Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endothelial cell adenylate cyclase: activation by catecholamines and prostaglandin I2+

Following incubation of intact vascular endothelial cells with 1 mM 3-isobutyl-1-methylxanthine, and isoproterenol or PGI₂, cyclic AMP levels increased 4- and 3-fold, respectively. Isoproterenol-stimulated adenylate cyclase activity of cell lysates was selectively inhibited by the β-adrenergic blocking agent propranolol. Catecholamines stimulated adenylate cyclase with the potency series: isoproterenol > epinephrine > norepinephrine. Prostaglandin did not stimulate adenylate cyclase activity in cell lysates, even in the presence of guanine nucleotides or following preincubation of the intact cells with prostaglandins prior to freeze-thaw lysis.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells.

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Calcium dependence of beta-adrenoceptor mediated cyclic AMP accumulation in human lymphocytes

In intact human lymphocytes, cyclic AMP accumulation in response to isoproterenol was inhibited by 5 mM EDTA, by deletion of calcium ions from the medium and by 1 mM lanthanum chloride, but not by 1 microM verapamil or by 10 microM nifedipine. A23187 caused a modest increase in cyclic AMP content. Exposure of lymphocytes to 5 microM 1-isoproterenol desensitized the cells to subsequent beta-adrenergic stimulation, reducing cyclic AMP accumulation. With higher concentrations of 1-isoproterenol (50 microM), receptor density was reduced as well. None of the above agents attenuated losses in agonist-stimulated cyclic AMP accumulation induced by treatment with 5 microM isoproterenol for 90 min. These data suggest that calcium ions, both those present in the extracellular medium and those bound to the plasma membrane, are required for isoproterenol-stimulation of adenylate cyclase. In addition, it appears that neither the presence of extracellular calcium ions nor full activation of adenylate cyclase are required for desensitization.     

Lipolysis and beta-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats

Adipocytes from spontaneously hypertensive rats demonstrated a blunted lipolytic response to isoproterenol and dibutyryl cyclic AMP. (-)-[3H]Dihydroalprenolol binding was examined in adipocytes from normotensive and spontaneously hypertensive rats. Increasing concentrations of isoproterenol decreased total (-)-[3H]dihydroalprenolol binding to intact cells from normotensive rats, and the efficacy of competition was decreased in adipocytes from spontaneously hypertensive rats. Scatchard analysis indicated that the number of (-)-[3H]dihydroalprenolol binding sites and the affinity of dihydroalprenolol binding were comparable between normotensive and spontaneously hypertensive rats. Isoproterenol- and Gpp(NH)p-stimulated adenylate cyclase activity was consistently depressed in adipocyte membranes from spontaneously hypertensive rats as compared to normotensive rats. No difference in fluoride-stimulated adenylate cyclase activity was observed. The blunted lipolytic and cyclic AMP response to isoproterenol in these cells suggest a postreceptor lesion of the lipolytic pathway (possibly the guanine nucleotide regulatory protein) in adipocytes from spontaneously hypertensive rats. The blunted lipolytic response to dibutyryl cyclic AMP suggests defective regulation of lipolytic enzymes at the protein kinase-hormone-sensitive lipase level.     

Large potentiation of agonist response in intact cells is produced by increases only in GTP-dependent adenylate cyclase activity

Treatment of cultured SV40-transformed normal rat kidney cells with the drug, 2-pyridine carboxylic acid, results in a pronounced potentiation in the ability of isoproterenol, prostaglandin E1, and cholera toxin to elevate cyclic AMP levels. With isoproterenol, the initial rate of cyclic AMP accumulation and the maximum cyclic AMP attainable are increased, and also the time of maximum cyclic AMP is prolonged. GTP-dependent adenylate cyclase activities are potentiated in crude membranes from the treated cells, but no evidence for alterations in cyclic nucleotide phosphodiesterase or release of cyclic AMP into the medium could be demonstrated. Results show that augmented adenylate cyclase activity alone, without changes in phosphodiesterase, can lead to dramatic alterations in cyclic AMP accumulation in response to cyclase agonists.     

Decreased Cyclic AMP Degradation in NG 108-15 Neuroblastoma × Glioma Hybrid Cells and S49 Lymphoma Cells Chronically Treated with Drugs that Inhibit Adenylate Cyclase

The increase in hormone-stimulated cyclic AMP accumulation observed in a variety of intact cells after chronic pretreatment with drugs that inhibit adenylate cyclase activity has been attributed to an increase in adenylate cyclase activity following withdrawal of the inhibitory drug. In NG 108-15 mouse neuroblastoma X rat glioma hybrid cells (NG cells) chronically treated with the muscarinic cholinergic agonist carbachol, we have found a significant decrease in the apparent degradation rate constant for cyclic AMP, in addition to an increase in the prostaglandin E1 (PGE1)-stimulated cyclic AMP synthesis rate in intact cells. In carbachol-pretreated NG cells that were stimulated with a maximally effective dose of PGE1, and that accumulated steady-state cyclic AMP concentrations fourfold or more higher than in control cells, the apparent rate constant for degradation was about 53% lower than the value for control cells. In carbachol-pretreated cells stimulated with a submaximal dose of PGE1 to yield a steady-state cyclic AMP concentration comparable to control cells, the apparent rate constant was 31% lower than the value for control cells. In S49 mouse lymphoma cells (S49 cells) chronically treated with an analog of the inhibitory agonist somatostatin, the first-order rate constant for cyclic AMP degradation in intact cells following isoproterenol stimulation was 29% lower than the value for control cells. Despite these changes in the kinetics of cyclic AMP degradation in intact NG cells and S49 cells, there was either no change or a minimal change (less than 10%) in phosphodiesterase activities assayed in extracts of cells chronically exposed to inhibitory drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTP gamma S) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.     

Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells.

The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.     

Predominance of beta-adrenergic over alpha-adrenergic receptor functions involved in phosphorylase activation in liver cells of cholestatic rats

Hepatocytes isolated from normal and cholestatic rats responded to adrenergic agonists and antagonists in a quite different manner. Much greater activation of glycogen phosphorylase was caused by phenylephrine, an alpha-agonist, than by isoproterenol, a beta-agonist, in normal rat hepatocytes, and vice versa in the cholestatic rat cells. Epinephrine activation of phosphorylase was antagonized more efficiently by phenoxybenzamine, an alpha-antagonist, than by propranolol, a beta-antagonist, in normal rats, whereas it was antagonized totally by propranolol but only partially by phenoxybenzamine in cholestatic rat hepatocytes. The number of alpha-adrenergic receptors, measured by [3H]prazosin binding to membranes, as well as alpha-receptor-mediated increases in 32Pi incorporation into phosphatidylinositol and in 45Ca efflux, were reduced in hepatocytes after induction of cholestasis. The reduction of these parameters of alpha-receptor-linked functions was associated with the reciprocal increase in the number of beta-receptors and enhancement of beta-receptor-mediated accumulation of cyclic AMP in cholestatic rat hepatocytes. The affinity of epinephrine for beta-receptors was higher in cholestatic rat cells than in normal rat cells; this difference in affinity was abolished by the addition of guanylylimidodiphosphate, indicating that induction of cholestasis rendered hepatic beta-receptors more tightly coupled to the GTP-binding protein. Thus, the cascade reactions arising from beta-receptors are predominant over those from alpha-receptors, eventually leading to glycogen breakdown in cholestatic rat hepatocytes, principally because of not only the elevated beta to alpha ratio of the membrane receptor density but also the tight coupling of beta-receptors to the adenylate cyclase system via the guanine nucleotide regulatory protein.     

Correction by dexamethasone treatment of the altered lipolytic cascade induced by adrenalectomy in rat adipocytes

The ability of glucocorticoid-treatment to reverse the metabolic alterations caused by adrenalectomy in rat adipocytes was studied. Correction of the enhanced adenosine antilipolytic effect and of the defect in lipolysis, cyclic AMP and adenylate cyclase responsiveness to guanine nucleotides were all achieved after a 24 h dexamethasone treatment, whereas correction of the defect in beta-adrenoceptor-density required a 48 h treatment. The latter treatment, however, failed to reverse the defect in both the adenylate cyclase catalytic activity and protein content per fat cell. These different kinetics of restoration indicate that correction by dexamethasone of the defective cyclic AMP and lipolytic responses on one hand and of the guanine nucleotide control of adenylate cyclase on the other one are two related phenomenoms.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but not adenosine 5'-monosulfate significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Evidence that metabolically active synaptosomes lack functional cyclic AMP-dependent protein kinase.

Cyclic adenosine monophosphate (cAMP)-mediated signal transduction was evaluated in synaptosomes prepared from rat brain cortex. Adenylate cyclase was responsive to known adenylate cyclase stimulators including peptides (CRH and VIP), catecholamines (norepinephrine and isoproterenol) and ligands that directly stimulate adenylate cyclase (forskolin). Cyclic AMP accumulation also increased approximately 2 to 3-fold, but none of the agonists was able significantly to activate cyclic AMP-dependent protein kinase (A-kinase) in cortical synaptosomes. However, in parallel studies with slices prepared from rat brain cortex, adenylate cyclase activity, cAMP accumulation and A-kinase activity were all stimulated by CRH, VIP, norepinephrine, isoproterenol and forskolin. These data suggest that, in intact synaptosomes, either the cellular machinery which facilitates binding of cAMP to the regulatory subunit of A-kinase is missing or the cAMP produced by adenylate cyclase is not accessible to A-kinase.     

Pertussis toxin blocks somatostatin's inhibition of stimulated cyclic AMP accumulation in anterior pituitary tumor cells

Somatostatin inhibits both forskolin and (-) isoproterenol-stimulated cyclic AMP accumulation in AtT-20 cells. Pretreatment of these cells with pertussis toxin prevents somatostatin's inhibitory effects on cyclic AMP production. This pretreatment also enhances the cyclic AMP response to forskolin and (-) isoproterenol without affecting basal cyclic AMP levels. The blockade of somatostatin's inhibitory effect was dependent both on the time of preincubation and concentration of pertussis toxin used. The rise in forskolin-stimulated cyclic AMP formation following pertussis toxin treatment preceded the blockade of somatostatin's inhibitory actions. The results suggest that somatostatin acts through an inhibitory guanine nucleotide regulatory protein to affect adenylate cyclase activity.     

Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase.

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.