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PubMed1986

cAMP augmentation of secretagogue-induced luteinizing hormone secretion.

Whether adenosine 3',5'-cyclic monophosphate (cAMP) acts as a mediator for luteinizing hormone-releasing hormone (LHRH) in either its immediate LH release action or in its self-priming action was investigated. Pituitary pieces from either proestrous or estrous rats were superfused in vitro in the presence of dibutyryl cAMP [(Bu)2cAMP], 8-bromo-cAMP (8BrcAMP), forskolin, or control for 2-3 h. For proestrous but not estrous pituitary pieces, a slight increase in base-line LH secretion rate occurred at approximately 70 min of exposure to elevated cAMP; in the same system LHRH caused an increase in LH secretory rate within 2 min in either proestrous or estrous tissue. In contrast to its ineffectiveness as a secretagogue, cAMP elevation resulted in a severalfold augmentation of both LHRH- and elevated K+-induced LH secretion from proestrous but not estrous pituitary pieces; for these experiments, superfusion with a cAMP analogue or forskolin for varying times preceded a 10-min pulse of either 8 nM LHRH or 47 mM K+. Augmentation was evident after 30 min of cAMP elevation; longer exposures were coincident with greater potentiation. Cycloheximide prevented (Bu)2cAMP augmentation of LHRH-induced secretion. These data show that cAMP does not mediate the immediate LH release action of LHRH, but cAMP does augment LHRH- or K+-induced LH secretion with characteristics in common with the self-priming action of LHRH.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Stretch-related changes in lung cAMP after partial pneumonectomy.

In rats, left pneumonectomy (PNX) initiates rapid compensatory hyperplastic growth of the right lung. Previous work indicated that increased polyamine uptake associated with post-PNX distortion or "stretch" of the remaining tissue and altered adenosine 3',5'-cyclic monophosphate (cAMP) metabolism may play a role in the early phase of the growth response. Evidence was sought to link these observations with the goal of understanding control of compensatory growth. At day 1 or 3 after left PNX in vivo, increased right lung cAMP was associated with activation of the cAMP-dependent protein kinase (PKa). Total PKa activity was unaffected, but the PKa activity ratio (-cAMP/+cAMP) doubled, providing evidence for PNX-associated activation of the kinase in vivo. Neither cAMP nor PKa was altered in sham-operated control lungs. To determine whether increased distension of the right lung after PNX might initiate these changes, lungs of unoperated animals were perfused 40 min in vitro, either without ventilation or with a distending constant positive airway pressure (CPAP) of 20 cmH2O. CPAP rapidly increased uptake of the polyamine spermidine (SPD; 1.5 microM), tissue cAMP, and the PKa activity ratio, in a manner similar to that observed after PNX. Both tissue cAMP and the kinase activity ratio increased with 1 microM forskolin (FSK), as expected, but SPD uptake was unaffected by FSK. FSK did not diminish the CPAP-associated elevations of SPD uptake (P less than 0.01) or cAMP (P less than 0.05), but the kinase was not further activated by CPAP with FSK present.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Expression of 25(OH)D3 24-hydroxylase in distal nephron: coordinate regulation by 1,25(OH)2D3 and cAMP or PTH.

Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1-34); 10 nM] was found to potentiate the effect of 1, 25(OH)2D3 on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1, 25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1, 25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3 in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Activation of protein kinase activity in pancreatic acini by calcium and cAMP.

Regulation of protein kinase activity by calcium and cAMP was investigated in cytosolic and particulate preparations from isolated mouse pancreatic acini. In cytosol, three protein kinase activities could be distinguished: a calcium-activated kinase activity that was increased by exogenous calmodulin (CaM) and abolished by treatment of cytosol with a phenothiazine-coupled resin, a calcium-activated kinase activity dependent on phosphatidylserine (PS), and cAMP-activated kinase activity. Phosphorylation of a Mr = 92,000 cytosolic protein was greatly increased by both CaM-dependent and cAMP-activated kinases, whereas PS-dependent kinase activity most heavily phosphorylated proteins of Mr = 62,000 and 40,000. In addition, these kinase activities demonstrated differences in specificity for exogenous protein substrates. CaM-and PS-dependent kinases were completely blocked by trifluoperazine; the inhibitor protein of cAMP-activated protein kinase selectively inhibited cAMP-activated kinase activity. Exogenous CaM decreased the concentration of free calcium for half-maximal activation of CaM-dependent kinase activity from 5.5 +/- 0.5 to 1.2 +/- 0.3 microM Ca2+; half-maximal activation of the PS-dependent and cAMP-activated kinase activities was achieved at 12 microM Ca2+ and 40-50 nM cAMP, respectively. In a particulate fraction depleted of endogenous CaM, CaM-dependent and cAMP-activated kinase activities were detected. In conclusion, this study demonstrates the existence of protein kinases in pancreatic acini which may be involved in the action of pancreatic regulatory agents that use calcium or cAMP as an intracellular messenger.

Animals↗
PubMed1987

Somatostatin inhibits cAMP-mediated cholinergic transmission in the myenteric plexus.

The mechanism by which somatostatin acts to modulate cholinergic transmission is not clear. In this study we investigated the role of the adenosine 3',5'-cyclic monophosphate (cAMP) system in mediating cholinergic transmission in the guinea pig myenteric plexus and examined the ability of somatostatin to alter acetylcholine (ACh) release stimulated by various cAMP agonists. Forskolin, 8-bromo-cAMP, vasoactive intestinal peptide (VIP), and cholera toxin each stimulated the release of [3H]ACh in a dose-related manner. Addition of theophylline enhanced the release of [3H]ACh stimulated by these cAMP agonists. In contrast 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, antagonized the action of forskolin, VIP, and cholera toxin but had no effect on that evoked by 8-bromo-cAMP. These observations suggest that cAMP may serve as a physiological mediator for ACh release from myenteric neurons. Somatostatin inhibited release of [3H]ACh evoked by various cAMP agonists in a dose-related manner. Maximal inhibition, observed in the presence of 10(-6) M somatostatin was 48 +/- 5, 47 +/- 9, and 43 +/- 12% of control for forskolin-, VIP-, and cholera toxin-evoked release of [3H]ACh. In contrast somatostatin at 10(-6) M inhibited only 20 +/- 5% of the release of [3H]ACh stimulated by 8-bromo-cAMP. Pretreatment with pertussis toxin antagonized the inhibitory effect of somatostatin on the release of [3H]ACh evoked by forskolin, VIP, or cholera toxin but had no effect on the inhibitory action of somatostatin on the release of [3H]ACh evoked by 8-bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Gastrin and carbachol require cAMP to elicit aminopyrine accumulation in isolated pig and rat parietal cells.

The role of endogenous adenosine 3',5'-cyclic monophosphate (cAMP) in the mechanisms of action of gastrin and carbachol on aminopyrine accumulation in isolated pig and rat parietal cells was investigated. In pig cells, pentagastrin (100 nM) alone stimulated aminopyrine accumulation, an action significantly reduced by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMP[S]; 100 microM). In rat cells, gastrin-17 (100 nM) was incapable of stimulating aminopyrine accumulation, but it potentiated the action of histamine (100 microM). Carbachol (10 microM) stimulated aminopyrine accumulation and potentiated the action of histamine, and its action was potentiated in a dose-dependent manner by Sp-adenosine 3',5'-cyclic monophosphothioate (Sp-cAMP[S]; a cAMP analogue) in both species. The effect of carbachol was dose dependently reduced by Rp-cAMP[S]. The basal cAMP in pig parietal cells was 3.5-fold higher than that in rat parietal cells. Histamine (100 microM) and 3-isobutyl-1-methylxanthine (IBMX; 100 microM) only slightly elevated the cAMP content (1.2- to 2.9-fold the basal level) in both pig and rat parietal cells. Their combination, however, increased the cAMP level by 8- to 38-fold, but it did not increase aminopyrine accumulation above that elicited by histamine alone. Gastrin did not alter the cAMP levels in parietal cells of either of the two species. Both gastrin and carbachol increased cytosolic free Ca2+ in enriched pig and rat parietal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopyrine↗
PubMed1995

EGF inhibits secretagogue-induced cAMP production and amylase secretion by Gi proteins in pancreatic acini.

In pancreatic acinar cells, the epidermal growth factor (EGF) receptor interacts with both cholera toxin- and pertussis toxin (PTX)-sensitive G proteins. In the present study, isolated rat pancreatic acini were used to investigate the effect of EGF on basal and secretagogue-induced adenosine 3',5'-cyclic monophosphate (cAMP) production and amylase release. EGF increased cAMP production and amylase release in pancreatic acini. However, cAMP accumulation and amylase release elicited by either vasoactive intestinal peptide (VIP) or forskolin were inhibited by EGF (17 nM). EGF inhibited the VIP-induced cAMP production and amylase release with a half-maximal effective concentration of 3 and 2 nM, respectively. EGF had no effect on the N6,2'-O-dibutyryladenosine-3',5'-monophosphate-stimulated amylase release, suggesting that the inhibitory effect of EGF on the VIP- and forskolin-induced cAMP production is due to inhibition of adenylyl cyclase. PTX pretreatment of the acini led to an increase of the basal, EGF-, and VIP-stimulated cAMP accumulation and amylase release, indicating that PTX-sensitive G proteins exert tonic inhibition of adenylyl cyclase even in the absence of agonist. In PTX-pretreated acini, the inhibitory effect of EGF on the VIP-induced cAMP production and amylase release was abolished. In conclusion, these results suggest that EGF inhibits secretagogue-induced cAMP production via activation of PTX-sensitive G proteins in rat pancreatic acini, whereas EGF-induced cAMP production and amylase release occurs via a PTX-insensitive pathway.

Adenylate Cyclase Toxin↗
PubMed1997

Effect of cholestasis on regulation of cAMP synthesis by glucagon and bile acids in isolated hepatocytes.

Previously, we have reported that bile acids can directly inhibit hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation through a protein kinase C (PKC)-dependent mechanism [Bouscarel, B., T.W. Gettys, H. Fromm, and H. Dubner. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. Therefore, the regulation of cAMP synthesis by glucagon and bile acids was investigated in hepatocytes isolated after 2-day ligation of the common bile duct in Golden Syrian hamsters. The bile acid concentration was increased 30-fold in the serum, whereas it was not significantly different in the bile of duct-ligated vs. sham-operated hamsters. The glycine/taurine and cholate/chenodeoxycholate ratios were significantly increased fourfold and sevenfold, respectively, only in the serum of bile duct-ligated hamsters. Ligation of the bile duct decreased the efficacy of glucagon-stimulated cAMP synthesis by 40-50% without changing its potency. This attenuation of cAMP synthesis, which was also observed with forskolin, remained in the absence of any detectable amount of bile acids in the hepatocytes. The decrease in glucagon-stimulated cAMP production was also not attributable to changes in either the affinity or the number of receptors for this hormone. The potency and efficacy of the bile acids to inhibit glucagon-induced cAMP formation was also reduced in bile duct-ligated hamsters. The inhibitory regulation of cAMP synthesis through angiotensin II was similarly diminished after bile duct ligation. Although the total expression of PKC-alpha was not affected, an increased translocation by 60% from the cytosol to the membrane fraction was observed in hepatocytes isolated after bile duct ligation. Therefore, during cholestasis and prolonged exposure of the liver to bile acids, both the stimulatory and inhibitory regulatory, mechanisms of cAMP synthesis are compromised in an irreversible manner because the effects persist even after isolation of the hepatocytes. This decreased regulation of cAMP synthesis is possibly mediated through PKC-alpha activation.

Angiotensin II↗
PubMed1989

Dopamine stimulates cAMP production in canine afferent arterioles via DA1 receptors.

Dopamine increases renal blood flow and dilates isolated afferent and efferent arterioles preconstricted with norepinephrine via dopamine 1 (DA1) receptors. DA1-receptor stimulation also results in dopamine-induced elevation of adenosine 3'5'-cyclic monophosphate (cAMP) in dog and rat renal arteries. The present study was undertaken to determine the effects of dopamine on cAMP accumulation in isolated canine superficial cortical afferent arterioles. The effect of Sch 23390, a specific DA1-receptor antagonist, on dopamine-stimulated cAMP accumulation was also examined. Forskolin (10(-5) M), a potent stimulator of adenylate cyclase, produced a greater than 11-fold increase in cAMP production compared with control. Dopamine produced a dose-dependent increase in cAMP accumulation in afferent arterioles at concentrations of 10(-4) M and 10(-6) M, Sch 23390 (2 x 10(-4) M) abolished dopamine (10(-4) M)-stimulated cAMP accumulation in afferent arterioles. The dopamine-induced increase in arteriolar cAMP accumulation was unaffected by propranolol (10(-4) M). Our results suggest that dopamine increases cAMP production in afferent arterioles via the DA1 receptor. Increased cAMP production may be responsible for dopamine-induced vasodilation in the afferent arteriole.

Animals↗
PubMed1993

Angiotensin II enhancement of hormone-stimulated cAMP formation in cultured vascular smooth muscle cells.

The mechanism by which angiotensin II (ANG II) potentiates hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation was studied in cultured rat vascular smooth muscle cells. Incubation of cells for 60 s with 100 nM ANG II produced a two- to threefold enhancement of cAMP stimulation when coupled with isoproterenol, prostaglandin I2, or adenosine. ANG II also enhanced cAMP formation when adenylyl cyclase was stimulated directly with forskolin or activated through the stimulatory guanyl nucleotide-binding protein (Gs) with cholera toxin. Forskolin stimulation was increased by only 40%, but cholera toxin-stimulated cAMP formation was doubled. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) enhanced isoproterenol-stimulated cAMP by 51%, but inhibitors of protein kinase activation had little effect on ANG II enhancement of cAMP production. However, use of PMA to cause feedback inhibition of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation blocked the effect of ANG II on agonist-stimulated cAMP formation, and the time course for this effect of PMA paralleled its inhibitory effect on Ins(1,4,5)P3 production. Furthermore, chelation of intracellular Ca2+ or treatment with calmodulin antagonists also diminished the synergism between ANG II and isoproterenol for cAMP stimulation. The results indicate that ANG II enhances cAMP formation in vascular smooth muscle cells by facilitating the interaction between activated Gs and adenylyl cyclase. In addition, the data suggest that this effect of ANG II is directly related to Ins(1,4,5)P3 stimulation and appears to involve a Ca(2+)-calmodulin-dependent mechanism.

Angiotensin II↗
PubMed1996

An analogue of cAMP mimics developmental change in neonatal rat ventricular myocyte sodium current kinetics.

During development, the voltage dependence of single rat ventricular sodium channels shifts to more negative potentials. This shift is mimicked by coculture of neonatal myocytes with sympathetic neurons or by a 96-h exposure to 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). The prolonged exposure to CPT-cAMP suggests that this is not a short-term modulatory effect on the sodium channel, but rather may reflect a trophic action. Here we examine the effect of CPT-cAMP using whole cell recording to investigate further the time period required for the effect. Sodium current was measured in a 50 mM NaCl bath solution at 20 +/- 1 degree C using the whole cell patch-clamp technique after exposure of myocytes to CPT-cAMP (0.25 mM) for 0,0.5,20, or 24 h. The relationship between the time constant of decay (tauh) of the sodium current and test voltage (V1) showed a shift to more hyperpolarizing voltages after exposure to CPT-cAMP for 24 h. In addition, the midpoint of the steady-state inactivation curve (V 1/2) was shifted from -75.8 +/- 1.1 mV (0-h exposure) to -83.3 +/- 1.6 mV (24-h exposure) (P < 0.05). Exposure for 0.5 h to CPT-cAMP did not alter the tauh or V 1/2 of the sodium current. However, exposure to CPT-cAMP for 20 h, followed by a 4-h washout period, produced an effect similar to that of the 24-h exposure. Thus the lack of effect of acute (0.5 h) exposure to CPT-cAMP and the persistence of the effect after washout of CPT-cAMP for 4 h suggest that adenosine 3',5'-cyclic monophosphate may play a trophic role in sodium channel development.

Animals↗
PubMed1997

cAMP induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle.

Recent studies indicate that vascular smooth muscle cells generate carbon monoxide (CO) via the action of heme oxygenase (HO). Because adenosine 3',5'-cyclic monophosphate (cAMP) is an important intracellular signaling molecule in the regulation of vascular cell function, we examined whether this second messenger modulates the expression of HO and the production of CO by rat aortic smooth muscle cells. Treatment of smooth muscle cells with the membrane-permeable cAMP derivative dibutyryl cAMP or with compounds that increase intracellular cAMP levels (isoproterenol and forskolin) resulted in a concentration- and time-dependent increase in the levels of HO-1 mRNA and protein, whereas the expression of HO-2 remained unchanged. Both actinomycin D and cycloheximide blocked the basal expression of HO-1 mRNA and protein and prevented the cAMP-mediated induction of HO-1. Incubation of platelets with cAMP-treated smooth muscle cells resulted in a significant increase in platelet cGMP concentration that was partially reversed by treatment of smooth muscle cells with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine or the HO blocker zinc protoporphyrin-IX. However, the combined addition of these two inhibitors to cAMP-treated smooth muscle cells or the addition of the CO and NO scavenger hemoglobin to platelets completely blocked the stimulatory effect on platelet cGMP levels. These results demonstrate that cAMP induces the expression of the HO-1 gene and stimulates the formation of CO and NO in vascular smooth muscle cells. The capacity of cAMP to induce the synthesis of guanylate cyclase-stimulatory CO from smooth muscle cells may represent a novel mechanism by which this nucleotide regulates vascular tone.

Animals↗
PubMed1997

Adenylyl cyclase isoforms and vasopressin enhancement of agonist-stimulated cAMP in vascular smooth muscle cells.

The influence of arginine vasopressin (AVP) on agonist-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated in vascular smooth muscle cells (VSMC) cultured from rat thoracic aorta. Incubation of VSMC with AVP for 60 s produced a 2- to 2.5-fold enhancement of isoproterenol-induced cAMP formation. AVP also increased cAMP stimulation by the prostaglandin I2 analogue iloprost. The effect of AVP to enhance agonist-stimulated cAMP formation was completely inhibited in cells pretreated with a selective antagonist of V1 vasopressin receptors but was not affected by blockade of V2 receptors. Inhibition of protein kinase C activation failed to alter the action of AVP to potentiate cAMP stimulation, but treatment of cells with calmodulin antagonists significantly attenuated this effect of the peptide. Moreover, depletion of Ca2+ stores with thapsigargin decreased AVP enhancement of isoproterenol-stimulated cAMP by > 70%. The action of AVP to increase cAMP stimulation was also demonstrated in freshly isolated strips of rat aorta where treatment with peptide produced a twofold increase in isoproterenol-stimulated cAMP formation. RNA blot analysis indicated expression in VSMC of mRNA encoding type III adenylyl cyclase, a Ca(2+)-calmodulin-sensitive isoform of the effector. Furthermore, when detergent-solubilized membrane extract was subjected to calmodulin affinity chromatography, a peak of adenylyl cyclase activity was identified which had affinity for calmodulin matrix in the presence of Ca2+. The results indicate that AVP activates V1 receptors in VSMC to enhance agonist-stimulated cAMP formation by a Ca(2+)-calmodulin-dependent mechanism and suggest that type III adenylyl cyclase may provide a focal point in the VSMC for cross talk between constrictor and dilator pathways.

Adenylyl Cyclases↗
PubMed2006

Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry.

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.

Adult↗
PubMed2003

cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase.

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of pulmonary vascular tone is currently an area of great interest. Normally, signaling mechanisms that elevate cAMP and guanosine-3',5'-cyclic monophosphate (cGMP) maintain the pulmonary vasculature in a relaxed state. Modulation of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BK(Ca) channel has been implicated in the development of pulmonary hypertension. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BK(Ca) channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 micro M), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and 8-4-chlorophenylthio (CPT)-cAMP (100 micro M), a membrane-permeable derivative of cAMP, opened BK(Ca) channels in single FHR PASMC. Treatment of FHR PASMC with 300 nM KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG) activity inhibited the effect of both forskolin and CPT-cAMP. In contrast, blocking PKA activation with 300 nM KT5720 had no effect on forskolin or CPT-cAMP-stimulated BK(Ca) channel activity. These results indicate that cAMP-dependent vasodilators activate BK(Ca) channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways, which suggests cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle and therefore, a unique signaling pathway for cAMP-induced pulmonary vasodilation.

Alkaloids↗
PubMed2002

Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells.

Mucosal and enterocyte IL-6 production is increased during sepsis and endotoxemia. Recent studies suggest that cAMP potentiates IL-6 production in endotoxin- or IL-1beta-stimulated enterocytes, but the molecular mechanisms are not known. We examined the role of the transcription factors NF-kappaB, activator protein (AP)-1, CCAAT/enhancer binding protein (C/EBP), and cAMP response element-binding protein (CREB) in cAMP-induced IL-6 production in cultured Caco-2 cells, a human intestinal epithelial cell line. In addition, the role of the protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein (MAP) kinase signaling pathways was examined. Treatment of the cells with IL-1beta increased IL-6 production and activated the IL-6 promoter in cells transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter. These effects of IL-1beta were significantly potentiated by cAMP. When the binding sites for the individual transcription factors in the IL-6 promoter were mutated, results indicated that all four transcription factors may be involved in the cAMP-induced activation of the IL-6 gene. Treatment of the Caco-2 cells with cAMP increased the DNA binding activity of CREB, C/EBP, and AP-1, but not NF-kappaB. By using specific blockers, evidence was found that both PKA and p38 MAP kinase (but not PKC or p42/44 MAP kinase) may be involved in the cAMP-induced potentiation of IL-6 production. The present results suggest that cAMP activates multiple transcription factors involved in the regulation of the IL-6 gene and that the activation of these transcription factors may at least in part explain why cAMP potentiates IL-6 production in stimulated enterocytes.

Blotting, Western↗
PubMed1989

Inhibitory modulation by cAMP of isoproterenol-induced prostacyclin synthesis in rabbit heart.

beta-Adrenergic receptor activation in heart is associated with enhanced production of adenosine 3',5'-cyclic monophosphate (cAMP) and prostaglandins (PG). The purpose of the present study was to test the hypothesis that cAMP mediates or modulates PG synthesis elicited by activation of beta-adrenergic receptors in the isolated, perfused rabbit heart. Infusion of 8-(4-chlorophenylthio) (cpt)-cAMP (100 microM), an analogue of cAMP, or stimulation of endogenous cAMP generation with forskolin (2 microM) resulted in a reduction of perfusion pressure and an increase in heart rate and contractility but had no effect on 6-keto-PGF1 alpha output. 6-Keto-PGF1 alpha production elicited by a bolus injection of isoproterenol (Isop) (475 pmol), however, was reduced by greater than 50% in the presence of these agents, cpt-cAMP was also found to inhibit 6-keto-PGF1 alpha output elicited by the calcium ionophore A23187 but not that in response to exogenous arachidonic acid. Perfusion with the adenosine analogue adenylate cyclase inhibitor PIA (1 microM) enhanced by twofold Isop-stimulated output of 6-keto-PGF1 alpha, whereas cAMP accumulation was prevented. Isop-stimulated production of 6-keto-PGF1 alpha was inhibited by 50% in the presence of the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine (50 microM), Ro 20-1724 (300 microM), or cilostamide (5 microM), whereas both basal and Isop-stimulated cAMP accumulations were enhanced by these agents. These data suggest that cAMP acts as an inhibitory modulator of PG synthesis in response to beta-adrenergic receptor activation in rabbit heart.

1-Methyl-3-isobutylxanthine↗
PubMed1997

cAMP and protein kinase A modulate cholinergic rapid eye movement sleep generation.

Cholinergic neurotransmission in the medial pontine reticular formation (mPRF) modulates rapid eye movement (REM) sleep generation. Microinjection of cholinergic agonists and acetylcholinesterase inhibitors into the mPRF induces a REM sleep-like state, and microdialysis data reveal increased mPRF levels of acetylcholine during REM sleep. Muscarinic cholinergic receptors (mAChRs) participate in REM sleep generation, and data suggest that mAChRs of a non-M1 subtype modulate REM sleep generation. The signal transduction pathway activated by m2 and m4 mAChRs involves a pertussis toxin-sensitive G protein, adenylate cyclase (AC), adenosine 3',5'-cyclic monophosphate (cAMP), and protein kinase A (PKA). Therefore, the present study tested the hypothesis that cAMP and PKA within the mPRF modulate the carbachol-induced REM sleep-like state. To test this hypothesis, the mPRF was microinjected with compounds known to facilitate the effects of cAMP (dibutyryl cAMP and 8-bromo-cAMP), stimulate PKA (Sp-cAMP[S]), and inhibit PKA (Rp-cAMP[S]). The results showed that compounds that fostered the intracellular effects of cAMP significantly decreased cholinergic REM sleep, while having no effect on spontaneously occurring REM sleep. These data are consistent with the recent finding that within the mPRF, AC and a pertussis toxin-sensitive G protein modulate cholinergic REM sleep generation. These new data suggest a modulatory role for pontine cAMP and PKA in cholinergic REM sleep regulation.

8-Bromo Cyclic Adenosine Monophosphate↗