Effects of oestrogen on adenylate cyclase system and glucose output in rat liver.

Effects of chronic oestrogen treatment on catecholamine- and glucagon-sensitive adenylate cyclase activity and glucose output in hepatocytes of castrated male rats were studied. In hepatocytes from male intact or castrated rats, the beta-adrenergic agonist isoprenaline did not stimulate adenylate cyclase activity and glycogenolysis, but glucagon markedly stimulated all these activities. Treatment of castrated animals with 17 beta-oestradiol for 7 days led to the appearance of beta-adrenergic-stimulated increases in both cyclic AMP generation and glucose output. The basal, glucagon- or fluoride-stimulated activities of adenylate cyclase of hepatic membranes prepared from oestrogen-treated rats were similar to those of control animals. Treatment with oestrogen did not influence the number or affinity of beta-adrenergic receptors. In hepatic plasma membranes from control rats, GTP failed to decrease the affinity of beta-adrenergic receptors for agonists, whereas the GTP-induced shift was apparently observed in those from oestrogen-treated animals. These results suggest that oestrogen is able to facilitate the coupling of hepatic beta-adrenergic receptors to the enzyme by increasing the effectiveness of receptor-guanine nucleotide regulation.     

Evidence for a second desensitized state of beta-adrenergic receptor with low affinity for beta-antagonists and normal reactivity towards beta-agonists in adipocyte membranes previously exposed to beta-antagonists.

When adipocyte membranes are successively exposed to (-)-propranolol or (+/- alprenolol at 25 or 4 degrees C, repeatedly washed and then assayed for (-)-[3H]dihydroalprenolol binding, the apparent number of beta-adrenergic binding sites is markedly decreased. Induction of this peculiar type of receptor desensitization does not require prolonged exposure of the membranes to the beta-adrenergic antagonists (half-time: 1 min), is stereospecific, concentration-dependent and almost complete with high concentrations of antagonists. p[NH]ppG, which reduces the affinity of fat cell beta-adrenergic receptors for agonists, does not prevent the antagonist-induced decrease in the receptor number. The magnitude of the desensitizating effect induced separately by (-)-isoproterenol and (-)-propranolol is not additive in membranes exposed to both drugs, suggesting that the receptors lost after exposure to agonists are the same sites as part of those lost after exposure to antagonists. However, contrary to the results found in membranes desensitized by agonists, adenylate cyclase activity remained fully responsive to catecholamines in membranes exposed to beta-antagonists. As shown by kinetic studies on (-)-[3H]dihydroalprenolol binding, this beta-antagonist-induced receptor desensitization is reversible after prolonged incubation. These data which have never yet been described in the other reported desensitizable beta-adrenergic systems, suggest that, when exposed to beta-antagonists, the fat cell beta-adrenergic receptors undergo a conformational change leading to a peculiar state which has low affinity for antagonists but behaves towards agonists as does the receptor in its resting state.     

Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines.

The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.     

Exfoliation of the beta-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed 'exosomes' into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, Gs, and the inhibitory, Gi, regulatory components) and beta-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of Gs or Gi in the exosomes was detected by ADP ribosylation using [alpha-32P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal Gs was almost as active as the membrane-derived Gs in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc- cells, which lack a functional Gs. The ability of exosomal Gs to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane Gs. The density of beta-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated beta-adrenergic receptors seem to be impaired in their ability to couple to and activate Gs. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the beta-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.     

Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni

Adenosine, acting via A1 adenosine receptors, can inhibit adenylate cyclase activity in adipocytes. To assess the effects of chronic adenosine agonist exposure on the A1 adenosine receptor system of adipocytes, rats were infused with (-)-phenylisopropyladenosine or vehicle for 6 days and membranes were prepared. Basal as well as isoproterenol-, sodium fluoride-, and forskolin-stimulated adenylate cyclase activities were significantly increased (approximately 2-fold) in membranes from treated animals. (-)-Phenylisopropyladenosine-mediated inhibition of forskolin-stimulated adenylate cyclase activity was significantly (p = 0.0001) attenuated in membranes from treated rats (20.1 +/- 2.1% inhibition) versus controls (31.6 +/- 2.3% inhibition). Prostaglandin E1-induced inhibition of forskolin-stimulated adenylate cyclase activity was also attenuated: 11.7 +/- 3.6 versus 23.2 +/- 4.6% (p = 0.001). Using the A1 adenosine receptor agonist radioligand (-)-N6-(3-[125I]iodo-4-hydroxyphenylisopropyl)adenosine, 32% fewer high affinity binding sites were detected in membranes from treated animals (p less than 0.04). Photoaffinity labeling with N6-2-(3-[125I]iodo-4-azidophenyl)ethyladenosine revealed no gross difference in receptor structure. The number of beta-adrenergic receptors as well as the percentage of receptors in the high affinity state as assessed by (-)-3-[125I]iodocyanopindolol binding were the same in both groups. In membranes from treated rats, the amount of [alpha-32P]NAD incorporated by pertussis toxin into the alpha subunit of the inhibitory guanine nucleotide regulatory protein (Ni) was decreased by 37 +/- 11%. Concurrently, the quantity of label incorporated by cholera toxin into the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Ns) was increased by 44 +/- 14% in treated membranes. Finally, the capacity of Ns solubilized from treated membranes to stimulate adenylate cyclase activity when reconstituted into cyc- S49 lymphoma cell membranes was enhanced by approximately 50% compared to control. Thus, heterologous desensitization, manifested by a diminished capacity to inhibit adenylate cyclase and an enhanced responsiveness to stimulatory effectors, can be induced in the A1 adenosine receptor-adenylate cyclase system of adipocytes. A decrease in Ni alpha subunit concomitant with an increase in Ns alpha subunit quantity and activity may represent the biochemical mechanism of desensitization in this system.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

Differential effects of cholera toxin on guanine nucleotide regulation of beta-adrenergic agonist high affinity binding and adenylate cyclase activation in frog erythrocyte membranes

The guanine nucleotide regulatory protein(s) regulates both adenylate cyclase activity and the affinity of adenylate cyclase-coupled receptors for hormones or agonist drugs. Cholera toxin catalyzes the covalent modification of the nucleotide regulatory protein of adenylate cyclase systems. Incubation of frog erythrocyte membranes with cholera toxin and NAD+ did not substantially alter the dose dependency for guanine nucleotide activation of adenylate cyclase activity. In contrast, toxin treated membranes demonstrated a 10 fold increase in the concentrations of guanine nucleotide required for a half maximal effect in regulating beta-adrenergic receptor affinity for the agonist (+/-) [3H]hydroxybenzylisoproterenol. The data emphasize the bifunctional nature of the guanine nucleotide regulatory protein and suggest that distinct structural domains of the guanine nucleotide regulatory protein may mediate the distinct regulatory effects on adenylate cyclase and receptor affinity for agonists.     

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells

The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The beta-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of beta-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 degrees C was not associated with a change in anisotropy.     

Subsensitivity of adenylate cyclase and decreased beta-adrenergic receptor binding after chronic exposure to (minus)-isoproterenol in vitro.

In vitro incubation of frog erythrocytes with (minus)-isoproterenol, 0.1 mM, at 23 degrees for 10 to 24 hours caused a 63% decline (rho less than 0.001) in the maximum (minus)-isoproterenol-stimulated adenylate cyclase activity in the erythrocyte membranes. Affinity for (minus)-isoproterenol as judged by the concentration which half-maximally stimulated the enzyme was not markedly altered. Basal enzyme activity and stimulation by fluoride or prostaglandin E1 remained unaltered. The number of beta-adrenergic receptor binding sites, assessed by binding studies with the beta-adrenergic antagonist (minus)-[3-H] alprenolol, declined by 50% (rho less than 0.005) in the (minus)-isoproterenol-treated cells. The binding affinity of the sites was not changed. Regulation of the concentration of functionally active beta-adrenergic receptors in membranes may be one of the mechanisms by which chronic exposure to catecholamines desensitizes tissues to beta-adrenergic stimulation.     

The hepatic adrenergic receptors

Summary The presence of both alpha- and beta-adrenergic receptors in liver designated the hepatic plasma membrane as a useful tool for the elucidation of the mechanisms by which the hormonal signal is transfered through the membrane via a coupling system to an amplifying entity. This process is well documented for the beta-adrenergic receptor which is linked to adenylate cyclase, whereby it modulates the cyclic AMP level. Much less is known about the alpha-adrenergic receptor.Recently, two factors have contributed to a renewed interest in alpha- and beta-adrenergic receptors in liver: i) The fact that activation of glycogenolysis in isolated liver parenchymall cells by epinephrine may be mediated by either alpha- or beta-adrenergic receptors, depending on the species or on the state of nutrition, and not only by beta-adrenergic receptors as previously thought. ii) The existence of specific adrenergic agonists and antagonists radiolabeled to a high specific activity which has permitted the characterization of adrenergic receptors in terms of nature, number, affinity and regulation.The present review will be devoted to the recent progress made in the physiological, pharmacological and biochemical characterization of alpha- and beta-adrenergic receptors in the liver.     

Functional desensitisation of beta-adrenergic receptors of avian erythrocytes by catecholamines and adenosine 3',5'-phosphate

Prolonged exposure to beta-adrenergic agonists of pigeon erythrocytes causes a reversible loss (70%) of catecholamine-stimulated adenylate cyclase activity without reduction in the number of beta-adrenergic receptors. In addition a less pronounced decrease in non-stimulated and NaF-stimulated adenylate cyclase activity (15-22%) is observed, appearing at different agonist concentrations and at a different rate. Dibutyryladenosine 3',5'-phosphate and the phosphodiesterase inhibitor methylisobutylxanthine partially mimick the action of the beta-adrenergic agonist, thus pointing to a possible role of adenosine 3',5'-phosphate in establishing desensitization. When adenylate cyclase from desensitized cells is stimulated with 5'-guanylyl-imidodiphosphate in the presence or absence of catecholamines the lag period preceding the attainment of maximal activity is extended. Likewise the rate of reversal by GTP or GTP of persistent activation of adenylate cyclase is slowed down. This is therefore interpreted to mean that the loss in hormonal stimulation on treatment of pigeon red blood cells with beta-adrenergic agonists is due to a delayed exchange of GDP against GTP on the regulatory GTP-binding protein. Furthermore, we conclude that events causing the refractory state in avian erythrocytes should occur at a site distal to the beta-adrenergic receptor.     

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.     

Cholesterol modulation of beta-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. beta-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. beta-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, beta-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. The increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn2+ and forskolin also demonstrated 50-80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Biochemical characterization of the BETA-adrenergic receptor of the frog erythrocyte

Summary The beta-adrenergic receptor which is coupled to adenylate cyclase in the frog erythrocycte plasma membrane provides a convenient model system for probing the molecular characteristics of an adenylate cyclase coupled hormone receptor. Direct radioligand binding studies with beta-adrenergic agonists and antagonists such as [³H]hydroxybenzylisoproterenol and [³H]dihydroalprenolol have shed new light on the biochemical properties of the receptor as well as on its mode of interaction with other components of the adenylate cyclase system. Agonist binding to the receptor induces a high affinity state of the receptor which can be selectively reverted to a low agonist affinity state by guanyl nucleotides. This agonist-induced high affinity state of the receptor appears to correspond to a receptor moiety which has larger apparent molecular weight and which is probably a complex of the beta-adrenergic receptor and nucleotide regulatory binding protein. Antagonists do not appear capable of inducing or stabilizing the formation of this high affinity receptor-nucleotide site complex.The beta-adrenergic receptors have been solubilized using the plant glycoside digitonin as the detergent and have been highly purified by biospecific affinity chromatography on an alprenolol-agarose affinity support. These highly purified receptor preparations retain all of the binding characteristics observed in the unpurified soluble receptor preparations.Remarkably, antibodies raised in rabbits against affinity chromatography purified preparations of the receptor, themselves bind beta-adrenergic ligands with typical beta-adrenergic specificity. Such antibodies which possess binding sites similar to those of physiological receptors provide useful model systems for further probing the molecular characteristics of beta-adrenergic binding sites.     

The effect of age and cholesterol on the rat lung beta-adrenergic system

To assess the influence of membrane lipid composition on beta-adrenergic receptor number and adenylate cyclase activity in aging, we investigated the effect of cholesteryl hemisuccinate on these parameters in lung membranes of 3-, 12-, and 24-month-old CDF (F-344) rats. When cholesteryl hemisuccinate (0.5 mg/ml) was incubated with lung membranes, beta-adrenergic receptor density was increased by 70%. This effect was the same for each age group studied and indicated that the density of both basal and CHS-sensitive receptors is unaltered in rat lung with age. Forskolin, NaF, p[NH]ppG, and isoproteronol-stimulated adenylate cyclase activity is 30% lower in lung membranes from aged rats. Since enzyme activity is affected by the lipid environment and membrane composition often changes with age, we assessed adenylate cyclase activity following cholesteryl hemisuccinate incorporation. There was up to a 75% decrease in adenylate cyclase activity following cholesteryl hemisuccinate incorporation in lung membranes in each of the three age groups. In untreated membranes, there was no significant difference in cholesterol or lipid phosphate content with age. These data suggest that cholesterol content does not account for alterations in senescent rat lung adenylate cyclase activity.     

Somatostatin induces translocation of the beta-adrenergic receptor kinase and desensitizes somatostatin receptors in S49 lymphoma cells

The beta-adrenergic receptor kinase is a cytosolic enzyme that specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta AR). Beta AR kinase appears to be translocated from the cytosol to the plasma membrane when kin- S49 lymphoma cells are incubated with either beta-adrenergic agonists or prostaglandin E1, both of which act through receptors which stimulate adenylate cyclase. We report here that brief (approximately 20 min) exposure of wild type S49 lymphoma cells to somatostatin (which inhibits adenylate cyclase) promotes the translocation of beta AR kinase to an extent comparable to that observed in the presence of the beta agonist isoproterenol or prostaglandin E1. Beta AR kinase activity can be measured using either beta AR or rhodopsin, the retinal receptor for light, as a substrate. The translocation process triggered by somatostatin is rapid, reversible, and is associated with somatostatin receptor desensitization. The latter is apparent as an attenuation of the inhibition by somatostatin of forskolin-stimulated adenylate cyclase activity in membranes of S49 cells preincubated in the presence of the peptide. These results strongly suggest that beta AR kinase is able to phosphorylate and desensitize both stimulatory and inhibitory adenylate cyclase-coupled receptors, thus emerging as a general kinase that regulates the function of different receptors in an agonist-specific fashion.     

Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.     

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.     

Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes.

Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.