Decrease In Adenylate Cyclase Activity Research Articles

Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

Inhibition of Camp-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/Mek/Erk and Ip3-Dependent Ca2+ Signaling in Atrial Myocytes

We previously reported in atrial myocytes that inhibition of PKA by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). Here, we determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 µM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 µM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 µM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in -PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in -PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In -PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in -PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) Is Dependent on PKC and IP3-Mediated Ca2+ Signaling in Atrial Myocytes

We reported that inhibition of adenylate cyclase (AC)/cAMP by cell attachment to laminin (+LMN) or pharmacological (KT5720) inhibition of cAMP-dependent protein kinase (PKA) in cells not attached to LMN (-LMN-PKA), activates β2-AR stimulation of ICa,L via cPLA2 signaling. The present study determined the role of PKC and IP3-mediated Ca2+ release in β2-AR/cPLA2 signaling. As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of L-type Ca2+ current (ICa,L) was unaffected by 10 μM AACOCF3 (cPLA2 inhibitor) in cells not attached to LMN (-LMN) but was significantly inhibited in +LMN and -LMN-PKA myocytes. Zinterol stimulation of ICa,L in -LMN-PKA myocytes was blocked by 5 μM U73122 (PLC inhibitor) and significantly inhibited by 4 μM chelerythrine (PKC inhibitor). Zinterol stimulation of ICa,L in -LMN myocytes was unaffected by inhibition of IP3-receptors (IP3Rs) by 2 μM 2-APB, but was significantly inhibited in +LMN and -LMN-PKA myocytes. Cells were cultured on LMN (24 hrs) with an adenovirus IP3 affinity trap to inhibit IP3-dependent Ca2+ signaling. Compared to control cells (β-gal), zinterol stimulation of ICa,L was significantly inhibited in cells infected with IP3 trap. Laser scanning confocal microscopy (fluo-4) revealed that zinterol stimulation of +LMN myocytes elicited local intracellular Ca2+ release events in 1 mM tetracaine (blocks RyR Ca2+ release), that were blocked by 2-APB. We conclude that inhibition of cAMP/PKA activates β2-AR stimulation of ICa,L via cPLA2 which is dependent on PKC and IP3-mediated Ca2+ signaling. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.

Inhibition of cAMP-Dependent Protein Kinase (PKA) Activates β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) in Atrial Myocytes

We reported that attachment of atrial myocytes to laminin (LMN) decreases adenylate cyclase (AC)/cAMP and increases β2-AR stimulation of L-type Ca2+ current (ICa,L). This study determined whether LMN enhances β2-AR signaling via a cAMP-independent mechanism, i.e. cPLA2 signaling. Atrial myocytes were plated on uncoated cover-slips (-LMN) or cover-slips coated with LMN (+LMN) (>2 hrs). As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of ICa,L was larger in +LMN than -LMN myocytes. In +LMN myocytes, zinterol stimulation of ICa,L was inhibited by 10 μM AACOCF3 (cPLA2 inhibitor), pertussis toxin or 10 μM BAPTA-AM (intracellular Ca2+ chelator). Stimulation of ICa,L by fenoterol (β2-AR/Gs agonist) was smaller in +LMN than -LMN myocytes. Arachidonic acid (AA; 5 μM) stimulated ICa,L in -LMN and +LMN myocytes similarly. Inhibition of PKA by either 5 μM H-89 or 1 μM KT5720 in -LMN myocytes mimicked the effects of +LMN myocytes to enhance zinterol stimulation of ICa,L, which was blocked by AACOCF3. In contrast, H-89 inhibited fenoterol stimulation of ICa,L, which was unchanged by AACOCF3. Inhibition of ERK1/2 by 1 μM U-0126 inhibited zinterol stimulation of ICa,L in +LMN myocytes and -LMN myocytes in which PKA was inhibited (KT5720). Western blots showed that inhibition of PKA (KT5720) in -LMN myocytes markedly increased zinterol phosphorylation of ERK1/2. We conclude that inhibition of AC/cAMP/PKA by cell attachment to LMN or PKA by pharmacological agents in -LMN myocytes switches β2-AR signaling from predominantly Gs/AC/cAMP/PKA to Gi/ERK1/2/cPLA2/AA. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.

The age-related decrease in catecholamine sensitivity is mediated by ß 1-adrenergic receptors linked to a decrease in adenylate cyclase activity in ventricular myocytes from male Fischer 344 rats

This study determined whether reduced sensitivity to catecholamines in aged myocytes resulted from deficits in the ß-adrenergic receptor (ß-AR) signaling pathway. Contractions and intracellular Ca 2+ were measured simultaneously in field-stimulated (2 Hz, 37 °C, fura-2) ventricular myocytes isolated from young adult (∼3 months) and aged (∼24 months) male Fischer 344 rats. Higher concentrations of a ß 1-AR agonist were required to increase contraction amplitudes in aged compared to younger cells; however, Ca 2+ transients were similar in both groups. There was no age-related difference in contraction or Ca 2+ transient amplitudes in response to a ß 2-AR agonist. The direct adenylate cyclase agonist forskolin caused smaller increases in contraction and Ca 2+ transient amplitudes in aged compared to younger cells. Phosphodiesterase inhibitors did not reverse the age-related deficit in positive inotropy caused by ß-AR stimulation. Direct measurement of cAMP showed significantly less cAMP formation in response to either ß-AR or adenylate cyclase stimulation in aged compared to younger cells. However, responses to dibutyryl cAMP were similar in young adult and aged myocytes, suggesting that events downstream of cAMP formation are not affected by age. The age-related decrease in catecholamine sensitivity is mediated by ß 1-ARs, resulting in a defect in cAMP production.

Reduced catecholamine sensitivity is related to a decrease in adenylate cyclase activity mediated by ß1‐adrenergic receptor signaling in aged ventricular myocytes

The aim of this study was to determine whether reduced sensitivity to catecholamines in aged myocytes results from deficits in ß‐adrenergic receptor (AR) signaling. Contraction amplitudes and intracellular Ca2+ were measured in field‐stimulated (2 Hz, 37°C, fura‐2) ventricular myocytes isolated from young adult (3 mos) and aged (24 mos) male Fischer 344 rats. The effect of a ß1‐AR agonist (but not a ß2‐AR agonist) on contraction amplitude was greater in young adult compared to aged myocytes; however, Ca2+ transient amplitudes were similar in both groups. To examine downstream effectors of ß‐AR signaling we assessed the activity of adenylate cyclase and the formation of cAMP. Aged myocytes had smaller increases in contraction and Ca2+ transient amplitudes in response to forskolin compared to young adult cells. In addition, direct measurement of cAMP showed an age‐related decrease in cAMP formation in response to forskolin. This reduction in adenylate cyclase activity is likely responsible for the age‐related decrease in catecholamine sensitivity as administration of dibutyryl cAMP showed no age bias on contraction or Ca2+ transient amplitude. Funding: CIHR & HSFNS.

Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain

Chronic dichlorvos exposure (6 mg/kg b.wt/day) for a period of 8 weeks resulted in significant reduction in body weight gain of the male Wistar rats. However, the dietary intake remained unchanged in experimental animals following dichlorvos treatment. Activity of the synthesizing enzyme of acetylcholine (ACh) ie, choline acetyltransferase, was found to be significantly increased and the activity of hydrolyzing enzyme, acetyl cholinesterase (AChE), was inhibited in all the three brain regions studied. Chronic dichlorvos treatment also caused significant reduction in both high affinity (HA) and low affinity (LA) choline uptake (CU), with maximal effect being observed in the brain stem followed by cerebellum and cerebrum. Muscarinic receptor binding was significantly decreased in brain stem and cerebellum as reflected in the decreased receptor number (B(max)), without any change in the binding affinity (K(d)) of the receptors. Dichlorvos treatment caused marked inhibition in cAMP synthesis as indicated by decreased adenylate cyclase activity as well as cAMP levels in cerebrum, cerebellum and brain stem. Our study shows that organophosphates may interact with muscarinic receptor-linked second messenger system and this could be a potential mechanism for the neurotoxic effects observed after repeated exposure to low levels of organophosphates, which are unexplainable on the basis of cholinergic hyperactivity.

Direct effects of IL‐15 on corticosterone secretion by rat adrenocortical cells

Cytokines might regulate the function of the hypothalamic-pituitary-adrenal axis. IL-15 is a potent non-T-cell-derived cytokine with IL-2-like activities. It has been shown that IL-15 can reverse the inhibition of glucocorticoids on PBMC. In vitro experiments were designed to assess the direct effect of IL-15 on corticosterone (CORT) secretion in the adrenal zona fasciculata-reticularis (ZFR) cells of male rats. Administration of IL-15 dose dependently decreased the basal and adrenocorticotropin-stimulated release of CORT and production of cAMP in ZFR cells. The stimulatory effect of forskolin (an adenylate cyclase activator) on CORT secretion and accumulation of cAMP in ZFR cells was attenuated by the administration of IL-15. However, 8-Br-cAMP (a cAMP analogue)-stimulated release of CORT was not affected by IL-15. Exogenous administration of IL-15 (10(-7) mol/L) significantly attenuated the pregnenolone (the substrate of 3beta-hydroxysteroid dehydrogenase)- or deoxycorticosterone (the substrate of 11beta-hydroxylase)-induced release of CORT. The results indicate that decrease of CORT secretion by IL-15 is in part because of (i) the decrease of adenylate cyclase activity and cAMP production and (ii) the inhibition of 3beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase activities in rat ZFR cells.

Mode of Action: Disruption of Brain Cell Replication, Second Messenger, and Neurotransmitter Systems During Development Leading to Cognitive Dysfunction—Developmental Neurotoxicity of Nicotine

Developmental exposure to nicotine in rats results in neurobehavioral effects such as reduced locomotor and cognitive function. Key events in the animal mode of action (MOA) include binding to the nicotinic cholinergic receptor during prenatal and/or early postnatal development. This leads to premature onset of cell differentiation at the expense of cell replication, which leads to brain cell death or structural alterations in regional brain areas. Other events include an initial increase followed by a decrease in adenyl cyclase activity, as well as effects on the noradrenergic, dopaminergic, and serotonergic neurotransmitter systems. Because the nicotine receptor is also present in the developing human brain and the underlying biology for DNA synthesis and cell signaling is comparable, this MOA is likely to be relevant for humans. Although the effects of nicotine exposure in developing humans is not well documented, nicotine exposure as a result of cigarette smoking during pregnancy is associated with several physiological and behavioral outcomes that are reminiscent of the effects of nicotine alone in animal models. As data become available with the advent of the use of the nicotine patch in pregnant humans, the question as to the relative importance of smoking per se versus nicotine alone may be determined.

Novel 5-HT7 receptor inverse agonists. Synthesis and molecular modeling of arylpiperazine- and 1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides.

A series of arylpiperazine- and 1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides was synthesized and evaluated for their interactions with the constitutively active 5-HT7 receptor. Effects on basal adenylate cyclase activity were measured using HEK-293 cells expressing the rat 5-HT7. All ligands produced a decrease of adenylate cyclase activity, indicative of their inverse agonism. Additionally, computational studies with a set of 22 inverse agonists, including these novel inverse agonists and inverse agonists known from literature, resulted in a pharmacophore model and a CoMFA model (R2 = 0.97, SE = 0.18). Docking of inverse agonists at the binding site of a model of the helical parts of the 5-HT7 receptor, based on the alpha carbon template for 7-TM GPCRs, revealed interesting molecular interactions and a possible explanation for observed structure-activity relationships.

The regulation of Enzyme IIA(Glc) expression controls adenylate cyclase activity in Escherichia coli.

During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions. In this study, the possible effect of H-NS on the cAMP level was investigated. In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity. This is caused by the reduced expression of crr, which encodes the Enzyme IIA(Glc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter. This leads to a twofold reduction in the global amount of Enzyme IIA(Glc), the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.

Deletion of CCK2Receptor in Mice Results in an Upregulation of the Endogenous Opioid System

Stimulation of the brain CCK2 receptor by the C-terminal octapeptide CCK8 of cholecystokinin (CCK) negatively modulates opioid responses. This suggests the existence of physiologically relevant interactions between endogenous CCK and opioid peptides, opening new perspectives particularly in the treatment of pain or drug addiction. CCK2 receptor-deficient mice were used to analyze the incidence of this gene invalidation on opioid system. Compared with wild-type mice, mutants exhibited the following: (1) a hypersensitivity to the locomotor activity induced by inhibitors of enkephalin catabolism or by morphine; (2) a spontaneous hyperalgesia to thermal nociceptive stimulus, which was reversed by previous administration of the NMDA antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], and a large reduction in analgesic effects of endogenous or exogenous opioids; and (3) a more severe withdrawal syndrome after chronic morphine treatment. As expected, stimulation of mu, delta, and D2 receptors on brain tissue of wild-type animals induced a dose-dependent decrease in adenylate cyclase activity, whereas a striking mirror effect was observed in mutants. All of these results suggest that the absence, in knock-out mice, of the negative feedback control on the opioid system, normally performed out by CCK2 receptor stimulation, results in an upregulation of this system. These biochemical and pharmacological results demonstrate the critical role played by CCK2 receptors in opioid-dependent responses.

Cardiac and vascular responses in deoxycorticosterone acetate-salt hypertensive rats.

1. Hypertension leads to ventricular hypertrophy and, eventually, to heart failure. The present study has investigated the functional consequences of deoxycorticosterone acetate (DOCA)-salt hypertension in rats by defining the inotropic, chronotropic and vascular responses to noradrenaline (NA; beta1-adrenoceptor agonist), forskolin (adenylate cyclase activator) and theophylline (phosphodiesterase inhibitor). 2. Administration of DOCA (25 mg, s.c., every 4th day) and excess salt (1% NaCl in drinking water) to uninephrectomized rats increased left ventricular wet weight by 35 and 71% after 4 and 8 weeks, respectively. Addition of KCl (0.4%) or CaCl2 (1%) in the drinking water for 4 weeks attenuated blood pressure increases, but not ventricular weight increases (46 and 28%, respectively). 3. Positive inotropic responses in papillary muscles from uninephrectomized rats to NA (-log EC50 6.73+/-0.38; n = 7), forskolin (-log EC50 6.15+/-0.31; n = 7) and CaCl2 (-log EC50 2.40+/-0.02; n = 14) were unchanged in hypertrophied left ventricles of DOCA and DOCA-CaCl2 rats, although maximal responses to NA were decreased in DOCA-KCI rats (1.2+/-0.6 mN, n = 8; DOCA-salt 2.9+/-0.5 mN, n = 6); theophylline was less potent in DOCA-salt rats. Positive chronotropic responses to NA, forskolin and theophylline in right atria and negative inotropic responses to carbachol in papillary muscles were unchanged. 4. Maximal vasoconstrictor responses to NA in thoracic aortic rings were reduced in DOCA-KCI rats to 2.4+/-0.9 mN (n = 5), but were increased in DOCA-CaCl2 rats to 26.6+/-2.2 mN (n = 7; DOCA-salt 7.8+/-2.2 mN, n = 9). Vasorelaxant responses to forskolin and theophylline were unchanged. 5. These results show that cardiac responses are only minimally affected during the development of DOCA-salt hypertension-induced hypertrophy, despite the reported decreases in adenylate cyclase activity, in these rats. This is in contrast with the decreased responses reported in other rat models of cardiac hypertrophy and in the failing human heart. Thus, hypertrophy in hearts of DOCA-salt hypertensive rats does not produce similar changes to the failing human heart.

Intermittent Warm Blood Cardioplegia Preserves Myocardial β-Adrenergic Receptor Function

Background. Left ventricular dysfunction is frequently observed in patients after hypothermic cardioplegic arrest, and often inotropic intervention is necessary for patients to be successfully weaned from cardiopulmonary bypass (CPB). A myocardial β-adrenergic receptor (βAR) desensitization has been noted to occur after hypothermic CPB in patients undergoing coronary artery bypass grafting. This randomized study was undertaken to determine the effect of cardioplegic solution temperature on cardiac βARs. Methods. Two groups of patients (20 patients in each) scheduled for elective coronary artery bypass grafting underwent CPB with either intermittent warm or cold blood cardioplegia. The density of the βARs, the proportion of β 1- to β 2-adrenergic receptors, and the βAR coupling capacity to adenylate cyclase were determined in specimens of the right atrial tissue at baseline, during CPB, and after discontinuation of CPB. Plasma concentrations of catecholamines were also measured in both arterial and coronary sinus samples. Results. In both cardioplegia groups, no significant modification in either the βAR density or the proportion of β 1- to β 2-adrenergic receptors was detected. However, a significant decrease in adenylate cyclase activity after stimulation with isoproterenol was observed in the cold blood cardioplegia group during CPB ( p < 0.01) and 30 minutes after its discontinuation ( p < 0.05). Moreover, a significant decrease in adenylate cyclase activity during CPB was detected in this group after stimulation with sodium fluoride ( p < 0.05), but this pattern was found to be completely reversed by 30 minutes after discontinuation of CPB. No modification in the basal or stimulated adenylate cyclase activity was observed in the warm blood cardioplegia group during or after CPB. Conclusions. Our results confirm the finding from previous studies of a cardiac βAR desensitization after hypothermic cardioplegic arrest, and provide evidence of the advantages of intermittent warm blood cardioplegia in preserving the autonomic sympathetic function of the heart.

Pharmacological basis for functional selectivity of partial muscarinic receptor agonists

Muscarinic receptor agonists activate phosphoinositide hydrolysis and adenylate cyclase in Chinese hamster ovary cells transfected with cDNAs encoding the human muscarinic m1 and m 3 receptors. Whereas carbachol activates similarly both receptor subtypes, 4-[3-chlorophenyl-carbamoyloxy]-2-butynyltrimethyl ammonium chloride (McN-A-343) preferentially activates the m1 subtype over m3, in regard to both phosphoinositide hydrolysis and adenylate cyclase activity. On the other hand, oxotremorine activates phosphoinositide hydrolysis to a similar extent in both cell lines, but it activates preferentially adenylate cyclase in m1 versus m3 receptor expressing cells. Relative to carbachol, both McN-A-343 and oxotremorine activate preferentially phosphoinositide hydrolysis over adenylate cyclase in both cell lines. Prolonged incubation of cells with either carbachol, McN-A-343, or oxotremorine down-regulated the m1 receptors. This was accompanied by a parallel decrease in adenylate cyclase activity, whereas phosphoinositide hydrolysis remained relatively high. Inactivation of the receptors by alkylation with acetylethylcholine mustard, or by blocking with atropine, reduced carbachol-stimulated adenylate cyclase activity more effectively than carbachol-induced phosphoinositide hydrolysis in both m1 and m3 receptor expressing cells. These findings imply that the receptor reserve in these cell lines is greater for phosphoinositide hydrolysis response than for adenylate cyclase response. Yet, the receptor reserve for each of these responses is similar in both m1 and m3 receptor expressing cells. Since the binding affinities of McN-A-343 and of oxotremorine to m1 and m3 receptors are very similar, and since both cell lines contain similar amounts of spare receptors, we propose that the preferential activation of muscarinic m1 over m3 receptor by partial agonists is related to differences in the abilities of the two receptor subtypes to undergo conformational changes following agonist binding. This hypothesis is supported by results showing that the muscarinic m1 but not m3 receptor exhibits two affinity states in a competition binding assay.

The guanine nucleotide-binding regulatory proteins (G proteins) in myocardium with ischemia.

Guanine nucleotide-binding regulatory proteins (G proteins) play a major role in the regulation of a number of physiological processes, such as stimulation or Inhibition of adenylate cyclase activity or gaiting of ionic channels. Myocardial ischemia could induce the changes in receptor-G protein signal transduction system in the heart. Therefore, this article will focus on the role and alterations of G proteins (especially, Gs and Gi) in myocardial ischemia. The Gi protein rapidly loses functional activity during very early myocardial ischemia. In contrast to Gi protein, the function of Gs protein during this phase has not been evaluated. Moreover, the changes in Gs protein after 30 min of ischemia are contradictory. However, the sensitization of the adenylate cyclase activity in the very early phase of acute ischemia is gradually replaced by a decrease in adenylate cyclase activity with prolonged ischemia. The decrease in the function and amount of Gs protein may be one of the factors that induce these changes. The function of Gs protein was also decreased in the canine hearts with ischemia and reperfusion. In contrast to ischemia and reperfusion, there are no significant alterations in G proteins and modulation of adenylate cyclase in the stunned myocardium. It has become increasingly evident that Gi protein may play an important role in the cardioprotective effects of preconditioning. When beta-adrenoreceptor densities are reduced in chronic myocardial ischemia, decreased in the amount and function of Gi protein and increased amount of Gs protein may play the role in preservation of the adenylate cyclase activity. These alterations in G proteins may play the important role in the myocardial function during myocardial ischemia.

Effect of essential fatty acid deficiency on forskolin binding sites, adenylate cyclase and cyclic AMP-dependent protein kinase activity, the levels of G proteins and ventricular function in rat heart

Three groups of rats were fed semi-purified diets. Diet I was deficient in essential fatty acids (EFAD), diet II was marginally deficient in essential fatty acids (MEFAD), and diet III contained adequate levels of essential fatty acids (control). After 9 weeks, some rats within each group were killed and cardiac membranes were prepared. Adenylate cyclase activity, [3H]forskolin binding sites, the levels of G proteins (Gi and Gs) and fatty acid composition of the membrane phospholipids were measured. Typical changes of EFA deficiency were observed in fatty acid composition of the membrane phospholipids. Adenylate cyclase activity was significantly lower in membranes of EFA-deficient rats than those of the controls. The MEFAD group gave intermediate values. Similar results were obtained with forskolin-stimulated activity with different concentrations of forskolin. Concentrations of the forskolin binding sites were also lower in the EFAD, but not in the MEFAD rats compared with the controls. There was no significant difference in forskolin binding affinities among the three groups. The decrease in adenylate cyclase activity in EFA-deficient rat heart was partially restored by feeding the control diet for 5 weeks to the EFAD or the MEFAD rats. The levels of Gi alpha and Gs alpha were not significantly different in cardiac membranes of rats fed the EFAD or the MEFAD diets from those of the control group. Lower adenylate cyclase activity in hearts of EFAD rats was also reflected in correspondingly lower activity of cAMP-dependent protein kinase. The results suggest an impairment of the cellular signalling pathway for the production of cAMP in rat heart during EFA deficiency. The beta-adrenergic response of isolated heart preparations obtained from rats fed the three diets was, however, not significantly altered.

Decreased Adenylate Cyclase Activity and Expression of G sα in Human Myocardium After Orthotopic Cardiac Transplantation

We studied several aspects of guanine nucleotide-stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained > or = 6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n = 12; P < .05), GTP gamma S-stimulated adenylate cyclase activity was decreased by 78% (n = 12; P < .05), Mn+2+forskolin-stimulated adenylate cyclase activity was decreased by 80% (n = 8; P < .05), and Mn+2-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n = 8, P < .05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of Gs alpha protein was decreased by 61 +/- 12% (n = 8; P < .001). There was no change in the level of Gi alpha as assessed by pertussis toxin-catalyzed ADP-ribosylation (n = 4; P = NS). With the use of the quantitative polymerase chain reaction, a 50 +/- 10% (n = 6; P < .001) reduction in the steady-state level of Gs alpha mRNA was observed. There was no change in the level of mRNA for Gi-3 alpha. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide-stimulated adenylate cyclase activity is decreased in parallel with decreased levels of Gs alpha protein and mRNA.

A comparative study of the role of adenylate cyclase in the release of adrenocorticotropin from the ovine and rat anterior pituitary

The interaction between corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) is important in the regulation of adrenocorticotropin (ACTH) release from the anterior pituitary (AP). CRF exerts its effect on the AP by activating the adenylate cyclase (AC) complex whereas AVP increases the turnover of phosphatidylinositol. In the rat and in man, CRF is the most potent ACTH secretagogue whereas AVP alone is only a weak agonist. Since recent studies in the sheep indicate a reversal of this order of potency, these studies were undertaken to test the hypothesis that a functional alteration of the AC in the ovine corticotrope might limit the ability of CRF to release ACTH from these cells. When rat AP cells were incubated with CRF, a dose-dependent increase in AC activity was observed. This effect was potentiated either by AVP or PMA, although neither agent alone altered AC activity. In contrast, CRF alone, or in combination with AVP or PMA, did not increase AC activity in ovine AP cells. Both cholera toxin (CT) and pertussis toxin (PT) caused a dose-dependent release of ACTH from rat and ovine AP cells, but the amount of ACTH released from the ovine AP cells by both agents was relatively reduced. In the ovine cells, however, AVP acted synergistically with CT or PT to markedly increase the release of ACTH to levels which approached those obtained when the rat AP cells were exposed to CT or PT alone. Forskolin increased AC activity in AP cells of both species, but to a much lower extent in ovine cells than in the rat cells. However, when the ovine cells were exposed to AVP, the AC response to forskolin became similar to the response observed in the rat cells when incubated with forskolin alone. Forskolin also released significantly less ACTH from the ovine AP cells, but AVP also acted synergistically with forskolin to greatly enhance the amount of ACTH released from these cells. Finally, 8-bromo-cyclic AMP produced a similar release of ACTH from both ovine and rat AP cells. We conclude that: (1) the decreased ability of CRF to increase ACTH release from the ovine AP reflects a net decrease in AC activity and cannot be ascribed to an ovine corticotropic resistance to cAMP; (2) the decreased activity of the ovine corticotropic AC complex may in turn reflect functional alterations at the level of both the G proteins and the catalytic subunit; (3) since AVP causes protein kinase C substrate phosphorylation in the ovine AP, AVP may increase AC activity in this tissue by phosphorylating the G proteins and/or the catalytic subunit.

Molecular cloning of a rat κ opioid receptor reveals sequence similarities to the μ and δ opioid receptors

By screening a rat brain cDNA library using a cloned mu opioid receptor cDNA as probe, a clone was identified that is very similar to both the mu and delta opioid receptor sequences. Transient expression of this clone in COS-7 cells showed that it encodes a kappa opioid receptor, designated KOR-1, which is capable of high-affinity binding to kappa-selective ligands. Treatment of transfected cell membranes with bremazocine, a kappa-selective agonist, resulted in a 53% decrease in adenylate cyclase activity, indicating that this kappa opioid receptor displays inhibitory coupling to adenylate cyclase. Thus, one member from each of the three opioid receptor types, mu, kappa and delta, has been molecularly cloned. Analysis of sequence similarities among these opioid receptors, as well as between opioid receptors and other G-protein-coupled receptors, revealed regions of sequence conservation that may underlie the ligand-binding and functional specificities of opioid receptors.