Gerald S. Levey

Activation of Adenyl Cyclase by Glucagon in Cat and Human Heart

The purpose of this investigation was to determine the direct effects of glucagon on adenyl cyclase activity in cat and human heart particles and to elucidate the role of the beta receptor in any glucagon-mediated activation of adenyl cyclase. At the peak of its dose-response curve, crystalline glucagon increased the conversion of AT32P to cyclic 3′, 5′-AM32P in particulate fractions of both cat and human heart homogenates by approximately 70%. The activation of adenyl cyclase was dose-related over a concentration ranging from 1 × 10−7M to 1 × 10−5M. Half maximal activity was observed at 8 × 10−7M. DL-propranolol, 1 × 10−5M, did not block the activation of adenyl cyclase produced by glucagon, 1 × 10−6M or 1 × 10−5M. However, the same concentration of propranolol blocked adenyl cyclase activation induced by norepinephrine, 1 × 10−6M and 1 × 10−5M. Combined maximal doses of glucagon and norepinephrine did not produce additive effects on the activation of adenyl cyclase. The failure of DL-propranolol to block the glucagon-mediated activation of adenyl cyclase suggests that there are at least two receptor sites in myocardial tissue responsible for the activation of adenyl cyclase, one responsive to glucagon and one to norepinephrine. Moreover, since combined maximal doses of glucagon and norepinephrine failed to produce an additive response, it appears that in the heart there is probably only a single adenyl cyclase enzyme responsive to these hormones.

Myocardial adenyl cyclase: activation by thyroid hormones and evidence for two adenyl cyclase systems

The mechanism responsible for the hyperdynamic circulatory state in hyperthyroidism has not been defined. Although certain cardiac manifestations resemble those caused by excessive adrenergic stimulation, recent evidence suggests that thyroid hormone exerts an effect on the heart that is independent of the adrenergic system. Since the inotropic and chronotropic effects of norepinephrine appear to be mediated by activation of adenyl cyclase, the possibility that thyroxine and triiodothyronine are also capable of activating adenyl cyclase was examined in the particulate fraction of cat heart homogenates.L-thyroxine and L-triiodothyronine increased the conversion of adenosine triphosphate-(32)P (ATP-(32)P) to cyclic 3,5-adenosine monophosphate-(32)P (3,5-AMP-(32)P) by 60 and 45% respectively (P < 0.01). A variety of compounds structurally related to the thyroid hormones, but devoid of thyromimetic activity did not activate adenyl cyclase: these included 3,5-diiodo-L-thyronine, L-thyronine, 3,5-diiodotyrosine, monoiodotyrosine, and tyrosine. D-thyroxine activated adenyl cyclase and half maximal activity was identical to that of the L-isomer. Although the beta adrenergic blocking agent propranolol abolished norepinephrine-induced activation of adenyl cyclase, it failed to alter activation caused by thyroxine. When maximal concentrations of L-thyroxine (5 x 10(-6) moles/liter) and norepinephrine (5 x 10(-5) moles/liter) were incubated together, an additive effect on cyclic 3,5-AMP production resulted. THIS INVESTIGATION DEMONSTRATES: (a) thyroid hormone is capable of activating myocardial adenyl cyclase in vitro and (b) this effect is not mediated by the beta adrenergic receptor. Moreover, the additive effects of norepinephrine and thyroxine suggest that at least two separate adenyl cyclase systems are present in the heart, one responsive to norepinephrine, the other to thyroid hormone. These findings are compatible with the hypothesis that the cardiac manifestations of the hyperthyroid state may, in part, be caused by the direct activation of myocardial adenyl cyclase by thyroid hormone.

Positive Inotropic Effects of Dibutyryl Cyclic Adenosine 3',5'-Monophosphate

The positive inotropic effects of catecholamines have been postulated to result from an increase in the intracellular level of cyclic AMP (adenosine 3′,5′-monophosphate) produced by activation of adenyl cyclase. Although lack of an inotropic effect by exogenously administered cyclic AMP has cast doubt on this hypothesis, cardiac cells are not readily permeable to cyclic AMP. The N6-2′-O-dibutyryl derivative of cyclic AMP is thought to enter cells more readily and is resistant to enzymatic degradation by phosphodiesterase. We examined the effects of cyclic AMP and its dibutyryl derivative on the contractile performance of isolated cat right ventricular papillary muscles. Cyclic AMP (1 × 10−4 to 5 × 10−3M) had no effect on papillary muscle function. However, dibutyryl cyclic AMP caused a concentration-dependent increase in isometric tension and rate of tension development, the threshold concentration being 5 × 10−4M. The increments in tension (4.5 ± 0.4 g/mm2) and rate of tension development (58.4 ± 5.4 g/mm2/sec) at peak concentration (3 × 10−3M) were similar to those found at peak norepinephrine concentration (10−5M). Dibutyryl cyclic AMP (10−3M) also caused a marked shift of the force-velocity curve upward and to the right. Although 10−6M propranolol depressed the inotropic effects of norepinephrine, it did not alter the contractile response to dibutyryl cyclic AMP. These findings are consistent with the hypothesis that the positive inotropic effects of catecholamines are mediated by cyclic AMP.

Solubilization of myocardial adenyl cyclase

Abstract The detergent, Lubrol-PX, solubilized approximately 90–100% of the particulate adenyl cyclase in cat left ventricular homogenates. The activity in the 12,000 × g supernatant was not sedimented at centrifugal speeds as great as 105,000 and 250,000 × g for 2 hours. Electronmicroscopy revealed a virtual absence of particulate material in the ultracentrifuge supernatants. Adenyl cyclase activity was not filterable by a 0.22 μ millipore filter. Sephadex chromatography indicated a molecular weight of approximately 100,000 to 200,000. The solubilzed enzyme was activated by sodium fluoride but not by the hormones which activate the particulate adenyl cyclase, norepinephrine, glucagon or thyroxine.

Activation of Renal Cortical Adenylate Cyclase by Circulating Immunoreactive Parathyroid Hormone Fragments

Three distinct immunoreactive species of parathyroid hormone (PTH) are present in human serum. One has an estimated mol wt of 9,500 and probably represents glandular hormone, the second 7,000-7,500 mol wt, and the third 4,500-5,000 mol wt. In order to assess the biological activity of these circulating forms of PTH, we determined their ability to activate renal cortical adenylate cyclase. The 9,500 mol wt and 4,500-5,000 mol wt fractions produced four- to sixfold increases in cyclic 3,5-AMP accumulation above control; the 7,000-7,500 mol wt fraction was inactive. None of the fragments had any effects on phosphodiesterase activity. Antiserum to bovine PTH did not block the activation of adenylate cyclase by either the gragments or bovine PTH. The data suggest that a large proportion of circulating immunoreactive human PTH is biologically active and that the biologically and immunologically active sites of the hormone are distinct.

Demonstration of adenyl cyclase activity in canine cardiac sarcoplasmic reticulum

Abstract A microsomal fraction from canine cardiac muscle that actively accumulates calcium has been found to exhibit adenyl cyclase activity. The enzyme responds in a characteristic manner to fluoride and norepinephrine. Electron microscopic studies of the microsomal fraction demonstrate that it contains intact membrane vesicles; the lack of any glutamic dehydrogenase activity suggests that no contaminating mitochondrial fragments are present. In view of its active calcium accumulating ability, this fraction is believed to represent fragments of sarcoplasmic reticulum. This report represents the first demonstration of adenyl cyclase in cardiac sarcoplasmic reticulum.

Activation of Myocardial Adenyl Cyclase by Histamine in Guinea Pig, Cat, and Human Heart

Histamine has positive inotropic and chronotropic effects on the heart which are not abolished by beta adrenergic-blocking agents. Since the positive inotropic and chronotropic effects of other hormones on the heart are thought to be mediated by cyclic 3,5-AMP, we examined the effect of histamine on adenyl cyclase in particulate preparations of guinea pig, cat, and human myocardium. Histamine at the peak of its dose-response curve, 3 x 10(-4)moles/liter, produced approximately a 300% increase in cyclic 3,5-AMP accumulation in the guinea pig, 60% in the cat, and 90% in the human heart particles. Half-maximal activity for the histamine mediated activation of adenyl cyclase in the guinea pig was 9 x 10(-6)moles/liter, almost identical with that observed for norepinephrine in the same preparation. DL-Propranolol, 1 x 10(-5)moles/liter, did not abolish the activation of adenyl cyclase produced by histamine but did abolish the activation produced by norepinephrine. In contrast, diphenhydramine hydrochloride, Benadryl, 8 x 10(-5)moles/liter, abolished the activation of adenyl cyclase by histamine but not that produced by norepinephrine. These data suggest that there are at least two receptor sites in guinea pig heart mediating the activation of adenyl cyclase, one responsive to histamine, the other to norepinephrine. In addition, combined maximal doses of histamine and norepinephrine produced completely additive effects on the activation of adenyl cyclase, which suggests that at least two separate adenyl cyclase systems are present in the heart, each responsive to one of these hormones. However, definitive proof would require physical separation of the two enzymes.

Adenyl cyclase and myocardial contractility.

Abstract The activity of many hormones can be related to their capacity to increase adenyl cyclase activity in their target organs. Evidence that the augmentation of myocardial contractility produc...

The effect of sulfonylurea drugs on rabbit myocardial contractility, canine purkinje fiber automaticity, and adenyl cyclase activity from rabbit and human hearts

Long-term clinical studies have associated tolbutamide therapy with an increased incidence of cardiovascular deaths. The effects of this and other sulfonylurea drugs on contractility and rate of isolated rabbit atria, automaticity of isolated dog Purkinje fibers, and adenyl cyclase activity in particulate preparations of rabbit and human hearts were studied. At concentrations that are attained clinically, tolbutamide (10 mg/100 ml) increased contractility of driven rabbit atria to 124+/-5% of control, acetohexamide (3.9 mg/100 ml) to 140+/-5%, chlorpropamide (8.3 mg/100 ml) to 139+/-6%, and tolazamide (3.1 mg/100 ml) to 119+/-6%. These effects were accentuated in the presence of 2.5 x 10(-4) M theophylline and were not blocked by 1 x 10(-5) M propranolol. Adenyl cyclase was activated by each of these drugs at concentrations below those which increase contractility. The drugs also increased the rate and slope of phase 4 depolarization in spontaneously beating Purkinje fibers, but did not alter the spontaneous rate of isolated rabbit atria. Since inotropic and chronotropic stimulation can be deleterious in some clinical settings, these findings may be of significance in interpretation of cardiovascular mortality data.

Principles of Membrane Receptor Physiology and Their Application to Clinical Medicine

The clinical involvement of membrane receptors for hormones, neurotransmitters, and other molecules of interest is reported with increasing frequency. In this review, the terminology of the hormone-receptor field is defined, and the current concepts of the participation of membrane receptors in the mechanism of hormone action are outlined. Several major developing concepts of receptor regulation and function, including spare receptors and down regulation of receptor number, provide a basis for understanding the physiology and pathophysiology of cellular sensitivity to specific hormones. The relation of these concepts to the pathophysiology of various metabolic disorders is discussed. Clinical disorders in which membrane receptors play a central role are reviewed, including Graves disease and myasthenia gravis, which involve antireceptor antibodies, and familial hypercholesterolemia and various conditions of hormone resistance that often involve a marked decrease in receptor number as the primary pathogenic factor. The significance of receptor physiology in other selected areas of clinical medicine is discussed.