The effects of hormones and prostaglandins on the calcium pools in cultured myocardial cells

Determinations of the calcium pools in myocardial cells in vitro have shown the existence of at least three pools of exchangeable calcium. Epinephrine and glucagon were found to produce significant increases in the size of the two slower exchanging pools. Prostaglandins E₁ and F_1α, also increased significantly calcium pool size whereas E₁ and F_2α did not; results which correlate well with the effects of the two former prostaglandins on intracellular cAMP levels. The results imply that these agents cause small, but significant, changes in the transmembrane exchange of calcium and large increases in the intracellular calcium pool. Effects which may involve the direct or indirect action of cAMP.     

Prostaglandin effects upon cyclic AMP levels, corticosterone output, and aldosterone output in the adrenal of the frog, Rana berlandieri forreri.

The mechanism of prostaglandin action in the adrenal of the frog (Rana berlandieri forreri) was evaluated in vitro. The prostaglandins evoked transient (PGA₂, PGB₁, PGE₂), continued (PGA₁, PGB₂, PGF_1α, PGF_2α), or no effects (PGE₁) upon cyclic AMP (cAMP) levels. Further, the cAMP levels were depressed (PGA₁, PGB₁, PGB₂), elevated (PGA₂, PGE₂, PGF_1α, PGF_2α), or unchanged (PGE₁) compared to the controls. Prostaglandins regulate cAMP levels in the frog adrenal. In addition, specific prostaglandins evoke specific effects in this regard. The frog adrenal corticosterone and aldosterone outputs are modulated by the prostaglandins. The modulations produced differ with the prostaglandins tested. The greatest adrenocortical sensitivity is to PGB₂ which evoked about a 13-fold increase in corticosterone output and a 5-fold increase in aldosterone output at 16 min. Only PGF_1α inhibited steroid output; the other prostaglandins stimulated steroid outputs to varying degrees. A close correlation of the prostaglandin-evoked corticosterone and aldosterone output responses with the cAMP changes was not present in the frog adrenal. The prostaglandins differentially affected cAMP levels and when compared to adrenocorticoid outputs, different prostaglandins produce different effects. The responses of the frog adrenal indicate that the mechanisms controlling adrenocortical function are more complex than originally visualized. In this regard, the site(s) of action (plasma membrane, mitochondrion, etc.) of a given factor must also be considered. Thus, the current concepts must be broadened to include a number of interacting factors among which are the cyclic nucleotides and the prostaglandins.     

Characterization of cyclic 3', 5'-nucleotide phosphodiesterase activity in an islet cell tumor of the syrian hamster

During the purification of cyclic nucleotide phosphodiesterase (diesterase) activity from an insulin-secreting islet cell tumor of the syrian hamster, two soluble diesterases (diesterase I and diesterase II) and a particulate diesterase were resolved. Based on gel filtration, the estimated molecular weights of diesterases I and II were approximately 180,000. All of the diesterases had ‘low’ (approximately 1 μM) and ‘high’ (10–20 μM) K_M's for both cAMP and cGMP. A heat-stable protein activator was partially separated from the soluble diesterases upon DEAE-cellulose chromatography. The activator enhanced the activity of the soluble diesterase 2$\text{1}\text{2}$-fold at cyclic nucleotide concentrations of 5 μM. No stimulation of diesterase activity was observed at higher (50–500 uM) substrate concentrations.The following compounds (in order of decreasing potency) inhibited both the soluble and particulate diesterases: papaverine, SQ 20009, xanthine derivatives, diphenylhydantoin, diazoxide, sulfonylureas, nicotinamide and catecholamines. Insulin, proinsulin, prostaglandins E₁ and F_1α, secretin, glucagon, colchicine, glucose, streptozotocin and imidazole were all without effect. Sulfhydryl-containing compounds and a number of amino acids enhanced the activity of soluble and paniculate diesterases. These investigations coupled with studies of adenylate cyclase and protein kinase activities, intracellular cyclic nucleotide levels and insulin secretion will provide a basis for understanding the role of cAMP in insulin secretion.     

Evidence of the receptor‐mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein‐coupled and PI3K signaling pathways

Abstract: Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α‐cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon‐producing mouse pancreatic α‐cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P‐PDOT abolished the glucagon‐stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild‐type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi‐ and Gαs‐coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin‐induced effect, indicating the physiological relevance of the Gαq‐coupled pathway. Our data point to the involvement of the phosphatidylinositol 3‐kinase signaling cascade in mediating melatonin effects in pancreatic α‐cells. In conclusion, these findings provide evidence that the glucagon‐stimulatory effect of melatonin in pancreatic α‐cells is melatonin receptor mediated, thus supporting the concept of melatonin‐modulated and diurnal glucagon release.     

Antioxidant inhibition of prostaglandin production by rat renal medulla.

Antioxidant effects upon renal production of both prostaglandins and cAMP were investigated using slices of rat inner medulla. Synthetic antioxidants were more potent inhibitors of prostaglandin production than were naturally occurring antioxidants. Synthetic compounds 2,7-naphthalenediol, and Santoquin^® (Ethoxyquin) caused a 60% inhibition of prostaglandin E₂(PGE₂) synthesis at a concentration of 0.01 mM. Ascorbic acid caused only a 30% inhibition at a concentration of 10 mM. Antioxidant inhibition of prostaglandin production was also observed following arachidonic acid addition. Antioxidants that reduced PGE₂ synthesis also reduced PGF_2α synthesis. Test agents found to reduce prostaglandin synthesis also lowered cAMP content. 2,7-Naphthalenediol elicited a dose-dependent decrease in both prostaglandin synthesis and cAMP content. While PGE₁ did not increase cAMP in control slices, the low cAMP level produced by Santoquin was increased to control values by PGE₁. Furthermore, Santoquin and 2,7-naphthalenediol did not alter arginine vasopressin-stimulated cAMP content. By contrast, inhibition of the arginine vasopressin stimulation by butylated hydroxyanisole suggested additional effects by this agent. These results are consistent with the hypothesis that endogenously produced PGE₂ can exert a hormonelike action in the inner medulla by increasing cAMP content. Advantages of the inner medullary slice system compared to homogenates for investigation of the actions of antioxidants or other agents thought to alter prostaglandin synthesis are discussed.     

Effects of α and β adrenoceptors on branchial cAMP level in seawater mullet, Mugil capito

At 21°, the basal level of branchial cAMP of Mugil capito is higher than that observed at 16°. Intraperitoneal injection of isoproterenol induces a great increase of branchial cAMP concentration which is completely abolished by simultaneous administration of propranolol. Phentolamine does not modify the response to isoproterenol. Since salbutamol, a β₂ stimulator, given in varied doses, is without effect on branchial cAMP level, the cAMP increase seems to be due to the activation of β₁ adrenoceptors. Phenylephine appears to stimulate both α and β receptors in the fish, since the addition of phentolamine potentiates the rise of the cAMP level whereas propranolol addition reduces it significantly. Another α stimulator, clonidine, provokes a branchial cAMP decrease which depends on the injected doses. These results show that α-adrenoceptor stimulation results in a reduction of cAMP concentration in the fish gill. Since phenylephrine and clonidine stimulate the α adrenoceptors, the gills of the mullet might hold α₁ and α₂ receptors. Further studies are necessary to characterize the subtypes of α and β adrenoceptors in fish.     

Glucagon secretion in relation to insulin sensitivity in healthy subjects

Aims/hypothesis The study evaluated whether glucagon secretion is regulated by changes in insulin sensitivity under normal conditions. Materials and methods A total of 155 healthy women with NGT (aged 53–70 years) underwent a glucose-dependent arginine-stimulation test for evaluation of glucagon secretion. Arginine (5 g) was injected i.v. under fasting conditions (plasma glucose 4.8±0.1 mmol/l) and after raising blood glucose concentrations to 14.8±0.1 and 29.8±0.2 mmol/l. The acute glucagon response (AGR) to arginine during the three glucose levels (AGR₁, AGR₂, AGR₃) was estimated, as was the suppression of baseline glucagon by the increased glucose. All women also underwent a 2-h euglycaemic–hyperinsulinaemic clamp study for estimation of insulin sensitivity. Results Insulin sensitivity was normally distributed, with a mean of 73.2±29.3 (SD) nmol glucose kg⁻¹ min⁻¹/pmol insulin l⁻¹. When relating the variables obtained from the arginine test to insulin sensitivity, insulin resistance was associated with increased AGR and with increased suppression of glucagon levels by glucose. For example, the regression between insulin sensitivity and AGR₂ was r=−0.38 (p<0.001) and between insulin sensitivity and suppression of glucagon levels by 14.8 mmol/l glucose r=0.36 (p<0.001). Insulin sensitivity also correlated negatively with insulin secretion; multivariate analysis revealed that changes in insulin sensitivity and insulin secretion were independently related to changes in glucagon secretion. Conclusions/interpretation The body adapts to insulin resistance by increasing the glucagon response to arginine and by increasing the suppression of glucagon levels by glucose. Hence, not only the islet beta cells but also the alpha cells seem to undergo compensatory changes during the development of insulin resistance.     

Prostaglandin E1-sensitive adenylate cyclase of rat liver plasma membranes

Adenylate cyclase of isolated rat liver plasma membranes is stimulated at least four-fold by prostaglandin E₁ (PGE₁) provided that EGTA and GTP are present in the assay system. The GTP-dependency is absolute, since in its absence the PGE₁ activation of adenylate cyclase is small and inconsistent. PGE₂ is much less effective while prostaglandins F_1α and F_2α are ineffective under all conditions tested. The stimulatory effect of PGE₁ is evident at concentrations around 0.5 uM, optimal stimulation requires 3 μM PGE₁.Several lines of evidence suggest that PGE₁ interacts with plasma membrane binding sites separate from those specific for glucagon and epinephrine. First, the effect of combinations of PGE₁ with glucagon or epinephrine at optimal concentrations are additive, while not additive is the combination PGE₁-NaF. Second, variations in the temperature of incubation between 25 and 45°C influence in a different way glucagon and PGE₁-sensitive cyclase. Glucagon stimulation continuously increases with the increase of temperature while PGE₁ stimulation increases up to 30°C then it levels off and decreases at 45°C. Third, propranolol which nearly completely blocks epinephrine stimulation is much less effective on PGE₁-sensitive cyclase. 0.1 mM phentolamine has little effect, if any, on hormonal sensitivity. Lubrol at very low doses (1 mg/100 ml) reduces the effect of epinephrine and glucagon, but increases that of PGE₁.     

Potentiation of alpha 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.

Treatment of isolated hepatocytes with the alpha 1-adrenergic agonist norepinephrine induced a dose-dependent increase in free cytosolic Ca2+, as judged by fluorescence increases, in cells loaded with the Ca2+ indicator (2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8 -bis [carboxymethyl]aminoquinoline (quin-2). Pretreatment with either glucagon or dibutyryl cAMP increased the rate and magnitude of the quin-2 fluorescence response in hepatocytes treated with submaximal doses of norepinephrine and increased the cell sensitivity such that a physiological concentration of norepinephrine (7.5 nM) was able to provoke a quin-2 fluorescence response. Similar enhancement of norepinephrine-induced phosphorylase activation and pyridine nucleotide reduction in isolated hepatocytes and Ca2+ efflux from the perfused liver was also observed in the presence of glucagon. These potentiated responses correlated with a cAMP-dependent increase (mediated by glucagon, dibutyryl cAMP, or forskolin) in the binding of [3H]norepinephrine or [3H]epinephrine to sites present on isolated hepatocytes bearing the characteristics of alpha 1-adrenergic receptors. The data suggest that a cAMP-dependent mechanism is involved in the regulation of alpha 1-agonist binding to liver cells and, thereby, in the control of hepatic carbohydrate metabolism in response to catecholamines.     

Metabolic effects of neutral red in rats

Summary Metabolic responses to neutral red administration were studied in normal and hypophysectomized rats. In normal animals neutral red causes a marked increase in blood glucose and lactic acid. The plasma glycerol increases very slightly, but significantly, whereas the plasma FFA do not show a significant increase. After DHE — an adrenergic blocking agent of hepatic glycogenolysis — neutral red induces a decreased but still evident hyperglycemic response. In hypophysectomized rats all metabolic responses to neutral red are abolished. These results suggest that neutral red causes hyperglycemia by releasing glucagon from A₂-cells, the glucagon in its turn causing the release of catecholamines from the adrenals. Both glucagon and catecholamines might be responsible for the lipolytic effect of neutral red. The influence of the hypophysis on these metabolic phenomena is discussed.Research supported by C.N.R. grant (70. 01729. 04 115. 1187)     

Studies on the influence of insulin on cyclic adenosine monophosphate in human vascular smooth muscle cells: dependence on cyclic guanosine monophosphate and modulation of catecholamine effects

Summary Insulin increases both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in human vascular smooth muscle cells (hVSMC) and attenuates noradrenaline-induced vasoconstriction. In the present study, we aimed at investigating in hVSMC: 1) the interrelationships between insulin-induced increases of cGMP and cAMP; 2) the insulin effect on the catecholamine modulation of cAMP. Catecholamines cause both vasoconstriction and vasodilation. Vasoconstriction is attributable to the reduced synthesis of cAMP in hVSMC through α₂-adrenoceptors and to direct effects on calcium fluxes through α₁-adrenoceptors; vasodilation is attributable to the increased synthesis of cAMP through β-adrenoceptors. In the present study, we determined the influence of insulin on cAMP in hVSMC incubated with or without: a) the inhibitor of guanylate cyclase methylene blue or the inhibitor of nitric oxide synthase N^G-monomethyl-l-arginine (l-NMMA); b) the β-adrenergic agonists isoproterenol and salbutamol; c) the physiological catecholamines noradrenaline and adrenaline; d) noradrenaline + the β-adrenergic antagonist propranolol or the α₂-adrenergic antagonist yohimbine; e) noradrenaline + methylene blue orl-NMMA. We demonstrated that: 1) the inhibition of the insulin-induced cGMP synthesis blunts the insulin-induced increase of cAMP; 2) insulin induces a significant increase of cAMP also in the presence of isoproterenol, salbutamol, noradrenaline and adrenaline: the combined effects of insulin and catecholamines were additive in some, but not in all the experiments; 3) insulin enhances the cAMP concentrations induced by noradrenaline also in the presence of α₂- or β-adrenergic antagonists; 4) in the presence of methylene blue orl-NMMA insulin does not modify the noradrenaline effects on cAMP.     

Different catecholamines induce different patterns of takotsubo-like cardiac dysfunction in an apparently afterload dependent manner

Background

Takotsubo cardiomyopathy (TCM) is characterized by regional left ventricular dysfunction that cannot be explained by an occlusive lesion in a coronary artery. Catecholamines are implicated in the pathogenesis of TCM but the mechanisms involved are unknown. Because the endogenous and the most commonly used exogenous catecholamines have well defined adrenoceptor subtype affinities, inferences can be made about the importance of each adrenoceptor subtype based on the ability of different catecholamines to induce TCM. We therefore studied which of five well-known catecholamines, that differ in receptor subtype affinity, are able to induce TCM-like cardiac dysfunction in the rat.

Methods

255 rats received intraperitoneally isoprenaline (β₁/β₂-adrenoceptor agonist), epinephrine (β₁/β₂/α-adrenoceptor agonist), norepinephrine (β₁/α-adrenoceptor agonist), dopamine (α/β₁/β₂-adrenoceptor agonist) or phenylephrine (α-adrenoceptor agonist). Each catecholamine was given in five different doses. We measured blood pressure through a catheter inserted in the right carotid artery and studied cardiac morphology and function by echocardiography.

Results

All catecholamines induced takotsubo-like cardiac dysfunction. Isoprenaline induced low blood pressure and predominantly apical dysfunction whereas the other catecholamines induced high blood pressure and basal dysfunction. In another set of experiments, we continuously infused hydralazine or nitroprusside to rats that received epinephrine or norepinephrine to maintain systolic blood pressure < 120 mm Hg. These rats developed akinesia of the apex instead of the base. Infusion of phenylephrine to maintain blood pressure > 120 mm Hg after isoprenaline administration prevented apical TCM-like dysfunction.

Conclusions

Catecholamine-induced takotsubo-like cardiac dysfunction appears to be afterload dependent rather than depend on stimulation of a specific adrenergic receptor subtype.     

Reversible Decrease of Surface ß2-Adrenoceptor Number and Response in Lymphocytes of Patients with Pheochromocytoma

To study the effect of chronic exposure to elevated plasma catecholamines on surface ß₂-adrenoceptor density, we measured these receptors in the lymphocytes of 9 patients with pheochromocytoma as well as in 27 healthy control subjects. Binding experiments were performed on intact lymphocytes using the hydrophilic ligand [³H]-CGP12177. Lymphocyte β₂-adrenergic response was also measured in three patients. ß₂-adrenoceptor density (p<0.01), and isoproterenol-stimulated increase in cAMP were reduced in patients with pheochromocytoma. Both parameters normalized (p<0.05) when patients were reevaluated 4 weeks after tumor removal, coinciding with normalization of plasma catecholamine levels. ß₂-adrenoceptor density inversely correlated to log of plasma epinephrine (r=-0.95, p<0.01) and to log of plasma norepinephrine (r=-0.58, p<0.05) in patients.

We conclude that chronic catecholamine excess induces a decrease of lymphocyte ß₂-adrenoceptor surface number and response that is reversible upon normalization of plasma catecholamine levels. This regulation is mainly dependent on plasma levels of the hormone epinephrine, but norepinephrine may also play a regulatory role at supraphysiological levels.     

Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. Here we report the cloning and characterization of cDNAs encoding rat and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor superfamily member expressed in the gut and closely related to the glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome 17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1 or related peptides, with increased cAMP production (EC₅₀ = 0.58 nM) and displayed saturable high-affinity radioligand binding (K_d = 0.57 nM), which could be displaced by synthetic rat GLP-2 (K_i = 0.06 nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo. These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.     

Early biochemical reactions in the lung of sheep exposed to asbestos: Evidence for cyclic AMP accumulation in bronchoalveolar lavage fluids

A conscious sheep model which allows sequential analysis of the bronchoalveolar milieu was used to investigate the early biochemical events following asbestos exposure. Segmental bronchoalveolar lavages (BAL) were performed in a group of 15 sheep exposed to repeated doses of UICC chrysotile B asbestos (0–128 mg) over a six month period. BAL cell population was analysed and various humoral components including total proteins, albumin, alpha₁-globulins (α ₁), alpha₂-globulins (α ₂), beta + gamma globulins (β+γ) and adenosine 3′,5′-cyclic monophosphate (cAMP) were determined in cell free supernates. All animals were also studied by pulmonary function tests (PF) and transbronchial lung biopsies (LB). Exposure to asbestos (up to 128 mg/month) did not cause any significant alteration of pulmonary functions and lung histology. However, analyses of the BAL effluent revealed that BAL fluids of sheep exposed to asbestos contained higher levels ofα ₁-globulins and cyclic AMP as compared to controls. Furthermore this phenomenon was observed without overall change of BAL cell population except for a significant eosinophilia in the asbestos-exposed animals. These data demonstrate that the early biochemical response of sheep exposed to asbestos is characterized by a significant eosinophilia and increases ofα ₁-globulins and cyclic AMP in the bronchoalveolar milieu. These biochemical events may be involved in the chronic inflammatory reaction to asbestos and possibly in the fibrogenic response.

     

The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells

The role of cyclic AMP in the induction of enzymes involved in estrogen and progestin biosynthesis in undifferentiated granulosa cells was investigated. When granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2 days in serum-free medium with aromatase substrate (10^?7M androstenedione) together with graded doses of FSH, prostaglandin E₂ (PGE₂), cholera toxin (CT), or dibutyryl cyclic AMP (Bu₂cAMP), there was a dose-related increase in estrogen (E) production. The induction of E production by saturating doses of FSH, PGE₂, CT and Bu₂cAMP required a lag phase of ～24 h, after which the E response increased sharply to maximum levels at day 3 and then declined gradually to day 5. Treatment for 24 h (day 0–1) with FSH, together with 1 μg/ml of either actinomycin D or cycloheximide, completely abolished the stimulatory action of FSH on E production. When the inhibitors were removed, the FSH-induced increases in E returned to near normal levels after a 24-h lag period. Similar effects of the inhibitors upon E production by CT, PGE₂ and Bu₂cAMP were observed. As with E, the production of progesterone and 20 α-dihydroprogesterone was markedly stimulated by FSH, PGE₂, CT and Bu₂cAMP and the results of the time course, dose response and inhibitor experiments were similar to those for E production.These results indicate that FSH induces the de novo synthesis of enzymes required for both estrogen and progestin biosynthesis by undifferentiated granulosa cells and suggest that this action is mediated by cyclic AMP.     

Antiadrenergic action of adenosine on ventricular myocardium in embryonic chick hearts

Adenosine, a physiological metabolite, is known to exert a negative inotropic effect on atrial muscle [5, 7], to impair atrioventricular conduction [2, 7, 17] and depress pacemaker activity [18]. In addition to such direct actions, adenosine is also known to modulate several of the actions of catecholamines in different tissues [9, 15, 16]. In ventricular muscle, adenosine attenuates the inotropic effect of catecholamines, but has no contractile effect in the absence of catecholamines [1, 6, 15]. Thus, adenosine appears to exert its functions in heart by direct [2, 5, 7, 18] and indirect (anti-adrenergic) [1, 11, 15] mechanisms.The isoproterenol-induced increase in ventricular contractility is associated with an increase in cyclic AMP (cAMP) [19]. Since isoproterenol, cAMP analogs, and cAMP itself (applied internally), increase the Ca²⁺-inward current (Isi) and slow action potentials, a causal relationship between cAMP and Isi has been proposed [10]. The ability of isoproterenol to restore excitability to partially depolarized cardiac fibers and/or enhance pre-existing slow action potentials is attributed to increase in cAMP that leads to an increase in the Isi.Adenosine decreased the cAMP levels of catecholamine-treated hearts [1, 6, 15] but unexpectedly adenosine had no inhibitory effect on slow action potentials obtained in elevated K⁺ (27 mM) plus 10^?7 to 10^?6 M isoproterenol solution [12, 13]. In the present study, we wanted to find out whether adenosine would attenuate ventricular slow action potentials enhanced by relatively low concentrations of isoproterenol in normal K⁺ solution. We set out to confirm in parallel experiments that adenosine also reduces the cAMP content of hearts exposed to isoproterenol.