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.     

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.     

Inhibitory action of tumor-promoting phorbol esters upon beta-adrenergic agonist stimulation of cyclic AMP accumulation in rat prostatic epithelial cells

The pretreatment of rat prostatic epithelial cells with 4 beta-phorbol 12-myristate 13-acetate resulted in an attenuation of beta-adrenergic stimulated cyclic AMP accumulation. The effect was dependent on time and concentration. The maximal extent of isoproterenol stimulation of cyclic AMP production was reduced by 35% after 15-min pretreatment with the phorbol ester at 25 degrees C. Since a similar action was exerted by other protein kinase C stimulators, present results suggest the involvement of this enzyme in a process of desensitization to beta-adrenergic agonists of the adenylate cyclase system in rat prostatic epithelium.     

α-Adrenergic inhibition of cyclic AMP accumulation in epithelial cells isolated from rat small intestine

The present work shows that α-adrenergic agonists induce the suppression of basal and hormone-stimulated cyclic AMP levels in rat intestinal epithelial cells. Epinephrine (100 μM) suppresses by 35% the cyclic AMP levels evoked by the vasoactive intestinal peptide (VIP). The adrenergic agent induces a similar percentage of inhibition at 15, 30 and 37°C. Addition of epinephrine 20 min prior to, on 5 or 20 min after VIP yields the same magnitude of inhibition as when performed together with the stimulus. The α-adrenergic agent does not alter the K_0.5 of VIP in stimulating cyclic AMP production but reduces its efficacy. Epinephrine also suppresses prostaglandin E₁- and E₂-stimulated cyclic AMP levels by about 35%. The lowest effective concentration of epinephrine required to suppress VIP-stimulated cyclic AMP levels is 0.1 μM, half-maximal (K_0.5) and maximal effects being observed at 5 and 100 μM, respectively. Norepinephrine has the same efficacy but a slightly lower potency (K_0.5 = 18 μM) than epinephrine. Phenylephrine acts as a partial agonist of very low potency; clonidine has very little intrinsic activity and antagonizes the inhibition by epinephrine. The inhibition of VIP-stimulated cyclic AMP levels is observed in the absence of any blocking agents. It is not affected by the β blocker propranolol, but is completely reversed with α blockers with the following order of potency: dihydroergotamine>yohimbine>phentolamine. Yohimbine is much more potent than prazosin, which only partially reverses the inhibition induced by epinephrine. It is concluded that α-adrenoreceptors of the α₂ subtype mediate the suppression of VIP-stimulated cyclic AMP levels in intestinal epithelial cells. This effect is likely to be due to the inhibition of adenylate cyclase within intact cells as epinephrine is able to reduce adenylate cyclase activity of intestinal epithelial cell plasma membranes.     

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.     

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells.

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.     

Involvement of cellular cyclic AMP in theophylline-induced sugar accumulation in chicken intestinal epithelial cells

We have studied the possible involvement of cellular cyclic AMP in theophylline-induced sugar gradient enhancement in isolated chicken enterocytes. Theophylline increases 3-O-methylglucose accumulation 3-fold after 30 min incubation. Exogenous cyclic AMP enhances sugar accumulation by 48%. Adenylyl cyclase inhibitor RMI 12 330A reduces theophylline-induced sugar gradients by 22% and theophylline-induced cyclic AMP levels by 24.5%. At the concentration used, RMI 12 330A has no effect on 3-O-methylglucose accumulation or basal cellular cyclic AMP. Since theophylline has a rapid inhibitory effect on Na+-independent sugar permeability, we conclude that the effects of the drug on sugar gradients are the result of its acting by both direct - surface membrane - and indirect - cyclic AMP mediated - mechanisms. The effect of theophylline and exogenous cyclic AMP on sugar accumulation is independent of external chloride.     

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)     

Alpha 1-adrenergic regulation of TSH-stimulated cyclic AMP accumulation in rat thyroid cells

Addition of epinephrine to cultured FRTL-5 rat thyroid cells led to a concentration-dependent reduction of TSH- and forskolin-stimulated cAMP accumulation. Clonidine, which preferentially activates the alpha 2-adrenoreceptor, had no effect on cAMP levels. The reduction of cAMP levels by epinephrine was selectively blocked by prazosin, an alpha 1-adrenoreceptor antagonist, but not by yohimbine, an alpha 2-adrenoreceptor antagonist. Pretreatment of FRTL-5 cells with pertussis toxin failed to abolish the inhibitory effect of epinephrine on cAMP accumulation. The bioactivity of the pertussis toxin preparation in this cell line was verified by its ability to ADP-ribosylate the alpha-subunit of the inhibitory guanine nucleotide regulatory protein, Ni, as well as its ability to abolish the inhibitory effect of N6-[L-2-phenylisopropyl]-adenosine on TSH-stimulated cAMP formation. The inhibitory effect of epinephrine on cAMP levels was dependent on Ca2+ and was reversed by 3-isobutyl-1-methylxanthine. Taken together, these results suggest that epinephrine reduces cAMP levels via alpha 1-adrenoreceptors. The failure of pertussis toxin to abolish this alpha-adrenergic effect is consistent with the conclusion that epinephrine-induced attenuation of cAMP accumulation occurs through activation of a Ca2+-calmodulin-sensitive phosphodiesterase and does not involve Ni or Ni-like proteins.     

Insulin as an activator of cyclic AMP accumulation in rat fat cells.

In rat fat cells incubated with lipolytic agents and insulin for 30 or 60 minutes the increase in cyclic AMP accumulation due to norepinephrine and theophylline or adenosine deaminase added during the last 2-5 minutes of the incubation period was much greater as compared to cells incubated in the absence of insulin. Protaglandin E1 or nicotinic acid were just as anti-lipolytic as insulin but prior incubation with these agents markedly decreased the subsequent rise in cyclic AMP accumulation due to late catecholamine addition. The ability of insulin to increase cyclic AMP accumulation appeared to be secondary to inhibition of lipolysis. These results indicate that prostaglandin E1 and nicotinic acid are inhibitors of cyclic AMP accumulation while insulin acts by another mechanism to reduce lipolysis.     

Activation of rat parotid low Km cyclic AMP phosphodiesterase by isoproterenol

Approximately 94% of rat parotid cyclic AMP phosphodiesterase activity measured at a substrate concentration of 0.1 microM cyclic AMP was found in the 100,000 X g supernatant while the remaining enzyme activity was in the particulate fraction. Incubation of parotid slices with 10 microM isoproterenol resulted in approximately 40% activation of the cyclic AMP phosphodiesterase activity of the 100,000 X g supernatant. The enzyme activity in the particulate fraction was unaffected. The activation resulted from an increase in the value of the Vmax while the apparent Km (0.51 microM) was unaffected. The concentration of isoproterenol required to give half-maximal activation was 0.34 microM. The activation was rapid, became significant after 2 min and reached maximum after 30 min incubation of the parotid slices with isoproterenol. The activation of the enzyme activity by isoproterenol could be blocked by propanolol but was unaffected by cycloheximide. Dibutyryl-cyclic AMP was also effective while phenylephrine and carbamylcholine were ineffective in increasing the activity of the enzyme.     

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.     

Activation of lipolysis and cyclic AMP accumulation in rabbit adipocytes by isoproterenol in the presence of forskolin or pertussis toxin

Adipocytes from rabbits are relatively insensitive to catecholamines or forskolin. However, the combination of catecholamines plus forskolin increased cyclic AMP accumulation and lipolysis much more than either agent alone. Pertussis toxin treatment also restored sensitivity to catecholamines. No defect in activation by catecholamines of adenylate cyclase was seen in isolated membranes incubated in the presence of GTP. Rabbit adipocytes appear to have an excess of the inhibitory guanine nucleotide binding protein (Ni). However, in plasma membranes this protein appeared to be relatively inactive as there was an activation of adenylate cyclase activity by catecholamines in the presence of GTP. These data suggest that in intact rabbit adipocytes catecholamines and forskolin are ineffective as stimulators of adenylate cyclase due to an excess of inhibitory guanine nucleotide binding proteins.     

Oxytocin and vasopressin have no effect on progesterone production and cyclic AMP accumulation by rat luteal cells in vitro

Enzymically dispersed luteal cells obtained from PMSG-hCG-treated immature pseudopregnant rats were incubated with oxytocin and vasopressin. In response to increasing doses of hCG the rat luteal cells produced progesterone and accumulated intracellular cAMP in a dose-dependent manner. A neuropeptide GnRH agonist (4 X 10(-6) M) produced a significant inhibition of hCG-stimulated progesterone production and of accumulation of intracellular cAMP. However, neither the basal nor the hCG-stimulated rate of progesterone production and level of intracellular cAMP was affected by the neurohypophysial peptides tested. Therefore, it is concluded that oxytocin and vasopressin do not have a direct action on steroidogenesis by rat luteal cells.     

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.     

Effect of cholera enterotoxin on calcium uptake and cyclic AMP accumulation in rat basophilic leukemia cells

Cholera enterotoxin (CT), at an optimal concentration of 2.38 X 10(-10) M, stimulated calcium uptake (P less than 0.01) and cyclic AMP accumulation (P less than 0.02) in cultured rat basophilic leukemia cells. No significant effect of CT on calcium release or cyclic GMP accumulation was detected. Pharmacologic and chemical agents which block calcium uptake or prostaglandin synthesis antagonized the effect of CT.     

Glucagon control of cyclic AMP accumulation in isolated intact rat liver parenchymal cells in vitro

Cyclic AMP levels were measured in suspensions of isolated rat liver parenchymal cells during incubation in vitro. Glucagon caused a rapid elevation of cyclic AMP content. With 1.4·10⁻⁶ M (5 μg/ml) of the hormone the levels increased about 10-fold during the first minute, thereafter the elevation was less rapid. Maximal values were reached at 5–10 min. Theophylline slightly increased the basal cyclic AMP levels, and markedly augmented the response to glucagon. Teh major part of the cyclic AMP was located within cells, but a siginificant fraction was present in the incubation medium, and the relative amount present extracellularly increased with incubation time. Significant elevation of the cyclic AMP levels was produced by glucagon 1.4·10⁻¹⁰M, and half-maximal stimulation occured at about 2·10⁻⁹ M. The initial rate of cyclic AMP accumulation was such more rapid in the parenchymal cells than in liver slices, and the maximal levels obtained were about 3 times higher (comparisons based on the finding that 1 mg liver tissue contains about 10⁵ parenchymal cells). It is concluded that preparations of parenchymal liver cells are useful in the study of glucagon actions on liver tissue.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Transformation-dependent effect of dibutyryl cyclic AMP on surface properties of rat cultured cells

The possible differential effect of dibutyryl cyclic AMP on the surface properties of ts-NT3-KR rat cells that express a normal phenotype at 37 degrees C and a transformed morphology and behavior at 33 degrees C has been studied. Electrophoretic examination of glycosylated macromolecules revealed a 350,000 dalton glycoconjugate in phenotypically normal cells but not in the corresponding samples from phenotypically transformed cells or in phenotypically "normal" cells rounded by exposure to the cyclic nucleotide. A decreased exposure of a major 100,000 dalton surface component characteristic of cells that expressed a transformed phenotype, was observed when the corresponding cultures were exposed to dibutyryl cyclic AMP. No change in the 230,000 dalton fibronectinlike molecule of phenotypically normal cells was apparent even in the corresponding cultures exposed to the cyclic nucleotide.