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Studies of the roles of calcium ions in anterior mesenteric portal vein Collins , Glenn Albert 1971

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STUDIES OF THE ROLES OF CALCIUM IONS IN ANTERIOR MESENTERIC PORTAL VEIN by GLENN ALBERT COLLINS B.Sc, University of British Columbia, 1968 A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in the Department of Pharmacology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April , 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the Uniyersity of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. o Department of The University of British Columbia Vancouver 8, Canada © O i . A preliminary report of part of this work was presented at the Canadian Federation of Biological Societies meetings held in Montreal during June, 1970. ABSTRACT There are two opposing schools of thought concerning the source of calcium ions for the i n i t i a t i o n and maintenance of contractions of smooth muscle. Bohr (1964) and Woodward et a l . (1970) believe that the calcium for the i n i t i a t i o n of contraction is released from bound intracellular stores, whereas Somlyo et a l . (1969) believe that most of the activator calcium comes from the extracellular f l u i d . It was f e l t that the determination of the source(s) and sink(s) of calcium ions in arteriolar smooth muscle would be required for an understanding of the control of peripheral blood pressure, and so experiments to obtain this information were carried out using the rabbit anterior mesenteric portal vein as a model of arteriolar smooth muscle. Spontaneous contractions of the vein stop within one minute after the addition of EGTA to the bath and can be returned by simply raising the extracellular calcium concentration. The addition of 1 mM MnC^, or raising the MgC^ concentration above 5 mM, or raising the CaC^ concen-tration above 10 mM a l l inhibit spontaneous activity; this inhibition can be rapidly reversed by the addition of appropriate amounts of EGTA. The removal of free extracellular calcium either by adding EGTA or placing the vein in calcium-free solution inhibits the responses to a l l agonists within five minutes. If the calcium concentration is reduced from 2.5 mM to 0.1 mM, the responses to agonists are greatly decreased. If one sets the response in normal Kreb's solution to each concentration of agonist equal to 100 per cent, then the relative reduction of responses in low calcium solution i s inversely proportional to both the potency and concentration of the agonist used. i i i . If, however, one produces contractions by adding calcium to tissues bathed in calcium-free solution containing noradrenaline, then the curves of the relative response versus calcium concentrations are independent of the concentration of noradrenaline. The addition of either EGTA or manganese to a tissue already contracted in response to any agonist produces a rapid relaxation to a decreased, but sustained tension. The degree of relaxation is proportional to the concentration of manganese or EGTA added. The addition of manganese is also able to inhibit the i n i t i a t i o n of responses to noradrenaline, KC1, serotonin, histamine and procaine. The inhibition by manganese of the responses to noradrenaline, KC1, and serotonin but not histamine and procaine can be reversed by increasing the extracellular calcium concentration. The addition of MnC^ or LaCl^ does not selectively inhibit a slow phase of the contraction to noradrenaline in the mesenteric portal vein as i t does in aorta (Van Breeman, 1969). The effect of adding MnCl^ is similar to the effect of decreasing extracellular calcium, in that the relative inhibition of response is inversely proportional to the potency and concentration of agonist used. The effect of altering pH is the same on contractions produced by each agonist tested; lowering the pH below 7.4 inhibits the responses, raising i t above pH 7.4 potentiates the responses. If the vein is placed in calcium-free solution containing EGTA for 10 minutes, the addition of CaC^ produces a contraction. The response to calcium is transient unless the f i n a l concentration of the added calcium is 5 mM or greater; at these higher calcium concentrations the iv. response is biphasic; an i n i t i a l transient response i s followed by a slow tonic response. The addition of stock solutions to produce f i n a l concentrations of I | | | | | 5 mM Mg or 0.5 mM Mn or 0.1 mM Ca in the bath after the EGTA treatment abolishes the transient responses to calcium but has l i t t l e I | effect on the tonic portion of a contraction produced by 10 mM Ca When these EGTA experiments are carried out in low sodium solution (17% of normal or less) the response to 2.5 mM Ca which is normally transient, becomes instead a sustained contraction. It i s concluded then that: 1. Calcium ions themselves are able to control the permeability of the membrane to calcium. 2. The i n i t i a t i o n of responses to a l l agonists probably involves the release of membrane-bound calcium and the influx of extracellular calcium. Differences in efficacy are probably due to differences in a b i l i t y to release the membrane-bound calcium. 3. A continued influx of calcium i s required to maintain a contraction produced by any agonist. 4. Manganese competes with calcium at a membrane site to inhibit the in i t i a t i o n of contraction, and to relax a tissue which is already contracted. 5. Responses to a l l agonists are potentiated at a pH greater than 7.4, and decreased below pH 7.4. 6. The relaxation process i n the mesenteric-portal vein seems to depend in some manner upon extracellular sodium ions. V. TABLE OF CONTENTS Page INTRODUCTION 1 I. Current concepts of striated muscle contraction 2 II. Current concepts of smooth muscle contraction 6 A. Contractile proteins of smooth muscle 6 B. Calcium and vascular smooth muscle 8 III. Anterior mesenteric portal vein as a model of arteriolar smooth muscle 10 STATEMENT OF THE PROBLEM 15 METHODS AND MATERIALS 19 I. MATERIALS 19 A. Tissue Preparation 19 B. Physiological Salt Solution 19 C. Drug Preparation 20 D. Apparatus 21 II. METHODS 24 A. Experiments in normal Kreb's solution 24 B. Experiments to study the interaction of extracellular calcium concentration and the responses to agonists 24 C. Experiments using Manganese chloride or Lanthanum chloride 25 D. Experiments involving chelation of bound calcium with EGTA 26 E. Experiments involving altered pH 27 v i . Page II. METHODS (Continued) F. Experiments involving zero sodium solution 27 RESULTS 28 I. Effects of altering the external calcium concentration 28 II. Effects of adding Manganese chloride to the Kreb's solution 35 III. Effects of EGTA treatment on the anterior mesenteric vein 40 DISCUSSION 43 I. Calcium and spontaneous activity 43 II. Calcium and the in i t i a t i o n of contractions to agonists 48 III. Calcium and sustained contractions 54 IV. Control of calcium influx 55 V. Effects of altering sodium concentration or pH 58 SUMMARY 60 BIBLIOGRAPHY 61 v i i . LIST OF FIGURES Figure 1 Organ bath apparatus. Figure 2 The effect of EGTA upon spontaneous activity of the anterior mesenteric portal vein. Figure 3 Effects of lowering the calcium concentration in the Kreb's solution upon the dose response curve to noradrenaline. Figure 4 Time course of contractions to 10 g/ml noradrenaline with various extracellular calcium concentrations. Figure 5 Time course of contractions to 10 ^ g/ml noradrenaline with various extracellular calcium concentrations. Figure 6 Time course of contractions to 80 mM KGl with various extracellular calcium concentrations. Figure 7 Effects of lowering extracellular calcium on the two phases of a contraction to 10 ^ g/ml noradrenaline. v i i i . Figure 8 E f f e c t of varying e x t r a c e l l u l a r calcium concentration on the phasic responses to various doses of noradrenaline. Figure 9 E f f e c t s of varying e x t r a c e l l u l a r calcium on noradrenaline contractions when the tissues were not pre-equilibrated with calcium. Figure 10 E f f e c t s of excess calcium on contractions to 10 g/ml noradrenaline. Figure 11 E f f e c t s of a l t e r i n g the e x t r a c e l l u l a r calcium concentration on the tonic portion of a maximum response to noradrenaline. Figure 12 Comparison of the e f f e c t s of lowering e x t r a c e l l u l a r calcium •~6 concentration on the responses to 10 g/ml noradrenaline and 40 mM KC1. Figure 13 Ef f e c t s of a l t e r i n g external calcium concentration on the responses to various doses of noradrenaline and KC1. Figure 14 E f f e c t s of manganese upon spontaneous a c t i v i t y . Figure 15 The e f f e c t s of manganese on spontaneous e l e c t r i c a l and mechanical a c t i v i t y of the mesenteric vein. Figure 16 I | The effect of 1 mM Mn on the dose-response curve to noradrenaline. Figure 17 I | Relative inhibitory effect of 1 mM Mn on the responses to various doses of noradrenaline. Figure 18 Relaxation of the response to 10 ^ g/ml noradrenaline by the addition of manganese (relaxation versus time). Figure 19 —6 Relaxation of responses to 10 g/ml noradrenaline by the addition of manganese (relaxation versus manganese concentration). Figure 20 Effect of increasing extracellular calcium on the inhibition of —6 responses to 10 g/ml noradrenaline by 1 mM MnCl^. Figure 21 Interaction between calcium and manganese and i t s effect on responses —6 to 10 g/ml noradrenaline. Figure 22 Effect of increasing extracellular calcium on the inhibition of responses to 40 mM KC1 by 1 mM MnC^. Figure 23 Interaction between calcium and manganese and i t s effect on responses to 40 mM KC1. X. Figure 24 ++ Effect of 1 mM Mn on the responses to 5-hydroxytryptamine and procaine. Figure 25 Responses to CaCl^ after calcium depletion with EGTA (tension versus time). Figure 26 Responses to CaC^ after EGTA treatment (tension versus calcium concentration). Figure 27 Effects of 1 mM MnC^ on the responses to CaC^ after calcium depletion of EGTA. Figure 28 Effects of 0.1 mM CaC^ on the responses to 10 mM CaC^ after depletion of calcium by EGTA. Figure 29 Effect of zero sodium solution on calcium responses after EGTA treatment. Figure 30 Effects of altering pH on the calcium responses after EGTA treatment. Figure 31 Effect of pH on responses to noradrenaline and KC1. x i . ACKNOWLEDGEMENTS The author thanks his supervisor, Dr. Morley C. Sutter, for his encouraging remarks and valuable criticism during the course of this work. The technical assistance of Mrs. Judy Teiser is greatly appreciated. The author also thanks Mrs. Judy Teiser and Miss Debbie Griswold for helping to prepare the figures, and Miss G i l l Kearney for typing and proof-reading this thesis. The author acknowledges his financial support by the Department of Pharmacology and the University of British Columbia. INTRODUCTION One of nature's most fascinating phenomena must surely be the ab i l i t y of some specialized striated muscles to undergo up to one thousand contraction-relaxation cycles in one second. When this is compared with the slow peristaltic movements produced by the contraction of most types of smooth muscle, i t is not so surprising that smooth muscle research has only recently begun to receive very much attention. This lack of attention is understandable but somewhat unjust, since although smooth muscle does not contract as rapidly as striated muscle, there i s certainly a greater diversity both in the functions of contraction of smooth muscle, and in the ways in which contractions can be initiated. The contractile force generated by smooth muscle can be-influenced by many endogenous compounds such as acetylcholine, adrenaline, histamine, serotonin, vasopressin, angiotensin, prostaglandins, etc. and also by electrical stimulation, whereas electrical excitation is the only known physiologically important means of in i t i a t i n g skeletal muscle contraction. The functions of smooth muscle contraction range from propelling food through the gastro-intestinal tract, to the control of blood pressure and shunting of blood, to forcing a reluctant foetus out into the world. Although smooth muscle research s t i l l lags behind, there is increasing evidence to show that differences between contractions of striated and smooth muscle may be due mainly to differences in the control of the excitation-contraction-relaxatibn cycle rather than differences in the molecular basis of contraction. With this in mind, 2. It may be useful to review the current concepts of striated muscle contraction and to compare this with the known facts of smooth muscle contraction. • I CURRENT CONCEPTS OF STRIATED MUSCLE CONTRACTION A striated muscle fiber or c e l l is composed of a large bundle of filaments or myofibrils running parallel to each other and perfectly aligned so that a cross section through the c e l l at any point would cut through the corresponding parts of each myofibril. It i s this highly ordered nature which leads to the formation of dark and light bands, and hence the term striated muscle. Using the electron microscope i t i s seen that the alternating dark and light bands are the result of an overlapping arrangement of thick and thin filaments. Carlsen, Knappeis and Buchtal (1961) showed that the degree of overlap decreased during passive stretch of the muscle and increased during contraction. This was the f i r s t evidence (using normal muscle) for the "sliding filament" hypothesis of H.E. Huxley and Hanson (1954) and A.F. Huxley (1956). This hypothesis held that co-ordinated interaction between the thick and thin filaments resulted in a sliding of the thin filaments along the thick filaments, resulting in shortening of the entire fiber. Hanson and H.E. Huxley (1955) provided evidence for the biochemical composition of. the thick and thin filaments. Using procedures which extracted myosin from the muscle they found that the thick filaments had disappeared while the thin filaments remained intact. 3. The energy required for contraction is provided by the hydrolysis of ATP. There is ATPase activity associated with myosin and i t has been postulated that the filaments slide together by a series of cross bridging interactions each of which involves the hydrolysis of one molecule of ATP. Although the sliding filament theory for contraction was quite well accepted, i t was not until 1960 that the processes involved in the i n i t i a t i o n and termination of the contraction began to be understood. Marsh (1951) found a muscle extract which was able to prevent the superprecipitation of actomyosin caused by ATP. Ebashi (1961) showed that this relaxing factor could accumulate calcium ions.in the presence of ATP and that various calcium chelating agents were also able to prevent actomyosin superprecipitation and to produce a relaxation of glycerinated muscle fibers. Electron microscopic studies of the Marsh relaxing factor indicated that i t was vesicular, and in fact contained triads which are character-i s t i c of the sarcoplasmic reticulum (Ebashi and Lipman, 1962). "It is rather ironic that recognition of the essential role of calcium ion in contraction has resulted mainly from the investigation into the mechanism of relaxation" (Ebashi and Endo, 1968). The discovery of the actual regulatory macromolecule with which calcium ions interact to i n i t i a t e contraction is credited to Ebashi. He noted that relatively crude preparations of actomyosin required both calcium ions and ATP to produce superprecipitation. If, however, the preparation of actin was further purified, the requirement for calcium ions was lost (Ebashi, 1963). Furthermore, while the ATPase of unpurified 4. actomyosin was inhibited by EDTA, the ATPase of purified "synthetic" actomyosin was unaffected. The contaminant of actin which endowed calcium sensitivity to the actomyosin was named native tropomyosin. Further purification and identification showed native tropomyosin to actually be composed of two proteins: (1) Troponin, a globular protein with molecular weight 80,000 which binds four calcium ions per molecule. (Ebashi, S., Ebashi, F., and Kodana, 1967) (2) Tropomyosin, a more filamentous protein with no a f f i n i t y for calcium, but which binds strongly to actin. (Ebashi and Endo, 1968) Endo et al (1966) used fluorescent antibodies to troponin and tropomyosin to show that these proteins were bound only to the thin filaments. It appears that troponin combines with tropomyosin to form native tropomyosin. The binding of native tropomyosin to actin produces an inhibitory effect on the interaction between myosin and actin and also inhibits actomyosin ATPase (Ebashi and Endo, 1968). The interaction of calcium with troponin reverses this inhibition. The source of the calcium ions for this de-repression process, and the means by which i t is released by an electrical impulse has been c l a r i f i e d by the combined techniques of histology, physiology and biochemistry. An internal membrane structure in striated muscle had been discovered by histologists prior to World War I, but was dismissed as an artifact by physiologists until they realized that such a system might allow the passage of an electrical signal into the interior of 5. the c e l l and therefore decrease the time required for activation of the entire fiber. This internal membrane structure consists of transverse tubules which are open to the extracellular space and wind around each of the individual f i b r i l s , and the sarcoplasmic reticulum which forms an elongated sac parallel to the muscle fibers. The sarcoplasmic reticulum is not directly connected to the extracellular f l u i d , but i t forms close junctions with the transverse tubules. (For a review of the morphology see Bianchi, 1968) Although i t has been known since the identification of the Marsh Factor that the sarcoplasmic reticulum can bind calcium, there is s t i l l very l i t t l e evidence that the calcium bound by the sarco-plasmic reticulum, or contained within i t , is the calcium which activates 45 the contractile process. Most of this evidence comes from Ca auto-radiography studies of Winegrad (1965, 1968, 1970). Using this technique he has shown that in a resting muscle most of the exchangeable calcium is stored at the Z line in the terminal cisternae (those parts of the sarcoplasmic reticulum which are in close apposition to the transverse tubules). During a sustained contraction there is a decrease in the calcium found at the Z line, and a large increase in calcium associated with the thin filaments (according to Ebashi Ts theory the calcium actually is bound to troponin). Stopping the electrical stimulation results in a relaxation of the muscle and a movement of calcium from the thin f i l a -ments to the longitudinal part of the sarcoplasmic reticulum. This is followed by a slower movement of calcium back into the terminal cisternae. 6. The present hypothesis concerning skeletal muscle contraction can be summarized as follows: an action potential coming down the motor nerve initiates (via acetylcholine) an action potential in the sarcolemma. The depolarizing current is then carried inward by the transverse tubules (Huxley and Taylor, 1958). This depolarization of the transverse tubules in some manner influences the terminal cisternae, such that their a f f i n i t y for calcium is reduced and free calcium is released. This calcium diffuses to i t s receptor sites on troponin which de-prepresses the actin-myosin interaction, allowing the ATPase to function, and the filaments to slide together. When the transverse tubules repolarize, the sarcoplasmic reticulum regains a higher a f f i n i t y for calcium. The calcium is bound or taken up by the longitudinal elements of the sarcoplasmic reticulum and diffuses back to the terminal cisternae. II CURRENT CONCEPTS OF SMOOTH MUSCLE CONTRACTION A. Contractile Proteins of Smooth Muscle Most researchers interested in the mechanisms of smooth muscle contractility have assumed that the molecular basis for smooth muscle contraction is similar to that in striated muscle, and have postulated that differences in the responses of the two muscle types are due to differences in the 'metabolism' of calcium. The evidence for this assumption is meagre and consists of comparisons of the properties of glycerinated fibers and isolated contractile proteins from the two types of muscle. 7. Fi l o , Ruegg and Bohr (1963) purified hog carotid artery acto-myosin and showed superprecipitation in 0.5 mM ATP under conditions in which skeletal muscle also undergoes superprecipitation. Their preparation also showed the same changes in viscosity due to ATP addition as those described by Mommaerts (1948) for an actomyosin preparation from striated muscle. Filo's actomyosin preparation also exhibited calcium and magnesium-activated ATPase activity. The total actomyosin content of the carotid artery is very low (about 10 mg/g tissue compared with 70 mg/g in skeletal muscle, F i l o , Bohr, Ruegg, 1965). The other major difference between actomyosin from smooth or striated muscle i s the condition necessary for i t s extraction. Smooth muscle actomyosin can be extracted by low ionic strength solution (0.05 M KC1) while extraction of actomyosin from skeletal muscle requires a higher ionic strength solution (0.6 M KC1). Ebashi et a l . (1966) showed that the amino acid composition of tropomyosin isolated from smooth muscle (chicken gizzard) or striated muscle were very similar. The smooth muscle however had a much higher tropomyosin: actomyosin ratio. F i l o et a l . (1965) compared the responses of glycerinated hog carotid arteries with glycerinated psoas muscles prepared under similar conditions. The responses of the two muscles to calcium ions were identical: threshold response at 10 ^ M calcium and maximum response —6 at 10 M calcium. The arterial preparation required at least 3 mM magnesium to be present in order to produce a response to added calcium. The psoas preparation required less than 1 mM magnesium to maintain sensitivity to calcium, and also produced a contraction when magnesium 8. was added in the absence of calcium. The greatest difference between smooth muscle and striated muscle, however, is the lack of an ordered structure of the contractile proteins in smooth muscle. Shoenberg (1966) using bovine arteries showed the presence of actomyosin in homogenates by viscosity and superprecipitation studies. Electron microscopy revealed only the presence of thin filaments, with characteristics similar to actin isolated from striated muscle. Shoenberg (1969) showed that myosin filaments could be prepared from chicken gizzard extracts in the presence of calcium and magnesium. She postulated that an increase in free calcium i n vivo could cause myosin to aggregate into filaments and allow the actin and myosin to interact as in skeletal muscle. She also points out that none of the earlier X-ray diffraction studies, which showed a lack of ordered structure in smooth muscle ( E l l i o t , 1964) had been done using isometrically contracting tissue. B. Calcium and Vascular Smooth Muscle Since i t is well known that calcium is required for vascular smooth muscle contraction, and since Ebashi has shown the presence of troponin in smooth muscle, i t is reasonable to assume until further evidence is available that the calcium ions bind to troponin and de-repress the actin-myosin interaction as in skeletal muscle. The problem for understanding contraction of smooth muscle then, is to determine the sources and sinks of this calcium. 9. The intracellular tubular network which i s so highly developed in striated muscle i s nearly absent in vascular smooth muscle. However, the surface area of smooth muscle plasma membrane i s greatly increased by numerous invaginations. These invaginations are usually described as spherical, about 450 X in diameter (Mathews and Gardner, 1966) and have been shown to communicate with extracellular f l u i d with the use of lanthanum chloride (Devine and Somlyo, 1970) or f e r r i t i n (Sutter, unpublished results). Recent electron microscope studies of rabbit anterior mesenteric vein using serial longitudinal sections (Sutter.unpublished results) have shown these vesicles to be about 400 X in diameter and up to 2000 X in, length. Almost a l l of the vesicles have an opening to the extra-cellular f l u i d v i s i ble in one or more of the sections. Rhodin (1962) calculated that these invaginations increase the membrane surface area by twenty five per cent. When one also considers the small diameter of smooth muscle c e l l s , resulting in a large surface area to volume ratio, and a small diffusion distance, i t seems possible that the smooth muscle plasma membrane with i t s invaginations may perform the same role as does the sarcoplasmic reticulum i n skeletal muscle. Thus i t can be postulated that electrical impulses or drugs could interact with the plasma membrane, releasing bound calcium into the cytoplasm and producing a contraction. The opposing theory of smooth muscle activation proposes that drugs or electrical impulses alter the permeability of the plasma membrane to calcium, and that the calcium ions which trigger the contraction come from the extra-cellular f l u i d . 10. Somlyo and Somlyo (1968b) distinguish between two types of vascular smooth muscle: phasic muscles in which action potentials are produced in response to drugs or electrical stimulation, (anterior mesenteric vein, mesenteric arterioles), and tonic muscles which respond with graded depolarizations (aorta, pulmonary artery). These would correspond to Bozler's (1948) single-unit (phasic) and multi-unit (tonic) classification of smooth muscle types. The differences between spike generating mesenteric veins and arteries and the gradedly responsive aorta include not only differences in elect r i c a l and contractile responses to drugs, but also differences in the effects of calcium depletion. The response of rabbit aorta to noradrenaline persists for an hour in calcium-free medium (Nash, Luchka and Jhamandas, 1966, Severson and Sutter, 1969), whereas rabbit anterior mesenteric veins continue to respond for only five to ten minutes after calcium withdrawal (Severson and Sutter, 1969). Somlyo and Somlyo (1968b) suggest that this difference is due to a greater calcium permeability in the aorta. I l l .ANTERIOR MESENTERIC PORTAL VEIN AS A MODEL  OF ARTERIOLAR SMOOTH MUSCLE Although a great amount of the research done on vascular smooth muscle has been done using aortic strips, i t is well established that small arteries and arterioles are the vessels which control peripheral vascular resistance and which would most l i k e l y be implicated in hypertension. 11. For this reason, the emphasis in this laboratory has been placed on the investigation of spike producing vascular smooth muscle, and in particular the anterior mesenteric portal vein. Steedman (1966) used micro-electrodes to measure spike potentials of arterioles In vivo in the rat mesentery. The average membrane potential was 39 millivolts with action potentials of up to 35 m i l l i -volts. She observed rhythmic slow wave depolarizations with spike potentials occuring at the peaks of the slow waves. Local admini-stration of adrenaline, noradrenaline or vasopressin,and stimulation of the splanchnic nerves a l l produced an increased amplitude of slow waves and increased frequency of action potentials. The anterior mesenteric portal vein exhibits essentially these same features both in vitro and in situ. Johansson and Ljung (1967) measured i n situ responses of rabbit and cat portal veins to splanchnic nerve stimulation and to adrenergic amines. The veins contracted spontaneously about 6-8 times per minute. Application of noradrenaline either locally or intravenously produced an increase in portal vein tension. Splanchnic nerve stimulation also produced an increase in tension, an< effect which could be inhibited by pretreatment with guanethidine (which should deplete catecholamine stores from nerve endings). Guanethidine had no effect on either spontaneous contractions or tension increases produced by noradrenaline; thus the contractions must be initiated by pacemaker cells in the smooth muscle layers. In vitro studies on the anterior mesenteric vein were f i r s t carried out by Cuthbert and Sutter (1964). They showed spontaneous contractions accompanied in one case by spike-like action potentials. 12. Sutter (1965) did a more complete study of the effects of drugs on this preparation. Spontaneous activity was invariably present in both longitudinal and spiral preparations of the anterior mesenteric vein. Noradrenaline and adrenaline both produced strong contractions —8 —5 with a threshold of 10 g/ml and maximum response at 10 J g/ml. These contractions were blocked by phentolamine or dihydroergotamine. Isopropylnoradrenaline, a predominantly beta adrenergic agonist, inhibited spontaneous contractions at low doses (10 ^ g/ml) and produced an i n i t i a l inhibition of spontaneous activity followed by a contraction at higher doses (10 ^ g/ml). The isolated anterior mesenteric veins contracted in response to the addition of acetylcholine, serotonin, or angiotensin. The acetylcholine response was inhibited by atropine but the response to serotonin could not be blocked by either methysergide or morphine. Following these i n i t i a l observations which indicated that the isolated anterior mesenteric portal vein would provide a model system for studying an arteriolar-like tissue, several investigators have tried to correlate changes in contractile activity produced by drugs or altered ionic composition with changes in membrane potential. The spontaneous activity of the portal vein in normal Kreb's solution is characterized by rhythmical phasic contractions, each of which is slightly preceded by a burst of action potentials (Cuthbert and Sutter, 1965, Axelsson et a l . , 1967, Holman et a l . , 1968). If the calcium concentration in the bath is not altered, there is a good correlation between spontaneous electrical and mechanical activities under most experimental conditions. 13. Holman et a l . (1968) showed that spontaneous activity ceases below 28° C and that raising the temperature in the range of 28°-38° C resulted i n parallel increases in the frequency of action potentials and associated contractions. Axelsson et a l . (1967) showed an i n i t i a l increase in both burst frequency and contraction in potassium-free solution, followed by a co-ordinated decrease in both. Substances which inhibit spontaneous contractions of the vein also inhibit action potentials. This effect was shown by Cuthbert and Sutter (1965) using theophylline and isopropylnoradrenaline, and by Rhodes and Sutter (1969) using diazoxide. It appears, therefore, to be impossible to dissociate spontaneous electrical activity and tension changes i f the calcium concentration in the bathing medium is not altered. Axelsson et a l . (1967) showed, however, that upon increasing the potassium concentration in calcium-free medium, spontaneous action potentials could be recorded without any accompanying changes in tension. The correlation between changes in electrical activity and tension due to the addition of drugs is not so good, however. Cuthbert and Sutter (1965) found that application of noradrenaline to vein strips produced an i n i t i a l increase in spike activity correlated with an increase in tension. However, after a few minutes, the electrical activity returned to normal while the tension remained elevated. Even more striking i s the fact that noradrenaline and other agonists can s t i l l produce a contraction when the vein is in a depolarized state, produced either by a potassium rich solution (Edman and Schild, 1962) or by ouabain (Mathews and Sutter, 1967). Somlyo and Somlyo (1968a) 14. have coined the term pharmaco-mechanical coupling for contractions produced by drugs, which do not require the additional step of membrane depolarization. Their theory of pharmaco-mechanical coupling may be summarized as follows: (i) The interaction of drug with membrane produces an increase in calcium permeability, perhaps due to the release of membrane bound calcium. ( i i ) The increased influx of calcium w i l l produce both a depolarization and contraction under normal conditions, but i f the c e l l is already depolarized the increased influx of calcium s t i l l produces a contraction. Thus Somlyo and Somlyo believe that excitatory drugs produce parallel changes in electrical and mechanical activities in smooth muscle, rather than the series depolarization-contraction linkage found in skeletal and cardiac muscles. This interesting hypothesis w i l l be delved into more deeply in the Discussion. 15. Statement of the Problem In order to understand the molecular basis for hypertension, i t w i l l be necessary to discover the means by which arteriolar resistance is controlled, i.e. how is the tension produced by arteriolar smooth muscle regulated? We are using the isolated anterior mesenteric portal vein as a model system since in vivo it;has many physiological and pharmacological properties similar to arterioles (see Introduction, Anterior Mesenteric Portal Vein . . .), and in this regard can provide far more relevant data than can the thoracic aorta. Experi-ments were carried out to try to determine the source(s) of calcium ions required for contractions of the anterior mesenteric portal vein to various agonists, and the means by which membrane permeability to calcium i s controlled. The experiments were of four main designs: 1. The effects of varying the extracellular calcium on both the time course and magnitude of contractions produced by various agonists. Although i t is well established that no contractions can be eli c i t e d in calcium-free solution (in the anterior mesenteric vein), there i s l i t t l e quantitative information relating contractile tension to extracellular calcium concentration. The relative importance of extracellular calcium for the i n i t i a t i o n of contractions produced by various agonists is also not known. Contractions of the mesenteric portal vein produced by most agonists are not phasic, and indeed may last for more than an hour. 16. Does the calcium which initiates the contraction remain in the cyto-plasm to allow the maintenance of contraction or is there a continuous turnover of calcium ions from source to sink? The effect of lowering the extracellular calcium on the sustained phase of the contraction was also tested. If there is no turnover of calcium ions during this phase then removing the extracellular calcium should have no effect. 2. The effects of adding manganese chloride (or lanthanum chloride) to the bathing medium on the spontaneous activity and the responses to agonists. If indeed the influx of calcium ions is necessary for the i n i t i a t i o n and/or maintenance of contraction of the vein, then the addition of an inhibitor of calcium influx should inhibit the i n i t i a t i o n of the contractile response and/or relax a vein which is already contracting. Sabatini-Smith and Holland (1969) showed that 0.1 mM manganese 45 ++ reduced the uptake of Ca by isolated rabbit atria to 25% of control. Hagiwara and Nakijimo (1966) and Bulbring and Tomita (1968) showed that manganese could block action potentials in barnacle muscle and guinea pig taenia c o l i respectively. Calcium ions are believed to be the chief current carrying species for the action potentials in both these muscles. Van Breemen (1969) showed that lanthanum chloride blocked the 45 ++ movement of Ca across an a r t i f i c i a l phospholipid-cholesterol membrane and also across the squid giant axon (Van Breemen and de Weer, 1970). 17. Thus the use of manganese or lanthanum as an inhibitor of calcium influx might also help to show the importance of extracellular calcium for the i n i t i a t i o n and/or maintenance of contractions. 3. The effects of removing some of the bound calcium using EGTA. Frankenhaeuser and Hodgkin (1957) showed that increasing calcium concentration decreased sodium permeability of the squid giant axon and shifted the curve of rate of rise of action potential versus membrane potential such that a greater depolarization was required to produce the same rate of rise. They referred to this as a stabilizing effect of calcium. Many people have suggested a similar role for calcium in smooth muscle, and Somlyo and Somlyo (1968) have even suggested (without evidence) that calcium may control i t s own permeability. It may be possible to show the existence of this "stabilizer calcium" by treating the tissues with EGTA; the permeability characteristics of the membrane should be greatly changed i f indeed stabilizer calcium has been removed. This change in membrane perme-ab i l i t y could be shown by the addition of various divalent cations. If the addition of calcium for example, produced a different effect after EGTA treatment then i t would without EGTA treatment, then i t is probable that the removal of bound calcium with EGTA has directly or indirectly "destabilized" the membrane. 4. The effects of altering pH and sodium concentration. Although i t has been shown that lowering the extracellular sodium concentration from 143 mM to 25 mM has very l i t t l e effect 18. on the amount of tension produced by noradrenaline or KC1 (Biamino and Johansson, 1970; Sutter, unpublished results), i t was thought that lowering the sodium concentration might have an effect on the responses of calcium depleted tissues (see #3 above). An inter-action between sodium and calcium fluxes was f i r s t reported by Baker et a l . (1969) with squid, giant axon and later by Glitsch et a l . (1970) with guinea-pig atria. Both the influx and efflux of calcium appeared to be linked with a movement of sodium ions in the opposite direction. If the influx of calcium in the vein is indeed carrier-mediated (as postulated by Glitsch for the guinea-pig atria), then decreasing the pH should decrease the a f f i n i t y of the carrier for calcium and hence inhibit contractile responses i f calcium influx i s necessary for contractions. The experiments with altered pH were done s t r i c t l y as a preliminary study since i t is impossible from this simple approach to determine the site(s) which is (are) affected 'by altering the pH. 19. METHODS AND MATERIALS I. MATERIALS A. . Tissue Preparation New Zealand white rabbits of either sex and weighing from 2.0 to 3.5.kg. were k i l l e d by a blow to the back of the neck. The abdominal wall was cut open, and after removing as much connective tissue as possible, the anterior mesenteric portal vein was removed from the point of entry into the l i v e r , to the branching of the anterior mesenteric vein. The vein was placed in a petri dish containing modified Kreb's solution (see section I. B) and any remaining connective tissue was dissected free. For organ bath experiments the vein was bisected longitudinally, but for sucrose gap experiments the vein was simply cut longitudinally to form a f l a t sheet. B. Physiological Salt Solutions Since many of the. experiments involved the addition of MnCl 2 or extra CaCl 2 which would precipitate as insoluble phosphates in ordinary Kreb's solution, the following solution lacking phosphate was routinely used. (1) Modified Kreb's Solutions NaCl, 118 mM; KC1, 4.7 mM; CaCl 2, 2.6 mM; MgCl 2 > 1.1 mM; NaHC03, 25 mM; glucose, 5.6 mM. The solution was bubbled with 95% 02~5% C0 2 and adjusted to pH 7.4 with HC1. 20. (2) Bicarbonate-free Kreb's Solution (This solution was used in experiments with LaCl^ to prevent I ^ C C O ^ ^ precipitation). Same composition as modified Kreb's solution except that the 25 mM NaHCO^ was replaced with 25 mM Tris(hydroxymethyl)aminomethane. The solution was bubbled with 100% 0^  and adjusted to pH 7.4 with HC1. (3) 16% Sodium Solution Same composition as modified Kreb's except the 118 mM NaCl was replaced by 118 mM Tris-chloride. The solution was bubbled with 95% 02~5% C0 2 and adjusted to pH 7.4 with HC1. (4) Sodium-free Kreb's solution Same composition as modified Kreb's except the 118 mM NaCl and 25 mM NaHCO^ were replaced with 143 mM Tris-chloride. The solution was bubbled with 100% 0 2 and adjusted to pH 7.4 with HC1. The physiological salt solutions were made up in large quantities without glucose and then the glucose (1.0 g/1) was added on the day of the experiment. C. Drug Preparations Stock solutions of drugs were prepared using d i s t i l l e d water or by using procedures outlined in the Merck Index of Chemicals and Drugs. 21. Drugs were obtained as follows: 1. L-Noradrenaline-D-bitartrate monohydrate, Mann Research Laboratories. 2. Phentolamine, Ciba. 3. Ouabain, Mann Research Laboratories. 4 . Serotonin creatinine sulphate monohydrate, Mann Research Laboratories. 5. Procaine Hydrochloride, K and K Laboratories. 6. Tetrodotoxin, Sankyo. 7. EGTA (ether glycol bis-amino tetraacetic acid), Geigy, Sigma. 8. EDTA (ethylene diamine tetraacetic acid), Fisher Scientific. A l l drug concentrations are expressed in terms of the f i n a l concentration of free base. D. Apparatus 1. Organ Bath The apparatus is shown in Figure." 1. 'it is a standard 18 ml muscle bath obtained from Glass Appliances Ltd. ,- Aberdeen Scotland. The warming c o i l also has an 18 ml capacity so that a complete change of bathing f l u i d can be effected without altering temperature. The temperature of the bathing f l u i d was maintained at 37° C by a continual circulation of warm water through the water jacket from a thermo-static a l l y controlled water pump. Aeration was accomplished by passing a small diameter polyethylene tube through the bottom drain tube and into the bottom of the central compartment. The end of the tubing was crushed with pliers and then pierced with a needle to produce a fine flow of bubbles over the tissue. FIGURE 1 Organ Bath Apparatus A Grass Model 7 Polygraph B Grass Model FT 03C Force Transducer C Water reservoir D Water pump E Heater F Pre-warming c o i l G Water jacket H Stopcocks J Aeration apparatus K Support rod for tissues L Bathing solution 22. The tissue was anchored at the bottom by a loop of surgical thread passed through the tissue and over a glass hook. The other end was connected to a Grass FT03C force displacement transducer which in turn was connected to.a Grass Model 7 Polygraph. The isometric contractions produced by veins in four separate organ baths were measured simultaneously by this method. Tension on the tissue could be altered by raising or lowering the force transducer using a rack and pinion. Resting tension was maintained at 500 mg. throughout the experiment by this method. Drugs were added to the bath in volumes of 0.18 ml or 0.36 ml using a 1 ml tuberculin syringe with a #22 needle. Drugs were usually washed out by emptying the bath from the lower drain tube and r e f i l l i n g ; this technique however produced an artifact in the tension record which could be eliminated by using an overflow washing technique. The tissue remained viable for at least eight hours when kept at 37° C in well oxygenated modified Kreb's solution. 2. Sucrose Gap Apparatus This apparatus consisted of a horizontal three-chambered bath made of lucite plastic, and allowed measurement of electrical activity and tension simultaneously. The tissue was attached by a loop of thread to a hook in the right chamber, then passed through the center chamber and then attached via a lucite plastic lever in the le f t chamber to a Grass FT03C force displacement transducer. The diffusion of ions between chambers was prevented by using rubber membranes placed on either side of the central chamber. 300 mM sucrose solution 23. (previously demitieralized with Rexyn 1-300 (Fisher Scientific) flowed slowly through the center compartment which kept the outer baths elec t r i c a l l y isolated from each other. The portion of tissue in the right compartment was depolarized by crushing with forceps or by bathing i t in 100 mM K^SO^ solution. The silv e r - s i l v e r chloride recording electrodes were made from 0.006 inch diameter silver wire, encased except for an 0.5 cm tip within a glass tube sealed with Pyseal cement (Fisher Scientific). The exposed tip was chlorided electrolytically using 100 mM KC1. The electrodes were fixed in place, one in each of the outer compartments, and were adjusted so that the electrode tips made direct contact with the tissue. The electrodes and the force transducer were connected to a Grass Model 7 Polygraph. If the ionic composition of the l e f t chamber was altered, a change in electrical potential between the electrodes could be measured which was observed even in the absence of livi n g tissue. This change in electrical potential is due to an alteration of the chemical activity of the ions involved in the silv e r - s i l v e r chloride electrode. To avoid this non-specific change in potential, the apparatus was designed so that any experimental compounds (MnCl2 for example) could be added to both outer compartments simultaneously. The tissue was maintained at 37° C by water circulating through tubing wrapped around the bath, or in a later design through channels which were an integral part of the apparatus. Both outer compartments were bubbled with 95% 02~5% C0 2 which also served to promote mixing of solutions. 24. II METHODS A. Experiments in Normal Kreb's Solution Non cumulative dose response curves were obtained from nor-adrenaline (10 ^  to 10 ^  g/ml), serotonin (10 ^  to 10 ^  g/ml), procaine (10~5 to 5 x 10~3 g/ml) and for KC1 (20-80 mM fi n a l concentration). The usual procedure involved starting with the threshold dose, allowing the tension to reach maximum, then washing and allowing at least five minutes after return to basal tension before challenging with the next higher dose. Occasionally the order of dosing was randomized to show that the response depended only on the concentration of drug, not on the order in which the dose-response curve was obtained. B. Experiments to study the interaction of extracellular calcium concentration and the responses to agonists. Similar non-cumulative dose response curves were obtained using Kreb's solution with the calcium concentration varied from less than -5 -2 10 M to 2 x 10 M. The lowest calcium concentration was achieved -4 by adding 10 M EGTA (ether glycol bis-amino tetraacetic acid) to the Kreb's solution (zero calcium Kreb's). The tissues were e q u i l i -brated for 30 minutes before testing, and then challenged with two test doses of agonist before the actual dose-response curve was obtained. A second type of experiment was done to study the importance of extracellular calcium. Fi r s t the tissues were soaked in calcium-free 25. solution un t i l no contractions could be el i c i t e d , then one of the following procedures was followed: (1) Calcium chloride solution was added to each bath (final concentration 0.1 mM to 10.0 mM) and the tissues were allowed to equilibrate for 5 minutes. The excitatory agent to be studied was then added and the tension produced was recorded. (2) The excitatory agent, was added to the tissues while they were s t i l l in calcium-free solution. Calcium chloride solution was then added to in i t i a t e a contraction. These two procedures were followed with noradrenaline and KC1 using several calcium concentrations to determine i f the order of addition had any effect on the tension produced. C. Experiments using Manganese chloride or Lanthanum chloride The protocol for obtaining dose-response curves in the presence of manganese or lanthanum was as follows: I | [ j | (1) control response without inhibitor (Mn or La ) (2) response to same dose of agonist after equilibrating for three minutes in the presence of manganese or lanthanum (3) second control response to show reversibility This same procedure was followed with each concentration of agonist. The experiments using Lanthanum chloride required bicarbonate free Kreb's solution to prevent Lanthanum carbonate precipitation. A second type of experiment using Manganese involved producing a contraction to an agonist in normal Kreb's solution, then adding 26. manganese (0.5-2.0 mM f i n a l concentration) and measuring the rate of relaxation. Experiments using manganese were routinely -4 performed in the presence of 10 M ascorbic acid to prevent manganese catalyzed oxidation of noradrenaline to noradrenachrome. D. Experiments involving chelation of bound calcium with EGTA The tissues were bathed in zero calcium Kreb's solution containing 1.0 mM EGTA for 10 minutes. After 10 minutes the tissues were washed twice with zero calcium Kreb's solution (containing 0.1 mM EGTA). The effects of adding various divalent cations to the calcium depleted tissues were then tested. Following this, the cycle of bathing in 1.0 mM EGTA and washing with zero calcium Kreb's solution was repeated before testing another cation solution. The effect of varying the concentration of EGTA in the solutions was tested and i t was found that raising the concentration from 1.0 to 5.0 mM had no effect on the responses to cations. It was also found that varying the EGTA in the washing solution from 0 to 1.0 mM had no effect on the responses. For this reason the standard procedure as given above used 1.0 mM EGTA for 10 minutes followed by two washing with zero calcium solution containing 0.1 mM EGTA. In the experiments which tested the inhibitory effect of manganese or lanthanum, these inhibitors were added after the washings with zero calcium solution; the tissues were allowed to equilibrate with the inhibitor for three minutes before adding the test solution of calcium or strontium. E. Experiments involving altered pH The tissues were equilibrated in the appropriate solution for 10 minutes before adding the agonist. The pH of the bathing solution was checked (if pH was the variable) by placing a micro pH electrode directly into the bath after the agonist had been added. Each agonist was tested several times with similar bathing solution u n t i l a consistent contraction was produced. F. Experiments involving zero sodium solution The tissues were equilibrated for two hours in zero sodium Kreb's before responses to agonists were tested. 28. RESULTS I. EFFECTS OF ALTERING THE EXTERNAL CALCIUM CONCENTRATION The maintenance of spontaneous activity in the rabbit portal vein is very dependent upon extracellular calcium. Within five minutes of exposure to calcium-free solution no further spontaneous tension changes occur, and this correlates with a marked decrease in electrical activity of the membrane as measured by the sucrose gap method. (Similar results were found by Cuthbert and Sutter, 1965.) Spontaneous activity can be halted even more rapidly by the addition of the calcium chelating agent EGTA to the bathing medium. Figure 2 shows the result of adding sufficient EGTA to chelate a l l of the extracellular calcium. Spontaneous activity ceased within two minutes, but could be restored to normal by the addition of excess calcium (2(A)). Figure 2(B) shows that the addition of excess MgCl 2 did not restore spontaneous activity, and therefore i t i s the specific chelation of calcium ions by the EGTA which has stopped spontaneous activity. The addition of SrCl 2 (2(C)) produced an increase in basal tension with superimposed spontaneous activity. Results similar to those shown in Figure 2 can be obtained by exposing the tissues to calcium-free solution without EGTA for 5 minutes and then adding the various divalent cations. However, under these conditions the return of spontaneous activity after readdition of calcium takes 10-15 minutes, whereas the addition of strontium causes an immediate return of activity similar to that B 3mM EGTA 4 m M MgCI^ I 1 0 0 m g 3 m M EGTA 1 min. FIGURE 2 The effect of EGTA upon spontaneous a c t i v i t y of the anterior mesenteric portal vein. Concentration of free calcium i n the solutions before addition of EGTA was 2.5 mM A. Readdition of CaCl 2 reversed the effect of EGTA and allowed return of normal spontaneous a c t i v i t y . B. The addition of MgCl 2 had no effect. C. Addition of SrCl- produced a large contraction. 29. shown in Figure 2(C). In order to try to determine the importance of bound calcium for drug induced contraction, the effects of lowering the external calcium concentration on responses to agonists were studied. After fifteen minutes in calcium-free solution none of the excitatory agents tested (noradrenaline, serotonin, histamine, procaine, potassium chloride) were able to produce a contraction even when given in what would normally be supramaximal doses. The effect of lowering the calcium concentration of the bathing medium upon the dose response curves to noradrenaline is shown in Figure 3. The response to noradrenaline i s limited by the extracellular calcium concentration up to 2.5 mM Ca .' If the calcium concentration is raised to 10 mM or greater (not illustrated) then the responses to noradrenaline are actually decreased relative to the response in 2.5 mM calcium. The dose response curves to a l l agonists tested were affected in the same general manner by alteration of the external calcium concentration. It appears therefore that the smooth muscle cells of the portal vein are very dependent on extracellular or rapidly exchange-able bound calcium for the i n i t i a t i o n of a contraction elici t e d in any manner. Figure 4 shows the effect of lowering external calcium on the time course of the responses to 10 "* g/ml noradrenaline. In a l l cases the tissues were bathed in calcium free medium unt i l no response to noradrenaline could be obtained. The dashed lines represent the responses to 10 g/ml noradrenaline obtained after 5 minutes 2.5 mM Co + + [ N O R A D R E N A L I N E D G./lVlL. FIGURE 3 Effects of lowering the calcium concentration in the Kreb's solution upon the dose response curve to noradrenaline. The response of each vein strip to 10 g/ml noradrenaline i n 2.5 mM I | Ca was set equal to 100 per cent. The strips were equilibrated for 30 minutes i n each solution and then challenged with 10 ^  g/ml noradrenaline u n t i l a constant contraction was produced before the dose response curve was obtained. 0.1 mM CoCI 2 125 mg 5min 0.2 mM CaCI2 FIGURE 4 Time course of contractions to 10 g/ml noradrenaline with various extracellular calcium concentrations. Each vein strip was washed in calcium-free medium until no contraction was obtained to noradrenaline. The dotted traces represent responses to noradrenaline after 5 minutes equilibration with Kreb's solution containing the calcium concentration indicated. The solid traces represent the responses obtained by f i r s t adding noradrenaline to calcium-free solution and then suddenly increasing the calcium concen-tration by adding concentrated CaCl_ solution to the bath via a syringe. 30. equilibration in bathing medium containing calcium concentrations as indicated. The solid lines represent the responses obtained by adding calcium to tissues bathed in calcium-free solution with 10 g/ml noradrenaline already in the bathing medium. The only difference between these two procedures is the sequence in which the. calcium and noradrenaline were added. The greatest effect of calcium, pre-incubation was seen at very low calcium concentrations (0.1 mM and 0.2 mM). At these concentrations of calcium, addition of 10 ^ g/ml noradrenaline produced a biphasic response; a phasic response of short duration, followed by a tonic contraction of decreased magnitude. Without pre-incubation with calcium, the phasic response was totally eliminated at these low calcium concentrations. Increasing the external calcium concentration had a greater effect on the tonic response than on the phasic response and at concentrations greater than 1 mM Ca , the tonic response was larger than the i n i t i a l response. With increased calcium concentration, the differential effect of pre-incubation with calcium also disappeared. Figure 5 shows the effects of lowering calcium upon the responses to 10 ^ g/ml noradrenaline. The tissues had been washed with calcium-—6 free solution u n t i l no response was produced by 10 g/ml noradrenaline. The dashed lines again represent the responses to noradrenaline after 5 minutes pre-incubation with the calcium concentration as indicated, and the solid lines represent the responses obtained by adding the noradrenaline before the calcium. With this low dose of noradrenaline there was no definite two phase response, and there was l i t t l e difference 0.1 mM 0.2 m M 0.5 m M 1.0 m M 50 mg 5 mi 500 mg [ r 2.5 m M 500 mg [ f 5.0 m M FIGURE 5 Time course of contractions to 10 ^ g/ml noradrenaline with various extracellular calcium concentrations. Experimental details as i n Figure 4. 50m 9 I 1 5 min. CCaCI2J O.ImM 0.2mM 0.5 mM l.OmM 2.5mM FIGURE 6 Time course of contractions to 80 mM KC1 with various extracellular calcium concentrations. Experimental details as i n Figure 4. 31. in the responses whether the tissues are pre-incubated with calcium or not. The results from similar experiments using 80 mM KC1 as the agent producing the contractile response are shown in Figure 6. Although the responses to 80 mM KC1 cannot be easily separated into phasic and tonic components, the 5 minute pre-incubation with calcium before addition of KC1 resulted in a greater rate of tension pro-duction, (even though the same fin a l tension could be produced i f the KC1 solution is added before the calcium). Very similar results were obtained using a lower concentration of KC1 (40 mM) although of course the tension produced was much smaller. Figures 7(A) and 7(B) show the effects of altering extra-cellular calcium on both the phasic and tonic responses to 10 g/ml noradrenaline. Each strip was placed in calcium-free medium unti l no response to noradrenaline could be measured, and then incubated for five minutes with the indicated calcium concentration before testing with noradrenaline. Each point represents the average response, and the bars represent the standard deviations of eight j | .vein strips.. The.response of each vein in 1.0 mM Ca was considered to be 100 per cent for the purpose of these graphs. The phasic response (7(A)) is proportional to the logarithm of the calcium concentration (up to 1.0 mM Ca ) whereas the tonic response (7(B)) is linearly proportional to calcium concentration within this same range. The tonic response becomes equal to magnitude to the phasic response when the extracellular calcium concentration is between 0.8 mM and 1.0 mM. When the calcium concentration i s raised further * 401 I 0 S Mi 4 I a B 0 X M l I I I .1 .» a I I I .« J . • C c«ci) • MM FIGURE 7 Effects of lowering extracellular calcium on the two phases of a contraction to 10 g/ml noradrenaline. The strips were washed i n calcium-free solution and then equilibrated with a given calcium concentration for 5 minutes before testing with noradrenaline. A Effect of low calcium on the phasic contraction B Effect on the tonic portion of the biphasic contraction (tension remaining 15 minutes after addition of noradrenaline) The v e r t i c a l bars indicate the standard errors of the mean (8 vein s t r i p s ) . 32. i t i s d i f f i c u l t to separate the response to noradrenaline into two distinct phases. The effects of lowering external calcium on the phasic responses to various doses of noradrenaline are shown in Figure 8. The tissues were equilibrated for 5 minutes in each of the solutions containing a particular amount of calcium before testing with noradrenaline. The maximum response obtained at each dose of noradrenaline was set equal to 100 per cent, therefore the curves represent relative effects of lowering external calcium on the response to each dose of noradrenaline. When the external calcium concentration was less than 0.5 mM, the responses to low doses of noradrenaline were greatly reduced, whereas the responses to high doses of noradrenaline were less affected. In the experiments shown in Figure 8, the addition of nor-adrenaline initiated the responses. However, i f the contractile response produced by noradrenaline is due simply to increased permeability to calcium, then i t should not matter in which order the noradrenaline and calcium are added, i.e. whether the addition of noradrenaline or calcium actually initiates the contraction. Figure 9 shows however that i f noradrenaline was added to calcium-free solution and then calcium was added later, the differential effect of decreasing extracellular calcium on the responses to various doses of noradrenaline (as seen in Figure 8) did not occur (under these conditions). The curves of response versus calcium concentration overlap for doses of noradrenaline from 10 ^  g/ml to 10 g/ml. Thus the order of calcium and FIGURE 8 Effect of varying extracellular calcium concentration on the phasic responses to various doses of noradrenaline. Each strip was washed in calcium-free solution u n t i l no further contraction could be e l i c i t e d by noradrenaline. CaC^ solution was added to the bath to give a f i n a l concentration of Ca"*-*" as indicated. After 5 minutes equilibration the strip was tested with one of the doses of noradrenaline. The strip was then washed i n calcium-free medium and re-equilibrated with calcium before testing with the next dose of noradrenaline. Each point represents the mean of 12 vein strips (6 animals). The vert i c a l bars represent the standard errors of the means. FIGURE 9 E f f e c t s of varying e x t r a c e l l u l a r calcium on noradrenaline contractions when the ti s s u e s were not p r e - e q u i l i b r a t e d with calcium. The t i s s u e s were washed i n calcium-free s o l u t i o n u n t i l no cont r a c t i o n could be e l i c i t e d by noradrenaline. Noradrenaline was then added. The contractions were produced by the a d d i t i o n of C a C ^ s o l u t i o n to achieve a f i n a l calcium concentration as i n d i c a t e d . 3 3 . noradrenaline a d d i t i o n i s important, and therefore a simple increase i n calcium permeability cannot be t o t a l responsible for the i n i t i a t i o n of a contraction produced by noradrenaline. A d d i t i o n a l e f f e c t s (upon noradrenaline responses) of a l t e r i n g the concentration of calcium i n the bathing medium are shown i n Figures 10 and 11. Figure 10(A) shows the t y p i c a l response to —5 ++ 10 g/ml noradrenaline i n 2.5 mM Ca . This concentration of noradrenaline produced a maximum response. When the calcium concentration was r a i s e d to 12.5 mM (10(B)), the response to nor-adrenaline was reduced a small but consistent amount. If however the calcium concentration was raised a f t e r the addi t i o n of noradrenaline, a supra-maximal response was obtained. These r e s u l t s are consistent with the e f f e c t s of raised calcium concentration upon spontaneous a c t i v i t y , i . e . when the calcium concentration was greater than 7.5 mM, spontaneous a c t i v i t y was abolished. This e f f e c t could be reversed by chelating some of the calcium with EGTA, I | or by returning to Kreb's s o l u t i o n containing 2.5 mM Ca The supramaximal response obtained by r a i s i n g the e x t r a c e l l u l a r calcium concentration during the tonic portion of a noradrenaline contraction could also be reversed by simply chelating the extra calcium with EGTA (Figure 11). Although the responses to a l l agonists are r a p i d l y abolished i n calcium-free s o l u t i o n , one might be able to le a r n something of the u t i l i z a t i o n of d i f f e r e n t calcium pools by examining the shapes of response versus calcium concentration curves f o r various agonists. —6 Figure 12 compares the r e l a t i v e responses to 10 g/ml noradrenaline 10 g/ml N A 3000 mg B lOmM 10 g/ml f 1 9 0 0 mg f 3500mg 3000mg lOn 10 g/ml FIGURE 10 Effects of excess calcium on contractions to 10 g/ml noradrenaline. A Contraction produced by 10 g/ml noradrenaline i n normal Kreb's solution. (2.5 mM Ca ) B Slight depressant effect of raising the calcium concentration to 12.5 mM before adding noradrenaline. C Supramaximal response produced by increasing the calcium concentration after the strip has reached peak tension i n normal Kreb's solution. 5 min FIGURE 11 Effects of altering the extracellular calcium concentration on the tonic portion of a maximum response to noradrenaline. 100-a CCoCI]] mM FIGURE 12 Comparison of the effects of lowering extracellular calcium concentration on the responses to 10 ^  g/ml noradrenaline and 40 mM KC1. The maximum response produced by each agonist i s set equal to 100 per cent. 34. and 40 mM KC1 with varying e x t r a c e l l u l a r calcium. The maximum response obtained with each agonist was set equal to 100 per cent. Thus when the e x t r a c e l l u l a r calcium concentration was below 0.5 mM, the response to 40 mM KC1 i s s i g n i f i c a n t l y reduced r e l a t i v e —6 to the response to 10 g/ml noradrenaline. However, these p a r t i c u l a r concentrations of noradrenaline and KC1 do not produce equal contractions i n normal Kreb's s o l u t i o n (2.5 mM Ca ). For t h i s reason s i m i l a r experiments were c a r r i e d out using various concentrations of both excitatory agents (Figure 13). This f i g u r e shows that the d i f f e r e n t i a l e f f e c t of lowering calcium on the responses to 10 ^ g/ml noradrenaline and 40 mM KC1 was rel a t e d to the r e l a t i v e potencies of the drug concentrations used rather than differences i n the mechanism of action of the two agents. In fa c t the r e l a t i v e dependency on e x t r a c e l l u l a r calcium shown i n Figure 13 has exactly the same rank order as the maximum contractions produced by the various drug concentrations; i . e . the greater the maximum contraction produced by a given concentration of drug, the le s s e f f e c t lowering external calcium had on the responses to that p a r t i c u l a r concentration of drug. This p a r a l l e l between drug potency, and dependence upon e x t r a c e l l u l a r calcium, also includes responses to serotonin and procaine. FIGURE 13 Effects of altering external calcium concentration on the responses to various doses of noradrenaline and KC1. Maximum response to each agonist concentration i s set equal to lOOper cent. Tissues were equilibrated for 30 minutes in Kreb's solution with the indicated calcium concentration before the responses to the various agonists were tested. 35. II. EFFECTS OF ADDING MANGANESE CHLORIDE TO THE KREB'S SOLUTION The second series of experiments designed to differentiate between the various sources of "activator calcium" involved the use of manganese. Manganese chloride has been shown to be an effective inhibitor of transmembrane calcium ion flux in several different tissues. Manganese inhibits action potentials in both barnacle muscle (Hagiwara and Naka, 1964) and guinea-pig taenia c o l i (Bulbring and Tomita, 1968). The current carrying species for the action potential in these tissues is believed to be calcium ions. Manga-' 45 ++ nese (0.1 mM) has also been shown to inhibit Ca uptake in rabbit atria (Sabatini-Smith and Holland, 1969). I | The addition of 0.5. mM Mn to normal Kreb's solution totally inhibited spontaneous activity of rabbit anterior mesenteric vein (Figure 14). This inhibition could be reversed by adding EDTA which has a very high a f f i n i t y for manganese (Martell, 1964), but could not be reversed by raising the extracellular concentration of calcium. The addition of-MnC^ blocked botht the spontaneous contractions and the associated membrane potential changes as measured by a sucrose gap apparatus (Figure 15). The delayed inhibitory action of manganese in the sucrose gap apparatus (cf. Figure 14) may be due to poor mixing which was necessary to reduce electrical artifacts produced by excess bubbling. This a b i l i t y of manganese to decrease membrane excitability and inhibit spontaneous activity i s also shared by magnesium and calcium FIGURE 14 Effects of manganese upon spontaneous a c t i v i t y . The tissues were bathed i n normal Kreb's solution containing 2.5 mM ++ Ca . The inhibitory effect of manganese was completely reversed by f EDTA which has a higher a f f i n i t y for manganese than for calcium or magnesium. A J U X I U I 1-0 m M K i n * * FIGURE 15 The effects of manganese on spontaneous e l e c t r i c a l and mechanical activity of the mesenteric vein. The tissue was placed i n a sucrose gap apparatus with which e l e c t r i c a l activity and tension can be measured simultaneously. The extended time period for complete inhibition of spontaneous activity may have been due i n part to poor mixing in the bath. 36. ions. Increasing the magnesium concentration to 5 mM or the calcium concentration to 10 mM had the same effect as the addition of 0.5 mM Manganese to normal Kreb's. The addition of manganese to normal Kreb's solution inhibited the responses produced by every agonist tested (noradrenaline, adrenaline, serotonin, histamine, procaine, and potassium chloride). Manganese produced a shift in the noradrenaline dose response curve as shown in Figure 16. The inhibition was not truly competitive since i t was non-surmountable and yet i t was not s t r i c t l y non-competitive either, since the dose of noradrenaline required for 50 per cent maximum response was shifted from 2.0 x 10 ^ g/ml to 3.5 x 10 ^ g/ml by the addition of manganese. The effect of manganese on ouabain treated veins was also tested. . After 90 minutes in 10 "* M ouabain, responses to nora-I | drenaline were slightly potentiated. The addition of 1 mM Mn produced the same relative inhibition of the responses to 10 ^  g/ml noradrenaline and 10 ^ g/ml noradrenaline as i t did in normal Kreb's solution. The addition of manganese had a much greater inhibitory effect on contractions produced by low doses of noradrenaline (Figure 17). Manganese also had a much greater effect on responses to low doses of noradrenaline when the responses were obtained as in Figure 9. i.e. the tissues were bathed in calcium-free solution with 0.1 mM ascorbic acid added; noradrenaline was added and then a contraction was produced by the addition of calcium to achieve a f i n a l concen-tration of 2.5 mM. 1 mM MnC^ was added before the noradrenaline during the experimental responses. It i s important to note that C N O R A D R E N A L I N E • g/m, FIGURE 16 I | The effect of 1 mM Mn on the dose-response curve to noradrenaline. The tissues were challenged with noradrenaline of a given dose, the peak tension was recorded and then the tissues were washed. Manganese chloride solution was added to the baths to give a f i n a l concentration of 1.0 mM. After one minute the tissues were again challenged with the same dose of noradrenaline. The tissues were then washed twice to remove the manganese and the above procedure was followed with each succeeding dose of noradrenaline. Although not shown in this figure, i t has been well established that two washings after treatment with manganese w i l l allow complete recovery of response to noradrenaline (95%-105% of the pre-manganese controls). FIGURE 17 Relative inhibitory effect of 1 mM Mn on the responses to various doses of noradrenaline. The experimental procedure was the same as in Figure 16 except that the tissues were equilibrated i n Kreb's solution containing the various concentrations of calcium before any response i s obtained. The control response for each concentration of calcium and noradrenaline was set equal to 100 per cent. The inhibitory effect of manganese was much greater for low doses of noradrenaline. 0.1 mM ascorbic acid was also present i n a l l bathing solutions. 37. ascorbic acid was used in these experiments. If i t is not added the manganese rapidly catalyzes the conversion of noradrenaline to noradrenochrome and hence lowers the effective concentration of drug. The addition of manganese did not have differential inhibi-tory effects on the biphasic response to 10 ^ g/ml noradrenaline. The phasic and tonic components of the contraction were reduced equally in the presence of ImM MnC^. A similar experiment was performed using lanthanum chloride as the inhibitor instead of manganese. Lanthanum is a much more potent inhibitor of noradrenaline contractions than manganese; 0.5 mM LaCJl^ i s equipotent as 5.0 mM Mnt^. However the effect of lanthanum was not reversible. Washing the tissues for one hour in normal Kreb's solution only partially restored the response to nor-adrenaline. In contrast, inhibition by manganese is completely reversed by a brief 5 minute washing in normal Kreb's. The addition of 0.5 mM LaCl^ to' the bath, like manganese, had no differential —6 inhibitory effect on the two; phases of the response to 10 g/ml noradrenaline. The addition of manganese to a vein strip during the tonic phase of a response to noradrenaline produced an immediate relaxation to a decreased, but sustained tension (Figure 18). The degree of relaxation was proportional to the logarithm of the concentration of manganese added (Figure 19). The relaxation produced by the manganese could be reversed by the addition of EDTA to bind the manganese, (Figure 18) or partially reversed by increasing the calcium concen-tration (not illustrated). 10'9/m. NA I 1 5 M I N . FIGURE 18 Relaxation of the response to 10 ^ g/ml noradrenaline by the addition of manganese (relaxation versus time). The tissue was bathed i n normal Kreb's solution (2.5 mM Ca ) with 1 mM ascorbic acid added to prevent the oxidation of noradrenaline to noradrenochrome. The relaxation produced by manganese could be reversed by EDTA as shown or by raising-sthe extracellular calcium concentration to 10 mM FIGURE 19 Relaxation of responses to 10 ^ g/ml noradrenaline by the addition of manganese (relaxation versus manganese concentration). The tissues were bathed i n normal Kreb's to which 1 mM ascorbic acid was added. The same relaxation was produced by a given concentration of manganese whether this concentration was achieved with one large addition or i n several steps with smaller additions. 38. This interaction between manganese and calcium ions and the effect of this interaction on the inhibition of noradrenaline responses by manganese is shown in Figures 20 and 21. The control response with each calcium concentration was set equal to 100 per cent so that the values shown in the two figures represent the relative inhibitory effects of manganese at various calcium concentration. Thus in Figure 20 i t can be seen that for any given concentration of manganese, the inhibitory effect can be decreased by raising the extracellular calcium concentration. The same data is replotted in Figure 21 using the reciprocals for both axes. If the curves are extrapolated (dotted lines) they a l l intersect the axis at approximately the same point (0.01 or 100 per cent of the control). This would indicate that the interaction between manganese and calcium is indeed competitive, or at least follows competitive kinetics. The competitive nature of the interaction between manganese and calcium is shown again in Figures 22 and 23 in which the excitatory agent i s 40 mM KC1. In Figure- 23 as in Figure 21, i f the curves are extrapolated to the ordinate they appear to intersect at 0.01. Theoretically i t should be possible to completely prevent the inhibitory action of manganese by sufficiently increasing the extracellular calcium concentration. However when the calcium concen-tration is raised above 10 mM, the excess calcium i t s e l f becomes inhibitory (see Figure 10) and so the inhibitory effect of manganese is potentiated rather than reduced. • I l l O i l 0.60 1.1S 3.5 S.0 K>.0 C c « c i s 3'«M FIGURE 20 Effect of increasing extracellular calcium on the inhibition of responses to 10 ^  g/ml noradrenaline by 1 mM MnC^. Each strip was equilibrated for 20 minutes in Kreb's solution containing calcium concentration as indicated. It was then challenged with 10 ^  g/ml noradrenaline several times u n t i l a consistant response was obtained. Manganese solution was added to give a f i n a l concentration from 0.5 mM to 2.0 mM as shown in the figures. Any given tissue was only tested with one particular manganese concentration. The calcium concentration i n the Kreb's was increased, and after equilibrating for 20 minutes, the procedure I | was repeated. As seen from the top curve (0 mM Mn ) the control response was always set equal to 100 per cent. C C . C I , ] m$A FIGURE 21 Interaction between calcium and manganese and i t s effect on responses to 10 ^ g/ml noradrenaline. Same data as in Figure 20 replotted as inverses to show the competitive nature of the calcium-manganese interaction. 0 T T T o.so 1.0 5.0 I 10.0 FIGURE 22 Effect of increasing extracellular calcium on the inhibition of responses to 40 mM KC1 by 1 mM MnCl^. The experimental procedure was the same as for Figure 20 except that —6 40 mM KC1 rather than 10 g/ml noradrenaline was used to produce a contraction. 3 0 J „..,„•• Interaction between calcium and manganese and i t s effect on responses to .40 mM KC1. 05 10 IS FIGURE 23 Same data as in Figure 22 replotted as inverses. 39. The relative inhibitory effect of 1 mM Mn on contractions produced by various concentrations of KC1 was also tested. The results were similar to those found for increasing doses of noradrenaline; the contractions produced by 20 mM KC1 were inhi-bited to a much greater degree than those produced by 80 mM KC1. To complete the study of the effects of manganese on the mesenteric vein, Figure 24 shows the inhibition of responses to I | serotonin and procaine by 1 mM Mn . The control response for each drug at each calcium concentration is set equal to 100 per cent. The addition of manganese completely inhibited the response to procaine and this inhibition could not be reversed by increasing the calcium concentration. The inhibition of serotonin responses was greater for the low dose of serotonin as with noradrenaline and KC1, and again increasing extracellular calcium lessened the inhibition. procaine. Control responses without manganese were set equal to 100 per cent. 40. III. EFFECTS OF EGTA TREATMENT ON THE ANTERIOR MESENTERIC VEIN As mentioned previously, placing the tissues in calcium-free medium (no added EGTA), results within ten minutes in the inabi l i t y to respond to any excitatory agent. Readdition of calcium immediately reverses this inhibition. The addition of 1 mM EGTA to the calcium-free Kreb's solution results in a faster inhibition of responses (less than 5 minutes). After the EGTA is removed, readdition of calcium produces a contraction. This was unexpected since no contraction was produced by the readdition of calcium to tissues bathed in calcium-free solution without EGTA. Figure 25 shows the responses obtained by readdition of calcium to tissues bathed for 10 minutes in calcium-free Kreb's solution to which 1 mM EGTA was added. After the 10 minute equilibration the tissues were washed twice with calcium-free solution containing 0.1 mM EGTA to remove excess chelating agent, and then concentrated CaC^ solution was added to the bath to produce a f i n a l calcium concentration as shown. The contractile response to the readdition of calcium was phasic unless the f i n a l calcium concentration was greater than 5 mM, in which case the response was biphasic. There was a rapid phasic response lasting about one minute, followed by a prolonged tonic contraction. The magnitude of the phasic contraction was proportional to the logarithm of the calcium concentration (Figure 26). The addition of SrC^ after EGTA treatment produced a contrac-tion followed by the immediate onset of spontaneous activity (similar to Figure 2(C)). The addition of either 5 mM MnCl0 or 5 mM MgCl 2.5mM 5.0 mM 10mM 20mM I I 1 m i n . FIGURE 25 Responses to CaCl 2 after calcium depletion with EGTA (tension versus time). Tissues were equilibrated i n zero calcium Kreb's. 1 mM EGTA was added to each bath and l e f t in for 10 minutes. After washing out the excess EGTA, CaCl 2 was added to give f i n a l calcium concentrations as shown. 1 1 — i — i i i 111 1 3 3 4 3 A 7 I 9 1 0 n j r » UN I H A T C N c a c l , ClMVlJ FIGURE 26 Responses to CaCl 2 after EGTA treatment (tension versus calcium concentration). The bars represent the standard errors of the mean (12 vein str i p s ) . The magnitude of the phasic contraction produced i s proportional to the logarithm of the calcium concentration. 41. had no direct effect, and in fact both ions had an inhibitory effect on the response to CaC^. Figure 27 shows the effect of adding 1 mM MnCl2 to the baths after the tissues have been subjected to the EGTA treatment. The top tracing (27(A)) i s the control response to 10 mM CaCl 2 < The addition of 1 mM MnCl 2 (27(B)) completely blocked the phasic contraction, but had no effect on the tonic component. This inhibitory effect of manganese was completely reversible (27(C)). The addition of 1 mM MnCl2 completely blocked the responses to low concentrations of CaCl^. The addition of MgCl 2 was also able to block the phasic response to CaCl,,, but 5 mM MgCl 2 was required to produce an inhibition equivalent to 0.5 mM MnCl 2 > The addition of a small amount of calcium (0.1 mM f i n a l free calcium) to the bathing medium after calcium depletion with EGTA had a result similar to the addition of 1 mM MnCl 2 (Figure 28(B)): the phasic component of the biphasic response is almost completely abolished. Lllttgau and Niedergerke (1958) showed that the tension produced by a frog heart was directly proportional to the calcium concentration and inversely proportional to the square of the sodium concentration. If this competition also occurs in the smooth muscle of the portal vein, then decreasing the extracellular sodium concen-tration should have an effect on these responses to calcium after EGTA treatment. CONTROL. 10 mM Mn** INHIBITION OF FAST RESPONSE 10 mM / Co** / 90 sac. after 1.0 mM Mn** B y T l i o o m g TIME CONTROL. 10 mM! _23«ec. FIGURE 27 Effects of 1 mM MnC^ on the responses to CaC^ after :ea±c±um rnVpVprriim by EGTA. Tissues were depleted of calcium by bathing for ten minutes w±trh TralTriin free Kreb's containing 1 mM EGTA. The EGTA was then washed :o:ut and calcium was added to give a f i n a l concentration of 10~mM. The airldlTirm of 1 mM Mn (27B) abolished the phasic component of the response. C O N T R O L A Co** I N H I B I T I O N O F F A S T R E S P O N S E FIGURE 28 Effects of 0.1 mM CaC^ on the responses to 10 mM CaC^ after depletion of calcium by EGTA. • Same experimental design as i n Figure 27. The addition of a low concentration of calcium p a r t i a l l y inhibited the phasic component of the response. 42. If the sodium concentration was decreased to 25 mM or less, the response to a low concentration of calcium (2.5 mM or less) after EGTA treatment was tonic rather than the normal transient contraction shown in Figure 25. Thus lowering the external sodium concentration had l i t t l e effect on the i n i t i a t i o n of contraction, but greatly extended the time course of contraction. The response to 10 mM CaC^ in zero sodium solution is shown in Figure 29. The response in zero sodium (29(B)) was similar to the control response (29(A)) except that spontaneous activity has been superimposed on the normal response. Figure 29(C)) shows a second control in normal calcium free Kreb's to show the reversibility of the effects of low sodium. The effects of altering the pH on these calcium contractions was also examined. Figure 30 shows the responses to 10 mM CaC^ after EGTA treatment under'•conditions of varying pH. Below pH 7.4 the slow tonic component is abolished. . As the pH is raised above 7.4, both phases of the contraction are increased with a slightly greater relative increase of the tonic phase. To see i f the effect of pH was specific to these calcium contractions or a generalized phenomenon, the effect of altering pH on the responses to noradrenaline and KC1 was also tested. (Figure 31) It appears that this effect of increased tension produced at high pH is a generalized phenomenon and probably related in some way to calcium metabolism, or to the interaction between actin and myosin. FIGURE 29 Effect of zero sodium solution on calcium responses after EGTA treatment. A Control response to 10 mM CaCl 2 after 10 minutes i n calcium-free solution with 1 mM EGTA (with 143 mM Na +). B Response to 10 mM CaCl 2 after 30 minutes i n sodium-free Kreb's solution with 1 mM EGTA added for the last 10 minutes. C Control response i n calcium-free Kreb's solution (with 143 mM Na +) to show r e v e r s i b i l i t y of effects of sodium-free solution. 190 7.2 7.3 7.4 pH 1 min. FIGURE 30 Effects of altering pH on the calcium responses after EGTA treatment. The tissues were bathed i n calcium-free, bicarbonate-free Kreb's solution with 25 mM Tris, and aerated with 100% 0^. The pH of the solution was adjusted with HC1 and the pH was checked again by putting a micro pH electrode directly into the organ baths. The tissues were bathed for 10 minutes at this pH with 2 mM EGTA added. The tissues were washed with Kreb's solution of the same pH and responses were then e l i c i t e d by the addition of CaC^ (final bath concentration: 10 mM). The small numbers on top give the tension produced (milligrams). 1 0 0 -PH FIGURE 31 Effect of pH on responses to noradrenaline and KCI. Tissues were equilibrated for 10 minutes i n bicarbonate-free Kreb's solution with a given pH and then tested with each concentration of agonist several times un t i l the response became constant. 43. DISCUSSION In this discussion we would like to show how our new evidence can be used in conjunction with previous work to elucidate the roles of calcium in regulating contractions of the rabbit anterior mesenteric portal vein. The aims of this discussion are to show that: (a) extracellular or loosely bound calcium are the only sources of activator calcium for contractions induced by any stimulus; (b) a continuous influx of calcium is required to maintain a contraction; (c) calcium permeability is controlled by membrane bound calcium. The discussion w i l l be arranged under four major headings: (1) Calcium and spontaneous activity. (2) Calcium and the i n i t i a t i o n of response to agonists. (3) Calcium and tonic contractions. (4) Control of calcium influx. I. Calcium and Spontaneous Activity Calcium ions appear to be the major current carrying species for action potentials in the mesenteric portal vein. The evidence for this comes from two types of experiments: (a) lowering extracellular sodium (the other ion most l i k e l y to 44. carry the current), or using an inhibitor of sodium influx have l i t t l e effect on spontaneous activity. (b) lowering extracellular calcium, or blocking calcium influx, totally inhibits spontaneous electrical and mechanical act i v i t i e s . The experimental details are as follows. The addition of tetrodotoxin had no effect on the spontaneous activity of the vein (Holman et a l . , 1968). Action potentials and phasic contractions of rat portal vein could be obtained for several hours in sodium free, sucrose substituted solution (Biamino and Johansson, 1970). This shows either that sodium is not necessary for action potentials, or at least that other ions are capable of substituting for sodium under these conditions. Moreover, we have found that spontaneous activity is rapidly abolished by adding EGTA, and can be returned to normal by raising the extracellular calcium concentration. Manganese which has been shown to block calcium mediated action potentials in barnacle muscle, (Hagiwara and Nakajimo, 1966) abolishes spontaneous activity of the mesenteric portal vein. The inhibition with manganese is rapidly reversed by EDTA (which does not cross c e l l membranes), showing that this action of manganese most likely i s on the c e l l membrane. Another criterion often used (Hagiwara and Naka, 1964; Bulbring and Tomita, 1968) to show the existence of calcium mediated action potentials is the a b i l i t y of other divalent cations to sustain action potentials in the absence of calcium. 45. Strontium chloride is able to support spontaneous electrical and mechanical activities in calcium-free solution (see also Severson and Sutter, 1969). This is unlikely to be due to release of bound calcium since we have shown that spontaneous activity can s t i l l be evoked i f strontium is added to tissues which have soaked in calcium-free solution with 1.0 mM EGTA for more than an hour. Thus i t appears that calcium ions can carry a significant portion of the depolarizing current during an action potential. Would the amount of calcium carrying the depolarizing current raise the intracellular calcium concentration sufficiently to activate the contractile machinery? This question can be answered by determining the actual transfer of charge which would occur during an action potential. The charge transferred equals the capacitance of the membrane multiplied by the amplitude of the action potential. Estimates for the capacitance of smooth muscle membrane (taenia coli) range from 1.5 uF/cm2 (Tomita, 1970) to 3.0 uF/cm2 (Tomita, 1966). The action potential in rabbit anterior mesenteric vein is about 19 mV (Cuthbert et a l . , 1965). A reasonable estimate for the c e l l diameter would be from 3 to 5 u (Burnstock, 1970). An action potential of 19 mV would require a charge transfer of: Q = CV (charge = capacitance x voltage) = (1.5 to 3.0 uF) x 19 mV —8 2 (2.9 to 5.8) x 10 coulombs/cm of membrane = (1.5 to 3.0) x 10 ^  moles Ca^/cm2 of membrane We now have to determine the volume and surface area of the average c e l l . However, smooth muscle cells are not uniform in shape, and so two extreme c e l l shapes w i l l be considered, on the assumption that the actual cells would have intermediate volumes and surface areas. (a) Assume the c e l l i s a cylinder: 2 Volume = 1fr 1 where r is the radius, 1 is the length Surface area = 2^1 (underestimate since micropinocytotic If we assume a l l of the charge is carried by calcium ions, then the change in intracellular calcium concentration during an action potential would be: vesicles would increase surface area) Rise in intracellular calcium = (influx per unit area) x surface area volume = (influx per unit area) x 2 r = (1.5 to 3.0) x 10 1 3 ) x 2 moles Ca^/cm2 (1.5 to 2.5) x 10~4 cm = 0.6 x 10 -6 M to 2.0 x 10 -6 M Ca - H -(b) Assume the c e l l i s a double cone: Volume = 2 x (frr 1) 3 Surface area = 2 x @Trl) Making calculations similar to those above: —6 Rise in intracellular calcium concentration = 0.9 x 10 M to 3.0 x 10~6 M. These two shapes, the cylinder and double cone, give the outside limits for the actual c e l l shape, and so even i f one ignores the presence of vesicles, a single action potential could raise the intra-cellular calcium concentration to at least 6 x 10 ^ M. Would this concentration of calcium produce a contraction? Since the binding constant of troponin for calcium ions is 6 x 10 M , (Ebashi et a l . 1967) a calcium concentration of 1.6 x 10 should half-saturate the troponin binding sites (and presumably give a half-maximal contraction). Using the skinned fiber preparation from frog skeletal muscle (better preparation than glycerinated fibers since the native tropomyosin is readily denatured by glycerination), Hellam and Podolsky (1966) showed a calcium threshold of 10 ^ M with the —6 | | maximum tension achieved with 7 x 10 M Ca Using either of the above c r i t e r i a , i t would appear that i f a sufficient portion of the depolarizing current is carried by calcium ions, that a single action potential (actually a multi-spike complex in the vein) would raise the intracellular calcium concentration sufficiently to account for the accompanying contraction. 48 . II. Calcium and the Initiation of Contractions  to Agonists Previous evidence showing the importance of extracellular or loosely bound calcium for contractions of the mesenteric portal vein comes from two types of experiments. F i r s t l y , contractions to a l l types of agonists are rapidly abolished: in calcium-free solution, (Cuthbert and Sutter, 1965, Severson and Sutter, 1969, Biamino and Johansson, 1970). Secondly, Somlyo et a l . (1969) have shown a good correlation between the maximum depolarization produced by a drug (excluding a depolarizing solution of KC1) and the maximum tension achieved. Somlyo then assumes that the degree of depolarization can ,act as an index of calcium permeability, and i f this is correct the maximum tension achieved by a drug is proportional to the increase in calcium permeability. In the absence of direct data to show the relative contribution of individual ionic species producing the depolarization, one can really only speculate that the variations in maximum depolarization are due to variations in ionic permeability including calcium permeability, which in turn leads to variations in maximum tension produced. We have also found that decreasing the extracellular calcium concentration rapidly and markedly decreased the responses to nor-adrenaline, adrenaline, KC1, serotonin, histamine, acetylcholine, and procaine. Since by definition tightly bound intracellular stores of calcium would not be so rapidly depleted, there are three possible hypotheses for these results. (1) Somlyo's theory is correct; the activator calcium for a l l contractions comes from the extracellular f l u i d or loosely bound surface stores. (2) The majority of the activator calcium is released from intracellular sites, but the influx of a small amount of calcium is necessary to release this bound calcium. (3) The presence of membrane bound calcium is required for contraction but no actual calcium influx i s required. The third hypothesis could be tested by measuring the rate of calcium exchange in the absence and presence of drugs. Although this has not been done using the mesenteric vein, the results from other types of smooth muscle show that the rate of calcium exchange does increase during contractions, and hence this third hypothesis is not li k e l y correct (Briggs and Melvin, 1961, Van Breemen and McNaughton, 1970). The problem then is to differentiate between the f i r s t two hypotheses. What does each hypothesis imply about the nature of excitation-contraction coupling? 1. Activator calcium comes from the extracellular f l u i d . This hypothesis implies that drugs can produce graded increases in calcium permeability in response to increased drug concentration. Each individual c e l l would therefore contract in a graded manner to increased drug concentration. 2. Activator calcium comes from bound intracellular stores and is released by the influx of a small amount of calcium. This hypothesis implies the presence of an intracellular bound calcium store, but does not imply that drugs can produce graded 50. increases in calcium permeability. What is the function of the calcium influx in this hypothesis? Under normal conditions, this influx would produce a depolarization and increase the intracellular calcium level. It is unlikely that the depolarization per se is necessary for excitation-contraction coupling since mesenteric veins which are f u l l y depolarized by KCI (Somlyo and Somlyo, 1968a) or ouabain (Mathews and Sutter, 1967) s t i l l contract in response to nor-adrenaline. This means that i t is the rise in intracellular calcium i t s e l f which is important for excitation-contraction coupling. This "calcium induced release of bound calcium" phenomenon has recently been shown by Endo et a l . (1970) to occur in skinned skeletal muscle fibers, thus raising this hypothesis for the i n i t i a t i o n of contractions, from mere speculation to a real possibility. The amount of calcium required to induce further caicium release was 10 M, which as they say was "usually below the threshold for contraction". Now assuming a rise in free intra-cellular calcium in mesenteric portal vein can release bound calcium as proposed by Endo for skeletal muscle, at some c r i t i c a l intra-cellular calcium concentration this phenomenon would become self-perpetuating and each individual c e l l should f i r e in an all-or-none manner. How do the data known" about the mesenteric vein, both from the present experiments and previous workers, f i t with these two hypotheses? Fir s t is there in fact a graded increase in calcium permeability with different drugs or increasing doses of drugs? (As would be required by hypothesis number one) 51. If one examines the frequency-distribution of membrane potentials of rabbit mesenteric vein, a normal frequency d i s t r i -bution is observed both in the absence and in the presence of drugs (Somlyo et a l . , 1969). When noradrenaline (10 ug/ml) or serotonin (20 ug/ml) was added there was a general shift towards lower membrane potentials and no bimodal distribution (indicating all-or-none depolarization) was seen. Noradrenaline produced a greater depolarization and also a greater contractile response than did serotonin. This means that smooth muscle cells have a normal s t a t i s t i c a l distribution with regard to electrical responses to drugs, and also that the ionic permeability of each individual muscle c e l l can be altered in a graded manner by different drugs. Presumably this would include a graded increase in the permeability to calcium. If this evidence does indeed demonstrate graded increases in calcium permeability, the criterion necessary for the f i r s t hypothesis is satisfied, but this does not rule out hypothesis number two. What evidence is there for a bound intra-cellular calcium store as required by the second hypothesis? F i r s t , smooth muscle cells have a poorly developed sarco-tubular system when compared with skeletal or cardiac muscle (Burnstock, 1970) and thus have less available intracellular calcium i f one assumes that the tubules are the primary intra-cellular calcium storage sites in smooth muscle. Second, in other types of smooth muscle which are believed to u t i l i z e intracellular calcium stores for contractions, biphasic responses are obtained in response to agonists (Bohr, 1964 using 52. rabbit aorta; Woodward, Bose and Innes, 1970 using cat spleen capsule). However, unless the calcium concentration is very low, we found that the tension produced by the mesenteric portal vein in response to agonists increases monotonically with time. These two pieces of data both argue against the release of intracellular bound calcium during contraction of mesenteric portal vein. There is one further bit of evidence which argues against the second hypothesis. The second hypothesis implies that each c e l l should f i r e in all-or-none manner. The graphs of tissue response versus calcium concentration (figures 8, 9, 12, 13) must then reflect the s t a t i s t i c a l variation of individual cells with regard to calcium permeability, or the s t a t i s t i c a l variation of c r i t i c a l intracellular calcium concentration required to i n i t i a t e the a l l -or-none contraction of each c e l l . Figure 9 shows that the statis-t i c a l variation i s very large; in fact i t ranges over at least a one hundred fold change in extracellular calcium concentration. Such a broad variation among cells from the same tissue would seem to be very unusual and leads one to suspect that individual cells are not contracting in an all-or-none manner, and that the second hypothesis is therefore not l i k e l y correct. Another major argument in smooth muscle research has been whether or not different agonists mobilize calcium from different sources. Hudgins and Weiss (1968) showed that procaine would relax rabbit aorta contracted in response to noradrenaline but not to 25 mM KC1. Van Breemen (1970) showed that lanthanum chloride had a greater inhibitory effect on contractions to KC1 53. than on noradrenaline induced contractions, again using rabbit thoracic aorta. Severson and Sutter (1969) showed that after placing the aorta in calcium free solution, responses to KCI were lost more quickly than those to noradrenaline. This result was also found by Hudgins and Weiss using equipotent doses of KCI and noradrenaline. The experiments with procaine prove very l i t t l e ; procaine certainly resembles noradrenaline more closely than i t does KCI, and so i t is hardly surprising that i t has more of an effect on noradrenaline contractions. The experiments with LaCl^ and calcium-free, solutions could be explained simply i f KCI and noradrenaline have, different effects on calcium permeability. To my knowledge no one has compared the effects of low calcium on KCI and noradrenaline induced contractions under equilibrium conditions using rabbit aorta. These arguments cast some doubts that different agonists mobilize calcium from different sources in the aorta. Experiments using the mesenteric portal vein are more clear cut in showing that different agonists do not u t i l i z e different sources of activator calcium. Severson and Sutter (1969) showed that there was l i t t l e difference in the rates at which KCI and noradrenaline responses were lost after exposure to calcium-free solution. We have shown in Figure 13 that equipotent doses of KCI and noradrenaline are equally affected by lowering the extracellular calcium concentration. Experiments using serotonin showed that responses to equipotent doses of this agonist were also equally affected by low extracellular calcium. 54. We also found that manganese chloride had an equal inhibitory effect on contractions to equipotent doses of noradrenaline, adrenaline, serotonin, histamine, KC1, and procaine. These experiments would indicate that i t is unlikely that different calcium sources are mobilized in the mesenteric portal vein for the contractions to various excitatory agents. It appears that contractions produced by a l l agonists are initiated by an increased calcium influx, and so differences in efficacy are explained by differences in the a b i l i t y of drugs to increase calcium permeability. III. Calcium and sustained contractions We have evidence that once the contraction has been initiated by this influx of calcium, a continuous influx of extracellular calcium is s t i l l necessary to maintain a tonic contraction. Figure 11 shows that the removal of extracellular calcium with EGTA results in a prompt relaxation to a lower tension. Also Figure 7(B) shows that the tonic tension is directly propor-tional to extracellular calcium concentration in the range from 0.1 to 1.0 mM. This l i k e l y means that there is. a continual calcium removal process and that during a sustained contraction the intra-cellular concentration is the result of a balance between calcium influx and removal. We have also shown that the addition of manganese to a vein contracted by noradrenaline (Figure 18) KC1, or serotonin produces a relaxation. This cannot be an effect of manganese on the contractile 55. proteins since EDTA rapidly reverses the inhibition (Figure 18). This data supports the hypothesis that a continued influx of calcium is required to maintain tension. The relaxation caused by addition of manganese can be partially reversed by raising extracellular calcium, showing that the relaxation i s probably due to a competition with calcium at the c e l l membrane. Thus i t appears that calcium ions for both the i n i t i a t i o n and maintenance of contractions in the mesenteric portal vein come from the extracellular f l u i d . How then i s the membrane permeability to calcium controlled? IV. Control of calcium influx The experiments involving calcium depletion with EGTA (Section III of Results) reveal several interesting facts about control of calcium permeability. The addition of calcium chloride has no effect on tissues bathed in normal Kreb's solution (containing 2.5 mM calcium). Bathing the tissues in calcium-free solution (no added EGTA) inhibits responses to a l l agonists, but the readdition of calcium s t i l l produces almost no response on i t s own. However, when the tissues are bathed for ten minutes in calcium-free solution containing 1 mM EGTA, the readdition of calcium produces a contraction. Thus the chelation of some bound calcium (presumably bound to the membrane) has made the membrane more permeable to added calcium. This function of membrane bound calcium in the vein is probably analogous to the stabilizing action of calcium described by Frankenhaeuser and Hodgkin (1957) for the squid giant axon. 56. Further evidence that calcium controls i t s own perme-ab i l i t y is shown in Figure 28. The addition of a small amount of calcium (0.1 mM f i n a l concentration) after EGTA treatment inhibits responses to large doses of calcium. The addition of either manganese or excess magnesium was also able to inhibit the response to calcium. This a b i l i t y of calcium to control i t s own influx was predicted by Somlyo and Somlyo (1968b). He believes that agonists i n i t i a t e contractions by releasing membrane bound calcium thus increasing calcium permeability. We have found evidence that agonists do indeed release membrane bound calcium (Figures 4 to 7). The transient portion of the biphasic response to added noradrenaline with 0.1 mM calcium in the bathing solution (Figure 4) must be due to the release of loosely bound calcium into the cytoplasm. If the transient response was due to simple influx of calcium (or release of intracellular calcium), then the same response should have been obtained by adding 0.1 mM calcium to tissues bathed in calcium free medium containing 10 g/ml noradrenaline. Further evidence that the transient response is due to release of membrane bound calcium comes from analysis of Figure 7. Figures 7(A) and 7(B) show quantitatively the effects of reduced extracellular calcium concentration on the two phases of the response to 10 g/ml noradrenaline. When the extracellular calcium concentration i s 1.0 mM, the; phasic and tonic responses are approximately equal in magnitude. If Figure 7(A) were replotted with a linear calcium scale, the curve would resemble 57. a rectangular hyperbola, indicative of a saturation phenomenon. This cannot be due to saturation of calcium binding sites on troponin since the tonic response (Figure 7(B)) is s t i l l increasing linearly when the extracellular calcium concentration is 1.0 mM. It must therefore represent a saturation of membrane binding sites for calcium. If the release of membrane bound calcium leads to increased calcium permeability, then drug efficacy can be explained by differences in the ab i l i t y of drugs to release this bound calcium. This idea is consistent with our obser-vation that as the concentration of agonist is decreased, the transient component of the contraction in low calcium (due to release of membrane bound calcium) was greatly decreased. For example, the transient response in low calcium i s not produced by the addition of 10 ^  g/ml noradrenaline (Figure 5) or by 40 mM KCI. As mentioned in the discussion of spontaneous activity, manganese appears to act on the c e l l membrane to prevent the influx of calcium. Manganese i s also able to inhibit the in i t i a t i o n of contraction to a l l agonists. The addition of 1 mM MnC^ to the Kreb's solution produced a non-surmountable inhibitory shift of the noradrenaline dose response curve (Figure 16) and also of the dose response curves to KCI and serotonin. Manganese also inhibited the responses to noradrenaline after the tissues had been in a -4 solution containing 10 M ouabain for 90 minutes. Thus manga-nese does not inhibit the contractile responses solely by 58. inhibiting action potentials, since this ouabain treatment renders the c e l l incapable of generating action potentials (Mathews and Sutter, 1967). The inhibitory effects of manganese are consistent with Somlyo's theory that excitatory drugs have some primary effect on the membrane which leads to parallel changes in tension and electrical activity. Manganese interferes with this primary effect, which is presumably the release of membrane bound calcium. The competitive nature of the interaction between manganese and calcium can be shown for contractions produced by either nor-adrenaline (Figure 20) or KC1 (Figure 22). The a b i l i t y of calcium to reverse the inhibitory effects of manganese is not always observed however. Raising the extracellular calcium w i l l not reverse the manganese induced inhibition of spontaneous activity or inhibition of contraction to procaine or low doses of nor-—8 adrenaline (10 g/ml). This is expected since we have shown that raising the calcium concentration can i t s e l f inhibit spontaneous activity and the response to procaine. Thus manganese appears to act as "super-calcium"to lessen the calcium permeability change produced by agonists and in this manner stabilize the membrane. V. Effects of altering sodium concentration or pH These last two types of experiments are really only preliminary observation. Studying the interactions between calcium ions and either hydrogen ions or sodium ions may provide more infor-mation about the nature of calcium binding sites and the importance of extracellular calcium for both the i n i t i a t i o n and maintenance of contractions. 59. The potentiation of contractile responses to a l l types of stimuli (Figure 31) is interesting. At least four possible explanations can be put forward for further experimentation: (1) an effect on the actin-myosin interaction independent of calcium ions; (2) an increased a f f i n i t y of troponin for calcium ions; (3) an increased influx of calcium; (4) a decreased efflux of calcium. The fact that lowering the extracellular sodium concentration I | changes a normally transient contraction (response to 2.5 mM Ca after EGTA treatment) into a sustained contraction, is also interesting and should be further studied with regard to a sodium-calcium exchange mechanism as described by Glitsch et a l . (1970) in guinea-pig atria. It is possible that this exchange may be important for the relaxation process in the mesenteric vein. Very recent experiments indicate that this is indeed true. Although small contractions may be produced by noradrenaline, adrenaline, KCI, serotonin, and acetylcholine after 90 minutes in zero sodium solution (Tris substitution), relaxation does not occur after washing out the drugs with zero sodium solution. Washing the tissues with normal Kreb's solution produces an immediate relaxation. 60. SUMMARY The rabbit anterior mesenteric portal vein which we are using as a model for arteriolar smooth muscle contracts in response to a wide variety of agonists. The response to each agonist appears to be initiated by the release of membrane bound calcium which produces an increased calcium permeability. We feel that the activator calcium for the contractile process comes solely from the extracellular f l u i d or loosely bound membrane stores. 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