Prognostic importance of the oxidized product of catecholamines, adrenolutin, in patients with severe heart failure.

OBJECTIVES: The purpose of this study was to assess whether adrenolutin, the inert product of the highly reactive molecules aminochromes, is increased in severe chronic heart failure and whether it is associated with a poor prognosis. BACKGROUND: Experimental evidence suggests that oxidative products of catecholamines, aminochromes, are more cardiotoxic than unoxidized catecholamines and may be increased in heart failure. METHODS: Adrenolutin was measured at baseline and at 1 and 3 months in 263 patients with chronic New York Heart Association class III or IV heart failure and a left ventricular ejection fraction of 22% +/- 7%. Adrenolutin levels were compared with normal levels, and their relation to prognosis was evaluated. RESULTS: Baseline adrenolutin was increased (55 +/- 90 pg/mL vs 8.4 +/- 9.1 pg/mL for control, P <.02) and remained increased at 1 month (49 +/- 65 pg/mL). During a mean follow-up of 309 +/- 148 days (22-609 days), 57 patients died. Baseline adrenolutin levels correlated with mortality rates by univariate and multivariate analyses (relative risk 1.06, 95% CI 1.01-1.10 for each 17.9-pg/mL rise, P =.032). Left ventricular ejection fraction (P =.013) and New York Heart Association class (P =.009) were the only other variables associated with survival. Age, sex, plasma creatinine, plasma N-terminal atrial natriuretic peptide, and plasma norepinephrine levels were not retained in our model. Adrenolutin levels 1 month after random assignment were not significantly correlated with total mortality rate (P =.061) but were correlated with mortality rate from low output (relative risk 1.14, 95% CI 1.06-1.22, P =.002). CONCLUSIONS: Plasma adrenolutin is increased in patients with heart failure and correlates with a poor prognosis independent of other important predictors of survival. This finding has potentially important pathophysiologic, prognostic, and therapeutic implications.

Biphasic changes in heart performance with food restriction in rats.

To examine effects of food restriction resembling very-low-calorie dieting on heart performance, normal rats were fed 25% of ad libitum food intake for 14 days. Although heart weight decreased (P < 0.05) after 5 days, left ventricular systolic pressure as well as rates of pressure development and fall were increased (P < 0.05) at 7 days and decreased (P < 0.05) after 14 days. Systolic and diastolic blood pressures were also increased from 5 to 7 days and decreased after 14 days. The increased hemodynamic performance of heart was associated with a raised plasma norepinephrine concentration, which peaked at day 7 of food restriction; epinephrine concentration was increased (P < 0.05) also at day 7. An increased catecholamine synthesis was indicated by the raised (P < 0.05) plasma dopamine beta-hydroxylase activity at 3 days, but this was decreased (P < 0. 05) at 14 days. The concentration of dopamine in the heart was increased (P < 0.05) at 2-14 days, of norepinephrine at 7-14 days, and of epinephrine at 10 and 14 days. Food restriction thus appears initially to be associated with an enhanced catecholamine influence on the heart and is followed by a depressed cardiac performance.

Beneficial effects of propionyl L-carnitine on sarcolemmal changes in congestive heart failure due to myocardial infarction.

OBJECTIVE: Earlier studies have revealed sarcolemmal (SL) defects in congestive heart failure due to myocardial infarction; however, the mechanisms of SL changes in the failing heart are poorly understood. Since congestive heart failure is associated with various metabolic abnormalities including a deficiency of carnitine, we examined the effects of propionyl L-carnitine, a carnitine derivative, in animals with congestive heart failure. METHODS: For this purpose, heart failure in rats was induced by occluding the coronary artery and 3 weeks later the animals were treated with 100 mg/kg (i.p. daily) propionyl L-carnitine for 4 weeks. The sham control group received saline injections. The animals were assessed for their left ventricular function. SL membranes were examined for Na(+)-K+ ATPase, Na(+)-Ca2+ exchange and adenylate cyclase activities. RESULTS: A marked improvement in the attenuated left ventricular function of the experimental animals was seen upon treatment with propionyl L-carnitine. The SL adenylyl cyclase activities in control, untreated failing hearts and treated failing hearts were 590 +/- 36, 190 +/- 22 and 320 +/- 21 pmol cAMP/mg/10 min, whereas the SL Na(+)-K+ ATPase activities were 35.7 +/- 2.8, 22.5 +/- 2.4 and 30.1 +/- 2.8 mumol Pi/mg/h, respectively. Furthermore, the SL Na(+)-dependent Ca(2+)-uptake activity, which decreased in the failing hearts (4.6 +/- 0.4 vs. 9.3 +/- 0.7 nmol Ca2+/mg/2 s for control), was improved (6.8 +/- 0.5 nmol Ca2+/mg/2 s) significantly following treatment with propionyl L-carnitine. CONCLUSION: These results indicate that metabolic therapy with propionyl L-carnitine may attenuate defects in the SL membrane and thus may improve heart function in congestive heart failure due to myocardial infarction.

Differential changes in sympathetic activity in left and right ventricles in congestive heart failure after myocardial infarction.

Although congestive heart failure subsequent to myocardial infarction is known to be associated with increased sympathetic activity, very little information regarding changes in the sympathetic nerves in the left and right ventricles at various stages after infarction is available. Male Sprague-Dawley rats were subjected to coronary artery ligation and studied 4 and 8 weeks later; these animals had mild and moderate stages of congestive heart failure. A sham group, without coronary ligation, was used as control. Four weeks after myocardial infarction, plasma and ventricular (left and right) epinephrine (EPI), unlike norepinephrine (NE), were markedly increased. Whereas plasma catecholamine (EPI and NE) levels were increased 8 weeks after infarction, NE concentration in the left ventricle was unchanged but EPI concentration was increased in comparison with sham control. The right ventricle showed an increased level of both NE and EPI 8 weeks after infarction. Measurement of the rate of change in the specific activity of NE (NE turnover) in the left and right ventricles 8 weeks after infarction revealed an increase in NE turnover in the left ventricle, without any changes in the right ventricle. The concentration of EPI, unlike NE, was increased in the kidney, spleen, and brain 8 weeks after coronary occlusion. These results are interpreted to mean that congestive heart failure caused by myocardial infarction is associated with differential changes in the status of sympathetic nerves in the left and right ventricles; sympathetic activity is increased only in the left ventricle, whereas the right ventricle may play an adaptive role by increasing catecholamine stores during the development of heart failure.

Differential changes in left and right ventricular adenylyl cyclase activities in congestive heart failure.

The status of beta-adrenergic receptors and adenylyl cyclase in crude membranes from both left and right ventricles was examined when the left coronary artery in rats was occluded for 4, 8, and 16 wk. The adenylyl cyclase activity in the presence of isoproterenol was decreased in the uninfarcted (viable) left ventricle and increased in the right ventricle subsequent to myocardial infarction. The density of beta1-adrenergic receptors, unlike beta2-receptors, was reduced in the left ventricle, whereas no change in the characteristics of beta1- and beta2-adrenergic receptors was seen in the right ventricle. The catalytic activity of adenylyl cyclase was depressed in the viable left ventricle but was unchanged in the right ventricle. In comparison to sham controls, the basal, as well as NaF-, forskolin-, and 5'-guanylyl imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activities were decreased in the left ventricle and increased in the right ventricle of the experimental animals. Opposite alterations in the adenylyl cyclase activities in left and right ventricles from infarcted animals were also seen when two types of purified sarcolemmal preparations were employed. These changes in adenylyl cyclase activities in the left and right ventricles were dependent on the degree of heart failure. Furthermore, adenosine 3',5'-cyclic monophosphate contents were higher in the right ventricle and lower in the left ventricle from infarcted animals injected with saline, isoproterenol, or forskolin in comparison to the controls. The results suggest differential changes in the viable left and right ventricles with respect to adenylyl cyclase activities during the development of congestive heart failure due to myocardial infarction.

Mechanisms of alterations in cardiac membrane Ca2+ transport due to excess catecholamines.

The occurrence of excessive catecholamine release is often associated with stress due to the lifestyle of Western societies. Contrary to the general thinking that excess catecholamines produce cardiotoxicity mainly via binding to adrenoceptors, there is increasing evidence that catecholamine-induced deleterious actions may also occur through oxidative mechanisms. In this overview it is shown that a high dose of isoproterenol induces a biphasic change in cardiac Ca2+ transport in the sarcolemma and in sarcoplasmic reticulum. Both sarcolemmal and sarcoplasmic reticular Ca2+-transport activities are initially increased to maintain Ca2+ homeostasis and then are impaired, which may be associated with the occurrence of intracellular Ca2+ overload. On the other hand, mitochondrial Ca2+-transport activities exhibited a delayed increase. Pretreatment with vitamin E partially prevented the deleterious changes in cardiac membranes as well as the depressed energetic status of the heart muscle cell. It is concluded that excess catecholamines affect Ca2+-transport mechanisms primarily via oxidation reactions involving free radical-mediated damage. Thus drug approaches that reduce circulating catecholamines and/or prevent their oxidation should prove beneficial. A combination therapy involving inhibitors of catecholamine release, blockers of adrenoceptors, and antioxidants may be indicated for stress-induced heart disease.

Effects of long-term dietary restriction on cardiovascular function and plasma catecholamines in the rat.

To examine the relationship between heart function and plasma catecholamines upon food restriction, normal adult rats were fed 12 g or 6 g food/day for 14 days and 12 g food/day for 28 days. Food-restricted rats exhibited bradycardia, hypotension, and decreased rates of cardiac contraction (+dP/dt) as well as relaxation (-dP/dt) at 14 (12 or 6 g food/day) and 28 (12 g food/day) days. Plasma norepinephrine and epinephrine levels were significantly elevated in the 6 g food/day group at 14 days, whereas in the 12 g food/day group, plasma norepinephrine was elevated at 14 days but was significantly decreased at 28 days. Heart norepinephrine and epinephrine concentrations were elevated at both 14 and 28 days of food restriction in the 12 g food/day group as well as at 14 days in the 6 g food/day group. Thus, dietary restriction appears to result in depressed indices of heart function, while the circulating levels of catecholamines were elevated at early stages.

Plasma neuropeptide Y in anxiety disorders: findings in panic disorder and social phobia.

The demonstration in preclinical studies that centrally administered neuropeptide Y (NPY) has anxiolytic effects had led to speculation that NPY may play a role in human anxiety disorders. We therefore decided to study plasma NPY levels in 22 patients with DSM-III-R anxiety disorders (11 with panic disorder and 11 with social phobia, generalized type) and 12 never psychiatrically ill comparison subjects. Under resting conditions, plasma NPY levels did not differ among the three diagnostic groups. Following hand immersion in ice water, plasma NE levels--but not NPY levels--increased immediately, but there were no significant differential diagnostic effects. These results are convergent with prior reports of normal sympathetic nerve activity in patients with anxiety disorders.

Mechanisms of cardiac cell damage due to catecholamines: significance of drugs regulating central sympathetic outflow.

A chronically increased rate of catecholamine release has various deleterious actions. Isoproterenol injections (80 mg/kg body weight) resulted in depressed Ca2+ transport in the sarcolemma (ATP-dependent Ca2+ uptake, Na(+)-dependent Ca2+ uptake) and sarcoplasmic reticulum (Ca2+ uptake) of rat heart. The formation of malondialdehyde owing to lipid peroxidation was increased. Pretreatment with vitamin E (10-25 mg/kg/day) strongly inhibited the membrane damage. The toxic effects of catecholamines arise most probably from their oxidation, and it is therefore important either to reduce the central sympathetic outflow or to prevent the oxidation. An inappropriately high sympathetic outflow is a typical feature of Western affluent societies, and is linked to psychosocial stress and hypercaloric nutrition. However, established pharmacologic interventions to reduce sympathetic outflow have proven not practicable because of marked side effects. Using radiotelemetry for monitoring cardiovascular parameters of spontaneously hypertensive rats treated with clonidine or moxonidine, we showed that clonidine, unlike moxonidine, resulted in rebound hypertension after drug withdrawal. Because the rebound blood pressure and the typical side effects of clonidine associated with low patient compliance are mainly mediated by alpha-adrenoceptors, it can be inferred that the I1-imidazoline agonist moxonidine does not exhibit the side effects commonly seen with clonidine and therefore represents a promising approach for reducing an inappropriately high central sympathetic outflow.

Characterization of adenylyl cyclase in heart sarcolemma in the absence or presence of alamethicin.

Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in Vmax as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn2+ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.

Measurement of adrenolutin as an oxidation product of catecholamines in plasma.

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (microM) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of 3H-adrenolutin after injection of 3H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.

Beneficial effects of verapamil in diabetic cardiomyopathy.

It has been suggested that the occurrence of an intracellular Ca2+ overload may result in the development of diabetic cardiomyopathy, which is associated with depletion of high-energy phosphate stores and a derangement of ultrastructure and cardiac dysfunction. Accordingly, the effects of verapamil, a Ca2+ antagonist, on cardiac function, ultrastructure, and high-energy phosphate stores in the myocardium were evaluated in rats made diabetic by an intravenous injection of streptozocin (65 mg/kg). Four weeks after the induction of diabetes, the animals were treated with three doses (2, 4, or 8 mg.kg-1.day-1) of verapamil for 4 wk until they were used for the measurement of different parameters. Untreated diabetic animals had slower heart rates, depressed rate of contraction and rate of relaxation, lower peak left ventricular systolic pressure, and elevated left ventricular diastolic pressure. All of these changes were significantly improved in diabetic rats receiving verapamil treatment. The beneficial effects of verapamil were more evident with higher doses (8 mg.kg-1.day-1) than with the lower doses (2 mg.kg-1.day-1). The diabetic animals also showed alterations in myocardial high-energy phosphate stores and exhibited evidence of ultrastructural damage; these abnormalities were improved by verapamil treatment without affecting their hyperglycemic status. Our results demonstrate that verapamil is capable of preventing diabetes-induced myocardial changes and support the involvement of Ca2+ in the cardiac pathology during diabetes.

Sarcolemmal Ca2+ transport in streptozotocin-induced diabetic cardiomyopathy in rats.

Heart sarcolemmal membranes were isolated by the sucrose density gradient method from rats with chronic diabetes induced by a streptozotocin (65 mg/kg iv) injection. Na+-dependent Ca2+-uptake activities were significantly depressed in diabetic sarcolemmal membranes; such alterations were evident at different incubation times and at different concentrations of Ca2+. Administration of insulin to diabetic rats normalized the Na+-dependent Ca2+-uptake activities. ATP-dependent Ca2+ accumulation and Ca2+-stimulated Mg2+-dependent ATPase, which represents Ca2+-pump mechanisms, were significantly depressed in sarcolemmal preparations for diabetic rats and these changes were also reversible upon insulin treatment. An increase in lysophosphatidylcholine and a decrease in phosphatidylethanolamine as well as diphosphatidylglycerol contents were observed in heart membranes isolated from diabetic rats but other phospholipids were unchanged. Cholesterol-to-phospholipid ratio was significantly increased in preparations from diabetic rats. These results indicate a depression in the ability of the cell to remove Ca2+ through Na+-Ca2+ exchange and Ca2+-pump mechanisms in sarcolemma, and these defects may contribute to the occurrence of intracellular Ca2+ overload and diabetic cardiomyopathy.

Norepinephrine storage, distribution, and release in diabetic cardiomyopathy.

The ability of hearts to store, distribute, and release norepinephrine (NE) was investigated in rats 8 wk after the induction of diabetes by an injection of streptozotocin (65 mg/kg iv). Chronic diabetes was associated with increased content and concentration of NE in heart and in other tissues such as kidney, brain, and spleen. Reserpine or tyramine treatment resulted in depletion of endogenous cardiac NE in control and diabetic rats. The depletion of NE stores at different times after a dose of reserpine was greater in diabetic hearts. On the other hand, NE stores in diabetic hearts were less sensitive than control hearts to low doses of tyramine but were more sensitive to high doses. The uptake of [3H]NE was greater in diabetic hearts in isolated perfused preparations. In comparison with the control values, diabetic hearts showed a decrease in [3H]NE in the granular fraction and an increase in the supernatant fraction. Diabetic hearts also showed an accelerated spontaneous release of [3H]NE. The increased cardiac NE and the uptake and release of NE in diabetic animals were reversible upon treatment with insulin. These results are consistent with the view that sympathetic activity is increased in diabetic cardiomyopathy and indicate that cardiac NE in diabetic rats is maintained at a higher level partly due to an increased uptake of released NE by adrenergic nerve terminals.

A biphasic change in contractile proteins during the development of cardiac hypertrophy in pigs.

A non-failing hypertrophy of the left ventricle was produced in the pig heart by supravalvular banding of aorta for 4, 8 and 12 weeks and the myosin and myofibrillar adenosine triphosphatase activities were measured. A significant increase in myosin Ca2+-ATPase activity was seen at 4 weeks of hypertrophy, but at 8 and 12 weeks this activity was significantly decreased compared to sham control. Similar changes were also seen in actin-activated myosin ATPase activities at 4, 8 and 12 weeks of hypertrophy. There were no changes in the K+- and NH4+-EDTA-stimulated ATPAse activities of myosin. Basal ATPase activities of myofibrils were decreased at 4 and 8 weeks of hypertrophy and there was no change in this activity at 12 weeks of hypertrophy. Ca2+ stimulated ATPase activity of myofibrils was significantly increased at 4 weeks, normal at 8 weeks and significantly reduced at 12 weeks of hypertrophy. The changes in ATPase activities were not due to any alterations of proteins by high concentrations of salts during the purification of myosin. The non-hypertrophied right ventricle from the banded animals did not show any change in the basal or Ca2+ stimulated myofibrillar ATPase activities. It is suggested that hypertrophy of the myocardium is accompanied by specific changes in the enzyme activities of the contractile proteins and the biphasic responses may correlate with the functional state of the myocardium subjected to a chronic increase in pressure.

Altered norepinephrine turnover and metabolism in diabetic cardiomyopathy.

Cardiac norepinephrine turnover and metabolism were examined in rats 8 weeks after the induction of chronic diabetes by an intravenous injection of streptozotocin (65 mg/kg). Cardiac norepinephrine concentration, norepinephrine turnover, and norepinephrine uptake were markedly increased in chronic diabetes in comparison with control values; these changes were reversible by 28-day insulin therapy. When the animals were exposed to cold for 6 hours, norepinephrine turnover rate constant increased in control and decreased in diabetic animals; cold exposure also increased norepinephrine concentration in diabetic hearts. Both cardiac norepinephrine concentration and turnover rate in diabetic rats were restored toward control values by ganglionic blockade with pentolinium. The conversion of [3H]tyrosine to [3H]catecholamine was enhanced and tyrosine hydroxylase as well as dopa decarboxylase activities were increased in diabetic hearts. The higher concentrations of [3H]normetanephrine and deaminated catechols indicated a faster metabolic rate of norepinephrine metabolism in hearts from diabetic rats; both monoamine oxidase and catechol-O-methyltransferase activities were also increased. The increased activities of the enzymes for the synthesis and metabolism of norepinephrine were not evident on treating the diabetic animals with insulin. These data not only support the view that chronic diabetes in rats is associated with increased sympathetic activity but also indicate that the cardiac norepinephrine concentration in diabetic rats may be maintained at a higher than normal level by an increased synthesis and uptake of norepinephrine in the adrenergic nerve terminals.

Adaptive changes in subcellular calcium transport during catecholamine-induced cardiomyopathy.

Rats were injected intraperitoneally with isoproterenol in dosage of 40 mg/kg body weight and heart microsomal and mitochondrial fractions were isolated 3, 9 and 24 h later. The heart/body weight ratio increased at 9 and 24 h after injection without any changes in the yield of subcellular organelles. Microsomal calcium uptake was significantly elevated at 3 h but returned to normal at 9 h and then became depressed 24 h post-injection. Mitochondrial calcium uptake was significantly increased 9 and 24 h after isoproterenol administration. Kinetic parameters of the calcium transport function indicated that the apparent affinity for Ca2+ remained unchanged, whereas Vmax values were altered in the experimental groups. Although there was no significant change in the phospholipid composition, the total phospholipid contents were increased (at 3, 9 and 24 h for microsomes; 3 and 9 h for mitochondria) in both types of organelles. The protein composition, as determined by gel electrophoresis, was altered in microsomes, but not in mitochondria. These results demonstrate rapid structural and functional changes in subcellular organelles. Such alterations may play an adaptive role in maintaining the intracellular calcium homeostasis during the development of catecholamine-induced cardiomyopathy.

Biochemical alterations in heart after exhaustive swimming in rats.

This study investigated alterations in glycogen, catecholamines, and the function of various subcellular membranes of the heart after exhaustive swimming in rats. The rats were exhausted by daily exercise over 1, 3, or 7 consecutive days. Glycogen content of the heart and three selected skeletal muscles was depleted after a single bout of exhaustive exercise. Repeated bouts of exhaustive swimming elicited a depletion of glycogen in only the plantaris and gastrocnemius skeletal muscles. Plasma norepinephrine and epinephrine levels were highly elevated, and cardiac concentrations of these hormones were significantly depleted immediately after all exercise sessions. Cardiac sarcoplasmic reticulum (SR) Ca2+ transport was depressed after a single exhaustive exercise period. After three exercise bouts SR Ca2+ accumulation remained depressed; however, mitochondrial Ca2+ transport was found to be augmented. If the exhaustive exercise protocol was continued up to seven days, only mitochondrial Ca2+ accumulation was depressed. Various parameters of sarcolemmal membrane function were observed to be unaltered after exhaustive exercise. These findings demonstrate that exhaustive swimming exercise in rats is capable of producing significant alterations in the Ca2+ transport capacity of the SR and mitochondrial membrane systems of the heart but is without apparent effect on the sarcolemmal membrane.

Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy.

The effects of insulin and thyroid hormone treatments on cardiac sarcoplasmic reticular function were investigated in chronic streptozotocin-induced diabetes in rats. ATP-dependent Ca2+ transport and Ca2+-stimulated ATPase activities were depressed significantly in microsomal samples from diabetic rats in comparison with control (P less than 0.05). This defect was seen at various times of incubation (1-20 min) and different concentrations of free Ca2+ (10(-7) to 10(-5) M Ca2+) and was accompanied by changes in the protein composition and phospholipid contents of the microsomal fraction. The defect in calcium transport in microsomal vesicles was not evident until 28 days after streptozotocin (65 mg/kg iv) injection, whereas increases in plasma glucose levels due to insulin-deficiency occurred within 3 days. All changes in function and composition of the sarcoplasmic reticulum were reversed by insulin administration to the diabetic rats. Although the plasma level of thyroid hormone was decreased in the diabetic rat, thyroid hormone treatment did not restore microsomal calcium transport in the diabetic animals. The results of this study provide some evidence that the depression in cardiac sarcoplasmic reticular calcium accumulation during diabetes is a consequence of insulin deficiency and associated chronic metabolic changes but the hypothyroid condition that accompanies experimental diabetes does not appear to play any role in this defect.

Role of phosphatidylinositol in basal adenylate cyclase activity of rat heart sarcolemma.

The adenylate cyclase activity and phospholipid composition were determined in rat heart sarcolemma after treating the membranes with a phosphatidylinositol-specific phospholipase C. Complete hydrolysis of phosphatidylinositol in sarcolemma was associated with a marked loss of the basal adenylate cyclase activity. The recombination of the supernatant with the phosphatidylinositol-depleted membranes was found to reactivate the adenylate cyclase activity. The soluble component(s) in the supernatant, which restored the adenylate cyclase activity, was thermolabile and precipitated by ammonium sulfate. Extensive hydrolysis of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in sarcolemma with a Clostridium welchii phospholipase C treatment did not affect the basal adenylate cyclase activity. These results suggest that phosphatidylinositol anchors component(s) essential for the expression of basal adenylate cyclase activity to the myocardial cell membrane.