Relaxation response of isolated canine veins to agents that act on the adenylate cyclase-cyclic AMP system: further investigation.

The present study was undertaken to clarify whether there is a deficiency in the relaxation mediated by the adenylate cyclase-cyclic AMP (cAMP) system in the portal vein as compared to the saphenous vein. Longitudinal strips of the portal vein and helical preparations of the saphenous vein were used. The relaxation response to various agents was examined under conditions such that the venous preparations were previously contracted by methoxamine in equipotent concentrations (EC80), i.e., 10(-6) M for portal vein and 10(-5) M for saphenous vein. The saphenous vein relaxed fully in response to isoproterenol but the portal vein relaxed only to 29% of the maximum relaxation induced by papaverine 10(-4) M. However, dibutyryl cAMP and 8-bromo-cAMP, membrane permeable derivatives of cAMP, 3-isobutyl-1-methylxanthine and papaverine, phosphodiesterase inhibitors, and forskolin, a direct stimulator of adenylate cyclase, relaxed portal and saphenous veins similarly though with quantitative differences. The results suggest that there is no profound deficiency in the adenylate cyclase-cAMP system but there may be a deficiency in the coupling between surface beta-adrenoceptors and adenylate cyclase or there may be a low density of beta-adrenoceptors in the portal vein.     

Heterogeneity of beta-adrenoceptor in canine veins: comparison among the facial, portal and saphenous veins

A study was made on the characteristics of beta-adrenoceptors in the isolated canine facial, portal and saphenous veins. Ring segments of the facial and saphenous veins and longitudinal strips of the portal vein were suspended in tissue baths containing Krebs solution oxygenated and maintained at 37 degrees C. They were moderately contracted with prostaglandin F2 alpha before examining their relaxation responses. The facial and saphenous veins fully relaxed to isoproterenol, while the portal vein relaxed to a small extent (20% of maximum relaxation) even in the presence of an alpha-adrenoceptor blockade. In contrast, both forskolin, a direct activator of adenylate cyclase, and membrane-permeable dibutyryl cyclic AMP similarly relaxed all the veins studied. Thus, the reduction of coupling between beta-adrenoceptors and the adenylate cyclase system may be involved in the decreased responsiveness of the portal vein to beta-adrenoceptor agonists. In addition, analyses of beta-adrenoceptor agonism and antagonism, using selective (beta 1: T-1583, beta 2: procaterol) and non-selective (isoproterenol) agonists as well as selective (beta 1: atenolol, beta 2: ICI 118,551) and non-selective (propranolol) antagonists, confirmed that beta-adrenoceptors in the canine facial vein are not homogeneous, with the beta 1-subtype predominating over the beta 2-subtype, and that the canine saphenous vein has a homogeneous population of the beta 2-subtype, as reported in the other species.     

Rate-limiting steps in isoproterenol and forskolin stimulated lipolysis

Using the flask-incubated fat cell system, effects of isoproterenol and forskolin on glycerol release, cyclic AMP levels and protein kinase were studied. Isoproterenol increased cyclic AMP levels, protein kinase activity and glycerol release over the same concentration range (10(-9) M to 10(-6) M). Forskolin also increased all three variables in a concentration-dependent manner (10(-7) M to 10(-4) M). The maximum response for each variable was significantly greater with forskolin than with isoproterenol. A combination of isoproterenol and forskolin resulted in an additional increase in cyclic AMP over forskolin alone, but no significant increase in protein kinase activity or glycerol release. These results support the concepts that the maximum lipolytic response to isoproterenol is limited by the accumulation of cyclic AMP and the maximum lipolytic response to forskolin is limited by some step distal to cyclic AMP production, possibly activation of protein kinase. At high concentrations of forskolin or with a combination of forskolin and isoproterenol, cyclic AMP levels were in excess of those needed to maximally activate protein kinase and lipolysis.     

Sumatriptan (GR43175) inhibits cyclic-AMP accumulation in dog isolated saphenous vein.

Sumatriptan (GR43175) contracts rings of dog isolated saphenous vein by an action at 5-HT1-like receptors. We have now examined the effects of sumatriptan on prostaglandin E2(PGE2)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in this tissue. Sumatriptan and 5-hydroxytryptamine (5-HT) produced a concentration-dependent inhibition of PGE2-stimulated cyclic AMP accumulation (EC50 values of 250 nM and 80 nM respectively), responses that were mimicked by 5-carboxamidotryptamine but not by U-46619 or methoxamine. The response to sumatriptan (1 microM) was antagonised by methiothepin (1 microM), but not by metergoline (0.1 microM), spiperone (1 microM) or ondansetron (GR38032, 1 microM). These results suggest that 5-HT1-like receptors which mediate contraction of the dog isolated saphenous vein are negatively coupled to adenylate cyclase in this preparation.     

Alterations in cardiac beta-adrenoceptor responsiveness and adenylate cyclase system by congestive heart failure in dogs

The effects of congestive heart failure on the physiological and biochemical functions of the cardiac beta-adrenoceptor-coupled adenylate cyclase system were studied in dogs with right heart failure produced by progressive pulmonary artery constriction and tricuspid avulsion. The cardiac inotropic response to dobutamine was attenuated in congestive heart failure, as determined by the right and left ventricular dP/dt responses. Adrenergic beta-receptor density, measured by [3H]dihydroalprenolol binding, was reduced in membrane fractions of the failing right ventricle, but not in the left ventricle. The functional activity of the adenylate cyclase system was studied in vitro by measuring the net cyclic AMP production following additions of isoproterenol, 5'-guanylylimidodiphosphate (Gpp(NH)p), forskolin, or manganese chloride, which act either directly on the beta-adrenergic receptors or on one of the post-receptor components of the adenylate cyclase system. Congestive heart failure reduced the net production of cyclic AMP by isoproterenol, Gpp(NH)p, and forskolin in both the right and left ventricles, but did not alter the effect of manganese chloride. Thus, beta-receptor down-regulation is chamber-specific, occurring only in the hemodynamically stressed right ventricle. In contrast, the post-receptor defect of the adenylate cyclase system occurred in both ventricles of the heart failure dogs. This decreased activation of adenylate cyclase by beta-agonists may be responsible, at least in part, for the diminished cardiac inotropic response to catecholamines in congestive heart failure.     

Anatomical differences in responsiveness to vasoconstrictors in the mesenteric veins from normal and portal hypertensive rats

The present study evaluates the effects of pre-hepatic portal hypertension, induced in rats by partial portal vein ligation, on the responsiveness of rostral (proximal) and caudal (distal) rings from the mesenteric vein. The anatomical origin of the sample influenced the response to vasoconstrictors in sham-operated animals, and this pattern of reactivity was specifically modified in portal-ligated rats. In veins from sham-operated rats, contraction induced by a submaximal concentration of KCl (60 mM) was greater in proximal than in distal rings. Vasopressin and 5-hydroxytryptamine contracted mainly distal rings, methoxamine showed a greater effect on proximal rings, and endothelin-1 and angiotensin-II contracted vein rings independently of their anatomical origin. In veins from portal hypertensive rats, responses to KCl (60 mM) were increased in distal rings, and all rings exhibited enhanced reactivity to vasopressin and 5-hydroxyptyptamine as well as attenuation of the response to methoxamine. Responses to endothelin-1 were decreased in proximal vein rings from portal hypertensive rats whereas responses to angiotensin-II were not influenced by the anatomical origin. Incubation with atropine, propranolol or indomethacin, did not modify the responses to vasopressin and 5-hydroxytryptamine in tissues from either sham-operated or portal hypertensive animals. Likewise, the hyporeactivity to methoxamine and endothelin-1 in rings from portal hypertensive rats persisted in the presence of the nitric oxide inhibitor N ^G-nitro-l-arginine methyl ester. These results suggest the physiological existence of anatomical differences in the responsiveness to vasoconstrictors throughout the mesenteric vein and that changes in the responsiveness of the mesenteric vein induced by portal hypertension are specific for each agonist and possibly result from individual variations at a receptor or post-receptor level.     

Cyclic adenosine 3',5'-monophosphate in rat cerebral cortical slices: effects of methoxamine and clonidine.

In incubated slices of cerebral cortex from Sprague-Dawley rats, methoxamine and clonidine have no effect on basal levels of cyclic AMP. Methoxamine effectively inhibits the noradrenaline-stimulated formation of cyclic AMP. The inhibitory constant for methoxamine was 12.6 mumol/l. In the presence of 100 mumol/l adenosine, methoxamine does not inhibit the activity of noradrenaline, but is capable to activate alpha-adrenergic receptors leading to enhanced formation of cyclic AMP. The mechanism by which adenosine alters adrenergic receptors to become methoxamine-sensitive is not known. Clonidine inhibits the effect of noradrenaline alone or in combination with adenosine on the cyclic-AMP-generating system. It does not, as reported earlier, enhance the activity of submaximal concentrations of the beta-adrenergic agonist isoproterenol. These data do not support the concept of adrenergic receptors which require both, alpha- and beta-stimulation for maximal activation of adenylate cyclase.     

Tachyphylaxis to beta-adrenoceptor agonists in guinea pig airway smooth muscle in vivo and in vitro.

Beta-Adrenoceptor tachyphylaxis was induced by incubating spirally cut guinea pig tracheas with isoproterenol (2.4 x 10(-7) M) for 20 min. This incubation reduced the relaxant effects of catecholamines but not of dibutyryl cyclic AMP, theophylline or sodium nitrite. Tracheas incubated with norepinephrine, phosphodiesterase inhibitors or cyclic nucleotides became tachyphylactic to isoproterenol. Pretreatment with indomethacin prevented induction of tachyphylaxis. Incubation with adenosine, methoxamine or sodium nitrite did not induce beta-adrenoceptor tachyphylaxis. When we gave isoproterenol intramuscularly to guinea pigs, airway sensitivity to aerosolized histamine was unchanged but the toxicity of parenterally administered histamine was increased. A prolonged treatment with isoproterenol reduced airway sensitivity to histamine aerosols; this reduced sensitivity was reversed by indomethacin. Thus, beta-adrenoceptor tachyphylaxis may not explain increased toxicity of parenteral histamine after isoproterenol treatment. Elevated levels of cyclic AMP and an increased synthesis of prostaglandins may result in diminished response to beta-receptor stimulation.     

Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes

Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.     

Influence of increase in osmotic pressure with sucrose on relaxation and cyclonucleotides levels in isolated rat aorta.

The influence of increases in osmolarity by addition of sucrose were investigated on relaxation and changes in adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in isolated rat aortic rings. Isoprenaline-mediated relaxations were attenuated in hypertonic (341+/-0.4 mOsmol) (mean+/-S.E.M.) solution. The concentration-response curve to isoprenaline was displaced to the right. The EC(50) (0.16+/-0.05 vs. 1.14+/-0.5 microM) significantly (n=6; P<0.05) increased without any changes to the maximum response. Hypertonic solution also attenuated methacholine-mediated relaxations resulting in a significant increase in the EC(50) (0. 28+/-0.04 vs. 0.52+/-0.04 microM) and reduced the maximal response (73+/-5% vs. 51+/-8%). In contrast, an increase in tonicity did not have any influence on sodium nitroprusside, forskolin or pinacidil concentration-response curves. Hypertonic solution also did not affect either basal cyclic AMP or cyclic GMP production. In addition, an increase in osmolarity did not affect isoprenaline-stimulated increases in the levels of cyclic AMP. However, an increase in the tonicity of Krebs solution significantly inhibited methacholine-stimulated (58%-34%) accumulation of cyclic GMP. The present data indicated that an increase in the tonicity of Krebs solution impaired endothelium-dependent relaxation and the associated increase in cyclic GMP production without affecting basal levels of this nucleotide. The inhibitory effects of high osmolarity on beta-adrenoceptor-mediated relaxation did not appear to be due to a reduction in cyclic AMP generation, or the result of inhibition of pinacidil-sensitive K(ATP)(+) channels. Moreover, an increase in the tonicity of Krebs solution did not influence relaxation induced by direct activation of adenylate cyclase or guanylate cyclase by forskolin and sodium nitroprusside, respectively.     

Desensitization of catecholamine-stimulated adenylate cyclase and down-regulation of beta-adrenergic receptors in rat glioma C6 cells. Role of cyclic AMP and protein synthesis

When exposed to the beta-agonist (-)-isoproterenol, rat glioma C6 cells exhibited a time-and concentration-dependent reduction in isoproterenol responsiveness (desensitization) and a loss of beta-adrenergic receptors (down-regulation). Other agents, such as dibutyryl cyclic AMP, isobutylmethylxanthine, and cholera toxin, all of which elevate intracellular cyclic AMP levels, also induced receptor down-regulation but at a much slower rate than isoproterenol. Loss of beta-receptors was detected with intact cells, cell lysates, and cell membranes. Receptor loss was accompanied by a reduction in isoproterenol-stimulated cyclic AMP production and adenylate cyclase activity. For a given amount of receptor loss, this reduction was much greater with isoproterenol than with other agents. In addition, the concentration of isoproterenol required for half-maximal stimulation of cyclic AMP production was increased in cells treated with isoproterenol but not with isobutylmethylxanthine or dibutyryl cyclic AMP. The affinity of beta-receptors for the agonist was also lower in membranes from cells treated with isoproterenol but not the other agents. Prior treatment of the cells with cycloheximide inhibited receptor loss by isoproterenol but did not prevent desensitization or reduced affinity of beta-receptors for the agonist. Cycloheximide also blocked the loss of receptors induced by dibutyryl cyclic AMP and, in addition, prevented a reduction in agonist-stimulated adenylate cyclase activity. We propose that desensitization is mediated in rat glioma C6 cells only by agonists and is not dependent on either cyclic AMP or protein synthesis. Down-regulation can be induced both by agonists and by cyclic AMP and does depend on protein synthesis. Thus, desensitization and down-regulation can occur independently.     

Effects of SQ 22536, an adenylyl cyclase inhibitor, on isoproterenol-induced cyclic AMP elevation and relaxation in newborn ovine pulmonary veins

The effects of inhibition of adenylyl cyclase on isoproterenol-induced relaxation were determined in isolated pulmonary veins of newborn lambs (7-12 days old). In veins constricted with endothelin-1, isoproterenol at concentrations < or = 3 x 10(-9) M had no effect on the cyclic AMP (cAMP) content but caused up to 56% relaxation. At higher concentrations (> or = 10(-8) M), isoproterenol elevated cAMP content and caused further relaxation. In veins constricted with endothelin-1 or U46619 (9,11-dideoxy-11, 9-epoxymethanoprostaglandin prostaglandin F2alpha), the cAMP elevation but not relaxation caused by isoproterenol was abolished by SQ 22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; an adenylyl cyclase inhibitor]. The effects of isoproterenol on vessel tension and cAMP content were inhibited by propranolol. Rp-8-CPT-cAMPS [8-(4-Chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer] and Rp-8-Br-PET-cGMPS [beta-phenyl-1, N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer], inhibitors of cAMP- and guanosine-3',5'-cyclic monophosphate (cGMP)-dependent protein kinases, respectively, attenuated relaxation caused by a cAMP analog but not that by isoproterenol. In the crude membrane preparations of pulmonary veins, an increase in the activity of adenylyl cyclase caused by isoproterenol was abolished by propranolol and SQ 22536. These results suggest that cAMP may not play a critical role in isoproterenol-induced relaxation of pulmonary veins of newborn lambs.     

A regional difference in the distribution of postsynaptic alpha-adrenoceptor subtypes in canine veins

The responsiveness of helical venous strips isolated from fifteen different sites in the body of dogs to relatively selective alpha 1- and alpha 2-adrenoceptor agonists was studied, as well as to a non-selective alpha-adrenoceptor agonist. Longitudinal strips of the portal and mesenteric veins and the inferior vena cava between the liver and the renal vein (segment C) were also investigated. All veins contracted to noradrenaline or phenylephrine whereas only seven veins responded significantly to clonidine: the saphenous, cephalic, jugular and femoral veins and longitudinal strips of the portal and mesenteric veins and the segment C of the inferior vena cava. The brachiocephalic, azygos, pulmonary and splenic veins and the superior vena cava and the supradiaphragmatic portion (segment A) and the infrarenal portion (segment D) of the inferior vena cava responded little to clonidine. Unlike the longitudinal strips, the helical strips of the portal and mesenteric veins and the segment C of the inferior vena cava did not respond to clonidine. According to the relative sensitivities to phenylephrine and clonidine, those veins which responded to clonidine could be divided into three groups. (1) The veins in which the sensitivity to phenylephrine was higher than to clonidine: longitudinal strips of the portal vein and segment C of the inferior vena cava, (2) the veins whose sensitivity to phenylephrine was lower than to clonidine: the saphenous, cephalic, femoral and external jugular veins, (3) the vein whose sensitivity to the two agonists was comparable: longitudinal strips of the mesenteric vein. Subtype characteristics were further analyzed in the saphenous vein and in the portal vein using prazosin, phentolamine and yohimbine as antagonists. Analysis of Schild plots to noradrenaline suggested that a mixed population of alpha-adrenoceptor subtypes might be present in the saphenous vein, whereas a rather homogeneous population of a single subtype might occur in the portal vein. The results of the antagonism experiment against phenylephrine and clonidine suggested that contractions of the saphenous vein are mediated by both alpha 1- and alpha 2-adrenoceptors whereas contractions of the portal vein are exerted mainly through alpha 1-adrenoceptors. The results suggest that there may be a distinct regional difference with respect to postsynaptic alpha- adrenoceptor subtypes in the canine venous system.     

Desensitization of guinea pig tracheal muscle preparation to beta-adrenergic stimulants by a preceding exposure to a high dose of catecholamines.

When a given concentration of a catecholamine was applied to guinea pig tracheal preparation contracted by 20 muM histamine or by 30 mM-K+-Tyrode's solution, constant relaxations were observed, if the relaxation was submaximal. When a high concentration of catecholamine, 200 times the ED50, was once applied, subsequent responses to beta-stimulants (ED80) was reduced by about 30-40%, in spite of repeated washings. The response was gradually recovered in 2 hr. Thus 45 muM epinephrine and 1 muM isoproterenol could cause desensitization to 0.65 muM EPINEPHRINE AND 0.03 muM isoproterenol, respectively. Epinephrine and isoproterenol could cause densensitization to isoprophenamine, a non-catechol beta-stimulant. Epinephrine did not affect the response to cyclic AMP, dibutyryl cyclic AMP, aminophylline and prostaglandin E1. This densensitization was not affected by phentolamine, normetanephrine nor by Ca2+ deprivation from the bathing solution. The mechanisms of the desensitization may relate to some step(s) between the receptor-drug interaction and cyclic AMP accumulation in the process of tracheal muscle relaxation induced by beta-stimulants.     

Effects of forskolin and isoproterenol on cyclic AMP and tension in the myometrium

The effects of forskolin and isoproterenol on total cyclic AMP accumulation and relaxation were compared in uterine segments from estrogen-treated rabbits and term-pregnant rats. In the rabbit, forskolin (0.2-5.0 microM) inhibited spontaneous and acetylcholine-induced contractions and increased cyclic AMP levels. At lower doses forskolin significantly inhibited contractions but had little effect on cyclic AMP (cAMP) levels. Isoproterenol (0.005-0.5 microM), doses equi-effective to those of forskolin for inhibition of contractions, did not alter cyclic AMP levels. In the rat, forskolin (0.5 microM) and isoproterenol (0.05 microM) inhibited spontaneous and potassium-induced contractions by 80%. This reduction was accompanied by an increase in cyclic AMP levels, but the increase was significantly greater with forskolin than with isoproterenol. We conclude that: (1) cAMP may mediate relaxation under certain conditions but it is not an obligatory mediator, (2) some form of compartmentalization of cyclic AMP might exist in the myometrium.     

Cross talk between receptors mediating contraction and relaxation in the arterioles but not the dilator muscle of the rat iris.

1. Sympathetic nerve stimulation causes contraction of the dilator muscle and the large arterioles of the iris via the activation of alpha 1B-adrenoceptors. We have investigated whether increases in adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and the activation of receptors in these tissues can modulate these nerve-mediated contractions. 2. Increasing intracellular cyclic AMP with dibutyryl cyclic AMP (1 mM), forskolin (50 microM) or isobutylmethylxanthine (100 microM) produced relaxation of both the dilator and the arterioles, abolished the nerve-mediated constriction of the arterioles, but potentiated the nerve-mediated contraction of the iris dilator. 3. Pretreatment of the preparations with cholera toxin, to activate Gs permanently, caused a dilatation of the arterioles and abolished the nerve-mediated constriction but had no effect on the dilator muscle. 4. The beta-adrenoceptor agonist, isoprenaline (1 microM), the adenosine-A1,-A2 agonist, N-ethylcarboxamidoadenosine NECA (100 nM), in the presence of the A1-selective antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 10 nM), and calcitonin gene-related peptide (CGRP, 10 nM) all separately caused a dilatation of the arterioles and abolished the nerve-mediated constriction, while only isoprenaline (1 microM) produced an effect on the dilator, i.e. a relaxation but a potentiation of the nerve-mediated contraction. These results suggest the presence of at least 3 types of receptor linked to Gs and an increase in cyclic AMP in the arterioles, i.e. beta-adrenoceptor, adenosine-A2 and CGRP, but only 1 Gs-linked receptor, i.e. beta-adrenoceptors, on the dilator muscle cells.2+ '     

Relationship between cyclic AMP-dependent protein kinase activation and Ca uptake increase of sarcoplasmic reticulum fraction of hog biliary muscles relaxed by cholecystokinin-C-terminal peptides

In hog terminal bile duct cholecystokinin peptides caused an activation of cyclic AMP-dependent protein kinase (A-PK) with cyclic AMP, followed by increase in Ca uptake of sarcoplasmic reticulum fraction (SR-F). By contrast, papaverine showed no activation of A-PK-induced Ca uptake by SR-F with cyclic AMP. The Ca uptake by SR-F was dependent on ATP and Mg², but the component phosphorylated was not the phosphoenzyme intermediate in Ca²-ATPase. The effect of Ca uptake was blocked by the inclusion of a protein inhibitor of A-PK. The correlation coefficient between cyclic AMP-dependent SR-F phosphorylation and stimulated Ca uptake by the phosphorylated SR-F was 0.731 (P < 0.01). These results suggest that one of the mechanisms by which CCK-4, CCK-8, and CCK-33 peptides relax isolated Oddi's sphincters of terminal bile ducts is activation of A-PK-induced Ca uptake by sarcoplasmic reticulum fraction and possibly also by plasma membrane.     

Acetylcholine modulation of phosphorylase and contractility in rat hearts exposed to anoxia or isoproterenol

The effects of acetylcholine (ACh) on glycogen phosphorylase activated by either a cyclic AMP-dependent (isoproterenol) or a cyclic AMP-independent (anoxia) mechanism were examined. Cyclic adenosine 3' : 5'-monophosphate and cyclic guanosine 3' : 5'-monophosphate content, protein kinase and glycogen phosphorylase activities, and the contractile force of isolated perfused rat hearts exposed to either isoproterenol or anoxia were determined. Isoproterenol (1.6 × l0^?7M) produced an increase in cyclic AMP, activated protein kinase and glycogen phosphorylase, and increased intraventricular pressure developed by the myocardium. Acetyleholine did not alter basal phosphorylase activity or contractility. Acetylcholine, infused concurrently with isoproterenol, produced an increase in cyclic GMP and a decrease in cyclic AMP as well as in protein kinase and phosphorylase activity. The effects of ACh on cyclic GMP and phosphorylase were observed at 10^?5 M. Exposure of isolated perfused hearts to anoxia decreased the intraventricular pressure developed. This negative inotropic effect was accompanied by an activation of glycogen phosphorylase that was independent of alterations in cyclic AMP or cyclic GMP. A high concentration of acetylcholine (10^?4 M) further diminished the contractile activity of the heart and abolished the activation of phosphorylase. These effects also occurred in the absence of alterations in cyclic AMP but were coincident with an elevation of cyclic GMP. A lower concentration of ACh (10^?5 M) infused during anoxia, however, elevated cyclic GMP without concurrent effects on cyclic AMP, protein kinase, phosphorylase or contractility. Thus, phosphorylase activated by a cyclic AMP-independent mechanism was not affected by doses of acetvlcholine that were capable of suppressing phosphorylase activated by a cyclic AMP-dependent mechanism. These data support the concept that a reduction in cyclic AMP may be involved in mediating the effects of ACh on catecholamine-stimulated phosphorylase in myocardial tissue. The data do not support a role for cyclic AMP in the inhibition of anoxia-stimulated phosphorylase activation and, thus, some other modulating factor may be operative under these conditions. The role of cyclic GMP in this response remains in question.     

Effect of adrenergic agonists on the cyclic AMP level in the liver and heart from normal and hypothyroid rats.

In order to clarify the mechanism of the different sensitivity of the adrenoceptors between normal and hypothyroid rats, cyclic AMP levels in the liver and heart were measured after the administration of phenylephrine, isoproterenol, epinephrine and methoxamine. Cyclic AMP increased in all cases, but the extent of its increment in the heart was much less than that in the liver. Phentolamine and propranolol showed only a partial inhibition of cyclic AMP elevation by the agonists in the liver from normal and hypothyroid rat. On the other hand, propranolol blocked completely the effect of the agonists on the heart from both groups. It was also observed that cyclic AMP increased in adrenalectomized rats after the injection of the adrenergic agonists. The basal activity of protein kinase in hypothyroid status was slightly lower than that in the normal, but this enzyme was stimulated in the presence of cyclic AMP in vitro. These results suggest that the function of beta-adrenoceptor remained normal even in hypothyroidism and responded well to isoproterenol and epinephrine. It is also indicated that the increased sensitivity of alpha-adrenoceptor to phenylephrine in the hy pothyroid atria previously observed is probably in part independent of the mechanism mediated by cyclic AMP.     

Norepinephrine stimulated cyclic AMP accumulation in rat limbic forebrain slices: partial mediation by a subpopulation of receptors with neither alpha nor beta characteristics.

Both isoproterenol and norepinephrine (NE) increase cyclic AMP in slices of the rat limbic forebrain and the responses are enhanced in the presence of the phosphodiesterase inhibitor RO 20-1724. However, even in the presence of RO 20-1724, no accumulation of cyclic AMP was observed after the addition of dopamine, serotonin or the alpha-agonists methoxamine and phenylephrine. This suggests that these agents do not activate adenylate cyclase in this preparation or that their respective receptors--unlike the beta-receptor--are not coupled to adenylate cyclase. Isoproterenol, which has a high affinity for this adenylate cyclase system but only 20-30% of the maximal activity of NE, does not interfere with the agonist activity of NE. Moreover, the effect of isoproterenol is not additive with that of NE thus suggesting that isoproterenol is acting on a subpopulation of NE receptors. The results indicate that two populations of NE receptors coupled to adenylate cyclase are present in slices of rat limbic forebrain: one which has beta-characteristics and the other with neither alpha- nor beta-characteristics based on agonist studies.