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Effect of isoproterenol on physiological and biochemical changes in euthyroid and hyperthyroid rat hearts Longhurst, Penelope Anne 1978

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EFFECT OF ISOPROTERENOL ON PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN EUTHYROID AND HYPERTHYRO 3D RAT HEARTS by PENELOPE ANNE LONGHURST B.Sc, University of London, England, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 19?8 © Penelope Anne Longhurst, 1978 In presenting th i s thes i s in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my writ ten permission. Department of Pharmaceutical Sciences The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date September' 22, 1978 -11-ABSTRACT. The effect of triiodothyronine pretreatment on the physiological and "biochemical properties of the rat heart was Investigated In a number of cardiac preparations. Pretreatment with triiodothyronine for three days produced an increased chronotropic effect in the isolated right atrium, and a decreased Inotropic effect In the isolated l e f t atrium and Langendorff heart. Following administration of Isoproterenol there was a dose-dependent increase in rate of the right atrium. The absolute increase in rate was similar In euthyroid, and hyperthyrold. tissues; the hjrperthyroId atria maintained a higher rate than the euthyroid atria, with no sign of supersensitivity. In the el e c t r i c a l l y stimulated l e f t atrium and right ventricle, Isoproterenol produced a dose-dependent Increase In tension. The absolute Increase in tension was slig h t l y greater In the euthyroid tissues than in the hyperthyrold tissues. In a l l three preparations, the contractile changes produced by Isoproterenol were accompanied by an increase, In phosphorylase activity which was similar In euthyroid and hyperthyrold animals. Five nanograms of Isoproterenol produced a similar increase In tension in euthyroid and hyperthyrold Langendorff rat hearts. The Increase in tension was accompanied by an increase In phosphorylase a activity. This effect of -111-Isoproterenol on phosphorylase activation was potentiated in the hyperthyroid hearts. The possibility that the potentiation of phosphorylase activation In the perfused. heart was a result of the Increase in coronary blood flow, noted in hearts from hyperthyroid animals, was investigated* Coronary blood flow was reduced in a group of hearts from hyperthyroid animals to the same level as that of hearts from euthyroid animals, and the phosphorylase-actlvating effect of isoproterenol was again tested. Under these conditions the phosphorylase-actlvating effect of isoproterenol was s t i l l enhanced. A DA study carried out on the rat right atrium showed 2 that beta-adrenoceptors in the euthyroid and hyperthyroid state were similar. The data obtained in the present study suggest that the actions of thyroid hormones on the heart do not result in a supersensitivity <to the chronotropic or inotropic effects of isoproterenol. In the Langendorff heart preparation a supersensitivity to the phosphorylase-actlvating effect of isoproterenol was detected, but this same potentiation could not be demonstrated In the right atrium or right ventricle. The reason for the absence of an isoproterenol-lnduced potentiation of phosphorylase activation in these two preparations, when i t can be readily demonstrated in the Isolated perfused heart, is not clearly understood but mayobe due to tissue damage Incurred during dissection. It is suggested that the greater resting rate of - i v -hyperthyroid myocardial tissue might be due to a direct action of the thyroid hormones on calcium movements in the slno-atrial node. -V-TABLE OF CONTENTS PAGE ABSTRACT 11 LIST OF FIGURES v i INTRODUCTION 1 Adrenal medullary involvement in thyrotoxicosis 1 Cardiac beta-receptor involvement 6 Cyclic AMP and phosphorylase involvement 8 Involvement of the parasympathetic nervous system 2k Direct actions of thyroid hormones 26 Muscle mechanics 30 Involvement of the peripheral blood vessels Jk Purpose of the investigation 37 MATERIALS AND METHODS 39 Methods 39 Preparation of at r i a 39 Preparation of ventricles k0 Preparation of Langendorff hearts ko Measurement of pA kZ 2 Measurement of coronary blood flox* Phosphorylase assay k-J S t a t i s t i c a l methods Materials k$ RESULTS ^6 DISCUSSION 77 BIBLIOGRAPHY 88 - v i -LIST OF FIGURES FIGURE PAGE 1. Glycogenolysis. 9 2. Electrode used for ventricle stimulation. hi 3. Effect,of three day pretreatment with 3»3'»5 - triiodothyronine on rat body weight. ^8 h. Effect of isoproterenol on rate of right atria from euthyroid and hyperthyroid rats. 50 5. Effect of Isoproterenol on % increase in rate of right atria from euthyroid and hyperthyroid rats. 53 6. Effect of isoproterenol on tension of l e f t atria from euthyroid and hyperthyroid rats while stimulating at 3 H z . 55 7'.Changes in rate following administration of 5xl0 ~ ° M isoproterenol to euthyroid and hyperthyroid rat right a t r i a . 58 8. Changes in % phosphorylase a following administration of 5xlO~ Y M isoproterenol to euthyroid and hyperthyroid rat right a t r i a . 60 9. A comparison of pAg values for propranolol in euthyroid and hyperthyroid rat right a t r i a . 63 10. Effect of isoproterenol on changes in tension of the right ventricle of euthyroid and hyperthyroid rats while stimulating at 3Hz. 65 11. Changes in tension following administration of 5xl0 ~ ° M Isoproterenol to the right ventricle of euthyroid and hyperthyroid rats while stimuli., ating at 3 H z . 68 12. Changes in % phosphorylase a following admin-istration of 5X10~"M isoproterenol to the right ventricle of euthyroid and hyperthyroid rats while stimulating at 3 H z . 70 13. Differences between euthyroid and hyperthyroid rat Langendorff hearts. 73 - v i i -FIGURE lk. Effect of changes In coronary blood flow on % phosphorylase a activation. - v i i i -ACKNOWLEDGMENTS I am deeply grateful to Dr. J. H. McNeill for his guidance and encouragement throughout this study. I would also like to thank the Canadian Heart Foundation for their financial support. - 1 -INTRODUCTION Adrenal Medullary Involvement in Thyrotoxlcosis. The association of toxic goitre with rapid heart rate has been known for several centuries. In 1825» Caleb Parry, a practitioner in Bath, England, described eight cases which he saw between 1786 and 1814. Other early practitioners associating a rapid pulse with thyroid enlargement were Flajani (1802), Graves ( I 8 3 5 ) , and von Basedow (18^0). In 1 8 6 5 i i t was suggested by Moore that the symptoms of thyrotoxicosis might be due to increased vasomotor or sympathetic actions. The introduction of the chemical synthesis of adrenaline In 1901 meant that the relationship between the thyroid gland and the adrenal medulla could be more closely Investigated. Finally in 1 9 0 8 , Kraus and Friedenthal demonstrated that adrenaline could produce tachycardia and some of the eye signs of thyrotoxicosis. In more recent studies there has been some support for this interaction between the two systems. Thier and co-workers (1962) showed an increase in heart rate over controls In hyperthyrold. rat atria. This increase was significantly reduced when the animals were pretreated with reserpine. Cravey and Gravenstein (1965) also showed that atria from hyperthyrold rats beat faster than those from euthyroid rats. Following adrenalectomy, the hyperthyrold atria rates were significantly reduced, but returned to their -2-i n i t l a l (nonadrenalectomlzed hyperthyroid) rates following corticosterone treatment. Lee, Lee and Yoo (19&5) showed using rabbit atria that atria taken from thyroxine-treated animals beat at a significantly higher rate than those taken from euthyroid animals. Following administration of noradrenaline the increases in rate and amplitude were greatest in atria from the hyperthyroid animals. However, the majority of evidence tends to suggest that this relationship Is much less straightforward than It seems. I n i t i a l l y Benfey and Varma(1963) showed that in spinal cats following treatment with triiodothyronine, intravenous adrenaline produced no greater effects on heart rate, blood pressure and contractile force than in control animals. Margollus and Gaffney (1965) found that in the intact dog graded doses of noradrenaline produced no change in absolute or percentage increases in heart rate of euthyroid or hyperthyroid animals, although the i n i t i a l heart rate was higher in the hyperthyroid dogs. Stimulation of the accel-erator nerve produced similar frequency-dependent absolute increases in heart rate in euthyroid and hyperthyroid dogs. Van der Schoot and Moran (1965) working with open chest dogs found an increased i n i t i a l heart rate in hyperthyroid animals as compared to controls. Adrenaline and noradrenaline changed the absolute and percentage increase in heart rate less in hyperthyroid than in euthyroid dogs. They also found less of an increase In contractile force in hyperthyroid than in euthyroid animals. In rat ventricle strips Van der Schoot - 3 -and Moran found that the i n i t i a l contractile force of the strips from hyperthryoid rats was significantly lower than those from euthyroid rats. The positive inotropic effect of noradren-aline was similar In both groups. With rat at r i a , the i n i t i a l contractile force was greater in euthyroid than in hyperthyrold animals. The positive Inotropic effect of noradrenaline was less in the hyperthyrold than in the euthyroid animals i f the response was expressed as grams tension. There was no significant difference between the two groups i f the response was expressed as percent change. The i n i t i a l rate of spon-taneous: contraction of a t r i a from hyperthyrold rats was greater than that from euthyroid rats. In the presence of noradrenaline, the absolute increase in rate was the same in both groups; but i f expressed as percent Increase was less in hyperthyrold than in euthyroid rats. Wilson and co-workers (1966) working with human volunteers, found that the major difference in the response of the euthyroid and hyperthyrold heart to isoproterenol was that the hyperthyrold heart had a higher i n i t i a l rate; otherwise the increments in heart rate to graded doses of isoproterenol in the two states did not di f f e r , and therefore the receptor sensitivity was unchanged. Following treatment with propranolol, isoproterenol did not produce a change in heart rate In either state. Aoki and co-workers (1967), also working with human volunteers found that daily administration of 500 ug of triiodothyronine produced the typical symptoms of thyrotoxicosis, including palpitation and pounding of the heart. Heart rate was significantly higher during the treatment regime than during the control periods. The response to nor-adrenaline infusion was similar during the two sessions. Cairoll and Crout (1967) showed that hyperthyroid rats developed an Increased metabolic rate and tachycardia. To test whether the tachycardia was due to increased adrenergic nerve activity or enhanced responsiveness of the pacemaker to noradrenaline, they studied the effect of propranolol on the resting heart rate of the unanesthetized rat. This produced less cardiac slowing in the hyperthyroid than in the euthyroid group, thus ruling out these two p o s s i b i l i t i e s . Atropine was added to check whether cholinergic input was deficient (in which case there should have been l i t t l e change); but there was a sharp increase in rate in both groups, Indicating that vagal input to the sino-atrial node was unaffected by the hyperthyroid state. Treatment with reserplne reduced the hyperthyroid heart rate to that of the control animals. If atropine was added after reserplne pretreatment, the depression of heart rate was reversed. , This suggested that reserplne could only "normalize" the hyperthyroid heart rate i f vagal function was intact, and led to their conclusion that there was no evidence to suggest that the hyperthyroid state enhanced either adrenergic neural a c t i v i t y or the sensitivity of cardiac beta-receptors to noradrenaline, but rather that the tachycardia was due to a direct effect of the thyroid hormones on the pacemaker c e l l . Thus, It can be clearly seen that treatment with thyroid hormones does raise resting heart rate above control values. Quite possibly i f the data presented in the early studies were re-examined, taking into account the differing baselines, no catecholamine supersensitivity would be seen. -6-Cardlac Beta Receptor Involvement. Cl i n i c a l l y , beta-adrehoceptor blocking agents have been used to treat patients with thyrotoxicosis for many years. Employed in this manner, they control nervousness, palpitations, tachycardia, Increased cardiac output, tremor and acute hyperthyrold c r i s i s ; but have no effect on the underlying hyperthyrold process i t s e l f ( Levey, 1976; McDevltt, 1976; Brit. Med. J., 1977). Kunos (1977) has suggested that since beta-adrenoceptor conversion seems to be promoted by an Increase In metabolic activity (Buckley and Jordon, 1970; Kunos and Nlckerson, 1976), changes in thyroid state could also alter adrenoceptor properties. Williams and co-workers (1976) showed, using the beta-3 adrenoceptor blocking agent H -dlhydroalprenolol, that there was a 100 percent Increase in the number of binding sites per milligram of protein In hyperthyrold myocardial membranes as compared to a similar preparation from euthyroid hearts. However the relative a f f i n i t i e s of isoproterenol In the two groups were practically identical. I n addition, Claraldi and Marinettl (1977) showed an increased number of beta-receptors and decreased number of alpha-receptors In heart ventricles from thyroxine-treated rats, without any change in the binding a f f i n i t y of the beta-receptor. Spauldlng and Noth (1975) reported that in hyperthyroidism there is a decrease in the amount of noradrenaline released from sympathetic nerve endings. It has therefore been suggested by Banerjee and Kung (1977) that this could support the hypothesis that the concentration of noradrenaline is important in regulation of beta-receptor density on mammalian myocardial membranes. Another possible theory of the mechanism of action of beta-adrenoceptor blocking agents in hyperthyroidism was put forward by Melander and co-workers in 1975. They showed that in hyperthyroid mice, thyroid hormone secretion could be Initiated by beta -, but not beta -adrenoceptor agonists. 2 1 The induction of secretion could be abolished by pretreatment with L-propranolol. However, no c l i n i c a l studies to date have demonstrated that beta-adrenoceptor blocking agents inhibit thyroid hormone secretion in hyperthyroid patients. Most recently; Kempson, Marinettl and Shaw (1978) showed that triiodothyronine and thyroxine Increase the number of beta-adrenoceptors in rat heart ventricle slices. These effects take place In two stages; the acute effect at 1.5 to two hours being a post-translatlonal event; and the chronic effect at fifteen hours being a transcriptional or translatlonal event. They suggest that there Is a pool of catecholamine receptors located within the cytoplasm of the ventricular cells which may move from the pool into the plasma membrane following thyroid treatment. -8-Cyclic AMP and Phosphorylase Involvement. Adenosine 3',5'-monophosphate (cyclic AMP), the enzyme which acts as a second messenger in many biological systems was f i r s t discovered by workers investigating the increased breakdown of glycogen to glucose in li v e r slices stimulated by adrenaline and glucagon. Sutherland and Rail (i960) found that adrenaline acted on a membrane-bound enzyme, adenylate cyclase which was responsible for formation of cyclic AMP from ATP in the presence of Mg , Cyclic AMP in turn accelerated conversion of inactive phosphorylase b to active phosphorylase a, by the steps shown in figure 1. This then resulted in breakdown of glycogen to glucose. In heart muscle the same reaction occurs, but the finishing product Is lactate. Inactivation of cyclic AMP was found to be brought about by a cyclic nucleotide phosphodiesterase, with subsequent formation of 5'-AMP (Sutherland and Hall, 1958). Cyclic GMP (guanosine 3',5'-monophosphate) is formed in a similar manner from guanylate cyclase and GTP, and is also broken down by a phosphodiesterase. Tissue levels of cyclic AMP depend on the balance between the activities of adenylate cyclase and phospho-diesterase. These acti v i t i e s may be stimulated or Inhibited by a number of drugs. The methylxanthlnes caffeine and theophylline; papaverine, nitroglycerine, hydralazine, and some diuretics have been reported to be phosphodiesterase - 9 -Adrenallne Glucagon adenylate ATP ++ > oAMP + PP1 cyclase Mg Inactive Protein Kinase PDE -> AMP -> Active Protein Kinase ATP + Dephospho-phosphorylase kinase (inactive) Phospho-phosphorylase b kinase (active) + ADP ATP + Phosphorylase b (inactive) Phosphorylase a (active ) + ADP Glycogen + Pi Glucose-l-Phosphate Glucose-6-Phosphate V Glucose + Pi (liver) Figure 1. GLYC 0 GENOLYSIS -10-inhlbitors ( 0 y e , 1975). Their use results in Increased cyclic AMP levels. The catecholamines; glucagon, ACTH, histamine, and. prostaglandins are some substances which activate adenylate cyclase (Butcher, 1968) and increase cyclic AMP levels. In the heart, cyclic AMP is thought to be the mediator of the inotropic response to catecholamines acting through the beta-adrenoceptor. It has been shown ( 0 y e et a l , 1964) that the inotropic response to adrenaline in perfused, hearts was preceded by a rapid rise in cyclic AMP levels, and followed, by a rise in phosphorylase a. Beta-adrenoceptor blocking agents have been shown to inhibit a l l the cardiac effects of the beta-agonists ( 0 y e , 1975). Other agents which cause an Inotropic response of the heart, such as glucagon, histamine, dlbutyryl cyclic AMP and some phosphodiesterase inhibitors have also been shown to Increase cyclic AMP levels prior to the inotropic effect. Pretreatment with beta-adrenoceptor blocking agents has no effect on the response to these drugs(Butcher, 1968; Drummond and Hemmlngs, 1972; McNeill and Muschek, 1972; McNeill, Brenner and Muschek, 1974). In heart muscle, the stimulation of glycogenolysis by these inotropic agents via cyclic AMP may provide some of the energy required to sustain the response, although i t cannot be necessary for e l l c i t a t l o n of the response since phosphorylase a levels only Increase following the peak of the inotropic response (Williamson and Schaffer, 1976). -11-In I n i t i a l studies, phosphorylase a, the enzyme which catalyzes the breakdown of glycogen was extensively studied. Hornbrook and Brody (I963) showed that chronic thyroxine treatment (for fourteen to twenty days) produced an increase In anaesthetized rat cardiac phosphorylase a levels, and a decrease in cardiac glycogen levels when compared to control animals. In a subsequent paper (Hornbrook et a l , 1965)• they showed that infusion of noradrenaline potentiated the increase in % phosphorylase a seen in hyperthyrold animals. In the hyperthyrold rats, following reserpine administration, there was again potentiation of the noradrenaline-induced increase in cardiac phosphorylase a, compared to euthyroid rats pretreated with reserpine; although in the absence of noradrenaline the % phosphorylase a values in euthyroid and hyperthyrold rats pretreated with reserpine were essentially the same. TMs suggested to them that the catecholamine-induced responses seen In the hyperthyrold animals were due to some form of modulation of the "myocardial metabolic adrenergic receptor". A series of studies by Hess and Shanfeld (1965)» showed that the Increase (from 30 to 50 percent) In cardiac phosphorylase a, brought about by chronic (more than three days) thyroxine pretreatment developed gradually over the time period studied, and that when treatment stopped, the phosphorylase a a c t i v i t y returned to control values in twenty days. Blood pressure and heart rate changes were shown to behave In a similar manner. In the presence of adrenaline, -12-% phosphorylase a increased in both euthyroid and hyperthyroid rat hearts, but the absolute increase (allowing for the higher basal act i v i t y in the hyperthyroid animals) was similar. They therefore concluded that thyroxine did not potentiate the effects of adrenaline on cardiac phosphorylase a. These results differ from those of Hornbrook and co-workers (1965), who did show noradrenaline potentiation. F i r s t l y , the % phosphorylase a values of Hess and Shanfeld in the absence of adrenaline (30 percent for euthyroid and 50 percent for hyperthyroid) are very high compared to Hornbrook's values (1^.2 and 32.8 percent respectively). Secondly, the doses of noradrenaline used by Hornbrook were not able to produce a significant increase in % phosphorylase a levels in the euthyroid animals over euthyroid controls; although in the hyperthyroid animals, the same doses did produce a significant increase in phosphorylase a. To test the effect of "antladrenergic" drugs on thyroid hormone pretreatment, Hess and Shanfeld studied the response of % phosphorylase a to acetylcholine, reserplne and pronethalol. They found that in euthyroid rats acetylcholine had l i t t l e effect on phosphorylase activity, but in hyperthyroid animals i t produced a significant decrease in % phosphorylase a compared to values in the presence of thyroxine alone; the levels being reduced to euthyroid control values. Pretreatment for five days with reserplne reduced the usual Increase in phosphorylase activ i t y seen in hyperthyroid hearts when compared to euthyroid controls. In euthyroid rats, reserplne produced no change in cardiac phosphorylase a activity. Pronethalol, a "beta-adrenoceptor "blocking agent produced no change in the phosphorylase ac t i v i t y of euthyroid rat hearts one minute after administration; although i t produced a significant decrease in heart rate. In hyperthyrold rats, pronethalol produced a significant decrease in cardiac phosphorylase a activity, as well as a decreased heart rate. These results seemed to indicate some relationship between the catecholamines and thyroid hormones, which at this time was postulated to be due to increased adenylate cyclase activity. McNeill and Brody (1968) used cardiac phosphorylase a as a marker to test whether thyroid hormones enhanced catecholamine actions by blocking uptake into the neuron. This was tested using cocaine, since i f hyperthyroidism produced blockade of catecholamine uptake, subsequent administration of cocaine would have no further effect. I n i t i a l l y , the hyperthyrold animals receiving cocaine had significantly higher % phosphorylase a values than those receiving triiodothyronine alone. Following infusion of noradrenaline, the hyperthyrold animals pretreated with cocaine again had significantly higher % phosphorylase a values than those receiving either triiodothyronine or cocaine alone. Tyramine is a sympathomimetic amine which ut i l i z e s the catecholamine uptake system to enter the sympathetic neuron; therefore its actions would be affected i f triiodothyronine -14-were to have an effect on catecholamine uptake. However, triiodothyronine pretreatment enhanced the effect of tyramlne on % phosphorylase a compared to controls, although in a later study (Young and McNeill, 197*0 i It was shown that in the guinea pig Langendorff heart, triiodothyronine did not enhance the phosphorylase activating, nor cyclic AMP increasing effect of tyramlne. It is therefore unlikely that pretreatment with thyroid, hormones decreases catecholamine uptake, In a follow-up study, McNeill, Muschek and Brody (I969) showed that while pretreatment of rats with triiodothyronine significantly increased cardiac phosphorylase a over oontrol values, "both in the absence and presence of adrenaline; the thyroid hormone treatment had l i t t l e effect on adenylate cyclase activity in the same hearts. Cyclic AMP levels in euthyroid and hyperthyroid hearts were also essentially similar. It was therefore apparent that the greater Increase in % phosphorylase a seen in the hyperthyroid animals was not due to any changes in adenylate cyclase activity. This was further supported by Sobel, Dempsey and Cooper (I969) who showed that adenylate cyclase activity x\ras similar in euthyroid and hyperthyroid rat hearts. However Levey and Epstein (1968) working with cat ventricle homogenates found that administration of triiodothyronine and thyroxine in vitro to the incubation mixture would increase adenylate cyclase activity as measured by cyclic AMP production, and that this could be achieved -15-wlthout any Inhibition of phosphodiesterase. A later study (Levey and Epstein, 1969) showed that in the presence of propranolol, triiodothyronine and thyroxine s t i l l Increased adenylate cyclase activity, although the same concentration of propranolol abolished the stimulatory effect of noradrenaline on adenylate cyclase. They also showed that the combination of maximal stimulatory doses of thyroxine and noradrenaline produced an additive effect on cyclic AMP production. This evidence would seem to point towards the existence of two separate adenylate cyclase systems; one responsive to thyroid hormones, and the other to noradrenaline with the a b i l i t y to be blocked by beta-adrenoceptor antagonists. McNeill, LaRochelle and Muschek (1971) were, however, unable to confirm these findings using adenylate cyclase from rat heart. Levey also carried out some studies (Levey, Skelton and Epstein, 1969) with euthyroid and hyperthyrold cats. They showed that when l e f t ventricle muscle was Incubated with nor-adrenaline, the effect on adenylate cyclase was similar In the two groups, although the control values of adenylate cyclase were significantly lower in hyperthyrold than in euthyroid animals. This is similar to McNeill's findings in the anaesthetized rat (McNeill et a l , I969), where adrenaline increased adenylate cyclase act i v i t y in a similar manner in euthyroid and hyperthyrold animals. The only difference being that although hyperthyrold control adenylate cyclase activity was less than euthyroid control adenylate cyclase a c t i v i t y , , i t was not significantly so. This is because the euthyroid ac t i v i t y in the Levey et a l experiment was much higher than in the study of McNeill et a l ; the hyperthyrold values being essentially the same. In the study carried out by McNeill, LaRochelle and Muschek (1971) i t was also shown that following pretreatment with triiodothyronine and theophylline, or triiodothyronine and tripelennamine; addition of noradrenaline resulted in an additive effect on % phosphorylase a, and that a combination of the three treatments resulted in a further enhancement. This indicated that the treatments enhanced the responses of phosphorylase by different mechanisms. In 1974, Young and McNeill showed in the rat Langendorff heart that although control values were similar, the phosphorylase-activating effect of noradrenaline was enhanced by triiodothyronine pretreatment, but that cyclic AMP levels at these doses were similar in both euthyroid and hyperthyrold animals. In order to determine more accurately the point in the phosphorylase activation cycle (figure 1) where the thyroid hormones act, Fraser, Hess and Shanfeld (1969) investigated phosphorylase b kinase, the enzyme responsible for the conversion of phosphorylase b to phosphorylase a. They found that although thyroxine pretreatment resulted In an increase in phosphorylase a and phosphorylase b kinase at pH 8.2 in anaesthetized rat hearts; there was no overall activation of the enzyme, since the acti v i t y at this pH is due to both active and Inactive forms. The ratio of ac t i v i t y -17-at pH 6.8 (active only) to pH 8.2 (active plus inactive) which was used as an index of activation, was in fact lower in hyperthyroid than in euthyroid hearts. No change in cyclic AMP levels was detected. The effect of thyroxine pretreatment differed from that of adrenaline administration, since following Injection of 2 ug per kg l.v. of adrenaline, there was an increase in cyclic AMP, phosphorylase a and phosphorylase b kinase activity. This therefore suggests that the mechanism for thyroid hormone-Induced elevation of cardiac phosphorylase a differs from that of adrenaline. It was postulated that since both electrical stimulation and ++ perfusion with a high Ca medium can also elevate phos-phorylase a without a concommitant increase in cyclic AMP or activation of phosphorylase b kinase, some mechanism Requiring calcium might be involved. Although most investigations were restricted to Interactions of thyroid hormones with catecholamines, McNeill and Schulze (1972) carried out a study on the interaction of histamine with triiodothyronine. They showed that histamine could increase cardiac phosphorylase a, and that this effect was potentiated by prior treatment with t r i -iodothyronine. The activating action of histamine was thought to be brought about by stimulation of adenylate cyclase at a different site than that for the catecholamines. A new hypothesis was put forward by Hornbrook and Cabral (1972) who, noted again that pretreatment of rats with triiodothyronine Increased cardiac phosphorylase a compared -18-to euthyroid rats, and that administration of noradrenaline produced a greater activation of phosphorylase a In hyperthyroid than in euthyroid rats although the contractile response was similar. They therefore proposed that the interaction of thyroid hormones and catecholamines occurred at a site other than the adrenoceptor. This would probably be somewhere in the biochemical chain between adenylate cyclase and phosphorylase (figure 1). They suggested that creatine phosphate (creatlne-P) might be Involved, since its levels were found to be lower in hyperthyroid than in euthyroid hearts. They also demonstrated a negative correlation between phosphorylase and creatine-P levels following administration of noradrenaline. McNeill (1977a,b) carried out a series of experiments to determine whether pretreatment with thyroid hormones sensitized a step in the activation of phosphorylase by cyclic AMP. Langendorff rat hearts were injected with dlbutyryl cyclic AMP and assayed for phosphorylase a at various times following its administration. Phosphorylase a levels increased steadily following injection of dlbutyryl cyclic AMP, reaching a maximum at eight minutes (McNeill, 1977a). At a l l times studied, activation was greatest in hearts from triiodothyronine-pretreated rats. When the Langendorff hearts were frozen five minutes after injection, dlbutyryl cyclic AMP was shown to produce a dose-dependent increase in phosphorylase a. Again, activation was greatest In hearts from triiodothyronine-pretreated animals (McNeill, -19-1977a,b). To test the hypothesis that dibutyryl cyclic AMP produced a greater response In hyperthyrold animals because more drug could enter the heart ce l l s , trltlated dibutyryl cyclic AMP was Injected and. the amount of activity retained in the heart af five minutes was measured. Hyperthyrold hearts retained significantly less ac t i v i t y than euthyroid hearts, thus discounting this theory (McNeill, 1977b). It was suggested that activation of protein kinase might be the key step in the supersensitivity of phosphorylase a in the hyperthyrold heart. The next component of the phosphorylase activation cycle to be investigated was accordingly cyclic AMP-dependent protein kinase, the enzyme responsible for conversion of inactive phosphorylase b kinase to its active form. Gibson, Tichonlcky and Kruh (1975) found that following triiodo-thyronine pretreatment, cytosol protein kinases from rat heart were not significantly different from those obtained from euthyroid animals, nor was cyclic AMP stimulation of these protein kinases affected by triiodothyronine pretreatment. However when non-hlstone proteins were extracted from cardiac nuclei of animals pretreated. with triiodothyronine, there was a rapid increase in protein kinase activity. Following administration of the hormone there was an increase in activity commencing at two hours, reaching a maximum after three days treatment, and. declining to control levels after one week of treatemnt. This increase in protein kinase a c t i v i t y was thought to represent an early step In the development of - 2 0 -cardiac hypertrophy, through a regulatory action on the genes responsible for RNA production. Katz and co-workers (1977) working with Langendorff hearts from rats pretreated with triiodothyronine for three days (at a three-fold higher dose than that used by Gibson and co-workers), found no difference in protein kinase ac t i v i t y from the 1 2 , 0 0 0 x g supernatant fraction, when compared to euthyroid controls. In the presence of noradrenaline there was a significant Increase In protein kinase ac t i v i t y in both groups of animals 5 the degree of activation being significantly less in hearts from hyperthyroid animals than in those from the controls. It therefore seems certain that the increase in phosphorylase a activity seen In hyperthyroid animals cannot be explained on the basis of a change in the supernatant fraction protein kinase activity. The supernatant fraction of Katz and co-workers may be similar to the cytosol fraction of Gibson and co-workers, since the two studies agree that these fractions showed no difference In protein kinase activation between the two groups. Although an increase in nuclear protein kinase activ i t y was seen by Gibson's group, this had returned to control values by one week of treatment with triiodothyronine. Since Katz used a three-fold greater dose of triiodothyronine; by three days, nuclear protein kinase levels might well have been low i f they had been studied. However, i t is very d i f f i c u l t to compare the two studies c r i t i c a l l y , due to the differences of dosage, and the different protein kinase -21-extract Ion methods used. Frlesen, Allen and Valadares (I967) showed that calcium was capable of activating cardiac phosphorylase a, and It has since been shown (Hartley and McNeill, 1976) that this activation can occur without any changes in cyclic AMP levels. In addition, the elevation in % phosphorylase a following calcium administration was shown to be greater in hearts from hyperthyrold than euthyroid rats, up to ten seconds after the peak in contractile force. By thirty seconds after the peak force change, % phosphorylase a levels from hyperthyrold hearts were significantly lower than those from euthyroid hearts. Propranolol had no effect on the calcium-induced increase in phosphorylase a in either group, indicating that the beta-adrenoceptor was not Involved. A study was also carried out by Aronson (1976); the results of which closely agree with Hartley and McNeill showing that although calcium did Increase cardiac phosphorylase a, there was l i t t l e difference between euthyroid and hyperthyrold animals. Nemecek and Hess (1974) carried out a study to determine the effect of altered sympathetic acti v i t y on the metabolic actions of the thyroid hormones. Groups of animals used were those treated with nerve growth factor (NGP), to increase sympathetic innervation; nerve growth factor antiserum (AS), to produce a permanent immunological sympathectomy; and 6-hydroxydopamine (6-OHDA), to destroy adrenergic nerve terminals reverslbly or Irreversibly. It was shown that the lmmunosympath-ectomlzed and NGF-treated mice s t i l l responded to triiodothyronine -22-pretreatment with tachycardia, Increased oxygen u t i l i z a t i o n and increased phosphorylase a activity; but the AS-treated mice did not develop cardiac hypertrophy. Increasing the density of sympathetic innervation with NGF resulted in potentiation of the trilodothyronlne-lnduced increases in heart rate. A drug, P-286, which blocks the release of catecholamines from AS-resistant chromaffin tissues such as the adrenal medulla, was used to eliminate responses due to circulating catecholamines. P-286 did not prevent either the increase in heart rate or % phosphorylase a seen following triiodothyronine pretreatment. Neonatal pre-treatment with AS combined with P-286 administration resulted in significant bradycardia, but no change in % phosphorylase a compared to euthyroid controls. Surgical demedullatlon and/or treatment with 6-OHDA prevented the thyroxine-lnduced increase in phosphorylase a, and decreased cardiac noradrenaline levels without affecting the tachycardia and Increased oxygen consumption and heart weight. It therefore seems that the thyroid hormone-induced Increases in cardiac phosphorylase a are dependent on the presence of catecholamines; while the calorlgenlc and chronotropic.effects are independent of the sympathetic nervous system. One interesting point to come out of the work on phosphorylase a is that i f the control % phosphorylase a is measured in an anaesthetized animal (Frazer et a l , 1969; Hess and Shanfeld, 1965? Hornbrook and Brody, 1963; Hornbrook et a l , 1965; McNeill and Brody, I9685 McNeill et a l , 1969; McNeill et a l , 1971; and Nemep.ek and Hess, 1974), the resting levels in the hyperthyrold. animals are significantly greater than in the euthyroid animals. However, i f control % phosphorylase a is measured, in the Langendorff heart preparation (Aronson, 1976} Hartley and McNeill, 1976$ Hornbrook and Cabral, 1972; McNeill, 1977a; McNeill and Schulze, 1972; and. Young and McNeill, 197*0, resting levels in the hyperthyrold. animals are not significantly different from the euthyroid, animal, indicating that phosphorylase a in the anaesthetized heart is probably being activated by endogenous catecholamines. After administration of exogenous catecholamines to both groups, there Is a significantly greater increase in % phosphorylase a In the hyperthyrold heart. Hess and Shanfeld (I965) showed that pretreatment with reserpine reduced the increased % phosphorylase a seen in anaesthetized, hyperthyrold rats compared to control euthyroid levels. Also Nemecek and Hess (197*0 showed that adrenal demedullatlon and chemical sympathectomy of thyroid hormone-pretreated animals, followed by anaesthetization and measurement of cardiac phosphorylase a levels did not produce the increase which they had. previously seen in thyroid hormone-pretreated animals compared to euthyroid controls. This evidence would support the notion that circulating catecholamines increase cardiac phosphorylase a levels In the anaesthetized, thyroid hormone-pretreated animal. - 2 k -Involvement of the Parasympathetic Nervous System. Differing responses between euthyroid and hyperthyroid heart tissues are also notlcable when agents which affect the parasympathetic nervous system are studied. Cairoll and Crout (1967), found that in thyroxine-treated rats, stimulation of the vagus at varying frequencies produced a decrease in heart rate which was less than that observed in euthyroid controls. A larger study by Frazer and Hess (1969) also showed that the bradycardia produced by vagal stimulation in hyperthyroid rats was less than In euthyroid controls. They also noted that the more thyrotoxic the rat became, the less the heart rate decreased in response to vagal stimulation. The bradycardia seen In the hyperthyroid hearts was not accompanied by any change in phosphorylase a_ levels. Myocardial acetylcholinesterase activity was measured to see i f there was Increased enzyme act i v i t y in the hyperthyroid animals resulting in a rapid breakdown of acetylcholine and a smaller response, but the levels were similar in euthyroid and hyperthyroid animals. To determine whether thyroxine pretreatment produced a decreased sensitivity of the cardiac muscarinic receptors, acetylcholine vras injected in vivo and caused an equal depression of cardiac phosphorylase a, heart rate and blood pressure in both groups of animals. A follow-up study (Hess and Bilder, 1972) showed that choline acetyltransferase (choline acetylase- the enzyme which catalyses the acetylation of choline) -25-acti v i t y was similar in both euthyroid and hyperthyrold rat hearts; thus ruling out any decrease in synthesis of acetylcholine in the hyperthyrold animals which might have accounted for the reduced bradycardia and phosphorylase activation seen in the hyperthyrold animals following vagal stimulation. They also showed that although free plasma calcium was lower in hyperthyrold than in euthyroid rats, this was not responsible for the decreased response to vagal stimulation, since lowering the free plasma calcium in normal rats by infusion with EDTA did not change the response normally seen in the euthyroid animals. Finally, choline Infusion in the hyperthyrold rats during stimulation of the vagus resulted in greater cardiac slowing than in euthyroid controls. Since the resting heart rate in both groups was unchanged by the choline infusion, the response of the hyperthyrold hearts to vagal stimulation is unlikely to be due to a muscarinic action of choline. From these findings i t was postulated, that thyroxine treatment might lower free extracellular choline, and/or inhibit uptake of choline into parasympathetic neurons. - 2 6 -Dlrect Action of Thyroid Hormones. An easy answer to the problem of why thyrotoxic hearts have different contractile characteristics than their euthyroid counterparts would be that the. thyroid hormones themselves have a direct action on the heart. Although i t has been demonstrated that single cardiac cells or heart fragments w i l l respond to thyroid hormones in vitro; few reports show a response by intact hearts or heart portions. The reason for this may be because the tachycardia and other manifestations of hyperthyroidism shown following in vivo administration of thyroid hormones do not become apparent until several hours or days later (Brewster et a l t 1956; Hess and Shanfeld, 1965; Hirvonen and Lang, 1962; Markowltz and Yater, 1932; Priestley, Markowltz and Mann, 1931; Skelton, Karch and Wildenthal, 1973; van der Schoot and Moran, 1965; Wildenthal, 1971, 1972; and Yater, 1931). Markowltz and Yater (1932) demonstrated that explanted heart fragments from two day chick embryos which were devoid - 5 of functioning nerve tissue responded to 3x10 M thyroxine with an Increased number of pulsations, although adrenaline and acetylcholine had no effect. This response could also be demonstrated In fragments from chick embryos up to nine days old, which had developed functioning nerve elements and were capable of responding to adrenaline and acetylcholine. More recently, Wollenberger (1964a,b; 1975) has shown that a twenty-four hour culture of chick embryo ventricle cells -7 responds to 6.6x10 M triiodothyronine within ten minutes, - 2 7 -reaching a maximal increase of twenty beats per minute and remaining at that rate u n t i l washed. This action could be blocked by concentrations of veratramlne and pronethalol which did not affect the rate of beat of the cultured heart cells in trilodothyronlne-free medium (Wollenberger, 1964a,b), but this may be due to an unspeclflc antagonistic effect. Vlldenthal (1972) working with cultured foetal mouse whole hearts could find no significant difference in rate -7 -6 between controls and hearts exposed to 5x10 M and 5x10 M triiodothyronine for 2^ to 3 hours or two days. However, i f the drug was added to the culture medium for four to ten days (Wlldenthal, 1971), a significant increase In rate over controls was observed, -6 In hearts exposed to 5x10 M triiodothyronine for two - 8 -7 days, 1x10 M and 1x10 M noradrenaline produced an increase in rate which was significantly greater than in the group -7 -6 exposed to 5x10 M triiodothyronine or controls. At 1x10 M noradrenaline, a l l groups showed a maximal increase in rate with no difference between them. Early workers showed that thyroxine had no effect on the frog Straub heart rate (Kalnlns, 1928), or on the anaesthetized dog heart rate (Coelno and Rocheta, 1929). Later work in the Isolated frog heart by Klelnfeld, Rosenthal and Stein (1958) showed that 0,2 mg of thyroxine or triiodothyronine produced a slight increase in heart rate? triiodothyronine being more potent than thyroxine. Higher doses produced a decrease in heart rate. The methods do not describe -28-how long the hearts were In contact with the hormones, but i t is unlikely that i t was greater than a few minutes. Harvey and - 5 MacRae (1931) showed that administration of 1x10 M thyroxine to turtle hearts had no effect on heart rate for up to several days of treatment. Rabbit langendorff hearts -7 -6 perfused with 5x10 M and 5x10 M triiodothyronine or thyroxine were also shown to be unaffected (Lewis and McEachern, 1931)* More recently, Lee, Lee and Yoo (1965) found that doses of -4 thyroxine up to 1x10 M had no effect on the rate of spon-taneously beating rabbit atria. Balrd and. Spilker (1970) - 5 - 5 reported that 1.4x10 M to 8.3x10 M triiodothyronine produced dose-related increases in the tension developed by Isometrlcally Contracting guinea pig l e f t a t r i a . At the peak, the tension increase averaged only 12 percent, but was s t a t i s t i c a l l y different from the tension increase at lower doses. Thyroxine -5 - 5 (1x10 M to 6x10 M) had no effect on inotropic activity. Conversely, Skelton, Karch and Wildenthal (1973) found that - 6 -4 neither 1x10 M to 1x10 M thyroxine nor triiodothyronine had any effect on the tension developed by isometrlcally contracting cat papillary muscle, or l e f t atria; or guinea pig l e f t atria during periods of observation of up to eight hours. Experimental conditions of the study were almost Identical to those of Baird and Spilker, but the results do not agree. No explanation was given for this difference. , Therefore, although thyroid hormones have been shown to affect heart rates of cultured isolated cardiac cells f a i r l y quickly; in Isolated mammalian hearts this cannot easily be -29-demonstrated. However, cultured foetal hearts exposed to thyroid hormones for periods of time longer than four days do demonstrate an increase in heart rate over controls, similar to in vivo Induction of tachycardia by thyroid hormones. This would tend to indicate that there may be membrane or ultrastruetural damage in the preparation of the c e l l cultures, resulting in an immediate tachycardia when the thyroid hormones are added to the medium, and there-fore that these preparations do not represent a truly physiological situation. -30-Muscle Mechanics. As well as affecting rate, tension development and enzyme content, thyrotoxicosis has been shown to affect the intrinsic contractile properties of the heart, and produce changes in the contractile protein content. In 1965» Inchlosa and Freedberg described experiments which demonstrated that pretreatment of rabbits with triiodothyronine or thyroxine produced a 35 percent hypertrophy of the ventricles of the heart accompanied by a 49 percent increase in the contractile protein actomyosin. Later work (Yazaki and Raben, 1975)» demonstrated that thyroxine treatment increased cardiac myosin ATPase ac t i v i t y (Ca -activated) over a period of two weeks. The cardiac myosin formed had altered enzymatic properties when compared to myosin extracted from normal rabbits, suggesting a structural difference. This difference was specific for the heart, since skeletal myosin was identical in the two groups. The synthesis of this new myosin might therefore mediate the thyroid hormone-induced changes in contractility. However, in the rat the properties were different. Cardiac myosin from thyroidectomlzed rats showed a pattern of activity similar to the myosin from euthyroid rabbits. With euthyroid and thyroxlne-treated rats, cardiac myosin activity was similar to that from thyroxlne-treated rabbits. Rat cardiac myosin therefore seems to be more sensitive to thyroid hormone than rabbit cardiac myosin; since the structural -31-changes, which in the rabbit required thyroxine treatment to occur, were observed in the rat even in the euthyroid state. Since analysis of the amino acid content and molecular weight of cardiac myosin demonstrate no significant changes between the hypothyroid and hyperthyrold state, i t is not yet possible to determine where the structural change occurs and what form i t takes, although i t is unlikely to be a major difference. The contractile properties of the myocardium have been more extensively studied than the mechanisms behind them. In 1967, Bucclno and co-workers found that Isolated papillary muscles from anaesthetized hyperthyrold cats demonstrated an augmented velocity of shortening and. rate of tension development, and decreased duration of active state compared to euthyroid muscles. Isometric tension was sl i g h t l y higher In the muscles from hyperthyrold animals and lower in the muscles from euthyroid animals. This was supported by Levey, Skelton and Epstein (I969), and Skelton, Su and Pool (1976). No significant difference was found between papillary muscle levels of creatine-phosphate and ATP levels in anaesthetized euthyroid and hyperthyrold cats. This differs from the results of Hornbrookaand Cabral (1972), who found that In both anaesthetized and perfused rat heart ventricles, creatine-phosphate levels were significantly lower in the hyperthyrold animal, ATP levels were similar in eu-thyroid and hyperthyrold perfused hearts, and lower in hyperthyrold non-perfused hearts. -32-Later work "by Buccino and co-workers (1968) showed that pretreatment with reserpine did not significantly alter the Increased velocity of shortening in either euthyroid or hyperthyrold cat papillary muscles. That i s , the velocity of shortening was s t i l l significantly increased in the hyperthyrold animals compared to euthyroid controls, and does not seem to dependlupon endogenous catecholamine stores. A series of studies by Strauer and Scherpe were carried out to measure various indices of contractility. They demonstrated an increase of both the maximum rate of l e f t ventricular pressure developemnt (dp/dt ) and pressure f a l l max (dp/dt ) In anaesthetized hyperthyrold cats compared to min euthyroid controls. The time Interval from the beginning of ventricular contraction to peak dp/dt was found to be max shortened in hyperthyroidism. These changes were considered by them to be evidence for a direct increase of contractility in hyperthyroidism (Strauer and Scherpe, 1975a). In cat papillary muscles, the isotonic contraction velocity (dl/dt ) max'; was significantly higher in the hyperthyrold state, as was the maximal isometric tension rise velocity (dT/dt ), (Strauer max and Scherpe, 1975b,d). Treatment with propranolol in vitro reduced the velocity values of Isotonic (dl/dt ) and Isometric max (dT/dt ) contraction in both euthyroid and hyperthyrold animals max in a dose-dependent manner. Hyperthyrold animals were much more responsive to the negative inotropic effect of propranolol, the f a l l In dl/dt being much greater In hyperthyrold than in max euthyroid tissues. Heart rate in vivo f e l l in a dose-dependent -33-manner following administration of propranolol to "both groups, but in hyperthyroid animals the f a l l in heart rate was much smaller than the f a l l in maximal pressure rise velocity of the l e f t ventricle (dp/dt ). The greater f a l l max of dp/dt in the hyperthyroid heart could explain the max increased responsiveness of the hyperthyroid papillary muscles. This decrease of contractility seen after use of propranolol, accompanied by a decrease in myocardial oxygen consumption (Strauer and Scherpe, 1975d), makes the use of propranolol in the c l i n i c a l situation quite appropriate (Strauer and Scherpe, 1975c). Therefore, myocardial tissues from hyperthyroid animals demonstrate an enhanced rate of tension development and a decreased time to peak tension. Whole hearts demonstrate an Increased rate of l e f t ventricular pressure development and pressure f a l l , resulting In an overall increase in contractility of both the contraction and relaxation phas e. -34-Involvement of the Peripheral Blood Vessels. Much of the early work on the effect of thyroid hormone pretreatment on peripheral blood flow Is not quantitative, but nevertheless as early as 1931* i t was noted that in four out of five rabbit Langendorff hearts, there was an increased coronary -6 blood, flow following perfusion with 3.2x10 M thyroxine. A decreased flow was seen in one heart. "These changes were not striking," (Lewis and McEachern, 1931). In 1933, Herrick and co-workers carried out experiments which demonstrated, "a tremendous Increase" in blood flow through the unanaesthetized dog femoral artery following ingestion of dessicated thyroid gland. From his data, a t-test performed shows the increase in blood flow following thyroid gland. ingestion to be significantly greater than that In control animals at P<0.05. The same group of workers (Essex ert a l , 1936), found that intravenous injection of thyroxine (lmg/kg) Into unanaesthetized dogs resulted in increased coronary blood flow forty-eight hours later, but no significant change at 42 twenty-four hours. Wurtman and co-workers, using K as a marker, determined that cardiac blood flow was increased, in hyperthyrold unanaesthetized rats. This Increase in blood flow through the heart paralleled the Increase in heart weight, so flow per unit mass remained unchanged (Wurtman et a l , I 9 6 3 ) . Administration of adrenaline increased, the percent of cardiac output delivered to the heart in a dose dependent manner In both euthyroid and hyperthyrold animals. Hyperthyrold hearts -35-exhiblted a steeper dose-response curve to adrenaline than the euthyroid hearts. This was probably due to Increased delivery of catecholamines to the heart (Wurtman et a l , 1964). Although thyroid hormone treatment did not significantly alter hind limb blood flow of cats and dogs; following adrenaline and noradrenaline infusion hyperthyroid animals demonstrated a less marked reduction of blood flow than did euthyroid animals (Zsoter, Tom and Chappel, 1964). In contrast, the vasoconstriction produced by angiotensin and vasopressin was not significantly affected, suggesting an enhanced sensitivity of beta-adrenoceptors. In summary, experimental hyperthyroidism produces tachycardia, accompanied by an increased rate of tension development, decreased time to peak tension and Increased velocity of shortening. Tension developed is reduced compared to euthyroid controls. Absolute increases in rate in response to catecholamines are similar in euthyroid and hyperthyroid hearts, but there is an augmented activation of phosphorylase a In the hyperthyroid animals. . No difference in act i v i t y of cyclic AMP, adenylate cyclase, protein kinase or phosphorylase b kinase has been detected between euthyroid and hyperthyroid animals; although in one study, creatine-P, a source of high energy phosphate, has been shown to be lower in hyperthyroid than in euthyroid hearts. Propranolol and other beta-adrenoceptor blocking agents -36-produce either no difference, or less slowing in the hyperthyroid heart when compared to the euthyroid heart. Reserplne pretreatment may reduce heart rate in hyperthyroid animals to that of the euthyroid controls, "but does not affect the noradrenaline-induced potentiation of phosphorylase a. Vagal stimulation of hyperthyroid hearts results In less bradycardia than is seen in euthyroid hearts at the same frequency. This bradycardia was not associated with changes in myocardial acetylcholinesterase or choline acetyltransferase levels. The cardiac manifestations of hyperthyroidism are unlikely to be due to an immediate direct action of thyroid hormones since few studies have demonstrated this in the intact heart. Finally in hyperthyroidism, peripheral blood flow has been shown to be increased, including flow through the coronary blood vessels. -37-Purpose of the Investigation. This study set out to Investigate contractile and enzymatic changes in the atria and right ventricle of the hyperthyrold rat heart. Previously the majority of work had. "been carried out on the Langendorff or intact heart, with few instances of studies where the separate portions were used (Cravey and Gravensteln, 1965; Their, Gravenstein and Hoffman, 1962? van der Schoot and Moran, 1965). We f e l t that It would "be interesting to follow the isoproterenol-induced increase in phosphorylase a in both euthyroid and hyperthyrold myocardial tissues and determine whether any differences could be seen. We also wished to determine the effect of using rate-controlled tissues, such as the l e f t atrium and right ventricle, where factors such as changes in rate and coronary blood flow could be controlled. Following studies In the right and l e f t a t ria and. right ventricle, where contractility changes occurred following isoproterenol administration that were significantly different between the two groups, but were not accompanied by significant differences in phosphorylase a activity, we decided, to work with the Langendorff heart preparation. It has been shown in the Langendorff heart preparation (Hornbrook and Cabral, 1972; McNeill and Schulze, 1972; and Young and McNeill, 197*0 and anaesthetized rat heart (Hess and Shanfeld, 1965; Hornbrook et a l , 1965; McNeill and Brody, I9685 McNeill, LaRochelle and Muschek, 1971; and McNeill, Muschek and Brody, 1969), that catecholamine -38-adminlstration results In an Increase In myocardial phosphorylase a, which is significantly higher in the hyperthyroid animals. It was therefore surprising that no differences in phosphorylase a activ i t y could be detected between the two groups in our study of atria and ventricles. To test whether the higher level of phosphorylase a found in the hyperthyroid whole heart was due to an Increased coronary blood flow, resulting in increased levels of catecholamine being made available for beta-adrenoceptor binding, a study was undertaken in which the perfusion pressure was decreased so coronary blood flow in the hyperthyroid hearts was now the same as that of the euthyroid hearts. The response of phosphorylase a to isoproterenol was then determined, to see i f phosphorylase a activation was now similar to "that of the euthyroid hearts. Finally, a study was carried out to determine whether the receptors through which the inotropic effect of isoproterenol was mediated In the right atrium, were the same in the euthyroid and hyperthyroid state. This study took the form of a pA test as described by Schlld (1957)» and utilized 2 propranolol as the antagonist. -39-MATERIALS AND METHODS. Methods. Male Wistar rats weighing 250-350g were used throughout the study and received food (Purina Lab Chow) and water ad libitum. Half of the animals were made hyperthyroid by subcutaneous injection of 3,3 ,5 -trliodo-L-thyronine (500 mg per kg) in alkaline saline, administered dally for three days. This treatment has previously been shown to make rats hyperthyroid (McNeill et a l , 1969). A l l studies carried out involved both euthyroid and hyperthyroid rats. The animals were pretreated with heparin sodium (8 mg per kg s . c ) , ten minutes prior to sacrifice. They were stunned by a blow to the head, and the hearts were rapidly removed. Preparation of Atria, Atria were dissected free by the method of Levy (1971), and suspended by the method of Katzung (I968) in organ baths 0 containing Chenoweth-Koelle solution at 37 , (Chenoweth and Koelle, 1946), The solution was aerated with 95$ oxygen, 5% carbon dioxide. Contractile force and rate were measured by means of a Palmer cl i p placed on the apex of the atrium and connected to a Grass force displacement transducer, and recorded on a Grass model 79D polygraph. Diastolic tension was adjusted to 1 g. The atria were allowed to equilibrate for 30 minutes before the drug was added. -40-Rlght atria were allowed to contract spontaneously. Left atria were driven at 1Hz with 5 millisecond square wave pulses, using one to four volts, by a Grass model S6 stim-ulator. Dose-response curves were measured, using the cumula-tive method, described by van Rossum and van der Brink (19o3)» and van Rossum (I963). Rates were measured 75-90 seconds after drug administration for right a t r i a . For l e f t a tria, the maxlmun tension attained was recorded. Preparation of Ventricles. Rat right ventricles were dissected, free from the l e f t ventricles and suspended in the organ baths of Chenoweth-Koelle 0 solution at 37 , aerated with 95$ oxygen, 5% carbon dioxide from an electrode designed in our laboratory, (Figure 2). Diastolic tension was adjusted, to l g . The ventricles were driven at 3Hz with 5 millisecond square wave pulses using one to six volts, by a Grass model S6 stimulator. Dose-response curves were obtained as described, above, and maxlmun tension attained was recorded. Preparation of Langendorff Hearts. Whole hearts were set up by the method of Langendorff ( I 8 9 5 ) , using a constant pressure apparatus similar to that described by Chenoweth and KoeTle (1946), at a reservoir height of 40cm of perfusate, Chenoweth-Koelle solution at 0 a reservoir temperature of 37.5 and aerated with 95$ oxygen, 5% carbon d.ioxld,e was used as perfusate. Diastolic tension was -41-Figure 2. Electrode used, for Ventricle Stimulation.  (Actual Size). Platinum Electrodes. -42-adjusted to l g . Contractile force and rate were measured by-means of a Palmer c l i p placed on the apex of the heart and connected to a Grass force displacement transducer,, and recorded on a Grass model 79 polygraph. Hearts were allowed to equilibrate unti l rate and contractile force were constant before drugs were added. For a l l studies, dose-response curves to isoproterenol were carried out I n i t i a l l y ; and a sub-maximal dose was chosen for a time-response study. To determine the biochemical responses; at various times following administration of the chosen sub-maximal dose, the atria and Langendorff hearts were frozen by clamping them with tongs previously chilled in a mixture of 2-methyl butane and dry ice (Wollenburger et-al, i 9 6 0 ) . The ventricles were rapidly removed from the bath and dropped into the 2-methyl butane 0 mixture. A l l samples were stored at -80 u n t i l assayed for phosphorylase. Measurement of pA 2 A pA study was carried out using right atria prepared as 2 described above, using the method of Schlld (1957). The agonist used was Isoproterenol, and the antagonist propranolol. Doses -7 -7 -6 of antagonist used, were 2x10 M, 5x10 M, and 1x10 M. ED 50 values were calculated for each tissue. From the pooled ED 50 values at each dose of antagonist, the pA values for euthyroid 2 and hyperthyrold rat right atria were calculated. Measurement of Coronary Blood Flow. Coronary blood flow determinations were carried out using the Langendorff heart technique described above. Care was taken to ensure that there were no leaks in the system, and the volume of perfusate leaving the heart per unit time was measured as described by Lewis and McEachern ( 1 9 3 D . This volume was taken as coronary blood flow. By raising or lowering the height of the reservoir the volume of perfusate leading the heart could be changed. Phosphorylase Assay. The phosphorylase assay used is a modification of the method of Corl and Cori (19^0). .".Liberation of inorganic phosphate during the synthesis of glycogen from glucose-l-phosphate is used as an Indicator of phosphorylase activity. The method followed is that of Diamond and Brody (19&5). Heart samples weighing 70-100 mg were homogenised using a Polytron (Brinkmann Instruments, Rexdale, Ont.) in 200 volumes of a solution containing 0.05M Tris buffer (pH 6.8), 0.001M EDTA, 0.01M NaF, and 0.3% serum albumin. A l l procedures 0 were carried out at 0-4 . After centrifugatlon of the homogenate at 10,000g for 10 minutes, 0.2 ml aliquots of the 0 supernatant were incubated at 37.5 • In test tubes containing 0.05M Tris buffer (pH 6.8), 0.k% glycogen, 0.01M glucose-l-phosphate, 0.001M EDTA, 0.01M NaF, and 0.3% serum albumin in a f i n a l volume of 1,0 ml. Duplicate samples of -44-the supernatant solutions were Incubated in the same reaction mixture containing, in addition.,AMP in a f i n a l concentration of 0.001M. The reaction was terminated by the addition of 2.0 ml of 10$ tri-chloroacetic acid. The samples were then centrifuged. at 2,500g for 10 minutes, and the supernatants assayed for Inorganic phosphate by the method, of Flske and SubbaRow (1925). The rate of liberation of inorganic phosphate was linear over the time studied in a l l tissues, and was proportional to enzyme concentration. Incubation times chosen were 16 minutes for the right ventricle and whole heart, and 30 minutes for the right and l e f t a t r i a . When the glycogen primer was omitted from the reaction mixture, the liberation of Inorganic phosphate was negligible. The amount of inorganic phosphate liberated in the absence of AMP represented, phosphorylase a, and the amount liberated In the presence of AMP represented total phosphorylase, Results are given as % phosphorylase a,, which Is; activity In the absence of AMP 1 n n a c t i v i t y in the presence of AMP St a t i s t i c a l Methods. Stat i s t i c a l analysis was done by the Student's t-test for unpaired, data. A probability of p<0.05 was taken as the criterion for significance. -45-Materlals. Drugs used were DL-Isoproterenol, DL-Propranolol HC1, i t and 3,3 ,5 -trliodo-L-thyronine, ( a l l from Sigma Chemical Corporation). -46-RESULTS . Effect of Three Day Pretreatment with 3*3 ,5 -Trllodo-L-Thyronine on Rat Body Weight. Control and test rats were weighed before pretreatment with drug or vehicle for three days, and again on day four, prior to sacrifice (figure 3). Control rats gained weight in a linear manner, from 275g to 2958 over the four day period. The weights on days three and four were significantly different from that on day one. Conversely the test rats did not show any significant Increase in weight, with a mean weight of 283g on both day one and day four. On day four, the control and hyperthyrold animal weights differed significantly from each other. Effect of Isoproterenol on Rate of Right Atria from  Euthyroid and Hyperthyrold Rats. Isoproterenol produced dose-related increases in rate in both euthyroid and hyperthyrold rat hearts (figure 4). In the absence of drug, the two groups had significantly different i n i t i a l heart rates. The hyperthyrold atria had significantly higher rates than the euthyroid atria throughout the extent of the isoproterenol dose-response curve. Despite the differing I n i t i a l heart rates, the absolute increase In rate following Isoproterenol administration was similar in both groups. -47-Fi£ure_Ji. Effect of Three Day Pretreatment with i t 3i3 i 5 -Trilodo-L-Thyronine on Rat Body Weight. Rats were weighed dally before pretreatment with drug or vehicle for three days, and again on day four, prior to sacrifice. Each point represents the mean weight (g) + S.E.M. of 38-42 observations. a Significantly greater than day one value at P-<0.05. b Euthyroid significantly greater than hyperthyroid at P<0.05. -48-Day of Treatment -if-9-Figure h. Effect of Isoproterenol on Rate of Right Atria from  Euthyroid and Hyperthyroid Rats. The plot depicts the effect of various doses of isoproterenol on rate in euthyroid and hyperthyroid rat right a t r i a . Rates were measured 75-90 seconds after drug administration. Each point represents the mean rate (beats per minute) -t- S.E.M. of 17-19 observations. A l l points were significantly different from the corresponding control value at P-<0.05. -50-540 r 500 4) •| 460 0) CL I 420 o DC 380 340 —.o euthyroid A A hyperthyroid ' i i I I I 1 C 9 8 7 6 5 4 Dose of Isoproterenol (—log M) - 5 1 -Effect of Isoproterenol on % Increase in Rate of Right Atria  from Euthyroid and Hyperthyrold Rats. When the data was expressed as absolute rate, the Increase in rate in response to Isoproterenol was the same in the two groups. However, plotting the same data in terms of % increase in rate put a different aspect on the results. In this form, the percent increase was significantly less in the hyperthyrold than in the euthyroid tissues (figure 5 ) . Effect of Isoproterenol on Tension of Left Atria from  Euthyroid and Hyperthyrold Rats while stimulating at 3Hz. Isoproterenol produced a dose-related Increase in tension in the l e f t a t ria from both euthyroid and hyperthyrold rats, as shown In figure 6. The tension development was greater in euthyroid than in hyperthyrold a t r i a , both in the absence and presence of Isoproterenol. This difference was -7 significantly greater at doses of isoproterenol over 1x10 M. -8 Changes in Rate Following Administration of jxlO M  Isoproterenol to Euthyroid and Hyperthyrold Rat Right Atria. -8 From the dose-response curve in figure 4 , 5x10 M isoproterenol was chosen as a sub-maximal dose for a time-response study. Rates were studied In euthyroid and hyperthyrold rat right atria at various times following administration of the drug. The difference in rate between euthyroid and hyperthyrold -52-Effect of Isoproterenol on % Increase In Rate of Right  Atria from Euthyroid and Hyperthyroid Rats. The plot depicts the effect of various doses of isoproterenol on the % increase in rate of right atria from euthyroid and hyperthyroid rats. Each point represents the mean % increase In rate + S.E.M. of 17-19 observations. a Euthyroid significantly greater than hyperthyroid at P<0.05. -53-i i i i i i C 9 8 7 6 5 Dose of Isoproterenol (—log M) -54-Figure 6. Effect of Isoproterenol on Tension of Left Atria from  Euthyroid and Hyperthyrold Rats while stimulating at 3Hz, The plot depicts the effect of various doses of Isoproterenol on the absolute tension of l e f t a t r ia from euthyroid and hyperthyrold rats. Maximal tension attained was recorded. Each point represents the mean tension (g) + S.E.M. of 17-20 observations, a Euthyroid significantly greater than hyperthyrold at P<0,05. -55-1.8 r 1.6 1.4 .1 1.2 C 1.0 0.8 0.6 *a «,—.*<> euthyroid A — — * A hyperthyroid 8 Dose of Isoproterenol (-log M) - 5 6 -atria was maintained throughout the time-response curve (figure 7). Rates of the hyperthyrold atria were s i g n i f i -cantly higher than those of the euthyroid atria at a l l times studied. Changes in % Phosphorylase a Following Administration of  5x10 M Isoproterenol to Euthyroid and Hyperthyrold Rat  Right Atria. The Increase in % phosphorylase a following admlnis-- 8 tration of 5x10 M isoproterenol in rat right atria is shown in figure 8 . In the absence of drug, % phosphorylase a levels were significantly greater i n the hyperthyrold at r i a , but this was not maintained when Isoproterenol was added to the bath. At a l l other times studied, no significant difference was' found between % phosphorylase a levels in euthyroid or hyperthyrold rat right a t r i a . However, In both groups, % phosphorylase a levels Increased significantly following administration of the drug, compared to the appropriate control; reaching maximal values at eighty to one hundred seconds. -57-Figure 7. -8 Changes In Rate Following: Administration of 5x10 M  Isoproterenol to Euthyroid and Hyperthyroid Rat Right Atria. The plot depicts the change in rate of euthyroid and hyperthyroid rat right a t r i a , at various times following administration of a sub-maximal dose of isoproterenol. Each point represents the mean rate (beats per minute) + S.E.M. of 5-88 observations. A l l points were significantly different from the corresponding control value at P<0.05. -58-Time (sees.) after injection of 5x10 M Isoproterenol -59-Flgure 8. Changes In % Phosphorylase a Following Administration of  5x10 M Isoproterenol to Euthyroid and Hyperthyrold Rat  Right Atria. The plot depicts the changes in % phosphorylase a content of euthyroid and hyperthyrold rat right a t r i a , frozen at various times following administration of a sub-maximal dose of Isoproterenol. Each point represents the mean % phosphorylase a t S.E.M. of 8-18 observations. a Hyperthyrold significantly greater than euthyroid at P<0.05. -60--61-A Comparison of pA Values for Propranolol in Euthyroid 2 and Hyperthyroid Rat Right Atria. The Schlld olot used to determine the pA values for 2 propranolol in euthyroid and hyperthyroid rat right a t r i a is shown in figure 9. There was no significant difference between the two values; euthyroid atria having a pA of 7.0996, and 2 hyperthyroid atria a pA of 7.1180. This Indicates that 2 the receptors involved in the inotropic response of the rat right atrium to isoproterenol are the same in euthyroid and hyperthyroid animals. Effect of Isoproterenol on Changes in Tension of the Right  Ventricle of Euthyroid and Hyperthyroid Rats while stimulating  at 3Hz. Isoproterenol produced a dose-related increase in tension in the right ventricles from both euthyroid and hyperthyroid rats as shown In figure 10. The tension developemnt was significantly greater in euthyroid than in hyperthyroid -6 -5 ventricles at doses of 1x10 M and 1x10 M Isoproterenol. In the absence of drug, the tension developed by the two groups of ventricles was indistinguishable. -62-Flgure 9» A Comparison of pA Values for Propranolol In Euthyroid 2 and Hyperthyrold Rat Right Atria. The plot depicts the log dose of antagonist versus log (DR-1) for (euthyroid and hyperthyrold rat right a t r i a . Each point represents the log (DR-1) calculated from the mean ED of 7-19 observations. 50 The lines were not significantly different from each other. -63-log dose antagonist -64-Flgure 10. Effect of Isoproterenol on Changes In Tension of the Right  Ventricle of Euthyroid and Hyperthyrold Rats while  stimulating at 3Hz. The plot depicts the effect of various doses of Isoproterenol on absolute tension of euthyroid and hyper-thyrold rat right ventricles, following stimulation at 3Hz. Maximal tension attained was recorded. Each point represents the mean tension (g) + S.E.M. of 5-44 observations. a Euthyroid significantly greater than hyperthyrold at P<0.05. -65-0 i i I I 1 I L C 9 8 7 6 5 4 Dose of Isoproterenol (-log M) -66--8 Changes In Tension Following Administration of 5x10 M  Isoproterenol to the Right Ventricle of Euthyroid and  Hyperthyroid Rats while stimulating at 3Hz. -8 From the dose-response curve in figure 10, 5x10 M Isoproterenol was chosen as a sub-maximal dose for a time-response study. Tension was studied in euthyroid and hyperthyroid rat right ventricles at various times following administration of the drug. In the absence of drug, the tension developed by the two groups was indistinguishable; but by fifteen seconds after administration of the drug the euthyroid ventricles had a significantly higher developed tension than the hyperthyroid ventricles (Figure 11) . This significant difference was maintained until forty seconds, although the euthyroid ventricles had consistently higher developed tensions at a l l times studied. Changes in % Phosphorylase a Following Administration of  5x10 M Isoproterenol to the Right Ventricle of Euthyroid  and Hyperthyroid Rats while stimulating at 3Hz. The response of % phosphorylase a a c t i v i t y to -8 administration of 5x10 M isoproterenol in rat right ventricles is shown in figure 12. In the absence of drug there was no significant difference between % phosphorylase a content in euthyroid or hyperthyroid -8 rat right ventricles. Following administration of 5x10 M -67-Flgure 11. -8 Changes In Tension Following Administration of 5x10 M  Isoproterenol to the Right Ventricle of Euthyroid and  Hyperthyrold Rats while stimulating at 3Hz. The plot depicts the change in absolute tension of euthyroid and hyperthyrold rat right ventricles, at various times following administration of a sub-maximal dose of isoproterenol. Each point represents the mean tension (g) ± S.E.M. of 7-55 observations. a Euthyroid significantly greater than hyperthyrold at P<0.05. -68-0 20 40 60 80 100 120 Time (sees.) after injection of 5x10 M Isoproterenol -69-Flgure 12. Changes In % Phosphorylase a Following Administration of  5x10 M Isoproterenol to the Right Ventricle of Euthyroid  and Hyperthyroid Rats while stimulating at 3Hz. The plot depicts the change in % phosphorylase a content of euthyroid and hyperthyroid rat right ventricles, frozen at various times following administration of a sub-maximal dose of Isoproterenol. Each point represents the mean % phosphorylase a + S.E.M. of 6-13 observations. There was no significant difference between the two groups at any time studied. -70-Time (sees.) following injection of 5x10" M Isoproterenol -71-lsoproterenol there was a small time-related increase in % phosphorylase a, but at no time studied was there a significant difference between levels in euthyroid or hyperthyrold tissues. Only In euthyroid ventricles were the levels of % phosphorylase a following drug administration significantly greater than in the absence of drug. Differences Between Euthyroid and Hyperthyrold Rat  Langendorff Hearts. Using the constant pressure Langendorff heart perfusion apparatus, i t was found that there was a significantly higher coronary blood flow in the hyperthyrold hearts than in the euthyroid hearts (figure 13A). Euthyroid hearts having a coronary blood flow of 3.72 + 0.177 ml per minute, and hyperthyrold hearts having a flow of 5.08 + 0.134 ml per minute. Associated with this, the hyperthyrold hearts also demonstrated a significantly higher wet weight (euthyroidi 1.00 + 0.034 gl hyper thyroid. 1 1.25 + 0.056 g) (figure 13B), and significantly higher resting heart rates (euthyroid! 197.54 + 4.826; hyperthyrold! 280.75 ± 7.019) (figure 13C), than the euthyroid hearts. Conversely, euthyroid hearts demonstrated a significantly greater developed tension than did the hyperthyrold hearts, with a value of 5.02 + 0.210 g against the hyperthyrold value of 4.18 + 0.116 g, (figure 13D). -72-Flgure 13. Differences Between Euthyroid and Hyperthyroid Rat  Langendorff Hearts. A a Differences in coronary blood flow ( ml per minute). Bt Differences in wet heart weight (g). Ci Differences in resting rate. Di Differences in basal tension development. The bars represent mean value + S.E.M. The number within the bar represents the n values. a Hyperthyroid significantly greater than euthyroid at P<0.05. b Euthyroid significantly greater than hyperthyroid at P<0.05. Rate (beats/m in) Volume of effluent (ml/min) -74-Effeet of Changes in Coronary Blood Flow on % Phosphorylase a  Activation. No significant difference in % phosphorylase a levels was found in Langendorff hearts from euthyroid and hyperthyroid rats perfused at the regular reservoir height of forty cm, and hyperthyroid hearts perfused at a reduced reservoir height so that the coronary "blood flow was reduced to that of the euthyroid hearts ± two S.E.M. (figure 14). In these same hearts "basal tension was greater in the euthyroid hearts than In the hyperthyroid hearts regardless of reservoir height (euthyroidi 5.05 i 0.436 g, n=8; hyperthyroid at regular reservoir heighti 4.68 ± 0.280 g, n=ll; hyperthyroid at reduced reservoir heighti 4.21 ± 0.229 g, n=13). Following the bolus injection of five ng isoproterenol, there was a significant increase in tension and % phosphorylase a in a l l treatment groups compared to the appropriate control in the absence of drug. The absolute increase in tension was similar in a l l groups; the hearts from euthyroid animals main-taining a greater tension development than those from hyper-thyroid animals (euthyroid: 7.38 ± 0.758 g, n=4; hyperthyroid at regular reservoir heighti 6.72 ± 0.505 S» n=6; hyperthyroid at reduced reservoir heighti 5.50 ± 0.257 g» n=7). The increase in % phosphorylase a In both hyperthyroid groups was significantly greater than the increase in hearts from euthyroid animals following administration of the drug, but not signif-icantly different from each other. -75-Figure 14. Effect of Changes In Coronary Blood Flow on % Phosphorylase a  Activation, The plot depicts the % phosphorylase a activ i t y in euthyroid and hyperthyrold rat hearts, perfused at the regular reservoir height, and In hyperthyrold rat hearts perfused at a reduced reservoir height} thirty seconds after injection of 5 ng of isoproterenol or vehicle. The bars represent mean % phosphorylase a + S.E.M. The number within the bar represents the n value, a 1 ;Significantly greater than control value at P<£0,05. b Hyperthyrold + drug significantly greater than euthyroid + drug at P< 0.05. c Euthyroid and hyperthyrold hearts perfused at regular reservoir height (40cm). d Hyperthyrold hearts perfused at reduced reservoir height. -76--77-DISCUSSION. To date, then, our results have shown that cardiac tissues from hyperthyroid rats differ from those of euthyroid rats in that they exhibit an Increased chronotropic and decreased inotropic response In their basal states. In response to Isoproterenol administration, in the right atrium there is a dose-related Increase in rate which is significantly greater in the hyperthyroid animal; in the l e f t atrium and right ventricle there is a dose-related increase in contractile force which is significantly greater in the euthyroid animal. No supersensitivity to these effects was noted In the hyper-thyroid tissues. The responses to isoproterenol, while accompanied by an increase in phosphorylase a activity, demonstrate no differences in enzyme act i v i t y between the two groups. In the Langendorff heart however; isoproterenol besides producing an inotropic response, Increased phosphorylase a to a significantly greater extent in the hyperthyroid animals. The greater resting rate and lower basal tension of the hyperthyroid Langendorff hearts Is accompanied by a significant increase in coronary blood flow as compared to euthyroid hearts. Reduction of the blood flow in the hyperthyroid hearts to the same level as In the euthyroid hearts had no significant effect on the phosphorylase activating effect of isoproterenol. The pA study which was carried out showed that any di f f e r -2 ence in the responses of euthyroid and hyperthyroid hearts to catecholamines (whether exogenous or endogenous) was not due -78-to any changes in receptor similarity, since the values obtained were practically identical In the two groups. It is well documented that treatment of animals with thyroid hormones produces a reduction in whole body weight and Increase in heart weight, resulting in an increased heart weight to body weight ratio when compared to euthyroid controls (Aronson, 1976; Calroli and Crout, 1967; Frazer, Hess and Shanfeld, 1969; Gibson, Tlchonicky and Kruh, 1975; Hornbrook and Cabral, 1972; Hornbrook et a l , 1965; Inchlosa and Freedberg, 1965; Levey, Skelton and Epstein, 1969; Margollus and Gaffney, 1965; McNeill, Muschek and Brody, 1969; Nemecek and Hess, 197^; Skelton, Su and Pool, 1976; van der Schoot and Moran, 1965; and Yazakl and Raben, 1975)• This was also found in the present study. Hyperthyrold rats did. not gain any overall weight during the four day treatment period in contrast to control rats which gained an average of twenty grams (figure 3 ). Wet heart weight was significantly greater in the hyperthyrold than in the euthyroid rats, by a factor of 25 percent (figure 13B). Since the heart weight and body weight studies were not carried out on the same animals, heart weight to body weight ratios were not determined, but would almost certainly have been increased in the hyperthyrold animals. The myocardial hypertrophy of hyperthyroidism has been shown to be accompanied by an enlargement of the myocardial cells; and an increase in the number, size and complexity of the mitochondria (DeGroot, 1972). Triiodothyronine has been -79-shown to augment the synthesis of both nuclear and cytosol li v e r proteins within five hours of administration to thyroidectomized rats (Bernal, Coleoni and DeGroot, 1978), and to cause mitochondrial swelling, probably due to Increased permeability of cations (Marzoev and Vladimirov, 1978). These findings could a l l account for the cardiac hypertrophy found following thyroid hormone treatment, Detection of loss of body weight or cardiac hypertrophy is an easy index of thyrotoxicosis to measure, Other indices which are often used Include increases In oxygen consumption (Calroll and Crout, 1967; Frazer, Hess and Shanfeld, 1969; Hornbrook and Cabral, 1972; Nemecek and Hess, 197^; and van der Schoot and Moran, I965)» or Increases in protein-bound, and total Iodine (BUcclno et a l , 1967; Levey, Skelton and Epstein, 1969; Margollus and Gaffney, 1965; and Melander et a l , 1975). A significant difference in body weight was found in our experiment after only four days, and was used as the Index of thyroid hormone activity. Right atria from hyperthyrold rats beat consistently faster than those from euthyroid rats (figure 4 ) , The mean rate of atria from hyperthyrold rats was 25 percent higher than for those from euthyroid animals. Following admin-istration of isoproterenol there was an Increase in rate In both groups, the hyperthyrold a t r i a maintaining consistently higher rates than the euthyroid at r i a . This is also well supported, in the literature (Cravey and Gravenstein, 1965; Lee, Lee and Yoo, 1965; Thler, Gravenstein and Hoffman, 1962; - 8 0 -and van der Schoot and Moran, I965). Although these inves-tigators used adrenaline and noradrenaline, the results were similar; the i n i t i a l heart rate was greater in atria from hyperthyroid rats than in those from euthyroid rats, and following catecholamine administration there was a dose-dependent increase in rate in "both groups. Atria from hyperthyroid rats maintained consistently higher rates than those from the euthyroid group. When the data were expressed as percent of maximal Increase, we found that the results were similar to those of van der Schoot and Moran (1965)» who showed that although the noradrenaline-Induced Increase in rate was similar in euthyroid and hyperthyroid rat atria (allowing for the different basal rates), when the data were expressed as percent of maximal Increase, the response in the hyperthyroid atria was less than in the euthyroid atria (figure 5). It therefore seems that, similar to the suggestion of Wilson e_t a l , (I966); the chief difference between ttie euthyroid and. hyperthyroid animals is the starting level of the heart rate. The Increase in intrinsic heart rate may be due to a direct action of the thyroid hormones on the pacemaker cells as was suggested by Cairoli and Crout (1967). Dratman (197*0 has suggested that the mechanism of action of thyroxine may be similar to that of tyrosine, (which thyroxine Is an amino acid analogue of). She postulated that thyroxine forms false neurohumours in adrenergic nerves, to be released instead of catecholamines (or with cat-echolamines) when the nerve Is stimulated, It has been -81-shown that In hyperthyroidism there are decreased tissue and blood levels of adrenergic neurotransmitters (Dratman, 1974), although the body as a whole behaves as i f there was increased sympathetic stimulation. Dratman suggests that this is due to lodothyronine-derived neurohumours of high biological a c t i v i t y which are released following nerve stimulation, and produce various manifestations of thyrotoxicosis. There is no direct evidence to support this hypothesis however. In our experiment with right atria there was definitely no supersensitivity development to the chronotropic effect of Isoproterenol In the hyperthyroid animal, since the Increase in rate of the euthyroid atria was slig h t l y more than that of the hyperthyrold atria (figure 4 ) . In the rat l e f t atrium, right ventricle and Langendorff heart,basal tension was greater in euthyroid tissues than in hyperthyrold controls. In the l e f t atrium, the euthyroid tissues had a mean basal tension which was 23 percent greater than that of the hyperthyrold tissues (figure 6 ) . Following administration of isoproterenol, there was a dose-related increase in tension in both groups; but the tension development in the euthyroid tissues became significantly greater than that in the hyperthyrold tissues. In the rat right ventricle there was no difference between basal tension In euthyroid and hyperthyrold tissues. Following administration of Isoproterenol, there was a dose-dependent increase in contractile force, euthyroid tissues having a -82-signlficantly greater tension development than hyperthyroid tissues (figure 10). These results would therefore rule out the possibility of thyroid hormone-Induced supersensitivity to the Inotropic effects of Isoproterenol in the rat l e f t atrium and right ventricle. The mean basal tension of euthyroid rat Langendorff hearts was significantly greater than that of hyperthyroid hearts (figure 13D). Dose-response curves to Isoproterenol were not performed, but doubtless the data would have been similar to that of the l e f t atrium and right ventricle since previous studies with other adrenergic amines have shown that no enhancement of the Inotropic response occurs in this preparation. Young and McNeill(1974) showed that noradrenaline produced a dose-dependent increase in tension and phosphorylase ;a ac t i v i t y in both euthyroid and hyperthyroid rat Langendorff hearts. They found no significant difference in force or cyclic AMP activation between the two groups, but phosphorylase a activation was potentiated In the hyperthyroid hearts. Van der Schoot and Moran (1965) suggested that the reduced inotropic force found in hyperthyroid cardiac tissues might be due to hypoxia of the myocardium, as a result of the increased oxygen comsumptlon induced in hyperthyroid animals. Dobson et a l (1974) carried out a study using Isolated perfused hearts, in sItu hearts, right ventricle strips and papillary muscles from guinea pigs. On the basis of comparison of several metabolic and physiologic parameters measured in these tissues, they concluded that the right ventricle was prone to protein loss due to damage during the dissection period, and to tissue hypoxia; - 8 3 -renderlng i t unsuitable for studies of the mechanisms of myocardial contractility. These factors would probably be accentuated to an even greater extent in a hypermetabolic state such as hyperthyroidism. In both the spontaneously contracting rat right atrium (figures 7 and 8), and. the e l e c t r i c a l l y driven rat right ventricle (figure 10 and 11)} a sub-maximal dose of -8 isoproterenol (In both cases, 5x10 M) produced a change in contractile force which was significantly different between euthyroid and hyperthyrold tissues, without producing a significant difference In phosphorylase a activity. However, in the rat Langendorff heart, 5 ng of isoproterenol produced a significantly greater increase in phosphorylase a in hyperthyroid than in euthyroid hearts. That this difference in phosphorylase a a c t i v i t y was not a result of the increased coronary blood flow detected in the hyperthyroid animals was demonstrated by the fact that reducing the coronary blood flow of the hyperthyrold hearts to that of the euthyroid hearts had no effect on the isoproterenol-lnduced phosphorylase activation (figure 14). In our study, coronary blood flow was measured by the oldest and easiest method available; collection and mea-surement of the effluent from the coronary blood vessels, following retrograde perfusion through the aorta. Other more sophisticated methods are available and are discussed In a book on coronary vasodilators by R. Charlier, (1961); as are other variations of the Langendorff method. In our -84-case the method used was the most practical, since i t was relatively easy to perform, and we did not require long-term measurements to be made. Other methods available include involvement with cinematography (Stehle, 1932), elaborate cannulations (Lu and Melville, 1950), radioactive methods 42 3 using K or H-labelled drugs (Wurtman, Kopln and Axelrod, 1963? Wurtman ejt a l , 1964), or the thermostromuhr method of Rein (Charlier, 1961; Essex, Baldes and Mann, 1936), in which a small cuff containing a diathermy unit and two thermocouples is fitted around a coronary artery. Part of the heat supplied by the unit is removed by the blood stream, providing a difference in temperature between the two thermocouples which is a function of the blood flow and can be recorded with a galvanometer, The advantage of this method is that i t can be used to record coronary blood flow continuously, and can be used in the normal unanaesthetlzed animal. Its disadvantages are that It requires surgical installation, a recovery period, and may be dislodgedj generally with fatal results. It Is possible that the apparent potentiation of phosphorylase activation seen in the Langendorff heart could be due to a number of factors. F i r s t l y , the physical process of dissection carried out to obtain the separate portions of the heart could damage c e l l s , although i t would appear l i k e l y that only a minority of cells would be affected, and that this is therefore unlikely to be a major cause of the discrepancy. Secondly, in the isolated organ bath, -85-penetratlon of the tissues by drug is unlikely to be as complete as when the drug is introduced into the coronary circulation, as is the case in the Langendorff heart. However, we demonstrated in the Langendorff heart that even when coronary blood flow was equal in the euthyroid and hyperthyroid. hearts there was s t i l l potentiation of the phosphorylase-activatlng effect of isoproterenol. It has recently been shown (Herd, 1978), that thyroxine directly affects the kinetics of calcium transfer across mitochondrial membranes. This change may be due to alteration of the number of ions transported per transfer cycle, altered mobility of the calcium in the membranes, and/or altered rate of release of the transport calcium. This action of thyroxine on calcium may also alter calcium-dependent metabolic processes such as ADP translocation, which is stiumlated in the presence of thyroid hormone, Friesen, Allen and Valadares (1967), and Hartley and McNeill (1976) showed that calcium can activate phosphorylase directly in the perfused heart; although the sensitivity of phosphorylase a to calcium is not changed by thyroid hormone treatment, pA studies (Schild, 1957) are widely used to quantify the 2 a f f i n i t y of antagonists for a receptor site, and also to characterize receptor types in vitro. The pA is defined as 2 the negative logarithm of the molar dose of antagonist which reduces the effect of a double dose of agonist to that of a single dose. In the present study we wished to determine i f the beta-adrenoceptors of the spontaneous rat right -86-atrium were the same In their a f f i n i t i e s for beta-agonists and antagonists whether the heart was obtained from a euthyroid or hyperthyroid animal. If pA values are the 2 same, then It is usually considered that the receptors are the same. From the data we present here (figure 9)» i t can be seen that the pA values for the receptor Interaction were 2 almost identical, regardless of the thyroid state. This would, tend to indicate that the difference in intrinsic rate and other cardiac manifestations of thyrotoxicosis are not due to any changes in receptor sensitivity, although i t is possible that changes in beta-receptor number do take place (Williams et a l , 1977). In summary, we have confirmed that catecholamine-induced phosphorylase activation is potentiated in the isolated perfused hyperthyroid rat heart. However, in Isolated portions of the heart no potentiation of phosphorylase activation in hyper-thyroid animals could be found. We have also determined that the effect is not due to the increased coronary blood flow found in the hyperthyroid hearts. A similar situation has been shown to occur with reserpine supersensitivity, McNeill and Schulze (1972) showed in the guinea pig Langendorff heart that pretreatment with reserpine for forty-eight hours, resulted in the development of supersensitivity to both the phosphorylase-activatlng and the inotropic effects of noradrenaline and histamine. However, in the guinea pig right atrium (Westfall and Fleming, 1968), and the rat ventricle strip (McNeill, unpublished data) supersensitivity to - 5 7 -catecholamlnes after reserplne pretreatment cannot be readily demonstrated. Westfall and Fleming suggested that the a b i l i t y to demonstrate reserpine-lnduced super-sensitivity In myocardial tissues is inversely related to the extent of mechanical manipulation of the muscle. This anomaly seems to be analogous to the situation found in the heart following pretreatment with thyroid hormones? which could also be due to damage caused during dissection of the various heart portions. Future studies could include investigation of phos-phorylase phosphatase, the enzyme which converts phosphorylase a back into the inactive phosphorylase b; to see If the po-tentiation of phosphorylase acti v i t y seen in hyperthyroid hearts is due to alterations in the ac t i v i t y of this enzyme. The method devised by Rail and Sutherland (1962) for estimation of phosphorylase phosphatase Is quite similar to that which we use for phosphorylase determinations (Diamond and Brody, 1965)» and could therefore be carried out. It would also be of interest to investigate electro-physiological changes in the sino-atrial node of the right atrium of euthyroid and hyperthyroid animals, and to determine the effect of calcium on the resting membrane potential and action potential In cardiac tissues from the two groups to determine whether the thyroid hormones alter the permeability of nodal tissue to calcium. 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