Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells

To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol-stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol-stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol-stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down-regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.     

Muscarinic cholinergic inhibition of cyclic AMP formation and adrenocorticotropin secretion in mouse pituitary tumor cells

Cholinergic muscarinic receptors were identified in AtT-20/D16-16 (AtT-20) cell membranes by receptor binding techniques and the effect of carbachol on basal and stimulated cyclic AMP formation and ACTH release was investigated. Carbachol markedly decreased the stimulatory effect of the adenylate cyclase activator, forskolin, on both cyclic AMP formation and ACTH secretion. Carbachol also reduced forskolin-stimulated adenylate cyclase activity. The stimulatory effects of (-) isoproterenol on cyclic nucleotide formation and ACTH secretion were also blocked by carbachol. The inhibitory effects of carbachol on (-) isoproterenol-stimulated cyclic AMP synthesis and ACTH secretion were reversed by the muscarinic antagonist, atropine, and not by the nicotinic antagonist, gallamine. These data suggest that in AtT-20 cells, inhibition of ACTH secretion may be regulated by activation of muscarinic receptors coupled negatively to adenylate cyclase.     

Comparison of the inhibition of insulin release by activation of adenosine and alpha 2-adrenergic receptors in rat beta-cells.

Rat islets were used to compare the mechanisms whereby adenosine and adrenaline inhibit insulin release. Adenosine (1 microM-2.5 mM) and its analogue N6(-)-phenylisopropyladenosine (L-PIA) (1 nM-10 microM) caused a concentration-dependent but incomplete (45-60%) inhibition of glucose-stimulated release. L-PIA was more potent than D-PIA [the N6(+) analogue], but much less than adrenaline, which caused nearly complete inhibition (85% at 0.1 microM). 8-Phenyltheophylline prevented the inhibitory effect of L-PIA and 50 microM-adenosine, but not that of 500 microM-adenosine or of adrenaline. In contrast, yohimbine selectively prevented the inhibition by adrenaline. Adenosine and L-PIA thus appear to exert their effects by activating membrane A1 receptors, whereas adrenaline acts on alpha 2-adrenergic receptors. Adenosine, L-PIA and adrenaline slightly inhibited 45Ca2+ efflux, 86Rb+ efflux and 45Ca2+ influx in glucose-stimulated islets. The inhibition of insulin release by adenosine or L-PIA was totally prevented by dibutyryl cyclic AMP, but was only attenuated when adenylate cyclase was activated by forskolin or when protein kinase C was stimulated by a phorbol ester. Adrenaline, on the other hand, inhibited release under these conditions. It is concluded that inhibition of adenylate cyclase, rather than direct changes in membrane K+ and Ca2+ permeabilities, underlies the inhibition of insulin release induced by activation of A1-receptors. The more complete inhibition mediated by alpha 2-adrenergic receptors appears to result from a second mechanism not triggered by adenosine.     

Adaptive increase in adenylyl cyclase activity in NG108-15 and S49 cells induced by chronic treatment with inhibitory drugs is not due to a decrease in cyclic AMP concentrations.

NG108-15 neuroblastoma x glioma hybrid cells and S49 lymphoma cells exhibit an enhancement in adenylyl cyclase activity after chronic treatment with receptor agonists that acutely inhibit the enzyme. Using agonists that activate five distinct inhibitory receptors in NG108-15 cells, we have found that there is a correlation between the extent of acute inhibition of prostaglandin E1 (PGE1)-stimulated cAMP accumulation and efficacy for induction of enhanced PGE1 stimulation of cAMP accumulation after chronic treatment and withdrawal. Chronic treatment with dideoxyadenosine, which acutely inhibits adenylyl cyclase activity by a mechanism independent or cell surface receptors or pertussis toxin-sensitive G proteins, did not induce enhanced PGE1 stimulation of cAMP accumulation in NG108-15 cells or forskolin stimulation of cAMP accumulation in S49 cells. While control basal cAMP concentrations were acutely decreased by carbachol in NG108-15 cells and by somatostatin in S49 cells, when the cAMP concentrations were maintained above the control basal values with a phosphodiesterase inhibitor, chronic treatment with these inhibitory drugs nonetheless resulted in enhanced cAMP responses in both NG108-15 and S49 cells. These results provide evidence that the initial decrement in cAMP concentrations caused by inhibitory drug is not the requisite signal for inducing the subsequent sensitization of adenylyl cyclase in NG108-15 and S49 cells but that activation of a pertussis toxin-sensitive G protein is involved in the development of this important adaptation.     

Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors

Abstract: Previous studies have suggested that activation of D2-like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1-like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2-like dopamine receptors. Both D1- and D2-selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K⁺ levels. The D1-selective agonists 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (Cl-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with ～10 µM Cl-APB and ～100 µM SKF-38393. Of the D2-selective agonists, bromocriptine was a more potent inhibitor of DMPP-stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by Cl-APB or SKF-38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2-selective, but not D1-selective, dopamine agonists. In addition, forskolin-stimulated adenylyl cyclase activity was inhibited by D2-selective, but not D1-selective, agonists. Neither D1- nor D2-selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP-stimulated Ca²⁺ uptake was inhibited by both D1- and D2-selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1-like and D2-like subfamilies was responsible for the observed inhibition. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1- and D2-selective agonists inhibit Ca²⁺ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.     

Antisecretory effects of somatostatin and vasopressin in the rat colon descendens in vitro

The effects of two hormones, vasopressin and somatostatin (SOM), on ion secretion in rat colon descendens were compared. Three modes for induction of epithelial secretion were used: neuronally mediated secretion due to electric field stimulation (EFS), Ca2+-dependent secretion elicited by carbachol, and cAMP-dependent secretion evoked either by a receptor-mediated mechanism elicited by vasoactive intestinal peptide (VIP) or by a direct activation of the adenylate cyclase by means of forskolin. Somatostatin inhibited ion secretion evoked by EFS (55-65%), carbachol (80%) and VIP (95%) in a dose-dependent manner. Maximal inhibition by SOM was observed at 10(-7) M. Somatostatin had, however, no effect on the secretory response to forskolin. The inhibition of the VIP effect could be attenuated by pretreatment with pertussis toxin. In contrast, vasopressin in concentrations as low as 0.025-0.25 U/liter decreased the secretory effects of EFS (55-75%) and carbachol (85%), but had no effect on cAMP-dependent secretion elicited either by VIP or forskolin. The results suggest that the antisecretory effect of vasopressin is mediated only by a block in the Ca2+ pathway, whereas SOM inhibits Ca2+-dependent secretion as well as receptor-mediated cAMP-dependent secretion. The interaction with the cAMP pathway is located at the step between stimulation of the receptor and activation of the adenylate cyclase and probably involves an Ni-protein.     

Forskolin alters sensitivity of the cAMP-generating system to stimulatory as well as to inhibitory agonists. A study with intact human platelets and guinea pig myometrium

1. In both the intact guinea pig myometrium and human platelets, cAMP accumulation was enhanced by prostaglandin I2 (prostacyclin, PGI2) and forskolin with potentiated responses in the simultaneous presence of both effectors. Under basal conditions, forskolin caused rises in platelet cAMP concentration through a single low-affinity interaction (Kapp = 90 microM) while in myometrium, activation involved both a low-affinity (Kapp = 10 microM) and a high-affinity (Kapp = 0.8 microM) component. The contribution of the high-affinity component could be reduced when endogenous PGI2 was decreased. In both tissues, the synergistic effect of forskolin in the presence of PGI2 was mediated by a single high-affinity interaction (Kapp = 0.3 microM). The data were consistent with a low-affinity interaction of the diterpene with the cyclase catalytic unit C generating the C...forskolin state and with a high-affinity interaction of the diterpene with the activated complex (stimulatory regulatory protein) and C generating the potentiated Gs-C...forskolin state. 2. Both norepinephrine in platelets and carbachol in the myometrium (via Gi, the inhibitory regulatory protein) inhibited PGI2-mediated cAMP accumulation (EC50 = 100 nM and 8 nM respectively). The persistently activated cAMP-generating system induced by cholera toxin in the myometrium was also susceptible to inhibition but the EC50 for carbachol was increased to 50 nM and the extent of inhibition was decreased. Forskolin-mediated effect in platelets was inhibited by norepinephrine as was the PGI2 response. By contrast, the synergistic state of the cyclase resisted the inhibitory action of norepinephrine and carbachol in platelets and myometrium respectively. In the myometrium, where the cAMP response due to forskolin alone partially involved some synergistic Gs-C ... forskolin species, carbachol at 50 microM elicited no more than 30% inhibition. Inhibition was partly improved (60% inhibition at 1 microM carbachol) when the contribution of the Gs-C species was decreased by lowering the concentration of local PGI2. Partial inhibition by norepinephrine was similarly observed in platelets under partial synergistic conditions. The data suggest that receptor-mediated inhibition of cAMP generation could be differentially expressed depending on the nature of the active species of the cyclase involved in the stimulatory responses.     

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: the possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.     

Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Effects of forskolin and analogues on nicotinic receptor-mediated sodium flux,voltage-dependent calcium flux,and voltage-dependent rubidium efflux in pheochromocytoma PC12 cells

1. Forskolin, a naturally occurring diterpene that activates adenylate cyclase, HL706, a water-soluble derivative of forskolin (6 beta-[(piperidino)acetoxy]-7-desacetylforskolin) that is less potent than forskolin in activating adenylate cyclase, and 1,9-dideoxyforskolin, an analogue that does not activate adenylate cyclase, were examined for effects on the nicotinic receptor-mediated 22Na+ flux, a high potassium-induced 45Ca2+ flux through L-type calcium channels, and a high potassium-induced 86Rb+ efflux through a calcium-dependent potassium channels in PC12 cells. 2. Forskolin and analogues at 30 microM completely blocked carbamylcholine-elicited flux of 22Na+ through the nicotinic receptor-gated channel. 1,9-Dideoxyforskolin had an IC50 value of 1.6 microM with forskolin and HL706 being two- to three fold less potent. 3. Forskolin and its analogues appear to be noncompetitive blockers of the neuronal nicotinic receptor-channel complex in PC12 cells, but unlike many noncompetitive blockers, did not markedly enhance desensitization. Instead, forskolin, but not HL706 or 1,9-dideoxyforskolin, slightly antagonized the desensitization evoked by high concentrations of carbamylcholine. N-Ethylcarboxamidoadenosine, an adenosine analogue that elevates cyclic AMP and 8-bromo-cyclic AMP had no effect on desensitization. 4. Forskolin, HL706, and 1,9-dideoxyforskolin in the presence of carbamylcholine inhibited the binding of a noncompetitive blocker, [3H]perhydrohistrionicotoxin, to the muscle-type nicotinic receptor-channel complex in Torpedo electroplax membranes with IC50 values of 20 microM. Forskolin had no effect on [3H]perhydrohistrionicotoxin binding in the absence of carbamylcholine, while HL706 and 1,9-dideoxyforskolin still inhibited binding in the absence of carbamylcholine. 5. Forskolin, but not HL706 or 1,9-dideoxyforskolin had a slight inhibitory effect on the binding of [125I]alpha-bungarotoxin to acetylcholine recognition sites in Torpedo membranes. 1,9-Dideoxyforskolin at 30 microM, but not forskolin or HL706, markedly inhibited depolarization-evoked 45Ca+ flux and 86Rb+ efflux in PC12 cells, suggesting that 1,9-dideoxyforskolin has nonspecific inhibitory effects on a variety of ion channels.     

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating.     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

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

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

Secretion of gastric mucus phospholipids in response to beta-adrenergic G protein-coupled receptor activation is mediated by SRC kinase-dependent epidermal growth factor receptor transactivation.

Recent advances in understanding the nature of cellular responses mediated by G protein-coupled receptor (GPCR) activation indicate that integration of the converging regulatory signals into functional cellular pathways requires epidermal growth factor receptor (EGFR) transactivation. In this study, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation in gastric mucus phospholipid secretion occurs with the involvement of EGFR. Using [14C]choline-labeled gastric mucosal cells in culture, we show that stimulatory effect of beta-adrenergic agonist, isoproterenol, on phospholipid release was subject to a dose-dependent suppression by EGFR kinase inhibitor, PD153035, as well as wortmannin, a specific inhibitor of PI3K. Both inhibitors, moreover, caused the reduction in the gastric mucosal cell phospholipid secretory responses to beta-adrenergic agonist-generated second messenger, cAMP as well as adenyl cyclase activator, forskolin. The gastric mucosal phospholipid secretory responses to isoproterenol, furthermore, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation, but not by ERK inhibitor, PD98059. The inhibition of ERK, moreover, did not cause attenuation in phospholipid secretory responses to cAMP and forskolin. The findings underline the central role of EGFR in mediation of gastric mucosal secretory processes, and demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of gastric mucosal phospholipid secretion in response to beta-adrenergic GPCR activation.     

cAMP protection of pancreatic cancer cells against apoptosis induced by ERK inhibition.

Large increases in cAMP concentration inside the cell are generally growth inhibitory for most cell lines of mesenchymal and epithelial origin. Moreover, recent data suggest a role of cAMP in survival of different cell types. Herein, the ability of forskolin (an adenylyl cyclase activator) and IBMX (3-isobutyl-1-methylxanthine) (a phosphodiesterase inhibitor) to modulate cell cycle progression and survival of human pancreatic cancer cells was evaluated. We showed that forskolin + IBMX inhibited serum-induced ERK activities, Rb hyperphosphorylation, Cdk2 activity, and p27(Kip1) downregulation and caused G1 arrest in MIA PaCa-2 cells. Furthermore, forskolin + IBMX protected pancreatic cells against apoptosis induced by prolonged inhibition of ERK activities by preventing Bcl-X(L) downregulation, activation of caspases 3, 6, 8, and 9, and PARP cleavage and by inducing Bad phosphorylation (ser112). Taken together, our data demonstrate for the first time that cAMP is an inhibitor of cell cycle progression and apoptosis in human pancreatic cancer cells.     

Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.     

Effects of Substance P on Nicotinic Acetylcholine Receptor Function in PC 12 Cells

The effects of substance P on the functioning of nicotinic acetylcholine receptors in PC12 cells were examined. Carbachol-stimulated 22Na+ uptake was used to assess the functional state of the nicotinic receptor. We found that incubation of the cells with substance P alone caused a loss of receptor function. Receptors recovered from this effect with a t1/2 of 0.94 +/- 0.10 min. Since receptors recovered from carbachol-induced desensitization at a significantly slower rate (t1/2, 1.77 +/- 0.21 min), it was concluded that the two inactive states are not kinetically equivalent. The effects of substance P on carbachol-induced loss of receptor activity were also examined. Substance P had no effect on a component of carbachol-induced loss of activity that was nonrecoverable (inactivation). However, substance P had several effects on the recoverable loss of activity induced by carbachol (desensitization). Substance P caused a shift to the left in the EC50 for carbachol-induced desensitization at equilibrium. If cells were simultaneously incubated with carbachol and substance P7-11, a low-potency analog of substance P, an increase in the rate of formation of a state of the receptor that was kinetically indistinguishable from the state induced by carbachol alone was observed. However, not all inhibition of nicotinic cholinergic function could be explained by an increased rate of formation of a desensitized receptor and it is concluded that substance P causes both enhanced desensitization and block of the nicotinic receptor-linked channel.     

Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

Role of adenylate cyclase in human T-lymphocyte surface antigen capping

Our recent studies indicated that capping of T3, T4 and T8 surface antigens on human T lymphocytes is augmented by interaction of adenosine with a purinergic receptor. We suggested that the T-cell capping process was mediated by an adenylate cyclase-coupled purinergic receptor that resulted in the generation of cAMP and occupancy of cAMP receptors. The present study was undertaken to examine whether activation of adenylate cyclase in the absence of purinergic stimulation is sufficient to regulate surface antigen capping. Treatment of T lymphocytes with forskolin or cholera toxin caused activation of adenylate cyclase and occupancy of intracellular types I and II regulatory subunits of protein kinase by cAMP, as demonstrated by photoaffinity labeling with [8-3H]N3-cAMP. Such treatment augmented the rate of capping of the T3, T4, and T8 antigens, which resulted in a significant decrement in the elapsed time to half-maximal capping of each antigen. These observations support the proposition that the normal T-lymphocyte capping mechanism of both T3+, T4+ (inducer/helper) and T3+, T8+ (suppressor) subsets can be augmented by activation of adenylate cyclase.