Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate

Immunologic activation of purified human lung mast cells (HLMC) and basophils with anti-IgE induced histamine release but failed to elicit any changes in cAMP levels. In contrast, histamine release and monophasic rises in cAMP were observed in both rat peritoneal mast cells (RPMC) challenged with concanavalin A (73% enhancement over basal cAMP 20 sec after activation) and a cultured mouse bone marrow-derived mast cell (PT18 cell line) passively sensitized with dinitrophenol-specific IgE and stimulated with antigen (39% increase above basal at 15 sec). The adenylate cyclase activators isoprenaline, prostaglandin E2 (PGE2), and forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) all induced elevations in cAMP levels in both basophils and HLMC. In basophils, PGE2 and isoprenaline produced approximately twofold increases in cAMP that were maximal at 1 min and decayed thereafter. Forskolin and IBMX produced threefold increases in cAMP that peaked 10 min after activation and persisted for up to 20 min. In HLMC, isoprenaline provoked a rapid monophasic fourfold increase in cAMP that was maximal at 1 min after addition. Levels of cAMP subsequently declined but remained significantly elevated over resting levels for up to 30 min. PGE2, forskolin, and IBMX all produced approximately threefold rises in HLMC cAMP that peaked around 5 min and persisted for 30 min. In both the basophil and HLMC, agonist-induced elevations in cAMP correlated well with the inhibition of mediator release. In basophils, the order IBMX greater than forskolin greater than PGE2 greater than isoprenaline held for both the inhibition of histamine and leukotriene C4 release and the augmentation of cAMP levels. In HLMC, individual agonists elevated cAMP levels to similar degrees and inhibited the release of histamine, leukotriene C4, and PGD2 to comparable extents, although the release of the arachidonate metabolites was generally more sensitive to the inhibitory actions of these agonists. These results suggest that elevations in cAMP, in both the basophil and HLMC, are associated with the inhibition of mediator release but not the initiation of the secretory process.     

Cyclic AMP antagonist Rp-cAMPS inhibits amylase exocytosis from saponin-permeabilized parotid acini.

Rp-cAMPS, the Rp-diastereomer of adenosine 3',5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp-cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp-cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken together with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini.     

Role of cyclic AMP in corticotropin releasing factor mediated ACTH release

To elucidate the role of cAMP in the secretion of ACTH, the effect of (1) three phosphodiesterase inhibitors, (2) forskolin, and (3) 8Bromo-cAMP, on CRF mediated ACTH release was studied in rat pituitary cell culture. The action of glucocorticoids on CRF induced cAMP accumulation and ACTH release was investigated. Isobutyl-methylxanthine (IBMX), caffeine, and forskolin augmented the release of ACTH induced from CRF 1.0 nM by 17%, 39%, and 20%, respectively. Also IBMX and caffeine potentiated CRF 10 nM stimulated ACTH release by 32% and 20%. Doses of forskolin and 8Bromo-cAMP, which alone stimulate large amounts of ACTH release, did not increase the amount of ACTH released from CRF 100 nM stimulated cells. Cortisol (500 nM) and corticosterone (500 nM) inhibited CRF induced intracellular cAMP by 39% and 26% while inhibiting pituitary ACTH release by 40% and 52%. In conclusion, cAMP plays an important role in the mechanism of ACTH secretion and it appears the final intracellular mechanism of CRF stimulated ACTH is via cAMP. Also, glucocorticoids exert their inhibitory influence prior to cAMP generation.     

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.     

Steroid-induced final maturation in brook trout (Salvelinus fontinalis) oocytes in vitro: the effects of forskolin and phosphodiesterase inhibitors

The effects of phosphodiesterase inhibitors and forskolin on steroid-induced germinal vesicle breakdown (GVBD) were investigated in brook trout (Salvelinus fontinalis) oocytes using an in vitro incubation technique. Follicles were first treated with a collagenase solution to remove the follicle wall. Denuded oocytes were examined, using scanning electron microscopy. In all experiments GVBD was induced by the use of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one. Cyclic adenosine 3',5'-monophosphate (cAMP) levels were measured (by protein-binding assay) in control and forskolin-treated oocytes. Collagenase treatment removed a majority of the follicle wall, as shown by scanning electron microscopy. Partially denuded (PD) oocytes were slightly more sensitive to steroid treatment than intact follicles (IF), as shown by ED50 values; but PD oocytes did not respond to gonadotropin (GTH) stimulation. Both 3-isobutyl-1-methyl-xanthine (IBMX) and SQ20,006 (Squibb) blocked GVBD, but IBMX was more inhibitory. Forskolin also blocked steroid-induced GVBD. Kinetics of inhibition studies were performed using IBMX, forskolin, and cycloheximide. IBMX and cycloheximide inhibited GVBD if added during the first 18 h following steroid stimulation, whereas forskolin blocked GVBD if added within 12 h after steroid treatment. Forskolin, at levels that block GVBD in vitro, significantly increased cAMP in both IF and PD oocytes, but the response of IF was greater than that of PD oocytes.     

Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase.

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Characterization of the 3',5'-cyclic adenosine monophosphate-mediated regulation of IL2 production by T cells and Jurkat cells.

The effect of cyclic AMP-elevating agents on mitogen-stimulated IL2 production was examined. Prostaglandin E2 (PGE2) inhibited IL2 production by human peripheral blood T cells stimulated with PHA. In contrast, PGE2 did not inhibit PHA-stimulated IL2 production by the human leukemic T cell line. Jurkat, and often slightly enhanced IL2 production by those cells. Other cyclic adenosine monophosphate (cAMP) elevating agents (forskolin, isoproterenol, and the cAMP analogue, dibutyryl cAMP) also inhibited lectin-stimulated IL2 production by T cells, but could not inhibit IL2 production by Jurkat cells. Of the cAMP-elevating agents examined, only cholera toxin (CT) inhibited IL2 production by both Jurkat cells and peripheral blood T cells. Although phorbol myristate acetate (PMA) greatly enhanced PHA-stimulated IL2 production by Jurkat cells. CT remained markedly inhibitory. The combination of PMA and the calcium ionophore, ionomycin, also induced IL2 production by Jurkat cells, and this was similarly suppressed by CT, suggesting that a step after initial second messenger generation was inhibited. A prolonged increase in intracellular cAMP levels was induced by CT in both T cells and Jurkat cells, but the maximal level and the length of elevation achieved in T cells were much less than those observed in Jurkat cells. In contrast, PGE2 caused only a modest and transient increase in intracellular cAMP levels in Jurkat cells compared to that noted with T cells. PGE2 induced a more marked and sustained increase in cAMP levels in Jurkat cells treated with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Moreover, in the presence of IBMX, PGE2 caused a marked inhibition of IL2 production by PHA-stimulated Jurkat cells. Differences in the capacity of PGE2 to induce cAMP could not be explained by disparities in the level of cAMP phosphodiesterase activity as this was comparable in Jurkat cells and in T cells. Thus, these observations indicate that IL2 production by both peripheral T cells and Jurkat cells can be modulated by cAMP-elevating agents. The data suggest that the diminished capacity of PGE2 to inhibit IL2 production by Jurkat cells reflects both a diminished capacity of PGE2 to induce increases in cAMP levels in these cells and an increase in the threshold of cAMP required to inhibit Jurkat cells.     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.     

Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland

Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP)-mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTPS in PT-treated membranes was much larger than that in normal membranes. ANP dose-dependently inhibited adenylate cyclase stimulated by GTPS in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10^–7M) inhibited cAMP accumulation stimulated by forskolin (10^–6M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells, amylase release stimulated by isoproterenol (10^–6M) and forskolin (10^–6M) were also depressed by ANP (10^–7M) by 27 and 30%, respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP-ribosylated 41-KDa protein by incubation of parotid membranes with PT and [-³²P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi. (Mol Cell Biochem)139: 53–58, 1994)     

Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: Interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.     

Effects of sodium ions and monensin on amylase secretion from rat parotid cells

The role of sodium ions in amylase secretion from rat parotid cells was studied using various Na+-free media and monensin. In a sucrose medium, amylase secretion was not stimulated by isoproterenol but was significantly stimulated by dibutyryl cAMP. In choline chloride and LiCl media, both isoproterenol and dibutyryl cAMP clearly evoked amylase release. Monensin itself elicited amylase secretion slightly, but significantly inhibited the secretion stimulated by isoproterenol or dibutyryl cAMP. The inhibitory effect of monensin was detectable even in choline chloride, LiCl and KCl media. These results indicate that sodium ions are not essential for amylase secretion from rat parotid cells and that the inhibitory effect of monensin is independent of influx of sodium ions or efflux of potassium ions.     

Spontaneous maturation in vitro of cumulus-enclosed rat oocytes is inhibited by forskolin

We have recently reported that the adenylate cyclase activator, forskolin, induces in the rat ovarian follicle both cAMP accumulation and oocyte maturation. We demonstrate here, on the other hand, that the spontaneous maturation in vitro of isolated rat cumulus-enclosed oocytes is inhibited by forskolin. The inhibitory effect of forskolin is dose dependent with an ED50 at 15 microM. Forskolin inhibition decreases gradually with time, being completely relieved by 20 h of culture. Methylisobutylxanthine significantly prolongs the duration of the inhibitory action of forskolin. In addition to its inhibitory effect on oocyte maturation, forskolin triggers the cumulus-oocyte complex to generate cAMP. Cyclic AMP accumulation is maximally stimulated by 100 microM of forskolin with an ED50 at 60 microM. The potency of the cumulus-oocyte complex to respond to forskolin in terms of cAMP accumulation decreases with time. The pattern of the decrease in the potency of the cumulus-oocyte complexes to generate cAMP corresponds with the relief of its inhibitory influence on the oocyte. These results indicate that inhibition of maturation of the cumulus-enclosed oocyte may be coupled to elevation of cAMP levels in the cumulus-oocyte complex. As isolated cumulus-free oocytes are not inhibited by forskolin, we suggest that in the cumulus-enclosed oocyte system, cAMP generated by the cumulus cells is apparently transferred to the oocyte and maintains it in a meiotically arrested state. Maturation in this system occurs upon relief of inhibition which results from cessation of cAMP generation by the cumulus cells.     

Benzyl alcohol increases membrane fluidity and modulates cyclic AMP synthesis in intact renal epithelial cells

To evaluate a possible modulation by membrane fluidity of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of the neutral local anesthetic, benzyl alcohol, on membrane fluidity and on basal and stimulated intracellular cAMP content in intact MDCK cells. Benzyl alcohol induced a dose-dependent decrease of lipid order which was measured by steady-state fluorescence anisotropy using trimethylammonium-diphenylhexatriene and propionyl-diphenylhexatriene as fluorescent probes. Benzyl alcohol induced a 2-fold increase in basal cAMP content, likely as a consequence of increased prostaglandin synthesis since this effect was abolished by indomethacin. The effect of benzyl alcohol on stimulated cAMP synthesis depended on the nature of the ligand: 10 mM benzyl alcohol increased significantly the stimulatory effect of prostaglandin E2, glucagon and forskolin but not of vasopressin. At higher concentrations (40 mM), benzyl alcohol did not affect significantly the glucagon-stimulated cAMP content, while it inhibited significantly the prostaglandin E2-, forskolin- and vasopressin-stimulated cAMP synthesis. The 40 mM benzyl alcohol-induced inhibition was reversed by 1 mM Mn2+, which is known to block the inhibitory GTP-binding protein Ni. These results suggest that: (i) the various components of the adenylate cyclase-cAMP system and their coupling are affected differently by changes in membrane fluidity, which might reflect differences in their lipid environment, (ii) changes in membrane fluidity can modulate responses of renal tubular cells to hormones, and thus tubular functions.     

Evidence for the involvement of cAMP-GEF (Epac) pathway in amylase release from the rat parotid gland

Amylase release from the rat parotid gland is mainly mediated in a cAMP-dependent protein kinase (PKA)-dependent manner. In the present study, amylase release mediated in cAMP-dependent and PKA-independent manners was investigated with a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF: Epac)-selective cAMP analogue, 8CPT-2Me-cAMP. The Epac was localized in the intracellular and the plasma membrane fractions. PKA activation by 8CPT-2Me-cAMP was 100-fold lower than that by cAMP. The amylase release (% of the total) from the intact parotid acinar cells was 16 and 3.6% by isoproterenol (1microM) and 8CPT-2Me-cAMP (200microM), respectively, and that from the saponin-permeabilized cells was 15 and 3% by cAMP (100microM) and 8CTP-2Me-cAMP (10microM), respectively. H-89 inhibited cAMP-induced amylase release, but did not inhibit 8CPT-2Me-cAMP-induced amylase release. These results indicated that amylase release by beta-adrenergic stimulation is mediated through both the cAMP/PKA and cAMP/Epac signal pathways.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Roles of potassium and chloride ions in cAMP-mediated amylase exocytosis from rat parotid acini.

The roles of potassium and chloride ions in cAMP-mediated amylase exocytosis were studied using intact and saponin-permeabilized parotid acini. Cyclic AMP-evoked amylase release from saponin-permeabilized parotid acini decreased markedly when KCl in the incubation medium was isoosmotically replaced by K-glutamate, NaCl, Na-isothionate, or mannitol. Quinidine and barium, K+ channel blockers, clearly inhibited amylase release from the permeabilized acini, but not from intact ones. The chloride channel blocker DPC (diphenylamine-2-carboxylate) also inhibited amylase release, while DIDS (4,4'-diisothiocyanostilben-2,2'-disulfonate) or bumetanide had little effect, if any, on the exocytosis. Hyperosmolarity with mannitol markedly reduced amylase release from permeabilized acini. These results suggest that potassium and chloride ions play important roles in cAMP-mediated amylase exocytosis, and that these ions act on secretory granules inside the acinar cells.