 9.01 x 10" 9 1.37 x 10" 8 3.72 x 10" •8 c 7.30 x 10~9 6.92 x 10" 9 8.99 x 10" 9 2.77 x 10" •8 d 1.73 x 10"8 1.17 x 10" 8 2.09 x 10" 8 5.01 x 10* 8 0.5 Hz 1 Hz 2 HZ: 3 Hz 4 Hz a 10 18 10 11 10 b 3.57 x 10"8 1.37 x 10" 8 1.70 x 10" 8 1.87 x 10" 8 6.03 x-10"9 c 2.32 x 10"8 8.99 x 10" 9 9.89 x 10" 9 1.19 x 10" 8 2.77 x 10"9 d 5.55 x 10"8 2.09 x 10" 8 2.91 x 10" 8 2.95 x 10" 8 1.31 x 10"8 1.5 mM 2.2 mM 4 mM 6 mM 8 mM a 12 18 18 11 14 b 3.44 x 10 - 8 1.37 x 10" 8 1.96 x 10" 8 6.11 x 10" 9 5.64 x 10"9 c 2.44 x 10"8 8.99 x 10" 9 1.53 x 10" 8 3.91 x 10" 9 4.11 x 10"9 d 4.86 x 10"8 2.09 x 10" 8 2.51 x 10" 8 9.53 x 10" 9 7.72 x 10"9 a: n value b: geometric mean E D ^ Q value ( M ) c: 95% confidence interval-lower limit d: 95% confidence interval-upper limit -219-Table XIII The Effect of Changing Temperature, Frequency of Stimulation and Calcium Concentration on the ED^ Q Value for Histamine in Guinea Pig Papillary Muscles (Data for figure 16 ). 25° 30° 37. 5° 42° a 10 9 9 15 b 3. 72 x 10"7 M 4. 26 x 10"7 8. 46 x lo" 7 8. 97 x 10"7 c 1. 92 x 10~7 2. 11 x 10"7 3. 90 x lo" 7 6. 29 x 10"7 d 7. 19 x 10~7 8. 58 x 10"7 1. 83 x lo" 6 1. 28 x 10"6 0.5 Hz 1 Hz 2 Hz 3 Hz 4 Hz a 8 9 7 13 8 b 4. 61 x 10"7 M 8. 46 x 10"7 7. 17 x lo" 7 8. 88 x 10"7 4 .60 x 10"7 c 2. 51 x 10"7 3. 90 x 10"7 4. 10 x IQ"7 ' 5. 49 x 10"7 1 .62 x 10"7 d 8. 46 x 10~7 1. 83 x 10"6 1. 25 x lo" 6 1. 44 x 10~6 1 .31 x 10"6 1.5 mM 2.2 mM 4 mM 6 mM 8 mM a 7 9 15 8 16 b 1. 06 x 10"6 8. 46 x 10"7 3. 83 x lo" 7 3. 99 x 10"7 5 .11 x 10"7 c 6. 20 x 10"7 3. 90 x 10~7 2. 41 x ID"7 2. 50 x 10~7 3 .64 x 10"7 d 1. 93 x 10"6 1. 83 x 10~6 6. 09 x lo" 7 6. 38 x 10"7 7 .17 x 10"7 a: n value b: geometric mean ED^ value c: 95% confidence interval - lower limit d: 95% confidence interval - upper limit -220-Table XIV The Effect of Changing the Calcium Concentration while Stimulating at 0.2 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Isoproterenol. (Data for figures 18 and 19 ). 37.5, 0 2 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0583* 0.1179 0.3182 ±.0110 (9) ±.0365 (7) ±.0400 (11) -9 10 M 0.0583 0.1179 0.3818 ±.0110 (9) ±.0365 (7) ±.0612 (11) 10" 8 M 0.1556 0.1214 0.9318 ±.0473 (9) ±.0355 (7) ±.0910 (11) .5 x 10" 8 M 0.3938 0.4500 1.7182 ±.1259 (E 0 ±.0893 (7) ±.1765 (11) 10" 7 M 0.5438 0.8643 2.2091 ±.1723 a 5) ±.1204 (7) ±.1540 (11) 5 x 10~7 M 0.6125 1.3786 2.3273 ±.2207 a J) ±.2385 (7) ±.1579 (11) 10" 6 M 0.7625 1.4429 2.4045 ±.2195 a 3) ±.2569 (7) ±.1613 (11) 5 x 10" 6 M 0.7750 1.4929 2.4000 ±.2184 a ±.2465 (7) ±.1612 (11) 10" 5 M 0.7938 1.5000 2.4091 ±.2107 a n ±.2582 (7) ±.1626 (11) -221-Table XIV, continued 10 4 M 0.8000 1.5429 2.4091 ±.2083 (8) ±.2467 (7) ±.1626 (11) "numbers indicate mean tension in g, - SEM (n) Table XV The Effect of Changing the Calcium Concentration while Stimulating at 0.5 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Isoproterenol (Data for figures 18 and 19 ). 37.5°, 0.5 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0361* 0.1500 0.2214 ±.0044 (9) ±.0264 (9) ±.0510 (7) -9 10 M 0.0389 0.1500 0.2214 ±.0044 (9) ±.0264 (9) ±.0510 (7) ,10~8 M 0.1167 0.1722 0.2929 ±.0232 (9) ±.0278 (9) ± .0550 (7) 5 x 10"8 M 0.3833 0.5722 0.8429 ±.0722 (9) ±.0936 (9) ±.2361 (7) 10~7 M 0.5778 0.9778 1.3143 ±.0921 (9) ±.1555 (9) ±.2978 (7) 5 x 10"7 M 0.7944 1.5944 1.7643 ±.1153 (9) ±.2650 (9) ±.3832 (7) 10"6 M 0.8938 1.6778 1.7357 ±.1163 (8) ±.2756 (9) ±.3560 (7) 5 x 10~6 M 0.9625 1.7000 ±.1295 (8) ±.3338 (7) 10"5 M 0.9813 1.7167 1.6500 ±.1271 (8) ± .2646 (9) ±.3331 (7) -4 10 M 1.0500 1.6563 1.6357 ±.1299 (8) ± .2809 (8) ± .3260 (7) n^umbers indicate mean tension in g, - SEM (n). Table XVI The Effect of Changing the Calcium Concentration while Stimulating at 1 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Isoproterenol. (Data for figures 18 and 19 ) 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0341* 0.2708 0.6000 ±.0038 (11) ±.0351 (12) ±.0866 (E 0 -9 10 M 0.0341 0.2750 0.5750 ±.0039 (11) ±.0396 (12) ±.0796 a 0 10"8 M 0.0705 0.4625 0.8750 ±.0150 (11) ±.0680 (12) ±.1048 (E 3) 5 x 10"8 M 0.3300 1.2100 1.8000 ±.0676 (10) ±.1917 (10) ±.1376 (E 3) 10"7 M 0.5250 1.5455 1.8375 ±.0964 (10) ±.2051 (11) ±.1413 (E 3) 5 x 10"7 M 0.7200 1.8111 1.7500 ±.1294 (1) ±.2245 (9) ±.1210 a 3) 10"6 M 0.7400 1.7450 1.6000 ±.1253 (10) ±.1866 (10) ±.1018 (E 3) 10"5 M 0.7850 1.6400 1.5000 ±.1331 (10) ±.1648 (10) ±.0886 0 3) -4 10 M 0.7900 1.6000 1.3875 ±.1352 (10) ±.1592 (10) ± .0895 a 3) •'numbers indicate mean tension in g, - SEM (n). -224-Table XVII The Effect of Changing the Calcium Concentration while Stimulating at 3 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Isoproterenol. (Data for figures 18 and 19 ) 37.5°, 3 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0750* 0.2917 0.3625 ± .0206 a 3) ± .0436 (6) ± .0557 (E 3) -9 10 M 0.0875 0.3000 0.3813 ± .0177 a 3) ± .0447 (6) . ± .0582 G 3) 5 x 10"9 M 0.4750 ± .0762 a 3) 10~8 M 0.1344 0.4500 0.5250 ±.0375 a O ± .0856 (6) ± .0750 (E 3) 5 x 10"8 M 0.3875 0.7917 0.6625 1 ± .0324 (E 0 ± .1114 ± .0999 (E 3) 10"7 M 0.4563 0.7250 0.5938 ±.0593 a 0 ± .1094 (6) ±.0988 (E 3) 5 x 10"7 M 0.5188 0.8900 0.5500 ±,0813 (E 0 ± .0872 (5) ± .0921 (E 3) 10"6 M 0.5250 0.7375 0.4750 ±.0785 (E 0 ±.1064 (4) ± .0694 (E 3) 5 x 10"6 M 0.4969 0.7250 0.4500 ±.0665 (E D ±.1164 (4) ± .0605 (E 3) 10"5 M 0.4500 0.6750 0.4500 ±.0612 (E 0 ±.1051 (4) ± .0543 (E 3) -4 10 M 0.4000 0.6000 0.4563 ±.0655 (E 0 ±.1137 (4) ±.0530 (E 3) -'numbers indicate mean tension in g, - SEM (n). Table XVIII -225-The Effect of Changing the Calcium Concentration while Stimulating at 0.2 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Histamine. (Data for figures 20 and 21 . ). 37.5°, 0.2 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0281* 0.1583 0.3050 ±.0031 a O ±.0375 (6) ± .0508 (10) 10"8 M 0.0281 0.1583 0.3150 ±.0031 (E 0 ±.0375 (6) ±.0527 (10) 10~7 M 0.0313 0.1667 0.3500 ±.0041 (E 0 ±.0357 (6) ±.0645 (10) 5 x 10"7 M 0.0375 0.1750 0.4250 ±.0047 (E 0 ±.0359 (6) ±.0804 (10) 10"6 M 0.0469 0.2250 0.5650 ±.0031 (E 0 ±.0382 (6) ±.1090 (10) 5 x 10"6 M 0.0844 0.3917 0.8800 ±.0133 (E 0 ±.0889 (6) ±.1506 (10) 10"5 M 0.1344 0.5417 1.0550 ±.0279 (£ ) ±.1357 (6) ±.1694 (10) 5 x 10~5 M 0.1875 0.6833 1.2350 ±.0479 (E 0 ±.1716 (6) ±.1839 (10) -4 10 M 0.1844 0.7000 1.2300 ±.0430 (E 0 ±.1839 (6) ±.1818 (10) 10"3 M 0.2125 0.7083 1.2550 ±.0581 (E 0 ±.1943 (6) ±.1947 (10) '''numbers indicate mean tension in g, - SEM (n). Table XIX The Effect of Changing the Calcium Concentration while Stimulating at 0.5 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Histamine. (Data for figures 20 and 21 )• 37.5°, 0.5 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0333* 0.1208 0.2188 ± .0042 (9.) ± .0208 (12) ± .0719 a 3) 10" 8 M 0.0333 0.1250 0.2313 ± .0042 (9) ± .0218 (12) ± .0773 a 3) 10" 7 M 0.0333 0.1292 0.2563 ± .0042 (9) ± .0226 (12) ± .0842 a 3) 5 x 10" 7 M 0.0441 0.1583 0.3313 ± .0081 (9) ± .0281 (12) ± .0906 a 3) 10" 6 M 0.0472 0.2250 0.4125 ± .0088 (9) ± .0471 (12) ± .1034 a 3) 5 x 10" 6 M 0.0889 0.4083 0.6313 ± .0232 (9) ± .0959 (12) ± .1239 a 3) 10~5 M 0.1500 0.5292 0.7685 ± .0354 (9) ± .1216 (12) ± .1464 a 3) 5 x 10" 5 M 0.1944 0.5563 0.8938 ± .0556 (9) ± .1754 (8) ± .1519 O 3) -4 10 M 0.2000 0.6500 0.8750 ± .0546 (9) ± .1314 (12) ± .1414 a 3) 5 x 10" 4 M 0.6417 ± .1328 (12) 10" 3 M 0.2055 0.6333 0.9000 ± .0592 (9) ± .1275 (12) ± .1473 a 3) -'numbers indicate mean tension in g, - SEM (n). -227-Table XX The Effect of Changing the Calcium Concentration while Stimulating at 1 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Histamine. Data for figures 20 and 21 ). 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0455* 0.3400 0.5500 ± .0111 (11) ± .0302 (15) ± .1018 (8) 10"8 M 0.0455 0.3233 0.6000 ±.0111 (11) ± .0280 (15) ± .1134 (8) 10"7 M 0.0523 0.3300 0.6938 ± .0152 (11) ± .0358 (15) ± .1174 (8) 5 x 10"7 M 0.6667 ± .1054 (6) 10"6 M . 0.1114 0.4433 0.9500 ±.0405 (11) ± .0497 (15) ± .1323 (8) 5 x 10~6 M 0.2068 0.7462 1.2563 ±.0578 (11) ± .0647 (13) ± .1539 (8) 10"5 M 0.2705 1.0500 1.3063 ±.0741 (11) ± .1022 (15) ± .1513 (8) 5 x 10~5 M 0.3409 1.2000 ±.0809 (11) ± .1258 (11) -4 10 M 0.3409 1.3100 1.3625 ± .0803 (11) ± .1233 (15) ± .1401 (8) 10~3 M 0.3182 1.1650 1.2625 ± .0767 (11) ± .1468 (10) ± .1209 (8) n^umbers indicate mean tension in g, - SEM (n). -228-Table XXI The Effect of Changing the Calcium Concentration while Stimulating at 3 Hz Frequency on the Dose-Response Curve of Rabbit Papillary Muscles to Histamine. (Data for figures 20 and 21 ). 37.5°, 3 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0718* 0.2833 0.3250 +.0145 (8 ) ± .0459 (6) ±.0845 tt 3) 10"8 M 0.0718 0.2667 0.3500 ±.0145 (8 ) ±.0511 (6) ±.0861 a 3) 10"7 M 0.0718 0.2833 0.3563 ±.0145 (6 ) ±.0459 (6) ±.0853 a 5 x 10"7 M 0.1125 0.3333 0.3938 ±.0241 (£ ) ±.0422 (6) ± .0868 tt 10"6 M 0.1750 0.3917 0.4188 ±.0283 (S ) ±.0455 (6) +.0876 a 3) 5 x 10~6 M 0.2563 0.5333 0.4500 ±.0333 (£ ) ±.0357 (6) ±.0964 tt 3) 10~5 M 0.2750 0.5500 0.4563 ±.0366 (£ ) ±.0447 (6) ±.0952 ({ 3) 5 x 10"5 M 0.3125 0.5500 0.4500 ±.0324 (£ ) ±.0516 (6) ±.0911 tt 3) -4 10 M 0.2938 0.5000 0.4125 ±.0305 (£ ) ±.0577 (6) ±.0811 (( 3) -4 5 x 10 M 0.4917 0.3813 ±.0523 (6) ±.0750 tt 3) 10"3 M 0.2625 0.4667 0.3813 ±.0324 (£ ) ± .0641 (6) ± .0744 tt 3) '''numbers indicate mean tension in g, - SEM (n). -229-Table XXII The Effect of Changing Frequency of Stimulation and Calcium Concentration on the BDF, and MDF to Isoproterenol and Histamine in Rabbit Papillary Muscles. (Data for figures 17, 19, 21).  BDF Isoproterenol MDF Histamine MDF 37 5° 0.2 Hz+ 0.5 mM 0.0441 + .0070 (17)* 0.8000 + .2083 (8) 0. 2125 + .0581 (8) 2.2 0.1365 + .0257 (13) 1.5429 + .2467 (7) 0. 7083 + .1943 (6) 6 0.3119 + .0312 (21) 2.4091 + .1626 (11) 1. 2550 + .1947 (10) 37 5° 0.5 Hz+ 0.5 mM 0.0434 + .0091 (19) 1.0500 + .1299 (8) 0. 2055 + .0592 (9) 2.2 0.1333 + .0163 (21) 1.7167 + .2646 (9) 0. 6500 + .1314 (12) 6 0.2200 + .0436 (15) 1.7643 + .3832 (7) 0. 8938 + .1519 (8) 37 •5°, 1 Hz+ 0.5 mM 0.0398 + .0059 (22) 0.7900 + .1352 (10) 0. 3409 + .0803 (11) 2.2 0.3093 + .0234 (27) 1.8111 + .2245 (9) 1. 3100 + .1233 (15) 6 0.5750 + .0649 (16) 1.8375 + .1598 (8) 1. 3625 + .1401 (8) 37 5° 3 Hz+ 0.5 mM 0.0828 + .0111 (16) 0.5250 + .0785 (8) 0. 3125 + .0324 (8) 2.2 0.2875 + .0302 (12) 0.8900 + .0872 (5) 0. 5500 + .1095 (6) 6 0.3438 + .0491 (16) 0.6625 + .0999 (8) 0. 4563 + .0952 (8) '''numbers indicate mean tension in g, - SEM (n). -230-Table XXIII The Effect of Changing Frequency of Stimulation and Calcium Concentration on the % Increase of Maximal Developed Force over Basal Developed Force in Rabbit Papillary Muscles. (Data for figure 22 ). 37 .5°, 0.2 Hz+ 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 1163 1505 771 - 200% (8) - 227% (7) - 100% (11) 2. Histamine 625 442 335 - 169% (8) - 177% (6) - 43% (10) 37 .5°, 0.5 Hz+ 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 3138 1308 844 - 454% (8) - 241 % (9) - 163% (7) 2. Histamine 600 524 534 - 233% (9) - 124% (12) - 106% (8) 37 .5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 2400 669 269 (10) + - 558% - 87% (10) - 46% (8) 2. Histamine 945 .'337 227 - 282% (11) - 30% (15) - 70% (8) 37 .5°, 3 Hz + 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 738 207 92 - 214 ( 8) - 32% (6) - 21% (8) 2. Histamine 394 146 51 - 108% (8) - 28% (6) - 9% (8) '''numbers indicate mean % increase - SEM (n). - 2 3 1 -Table XXIV The Effect of Changing Frequency of Stimulation and Calcium Concentration on the ED,-n Value for Isoproterenol in Rabbit Papillary Muscles. (Data for figure 2 3 ) . : 37 5 ° 0 .2 Hz+ 0 .5 mM 2.2 mM 6 mM a 8 7 11 b 1.42 x l o " 7 8 .45 x l O " 8 2.35 x 1 0 " 8 c 4 . 8 4 x l o " 8 5.76 x l O " 8 . 1 .47 x 1 0 - 8 d 4 . 2 0 x I Q " 7 1.24 x l O " 7 3.76 x 1 0 - 8 37 5 ° 0 .5 Hz+ 0 .5 mM 2.2 mM 6 mM a 8 9 7 b 1.03 x lo " 7 1.03 x l O " 7 6.28 x 1 0 - 8 c 5.35 x l O " 8 7.17 x l O " 8 4 . 0 7 x 1 0 - 8 d 2.00 x l o " 7 1.47 x l O " 8 9 .70 x 1 0 " 8 37 5 ° 1 Hz+ 0.5 mM 2.2 mM 6 mM a 10 11 I 3 b 6.41 x l O " 8 3.58 x l O " 8 1.50 x 1 0 - 8 c 4 . 7 4 x l O " 8 2.61 x l O " 8 1.28 x 1 0 - 8 d 8.67 x l O " 8 4 . 9 1 x l O " 8 1.75 x 1 0 " 8 37 5 ° 3 Hz+ 0 .5 mM 2.2 mM 6 mM a 8 6 8 b 2.08 x l O " 8 1.57 x l O " 8 6 .12 x 1 0 " 9 c 1.20 x io- 8- 5.83 x l O " 9 3.63 x 1 0 " 9 Q d 3.62 x l O " 8 4 . 2 5 x l O " 8 1.03 -o x 10 a: n value b: geometric mean ED^ ^ value (m) c: 95% confidence interval - lower limit d: 95% confidence interval - upper limit -232-Table XXV The Effect of Changing Frequency of Stimulation and Calcium Concentration on the ED^ Q Value for Histamine in Rabbit Papillary Muscles. (Data for figure 24). 37.5 , 0.2 Hz+ •• 0.5 mM 2.2 mM 6 mM a 8 6 10 b 6.52 x 10"6 M 5.93 x 10 - 6 3.54 x 10~6 c 2.25 x 10"6 M 4.73 x 10"6 2.53 x 10"6 d 1.89 x 10"5 M 7.43 x 10~6 4.95 x 10~6 37.5°, 0.5 Hz+ 0.5 mM 2.2 mM 6 mM a 9 12 8 b 5.64 x 10"6 5.64 x 10~6 3.30 x 10"6 c 3.14 x 10"6 3.61 x 10"6 2.01 x 10~6 d 1.01 x 10"5 8.8 x 10"6 5.43 x 10~6 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM a 11 15 8 b 4.76 x 10"6 5.02 x 10 _ 6 9.14 x 10~7 c 2.48 x 10"6 4.12 x 10 - 6 6.18 x 10~7 d 9.12 x 10"6 6.12 x 10"6 1.35 x 10 - 6 37.5°, 3 Hz+ 0.5 mM 2.2 mM 6 mM a 8 5 8 b 1.58 x 10~6 1.16 x 10"6 3.00 x 10 - 7 c 8.89 x 10~7 4.08 x 10 - 7 7.58 x 10~8 d 2.81 x 10"6 3.28 x 10~6 1.19 x 10 - 6 a: n value b: geometric mean ED^ Q value (m) c: 95% confidence interval - lower limit d: 95% confidence interval - upper limit -233-Table XXVI The Effect of Changing the Calcium Concentration While Maintaining the Temperature at 25° on the Dose-Response Curve for Isoproterenol in Rabbit Papillary Muscles. (Data for figures 26, 27). 25°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.4250* 0.5188 1.9571 ± .0704 (6) ±.0945 (E 3) ±.1378 (7) -9 10 M 0.4917 0.5438 2.1571 ±.0898 (6) ±.0979 (c 3) ±.1510 (7) 10"8 M 0.6917 0.6938 2.5000 ±.1417 (6) ±.1307 a 3) ±.2573 (7) 5 x 10"8 M 0.7333 0.7750 2.4857 ±.1569 (6) ±.1199 a 3) ±.2939 (7) 10"7 M 0.7583 0.8000 2.4429 ±.1800 (6) ±.1176 a 3) +.2943 (7) 5 x 10~7 M 0.8583 0.8188 2.3714 ±.1873 (6) ±.1157 (E 3) ±.2974 (7) 10"6 M 0.8667 0.8250 2.3714 ±.1896 (6) ±.1134 (6 O ± .2974 (7) 5 x 10"6 M 0.9000 0.8188 2.3571 ±.1884 (6) ± .1149. (E O ±.3054 (7) 10"5 M 0.9167 0.8188 2.3143 ±.1801 (6) ± .1130 (E ) ± .3066 (7) -4 10 M 0.9167 0.7938 2.3571 ± .1801 ± .1122 (E 0 ± .3007 (7) n^umbers indicate mean tension in g, - SEM (n). Table XXVII -234-The Effect of Changing the Calcium Concentration While Maintaining the Temperature at 37.5° on the Dose-Response Curve for Isoproterenol in Rabbit Papillary Muscles. (Data for figures 26, 27). 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.5188* 0.2708 0.1357 +.0945 tt 3) ±.0351 (12) ±.0459 (7) -9 10 M 0.5438 0.2750 0.1357 ±.0979 tt 3) ± .039.6. ±.0459 (7) 10"8 M 0.6938 0.4625 0.2357 ±.1307 tt 3) ±.0680 (12) ±.0688 (7) 5 x 10~8 M 0.7750 1.2100 0.5071 ±.1199 tt 3) ±.1917 (10) ±.0592 (7) 10"7 M 0.8000 1.5455 0.7929 ±.1176 (E 3) ±.2051 (11) ±.0812 (7) 5 x 10"7 M 0.8188 1.8111 1.1786 ±.1157 tt 3) ±.2245 (9) ±.0899 (7) 10~6 M 0.8250 1.7450 1.2429 ±.1134 tt 3) ±.1866 (10) ±.1131 (7) 5 x 10~6 M 0.8188 1.2286 ±.1149 tt 3) ±.1149 (7) 10 - 5 M 0.8188 1.6400 1.1643 ±.1130 (E 3) ±.1648 (10) ± .1169 (7) -4 10 M 0.7938 1.6000 1.0786 ±.1128 (E ) ±.1592 (10) ±.1051 (7) ''numbers indicate mean tension in g - .SEM (n). -235-Table XXVIII The Effect of Changing the Calcium Concentration While Maintaining the Temperature at 42° on the Dose-Response Curve for Isoproterenol in Rabbit Papillary Muscles. (Data for figures 26, 27). 42°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0750s 's 0.1357 0.8000 ±.0106 ( 3) ±.0459 (7) ±.1617 ( 3) -9 10 M 0.1531 0.1357 0.8188 ±.0540 0 3) ±.0459 (7) ±.1711 0 3) 10"8 M 0.3188 0.2357 1.0063 ±.1195 (S 3) ±.0688 (7) ±.1881 a 3) 5 x 10"8 M 0.9250 0.5071 1.7125 ±.1658 0 3) ±.0592 (7) ±.2546 0 3) 10"7 M 1.2000 0.7929 1.6875 ±.1118 a 3) ±.0812 (7) ±.2482 G 3) 5 x 10 - 7 M 1.6625 1.1786 1.6000 ±.1362 a 3) ±.0899 (7) ±.2478 (i 3) 10~6 M 1.6750 1.2429 1.4876 ±.1386 a 3) ±.1131 (7) ±.2386 (c 3) 5 x 10~6 M 1.6375 1.2286 1.4000 ±.1322 a 3) ±.1149 (7) ±.2136 a 3) 10"5 M 1.5750 1.1643 1.3500 ±.1206 a 0 ±.1169 (7) ±.2i49 a 0 -4 10 M 1.4875 1.0786 1.2938 ±.1288 (6 ) ±.1051 (7) ±.1967 (c ) ''numbers indicate mean tension in g, - SEM (n). -236-Table XXIX The Effect of Changing the Calcium Concentration While Maintaining the Temperature at 25° on the Dose-Response Curve for Histamine in Rabbit Papillary Muscles. (Data for figures 28, 29). 25°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.4750 0.5250 1.9688 ±.0692 (10) ±.1217 G 3) ±.1864 ( 3) l O - 8 M 0.4900 0.5938 1.9875 ±.0809 (10) ±.0937 G 3) ±.1865 G 3) 10"7 M 0.5150 0.6188 2.1000 ±.0879 (10) ±.1043 G 3) ±.2130 G 3) 5 x 10"7 M 0.5450 0.6750 2.1500 ±.0899 (10) ±.1126 G 3) ±.2035 G 3) 10"6 M 0.5800 . 0.7563 2.2875 ±.0972 (10) ±.1304 G 3) ±.2048 G 3) 5 x 10"6 M 0.6950 0.8348 2.4500 ±.1097 (10) ±.1492 G 3) ±.2220 G 3) 10"5 M 0.7700 0.8438 2.6688 ±.1181 (10) ±.1492 G 3) ±.2466 G 3) 5 x 10~5 M 0.8500 0.8500 2.7688 ±.1230 (10) ±.1411 G 3) ±.2962 (E 3) -4 10 M 0.8500 0.8375 2.6563 ±.1245 (10) ±.1429 G 3) ±.2386 (E 3) 5 x 10~4 M 0.8375 2.6375 ±.1429 G O ±.2360 (E O 10"3 M 0.8500 0.8125 2.6063 ±.1218 (10) ±.1339 G 3) ±.2280 (E O "numbers indicate mean tension in g, - SEM (n). -237-Table XXX The Effect of Changing the Calcium Concentration While Maintaining the Temperature at 37.5° on the Dose-Response Curve for Histamine in Rabbit Papillary Muscles. (Data for figures 28, 29) 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.04555' ±•.0111 (11) 0.3400 ± .0302 (15) 0.5500 ± .1018 (15) 10"8 M 0.0455 ± .0111 (11) 0.3233 ± .0280 (15) 0.6000 ± .1134 (15) 10~7 M 0.0523 ± .0152 (11) 0.3300 ± .0358 (15) 0.6938 ± .1174 (15) 5 x 10~7 M 0.6667 ± .1054 (15) 10"6 M 0.1114 ± .0405 (11) 0.4433 ± .0497 (15) 0.9500 ± .1323 (15) 5 x 10"6 M 0.2068 ± .0578 (11) 0.7462 ± .0647 (13) 1.2563 ± .1539 (15) 10~5 M 0.2705 ± .0741 (11) 1.0500 ± .1022 (15) 1.3063 ± .1513 (15) 5 x 10~5 M 0.3409 ± .0809 (11) 1.2000 ± .1258 (11) -4 10 M 0.3409 ± .0803 (11) 1.3100 ± .1233 (15) 1.3625 ± .1401 (15) 10"3 M 0.3182 ± .0767 (11) 1.1650 + .1468 (10) 1.2625 ± .1209 (15) "numbers indicate mean tension in g, - SEM (n). -238-Table XXXI The Effect of Changing the Calcium Concentration While Maintaining the Temperature at 42° on the Dose-Response Curve for Histamine in Rabbit Papillary Muscles. Data for figures 28, 29). 42°, 1 Hz+ 0.5 mM 2.2 mM 6 mM Control 0.0906* 0.1643 0.8600 ±.0149 tt 3) ±.0433 (7) ±.1024 (10) 10"8 M 0.0906 0.1643 0.8600 ±.0149 tt 3) ±.0433 (7) ±.1024 (10) 10~7 M 0.0906 0.1643 0.8750 ±.0149 tt 3) ±.0433 (7) ±.1009 (10) 5 x 10~7 M • 0.1063 0.2000 0.8850 ±.0199 tt 0 ±.0450 (7) ±.0997 (10) 10"6 M 0.1563 0.2571 0.9150 ±.0346 C E 3) ±.0481 (7) ±.1014 (10) 5 x 10"6 M 0.3344 0.4429 1.0350 ±.1166 (E 3) ±.0631 (7) ±.1044 (10) 10"5 M 0.6219. 0.6571 1.1400 ±.1663 (E 3) ±.0827 (7) ±.1110 (10) 5 x 10"5 M 0.8719 0.8071 1.3100 ±.1546 (E 3) ±.0991 (7) ±.1301 (10) -4 10 M 0.9063 0.8214 1.2950 ±.1304 (E 3) ±.0837 (7) ±.1301 (10) 5 x 10"4 M 0.8969 0.7929 ±.1265 (E 3) ±.0834 (7) 10~3 M 0.9469 0.8571 1.2900 ±.1296 (E O ±.0889 (7) ± .1197 (10) '''numbers indicate mean tension in g - SEM (n). -239-Table XXXII The Effect of Changing Temperature and Calcium Concentration on the BDF, and MDF to Isoproterenol and Histamine in Rabbit Papillary Muscles. (Data for figures 25, 27, 29). BDF Isoproterenol MDF Histamine MDF 25°, 1 Hz+ 0. 5 mM . 0. 4382 + 0499 (17)* 0.9167 + .1801 (6) 0. 8550 + .1230 (10) 2. 2 0. 5531 + .0659 (16) 0.8250 + .1134 (8) 0. 8500 + .1411 (8) 6 1. 9633 + .1143 (15) 2.5000 + .2573 (7) 2. 7688 + .2962 (8) 37. 5C > 3 1 Hz 0. 5 mM 0. 0398 + .0059 (22) 0.7900 + .1352 (10) 0. 3409 + .0803 (11) 2. 2 0. 3093 + .0234 (27) 1.8111 + .2245 (9) 1. 3111 + .1233 (15) 6 0. 5750 + .0649 (16) 1.8375 + .1413 (8) 1. 3625 + .1401 (8) 42°, 1 Hz 0. 5 mM 0. 0828 + .0090 (16) 1.6750 + .1386 (8) 0. 9063 + .1304 (8) 2. 2 0. 1500 + .0306 (14) 1.2429 + .1131 (7) 0. 8571 + .0889 (7) 6 0. 8333 + .0890 (18) 1.7125 + .2546 (8) 1. 3100 + .1301 (10) n^umbers indicate mean tension in g, - SEM (g). -240-Table XXXIII The Effect of Changing Temperature and Calcium Concentration on the % Increase of Maximal Developed Force over Basal Developed Force in Rabbit Papillary Muscles. (Data for figure 30). 25°, 1 Hz+ 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 133 + 57% (6) 74 + 16% (8) 35 - 11% (6) .'2. Histamine 97 48 47 - 23% (10) - 9% (8) - 19% (8) 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 2400 - 558% (10) 699 - 87% (10) 269 - 46% (8) 2. Histamine 945 337 227 - 282% (11) - 30% (15) - 70% (8) 42°, 1 Hz+ 0.5 mM 2.2 mM 6 mM 1. Isoproterenol 3025 - 911% (8) 1312 - 375% (7) 155 - 33% (8) 2. Histamine 1310 591 63 - 300% (8) - 124% (7) - 15% (10) "numbers indicate mean % increase - SEM (n). -241-Table XXXIV The Effect of Changing Temperature and Calcium Concentration on the EDr n Value for Isoproterenol in Rabbit Papillary Muscles. (Data for figure 31). 25°, 1 Hz+ 0.5 mM 2.2 mM 6 mM a 6 8 6 b 1.62 x 10"8 1.03 x 10 - 8 1.60 x 10" •9 c 5.53 x 10"9 5.27 x 10"9 3.63 x 10" -10 d 4.76 x 10~8 2.03 x 10"8 7.04 x 10" -9 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM a 10 9 8 b 6.41 x 10"8 3.58 x 10"8 1.50 x 10" -8 c 4.74 x 10"8 2.61 x 10~8 1.28 x 10" -8 d 8.67 x 10"8 4.91 x 10"8 1.75 x 10" -8 42°, 1 Hz+ 0.5 mM 2.2 mM 6 mM a 8 7 8 b 4.22 x 10"8 8.13 x 10"8 .1.58 x 10" -8 c 1.98 x 10"8 5.94 x 10~8 9.80 x 10" -9 d 8.97 x 10"8 1.11 x 10"7 2.55 x 10" -8 a: n value b: geometric mean ED[-Q value (m) c: 95% confidence interval -. lower limit d: 95% confidence interval - upper limit -242-Table XXXV The Effect of. Changing Temperature and Calcium Concentration on the EDj-n Value f o r Histamine i n Rabbit P a p i l l a r y Muscles. (Data f o r figure 32). 25°, 1 Hz+ 0.5 mM 2.2 mM 6 mM a 10 8 8 b 2.73 x l O " 6 6.00 x l O " 7 9.84 x 1 0 - 7 c 1.11 X l O " 6 3.56 x l O " 7 3.87 x 1 0 - 7 d 6.67 x l O " 6 1.01 x l O " 6 2.51 x 1 0 - 6 37.5°, 1 Hz+ 0.5 mM 2.2 mM 6 mM a 11 15 8 b 4.76 x l O " 6 5.02 x l O " 6 9.14 x 1 0 - 7 c 2.48 x l O " 6 4.12 x l O " 6 6.18 x 1 0 - 7 d 9.12 x l O " 6 6.12 x l O " 6 1.35 x 1 0 - 6 42°, 1 Hz+ 0.5 mM 2.2. mM 6 mM a 8 7 10 b 9.49 x l O " 6 5.98 x l O " 6 6.02 x 1 0 - 6 c 4.67 x l O " 6 4.42 x l O " 6 3.49 x 1 0 - 6 d 1.28 x l O " 5 8.09 x l O " 6 1.04 x 1 0 - 5 a: n value b: geometric mean E D ^ value (m) c: 95% confidence i n t e r v a l - lower l i m i t d: 95% confidence i n t e r v a l - upper l i m i t -243-Table XXXVI The Effect of 60. Minutes Pretreatment with D600 on the Dose-Response Curves of Isoproterenol and Histamine in Rabbit Papillary Muscles. (Data for figure 33). Isoproterenol Isoproterenol + Histamine Histamine + 5 x 10 - 7 M D600 5 x 10 7 D600 Control -9 .10 M 5 x 10 - 9 M 0.8188* ±.2276 (8) 0.8750 ±.2293 (8) 1.0688 ±.2737 (8) 0.2850 ±.0687 (10) 0.3450 ±.0871 (10) 0.4450 ±.1257 (10) 0.5475 ±.1117 (10) 0.2944 ± .1461 (9) 10 M 5 x 10 M 10 7 M 5 x 10 7 M 10 6 M 5 x 10 6 M 10 5 M 5 x 10 - 5 M -4 10 M 5 x 10 4 M 1.3688 ±.2864 (8) 2.1625 ±.3000 (8) 2.3875 ±.2416 (8) 2.5313 ±.2179 (8) 2.4813 ±.2101 (8) 2.3750 ±.1946 (8) 2.2875 ±.1827 (8) 2.2000 ±.1176 (8) 0.7300 ±.2036 (10) 1.4350 ±.3061 (10) 2.0350 ±.3208 (10) 2.4400 ±.3077 (10) 2.5750 ±.3165 (10) 2.6400 ±.3128 (10) 2.6750 ±.3067 (10) 0.5400 ±.1059 (10) 0.3100 ±.1576 (9) 2.7100 ±.3020 (10) 0.5500 ± .1119 0.6050 ±.1198 0.7050 ± .1343 0.9050 ± .1546 1.0800 ± .1611 1.2100 + .1665 1.2450 ±.1601 1.2400 ±.1622 (10) (10) (10) (10) (10) (10) (10) 0.3167 ±.1605 (9) 0.3806 ±.1872 (9) 0.4250 ±.2022 (9) 0.6139 ±.2684 (9) 0.7639 ±.3044 (9) 0.0194 ±.3284 (9) 0.9806 ±.3493 (9) 1.0875 ±.3548 (8) -244-Table XXXVI continued 10~3 M 1.2750 1.1438 ± .1682 (10) ± .3191 (8) ED,n (M) 2.04 x 10"8 5.66 x 10"8 5.43 x 10 _ 6 9.95 x 10 _ 6 50 95% CI (lower) 95% CI (upper) 1.07 x 10 8 3.44 x 10 8 3.36 x 10 - 6 2.84 x 10~6 3.90 x 10 8 9.29 x 10 - 8 8.78 x 10~6 1.04 x 10~5 n^umbers indicate mean tension in g - SEM (n). -245-Table XXXVII 14 C - Sorbitol Measurement of the Extracellular Space of Rabbit Papillary Muscles. (Data for figure 34). Time in Split Whole 1 UC-Sorbitol Tissues' Tissues (min) 30 0.4463* 0.4011 +,0276 (2) ±.0216 (3) 60 0.6752 0.4263 ±.0890 (2) ±.5311 (2) '''numbers indicate the mean ECS - SEM (n). -246-Table XXXVIII 14 C - Sorbitol Measurement of the Extracellular Space of Rabbit Left Atrial and Right Ventricle Strips. (Data for figure 35). Time in 1 4C-Sorbitol (min) • Right Ventricle Strips Left Atrial Strips 30 0.3811* 0.4773 ±.0101 (4) ±.0099 (3) 60 0.4261 0.4665 ±.0253 (3) ±.0154 (3) -'numbers indicate the mean ECS - SEM (n). -247-Table XXXIX 14 C - Sorbitol Measurement of the Extracellular Space of Guinea Right Ventricle Strips. (Data for figure 36). Time in 1 4C-Sorbitol (min) 15 30 mean ECS ± SEM (n) 0.3138 ± .0255 (4) 0.3509 ± .0330 (4) -248-Table XXXXa Determination of Ca Content of Rabbit Right Ventricle Strips,with Time using Different Quenching Solutions. (Data for figure 37). Time (min) Isotonic Sucrose Isotonic Choline 0 Ca, 0 Mg, 140 mM Na Control 0 2548.93* 3008.45 2541.10 2363.79 10 2045.48 2046.40 1604.20 1398.24 20 1878.78 1701.60 1318.40 1096.49 30 1771.08 1508.50 1127.85 915.79 40 1689.38 1374.70 1000.75 798.09 50 1631.53 1273.10 906.75 706.89 60 1581.13 1204.30 836.00 635.54 70 1537.48 1150.70 779.85 576.94 80 1500.58 1105.40 735.90 529.84 90 1467.58 1068.45 700.00 490.14 100 1438.78 1038.25 665.80 455.29 110 1412.73 1011.60 641.25 424.74 120 1388.48 988.30 618.05 398.94 130 1364.48 967.05 596.95 375.79 140 1341.98 938.85 579.20 355.69 Tissue 1322.93 930.35 562.30 337.84 n^umbers indicate tissue Ca content in ymoles/kg wet weight from duplicate samples. A l l solutions at 0° unless otherwise indicated. -249-Table XXXXb 45 Determination of Ca Content of Rabbit Right Ventricle Strips with Time using Different Quenching Solutions. (Data for figure 37). Time (min) 0 Ca, 0 Ca, 0 Ca, 10 mM Mg 0.5 mM 10 mM La Control, 37 EGTA , 0 2130.66''' 2176.12 2025.80 2701.80 10 1157.36 1275.12 1233.00 1168.85 . 20 881.36 982.22 954.35 756.35 30 735.11 821.52 792.55 543.40 40 644.96 710.77 676.85 404.45 50 585.46 628.27 585.15 312.65 60 537.01 562.52 508.80 250.85 70 500.11 511.22 445.20 203.60 80 469.16 469.82 391.55 166.45 90 444.26 433.17 345.60 139.15 100 424.96 399.77 304.85 118.05 110 405.71 370.97 267.70 100.70 120 390.41 347.42 232.90 86.70 130 377.21 325.37 205.50 75.20 140 363.06 306.37 179.30 65.35 Tissue 352.11 289.17 157.25 56.55 '''numbers indicate 45_ tissue Ca content in ymoles/kg wet weight from duplicate samples. A l l solutions at 0 unless otherwise indicated. -250-Table XXXXIa Compartmental Analysis of the Release of Ca from Rabbit .Right Ventricle Strips. (Data for Figure 38). A: At 0° Time (min) 45 Ca Content ( ymole/kg) Ae" b t A 0 2363.79 839.07 1524.72 10 1398.24 788.63 609.61 20 1096.49 741.22 355.27 30 915.79 696.66 219.13 40 •' 798.09 654.78 143.31 50 706.89 615.41 91.48 60 635.54 578.42 57.12 70 576.94 543.64 33.30 80 529.84 510.96 18.88 90 490.14 480.24 9.90 100 455.29 451.37 3.92 110 429.74 424.24 120 398.94 398.73 130 375.79 374.76 140 355.69 352.23 Tissue 337.84 331.06 r -0.9990 -0.9943 -k = b -0.0062 -0.0531 a 6.7323 7.0501 (839.07) (1152.97) -251-Table XXXXIb Compartmental Analysis of the Release of 4 5Ca from Rabbit Right Ventricle Strips. . B: At 37° (Data for figure 38).'-• Time (min) ^Ca Content ( ymole/kg) Ae"b t A 0 2701.80 534.86 2166.94 10 1168.85 459.90 708.95 20 756.35 395.44 360.91 30 543.40 340.02 203.38 40 404.45 292.37 112.08 50 312.65 251.39 61.26 60 250.85 216.16 34.69 70 203.60 185.86 17.7.4 80 166.45 159.81 6.64 90 139.15 137.41 1.74 100 118.05. 118.16 110 100.70 101.60 120 86.70 87.36 130 75.20 75.11 140 65.35 64.59 Tissue 56.55 55.53 r -0.9995 -0.9998 -k = b -0.0151 -0.0605 a 6.2820 7.1389 (534.86) (1260.04) -252-Table XXXXJI Determination of Ca Content of Rabbit Right Ventricle Strips with Time using Modified Chenoweth-Koelle Solutions. (Data for figure 39). Time (min) Control, 37° 0 Ca, 10 mM Mg 10 mM Mg, 140 mM Choline 0 3529.35* 3920.73 5605.95 10 1632.75 2336.38 3663.05 20 1130.85 1825.43 2910.00 30 852.70 1521.93 2463.40 40 671.45 1305.83 2121.15 50 529.20 1143.93 1862.65 60 426.15 1018.53 1653.85 70 346.45 918.18 1470.35 80 284.75 835.13 1318.90 90 236.35 768.63 1188.95 100 199.40 711.43 1076.80 110 168.10 665.23 979.00 120 141.15 623.43 891.40 130 118.40 588.78 813.50 140 100.70 557.08 744.50 Tissue 86.25 530.88 682.70 '"'numbers indicate tissue Ca content in ymoles/kg wet weight from duplicate samples. A l l solutions at 0° unless otherwise indicated. -253-Table XXXXIII. Determination of Ca Content of Guinea Pig Right Ventricle Strips with Time using Modified Chenoweth-Koelle Solutions. (Data for figure 40). Time (min) Control 0 Ca, 10 mM Mg Control, 37° 0 4660.98* 3790.59 4129.09 10 2946.78 2197.64 1645.04 20 2341.08 1722.09 996.84 30 1956.98 1453.94 669.99 40 1675.58 1276.64 471.49 50 1463.98 1146.49 354.19 60 1297.78 1044.54 267.09 70 1153.48 961.84 215.79 80 1039.68 894.69 180.09 90 942.28 839.64 154.24 100 861.58 790.74 136.19 110 793.88 750.79 122.59 120 732.98 715.64 112.09 130.' 681.58 685.79 103.64 140 638.28 659.34 96.89 Tissue 601.38 635.64 92.49 '''numbers indicate tissue Ca content in limoles/kg wet weight from duplicate samples. A l l solutions at 0° unless otherwise indicated. -254-Table XXXXIV 45 Measurement of Ca Uptake into Quiescent Rabbit Right Ventricle Strips. (Data for figure 41). Time 4 5Ca in (min) 450 ^ Ca content*-1 10 335.6 ± 9.1 (3) 30 884.1 ± 39.6 (3) 60 1227.3 ± 52.7 (3) 90 1283.6 ± 70.7 (2) 120 1336.7 ± 22.4 (2) '•numbers indicate tissue Ca content in ymoles/kg wet weight - SEM (n). -255-Table XXXXV Measurement of Ca Uptake by Quiescent Tissues over 2-10 Minutes. (Data for figure; 42). Time Control 10"6 M 126 mM K 126 mM K+ (min) Isoproterenol —6 10 M Isoproterenol 2 145.8* 116.6 322.0 265.2 ±5.2 (2) ± 6.7 (2) - (1) ± 11.7 (2) 4 279.7 244.9 283.7 364.2 ± 5.5 (2) ± 5.8 (2) ± 60.2 (2) ± 2.4 (2) 6 339.8 318.9 431.4 644.7 ±6.7 (2) ±14.9 (2) ±10.4 (2) ± 100.9 (2) 8 433.2 425.3 681.9 776.7 ±5.9 (2) ±31.6 (2) ± 200.8 (2) ± 6.3 (2) 10 511.8 442.1 860.8 834.5 ±41.6 (2) ±4.5 (2) ± 52.1 (2) ± 16.4 (2) '-numbers indicate tissue Ca content in y moles/kg wet weight - SEM (n). -256-Table XXXXVI • 45 Measurement of Ca Uptake by Quiescent Tissues After 80 Minutes Loading Followed by 10 Minutes Treatment. (Lata for figure 43). N Normal CK 126 mM K - CK Control 1167.16* 1720.75 ± 83.66 (9) ± 58.82 (8) lO" 6 Isoproterenol 1454.30 1959.80 ± 49.14 (4) ± 71.45 (4) lO" 6 Histamine 1162.65 1611.72 ± 110.22 (8) ± 68.32 (6) ''"numbers indicate tissue Ca content in ymoles/kg wet wei, - SEM (n). -257-Table XXXXVII 45 Measurement of Ca Uptake by Stimulated Tissues over 2-4 Minutes. (Data for figure 44). Time (min) Control 10"6 M 10~6 M Isoproterenol Histamine 2 115.95 160.21 116.71 ± 9.02 (14) ± 11.71 (7) ± 10.32 (9) 4 239.63 258.76 222.84 ±17.80 (15) ± 22.68 (8) ± 20.84 (8) '''numbers indicate tissue Ca content in - SEM (n). y moles wet weight -258-Table XXXXVIII 45 Measurement of Ca Uptake by Stimulated Tissues After 80 Minutes Loading Followed by 10 Minutes Treatment. (Data for figure 45). Normal CK 126 mM K - CK Control 1120.39* 1428.10 ±135.12 (?) ±107.83 (7) 10"6 M Isoproterenol 1316.42 2121.10 ±228.14 (6) ±493.19 (3) 10"5 M Histamine 1052.00 1530.78 ±138.23 (5) i ±404.75 (4) n^umbers indicate tissue Ca content in ymoles/kg wet weight - SEM (n). PUBLICATIONS Barrett, A.J., Weller, E., Rozengurt, N., Longhurst, P. and Humble, J.G. Amidopyrine agranulocytosis: Drug inhibition of granulocyte colonies i n the presence of patients' serum. Brit. Med. J. 2: 850-851, 1976. Barrett, A.J., Longhurst, P., Long, D., Sneath, P., Humble, J.G. and Watson, G. Some observations on circulating C.F.U. i n man. in : Lo Stuio i n vitro delle cellule staminali. II incontrol nazionale d i ematologia sperimentale. eds. M.A. Brunelli, B.P. Bagnara and C. Castaldini. pp. 185-189, Esculapio, Bologna, 1976. Barrett, A.J., Longhurst, P., Sneath, P. and Watson, J.G. Mobilization of CFU-C by exercise and ACTH induced stress i n man. Exp. Hemat. 6: 590-594, 1978. Barrett, A.J., Longhurst, P., Rosengurt, N., Hobbs, J.R. and Humble, J.G. Crossreaction of antilymphocyte globulin with human granulocyte colony-forming c e l l s . J. Clin. Pathol. 31: 129-135, 1978. Longhurst, P.A. and McNeill, J.H. Isoproterenol effects in isolated heart preparations from normal and hyperthyroid rats. Proc. West. Pharmacol. Soc. 22: 415-418, 1979. Longhurst, P.A. and McNeill, J.H. Effect of isoproterenol on phosphorylase activation i n the hyperthyroid rat heart. Can. J. Physiol. Pharmacol. 57: 567-573, 1979. Longhurst, P.A. and McNeill, J.H. The influence of frequency of stimulation and calcium concentrations on the contractile force of the rabbit papillary muscle. Proc. West. Pharmacol. Soc. 23: 21-24, 1980. Polanin, A., Longhurst, P.A. and McNeill, J.H. Comparison of histamine receptors i n l e f t and right rabbit a t r i a . Proc. West. Pharmacol. Soc. 23: 49-52, 1980. Longhurst, P.A., and McNeill, J.H. Species variation i n cardiac hista-mine receptors. In: New Cardiotonic Agents, edited by S. Shibata and L.E. Bailey. Medical Publishers and Importers, Igaku-Shoin Ltd. Tokyo. In Press. Longhurst, P.A., Verma, S.C, and McNeill, J.H. Rabbit cardiac histamine receptors and cyclic AMP. Life Sciences (submitted). ABSTRACTS McNeill, J.H., Longhurst, P.A. and Verma, S.C. The effect of isoproterenol on relaxation, cyclic AMP (cAMP) and phosphorylase (P) in isolated rat uterus. Fed. Proc. 34: 818, 1975. Barrett, A.J., Longhurst, P.A., Newton, K.A., and Humble, J.G. Treatment of a patient with aplastic anaemia with lithium carbonate. Exp. Herrat. 4: (Suppl.) 42, 1976. Barrett, A.J., Longhurst, P.A. and Humble, J.G. Investigation of the stim-ulation of colony formation produced by lithium. Exp. Hemat. 4: (Suppl.) 53, 1976. Barrett, A.J., Longhurst, P.A. and Humble, J.G. Attempts at removal of cross-reaction of ALG and ATG with human CFU's by absorption. Exp. Hemat. 4: (Suppl.) 98, 1976. Barrett, A.J., Longhurst, P.A. and Humble, J.G. Changes in peripheral blood CFU during splenic irradiation in a patient with CGL. Exp. Hemat. 4: (Suppl.) 151, 1976. Longhurst, P.A. and McNeill, J.H. Response of guinea pig (GP) papillary muscle to histamine differs from that of left atrium. Pharmacologist 21: 265, 1979. Longhurst, P.A., van Breemen, C, and McNeill, J.H. Measurement of 45-Ca uptake after treatment with isoproterenol and histamine in rabbit ventricular muscle. Fed. Proc. 40: 730, 1981. Longhurst, P. A., Verma, S.C, and McNeill, J.H. Contractile and cyclic AMP activating effects of histamine in rabbit heart. Pharmacologist 23:(In press) 1981. 

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