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.     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

Similarities and differences in phorbol ester- and luteinizing-hormone-induced desensitization of rat tumour Leydig-cell adenylate cyclase.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was shown to mimic luteinizing hormone (LH; lutropin) in causing desensitization of LH-mediated cyclic AMP production in tumour Leydig cells. However, there were differences between LH- and TPA-induced desensitization: (1) TPA induced a more rapid effect than LH; (2) adenosine did not inhibit TPA-induced desensitization, whereas it completely inhibited the LH-induced desensitization; (3) adenylate cyclase activity in plasma membranes from TPA-desensitized cells was not decreased, whereas similar preparations from LH-desensitized cells lost their response to LH and to LH plus guanosine 5'-[beta gamma-imido]triphosphate; TPA-, but not LH-, treated cells had a decreased capacity to respond to cholera toxin and forskolin. These results indicate that LH and phorbol esters induce desensitization of adenylate cyclase in rat tumour Leydig cells by different mechanisms.     

Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.     

Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells

The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.     

Bacterial adenylate cyclase increases cyclic AMP and hormone release in pituitary tumor cells

Calmodulin-activated, adenylate cyclase toxin, a virulence factor produced by the human respiratory pathogen Bordetella pertussis, elicits marked accumulation of cyclic AMP in cell lines from rat pituitary tumors. This effect is associated with and apparently responsible for an enhanced release of prolactin and/or growth hormone from GH3, GH4C1 and 235-1 cells. The utility of this novel toxin in probing cyclic AMP-mediated responses is supported by these observations and studies with pertussis and cholera toxins.     

Protein kinase C potentiates isoproterenol-mediated cyclic AMP production without modifying the homologous desensitization process in J774 cells

The J774 murine macrophage cells possess a beta 2-adrenergic receptor coupled to adenylate cyclase, which can be regulated by homologous desensitization. Stimulation of protein kinase C by phorbol esters or oleoyl acetyl glycerol potentiates two-to-threefold the isoproterenol-induced cyclic AMP accumulation. These promoters act at a post-receptor level, since the number and affinity of the beta-adrenergic receptors, measured by use of the hydrophilic ligand [3H]CGP-12177, are not modified. In addition, the effect of cholera toxin is similarly increased and pretreatment of the cells with pertussis toxin prevents the action of phorbol esters. On the other hand, these promoters are ineffective on isoproterenol-induced desensitization and the rates of receptor segregation and recovery remain unchanged. Therefore, protein kinase C modulates the isoproterenol-stimulated adenylate cyclase, whereas it is inactive on the homologous desensitization process.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

Consequences of hormone-induced desensitization of adenylate cyclase in intact cells.

A hypothesis on the role of the hormone-induced desensitization of adenylate cyclase is proposed. It is suggested that the desensitization process could provide the cell with a highly efficient cyclic AMP system for transmitting hormone stimulus without requiring a large energy consumption. Theoretical considerations show that in fact the desensitization phenomenon allows the cyclic AMP system to present a good compromise between the efficiency and economy requirements of the cells.     

Inhibitory effect of clonidine upon adenylate cyclase activity,cyclic AMP production,and insulin release in rat pancreatic islets

Conflicting opinions were recently expressed concerning the possible effect of ₂-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca²⁺), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.     

Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

Galanin inhibits adenylate cyclase of rat brain membranes.

The ubiquitous neuropeptide, galanin, strongly inhibits adenylate cyclase in rat brain membranes. While basal enzyme activity was not altered, galanin from 10(-11) M to 5 x 10(-7) M decreased forskolin- and VIP-stimulated adenylate cyclase with a half-maximal effect being elicited by 0.7 nM neuropeptide and a maximal 80% inhibition of the enzyme activity. The galanin fragments (2-29) and (1-15) dose-dependently inhibited the forskolin-stimulated adenylate cyclase, while the fragments (3-29) and (10-29) were found inactive. These results indicate that the regulatory action of galanin in the central nervous system involves the coupling of galanin receptors to the inhibition of the adenylate cyclase system.     

Evidence for the functional coupling of cyclic AMP in MA-10 mouse Leydig tumour cells

A number of studies have indicated that increased production of steroids can be obtained with doses of tropic hormone which do not result in detectable increases in intracellular cAMP. It has been suggested that this may be a result of compartmentalization or functional coupling of cAMP generated by hormone-receptor interactions to specific steroid producing pathways in the cell. In the present study we have stimulated the MA-10 mouse Leydig tumour cell with hCG, dibutyryl cAMP (dbcAMP) and forskolin to determine if functional coupling of cAMP occurs. Treatment with hCG, dbcAMP and forskolin all resulted in significant increases in the production of progesterone, the major steroid produced in these cells. Stimulation with hCG followed by 2D-PAGE analysis of the proteins resulted in the appearance of two proteins in the 30,000 molecular weight range (pI 6.8 and 6.6) and two in the 25,000-27,000 region (pI 5.9-6.0). Stimulation with dbcAMP or forskolin resulted in the appearance of the same proteins seen with hCG, but also in the appearance of two additional proteins, also having molecular weights of approximately 30,000 (pI 6.3 and 6.1). These data indicate that cAMP generated via hCG stimulation, whilst able to generate similar amounts of progesterone, does not stimulate the synthesis of the same proteins as does cAMP added exogenously or generated through indiscriminate activation of adenylate cyclase activity. Thus, it would appear that the gonadotropin activated pathway generates cAMP which remains functionally compartmentalized within the cell.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.