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PubMed1984

Deletion analysis of the CAP-cAMP binding site of the Escherichia coli lactose promoter.

S1 nuclease was used to generate a series of deletions which extend into the CAP-cAMP binding site from upstream of the Escherichia coli lactose operon promoter (lacP). Deletion and insertion mutations were also created which changed the spacing region between the CAP-cAMP binding site and the lacP -35 region. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. The results show that sequence information prior to 74 base pairs (-74) upstream from the transcription start site (designated as +1) is not necessary for the full activation of the lac promoter by the CAP-cAMP complex. However, the deletion which extends to the -71 position retains only one third of the promoter activity in the presence of the CAP-cAMP complex. Removal of one symmetrical element from the two fold symmetry in the CAP-cAMP binding site abolished the CAP-cAMP stimulation of the lac promoter. Spacer mutations which increase by one base pair or decrease by two base pairs the length of the spacing region between the CAP-cAMP binding site and the lacP -35 region drastically reduced the CAP-cAMP stimulation of the lac promoter. This suggests that the distance between the lac promoter transcription start site and CAP-cAMP binding site is crucial for the function of the lac promoter, despite the fact that this distance varies in other E. coli promoters positively regulated by CAP-cAMP. A deletion which extends to the -59 position results in a two fold enhanced expression of lac in the absence of CAP-cAMP. This is consistent with the existance of a competitive RNA polymerase binding site in this region which would normally act to inhibit RNA polymerase binding.

Base Sequence↗

PubMed1998

Ketamine inhibits the proinflammatory cytokine-induced reduction of cardiac intracellular cAMP accumulation.

UNLABELLED: The proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), are increased in heart failure and sepsis, clinical conditions for which the IV anesthetic ketamine is useful. The proinflammatory cytokines cause beta-adrenergic receptor (betaAR) hypofunction secondary to reduced function of the enzyme adenylylcyclase (AC). In this study, we evaluated the effect of ketamine alone, TNF-alpha and IFN-gamma, and ketamine plus TNF-alpha and IFN-gamma, on isoproterenol (ISO, a betaAR agonist) and forskolin (FSK, an activator of AC)-induced intracellular accumulation of cAMP. An in vitro culture of a rat heart cell line (H9c2) was labeled with [3H]adenine to produce [3H]ATP, and we measured the intracellular accumulation of [3H]cAMP after stimulation with ISO or FSK to convert the [3H]ATP to [3H]cAMP. Pretreatment with either cytokine alone did not significantly affect ISO or FSK-induced intracellular cAMP accumulation, whereas the combination of TNF-alpha and IFN-gamma caused a significant (P < 0.05 compared with untreated cells) reduction. Pretreatment with ketamine caused a significant (P < 0.05 compared with untreated cells) increase in ISO or FSK-induced cAMP accumulation. Pretreatment of the H9c2 cells with ketamine, plus the combination of TNF-alpha and IFN-gamma, inhibited the reduction of ISO or FSK-induced intracellular cAMP accumulation caused by the proinflammatory cytokines alone. These results demonstrate that the combination of the proinflammatory cytokines TNF-alpha and IFN-gamma reduce poststimulation (ISO or FSK) intracellular cAMP accumulation. This action of the proinflammatory cytokines is consistent with the observation of betaAR hyporesponsiveness to betaAR agonist therapy in sepsis and heart failure. Ketamine augments the poststimulation cAMP accumulation in H9c2 cells while inhibiting the cytokine-induced reduction of cAMP accumulation. This may partly explain the improvement in cardiac function after ketamine use in clinical conditions known to have increased systemic levels of proinflammatory cytokines, such as sepsis and heart failure. IMPLICATIONS: Tumor necrosis factor-alpha and interferon-gamma reduced poststimulation intracellular cAMP levels, whereas ketamine inhibits this action of the proinflammatory cytokines. Because cAMP is the second messenger for the beta-adrenergic receptor, this may be a mechanism for improved blood pressure and cardiac output in sepsis and heart failure after ketamine use.

Adrenergic beta-Agonists↗

PubMed1985

Is there a role for cAMP and adenyl cyclase?

The validity of the hypothesis that cyclic AMP (cAMP) is the 2nd messenger for cell activation was reexamined. Some enzymological aspects of adenyl cyclase (AC) should cause concern: the lack of data on the stoichiometry of cAMP formation in mammalian systems and the unusual enzymatic properties, as well as lack of end-product inhibition and linearity. Furthermore, adenyl cyclase assays may lack precision and accuracy; this may depend on the organ studied. There are also problems of artefactual formation of cAMP during the work-up of cAMP extracts. Thus CrP, Pi, or ATP might influence this process and the actual measurement of cAMP, but solid data are apparently not available. Although a hormone-sensitive AC system has now been reconstituted from pure beta-adrenergic receptor, guanine nucleotide regulatory protein (Ns), and from bovine brain, the sensitivity to isoprenaline was very low and many questions remain--questions about the role of ions and Ns, in particular. The assumption that cAMP is the sole 2nd messenger is questioned since other nucleotides (AMP, ADP) and adenosine may change even more during hormone stimulation, and these compounds can also modulate protein kinase at concentrations often observed in vivo. Doubt over cAMP's role also stems from the observation that basal cAMP levels are sufficiently high to stimulate maximally protein kinase. Discrepancies between cAMP formation and lipolysis during isoprenaline or forskolin stimulation have been observed, and could indicate either compartmentalization of cAMP or alternatively disprove the cAMP hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1999

Elevated cAMP suppresses muscarinic inhibition of L-type calcium current in guinea pig ventricular myocytes.

We investigated the effect of carbachol (CCh) on L-type Ca2+ current (ICa(L)) enhanced by dialyzed adenosine 3',5'-cyclic monophosphate (cAMP) and/or bath-applied 3-isobutyl-1-methylxanthine (IBMX) in guinea pig isolated ventricular myocytes. At pipette concentrations ([cAMP]pip) from 30 microM to 1 mM, cAMP increased ICa(L) to 25.8 +/- 0.9 microA/cm2 (682 +/- 24.8% increase above control). CCh (100 microM) did not inhibit ICa(L) at any [cAMP]pip. IBMX, a nonselective phosphodiesterase (PDE) inhibitor, increased ICa(L) maximally at 300 microM IBMX (17.9 +/- 0.7 microA/cm2; 449 +/- 20% increase). CCh (100 microM) inhibited ICa(L) by 92 +/- 9.5% at 30 microM IBMX and 78 +/- 4.6% at 100 microM IBMX; this effect was reduced or absent at higher IBMX concentrations (300 and 1,000 microM). Coadministration of cAMP and IBMX also progressively suppressed inhibition by CCh. CCh had a negligible effect on ICa(L) at 750 microM IBMX in the absence of pipette cAMP and at 50 microM IBMX in the presence of 100 microM [cAMP]pip. ACh-activated K+ current (IK(ACh)) was unchanged in atrial myocytes dialyzed with 100 microM cAMP; this excludes a phosphorylation-dependent desensitization of the muscarinic receptor (mAChR) or Gi by cAMP. LY83583 (100 microM), an inhibitor of cyclic guanosine monophosphate (cGMP) production, attenuated inhibition of ICa(L) by CCh in the presence of IBMX. 8-Bromo-cGMP (8-Br-cGMP), an activator of cGMP-dependent protein kinase (PKG), mimicked CCh in its actions on ICa(L) raised by both cAMP (no significant change) and IBMX (49 +/- 5.1% inhibition). Okadaic acid, an inhibitor of type 1 and 2A phosphatases, blocked inhibition of IBMX-stimulated ICa(L) by either CCh or 8-Br-cGMP. Thus the ability of CCh to inhibit ICa(L) appears caused by cGMP/PKG activation of an okadaic acid-sensitive protein phosphatase, and elevated levels of cAMP protect against this action.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA).

Cell movement and cell-type-specific gene expression during Dictyostelium development are regulated by cAMP, which functions both as an extracellular hormone-like signal and an intracellular second messenger. Previous data indicated that aca- mutants, which lack adenylyl cyclase activity, fail to aggregate and do not express cell-type-specific genes. We show here that overexpression of ACG, a constitutively active adenylyl cyclase, which in wild-type cells is only expressed during spore germination, partially restores the coordination of cell movement and completely restores developmental gene expression. The aca- cells can also be induced to develop into viable spores by synergy with wild-type cells and, furthermore, form small but normal fruiting bodies, after a developmentally relevant regimen of stimulation with nanomolar cAMP pulses followed by micromolar cAMP concentrations. 2'-Deoxy cAMP, a cAMP analog that activates the cell-surface cAMP receptors but not cAMP-dependent protein kinase (PKA), also induces fruiting body formation as well as expression of prespore-specific and prestalk-enriched genes in aca- cells. Intracellular cAMP levels were not altered in aca- cells after stimulation with 2'-deoxy cAMP. Our data indicate that ACA is not required to provide intracellular cAMP for PKA activation but is essential to produce extracellular cAMP for coordination of cell movement during all stages of development and for induction of developmental gene expression.

Adenylyl Cyclases↗

PubMed1976

Relation between the inhibition of aggregation and the concentration of cAMP in human and rat platelets.

Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.

Adenosine↗

PubMedDate not supplied

Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.

Carrier Proteins↗

PubMed1983

Inhibition of prostaglandin biosynthesis in renal (MDCK) cells by cAMP.

Cultured renal tubular cells (MDCK) have many of the biological properties of renal medullary tubular epithelial cells, including the ability to synthesize prostaglandin E2 (PGE2) as the major arachidonate metabolite. The hypothesis that adenosine 3',5'-cyclic monophosphate (cAMP) regulates prostaglandin synthesis in these cells was investigated by using cAMP, two degradation-resistant cAMP analogues [8-bromo-cAMP (8-BrcAMP) and N6,O2'-dibutyryl cAMP (DBcAMP)], and a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). These agents inhibited basal-, calcium ionophore (A23187)-, or bradykinin-stimulated PGE2 biosynthesis by MDCK cells. The observed inhibition was dose- and time-dependent and could be reversed after 30 min of incubation in the absence of inhibitor. IBMX dose-dependently increased intracellular and extracellular cAMP levels by severalfold, suggesting that it was inhibiting prostaglandin biosynthesis by increasing cellular cAMP levels. Vasopressin, which stimulated cAMP levels by less than two-fold, did not inhibit prostaglandin synthesis. 8-BrcAMP and N6,O2'-DBcAMP inhibited A23187- or bradykinin-stimulated release of [3H]arachidonate from prelabeled cells, suggesting that cAMP inhibited acylhydrolase activity. Moreover, 8-BrcAMP also inhibited the conversion of exogenous arachidonate to PGE2 in intact cells and in a subcellular fraction containing prostaglandin synthetase activity, suggesting that cAMP inhibited cyclooxygenase and/or PGE2 isomerase activity. cAMP thus appears to regulate prostaglandin biosynthesis in MDCK cells by modulating the activity of two or more of the enzymes involved in the biosynthetic process.

1-Methyl-3-isobutylxanthine↗

PubMed1983

Cholinergic potentiation of isoproterenol-induced cAMP level in sweat gland.

Although methacholine (MCh) and a Ca2+ ionophore A23187 do not enhance the tissue adenosine 3',5'-cyclic monophosphate (cAMP) level by themselves, they markedly potentiate isoproterenol (ISO)-induced tissue cAMP accumulation in isolated simian eccrine sweat glands in a dose-dependent manner. The agonist concentration producing 50% of the maximal response of such a potentiated cAMP accumulation was 2.1 X 10(-7) M for MCh, 2.5 X 10(-7) M for ISO, and 2.9 X 10(-6) M for A23187. Unlike cAMP accumulation induced by ISO alone, MCh-stimulated ISO-induced cAMP accumulation is dependent on extracellular Ca2+. MCh- plus ISO-induced cAMP level is tripled by 10(-2) M theophylline (TH), and while the ISO-induced cAMP level was also elevated by TH, it was not enhanced to the level of ISO-plus MCh-induced cAMP accumulation, indicating that phosphodiesterase inhibition is not the major mechanism for the augmentative effect of MCh. The augmentative effect of MCh was seen only in the secretory portion, whereas that of A23187 was seen in both the secretory portion and the duct. The data suggest that MCh-induced augmentation of ISO-stimulated cAMP accumulation is due to increased cAMP formation, not decreased cAMP degradation, and that it may be mediated by an elevated intracellular Ca2+.

Animals↗

PubMed1986

Eicosonoid metabolism and beta-adrenergic mechanisms in coronary arterial smooth muscle: potential compartmentation of cAMP.

Beta-Adrenergic relaxation in bovine coronary arteries is enhanced by inhibition of eicosonoid metabolism and inhibited by its stimulation. We investigated the interaction between eicosonoid metabolism and beta-adrenergic mechanisms by studying the effect of perturbations of eicosonoid metabolism on vascular adenosine 3',5'-monophosphate (cAMP) content and the cAMP-dependent relaxation of isometric force and activation of glycogen phosphorylase. KCl (35 mM) elicited a contraction, activated phosphorylase, and slightly decreased cAMP content. Isoproterenol (10(-7) M) relaxed the KCl contraction, further increased phosphorylase activity, and increased cAMP. Neither indomethacin (5 X 10(-6) M) nor arachidonic acid (3 X 10(-5) M) affected the KCl contraction, but arachidonic acid increased both cAMP and phosphorylase activity and indomethacin decreased cAMP. Indomethacin potentiated the relaxation induced by isoproterenol but inhibited the activation of phosphorylase and had no effect on the isoproterenol-induced increase in cAMP. Arachidonic acid, on the other hand, inhibited the isoproterenol-induced relaxation but potentiated both the increases of phosphorylase activity and cAMP. Thus neither relaxation nor phosphorylase activity was related in a straightforward manner to the total cAMP content. A direct relation between cAMP, relaxation, and phosphorylase can be reconciled with the antiparallel effects of alterations of eicosonoid metabolism observed in this study by a proposed model in which the effects of cAMP are assumed to be functionally compartmentalized.

Animals↗

PubMed1992

cAMP-dependent protein kinase mediates hydrosmotic effect of vasopressin in collecting duct.

The role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in mediating the hydrosmotic effect of vasopressin in in vitro microperfused rabbit cortical collecting ducts (CCDs) was examined. We measured PKA substrate phosphorylation and water permeability [hydraulic conductivity (Lp) = 10(-7) cm.atm-1.s-1], stimulated by substituted cAMP analogues selective for a unique cAMP binding site (site A or B) on PKA regulatory subunit (R). Synergy between site A- and site B-selective analogues suggests involvement of PKA, because both sites must be occupied for R to dissociate from the catalytic subunit (C), allowing phosphorylation to proceed. As single agents, the site B-selective analogues 8-(4-chlorophenylthio)-cAMP (8-CPT) and 8-thiomethyl-cAMP (8-SCH3) were at least two orders of magnitude more potent than the site A-selective analogues N6-monobutyryl-cAMP (N6-mono) or N6-benzoyl-cAMP (N6-benz). Combinations of subthreshold concentrations of two site A analogues (N6-mono+N6-benz) or two site B-selective analogues (8-CPT + 8-SCH3) failed to significantly increase protein phosphorylation or water permeability. In contrast, combination of a site A plus site B analogue synergistically stimulated both protein phosphorylation and Lp. Rp-cAMPS, an inhibitor of cAMP binding to PKA, reduced both vasopressin (41% inhibition)- and cAMP (56% inhibition)-stimulated water permeability. H-89 (50 microM), an inhibitor of PKA kinase activity, also blocked cAMP-stimulated water permeability (90% inhibition). These findings suggest that vasopressin-induced water permeability in the rabbit CCD is mediated by PKA.

Animals↗

PubMed1996

cAMP- and swelling-activated chloride conductance in rat hepatocytes.

An outwardly rectifying Cl- conductance was identified in primary isolated rat hepatocytes, and the whole cell patch-clamp technique was used to characterize its properties and mechanisms of activation. With symmetrical Cl(-)-containing solutions on both sides and adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM) in the pipette solution, a large outwardly rectifying conductance (1,014 +/- 153 pS/pF, n = 20) developed in all cells within 3 min. This cAMP-activated conductance was highly anion selective and slowly inactivated at voltages > 80 mV. It was completely inhibited by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM, n = 6) and partially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM, n = 7). It displayed a halide selectivity of I- > Br- > Cl-. In the absence of cAMP, a functionally similar conductance was activated by cell swelling. Reduction of bath osmolality from 300 to 250 mosmol/kg increased membrane conductance from 64 +/- 16.4 to 487 +/- 23 pS/pF (n = 4). This swelling-activated conductance was also highly anion selective and had identical halide selectivity and blocker sensitivity as the cAMP-activated conductance. Although cell swelling was not necessary for cAMP activation, cell shrinkage with hyperosmotic bath (350 mosmol/kg), either before or after exposure to cAMP, inhibited the cAMP-activated conductance. By the determination of conductance as a function of bath osmolality in the presence and absence of cAMP, it was observed that cAMP shifted the osmotic set point for conductance activation without changing either the maximum or minimum conductance. In conclusion, both cAMP and cell swelling activate a large outwardly rectifying Cl- conductance in rat hepatocytes. Its ionic selectivity and sensitivity to channel blockers are identical to those seen for swelling-activated Cl- conductances in many cell types. The conductive properties are not those of cystic fibrosis transmembrane conductance regulator-mediated Cl- conductance. cAMP appears to activate this conductance by altering the volume set point of a swelling-activated channel.

Animals↗

PubMed2002

Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP.

cAMP-mediated stimulation of hepatic bile acid uptake is associated with dephosphorylation and translocation of Na+-taurocholate (TC) cotransporting peptide (NTCP) to the plasma membrane. Although translocation of NTCP may be facilitated by dephosphorylation, the mechanism of dephosphorylation is unknown. The ability of cAMP to translocate and dephosphorylate NTCP is, in part, dependent on cAMP-mediated increases in cytosolic Ca2+ concentration ([Ca2+]), indicating that a Ca2+/calmodulin-dependent protein phosphatase (PP2B) may be involved. Thus we studied the role of PP2B using the inhibitor cypermethrin (CM). Freshly isolated hepatocytes were pretreated with 1-5 nM CM for 30 min followed by 15 min incubation with 10 microM 8-(4-chlorophenylthio)cAMP. CM (5 nM) and FK-506 (5 microM) inhibited cAMP-stimulated TC uptake by 80 and 75%, respectively, without affecting basal TC uptake. CM also reversed cAMP-mediated NTCP dephosphorylation and translocation to 80 and 15% of the basal level, respectively. cAMP stimulated PP2B activity by 60%, and this effect was completely inhibited by 5 nM CM. PP2B dephosphorylated NTCP immunoprecipitated from control but not from cAMP-treated hepatocytes. The effect of CM was not due to any changes in cAMP-mediated increases in cytosolic [Ca2+] or decreases in mitogen-activated protein kinase (extracellular regulated kinases 1 and 2) activity. Taken together, these results suggest that cAMP dephosphorylates NTCP by activating PP2B in hepatocytes, and PP2B-mediated dephosphorylation of NTCP may be involved in cAMP-mediated NTCP translocation to the plasma membrane.

Animals↗

PubMed1980

Effect of glucagon on bile cAMP secretion.

The effect of bile flow and adenosine 3',5'-cyclic monophosphate (cAMP) secretion of substances that alter cAMP metabolism in other systems was evaluated. The experiments were performed on awake dogs with chronic biliary and gastric fistulas and on anesthetized rabbits prepared with acute bile fistulas. In all experiments the enterohepatic circulation was maintained by intravenous bile salt administration. In dogs, glucagon increased bile flow and bile cAMP secretion with 4 micrograms . kg-1 . h-1 increasing bile flow from control values of 273 +/- 27 to 413 +/- 6 microliters/min, whereas bile cAMP concentration rose from 3.9 +/- 0.9 to 12.1 +/- 1.1 nmol/ml. Theophylline in dogs increased bile flow while cAMP secretion decreased. Evaluation of the effects of theophylline on glucagon-stimulated bile flow suggest that both agents act by the same mechanism; however, theophylline did not significantly alter glucagon-stimulated increases in bile cAMP. In rabbits, glucagon increased bile cAMP secretion while bile flow was not significantly changed. Hormonal production of increased systemic or hepatic cAMP can increase bile cAMP unassociated with changes in bile flow. Based on the measurement of cAMP in bile the increase in glucagon-stimulated bile flow produced by theophylline is not cAMP mediated.

Animals↗

PubMed1982

Histamine, cAMP, and activation of piglet gastric mucosa.

The involvement of cAMP as a second messenger for histamine-induced H+ secretion was studied in a physiologically active, in vitro preparation of piglet gastric mucosa. During the first 5--10 min of stimulation with either histamine or the cAMP phosphodiesterase inhibitor 3-isobutyl-1,4-methylxanthine (IBMX), increases (greater than or equal to 5-fold) in tissue cAMP content [(c-AMP]) were well correlated with the characteristic decrease in transepithelial resistance (R); these changes precede H+ secretion by several minutes. Control experiments indicate that, during these treatments, tissue [cAMP] is dominated by the [cAMP] of oxyntic cells alone; change in R and H+ are also related to activity of these cells alone. At the steady state (45 min), histamine and IBMX caused equivalent increases in H+ and decreases in R, but [cAMP] was markedly different in the two cases. With IBMX [cAMP] was elevated at least fivefold, whereas with histamine [cAMP] was less than or equal to 50% above resting levels. The tissue is also stimulated by exogenous additions of dibutyryl cAMP. A histamine-sensitive adenylate cyclase was present in isolated, purified oxyntic cells. The histamine sensitivity of the cyclase was very similar to that which the intact tissue exhibits for histamine-induced changes in H+ and R. The cyclase activity was blocked by cimetidine but not by promethazine. We conclude that during stimulation histamine activates a histamine (H2)-sensitive adenylate cyclase of oxyntic cells, and there is a rapid increase in cellular [cAMP] that is involved in activation of H+ transport and other associated changes of oxyntic cells. An active phosphodiesterase is responsible for reducing [cAMP] to a level much below the "peak" value. Other cellular factors (e.g. protein kinases and Ca2+-calmodulin) must also be involved in the maintenance of the stimulated state of oxyntic cells.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Mechanisms of cAMP-mediated relaxation of distal circular muscle in rabbit colon.

Photolytic release of free adenosine 3',5'-cyclic monophosphate (cAMP) from its caged form was used to evaluate the physiological role of several proposed mechanisms of cAMP-mediated relaxation of circular smooth muscle in the distal rabbit colon. Photolysis of caged cAMP produced a rapid relaxation of bethanechol-contracted distal circular muscle strips that was dependent on ultraviolet exposure time. An increase in release of free cAMP, associated with increased ultraviolet exposure, was confirmed with high-performance liquid chromatography. Vanadate (an ATPase inhibitor) (3 mM) caused a 48% decrease in cAMP-mediated relaxation, while ouabain and a zero K+ bath solution failed to affect relaxation. cAMP-mediated relaxation of KCl-contracted strips was significantly less effective than that of bethanechol-contracted strips. Although this finding suggested that cAMP-mediated relaxation may involve K+ channel modulation, specific (glibenclamide, charybdotoxin) and nonspecific (TEA) K+ channel blockade failed to affect cAMP-mediated relaxation of bethanechol-contracted strips. The photolytic release of cAMP failed to relax Ca(2+)-contracted saponin skinned muscle strips. These studies suggest 1) modulation of Ca2+ pumps plays an important role in this model of relaxation of distal colonic circular muscle in the rabbit colon, 2) modulation of the Na+ pump or sarcolemmal K+ channels may not play an important physiological role in relaxation induced by a rapid rise in intracellular cAMP, and 3) cAMP does not seem to have a significant physiological effect on the Ca2+ sensitivity contractile apparatus.

Animals↗

PubMed1996

Differential regulation of hepatocyte DNA synthesis by cAMP in vitro in vivo.

Adenosine 3',5'-cyclic monophosphate (cAMP) prevents epidermal growth factor (EGF)-induced DNA synthesis in many types of cultured cells, including hepatocytes, but its effects on cellular proliferation in vivo are unknown. This study compares the effects of supplemental cAMP on hepatocyte proliferation induced in vivo by 70% partial hepatectomy (PH) and in vitro by EGF and determines the effects of cAMP on AP-1, a family of growth-regulatory transcription factors, and the kinase cascades that normally activate AP-1. Although injection of dibutyryladenosine 3',5'-cyclic monophosphate (30 mg/kgip) at the time of PH increased liver cAMP concentrations at least 100-fold for several hours, it did not inhibit hepatic incorporation of [3H]thymidine or proliferating cell nuclear antigen expression 24 h after PH. cAMP treatment led to a complete inhibition of extracellular signal-related kinase (ERK) activity and transiently reduced NH2-terminal Jun nuclear kinase (JNK) activity after PH but did not decrease the expression of c-jun mRNA or protein. Consistent with the known cAMP stimulation of jun-B in cultured cells, cAMP treatment increased jun-B mRNA, protein, and DNA binding activity post-PH. Surprisingly, cAMP treatment enhanced Raf kinase activity after PH in rats. In primary hepatocyte cultures, supplemental cAMP inhibited JNK and ERK activity, total AP-1 and c-Jun transcriptional activities, and DNA synthesis. Thus elevated cAMP inhibited ERK and JNK activity in culture and in vivo and inhibited hepatocyte proliferation in culture but not in vivo. This suggests that in vivo mechanisms compensate for cAMP inhibition of certain growth-related signaling cascades and emphasizes potential risks of extrapolating from simple cell culture systems to explain physiology in intact animals.

Animals↗

PubMed1999

Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp.

cAMP stimulates Na(+)-taurocholate (TC) cotransport by translocating the Na(+)-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70(S6K) activity. However, rapamycin, an inhibitor of p70(S6K), failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70(S6K). Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

Androstadienes↗