Role Of cAMP Research Articles

Adenosine 3',5'-cyclic monophosphate increases processing of amyloid precursor protein (APP) to beta-amyloid in neuroblastoma cells without changing APP levels or expression of APP mRNA.

Beta-Amyloid (Abeta), a 39-43 residue peptide generated by splicing of the amyloid precursor protein (APP), is one of the major components of senile plaques which are the hallmark of Alzheimer's disease (AD); and therefore, a role of Abeta in neuronal degeneration has been proposed. The factors which regulate the levels of Abeta have not been fully identified. Since an elevation of the intracellular levels of adenosine, 3', 5'-cyclic monophosphate (cAMP) in neuroblastoma cells (NB) induces terminal differentiation, and since these differentiated NB cells undergo spontaneous degeneration, the role of cAMP in the regulation of Abeta levels in these cells have been investigated. In order to determine the specificity of the effect of cAMP on nerve cells, rat glioma cells (C-6) were investigated in a similar manner. Results showed that an elevation of the levels of cAMP in NB cells enhances the intensity of Abeta immunostaining without changing the levels of APP or APP mRNA. This suggests that the rate of processing of APP to Abeta increases following an elevation of cAMP level in NB cells. Data also revealed that an elevation of cAMP level in glioma cells did not alter the intensity of staining with APP or Abeta.

Regulation of cAMP-dependent protein kinase during appressorium formation in Magnaporthe grisea

The rice blast fungus, Magnaporthe grisea, requires formation of an appressorium, a dome-shaped and highly melanized infection structure, to infect its host. cAMP was identified as an important second messenger in signaling systems for appressorium formation in this fungus. To understand further the role of cAMP in infection-related morphogenesis, cAMP-dependent protein kinase activity was measured during appressorium formation and germination of conidia. Much higher activity of protein kinase was detected in germlings forming appressoria on the hydrophobic surface of GelBond than germlings growing vegetatively on a hydrophilic surface. In liquid culture, protein kinase activity increased during conidial germination, and peaked at 8 h after incubation before declining. Two transformants which develop small non-functional appressoria, and which lack cpkA, a gene encoding the catalytic subunit of protein kinase, did not show protein kinase activity. These data suggest that protein kinase has significant roles in the regulation of functional appressorium formation in M. grisea.

Inhibition of Tumor Cell Proliferation by Type IV Collagen Requires Increased levels of cAMP

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the α3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 µg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 µgg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the α3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10µg/ml. Treatment of the α3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence α3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%.Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the α3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its α3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).

Developmental competence of bovine oocytes is stimulated by low doses of dibutyryl-cAMP during cycloheximide-induced block of meiosis

The time that precedes the resumption of meiosis is crucial for the acquisition of oocyte developmental competence. However, at present, the mechanisms regulating this process are largely unknown though their understanding would enhance our capacity to improve the quality of in vitro produced embryos. This study was designed to assess the role of cAMP in the acquisition of oocyte developmental competence before meiotic resumption. To achieve this, bovine cumulusoocyte complexes (COCs) were reversibly maintained in meiotic arrest for 6 h by a protein synthesis inhibitor, cycloheximide (CHX), and during this period the intracellular cAMP concentration were modulated with different concentrations of a cAMP analog, dibutyryl-cAMP (dbcAMP) before being matured under standard conditions with serum and gonadotropins. COCs were isolated from ovaries recovered at slaughter by aspirating 2-5 mm follicles in TALP Hepes alone or supplemented with 2 gg/ml of CHX. COCs with compact cumulus layers and homogeneous cytoplasm were selected and incubated for 6 h with or without CHX and various concentrations of dbcAMP. At the end of culture COCs were washed and placed in M199 supplemented with 10% FCS, 1 gg/ml FSH and 5 pg/ml LH for 24 h'then fertilized for 16 h and cultured for 5 d in the presence of bovine oviductal epithelial cells. Frequency of cleavage, morula and blastocyst formation were determined 2 and 6 d post IVF (p.i.) respectively.

Apoptosis in developing retinal tissue.

The mechanisms of apoptosis are strongly dependent on cell-cell interactions typical of organized tissues. Experimental studies of apoptosis using a histotypical preparation of retinal explants are reported in the present article. We found that various characteristics of apoptosis are selectively associated with retinal cell death depending on cell type, stage of maturation, and means of induction of apoptosis. Among these were: (1) the requirements of protein synthesis; (2) the role of cAMP; (3) the expression of certain apoptosis-associated proteins; and (4) the sensitivity to excitotoxicity, modulation of protein phosphatases and calcium mobilization. Dividing cells undergo apoptosis in response to several inducers in specific phases of the cell cycle, and in distinct regions within their pathway of interkinetic nuclear migration. Recent post-mitotic cells are selectively sensitive to apoptosis induced by blockade of protein synthesis, while both proliferating and differentiated cells are more resistant. We also studied the association of several proteins, some of which play critical roles in the cell cycle, with both differentiation and apoptosis in the retinal tissue. Detection of cell cycle markers did not support the hypothesis that retinal cells re-enter the cell cycle on their pathway to apoptosis, although some proteins associated with cell proliferation re-appeared in degenerating cells. The transcription factors c-Jun, c-Fos and c-Myc were found associated with apoptosis in retinal cells, but their sub-cellular location in apoptotic bodies is not consistent with their canonical functions in the control of gene expression. The bifunctional redox factor/AP endonuclease Ref-1 and the transcription factor Max are associated with progressive cell differentiation, and both are down-regulated during cell death in the retina. The data suggest that Ref-1 and Max may normally function as negative modulators of retinal apoptosis. The results indicate that nuclear exclusion of transcription factors and other important control proteins is a hallmark of retinal apoptosis. Histotypical explants may be a choice preparation for the experimental analysis of the mechanisms of apoptosis, in the context both of cell-cell interactions and of the dynamic behavior of developing cells within the organized retinal tissue.

The role of cyclic AMP and protein kinase A in stimulation of neutrophil migration by endothelins.

The role of cAMP and cAMP-dependent protein kinase A (PKA) in endothelin (ET)-stimulated migration of human neutrophils was studied. Endothelins caused an increase in neutrophil migration when they were applied in low (nanomolar) concentrations; stimulation of migration was either predominantly chemokinetic (ET-1) or chemotactic (ET-2, ET-3). All endothelins, at concentrations which gave maximal stimulation of migration, caused an increase of cAMP level. Two inhibitors of adenylate cyclase, MDL-12330A and SQ-22536, completely inhibited migration activated by ET-1, ET-2 or ET-3, indicating that cAMP generation played a decisive role in endothelin-activated migration. The role of PKA in endothelin-activated migration was considered. Two specific antagonists of PKA strongly inhibited endothelin-activated migration. KT-5720, an inhibitor of PKA, also inhibited ET-activated migration, but only when electroporated cells were used. The results suggest that the effect of cAMP on endothelin-activated migration was mediated by PKA.

A Role for cAMP in Long-Term Depression at Hippocampal Mossy Fiber Synapses

Mossy fiber synapses on hippocampal CA3 pyramidal cells, in addition to expressing an NMDA receptor–independent form of long-term potentiation (LTP), have recently been shown to express a novel presynaptic form of long-term depression (LTD). We have studied the mechanisms underlying mossy fiber LTD and present evidence that it is triggered, at least in part, by a metabotropic glutamate receptor–mediated decrease in adenylyl cyclase activity, which leads to a decrease in the activity of the cAMP-dependent protein kinase (PKA) and a reversal of the presynaptic processes responsible for mossy fiber LTP. The bidirectional control of synaptic strength at mossy fiber synapses by activity therefore appears to be due to modulation of the cAMP-PKA signaling pathway in mossy fiber boutons.

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy.

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.

The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascaridia galli

AF3 (AVPGVLRFamide) is an endogenous RFamide-like peptide isolated from the parasitic nematode Ascaris suum. It has a potent and long lasting excitatory effect in A. suum and Ascaridia galli. This is mediated by a mechanism independent of the nicotinic-like acetylcholine (ACh) receptor, which mediates excitatory transmission at the neuromuscular junction of both nematodes. In addition, AF3 has been found to sensitise A. suum muscle to the contractile effect of ACh. In this study, the involvement of the second messenger cAMP in mediating the action of AF3 on the somatic musculature of A. suum and A. galli has been investigated. Two approaches have been used; the effects of drugs which raise intracellular cAMP levels on the contractile responses to AF3 have been examined and biochemical assays have been used to measure the effects of AF3 on cAMP levels. AF3 contractions were inhibited in A. suum by 10 μM forskolin (by 22% of control; P<0.05; n=9) and by 500 μM isobutylmethylxanthine (IBMX, by 27% of control; P<0.001; n=6). AF3 decreased cAMP concentrations in A. suum somatic muscle (basal, 1721±134 pmol mg −1 protein; with 1 μM AF3, 1148±133 pmol mg −1 protein; P<0.05, n=5). AF3 (1 μM) also reduced the 10 μM forskolin induced potentiation of cAMP concentrations in A. suum (forskolin 3242±471 pmol mg −1 protein; forskolin and AF3, 1524±143 pmol mg −1 protein; P<0.001, n=6) and A. galli (forskolin 291±32 pmol mg −1 protein, forskolin+AF3, 185±12 pmol mg −1 protein; P<0.05, n=5). These data suggest that in both nematodes the contractile effect of AF3 is, at least in part, regulated by cAMP.

Pathophysiological role of cAMP in process of interaction between macro- and microorganisms

Pathophysiological role of cAMP in process of interaction between macro- and microorganisms

Role of cAMP and neuronal K+ channels on alpha 2-AR-induced inhibition of ACh release in equine trachea.

To investigate the effects of changes in intracellular cAMP on alpha 2-adrenoceptor (AR)-induced inhibition of airway acetylcholine (ACh) release, we examined the effects of the alpha 2-AR agonist clonidine on electrical field stimulation-evoked ACh release from equine tracheal parasympathetic nerves before and after treatment with 8-bromo-cAMP or forskolin. We also tested whether charybdotoxin (ChTX)- or iberiotoxin (IBTX)-sensitive Ca(2+)-activated K+ channels mediate alpha 2-AR-induced inhibition by examining the effect of clonidine in the absence and presence of ChTX or IBTX on ACh release. The amount of released ACh was measured by HPLC coupled with electrochemical detection. Clonidine (10(-7) to 10(-5) M) dose dependently inhibited ACh release before and after treatment with 8-bromo-cAMP (10(-3) M) or forskolin (3 x 10(-5) M). ChTX and IBTX, both at the concentration of 5 x 10(-7) M, significantly increased ACh release; however, they did not alter the magnitude of clonidine-induced inhibition. These results indicated that in equine tracheal parasympathetic nerves, alpha 2-AR-induced inhibition of ACh release is via an intracellular cAMP-independent pathway. Activation of both ChTX- and IBTX-sensitive Ca(2+)-activated K+ channels inhibits the electrical field stimulation-evoked ACh release, but these channels are not involved in the alpha 2-AR-induced inhibition of ACh release.

Seasonal influences on the functional expression of the endogenous L-type Ca2+ channels in Pleurodeles oocytes: role of cAMP?

We have investigated the seasonal regulation of the Ba current (IBa) through the L-type Ca2+ channels in Pleurodeles oocytes and its relation with the intracellular cAMP level. IBa-density remained relatively constant from January to February, increased from March to April (with a maximum increase in March) and decreased from May to July. While the permeable cAMP analogue 8Br-cAMP reduced IBa during the breeding season, it was without effect during the resting season. However, IBa recorded during the two seasons was increased by a dihydropyridine agonist Bay K 8644 and blocked by cadmium. The main conclusion is that the seasonal reduction in the L-type Ca2+ current amplitude may be correlated with a high intracellular cAMP level and may account for the inability of oocytes to mature during the resting season.

Multiple Intracellular Pathways Interfere with the Activation of a CPP32-like Protease Induced by Serum Deprivation of AKR-2B Cells

As previously described, confluent AKR-2B fibroblasts rapidly disintegrate upon removal of serum. Platelet-derived growth factor isoforms AB or BB (PDGF-AB, -BB) added immediately after serum deprivation caused complete survival of the cells without initiating proliferation (Simmet al.,1994,J. Cell. Physiol.160, 295). Here the role of cAMP as a protective agent was investigated by using forskolin or 8-Br-cAMP. Both reagents afforded high cellular protection. The phorbolester TPA, an activator of protein kinase C isoforms, also exerted a high protection against cell death (ED50= 7 nM). Unexpectedly colchicine (ED50= 1.5 μM) an inhibitor of tubulin polymerization also protected cells from death. The protective effects of PDGF-BB and TPA were dependent on protein synthesis as indicated by their complete suppression by cycloheximide (CHx). Surprisingly, forskolin and 8-Br-cAMP remained effective even in the presence of CHx. Detailed studies of several signalling pathways were performed. These investigations showed no interference between PDGF-BB and cAMP-dependent pathways at the early stage of signal transduction. As previously described, the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone (YVAD-cmk) protected cells from death (Simmet al.,1997,J. Cell Sci.110, 819–828). As shown here, a substantial protection was also achieved by the addition of two other caspase inhibitors: asp-glu-val-asp-aldehyde (DEVD-cho; ED50= 100 μM) and benzoylcarbonyl-asp-glu-val-asp-chloromethylketone (Z-DEVD-cmk; ED50= 100 μM). The activity of caspases was studied using either tyr-val-ala-asp-aminomethylcoumarine (YVAD-amc) or aspglu-val-asp-aminomethylcoumarine (DEVD-amc) as substrates. There was no activation of a YVADase, whereas as pronounced increase in DEVDase activity was found with a maximum 3 h after serum removal. Cross competition experimentsin vitroshowed that the latter activity is inhibited also by low concentrations of YVAD-cmk (300–600 nM), suggesting that both inhibitors inactivated the same target protease. Remarkably all tested protective reagents lead to an inhibition of the DEVDase activity in intact cells. Since these reagents act via distinct intracellular pathways, the existence of a regulatory element upstream of the DEVDase is proposed which integrates signals from a variety of pathways.

Inhibition of cell growth by CCK requires activation of cyclic AMP signaling in HEK-293 cells

We have previously demonstrated that cholecystokinin (CCK) stimulates significant cAMP accumulation in HEK-293 cells expressing human CCK-A receptors while causes little of no significant cAMP response via CCK-B receptors. Substitution of Leu-81 to Art, or Ser-82 to Asn in the first intraceUnlar loop (ICL-1) of CCK,BR, resulted in significant increases in cAMP production. Aim: The role of cAMP in CCK-mediated cell growth and proliferation has not been fully characterized in HEK-293 cells. To further explore the functional ramifications of cAMP signaling induced by CCK in this cell system, we examined the mitogenic response of CCK-AR or the ICL-1 CCK-BR mutants cell lines to CCK. Methods: Cells grown on multi-well plates were treated with CCK at varying concentrations in serum-free DME/FI2 media for DNA synthesis, or in 0.5% fetal bovine serum for cell numbers. Forskolin alone or in conjunction with 1 mM IBMX was used as positive control of cAMP activation. DNA synthesis was measured by tritiated-thymidine incorporation into TCA-insoluble fraction after 24 h. Cell numbers were counted by microscopic examination of viable cells stained by trypan blue over a 7 day period. Results and Conclusion: CCK inhibited DNA synthesis in cells expressing CCK-AR and CCK-BR ICL-I mutants capable of cAMP production in a dose dependent manner. In contrast, CCK-BR wild type or mutant L85M that exhibited little or no cAMP increase were not sensitive to inhibition by CCK up to 100 nM. Cell numbers were significantly reduced by CCK in CCK-AR, CCK-BR mutants LS1R and $82N, to 8, 30 and 15% of their respective controls after 7 days. Forskolin up to 10 tiM alone had no significant inhibitory effect but caused dose-depandant inhibition of DNA synthesis in the presence of 1 mM IBMX in all HEK-293 cell lines tested. In addition, combination of CCK and forskolin]IBMX caused significent inhibition of DNA synthesis by wild type CCK-BR cells. These data suggest that inhibition of cell growth by CCK in transfected HEK-293 cells may require the involvement of cAMP and its down-stream signals.

Role of cAMP, cGMP, and protein kinase C in regulation of calcium current through the L-type calcium channels in the electroexcitable membrane of smooth muscle cells

Regulation of calcium current through L-type calcium channels (I Ca,L) of the guinea pigtaenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced theI Ca,L amplitude in a dose-dependent manner. Dose-dependent suppression ofI Ca,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects onI Ca,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reducedI Ca,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase inI Ca,L. 8-Bromo-cAMP (1 μM) increasedI Ca,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS onI Ca,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pigtaenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase theI Ca,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increasesI Ca,L (probably through activation of PKA). PKG apparently mediatesI Ca,L drops evoked by cAMP at high concentrations and by cGMP.

The role of cAMP in the frequency-dependent changes in contraction of guinea-pig cardiomyocytes.

beta-Receptor desensitisation, low basal cAMP, and a negative force-frequency relationship are characteristic changes in human heart failure. Isolated cardiomyocytes from noradrenaline-treated guinea pigs also show these features. We tested the hypothesis that low basal cAMP underlies the loss of contractile response to increasing stimulation frequency in this model. Isolated cardiomyocytes were obtained from noradrenaline-treated (NA) and sham-operated (SHAM) guinea pigs. They were stimulated from 0.1-2 Hz and contraction amplitude was monitored with a video edge-detection system. NA cells had less positive amplitude-frequency responses (AFR) compared to SHAMs at 2 mM (P = 0.002, n = 17), or midrange Ca2+ concentrations (EC40-EC60) (P < 0.001, n = 13). When the cAMP agonist, 8-CPT-cAMP (CPT, 10 microM) or high Ca2+ (above EC75) was added to NA cells the AFR was normalised to that of SHAM myocytes (NA vs. SHAM P = ns). In control experiments the cAMP antagonists, Rp-cAMPS (Rpc) and Rp-8-CPT-cAMPS (Rp8, 100 microM), blocked the positive inotropic effects of CPT at 0.5 Hz (control pD2 = 4.36 +/- 0.06, Rp8 pD2 = 3.68 +/- 0.08, P < 0.0001), n = 6 paired). Rpc (100 microM) completely but reversibly blocked the effect of maximal isoprenaline in control experiments (P < 0.0001). Neither antagonist reduced the AFR compared to time-matched controls (P = ns, n = 6). Blockade of SERCA2a with thapsigargin resulted in a significant reduction in the AFR (ANOVA P < 0.0001). The results are consistent with sarcoplasmic reticulum (SR) function being a more important determinant of the amplitude-frequency relationship than tonic levels of cAMP under basal conditions. Reversal of AFR depression by CPT may result from stimulation of SR Ca2+ uptake.

Inhibition of forskolin-induced sensitisation of frog skin nociceptors by the cyclic AMP-dependent protein kinase A antagonist H-89

The role of cAMP and protein kinase A (PKA) in nociceptor sensitisation has been studied in frog skin in vitro. Multifibre nerve responses during noxious heating were enhanced by adding forskolin, an agent that elevates cAMP. H-89, a PKA antagonist, blocked the increased responses due to forskolin, but did not affect baseline responses. Thus, cAMP appears to act via PKA when sensitising nociceptors in frog skin.

Possible role of cAMP, cGMP and [Ca 2+] i during NANC relaxation in the cat airway smooth muscle

To investigate the possible role of cyclic nucleotides and [Ca 2+] i in non-adrenergic non-cholinergic (NANC) relaxations evoked by electrical field stimulation (EFS) in the cat airway, we studied the effects of specific phosphodiesterase (PDE) type IV or V inhibitors on the biphasic-NANC relaxations, the correlation between NANC relaxation and changes in intracellular levels of cAMP and cGMP ([cAMP] i and [cGMP] i), and measured changes in [Ca 2+] i during the NANC relaxation. EFS (repetitive stimuli at 20 Hz, 1 or 4 ms pulse duration, 50 V) applied to the bronchial smooth muscle during contraction induced by 5-HT (10 −5 M) in the presence of atropine (10 −6 M) and guanethidine (10 −6 M) elicited biphasic NANC relaxations. Zaprinast (>3×10 −7 M), a specific PDE type V inhibitor, preferentially enhanced the amplitude of the first component of the NANC relaxations. However, rolipram (>3×10 −7 M) enhanced both the first and second component of the NANC relaxation to a similar extent. In the trachea, EFS evoked monophasic NANC relaxation accompanied by a concomitant accumulation of [cAMP] i and [cGMP] i. Pretreatment with rolipram (3×10 −6 M) enhanced the accumulation of [cAMP] i and amplitude of NANC relaxation evoked by EFS. However, zaprinast did not affect the amplitude of NANC relaxation although it significantly increased the levels of [cGMP] i. N ω -Nitro- l-arginine methylester ( l-NAME; 10 −5 M) completely suppressed the accumulation of [cGMP] i but only partly suppressed the NANC relaxation evoked by EFS. In contrast, EFS significantly enhanced [cAMP] i in the presence of l-NAME. During NANC relaxation, time-dependent decrease in [Ca 2+] i occurred, which was partly suppressed by l-NAME. These results indicate that NANC relaxation is associated with concomitant accumulation in both [cAMP] i and [cGMP] i, and decrease in [Ca 2+] i. However, the timing of the action of [cGMP] i and [cAMP] i in NANC relaxations differs in the central and peripheral airway.

Involvement of the cyclic AMP‐protein kinase a pathway in gap junctional communication in preimplantation mouse embryos

In this study, we have examined the role of cAMP in gap junctional communication (GJC) in preimplantation mouse embryos. GJC was monitored by Lucifer Yellow (LY) injected into one blastomere of compacted embryos. The speed of GJC was defined as the time taken for the last blastomere of the embryo to become visibly fluorescent. The median time for 8‐cell embryos (140 sec) was similar to that for 16‐cell (135 sec). To determine whether cAMP and cAMP‐dependent protein kinase (PKA) are involved in the regulation of GJC, the effects of PKA inhibitor (H8) and cAMP analogues (Rp‐cAMP and 8‐Br‐cAMP) on dye transfer between blastomeres of compacted embryos were examined. Some of the embryos treated with either H8 or Rp‐cAMP failed to transfer LY to all blastomeres within 10 min. In contrast, 8‐Br‐cAMP speeded up fluorescent dye transfer. The median time to fill all blastomeres with LY was 140 sec in untreated controls and 90 sec in siblings treated with 8‐Br‐cAMP. Inhibition of PKA by H8 or Rp‐cAMP induced delay o...

Role of cAMP and calcium influx in endothelin-1-induced ANP release in rat cardiomyocytes.

The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.