Catecholamine-induced desensitization in turkey erythrocytes: cAMP mediated impairment of high affinity agonist binding without alteration in receptor number

Desensitization of turkey erythrocyte adenylate cyclase by exposure of these cells to the beta-adrenergic agonist isoproterenol leads to a decrease in subsequent adenylate cyclase stimulation by isoproterenol, F-, or Gpp(NH)p without any apparent loss or down regulation of receptors (B.B. Hoffman et al. J. Cyclic Nucl. Res. 5: 363-366, 1979). We now report that the desensitization is associated with a functional "uncoupling" of the beta-adrenergic receptor. This is evidenced by an impaired ability of receptors to form a high affinity, guanine nucleotide sensitive complex with agonist as assessed by computer analysis of radioligand binding data. The changes in adenylate cyclase responsiveness as well as the alterations in receptor affinity for agonists are reproduced by incubation of turkey erythrocytes with the cAMP analog 8-Bromo-adenosine 3':5'- cyclic monophosphate. These findings suggest that one possible mechanism for the development of desensitization in adenylate cyclase systems may be a cAMP mediated alteration of a component(s) of the beta-adrenergic receptor-adenylate cyclase complex which results in impaired receptor-cyclase coupling.     

Iloprost-induced desensitization of the prostacyclin receptor in isolated rabbit lungs

Background

The rapid desensitization of the human prostacyclin (IP) in response to agonist binding has been shown in cell culture. Phosphorylation of the IP receptor by protein kinase C (PKC) has been suggested to be involved in this process.

Methods and results

In this study we investigated the vasodilatory effects of iloprost, a stable prostacyclin analogue, in perfused rabbit lungs. Continuous infusion of the thromboxane mimetic U46619 was employed to establish stable pulmonary hypertension. A complete loss of the vasodilatory response to iloprost was observed in experiments with continuous iloprost perfusion, maintaining the intravascular concentration of this prostanoid over a 180 min period. When lungs under chronic iloprost infusion were acutely challenged with inhaled iloprost, a corresponding complete loss of vasoreactivity was observed. This desensitization was not dependent on upregulation of cAMP-specific phosphodiesterases or changes in adenylate cyclase activity, as suggested by unaltered dose-response curves to agents directly affecting these enzymes. Application of a prostaglandin E1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) or the PKC inhibitor bisindolylmaleimide I (BIM) enhanced the vasodilatory response to infused iloprost and partially prevented tachyphylaxis.

Conclusion

A three-hour infusion of iloprost in pulmonary hypertensive rabbit lungs results in complete loss of the lung vasodilatory response to this prostanoid. This rapid desensitization is apparently not linked to changes in adenylate cyclase and phosphodiesterase activation, but may involve PKC function and co-stimulation of the EP1 receptor in addition to the IP receptor by this prostacyclin analogue.     

Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

Forskolin and 8-cyclopentyltheophylline synergistically facilitate the neuronal activity in the CA2 area of rat hippocampus via cAMP and non-cAMP cascades

High level of adenosine A1 receptor-like immunoreactivity has been found in the CA2/CA3a region of adult rat hippocampus, but its roles in the neuronal activity or signal propagation in hippocampus and its intracellular cascade remain to be studied. In this study, we examined the relation between adenosine-3',5'-cyclic monophosphate (cAMP) cascade and suppression of synaptic transmission by endogenous adenosine through adenosine A1 receptor in the CA2 area. In transverse hippocampal slice, maximal electrical stimulation of the hilus region (0.6 mA) only evoked small population spikes (PSs) in the CA2 area (0.5 mV). In the presence of forskolin (20 micromol/L), a direct adenylate cyclase activator, PSs in CA2 were increased to 1.1 mV. When 8-cyclopentyltheophylline (8CPT, 2 micromol/L), an adenosine A1 receptor antagonist, was added in the presence of 20 micromol/L forskolin, PSs with an average amplitude of 4.7 mV were recorded in the CA2 area, much higher than the sum of the amplitude of PSs in the presence of forskolin and 8CPT alone. To test whether this synergistic potentiation results from the additive activation of cAMP cascade, the cAMP content in hippocampal slices was measured with enzyme immunoassay (EIA). Results showed that 8CPT did not increase the cAMP content in CA2 with or without forskolin. Co-application of forskolin and Ro 20-1724, a cAMP-specific phosphodiesterase-IV inhibitor, only increased PSs in CA2 to 1.3 mV but increased cAMP content by 4.4 times. On the other hand, co-application of 8CPT and 1, 9-dideoxyforskolin, a forskolin analog which has no effect on adenylate cyclase, did not mimic the synergistic effect of 8CPT and forskolin on PSs in CA2. These results indicate that up-regulation of adenylate cyclase activity and inhibition of adenosine A1 receptor activity synergistically facilitate the neuronal activity in the CA2 area and the effect of adenosine A1 receptor antagonist is via non-cAMP cascade. These data also suggest that acting on adenosine A1 receptors, endogenous and extragenous adenosine/adenosine A1 agonist(s) inhibit neuronal activity through different pathways.     

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Prostaglandin endoperoxide/thromboxane A2 receptor desensitization. Cross-talk with adenylate cyclase in human platelets.

Platelet activation by the prostaglandin endoperoxide (PGH2)/thromboxane (Tx) A2 analog, U46619, involves stimulation of phospholipase (PL) C and an increase in intracellular calcium via distinct receptor subtypes. Agents which stimulate adenylate cyclase inhibit platelet function. We demonstrate that PGH2/TxA2 receptor desensitization is associated with enhanced stimulation of platelet cyclic AMP by the prostacyclin analog, iloprost and by forskolin. Sensitization of adenylate cyclase is mediated via the PGH2/TxA2 receptor subtype which activates PLC, as it is blocked by the specific antagonist, GR32191 (Takahara, K., Murray, R., FitzGerald, G. A., and Fitzgerald, D. J. (1990) J. Biol. Chem. 265, 6838-6844). This effect is not observed in platelets desensitized with thrombin or platelet activating factor and is not mediated by protein kinase C. Prior exposure of platelets to platelet activating factor results in much greater desensitization of PGH2/TxA2-induced aggregation (mean 64%) compared with calcium stimulation (mean 18%), consistent with selective heterologous desensitization of the PLC-linked PGH2/TxA2 receptor subtype. Platelet activation by PGH2/TxA2 is a tightly regulated process, involving both homologous desensitization of at least two receptor subtypes and sensitization of the platelet adenylase cyclase system.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

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.     

Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. Kinetics and concentration dependence

cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylatecyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of cAMP, and cAMP binding to surface receptors and cAMP-induced activation of adenylate cyclase were measured. cAMP could induce maximally 65% loss of binding activity and complete desensitization of cAMP-stimulated adenylate cyclase activity. Half-maximal effects for down-regulation were observed at 50 nM cAMP and for desensitization at 5 nM cAMP. Down-regulation was rapid with half-times of 4, 2.5, and 1 min at 0.1, 1, and 10 microM cAMP, respectively. Similar kinetic data have been reported for desensitization (Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561). Down-regulation and desensitization were not reversible at 0 degrees C. Down-regulation reversed slowly at 20 degrees C with a half-time of about 1 h. Resensitization of adenylate cyclase was biphasic showing half-times of 4 min and about 1 h, respectively; the contribution of the rapidly resensitizing component was diminished when down-regulation of receptors was enhanced. These results suggest that cAMP-induced down-regulation of receptors and desensitization of adenylate cyclase stimulation proceed by at least two steps. One step is rapidly reversible, occurs at low cAMP concentrations, and induces desensitization without down-regulation, while the second step is slowly reversible, requires higher cAMP concentrations, and also induces down-regulation.     

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.     

Enhancement of adenosine A2 and prostaglandin E1 receptor-mediated cAMP generation by prior exposure of Swiss 3T3 fibroblasts to Ca2+-mobilizing receptor agonists or phorbol ester. Activation of protein kinase C triggers increases in the receptor density in cell membranes

Production of cAMP in response to adenosine A2 or prostaglandin E1 receptor stimulation was, but the production induced by a beta-adrenergic agonist or forskolin was not, enhanced by prior exposure of Swiss 3T3 fibroblasts to agonists of Ca2+-mobilizing receptors or phorbol ester for 3 h. The enhancement reflected potentiation of the receptor-coupled activation of adenylate cyclase and the 2-fold increase in the adenosine A2 receptor number in membranes under these conditions. No enhancement was observed, however, when the medium used for the prior exposure was further supplemented with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporin, inhibitors of protein kinase C, neither of which affected the cAMP responses of the nonexposed cells. It is very likely, therefore, that activation of protein kinase C triggers the increase in certain receptor density in membranes, thereby enhancing the receptor-coupled cAMP-generating responses. The physiological significance of such cross-talk between cellular signaling systems is discussed in comparison with similar previous observations.     

Isoproterenol-induced desensitization of adenylate cyclase in human astrocytoma cells. Relation of loss of hormonal responsiveness and decrement in beta-adrenergic receptors.

Incubation of human astrocytoma cells (1321N1) with low concentrations of isoproterenol results in a specific loss of responsiveness to catecholamines as evidenced by a decreased accumulation of cAMP in intact cells, a reduction in isoproterenol-stimulated adenylate cyclase activity, and a decrease in beta-adrenergic receptor density, as measured by the specific binding of 125I-hydroxybenzylpindolol. The kinetics of desensitization suggest the involvement of two different reactions. The initial reaction involves a rapid loss of adenylate cyclase activity with little loss of beta-adrenergic receptors. Subsequently, a slower reaction results in the loss of measurable beta-adrenergic receptors. The degree of loss of both parameters was similar after 24 h of desensitization. It is concluded that the loss of beta-adrenergic receptors is an event that occurs as a result of the initial uncoupling of the beta-receptor-linked adenylate cyclase.     

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.     

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.     

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.     

Role of adenylate cyclase in human T-lymphocyte surface antigen capping

Our recent studies indicated that capping of T3, T4 and T8 surface antigens on human T lymphocytes is augmented by interaction of adenosine with a purinergic receptor. We suggested that the T-cell capping process was mediated by an adenylate cyclase-coupled purinergic receptor that resulted in the generation of cAMP and occupancy of cAMP receptors. The present study was undertaken to examine whether activation of adenylate cyclase in the absence of purinergic stimulation is sufficient to regulate surface antigen capping. Treatment of T lymphocytes with forskolin or cholera toxin caused activation of adenylate cyclase and occupancy of intracellular types I and II regulatory subunits of protein kinase by cAMP, as demonstrated by photoaffinity labeling with [8-3H]N3-cAMP. Such treatment augmented the rate of capping of the T3, T4, and T8 antigens, which resulted in a significant decrement in the elapsed time to half-maximal capping of each antigen. These observations support the proposition that the normal T-lymphocyte capping mechanism of both T3+, T4+ (inducer/helper) and T3+, T8+ (suppressor) subsets can be augmented by activation of adenylate cyclase.     

Pertussis toxin prevents homologous desensitization of adenylate cyclase in cultured renal epithelial cells

The biochemical mechanisms of adenylate cyclase desensitization in arginine vasopressin-responsive epithelial cells remain unclear. Preincubation of cultured rabbit renal cortical collecting tubular cells with arginine vasopressin leads to a 30-100% decline in arginine vasopressin-stimulated adenylate cyclase activity. This loss of adenylate cyclase activity is time- and arginine vasopressin concentration-dependent. Preincubation with arginine vasopressin does not result in significant changes in basal, NaF-, forskolin-, isoproterenol- or cholera toxin-stimulated adenylate cyclase activity. Preincubation of cells with chlorophenylthio-cAMP, forskolin, and cholera toxin does not result in loss of arginine vasopressin-stimulated adenylate cyclase activity. Since products of cyclo-oxygenase inhibit arginine vasopressin action, cells were preincubated with indomethacin. Arginine vasopressin-induced adenylate cyclase desensitization is not reversed by indomethacin. By contrast, incubation with pertussis toxin prevents arginine vasopressin-induced adenylate cycle desensitization. These data demonstrate that arginine vasopressin induces homologous desensitization in membranes from cultured rabbit cortical collecting tubular cells and suggest that this desensitization is mediated, at least in part, by pertussis toxin substrate. These observations provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.