Difference in sensitivity to alkaline phosphatase treatment between rat reticulocyte membranes in which β-adrenoceptor desensitization was induced by isoproterenol,dibutyryl cAMP and phorbol ester

The effect of alkaline phosphatase (3.1.3.1) on desensitization of β-adrenoceptor-responsive adenylate cyclase and the role of phosphorylation in desensitization were examined. Treatment of rat reticulocytes with isoproterenol, dibutyryl cAMP and tetradecanoyl phorbol acetate (TPA) caused the desensitization of β-adrenoceptor-coupled adenylate cyclase. When the membranes from dibutyryl cAMP- and TPA-desensitized cells were incubated with alkaline phosphatase for 60 min at 30°C, pH 8.0, the desensitization of isoproterenol-stimulated adenylate cyclase was markedly attenuated in both preparations. When the membranes from isoproterenol-desensitized cells were treated with alkaline phosphatase under the same conditions, the attenuation of the desensitization of alkaline phosphatase was less than in the case of treatment with dibutyryl cAMP or TPA. In other words, isoproterenol-induced desensitization was more resistant to alkaline phosphatase treatment. Isoproterenol- and dibutyryl cAMP-induced desensitization of NaF-stimulated adenylate cyclase were also attenuated by alkaline phosphatase treatment. Although the stability of the G_s-catalytic unit complex of adenylate cyclase was reduced by isoproterenol treatment, the reduction of stability was also decreased by alkaline phosphatase treatment.     

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.     

Glucagon-induced refractoriness of hepatocyte adenylate cyclase: comparison of homologous and heterologous components and evidence against a role of cAMP

Exposure of cultured hepatocytes to glucagon leads to a partial refractoriness of the adenylate cyclase both to glucagon (homologous desensitization) and to isoproterenol (heterologous desensitization). In contrast, isoproterenol produces a very strong homologous desensitization but almost no heterologous desensitization. The present study compared the pattern of the homologous and heterologous components of glucagon-induced desensitization in these cells, particularly during the first 4 hours, and examined the role of cyclic 3',5'-adenosine monophosphate (cAMP) in the mechanism of refractoriness development. The decrease in glucagon-sensitive and isoproterenol-sensitive adenylate cyclase activities were closely parallel with respect to the extent, the time course and the dose required. 8-Bromoadenosine 3',5'-monophosphate (8-Bromo-cAMP) also reduced the hormone-responsive adenylate cyclase activity, but this effect developed more slowly than the desensitization after glucagon treatment. No consistent relationship was found between cAMP levels and induction of hormone refractoriness when the cells were exposed to glucagon, isoproterenol, cholera toxin or forskolin. Furthermore, addition of 0.5 mM 3-isobutyl-1-methylxanthine) (IBMX) which strongly amplified the cAMP response, did not potentiate the glucagon-induced desensitization of either glucagon-sensitive or isoproterenol-sensitive adenylate cyclase activity. Taken together, the results suggest that homologous and heterologous desensitization of the adenylate cyclase developing after glucagon exposure occur by similar (agonist-non-specific) mechanisms which do not involve cAMP.     

Desensitization and resensitization of adrenomedullin-sensitive receptor in rat mesangial cells

Adrenomedullin is a potent adenylate cyclase activator and a vasodilatory peptide, that has anti-proliferative and apoptotic effects in rat mesangial cells. The present study was designed to determine the mechanisms of desensitization and resensitization of adrenomedullin-sensitive receptor in mesangial cells. Adrenomedullin caused a rapid desensitization of cAMP response evident within 5 min that was almost complete by 1 h of treatment. Pretreatment of cells with forskolin, that activates protein kinase-A by direct activation of adenylate cyclase, also caused adrenomedullin receptor desensitization. In addition, H89 [?N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride?], a potent protein kinase-A inhibitor inhibited adrenomedullin-induced desensitization of cAMP response. Adrenomedullin also caused desensitization of isoproterenol- and epinephrine-mediated cAMP accumulation. Furthermore, adrenomedullin induced cross-desensitization of endothelin-stimulated inositol phosphate accumulation. The attenuated cAMP response of adrenomedullin was restored to original levels within 2 h of agonist removal. This resensitization response was blocked by treatment with okadaic acid, a protein phosphatase (protein phosphatase-1/protein phosphatase-2A) inhibitor, during the 2 h resensitization period, indicating that protein phosphatase-1/protein phosphatase-2A may be involved in the resensitization of the adrenomedullin-sensitive receptor. We demonstrate for the first time in rat mesangial cells that the adrenomedullin-sensitive receptor undergoes heterologous desensitization and resensitization, and that it likely involves protein kinase-A and protein phosphatase-1/protein phosphatase-2A, respectively.     

Protein kinases induce isoproterenol desensitization of beta-adrenoceptor-coupled adenylate cyclase system: significance of receptor occupancy

Treatment of rat reticulocytes with isoproterenol resulted in dose- and time-dependent desensitization of adenylate cyclase to beta-adrenoceptor agonist stimulation. Cyclic AMP-dependent protein kinase was possibly involved in this desensitization. Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), also induced desensitization to beta-adrenoceptor agonists, indicating the possible involvement of protein kinase C. The addition of a beta-adrenoceptor agonist enhanced the desensitizing effect of dibutyryl cyclic AMP or TPA. T1/2 (time for induction of half maximal desensitization) was decreased from 30 to 2.5 min in accordance with an increase in the concentration of the agonist. The extent of the desensitization was also increased by addition of the agonist. The potency with which the increase was induced was compatible with the potency for binding of the agonist to beta-adrenoceptors (KD values). These results suggest that cyclic AMP- or phorbol ester-induced desensitization is enhanced by receptor occupation by beta-adrenoceptor agonists.     

Beta-adrenergic receptor levels and function after growth of S49 lymphoma cells in low concentrations of epinephrine.

Growth of S49 wild-type (WT) lymphoma cells for 24 hr with 3 nM epinephrine produces a very pronounced attenuation of cAMP accumulation in response to subsequent challenges with much higher concentrations of the catecholamine [Mol. Pharmacol. 36:459-464 (1989)]. We report here the effects of this treatment, in S49 WT, cyc-, and kin- cells, on the responsiveness of adenylate cyclase in partially purified membranes. The desensitization of adenylate cyclase in the S49 WT cells after 24-hr treatment was homologous, in that only responses to epinephrine were attenuated. The EC50 for epinephrine stimulation of adenylate cyclase was 54 +/- 8% (mean +/- standard error) higher in treated cells than in controls, and there was a 32 +/- 3% reduction in Vmax at supramaximal epinephrine concentrations. The treatment also caused a 34 +/- 9% reduction in the levels of the beta-adrenergic receptor (beta AR), which was of a sufficient magnitude to account for the homologous desensitization seen. The 24-hr treatment had similar effects in S49 kin- cells, where we observed a 28 +/- 4% decrease in Vmax, a 35 +/- 6% increase in EC50 for epinephrine stimulation of adenylate cyclase, and a 25 +/- 3% decrease in beta AR. In contrast, the 24-hr treatment had no measurable effect on adenylate cyclase activity in S49 cyc- cells. That is, the responsivity of adenylate cyclase reconstituted with Gs from S49 WT cells was not attenuated, although beta AR levels were significantly decreased. The desensitization of S49 cells with the 24-hr treatment was additive with that mediated by the cAMP-dependent protein kinase (cAPK). Further, unlike the cAPK-mediated attenuation, it was relatively insensitive to the levels of free Mg2+ in the adenylate cyclase reaction mixture. The characteristics of the desensitization produced by 24-hr treatment with 3 nM epinephrine, together with the observation that it is similar in S49 WT and kin- cells, demonstrates that the process in WT cells is, at least in part, independent of the rapid cAPK-mediated desensitization. It is also most likely that it is unrelated to the rapid cAMP-independent processes involving sequestration/internalization or the beta AR kinase, because those mechanisms require much higher receptor occupancies than the 0.2% occurring with 3 nM epinephrine. Thus, 24-hr treatment appears to produce attenuation of adenylate cyclase by causing down-regulation of beta AR, without involving any other known form of desensitization.     

Homologous vs. heterologous desensitization of the adenylate cyclase system in heart cells

Exposure of cultured heart muscle cells to noradrenaline led to a decrease in the effects of isoproterenol and prostaglandin E1 on cAMP formation and contraction velocity. However, heterologous desensitization, as measured by prostaglandin E1 stimulation, only occurred at higher noradrenaline concentrations than homologous desensitization (isoproterenol stimulation). As the defects of the adenylate cyclase system in heart failure are attributed to noradrenaline-induced desensitization, it is concluded from the results that, in comparison to the subsensitivity to beta-adrenoceptor agonists in failing human hearts, a decrease in the responsiveness to other receptor-dependent adenylate cyclase stimulators should also occur but only at higher degrees of heart failure.     

Reduction in the stability of the Gs-catalytic unit complex of adenylate cyclase in isoproterenol-induced heterologous desensitization

Treatment of rat reticulocytes with isoproterenol resulted in the heterologous desensitization of adenylate cyclase, causing a reduction in NaF/AlCl3- and guanylyl 5'-imidodiphosphate (Gpp(NH)p)-stimulated activities as well as in activity stimulated by beta-adrenoceptor agonists. Desensitization was also induced by dibutyryl cyclic AMP and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of cyclic AMP-dependent protein kinase (protein kinase A), prevented the isoproterenol-induced desensitization, suggesting the involvement of protein kinase A in the desensitization. Gs in the desensitized cell membrane was activated by treatment with Gpp(NH)p or NaF/AlCl3 in the presence of Mg2+. The activated state, the Gs-C complex, was gradually shifted to the basal state, i.e. the Gs-C complex was dissociated by removal of Mg2+. The rate constant of the dissociation was increased in the desensitized cell membranes (0.074 min-1) as compared with that in the control cell membranes (0.022 min-1). The half life of the Gs-C complex, calculated from the rate constant, was decreased during the process of desensitization. The results indicate that reduction in the stability of the Gs-C complex is related to heterologous desensitization. Agents which increase the level of cyclic AMP in the cell also reduced the stability of the complex. The potency of such an effect was in the following order; isoproterenol approximately NaF/AlCl3 (k = 0.071 min-1) greater than forskolin (0.039 min-1) greater than Mn2+ approximately dibutyryl cyclic AMP (0.030 min-1). The former two activate Gs and form Gs-C complex, while the latter two cause intracellular cyclic AMP accumulation without activation of Gs. It may be deduced from this order that the reduction of the stability depends not only on the increase in the cyclic AMP level but also on the level of Gs-C complex formed.     

Epidermal growth factor activation of rat parotid gland adenylate cyclase and mediation by a GTP-binding regulatory protein.

Injection of rats with a single dose of epidermal growth factor (EGF) or isoproterenol increased parotid gland acinar cell levels of cyclic AMP (cAMP) significantly above control basal concentrations (34, 177 and 11.5 pmol/g tissue/100 g body weight, respectively). Following a chronic regimen of isoproterenol (3 days), EGF, bovine galactosyltransferase (Gal Tase, EC 2.4.1.22) and isoproterenol increased cAMP levels, albeit to a lower level than observed for the single dose (21, 17 and 51 pmol, respectively). Using isolated parotid gland membranes, EGF and bovine galactosyltransferase also stimulated adenylate cyclase (EC 2.7.4.3) activity in a concentration-dependent manner. Introduction of the beta-adrenergic receptor antagonist propranolol blocked isoproterenol-stimulated adenylate cyclase activity and cAMP accumulation, but not that observed with EGF or the transferase treatment. Growth factor-stimulated adenylate cyclase activity required the presence of the guanosine triphosphate (GTP) analogue, guanyl-5'-imidodiphosphate (p[NH]ppG), while cAMP accumulation could additionally be blocked by introducing the GDP analog, guanosine 5'[beta-thio]diphosphate (GDP[S]). The ability of EGF to activate adenylate cyclase was not affected by pretreatment of acinar cell membranes with pertussis toxin, whereas pretreatment with cholera toxin eliminated EGF-stimulated cyclase activity. The experimental results presented here expand to the parotid gland our knowledge of the ability of EGF to stimulate the cAMP second messenger signalling pathway via a G-binding regulatory protein, by a mechanism independent of beta-adrenergic receptor activation.     

Growth of S49 cells in low concentrations of beta-adrenergic agonists causes desensitization

Epinephrine at concentrations approximating circulating levels in resting subjects produced significant desensitization in wild type S49 lymphoma cells after long term treatment. Desensitization by such low levels of catecholamines was measured by examining subsequent responses of the cells to higher agonist concentrations and was quantified by comparing the integral cAMP accumulations with time in naive and epinephrine-treated cells challenged with the higher epinephrine concentrations. The cells were significantly desensitized after 8 hr of treatment with 3 nM epinephrine or 3 nM terbutaline and were essentially maximally refractory after 24 hr. The 3 nM epinephrine treatment resulted in a small right shift of the EC50. Responses to epinephrine were partially restored by incubating desensitized cells for 8 hr or longer in growth medium that was free of epinephrine. The attenuation of cAMP responses was largely specific, in that the decrease in the response to prostaglandin was small and the response to forskolin was unchanged. This, together with small increases in cAMP destruction in cell-free preparations from treated cells, suggested that higher phosphodiesterase activity contributed in a minor way to the desensitization. However, the response of the adenylate cyclase system to epinephrine was dramatically attenuated, and very significant changes in the properties of the beta-adrenergic receptors were also obvious. That is, the number of binding sites for epinephrine was reduced by about 65% while the number of sites for [125I]iodocyanopindolol was unchanged. The affinity for the radioactive ligand was significantly reduced. Wild type S49 cells remained viable after several days of continuous treatment with 3 nM epinephrine or terbutaline but responded to subsequent increases in cellular cAMP levels with the expected growth arrest and cytolysis. Involvement of cAMP-dependent protein kinase in this type of desensitization was suggested by the observation that S49 kincells were not desensitized by long term incubation with 3 nM epinephrine. Further, low concentrations of dibutyryl cAMP mimicked the effect of low level epinephrine treatment. We conclude that circulating levels of epinephrine in intact animals are sufficiently high to cause desensitization in cells with sensitivities to the catecholamines in the same range as that of the S49 lymphoma cell in vitro. We would predict that cells with those characteristics would always be at least partially desensitized in vivo.     

Cholinergic antagonism of beta-adrenergic stimulated action potentials and adenylate cyclase activity in rabbit ventricular cardiomyocytes

The cholinergic antagonism of beta-adrenergic stimulation was examined by measuring adenylate cyclase activity and calcium-mediated action potentials in isolated ventricular cardiomyocytes of adult rabbits. The beta-adrenoceptor agonist isoproterenol and the direct adenylate cyclase activator forskolin increased adenylate cyclase activity in homogenates of the myocytes. The cholinergic agonist carbachol (10 nM-100 microM) inhibited in a concentration dependent manner basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase activity. The carbachol effect on basal adenylate cyclase activity was antagonized by atropine (10 microM). In parallel experiments using intact cardiomyocytes, calcium action potentials were elicited by intracellular depolarizing current pulses in partially depolarized preparations. These action potentials were prolonged by isoproterenol, forskolin and dibutyryl cyclic AMP. Acetylcholine reversibly inhibited the prolongation of the action potential induced by isoproterenol and forskolin but not dibutyryl cyclic AMP. These results suggest that cholinergic agonists modulate the increase in the calcium current elicited by isoproterenol and forskolin in isolated ventricular cardiomyocytes by inhibiting adenylate cyclase activity.     

Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein alpha-subunits

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 microM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of beta-adrenoceptor-independent adenylate cyclase by guanosine-5'-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. Gi, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the Mr approximately 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified beta gamma-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of beta gamma-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-Gi alpha-antibodies demonstrated a marked increase in the amount of Gi alpha in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-beta-antibodies showed that the level of the beta-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of alpha-subunits of Gi-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.     

The difference between methadone and morphine in regulation of delta-opioid receptors underlies the antagonistic effect of methadone on morphine-mediated cellular actions.

To investigate the cellular and molecular basis for using methadone in substitution therapy for morphine addiction, the difference between methadone and morphine in causing desensitization of delta-opioid receptors was examined, and the effects of methadone pretreatment on opiate-induced inhibition of forskolin-stimulated cAMP accumulation was studied. Methadone substantially attenuated the ability of [D-Ala2,D-Leu5]enkephalin (DADLE), morphine and methadone to inhibit forskolin-stimulated cAMP accumulation. Methadone was able to block the morphine-induced compensatory increase in intracellular cAMP levels and naloxone-precipitated cAMP overshoot after chronic exposure to morphine. The protein kinase inhibitor (1-5-isoquinolinesulfony)-2-methylpiperazine) (H7) could significantly block the chronic methadone treatment-induced loss of the ability of DADLE to inhibit adenylate cyclase. The protein kinase inhibitor chelerythrine was able to block the acute methadone treatment-induced loss of the ability of DADLE to inhibit adenylate cyclase. In contrast, morphine did not cause a substantial desensitization of the delta-opioid receptor. These results indicate that methadone is different from morphine in its regulation of the delta-opioid receptor. In addition, these results also indicate that the mechanisms of delta-opioid receptor desensitization induced by acute and chronic methadone treatment are different.     

Homologous desensitization of the beta-adrenergic receptor. Functional integrity of the desensitized receptor from mammalian lung

Previous work has demonstrated that injection of rats with isoproterenol is rapidly (10 min) followed by the development of a homologous form of desensitization of the beta-agonist-coupled adenylate cyclase in lung membranes. Half the receptor pool becomes sequestered in a light membrane fraction while the other half remains in the plasma membranes but becomes functionally uncoupled. In the present work we sought to assess whether "local sequestration" of the functionally intact receptor away from the effector adenylate cyclase in the plasma membrane contributes to the uncoupling of the beta-adrenergic receptor observed in the plasma membranes. We tested the functionality of the desensitized beta-adrenergic receptor in three different ways. We reconstituted the affinity chromatography purified control and "desensitized" receptors with pure Ns from human erythrocytes and assessed the ability to induce GTPase activity in Ns. Both control and desensitized beta-adrenergic receptors stimulate similar levels of GTPase activity in Ns (852 +/- 38 versus 738 +/- 49 fmol of Pi released/30 min (p greater than 0.05, n = 4). To further assess the relative ability of control and desensitized beta-adrenergic receptors to couple to another source of Ns we fused reconstituted beta-adrenergic receptors to Xenopus laevis erythrocytes, which contain Ns and adenylate cyclase but essentially no beta-adrenergic receptors. The functional interactions of control and desensitized beta-adrenergic receptor with the adenylate cyclase system of the acceptor cells was assessed by measuring the beta-agonist-stimulated adenylate cyclase activity and the agonist-induced formation of the high affinity state of the beta-adrenergic receptor (RH). Again both control and desensitized beta-adrenergic receptors appeared to interact with Ns to the same extent. To test if a local sequestration of the beta-adrenergic receptor away from Ns within the plasma membrane might contribute to the uncoupling of the beta-adrenergic receptors during desensitization, plasma membranes from control and desensitized lungs were treated with the fusogen polyethylene glycol to disrupt any compartmentalization of protein components within the plasma membrane. After polyethylene glycol treatment the previously uncoupled beta-adrenergic receptors could be recoupled to Ns as assessed by the formation of RH in agonist competition curves. These data suggest that in marked contrast to the heterologous type of desensitization, homologous desensitization may involve a local sequestration of a functionally intact beta-adrenergic receptor away from the adenylate cyclase effector system.     

Differential modulation of the adenylate cyclase/cyclic AMP stimulatory pathway by protein kinase C activation in rat adipose tissue and isolated fat cells. Influence of collagenase digestion.

Exposure of rat epididymal fat pad to phorbol 12-myristate 13-acetate (TPA), an activator of protein kinase C, results in an 85% increase in isoproterenol-stimulated cyclic AMP (cAMP) accumulation, an effect which was antagonized by H7, a protein kinase C inhibitor. This promoting action of TPA appears to be related to (i) an increase in the catalytic activity of adenylate cyclase, (ii) an increase in the maximal response of adenylate cyclase to fluoride and guanylimidodiphosphate (GppNHp) with no change in the EC50 value for GppNHp, and (iii) a reduction of the isoproterenol-stimulated low-Km cAMP phosphodiesterase activity present in the 30,000 g pellet of fat pad homogenates. In contrast with fat pads, exposure of isolated rat fat cells to TPA failed to influence their adenylate cyclase response to GppNHp and their cAMP accumulation and lipolysis. However, the other alterations caused by TPA in fat pads were still observed in fat cells. These results suggest that (i) the major alteration responsible for the promoted isoproterenol-stimulated cAMP response observed in fat pads after exposure to TPA is an increased interaction between the alpha s subunit of Gs and the catalytic site of adenylate cyclase and (ii) this increased interaction is dependent on protein kinase C activation and is abolished by collagenase digestion.     

低pH处理对阿片受体激动剂调节cAMP第二信使系统的作用

目的;研究不同的阿片类物质对ｃＡＭＰ第二信使系统不同作用的机制。方法;用低ｐＨ方法失活ＧＳ蛋白,用蛋白竞争法测ＮＧ１０８１－５细胞膜腺苷酸环化酶活性。结果;与二氢埃托啡和埃托啡不同,低ｐＨ处理明显增加吗啡和美沙酮对正常和自身慢性处理细胞的膜ＡＣ活性的抑制作用。     

Phorbol ester regulates stimulatory and inhibitory pathways of the hormone-sensitive adenylate cyclase system in rat reticulocytes

Treatment of rat reticulocytes with tetradecanoyl phorbol acetate (TPA), a tumor-promoting phorbol ester which activates protein kinase C, resulted in an about 50% decrease in the stimulation of adenylate cyclase activity by a subsequent challenge with a beta-adrenoceptor agonist. This phenomenon mimics agonist-induced desensitization. This decline is due to a reduction in the Vmax of the adenylate cyclase system rather than to a change in affinity to the agonist. The beta-adrenoceptor number was not changed while the KD for an agonist but not for an antagonist was increased by TPA treatment. Prostaglandin E1 (PGE1) plus GTP, NaF plus AlCl3, and guanylyl-5'-imidodiphosphate (GppNHp) regulated adenylate cyclase activity in a biphasic manner, i.e. stimulation at lower concentrations and inhibition at higher concentrations. The same treatment also caused a dose- and time-dependent reduction of the inhibitory phase of the PGE1/GTP action but did not affect the inhibitory phase of GppNHp and NaF/AlCl3 actions. Pertussis toxin (IAP) treatment caused a reduction of the inhibitory phase of PGE1/GTP action similar to that caused by TPA treatment. No synergistic effect was observed when the cells were treated with TPA and IAP simultaneously. These results suggest that TPA treatment impairs the coupling between PGE1 receptor and Gi rather than enhances that between PGE1 receptor and Gs. Protein kinase C was involved in the regulation of hormone-sensitive adenylate cyclase, the beta-agonist-induced stimulatory pathway and the PGE1-induced inhibitory pathway in rat reticulocytes, since other phorbol esters and diacylglycerol, which activate this kinase, caused the same response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the adenylate cyclase system by catecholamines in the rat prostate

Summary Tolerance to catecholamines in the beta-adrenergic and dopaminergic receptor-adenylate cyclase system from the rat prostatic membrane has been studied. Preincubation of the tissue with isoproterenol produced not only a decrease in beta-adrenergic receptors and its sensitive adenylate cyclase activity, but also a reduction in dopaminergic response. Similarly, exposure to dopamine resulted in a decline both of isoproterenol- and dopamine-stimulated adenylate cyclase activity. A beta adrenergic antagonist, propranolol, exclusively blocked desensitization in the beta-adrenergic and dopaminergic adenylate cyclase system caused by isoproterenol, while dopamine-induced refractoriness was prevented by a dopaminergic antagonist, haloperidol. These results suggest that desensitization of the beta-adrenergic and dopaminergic adenylate cyclase system begins on receptor-occupation by its specific agonist.This research was supported by Grant No. 377096 from the Ministry of Education, Science and Culture, JapanThe postgraduate fellow from Department of Neuropsychiatry, St. Marianna University School of Medicine     

低pH处理对阿片受体激动剂调节cAMP第二信使系统的作用(英文)

研究不同的阿片类物质对ＣＡＭＰ第二信使系统不同作用的机制．方法：用低 ｐＨ方法失活 Ｇｓ蛋白,用蛋白竞争法测ＮＧ１０８－１５细胞膜腺苷酸环化酶（ＡＣ）活性．结果：与二氢埃托啡（ＤＨＥ）和埃托啡（Ｅｔｏ）不同,低ｐＨ处理明显增加吗啡（ Ｍｏｒ）和美沙酮（Ｍｅｔ）对正常和自身慢性处理细胞的膜ＡＣ活性的抑制作用．与Ｍｏｒ等不同,ＤＨＥ和Ｅｔｏ对自身慢性处理细胞的膜ＡＣ活性抑制作用降低不明显,纳洛酮催促, ＤＨＥ和Ｅｔｏ慢性处理细胞的膜ＡＣ活性也未见显著反跳性升高．低ｐＨ处理使纳洛酮催促的Ｍｏｒ慢性处理细胞的膜ＡＣ活性反跳性升高作用消失。结论：不同的阿片类ＣＡＭＰ信使系统的不同作用与它们对Ｇｓ功能调节差异有关,Ｇｓ与阿片类物质的耐受和依赖密切相关。     

Effects of ketoconazole on progesterone and cAMP production in MA-10 mouse Leydig tumor cells

The effects of ketoconazole (KCZ) on secretion of progesterone and cAMP in Leydig cells were investigated in vitro. MA-10 mouse Leydig tumor cells were used to conduct the experiments. KCZ significantly inhibited the progesterone production from MA-10 cells in a dose dependent fashion between 2 and 20 microM among 1, 2 and 3 h of incubation. There was a statistically significant difference in luteinizing hormone (LH) stimulated progesterone production inhibited by 2 and 20 microm KCZ treatment compared to the control. The effect of KCZ on progesterone biosynthesis in MA-10 cells was mediated by cAMP, since KCZ suppressed basal and LH stimulated cAMP production and content within the same dose range. The stimulatory effects of forskolin and sodium fluoride on the adenylate cyclase system were also inhibited by KCZ. Moreover, dibutyryl cAMP blocked the inhibitory effect on steroidogenesis of KCZ in MA-10 cells. These data indicated that KCZ induced the inhibition of a catalytic component of adenylate cyclase holoenzyme in MA-10 mouse Leydig tumor cells.