Regulation of GH3 pituitary tumour-cell adenylate cyclase activity by activators of protein kinase C.

The influence of protein kinase C (PKC) activation on cyclic AMP production in GH3 cells has been studied. The stimulation of cyclic AMP accumulation induced by forskolin and cholera toxin was potentiated by 4 beta-phorbol 12,13-dibutyrate (PDBu). Moreover, PDBu, which causes attenuation of the maximal response to vasoactive intestinal polypeptide (VIP), also induced a small right shift in the dose-response curve for VIP-induced cyclic AMP accumulation. PDBu-stimulated cyclic AMP accumulation was unaffected by pretreatment of cells with pertussis toxin or the inhibitory muscarinic agonist, oxotremorine. PDBu stimulation of adenylate cyclase activity required the presence of a cytosolic factor which appeared to translocate to the plasma membrane in response to the phorbol ester. The diacylglycerol-generating agents thyroliberin, bombesin and bacterial phospholipase C each stimulated cyclic AMP accumulation, but, unlike PDBu, did not attenuate the stimulation induced by VIP. These results suggest that PKC affects at least two components of the adenylate cyclase complex. Stimulation of cyclic AMP accumulation is probably due to modification of the catalytic subunit, whereas attenuation of VIP-stimulated cyclic AMP accumulation appears to be due to the phosphorylation of a different site, which may be the VIP receptor.     

Phorbol 12,13-Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat

Phorbol 12,13-dibutyrate (PDBu) increased the production of 3,4-dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half-maximal at a concentration of approximately 0.1 microM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+-free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8-Br-cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 microM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion.     

Regulation of GH3-cell function via adenosine A1 receptors. Inhibition of prolactin release, cyclic AMP production and inositol phosphate generation.

We examined the mechanism by which adenosine inhibits prolactin secretion from GH3 cells, a rat pituitary tumour line. Prolactin release is enhanced by vasoactive intestinal peptide (VIP), which increases cyclic AMP, and by thyrotropin-releasing hormone (TRH), which increases inositol phosphates (IPx). Analogues of adenosine decreased prolactin release, VIP-stimulated cyclic AMP accumulation and TRH-stimulated inositol phospholipid hydrolysis and IPx generation. Inhibition of InsP3 production by R-N6-phenylisopropyladenosine (R-PIA) was rapid (15 s) and was not affected by the addition of forskolin or the removal of external Ca2+. Addition of adenosine deaminase or the potent adenosine-receptor antagonist, BW-A1433U, enhanced the accumulation of cyclic AMP by VIP, indicating that endogenously produced adenosine tonically inhibits adenylate cyclase. The potency order of adenosine analogues for inhibition of cyclic AMP and IPx responses (measured in the presence of adenosine deaminase) was N6-cyclopentyladenosine greater than R-PIA greater than 5'-N-ethylcarboxamidoadenosine. This rank order indicates that inhibitions of both cyclic AMP and InsP3 production are mediated by adenosine A1 receptors. Responses to R-PIA were blocked by BW-A1433U (1 microM) or by pretreatment of cells with pertussis toxin. A greater amount of toxin was required to eliminate the effect of R-PIA on inositol phosphate than on cyclic AMP accumulation. These data indicate that adenosine, in addition to inhibiting cyclic AMP accumulation, decreases IPx production in GH3 cells, possibly by directly inhibiting phosphoinositide hydrolysis.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

The vasoactive intestinal peptide receptor-effector system is developmentally regulated in rat jejuno-ileal epithelial cells

The specific binding of vasoactive intestinal peptide (VIP) to its specific receptors as well as the stimulatory effect of the neuropeptide on cyclic AMP accumulation were studied in jejuno-ileal epithelial cells from 14-, 20- and 60-day-old rats. The potency and specificity of the VIP receptor-effector system did not vary during development. However, the concentration of VIP receptors and the efficiency of VIP stimulation of cyclic AMP generation increased from suckling to adult conditions, and VIP levels in jejuno-ileal tissue followed a parallel course.     

Ileal vasoactive intestinal peptide (VIP) levels and VIP receptor/effector system in ileal epithelial cells after colectomy in the rat

The interaction of VIP (binding to specific receptors and stimulation of cyclic AMP accumulation) with ileal epithelial cells and the levels of the neuropeptide in the ileal segment were determined after colectomy (removal of cecum and colon followed by ileorectal anastomosis) in the rat. The number of VIP receptors (but not the affinity) and the efficiency (but not the potency) of the neuropeptide upon stimulation of cyclic AMP accumulation in ileal epithelial cells increased 21 (but not 7) days after colectomy, whereas VIP ileal levels followed an inverse pattern. These changes could be interpreted in terms of a consequence or a cause of some of the phenomena that appear after colectomy, i.e., chronic watery diarrhea.     

α2-Adrenoceptors Mediate Inhibition of Cyclic AMP Production in the Spinal Cord After Stimulation of Cyclic AMP with Forskolin but Not After Stimulation with Capsaicin or Vasoactive Intestinal Peptide

In slices obtained from the ventral and the dorsal guinea pig spinal cord both forskolin and vasoactive intestinal peptide (VIP) caused a dose-dependent stimulation of the production of cyclic AMP. By contrast capsaicin stimulated cyclic AMP formation only in the dorsal cord; no effect was observed in the ventral cord. The alpha 2-adrenergic agonist UK-14,304 dose-dependently inhibited the production of cyclic AMP in both the dorsal and ventral aspects of the cord when the formation of cyclic AMP had been stimulated with 3 microM forskolin, the maximal inhibition amounting to 25-32%. Also the basal (i.e., unstimulated) production of cyclic AMP was inhibited, the inhibition amounting to about 16-18%. However, after stimulation of cyclic AMP formation in the dorsal cord with capsaicin, UK-14,304 was virtually ineffective in inhibiting the accumulation of cyclic AMP. Also, when the formation of cyclic AMP was stimulated with VIP, UK-14,304 was virtually ineffective in inhibiting the formation of cyclic AMP both in the ventral and the dorsal parts of the cord. When cyclic AMP production had been stimulated with forskolin the ability of UK-14,304 to inhibit the formation of cyclic AMP was not attenuated by capsaicin, either in the ventral or in the dorsal cord. The results are discussed with the notion that cyclic AMP inhibitory spinal cord alpha 2-adrenoceptors are located on cells accessible to stimulation of cyclic AMP with forskolin but not with capsaicin or VIP.     

Interaction of Neuropeptides and Cultured Glial (Müller) Cells of the Chick Retina: Elevation of Intracellular Cyclic AMP by Vasoactive Intestinal Peptide and Glucagon

Vasoactive intestinal peptide (VIP) and, to a lesser extent, glucagon were found to increase intracellular cyclic AMP rapidly in cultured glial (Müller) cells of the chick embryo retina. Although VIP elicited higher cyclic AMP accumulation than glucagon at each concentration tested, the half-maximal concentrations were similar, i.e., 6 X 10(-8) M for VIP and 8 X 10(-8) M for glucagon. Secretin had a minimal effect on cyclic AMP accumulation even at a very high (5 X 10(-6) M) concentration. Several other peptide and nonpeptide putative agonists also had little effect on cyclic AMP accumulation. The cultured Müller cell may thus be a useful model for examining VIP and glucagon effects on glial elements of the CNS.     

Effect of gastroduodenostomy on intestinal vasoactive intestinal peptide (VIP) levels, and VIP binding and VIP stimulation of cyclic AMP in intestinal epithelial cells from rat

The concentration of VIP in duodenum and jejunum as well as the interaction of VIP (binding and stimulation of cyclic AMP accumulation) with epithelial cells from both gut segments were studied in rats after surgical bypass of the pylorus by gastroduodenostomy. Duodenal VIP concentration increased in rats with gastroduodenostomy as compared to sham-operated animals. The binding capacity (but not the affinity) of VIP binding sites and the efficiency (but not the potency) of VIP on cyclic AMP accumulation decreased in the condition of gastroduodenostomy. However, no modifications in either VIP concentration and interaction could be seen at the jejunal level.     

Interaction of vasoactive intestinal peptide with rat small intestinal epithelial cells after intestinal resection

Specific binding of vasoactive intestinal peptide (VIP) and VIP-stimulated c y c l i c AMP accumulation were studied in small intestinal epithelial cells (both of crypt and villous levels) 3, 7 and 14 d after a 60% resection of the small intestine . The affinity, but not the binding capacity, of VIP receptors decreased during the adaptive hyperplastic response. Basal cyclic AMP levels were similar in cells of both control and resected rats. Resection induced a decrease of potency, but not of efficiency, of VIP on the stimulation of cyclic AMP accumulation.     

Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum

The effects of secretin and vasointestinal peptide (VIP) on the production of cyclic AMP have been studied in gastric glands isolated by means of EDTA from rat fundic and antral mucosa. (1) In gastric fundus, secretin and VIP caused a time- and temperature-dependent stimulation of cyclic AMP production that was maximal when the test agents were incubated for 60 min at 20 degrees C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. The dose-response curve was monophasic for both peptides, the production of cyclic AMP being sensitive to 10(-10) M secretin and to 5 . 10(-8) M VIP. Half-maximal stimulation was obtained with 2.9 10(-9) M secretin or 2 . 10(-7) M VIP and the maximal stimulation represented a 21-fold and a 19-fold increase above control for secretin and VIP, respectively. Histamine also stimulated cyclic AMP production, with a Km of about 5 . 10(-4) M. No additive effect on cyclic AMP production was oberved when secretin and VIP were simultaneously added at maximally active concentrations, while an additive effect was observed when secretin and histamine were added together. (2) In gastric antrum, the characteristics of the secretin- and VIP-stimulated cyclic AMP production were similar to those observed in gastric fundus. Histamine nevertheless failed to stimulate the formation of cyclic AMP in antral mucosa. (3) These data demonstrate the existence of a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum and suggest that VIP operates through this system. (4) It is proposed that the pepsinogen- and/or mucous-secreting cells are implicated in the regulation of cyclic AMP production by secretin in gastric glands of the rat.     

Vasoactive-intestinal-polypeptide-stimulated adenosine 3'',5''-cyclic monophosphate accumulation in GH3 pituitary tumour cells. Reversal of desensitization by forskolin.

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.     

Somatostatin inhibits VIP- and isoproterenol-stimulated cyclic AMP accumulation in rat prostatic epithelial cells

The dual regulation of cyclic AMP accumulation was studied in rat prostatic epithelial cells incubated with somatostatin, vasoactive intestinal peptide (VIP), and the beta-adrenergic agent isoproterenol. Somatostatin noncompetitively inhibited the stimulatory effect of VIP and isoproterenol, but it did not alter basal cyclic AMP levels. In addition to the multifactorial regulation of the cyclic AMP system in rat prostatic epithelium, these results suggest that somatostatin may play a physiological role at this level.     

Stimulation of guanosine 3',5'-cyclic monophosphate accumulation in rat anterior pituitary gland in vitro by synthetic somatostatin

Synthetic somatostatin stimulated cyclic GMP accumulation with dose dependency (10 ng/ml – 10 μg/ml in a dose examined) in rat anterior pituitary gland in vitro. The stimulation of cyclic GMP levels in the gland was observed after 2 min incubation with somatostatin. Cyclic AMP production induced by TRH or PGE₁ was supressed by this GH release inhibiting factor, while cyclic GMP concentration in the gland was elevated. The present results seem to suggest that inhibitory effect on GH release by somatostatin in anterior pituitary gland is mediated through change in concentration of cyclic AMP and cyclic GMP in the target cells.     

Effects of age and androgens upon functional vasoactive intestinal peptide receptors in rat prostatic epithelial cells

The specific binding of vasoactive intestinal peptide (VIP) and the stimulatory effect of VIP upon cyclic AMP accumulation in isolated epithelial cells of rat ventral prostate were age dependent. The number of VIP receptors decreased but the efficiency of VIP on cyclic AMP accumulation increased in prostatic epithelium when considering the periods 35-65 days and 3-6 months. Since these features could be related to the known age-related decrease of androgen and androgen-receptor levels, we studied the effect of testosterone and its 5 alpha-reduced metabolite dihydrotestosterone upon both steps of VIP action. The two steroid hormones exerted a non-competitive inhibition on VIP-induced cyclic AMP accumulation but did not modify VIP binding to its specific receptors. This modulatory effect of androgens might involve their interaction with specific sites on the cell membrane leading to modifications of membrane activities including adenylate cyclase, as has been suggested by an increasing number of recent reports.     

Characterization and age dependence of the stimulatory effect of VIP on cyclic AMP accumulation in rat Leydig cells

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to stimulate cyclic AMP accumulation in Leydig cells isolated from rat testis. The effect was dependent on time, temperature and cell concentration. At 15° half-maximal and maximal stimulation were observed at about 1 and 100 nM VIP, respectively. The interaction was specific since an order of potencies chicken VIP> rat VIP> secretin>glucagon and no effect of neurotensin and substance P were obtained. The efficiency of VIP was lower in pubertal rats and then increased in young-adult and adult animals. These results together with the known presence of VIP in the testis support the idea that VIP may be involved in the regulation and function of Leydig cells during development.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15 degrees C, the response occurred in the 1 X 10(-10)-10(-7)M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1 X 10(-6)M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

Lindane effect upon the vasoactive intestinal peptide receptor/effector system in rat enterocytes

Isolated rat enterocytes exposed to the insecticide lindane (the gamma-isomer of hexachlorocyclohexane, HCCH) showed an important decrease in the efficiency of the neuropeptide vasoactive intestinal peptide (VIP) upon the stimulation of cyclic AMP accumulation. The effect of lindane was time- and dose-dependent, optimal conditions being reached after 5 min incubation of cells at 25 degrees C with 0.5 mM of this organochlorine compound. Lindane action exhibited an important degree of specificity since the isomer alpha-HCCH and endrin reproduced the same inhibitory pattern but beta-HCCH and dieldrin were inactive. The inhibition of VIP-induced cyclic AMP accumulation could not be explained by a lindane-dependent reduction in the binding of VIP to its specific receptors. Among various possibilities, the results suggest the modification of membrane fluidity by lindane and/or the activation of Ca2+-dependent protein kinase C by this compound leading to phosphorylation of Gs/adenylate cyclase.