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.     

Adenovirus type 12 transformation involves loss of beta-adrenergic receptors and isoproterenol responsiveness.

The responsiveness of a growth-regulated rat 3Y1 cell line and five clones of 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 to the catecholamine hormone (-)-isoproterenol was studied. The untransformed cells contained beta-adrenergic receptors characterized by specific binding of the beta-adrenergic receptor antagonist (-)-[3H]dihydroalprenolol, a 9- to 12-fold increase in cyclic AMP production in intact cells after incubation with 10 microM (-)-isoproterenol, and significantly increased adenylate cyclase (ATP pyrophosphatelyase [cyclizing], EC 4.6.1.1) activity in the presence of the hormone. In contrast, (-)-isoproterenol (10 to 100 microM) had no apparent effect on cyclic AMP production or the basal adenylate cyclase activity in the transformed cell lines. Binding studies revealed that untransformed cells contained approximately 19,400 beta-adrenergic receptor sites per cell. Three transformed cell clones tested showed a three- to fourfold loss of beta-adrenergic receptors.     

Down-regulation of gonadotropin and beta-adrenergic receptors by hormones and cyclic AMP

Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.     

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)     

Beta-adrenergic activity of (+/-)-hydroxybenzylisoproterenol in isolated rat fat cells and hepatocytes

The pharmacology of (+/-)-hydroxybenzylisoproterenol with respect to stimulation of cyclic AMP accumulation by isolated rat fat cells and liver cells was examined. (+/-)-Hydroxybenzylisoproterenol was found to be a full agonist and twice as potent as (-)-isoproterenol in liver cells, and equipotent to (-)-isoproterenol in fat cells with regard to stimulating cyclic AMP accumulation. A study of the ability of this catecholamine to stimulate adenylate cyclase activity of broken-cell preparations revealed that (+/-)-hydroxybenzylisoproterenol was equipotent to (-)-isoproterenol in liver cell homogenates, while 3- to 4-fold more potent than (-)-isoproterenol in fat cell ghost membranes. (+/-)-Hydroxybenzylisoproterenol was also found to be as potent as (-)-isoproterenol in stimulating cyclase activity of S49 mouse lymphoma cell membranes. Competition studies of specific [125I]iodohydroxybenzylpindolol binding to liver cell membranes revealed a Kd of 10 nM for (+/-)-hydroxybenzylisoproterenol and 25 nM for (-)-isoproterenol binding to the liver beta-adrenergic receptor. Competition studies of specific (-)-[3H]dihydroalprenolol binding to fat cell membranes indicated a similar affinity of these sites for both (+/-)-hydroxybenzylisoproterenol and (-)-isoproterenol. The guanyl nucleotide Gpp(NH)p induced a shift in the curve for competition of (-)-[3H]dihydroalprenolol binding by (-)-isoproterenol to the right, but failed to do so when (+/-)-hydroxybenzylisoproterenol was the competing agonist. Properties of (+/-)-[3H]hydroxybenzylisoproterenol binding to fat cell or liver cell membranes were inconsistent with those expected of adenylate cyclase coupled beta-adrenergic receptors.     

Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin.

Rat C6-2B astrocytoma cells responded to cholera toxin treatment with an 8-fold increase in intracellular cyclic AMP concentrations. Cyclic AMP levels began to rise 60--90 minutes after addition of the toxin and reached maximal concentrations in 3 hours. Cells exposed to cholera toxin and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), displayed an increase in cyclic AMP of 15-fold. The peak isoproterenol response was reduced 80--90% in cells previously treated with cholera toxin. Cholera toxin-induced refractoriness was time dependent and was not altered by concurrent treatment with propranolol. Prolonged exposure of the cells to isoproterenol reduced the cyclic AMP response to cholera toxin by 80%. MIX augmented both cholera toxin-induced refractoriness and isoproterenol-induced refractoriness. Cycloheximide inhibited the full development of refractoriness to both cholera toxin and isoproterenol. These results indicate that C6-2B cell refractoriness to cholera toxin is mediated by cyclic AMP and requires new protein synthesis. Refractoriness in C6-2B cells does not appear to be agonist-specific and probably involves a common locus of action on adenylate cyclase beyond that of the membrane receptors for cholera toxin and isoproterenol.     

Receptor-associated changes of the catecholamine-sensitive adenylate cyclase in glioma cells doubly transformed with Moloney sarcoma virus

A doubly transformed rat glioma cell line, designated C6V-1, was obtained from rat glioma C6 cells by infection with a rat-adapted variant of Moloney sarcoma virus (MSV-M-os). The C6V-1 cells show karyotypic changes in chromosome number (43) and structure, while C6 cells possess a normal male karyotype. C6V-1 and C6 cells were employed for characterization of a receptor-adenylate cyclase system of the surface membrane. C6V-1 cells showed lower adenylate cyclase activity than that of C6 cells, though the apparent Km for ATP in both types of cells was the same. The maximal stimulation of adenylate cyclase by isoproterenol was significantly reduced, and Kact for isoproterenol was approximately 18-fold lower in C6V-1 cells. When the concentration of beta-adrenergic receptors was measured by various concentrations of [3H] dihydroalprenolol (DHA), the maximal binding sites of C6 and C6V-1 cells were 760 and 230 fmol/mg protein, respectively, without any changes in the association constant for DHA. The concentration of isoproterenol required for 50% displacement of the [3H] DHA binding (Kd) was the same (around 1.5 X 10(-6)M) in both cells, measured in the presence of GTP. Thus the 19-fold drop in the Kd/Kact ratio in C6V-1 cells suggests an incomplete coupling of beta-receptors to adenylate cyclase. Cyclic AMP phosphodiesterase activity and cAMP content in C6V-1 were lower than in C6 cells. Mitochondrial monoamine oxidase and cytosomal enolase activities, however, were somewhat higher in C6V-1 cells. The results indicate that a set of changes in the receptors and in the cyclic AMP system of C6V-1 is one of the specific alterations by transformation, even though those may not be the cause of cell transformation.     

Cell cycle changes in the adenylate cyclase of C6 glioma cells

The adenylate cyclase of C6 glioma cell cultures was characterized for sensitivity to the beta-adrenergic agonist isoproterenol, as well as fluoride, and GTP as a function of the cell cycle. The mitotic phase of the cell cycle was emphasized because both the basal cellular cyclic AMP level and the intact C6 cell's capacity to accumulate cyclic AMP in response to isoproterenol decreased during mitosis. Basal and stimulated adenylate cyclase activities in mitotic cells were decreased relative to the enzyme activities in the G1, S, and G2 phases of the cell cycle. Analysis of the beta-adrenergic receptor using the radioligand(-)[3H]dihydroalprenolol showed that neither ligand affinity nor receptor density changed during the cell cycle, indicating that the reduced adenylate cyclase activity of the mitotic C6 cell was not caused by alterations in this hormone receptor. The reduction in the mitotic cell's basal adenylate cyclase activity was more prominent than the decrease in isoproterenol-, fluoride, or GTP-stimulated activities suggesting that the effectiveness of these enzymes activators (i.e., the efficiency of the coupling mechanism) was not attenuated during mitosis. These studies indicate that the intrinsic catalytic capacity (not the beta-adrenergic receptor or the coupling mechanism) of the C6 adenylate cyclase complex is reduced during mitosis and contributes to the mitotic cell's inability to accumulate and maintain the cyclic AMP concentration at the interphase level.     

Properties of beta-adrenergic receptors in untreated and butyrate-treated Hela cells.

HeLa cells contain receptors on their surface which are beta-adrenergic in nature. The binding of (-)-[3H]dihydroalprenolol is rapid, reversible, stereospecific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (-)-isoproterenol greater than (-)-epinephrine greater than (-)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5'-ylimidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (-)-isoproterenol and Gpp(NH)p leads to approximately additive rather than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5mM sodium butyrate there is an increase in the number of beta-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (-)-isoproterenol to activate adenylate cyclase. Other properties of the beta-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

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.     

Reduction in beta-adrenergic response of cultured glioma cells following depletion of intracellular GTP.

1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected. 2. The adenosine 3':5'-monophosphate (cyclic AMP) response of C6 glioma cells to the beta-adrenergic stimulant, (+/-)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (+/-)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid. 3. The adenylate cyclase response to (+/-)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP. 4. The adenylate cyclase response to (+/-)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic-acid-treated C6 cells gave similar adenylate cyclase responses to (+/-)isoprenaline in the presence of GTP. 5. It is concluded that mycophenolic acid may depress the beta-adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.     

Alcohol potentiation of isoproterenol-stimulated cyclic AMP accumulation in rat parotid

The ability of beta-adrenergic agonists to elevate rat parotid cyclic AMP concentrations is potentiated greatly by certain organic solvents. Propanol was found to be more effective than other tested solvents. Propanol stimulated adenylate cyclase and inhibited low Km cyclic AMP phosphodiesterase activities however the magnitude of effect upon these enzyme activities probably does not account for the potentiation of cyclic AMP accumulation observed in intact cells.     

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.     

Rapid agonist-induced decrease of 125I-pindolol binding to beta-adrenergic receptors. Relationship to desensitization of cyclic AMP accumulation in intact heart cells

Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment.     

Cholinergic inhibition of catecholamine-stimulable cyclic AMP accumulation in murine atria.

Carbachol antagonizes isoproterenol-stimulable cyclic AMP accumulation in mouse atria by direct activation of cardiac muscarinic receptors. Inhibition by carbachol occurs rapidly and is completely reversed when the drug is removed. Neither nitroprusside nor 8-bromo-cyclic GMP mimics the actions of carbachol and low concentrations of carbachol block cyclic AMP accumulation without increasing the intracellular cyclic GMP content. Carbachol does not block cyclic AMP accumulation by activating phosphodiesterase since it is fully effective in the face of marked phosphodiesterase inhibition, nor does it appear to inhibit the catalytic activity of adenylate cyclase since it does not decrease either basal or cholera toxin-stimulated cyclic AMP accumulation. The interaction between carbachol and isoproterenol is not competitive, since cholinergic inhibition cannot be surmounted by increasing concentrations of isoproterenol. The site of muscarinic action therefore appears to involve the mechanisms coupling the hormone-receptor complex to adenylate cyclase. This site is distinct from that of cholera toxin action since there is no antagonism between the effects of cholera toxin and carbachol on cyclic AMP metabolism in the atrium.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

Studies of cAMP metabolism in cultured hepatoma cells: presence of functional adenylate cyclase despite low cAMP content and lack of hormonal responsiveness.

The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined. The concentration of cAMP in BRL cells (approximately 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (approximately 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. binding studies with [125 I]-iodohydroxybenzylpindolol, a specific beta-adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 beta-adrenergic receptors whereas BRL cells contain 2-5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phosphodiesterase activities.     

Down-regulation of protein kinase C in rat glioma C6 cells: effects on the beta-adrenergic receptor-coupled adenylate cyclase

Continuous exposure of rat glioma C6 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a time and dose dependent loss of [3H]phorbol dibutyrate binding sites and protein kinase C activity. Thus, by 24 h, the cells were essentially depleted of protein kinase C activity. In agreement with previous studies, TPA treatment caused a reduction in isoproterenol-stimulated adenylate cyclase activity and a sequestration of beta-adrenergic receptors. Cells were treated with TPA for 24-48 h to completely down-regulate protein kinase C and then exposed to isoproterenol. Agonist-mediated desensitization of adenylate cyclase and sequestration of beta-adrenergic receptors occurred at similar rates in control and TPA-treated cells. In addition, agonist-mediated down-regulation of beta-adrenergic receptors was not impaired by the absence of protein kinase C activity. Although both agonists and phorbol esters cause desensitization of the beta-adrenergic receptor-coupled adenylate cyclase, agonist-mediated events can occur independently of protein kinase C.     

Enhancement of hormonal stimulation in intact cells. Potentiation of GTP-dependent activation of adenylate cyclase.

The ability of prostaglandin E1 (PGE1) and cholera toxin to increase cyclic AMP levels is potentiated 6-fold when normal rat kidney (NRK) cells are treated with picolinic acid or histidinol, or grown in isoleucine-deficient medium. The response to (-)-isoproterenol is increased 2-fold in NRK cells treated with picolinic acid but not in cells subjected to isoleucine deprivation. The increase in agonist responsiveness is time-dependent, reaches its maximum at 40 h, and is quickly reversed following removal of picolinic acid or addition of medium with normal amounts of isoleucine. The cholera toxin response is also increased about 7-fold in simian virus 40-transformed NRK cells and Moloney sarcoma virus-transformed NRK cells treated with picolinic acid. GTP-stimulated, but not fluoride-stimulated, adenylate cyclase activities are increased in membranes from NRK cells treated with picolinic acid or starved for isoleucine, indicating that the increased response is due, at least in part, to a specific potentiation of GTP-dependent functions of the adenylate cyclase system. The results demonstrate that GTP-dependent events in hormonal stimulation of adenylate cyclase can be altered in intact cells to modulate hormonal enhancement of cyclic AMP production.