Desensitization Of Adenylate Cyclase Research Articles

Down-regulation of adenylate cyclase coupled to adrenomedullin receptor in vascular smooth muscle cells

Adrenomedullin activates receptor-mediated adenylate cyclase to cause vasorelaxation. To elucidate whether desensitization of adenylate cyclase coupled to vascular adrenomedullin receptors occurs, we studied the adenylate cyclase activity after treatment with rat adrenomedullin in cultured rat aortic vascular smooth muscle cells. Cyclic AMP(cAMP) generation induced by adrenomedullin was markedly decreased by pretreatment with adrenomedullin: a maximal reduction (∼80%) was induced after 2 h and persisted during 24 h. Desensitization was independent of protein kinase A, protein kinase C, protein tyrosine kinase or receptor sequestration, because pretreatment with either isoproterenol, forskolin, tetradecanoylphorbol acetate, cytochalasin D, or colchicine did not affect the adrenomedullin-stimulated cAMP response. Furthermore, preincubation with inhibitors for these protein kinases prior to pretreatment with adrenomedullin failed to affect the adrenomedullin-induced decrease in cAMP response following the second stimulation with adrenomedullin. The present results provide the evidence for the existence of desensitization of adenylate cyclase coupled to vascular adrenomedullin receptors.

The cardiac beta-adrenoceptor-mediated signaling pathway and its alterations in hypertensive heart disease.

Hypertension-induced cardiac hypertrophy is a predictor of the development of cardiac failure. It is unknown which cellular markers contribute to the progression from compensated hypertrophy to failure. In heart failure, several signal transduction defects leading to adenylate cyclase desensitization have been demonstrated, such as beta-adrenoceptor downregulation, increase of inhibitory G protein expression, and uncoupling of beta-adrenergic receptors, presumably by an increase of receptor kinase activity. In hypertensive heart disease, most studies have been performed in rat models of hypertension. As in heart failure, heterologous adenylyl cyclase desensitization occurs. The mechanisms are often different between the heterogeneous models for acquired and genetic hypertension, but Gi protein alterations and beta-adrenoceptor downregulation have been observed frequently. The underlying mechanism for desensitization is most likely a sympathetic activation in established hypertension rather than genetic alterations of signal transduction proteins. The data available suggest that beta-adrenergic desensitization could represent a mechanism that contributes to the progression from hypertrophy to failure. The key question remains whether those hypertensive patients who develop heart failure are more prone to beta-adrenergic desensitization or whether early intervention to reduce sympathetic activity is more effective in preventing or delaying the transition from compensated hypertrophy to overt failure.

Desensitization of cyclic adenosine 3,5′-monophosphate response to thyrotropin in normal and primary or metastatic papillary thyroid cancer cells in vitro

Desensitization is an important physiologic process resulting in a decreased functional response after continuous or repeated stimulation. Prior exposure of normal human thyroid tissue to thyrotropin either in vivo or in vitro causes desensitization of adenylate cyclase. Little is known, however, about whether the thyrotropin-adenylate cyclase-cyclic adenosine 3,5'-monophosphate (cAMP) signal transduction system desensitizes in human thyroid carcinomas. Failure to desensitize could result in increased growth or metastases. Cell cultures from Chinese hamster ovary (CHO) cells transfected with normal human thyrotropin receptor (hTSHr) and thyroid neoplasms including one papillary carcinoma and one papillary lymph node metastases were evaluated for desensitization. CHO cells were stably transfected with plasmid DNA containing hTSHr. Cells were incubated with thyrotropin (10 mU/ml) for different periods (1 to 24 hours). A second incubation (30 minutes) was done with and without thyrotropin in medium including 3-isobutyl-1-methylxanthine (1 mmol/L). Intracellular cAMP accumulation was determined by means of radioimmunoassay. Maximal stimulation and desensitization to thyrotropin were observed at 30 minutes and 4 hours, respectively. The cAMP response to a second incubation with thyrotropin was 52% and 48% lower than the initial response to thyrotropin in CHO-hTSHr and papillary thyroid cancer cells, respectively (p < 0.001). A papillary carcinoma lymph node metastases had an increased basal cAMP level and also an increased cAMP level in response to thyrotropin stimulation (227%) but failed to desensitize (p < 0.001). Desensitization of the cAMP response to thyrotropin occurred in a papillary thyroid cancer but not in a metastatic thyroid cancer. Failure to desensitize might play a role in tumor progression.

Interaction of lipopolysaccharide endotoxin produced from escherichia coli with d-tubocurarine at the nicotinic 2 receptor and adenosine 3′:5′ cyclic monophosphate during physiological contraction in skeletal muscle

In this report the murine model of endotoxicosis was used to evaluate hyposensitivity to the neuromuscular relaxant d-tubocurarine (dTC). This hyposensitivity was expressed in terms of a decreased potency to dTC. A rightward shift of the dose-response curve due to endotoxin was observed. Mice were subjected to cumulative intraperitoneal doses of Escherichia coli endotoxin over a 2-wk period. The interaction between endotoxin and dTC was examined during an acute (1 wk) and chronic (2 wk) period of endotoxicosis. Muscle twitch analyses were performed and samples of gastrocnemius muscle were assayed for adenosine 3′:5′ cyclic monophosphate (cAMP) by [ 125I]radioimmunoassay. A parallel shift in the dose-response curve occurred in the endotoxin group subjected to doses corresponding to one-third the dose evoking 50% lethality for 2 wk. Both skeletal muscle tension and cAMP levels decreased as cumulative endotoxin doses increased. A relationship between decreasing cAMP levels and increasing dTC and effective dose required to achieve 50% muscle paralysis values was thought to be evoked by the agonistic activity of E. coli endotoxin leading to desensitization of adenylate cyclase. The perturbations of the classical second messenger cAMP system by endotoxin may be responsible for the skeletal muscle dysfunction observed in immunocompromised patients.

Role of protein kinase C on the acute desensitization of renal cortical adenylate cyclase to parathyroid hormone

The mechanisms of adenylate cyclase desensitization to parathyroid hormone are still unclear. Current evidence suggest that the signal generated after PTH binding to receptors results in activation of adenylate cyclase and stimulation of phospholipase C with subsequent activation of protein kinase C. Recent studies have suggested a role of protein kinase C on the regulation of the PTH-dependent receptor-adenylate cyclase system in cultured cells. Therefore, the present studies were conducted to examine the role of protein kinase C on the desensitization of canine renal cortical adenylate cyclase after an acute exposure in vivo to PTH. A group of normal dogs were treated with a single intravenous injection of 1 microgram/k of syn bPTH (1-34) or Nle bPTH (3-34). Ten minutes later, animals were subjected to bilateral nephrectomy and the kidney cortex processed for preparations of basolateral membranes for determinations of adenylate cyclase activity, as well as membrane and cytosolic fractions for analysis of protein kinase C activity. Animals not treated with PTH were used as controls. PTH administration in vivo resulted in a 46.9 +/- 9.3% decrease in maximal adenylate cyclase activity in vitro in response to syn bPTH (1-34) (P < 0.001). Likewise, PTH binding as measured with 125I-Nle8,18,Tyr34-bPTH (1-34)NH2 showed a 40 +/- 3% decrease. This alterations were associated with a marked translocation of protein kinase C from the cytosol to the membrane. Thus, protein kinase C activity in membrane fractions increased from 160.6 +/- 44.8 pmol Pi/min in controls to 500.4 +/- 123 in PTH treated dogs (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term exposure of intact ventricular myocytes to isoproterenol results in desensitization of adenylate cyclase

We have examined the effects of short-term exposure to low (50 nM) levels of the β-adrenoceptor agonist isoproterenol on the ability of intact rat ventricular myocytes to increase their intracellular levels of cyclic AMP (cAMP) in response to subsequent isoproterenol exposure. Exposing freshly isolated, nonstimulated myocytes (which contained 3.5 ± 0.3 pmol cAMP/mg protein) to isoproterenol resulted in rapid, dose-dependent increases in cAMP formation; 5-min exposure raised intracellular cAMP content to 6/5 + 0.7 pmol/mg protein and 5.4 ± 0.9 pmol/mg protein respectively, in the absence and presence of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imiazolidin-2-one (RO 20-1724). In myocytes incubated for 5–20 min with isoproterenol, washed twice to remove the agonist, and then rechallenged for 20 min with the agonist in the presence of RO 20-1724, a 10-min preexposure to the agonist resulted in a significant reduction (≈ 20%; P < 0.05) in the receptor's responsiveness to the rechallenge, and increasing the preexposure time to 20 min resulted in the maximum attenuation in responsiveness (≈ 30%; P < 0.001). Myocytes preincubated with forskolin (1 μM), which directly activates adenylate cyclase contained 9.0 ± 0.7 pmol cAMP/mg protein after 5 min exposure, and up to 20 min preexposure to forskolin did not alter the cells' responsiveness to subsequent challenge with isoproterenol. The results of this study indicate that exposing freshly isolated rat ventricular myocytes to nanomolar levels of isoproterenol for as little as 10 min desensitizes them to subsequent challenge with agonist. The process appears to be homologous, since it was not observed when forskolin was used to elevate cAMP levels in the initial incubation.

Rapid desensitization of parathyroid hormone dependent adenylate cyclase in perifused human osteosarcoma cells (SaOS-2)

The pulsatile but not the continuous application of parathyroid hormone (PTH) increases bone mass in vivo. To study the effects of intermittent hormonal administration on bone-derived cells in vitro, we established a perifusion system using the human osteosarcoma cell line SaOS-2. Cells were grown in suspension culture attached to collagen beads and were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1–34) resulted in a dose-dependent increase of cAMP release by SaOS-2 cells into the effluent medium. Cyclic AMP accumulation was rapidly desensitized by approx. 80% after 30 min of continuous exposure to PTH(1–34) (10 −7 M), while cells remained responsive to forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental setting was dose-dependent (EC 50 = 1 · 10 −10 M PTH(1–34)). Neither 8Br-cAMP (2 · 10 −4 M) nor PMA (1 · 10 −7 M) had an effect on the PTH(1–34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [ 125I]-[Tyr 36]hPTHrP(1–36)amide binding to SaOS-2 cells was decreased by 60–70% by PTH(1–34) (1 · 10 −6 M), bPTH(1–84) (1.8 · 10 −6 M) and bPTH(3–34) (2 · 10 −6 M), whereas 8Br-cAMP (2 · 10 −4 M) had no effect on radioligand binding. PMA (1 · 10 −7 M) appeared to slightly increase [ 125I]PTHrP binding. This observation is consistent with a small (3-fold) increase in PTH-induced cAMP release as a result of PMA pre-treatment. Receptor internalization was dose-dependent (EC 50 = 3 · 10 −7 M PTH(1–34)). The maximal effect occurred after 10–30 min and was largely reversible within 2 h. Monensin (3 · 10 −5 M) inhibited the recovery from receptor internalization. We conclude that a perifusion system using SaOS-2 cells is a suitable model to study the effect of discontinuous application of PTH on cAMP release. A rapid, homologous desensitization of PTH(1–34) stimulated cAMP accumulation has been observed that does not appear to involve protein kinase A or C.

Desensitization of adenylate cyclase in Chinese hamster ovary cells transfected with human thyroid-stimulating hormone receptor.

Desensitization or decreased response to the same (homologous) or other stimuli (heterologous) is a well known process. Homologous desensitization to TSH has been demonstrated in normal thyroid tissue. Chinese hamster ovary cells (CHO) transfected with normal human TSH receptor (hTSHR) DNA, in contrast, have been reported not to desensitize. The purpose of our investigation was to determine whether CHO cells transfected with hTSHR desensitize in response to TSH and postreceptor stimulation. CHO cells were stably transfected with plasmid DNA containing hTSHR; nontransfected CHO cells served as the control. TSH (10 mU/ml), 5'-beta,gamma-imido-triphosphate [Gpp(NH)p; 0.1 mM], sodium fluoride (NaF; 10 mM), forskolin (10 microM), and (Bu)2cAMP (100 microM) were used to determine whether homologous or postreceptor heterologous desensitization of adenylate cyclase activity occurred in CHO-transfected cells. Intracellular cAMP accumulation was determined by RIA. Cells were incubated with TSH (to stimulate TSH receptor), Gpp(NH)p, NaF (to stimulate G-protein), forskolin (to stimulate adenylate cyclase activity), and (Bu)2cAMP (nonmetabolized cAMP analog). A second incubation was carried out with TSH (10 mU/ml). Maximal desensitization to either TSH or postreceptor stimulation was observed at 2 h. When transfected CHO cells were preexposed to TSH (10 mU/ml) for 4 h, even the smallest dose of TSH (0.001 mU/ml) caused desensitization. All substances that increased the intracellular cAMP concentration, such as TSH, Gpp(NH)p, NaF, forskolin, and (Bu)2cAMP, caused desensitization. The decrease in the cAMP response to TSH added in the second incubation was 63% less than the initial response to TSH or to postreceptor stimulation (P = 0.0001). In conclusion, desensitization of hTSHR-transfected CHO cells occurs in response to both receptor and postreceptor stimulation that increase cAMP levels. Because hTSHR transfected CHO cells desensitize, no specific thyroid factor(s) other than increased levels of cAMP is required.

Desensitization of adenylate cyclase in Chinese hamster ovary cells transfected with human thyroid-stimulating hormone receptor

Desensitization of adenylate cyclase in Chinese hamster ovary cells transfected with human thyroid-stimulating hormone receptor

Regulation of hepatocyte adenylate cyclase by amylin and CGRP: a single receptor displaying apparent negative cooperatively towards CGRP and simple saturation kinetics for amylin, a requirement for phosphodiesterase inhibition to observe elevated hepatocyte cyclic AMP levels and the phosphorylation of Gi-2.

Challenge of intact hepatocytes with amylin only succeeded in elevating intracellular cyclic AMP levels and activating phosphorylase in the presence of the cAMP phosphodiesterase inhibitor IBMX. Both amylin and CGRP similarly activated adenylate cyclase, around 5-fold, although approximately 400-fold higher levels of amylin were required to elicit half maximal activation. Amylin activated adenylate cyclase though apparently simple Michaelien kinetics whereas CGRP elicited activation by kinetics indicative of apparent negative co-operativity. Use of the antagonist CGPP(8-37) showed that both CGRP and amylin activated hepatocyte adenylate cyclase through a common receptor by a mnemonical mechanism where it was proposed that the receptor co-existed in interconvertible high and low affinity states for CGRP. It is suggested that this model may serve as a paradigm for G-protein linked receptors in general. Amylin failed to both stimulate inositol phospholipid metabolism in hepatocytes and to elicit the desensitization of glucagon-stimulated adenylate cyclase. Amylin did, however, elicit the phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes and prevented the action of insulin in reducing the level of phosphorylation of this G-protein.

Growth of S49 wild type cells in 3 nM epinephrine increases cyclic AMP phosphodiesterase activity

In this communication we report that intact cell measurements of cAMP decay have shown an increase averaging over 80% in the cAMP decay constant (k dy) in intact S49 WT cells following a 24 hour growth in 3 nM epinephrine (24 hr/3 nM epinephrine). A comparable percentage increase was seen in cell free PDE activities from cells similarly treated. The enhanced PDE activity and k dy acre detectable after 6 hours of incubation with 3 nM epinephrine, and were maximal by 24 hours of incubation. Lysate PDE activity returned to control levels within 24 hours after the cells were transferred to epinephrine-free growth medium. The increased PDE activity present in S49 WT cells after 24 hr/3 nM epinephrine was the result of increased concentrations of the enzymes already present or the expression of very similar enzymes, rather than the expression of enzymes with markedly different characteristics. Firstly, the apparent K m values for the PDE's in lysates from control and 24 hr/3 nM epinephrine cells were both in the region of 1 μM. Secondly, PDE activities in lysates from 24 hr/control and 24 hr/3 nM epinephrine S49 WT cells showed similar sensitivities to PDE inhibitors. There was support for the hypothesis that the increase in PDE activity in 24 hr/3 nM epinephrine S49 WT cells is dependent on the presence of cAPK. That is, although 24 hr/3nM epinephrine caused a distinct homologous desensitization of adenylate cyclase in S49 kin cells, neither k dy nor lysate PDE activity were affected. Additionally, 24 hours incubation of S49 WT cells with 3 μM dibutyryl cAMP resulted in increased PDE activity. Finally, the stimulatory effect of 24 hr/3 nM epinephrine on PDE activity was inhibited in the presence of cycloheximide, suggesting the involvement of protein synthesis in the process. This study shows that prolonged treatment with very low concentrations of epinephrine results in an increase in PDE activity which had a significant effect on cAMP accumulation. The increases were quantified by intact cell and cell free measurements, with kinetic data which were consistent with the hypothesis that the increased PDE activity in 24 hr/3 nM epinephrine cells reflected an increase PDE synthesis which was dependent on activation of the cAPK.

Homologous desensitization of calcitonin receptors and calcitonin-dependent adenylate cyclase in T47D cells

Prolonged use of calcitonin (CT) in vivo leads to a loss of biological response (escape). To understand the molecular basis of this phenomenon, we examined desensitization of calcitonin receptors and down-regulation of adenylate cyclase response to CT in T47CD cells after a preincubation with CT. Preincubation with salmon or human CT (sCT, hCT) for 3 h led to a concentration-dependent loss of [125I]-sCT binding and a similar loss of adenylate cyclase response to a maximal stimulatory dose of sCT. At the same time there was an increased basal activity of the adenylate cyclase. After 24 h preincubation with sCT, basal cAMP levels fell considerably but not to basal levels. Time course experiments showed a delayed decline of maximally sCT-stimulated cAMP levels, which started after 1 h, while binding values declined over the first 60 min to one-third of the original values. Upon removal of CT from the medium, recovery of hormone binding occurred in parallel with the recovery of the adenylate cyclase response to sCT. T47D cells incubated with 25 mumol/l monensin, a lysosomal inhibitor, showed a persistent [125I]-sCT binding after removal of hormone, consistent with diminished intracellular receptor degradation. However, despite persistent binding to the cells, basal and stimulated cAMP levels dropped in the same manner as seen in controls. Our experiments support the view that tight binding of CT to its receptor stimulates adenylate cyclase in T47D cells, until receptors are removed by internalization. The physiological role of internalization of the CT receptor might be to end continuous stimulation of the adenylate cyclase, which occurs after binding.

Desensitization of adenylate cyclase and increase of Gi alpha in cardiac hypertrophy due to acquired hypertension.

The present study investigated whether reduced adenylate cyclase activity and an increase in inhibitory guanine nucleotide binding proteins (Gi alpha), which have been observed in the failing human heart, already occur in myocardial hypertrophy before the stage of heart failure. In membranes of hypertrophic hearts from rats with different forms of experimentally induced hypertension without heart failure (one-kidney, one clip rats, deoxycorticosterone-treated rats, and rats with reduced renal mass), basal as well as isoprenaline-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylate cyclase activity was reduced. The activity of the catalyst was depressed in deoxycorticosterone but unchanged in one-kidney, one clip and reduced renal mass compared with controls. The number of beta-adrenergic receptors was similar in all groups. Radioimmunological quantification of Gi alpha proteins revealed an increase by 73% in one-kidney, one clip, 67% in reduced renal mass, but only 20% in deoxycorticosterone compared with sham-operated, age-matched control rats. The increase of Gi alpha was accompanied by smaller changes of pertussis toxin-induced [32P]ADP-ribosylation of a 40-kd membrane protein. It is concluded that Gi alpha contributes to the reduced adenylate cyclase activity in cardiac hypertrophy in one-kidney, one clip and reduced renal mass and to a smaller extent in deoxycorticosterone. It is suggested that an enhanced expression of Gi alpha could occur not only in severe heart failure but also in cardiac hypertrophy and could, therefore, contribute to myocardial depression and progression of disease in heart failure. In addition, Gi alpha might represent an important regulatory mechanism for cardiac adenylate cyclase activity and thus, might play an important role in various cardiac diseases.

Heterologous desensitization of adenylate cyclase in renal and desoxycorticosterone-induced hypertensive cardiomyopathy

Heterologous desensitization of adenylate cyclase in renal and desoxycorticosterone-induced hypertensive cardiomyopathy

Prostaglandin-concentration-dependent desensitization of adenylate cyclase in human erythroleukaemia (HEL) cells is abolished by pertussis toxin and enhanced by induction by dimethyl sulphoxide

Prostaglandin-regulated cyclic AMP metabolism in human erythroleukaemia (HEL) cells was similar to that previously described in platelets [Ashby (1989) Mol. Pharmacol. 36, 866-873], displaying prostaglandin-concentration-dependent desensitization that could be explained by the presence of separate stimulatory and inhibitory prostaglandin receptors. Pertussis toxin abolished prostaglandin-concentration-dependent desensitization, indicating that the process is mediated through a pertussis toxin-sensitive GTP-binding protein. Treatment of HEL cells for 4 days with the inducer dimethyl sulphoxide enhanced prostaglandin-concentration-dependent desensitization, but did not alter the initial rate of cyclic AMP synthesis or the amount of Gi2 alpha measured by immunoblotting, suggesting that the inhibitory receptor was selectively induced by changing the cells to a more platelet-like form.

Reducing reagent-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of an intrinsic agonist (cAMP) to specific receptors on the cell surface induces transmembrane signals for activation and desensitization (adaptation and down regulation) of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. It is generally believed that dithiothreitol (DTT) induces the activation through interaction between the receptor and gradually accumulated cAMP, since DTT is known to inhibit cAMP-phosphodiesterase which degrades cAMP. In the present paper, we investigated the mechanism of activation of adenylate cyclase by the thiol-reducing agents, DTT and 2,3-dimercapto-1-propanol (BAL). We found that BAL activated adenylate cyclase transiently even under conditions where the intrinsic agonist supersaturated the cAMP-receptors and competitively inhibited phosphodiesterase. This result is inconsistent with the generally accepted notion. We conclude that BAL has an independent effect from those of the intrinsic agonist (cAMP) and phosphodiesterase in activation of adenylate cyclase. Since BAL could induce activation just after the activation induced by a supersaturating concentration of the intrinsic agonist had ceased, the independent effect of BAL is not a simple enhancement of the cAMP-induced activation. Our result also suggests that the cAMP-induced adaptation (but not down regulation) suppresses the BAL-induced activation while BAL itself does not induce adaptation to cAMP or BAL. We propose that the thiol-reducing reagent induces or modifies the transmembrane activation signal for adenylate cyclase.

Treatment of intact hepatocytes with synthetic diacyl glycerols mimics the ability of glucagon to cause the desensitization of adenylate cyclase

Incubation of intact hepatocytes with either of the synthetic diacyl glycerols 1-oleoyl-2-acetyl glycerol (OAG) or dihexanoyl glycerol (DHG) caused the transient uncoupling of the ability of glucagon to stimulate adenylate cyclase in membranes prepared from those cells. No change occurred in either the activity of the catalytic unit of adenylate cyclase or the coupling of G 5 to adenylate cyclase. Diacyl glycerol action appeared to mimic glucagon-mediated desensitization of adenylate cyclase, suggesting that protein kinase C activation may provide the molecular trigger for glucagon desensitization.

Rapid desensitization of the parathyroid hormone-responsive adenylate cyclase in isolated renal cortex microvessels: T. Massfelder and J.J. Helwig. Labo Physio Rénale, Hospices Civils, Université Louis Pasteur, Strasbourg, France

Rapid desensitization of the parathyroid hormone-responsive adenylate cyclase in isolated renal cortex microvessels: T. Massfelder and J.J. Helwig. Labo Physio Rénale, Hospices Civils, Université Louis Pasteur, Strasbourg, France

Modulation of Gs activity by phorbol myristate acetate in rat hepatocytes

Activation of protein kinase C promotes heterologous desensitization of hepatic adenylate cyclase. The basis for this desensitization was explored by use of a strategy with several independent approaches. Although not influencing the amount of forskolin-stimulated adenylate cyclase activity (catalyst), treatment with phorbol 12-myristate 13-acetate (PMA) decreased adenylate cyclase activation in response to either sodium fluoride or guanylyl imidodiphosphate [Gpp(NH)p]. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cholera toxin-treated hepatocytes and both the basal and GTP-stimulated adenylate cyclase activity of membranes from toxin-treated cells displayed a marked reduction in response to PMA. The ability of cholate extracts of hepatocyte membranes to reconstitute beta-adrenergic-stimulated adenylate cyclase activity of membrane of S49 mouse lymphoma cyc- cells was reduced by treatment with PMA. Cholera toxin-catalyzed labeling of Gs alpha-subunits was likewise diminished by phorbol ester treatment. Immunoblots of membranes from control or PMA-treated hepatocytes showed no difference in the amount of Gs alpha. Immunoprecipitation studies failed to detect phosphorylation of this G protein alpha-subunit. The data demonstrate that PMA induces an alteration in the functional status of Gs without altering the amount of this transmembrane signaling element. The alteration in Gs function may play a significant role in heterologous desensitization.

In vivo adaptive control of β-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands

1. Adaptation of β-adrenergic receptors (β-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33°C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of β-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [ 3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between β-AR and GTP binding protein(s).