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.     

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

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

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.     

Gi affects the agonist-binding properties of beta-adrenoceptors in the presence of Gs

Pertussis-toxin-catalyzed ADP-ribosylation of Gi in S49 membranes, but not in S49AC- membranes, which lack Gs, induces a threefold reduction of isoproterenol affinity to the beta-adrenoceptors. A similar treatment of turkey erythrocyte membranes, which are devoid of functional Gi, has no effect on beta-agonist affinity to their beta-adrenoceptors. Non-hydrolyzable analogs such as GTP[S] induce a larger decrease in beta-adrenoceptor affinity in S49 cells towards the agonist isoproterenol as compared to pertussis-toxin-catalyzed ADP-ribosylation of Gi. These results suggest that Gi affects beta-adrenoceptor affinity to its agonist and that this interaction requires the presence of Gs. It seems, therefore, that Gi physically interacts with Gs to exert its effects on the receptor and probably on adenylate cyclase as well. Our ability to detect (a) the effect of pertussis-toxin-catalyzed ADP-ribosylation in S49 cells on beta-agonist affinity and (b) the quantitative difference between the effect of pertussis toxin (approx. threefold) and GTP[S] (fivefold to sevenfold) depends on the use of a simple but rigorous method to study in detail the affinity of beta-agonists to their receptors. This method seems to be superior to the analysis of displacement curves as a means to examine receptor-ligand interactions.     

Agonist regulation of beta-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants

Agonist-promoted down-regulation of beta-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of beta-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 +/- 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the beta-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gs alpha (H21a, unc, cyc-) and a single variant lacking cAMP-dependent protein kinase activity (kin-). H21a (Gs alpha coupled to receptor, but not to adenylate cyclase), unc (Gs alpha uncoupled from receptor), and cyc- (lacking Gs alpha) variants accumulated cAMP and down-regulated beta AR mRNA in response to forskolin. In unc and cyc- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of beta-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gs alpha (cyc-) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.     

Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

Catecholamine-Sensitive Adenylate Cyclase in the White Perch (Roccus americanus) Retina: Evidence for β-Adrenergic and Dopamine Receptors Linked to Adenylate Cyclase

We have examined the catecholamine-sensitive adenylate cyclase in the retina of the white perch (Roccus americanus). Both dopamine and the beta-adrenergic agonist isoproterenol stimulate cyclic AMP accumulation in this retina, but serotonin, an indoleamine, and phenylephrine, an alpha-adrenergic agonist, had no effect. The stimulation of adenylate cyclase by isoproterenol is more potent and effective than that of dopamine. The effects of dopamine and isoproterenol are mediated via independent dopamine and beta-adrenergic receptors. Haloperidol, a dopamine antagonist, blocks the stimulatory effect of dopamine but not of isoproterenol. Conversely, propranolol, a beta-adrenergic antagonist, blocks the stimulatory effect of isoproterenol but not of dopamine. The effects of dopamine and isoproterenol are not additive. In fractions of purified horizontal cells we found evidence for dopamine receptors linked to adenylate cyclase but did not find evidence for the presence of cyclase coupled beta-adrenergic receptors. The cellular location of the beta-adrenergic receptors is unknown. Our findings demonstrate the existence of both beta-adrenergic and dopamine receptors coupled to adenylate cyclase in the white perch retina. However, we did not find either epinephrine or norepinephrine, endogenous ligands of the beta-receptor, to be present in retinal extracts subjected to HPLC.     

Pertussis toxin treatment in vivo is associated with a decline in G-protein beta-subunits

The effects of pertussis toxin on the steady-state levels of G-protein alpha- and beta-subunits were investigated both in vitro and in vivo. The steady-state level Go alpha, a major substrate for pertussis toxin-catalyzed ADP-ribosylation, was unaltered by pertussis toxin treatment for periods up to 100 h for 3T3-L1 cells in culture or up to 3 days in vivo. In 3T3-L1 cells pertussis toxin treatment did not alter levels of Gs alpha-subunits; in S49 cells the level of Gs alpha-subunits declined moderately following by pertussis toxin treatment. The steady-state levels of G beta-subunits, in contrast, were found to decline to less than 50% of the normal cellular complement following pertussis toxin treatment in vitro and in vivo. Inhibitory control of adenylate cyclase, pertussis toxin-catalyzed ADP-ribosylation of Gi alpha and Go alpha, and the GTP-dependent shift in agonist-specific binding to beta-adrenergic receptors were attenuated or abolished within 5 h of pertussis toxin treatment, representing "early" effects of the toxin. Stimulatory regulation of adenylate cyclase, in contrast, displayed a progressive enhancement that was first observed 4 h after pertussis toxin treatment, increasing thereafter up until 100 h, the last time point measured. This progressive enhancement of the stimulatory pathway of adenylate cyclase was not manifest at the level of stimulatory receptors, since the Kd and Bmax for one such receptor, the beta-adrenergic receptor, were shown to be unaltered in toxin-treated cells. Furthermore, the potentiation of stimulation of adenylate cyclase was observed in cells stimulated by the beta-adrenergic agonist isoproterenol and PGE1 alike. The progressive enhancement of the stimulatory pathway correlated best with the decline in G beta-subunit levels that occurs following pertussis intoxication. The changes in both of these parameters occur "late" (12-48 h), as compared to the early events that occur within 5 h. Pertussis toxin action appears to be composed of two, temporally distinct, groups of effects. Pertussis toxin-catalyzed ADP-ribosylation of G alpha-subunits, attenuation of the inhibitory regulation of adenylate cyclase, and attenuation of the ability of GTP to induce an agonist-specific shift in receptor affinity are members of the early group of effects. The second group of late effects includes the decline in G beta-subunit levels and the progressive enhancement of the stimulatory pathway of adenylate cyclase. This enhanced stimulatory control at these later times cannot be explained by the attenuation of the inhibitory pathway occurring early, but rather appears as G beta-subunit levels decline.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.     

Prolonged activation of inhibitory somatostatin receptors increases adenylate cyclase activity in wild-type and Gs alpha-deficient (cyc-) S49 mouse lymphoma cells.

Many cells develop enhanced adenylate cyclase activity after prolonged exposure to drugs that acutely inhibit the enzyme and it has been suggested that this adaptation may be due to an increase in Gs alpha. We have treated wild-type and Gs alpha-deficient cyc- S49 mouse lymphoma cells with a stable analogue (SMS 201-995) of the inhibitory agonist somatostatin. After incubation with SMS for 24 h, the forskolin-stimulated cAMP synthetic rate in intact cyc- cells was increased by 76%, similar to the increase found in the wild-type cells. Forskolin-stimulated adenylate cyclase activity in the presence of Mn2+ was also increased in membranes prepared from SMS-treated cyc- cells; however, guanine nucleotide-mediated inhibition of adenylate cyclase activity was not changed despite a small decrease in inhibitory Gi alpha subunits detected by immunoblotting. Pretreatment of cyc- cells with pertussis toxin prevented SMS from inducing the enhancement of forskolin-stimulated cAMP accumulation in intact cells. After chronic incubation of cyc- cells with SMS, exposure to N-ethylmaleimide, which abolished receptor-mediated inhibition of cAMP accumulation, did not attenuate the enhanced rate of forskolin-stimulated cAMP synthesis compared to N-ethylmaleimide-treated controls. These results with cyc- cells demonstrate that an adaptive increase in adenylate cyclase activity induced by chronic treatment with an inhibitory drug can occur in the absence of expression of Gs alpha.     

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

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

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.     

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.     

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.     

Cholesterol modulation of membrane fluidity and VIP receptor/effector system in rat prostatic epithelial cells.

Treatment of rat prostatic epithelial cells with cholesteryl hemisuccinate (ChH) resulted in a time- and dose-dependent inhibition of the stimulatory effect of the neuropeptide vasoactive intestinal peptide (VIP) on cyclic AMP accumulation, with a 40% decrease in the response to a maximally effective VIP concentration. Cell treatment with ChH led also to a similar blocking of isoproterenol (a beta-adrenergic agonist) action but did not modify forskolin (which is assumed to act directly on the catalytic unit of adenylate cyclase) activity upon cyclic AMP levels. The levels of the transduction protein Gs were similar in membranes from both control and ChH-treated cells as suggested by experiments on cholera toxin-catalyzed ADP-ribosylation. The inhibitory effect of ChH was accompanied by an increase of membrane microviscosity as estimated by measurements of fluorescence polarization. Experiments on VIP binding indicated that increasing cholesterol concentration in the plasma membrane led to a higher VIP binding capacity without changes in the affinity of VIP receptors. These data suggest that membrane cholesterol incorporation diminishes the coupling efficiency between adenylate cyclase and the VIP-receptor complex or other receptor systems (i.e., desensitization) due to an increase of plasma membrane rigidity.     

Increase in Gs and cyclic AMP generation in HIT cells. Evidence that the 45-kDa alpha-subunit of Gs has greater functional activity than the 52-kDa alpha-subunit

Cyclic AMP accumulation in response to forskolin, cholera toxin, or isoproterenol is dramatically increased in HIT T-15 cells, a clonal cell line of Syrian hamster pancreatic islet beta cells, as a function of passage number. Forskolin and cholera toxin elevate cyclic AMP levels 5- to 10-fold higher in later passages (87-100) than in earlier passages (70-80). A similar phenomenon is observed with isoproterenol (10 microM) which increases cyclic AMP levels 56-fold in older HIT cells (passage 94), whereas only marginally stimulating cyclic AMP production in younger cells (passage 70-82). To determine whether a change in the stimulatory or inhibitory guanine nucleotide regulatory proteins, Gs or Gi, was responsible for these observations, ADP-ribosylation of HIT cell membranes with cholera toxin and pertussis toxin was examined. All passages contained two cholera toxin substrates at 52 and 45 kDa. The amount of 52 kDa did not appear to change with passage number, but the amount of 45 kDa increased in the later passages (89 and 94). The ratio of 45 to 52 kDa cholera toxin substrate, as determined by densitometric analysis, increased from 0.1 in passages 70, 75, and 82 to 0.45 at passage 89. No passage related changes in a 40-kDa pertussis toxin substrate were observed. An increase in the amount of the 45-kDa alpha-subunit of Gs was confirmed on immunoblots using antisera specific for the alpha-subunits of Gs. The amount of functional Gs present in various HIT cell passages was examined by determining the extent to which extracts from HIT cell membranes reconstituted guanine nucleotide-sensitive adenylyl cyclase in S49 cyc- membranes. Extracts derived from passage 94 reconstituted three to four times more adenylyl cyclase activity in cyc- membranes than extracts from passages 70, 75, and 82. These data indicate that an increase in functional Gs in later passages may be the underlying cause for the increased responsiveness to isoproterenol and forskolin in later passages. These data also suggest that functional differences exist between the Gs alpha-subunits, with the smaller 45-kDa subunit being more efficacious in coupling to cyclic AMP synthesis than the larger 52-kDa subunit. This is a departure from the commonly held view that the two subunits have similar efficacies in stimulating adenylyl cyclase.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additive effect of VIP or isoproterenol on forskolin-stimulated cyclic AMP accumulation in rat prostatic epithelial cells

The effects of forskolin alone or in combination with vasoactive intestinal peptide (VIP) and the beta-adrenergic agonist isoproterenol on cyclic AMP accumulation in epithelial cells of rat ventral prostate were examined. Forskolin stimulated cyclic AMP in a time- and temperature-dependent manner. At 15 degrees C, forskolin behaved as a highly potent and relatively efficient stimulatory agent. The combination of forskolin with maximal doses of VIP or isoproterenol were purely additive. These results suggest that forskolin might act directly upon the catalytic subunit of adenylate cyclase in this particular class of cells.     

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.