Adenylate Cyclase In Membranes Research Articles

Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm

The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27–29%) and acrosome-reacted sperm (23–26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.

Steroid-membrane-adenylate cyclase interactions during Xenopus laevis oocyte meiosis reinitiation: a new mechanism of steroid hormone action.

Progesterone acts physiologically on Xenopus laevis oocyte meiosis reinitiation in interacting with plasma membrane. Hormonal studies indicate a specific "receptor-like" structure. The involvement of membrane adenylate cyclase activity which is selectively decreased by progesterone is described in intact oocytes as well as in a cell-free membrane containing system. The participation of Ca2+ changes and of phospholipid components of membrane is analyzed. We discuss the possibility that the progesterone-oocyte system is a model for understanding other steroid hormone activities in other target cells.

Two independent effects of cortisol on chicken liver.

Activities of two key enzymes of glycogen metabolism have been measured after an acute administration of cortisol to 3d-old chickens. Glycogen synthase activity is enhanced 2-3 hours after a cortisol injection, and this activation is blocked by use of protein synthesis inhibitors. Glycogen phosphorylase activity is enhanced at an early stage, and this effect is not suppressed by protein synthesis inhibitors. Liver cAMP levels are not increased concomitantly with this early activation of glycogen phosphorylase; indeed they are depleted. These results point to the existence of an effect of cortisol previous to and independent of its nuclear interaction, and not mediated by an activation of the membrane adenylate cyclase.

The Biochemical Mechanisms of the Stimulating Action of Estradiol on the Growth of Mammary Tumours1)

The mechanisms of stimulating effect of estradiol on the growth of hormone-dependent mammary tumours in experimental animals were studied. Estradiol receptors were detected in plasmatic membranes of estradiol-dependent tumour cells. Estradiol-independence of tumours was found to be a result of the impairment of the estradiol receptors in the plasmatic membranes, intracellular receptors, or of their ability to interact with the chromatin of the target cells. It was shown that the interaction of receptors of plasmatic membranes of hormone-dependent tumour cells with estradiol results in activation of the membrane adenylate cyclase and in a short-term rise of a cAMP content. A treatment of the hormone-dependent tumours with ovarian hormones brings about an increase in the activity of the cAMP-dependent protein kinases in cell nuclei, which points to their translocation from cytosol. Under effect of estradiol the phosphorylation of nuclear proteins at the serine, threonine and tyrosine residues increases.

Cyclic AMP formation in rat bone and kidney cells is stimulated equally by parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34.

Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of humoral hypercalcemia of malignancy. It interacts with the PTH receptor and has therefore a nearly identical effect on bone cells as PTH. However, PTHrP is thought to be less potent than PTH in stimulating adenylate cyclase in canine renal membranes, leading to the hypothesis of a differential efficiency in signal transduction by PTHrP with respect to bone vs kidney. In a homologous model with intact osteoblast-like cells (UMR 106) and primary kidney cells, both from the rat, we have tested N-terminal peptide fragments, based on the rat amino acid sequence 1-34, of PTH and PTHrP. Compared with PTHrP(1-34), rat PTH(1-34) had a similar relative potency in bone cells (85%) and in kidney cells (140%) in its ability to stimulate adenylate cyclase. Human PTH(1-34) was 5.6- to 6.5-fold less potent than rat PTH(1-34) in both cell types. In human osteoblast-like cells (SaOS-2), rat and human PTH were essentially equally potent compared to PTHrP(1-34) (identical sequence in rat and human) in stimulating cAMP accumulation. In conclusion, our study revealed the equipotency of rat PTH(1-34) and PTHrP(1-34) in stimulating intracellular cAMP formation in a homologous system of rat bone and kidney cells. There seemed to be no unique signal transduction mechanism of PTHrP to the adenylate cyclase in rat kidney cells compared with bone cells.

CAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.

Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts

Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA. The expression of VIP was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT-PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin-6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists. VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC1) was the dominant AC-coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC2 was dominant. Tumor necrosis factor alpha-treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC1- and VPAC2-specific agonists in OA FLS and RA FLS, respectively. VIP expression is down-regulated in RA and in tumor necrosis factor alpha-treated FLS, suggesting that down-regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC2 mediates the antiinflammatory effects of VIP, suggesting that VPAC2 agonists may be an alternative to VIP as antiinflammatory agents.

Acute β-blockade prevents myocardial β-adrenergic receptor desensitization and preserves early ventricular function after brain death

Beta-adrenergic receptor desensitization through activation of the G protein-coupled receptor kinase 2 is an important mechanism of early cardiac dysfunction after brain death. We hypothesized that acute beta-blockade can prevent myocardial beta-adrenergic receptor desensitization after brain death through attenuation of G protein-coupled receptor kinase 2 activity, resulting in improved cardiac function. Adult pigs underwent either sham operation, induction of brain death, or treatment with esmolol (beta-blockade) for 30 minutes before and 45 minutes after brain death (n = 8 per group). Cardiac function was assessed at baseline and for 6 hours after the operation. Myocardial beta-adrenergic receptor signaling was assessed 6 hours after operation by measuring sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, and G protein-coupled receptor kinase 2 expression and activity. Baseline left ventricular preload recruitable stroke work was similar among sham, brain death, and beta-blockade groups. Preload recruitable stroke work was significantly decreased 6 hours after brain death versus sham, and beta-blockade resulted in maintenance of baseline preload recruitable stroke work relative to brain death and not different from sham. Basal and isoproterenol-stimulated adenylate cyclase activities were preserved in the beta-blockade group relative to the brain death group and were not different from the sham group. Left ventricular G protein-coupled receptor kinase 2 expression and activity in the beta-blockade group were markedly decreased relative to the brain death group and similar to the sham group. Beta-adrenergic receptor density was not different among groups. Acute beta-blockade before brain death attenuates beta-adrenergic receptor desensitization mediated by G protein-coupled receptor kinase 2 and preserves early cardiac function after brain death. These data support the hypothesis that acute beta-adrenergic receptor desensitization is an important mechanism in early ventricular dysfunction after brain death. Future studies with beta-blocker therapy immediately after brain death appear warranted.

A cell-physiological description of GE, a GTP-binding protein that mediates exocytosis.

Introduction of GTP gamma S or other non-metabolic analogues of GTP into permeabilized myeloid granulocytes (mast cells, eosinophils, neutrophils) constitutes a sufficient stimulus to induce exocytosis. We concentrate on mast cells. Exocytosis from cells permeabilized in isotonic glutamate solution proceeds in the absence of ATP and at exceedingly low levels (< 10(-9) M) of Ca2+. Mg2+ strongly promotes GTP gamma S-induced exocytosis but this requirement can be spared and then obliterated by lifting Ca2+ through 10(-7) to 10(-6) M. GTP provides only a modest support to exocytosis but becomes almost equipotent with GTP gamma S when Mg2+ is excluded. Ca2+ alone is unable to induce exocytosis. We envisage that the terminal stage of exocytosis (membrane fusion) requires activation of GE, a putative GTPase so far undefined as a molecular entity. Ca2+, presumed to act through a Ca(2+)-binding protein (CE, also undefined) supports exocytosis by promoting the exchange of guanine nucleotides on GE. In the absence of Mg2+ the onset of exocytosis is characterized by delays that have concentration-dependent (binding) and independent components. The latter are sensitive to the identity of the stimulating nucleotide (GTP < GTP gamma S < Gpp [NH]p) and may reflect activation of GE. The activation by Ca2+ and Mg2+ and the delays preceding onset of GTP gamma S-triggered exocytosis are reminiscent of the action of glucagon and Mg2+ in the activation of adenylate cyclase in hepatocyte membranes. The cell-physiological description predicts GE to be an alpha beta gamma heterotrimeric GTP-binding protein with functional similarity to GS.

Role of the β-adrenergic receptor kinase in myocardial dysfunction after brain death

Significant cardiac dysfunction after brain death leading to exclusion from procurement for cardiac transplantation is seen in up to 25% of potential organ donors in the absence of structural heart disease. The cause includes uncoupling of the myocardial beta-adrenergic receptor signaling system. The mechanism, however, has not yet been described. This study investigates our hypothesis that brain death causes acute activation of the betaAR kinase and leads to desensitization of myocardial beta-adrenergic receptors and impaired ventricular function.Adult pigs underwent a sham operation or induction of brain death by means of subdural balloon inflation (n = 8 in each group). Cardiac function was assessed by using sonomicrometry at baseline and for 6 hours after the operation. beta-Adrenergic receptor signaling was assessed at 6 hours after the operation by measuring myocardial sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, beta-adrenergic receptor kinase expression, and activity.Induction of brain death led to significantly decreased left ventricular systolic and diastolic function. Basal and isoproterenol-stimulated adenylate cyclase activity was blunted in the brain dead group compared with the sham-operated group (28.3 +/- 4.3 vs 48.3 +/- 7.6 pmol of cyclic adenosine monophosphate.mg(-1) x min(-1) [P = .01] and 54.8 +/- 9.6 vs 114.5 +/- 18 pmol of cyclic adenosine monophosphate x mg(-1) x min(-1) [P < .02]). There was no difference in beta-adrenergic receptor density between the brain dead and sham-operated groups. Myocardial beta-adrenergic receptor kinase expression was 3-fold greater in the brain dead versus sham-operated groups, and membrane beta-adrenergic receptor kinase activity was 2.5-fold greater in the brain dead group compared with that seen in the sham-operated group.Induction of brain death leads to significant left ventricular dysfunction in this porcine model. Cardiac beta-adrenergic receptors are clearly uncoupled after brain death, and our data suggest that the mechanism is acute increase of myocardial beta-adrenergic receptor kinase activity, leading to beta-adrenergic receptor desensitization and ventricular dysfunction.

Mechanism of galanin-inhibited insulin release

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP-and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.

On minimum cyclic AMP formation rates associated with positive inotropic effects mediated through ?1-adrenoceptors in kitten myocardium

The agonist (-)-denopamine was used as a tool to study relationships between pharmacological effects and adenylate cyclase stimulation mediated through beta 1-adrenoceptors. 1. (-)-Denopamine was a full agonist in kitten papillary muscles (force), kitten left atria (force) and kitten and guinea-pig atria (sinoatrial frequency). (-)-Denopamine was a strong partial agonist in guinea-pig tracheae (relaxation). None of these effects was influenced by blockade of beta 2-adrenoceptors. beta 1-Adrenoceptors mediated all effects of (-)-denopamine in atria and effects of low (-)-denopamine concentrations in papillary muscles and tracheae, as assessed with beta 1-selective antagonists. 2. High (-)-denopamine concentrations caused positive inotropic effects in papillary muscles and tracheorelaxant effects that were resistant to blockade by beta 1-, beta 2- and alpha-adrenoceptor antagonists (non-adrenergic effects). 3. (-)-Denopamine stimulated the adenylate cyclase of membranes derived from kitten ventricle and calf tracheal cells with an intrinsic activity of 0.3 and 0.2, respectively, compared to catecholamines. The contribution of beta 1- and beta 2-adrenoceptors to cyclase stimulation was assessed by selective blockade. Cyclase stimulation through beta 2-adrenoceptors by (-)-denopamine was 12% in ventricle and 82% in trachea but is not associated with positive inotropic effects and tracheal relaxation. 4. (-)-Denopamine exhibited only a 2- to 5-fold selectivity for beta 1-adrenoceptors compared to beta 2-adrenoceptors, as estimated consistently from binding assays and blockade of cyclase stimulation in myocardial and tracheal cell membranes. 5. Desensitization of kitten ventricular tissues, caused by a 3 h exposure to 30 mumol/l (-)-isoprenaline followed by 5 h washout, reduced the inotropic sensitivity of papillary muscles without decreasing the maximum inotropic effects of (-)-denopamine. In desensitized tissues, the nonadrenergic effect contributed by 30% to the maximum inotropic effect of (-)-denopamine. 6. In membranes, derived from desensitized tissues, the maximum adenylate cyclase stimulation induced by (-)-isoprenaline, (-)-denopamine and xamoterol was reduced to 1/2 of the corresponding stimulations observed in membranes from sham desensitized tissues. The density of beta-adrenoceptors, assessed with 3H-(-)-CGP 12,177, was not changed by the desensitization procedure suggesting that part of the receptors was uncoupled from the adenylate cyclase. The partial inotropic agonist xamoterol, which has an intrinsic activity of 0.5 in non-desensitized tissues, failed to cause positive inotropic effects in desensitized papillary muscles suggesting that not all cyclic AMP possesses inotropic relevance.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of beta3-adrenergic receptors in the action of a tumour lipid mobilizing factor.

Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10−7–10−5 M) of the specific β3-adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOK1 cells transfected with the human β3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a β3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the β3-adrenoceptor showed a high affinity binding site with a Kd value 78±45 nM and a Bmax value (282±1 fmole mg protein−1) comparable with that of other β3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a β3-adrenoceptor.British Journal of Cancer (2002) 86, 424–428. DOI: 10.1038/sj/bjc/6600086 www.bjcancer.com© 2002 The Cancer Research Campaign

Cancer anorexia and cachexia

Patients with cancer cachexia experience a profound wasting of adipose tissue and lean body mass. Anorexia, although often present, is insufficient to account for tissue wasting because 1) cachexia involves massive depletion of skeletal muscle that does not occur during anorexia, 2) nutritional supplementation cannot replenish the loss of lean body mass, 3) cachexia can occur without anorexia, and 4) food intake might be normal for the lower weight of the cancer patient. Anorexia can arise from 1) decreased taste and smell of food, 2) early satiety, 3) dysfunctional hypothalamic membrane adenylate cyclase, 4) increased brain tryptophan, and 5) cytokine production. Appetite stimulants such as cyproheptadine, medroxyprogesterone acetate, and megestrol acetate do not significantly improve lean body mass. Tumor products might be more important in the development of cachexia. Cachectic patients excrete in their urine a lipid-mobilizing factor that directly stimulates lipolysis in a cyclic AMP–dependent manner and increases energy expenditure. Loss of skeletal muscle in cachexia is caused by upregulation of the ubiquitin–proteasome catabolic pathway. Cachexia-inducing tumors elaborate a sulfated glycoprotein, which directly initiates protein catabolism in skeletal muscle. The action of this proteolysis-inducing factor is attenuated by the polyunsaturated fatty acid eicosapentaenoic acid, which is also effective in preventing loss of skeletal muscle in cancer patients. Antagonists of tumor catabolic factors will provide important new agents in the treatment of cancer cachexia.

Inhibition of adenylate cyclase activity by 5-aminolevulinic acid in rat and human brain.

The effect of the haem precursor 5-aminolevulinic acid (ALA) on the production of cyclic adenosine-monophosphate (cAMP) by rat cerebellar membranes was investigated. It was found that ALA dose-dependently decreased cAMP levels (maximal inhibition of 38%, at 1 mM), due to an inhibition of basal adenylate cyclase activity. ALA also inhibited fluoride- and Gpp(NH)p-stimulated, but not the forskolin-stimulated adenylate cyclase activity. 5-Aminovaleric acid (an inhibitor of GABA B receptors) did not prevent the inhibition, indicating that it was not mediated by the activation of the G i-protein coupled GABA B receptor. In addition, the nucleotide binding site of G-protein appeared not to be affected by ALA since it did not inhibit [ 3H]Gpp(NH)p binding to our membrane preparation. Antioxidants (glutathione, ascorbate and trolox) completely prevented the inhibition indicating that ALA effect was mediated by an oxidative damage of adenylate cyclase. ALA also inhibited the activity of adenylate cyclase in membranes isolated from rat cortex and striatum and from human cortex. These results may be of value in understanding the neurochemical mechanisms underlying the neurotoxic effects of ALA.

Role of the cyclic AMP-dependent pathway in free radical-induced cholesterol accumulation in vascular smooth muscle cells

We have previously reported that free radical-treated vascular smooth muscle cells (SMC) lead to cholesterol accumulation in vitro. In the current study, we investigated the effects of oxidative stress on cyclic AMP concentration and cAMP-dependent enzymes involved in cholesterol homeostasis in A7r5 cells. Under our conditions of a mild oxidative stress, namely with no change in cell viability, we found that free radicals, initiated using azobis-amidinopropane dihydrochloride (AAPH), resulted in a dose-dependent decrease in cellular cAMP which was opposed by vitamin E preincubation. Although the addition of adenylate cyclase activators (carbacyclin and forskolin) increased cAMP levels it did not succeed in restoring the AAPH-induced decrease. The oxidative stress-induced increase in activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and of acyl coenzyme A: cholesterol acyltransferase and the decrease in neutral cholesteryl ester hydrolase activity were suppressed by addition of dibutyryl cAMP. Taken together, these results strongly suggest that free radicals reduce cAMP concentrations by altering cell membrane adenylate cyclase activity. The changes of cAMP-dependent enzymes induced by oxidative stress resulting in cholesterol accumulation might be one of the processes leading to SMC-derived foam cells depicted in atheroma plaque. Moreover, if extrapolated to in vivo, these data may explain in part the beneficial effects of antioxidants in the reduction of cardiovascular diseases.

Negative regulation of α2-adrenergic receptor-mediated Gi signalling by a novel pathway

Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.

Negative regulation of α2-adrenergic receptor-mediated Gi signalling by a novel pathway

Chinese hamster ovary (CHO) cells stably expressing α2 adrenergic receptor (α2AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The α2AAR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of α2AAR-mediated inhibition was not observed when α2AAR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to Gi-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates Gi signalling in a protein-kinase-C-dependent manner.

Alterations of G-protein and adenylate cyclase signaling in rat liver during the progression of sepsis.

Changes in the protein level of various subunits of G-protein and the activity of adenylate cyclase in rat liver plasma membranes during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that the protein levels of Galphai-2 and Galphai-3 were unchanged during the early hypermetabolic (hyperglycemic) phase (9 h after CLP), whereas Galphaai-2 and Galphaai-3 were increased by 32.4 and 59.1%, respectively, during the late hypometabolic (hypoglycemic) phase (18 h after CLP) of sepsis. The protein levels of Galphas and Gbeta remained unaltered during both the early and the late phases of sepsis. The activity of adenylate cyclase remained unchanged during the early phase, whereas it was decreased by 26% (p < .05) during the late phase of sepsis. Since the G-protein/adenylate cyclase signaling system mediates hormonal control of hepatic glucose metabolism, the observed increases in the Galphai-2 and Galphai-3 protein levels coupled with a decrease in the activity of adenylate cyclase may contribute to the development of the hypoglycemia during the late stage of sepsis.

Constitutive inhibitory action of muscarinic receptors on adenylate cyclase in cardiac membranes: effects of atropine, S-(−)-hyoscyamine, and R-(+)-hyoscyamine

Constitutive inhibitory action of muscarinic receptors on adenylate cyclase in cardiac membranes: effects of atropine, S-(−)-hyoscyamine, and R-(+)-hyoscyamine