Presence Of Inhibitor 3-isobutyl-1-methylxanthine Research Articles

Intrauterine growth restriction is associated with sex-specific alterations in the nitric oxide/cyclic GMP relaxing pathway in the human umbilical vein

IntroductionIntrauterine growth restriction (IUGR) is a leading cause of perinatal mortality and morbidity, and is linked to an increased risk to develop chronic diseases in adulthood. We previously demonstrated that IUGR is associated, in female neonates, with a decreased nitric oxide (NO)-induced relaxation of the umbilical vein (UV). The present study aimed to investigate the contribution of the smooth muscle components of the NO/cyclic GMP (cGMP) pathway to this alteration. MethodsUVs were collected in growth-restricted or appropriate for gestational age (AGA) human term newborns. Soluble guanylyl cyclase (sGC) and cGMP-dependent protein kinase (PKG) were studied by Western blot, cGMP production by ELISA and cyclic nucleotide phosphodiesterases (PDEs) activity using a colorimetric assay. Contribution of PDEs was evaluated using the non-specific PDEs inhibitor 3-isobutyl-1-methylxanthine (IBMX) in isolated vessel tension studies. ResultsNO-induced relaxation was reduced in IUGR females despite increased sGC protein and activity, and some increase in PKG protein compared to AGA. In males, no significant difference was observed between both groups. In the presence of IBMX, NO-stimulated cGMP production was significantly higher in IUGR than AGA females. Pre-incubation with IBMX significantly improved NO-induced relaxation in all groups and abolished the difference between IUGR and AGA females. ConclusionIUGR is associated with sex-specific alterations in the UV's smooth muscle. The impaired NO-induced relaxation observed in growth-restricted females is linked to an imbalance in the NO/cGMP pathway. The beneficial effects of IBMX suggest that PDEs are implicated in such alteration and they could represent promising targets for therapeutic intervention.

The β3 -adrenoceptor agonist mirabegron increases human atrial force through β1 -adrenoceptors: an indirect mechanism?

Mirabegron has been classified as a β3 -adrenoceptor agonist approved for overactive bladder syndrome. We investigated possible cardiac effects of mirabegron in the absence or presence of β-adrenoceptor subtype antagonists. In view of its phenylethanolamine structure, we investigated whether mirabegron has indirect sympathomimetic activity by using neuronal uptake blockers. Right atrial trabeculae, from non-failing hearts, were paced and contractile force measured at 37°C. Single concentrations of mirabegron were added in the absence or presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), β3 (L-748,337), β1 (CGP 20712A), β2 (ICI 118,551) -adrenoceptor antagonists, neuronal uptake inhibitors desipramine or phenoxybenzamine. Mirabegron significantly increased contractile force in human right atrium (1μM, 7.6±2.6%, n=7; 10μM, 10.2±1.5%, n=22 compared with (-)-isoprenaline P<0.05). In the presence of IBMX, mirabegron (10μM) caused a greater contraction. L-748,337 (100nM) had no effect on the increase in contractile force caused by mirabegron (10μM). In contrast, mirabegron (10μM) reduced contractile force in the presence of CGP 20712A, which was not affected by L-748,337 (100nM) or ICI 118,551 (50nM). Mirabegron (10μM) also reduced contractile force in the presence of desipramine or phenoxybenzamine. Mirabegron increases human atrial force through β1 - but not β3 -adrenoceptors. Desipramine and phenoxybenzamine block neuronal uptake and conceivably prevent mirabegron from releasing noradrenaline. A non-specific cardiodepressant effect is not mediated through β3 (or β2 )-adrenoceptors, consistent with lack of β3 -adrenoceptor function on human atrial contractility.

Involvement of the cAMP messenger system and extracellular Ca 2+ during hyposmotically-induced prolactin release in the Mozambique tilapia

In accord with its role in freshwater osmoregulation, prolactin (PRL) release from the tilapia pituitary is stimulated by small, physiologically relevant reductions in plasma osmolality, a response that is mediated by an acute influx of intracellular Ca 2+ through stretch-activated Ca 2+channels. In the present study, the role of the calcium and cyclic AMP (cAMP) messenger system in the transduction of a response to a hyposmotic stimulus was examined using dispersed PRL cells and PRL cell membrane preparations from freshwater-acclimated tilapia. When PRL cells were treated with the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (100 μM), significant increases in cAMP levels and PRL release were observed at 1 h. Exposure to reduced medium osmolality (300 mOsmolal) in the presence of IBMX further augmented PRL release. Depletion of Ca 2+ from the incubation medium blocked PRL release even in the presence of IBMX. By contrast, exposure of PRL cells to cholera toxin (CTX), an activator of adenylyl cyclase (AC), stimulated PRL release and cAMP accumulation in both the presence and absence of extracellular Ca 2+. On the other hand, treatment with the Ca 2+ ionophore A23187, which elicits a large rise in intracellular free Ca 2+, reduced cAMP accumulation. Likewise, the AC activity of a PRL cell membrane preparation was reduced as extracellular Ca 2+ concentration increased from 0.1 to 1 μM. These results indicate that: (1) the stimulation of PRL release and cAMP formation by a fall in extracellular osmolality are Ca 2+-dependent; (2) large increases in intracellular Ca 2+ attenuate cAMP formation; (3) direct agonists of cAMP messenger system, such as cholera toxin, however, stimulate PRL release independently of the extracellular Ca 2+. These findings add to the evidence that the osmosensitive response of the tilapia PRL cell is mediated through a Ca 2+-dependent mechanism. Nevertheless, the present findings also suggest that tilapia PRL cells have the ability to rapidly augment release PRL both via a Ca 2+-dependent manner and via a cAMP-dependent pathway in the absence of extracellular Ca 2+.

Parathyroid Hormone Receptor Trafficking Contributes to the Activation of Extracellular Signal-regulated Kinases but Is Not Required for Regulation of cAMP Signaling

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.

Guinea-pig lung adenylyl and guanylyl cyclase and PDE activities associated with airway hyper- and hypo-reactivity following LPS inhalation

The relationships between changes in in vivo airway reactivity and levels cyclicAMP and cyclicGMP were determined in guinea-pig lungs after exposure to inhaled lipopolysaccharide (LPS). After LPS (30 μg.ml −1, 1 h), guinea-pigs displayed in vivo airway hyperreactivity (AHR) at 1 h and hyporeactivity (AHOR) at 48 h, to inhaled (20 s) histamine (1 or 3 mM, respectively). Isoprenaline-stimulated cAMP or SNAP-stimulated cGMP were determined in the lungs isolated from guinea-pigs exposed to LPS inhalation to determine whether there was a relationship between AHR or AHOR and adenylyl/guanylyl cyclase and phosphodiesterase (PDE) activities. Assays were performed in the absence and presence of the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Levels of cAMP and cGMP in its presence indicated adenylyl and guanylyl cyclase activities, respectively. The difference between cAMP and cGMP levels, in the absence and presence of IBMX, reflected relevant PDE activity. In vivo AHR was associated with increased PDE activity towards cAMP and cGMP (67 and 278%, respectively) and also increased adenylyl (47%) and guanylyl (210%) cyclase activities. In vivo AHOR at 48 h after LPS inhalation was also associated with raised cyclase activity ( p < 0.05), whereas relevant PDE activity declined by 79 and 68%, compared with 48 h after vehicle. Although net stimulated cGMP levels increased during AHR and AHOR and net stimulated cAMP increased during AHOR, our index of PDE activity increased during AHR and decreased during AHOR. These results therefore support the rationale for the use of PDE-inhibitors in the treatment of respiratory diseases associated with AHR.

Irsogladine, an anti-ulcer drug, suppresses superoxide production by inhibiting phosphodiesterase type 4 in human neutrophils

Neutrophil superoxide production is implicated in the pathogenesis of gastric mucosal damage induced by various ulcerative agents and Helicobacter pylori infection. We investigated here the effects of an anti-ulcer drug irsogladine [2, 4-diamino-6-(2, 5-dichlorophenyl)-s-triazine maleate] on cAMP formation in isolated human neutrophils. The cAMP level in human neutrophils was elevated by a phosphodiesterase (PDE) type 4 selective inhibitor rolipram, but not by any inhibitors of PDE1, PDE2 and PDE3. Irsogladine also increased cAMP formation in a concentration-dependent manner in neutrophils. A non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) alone significantly increased cAMP level, whereas irsogladine was unable to further increase cAMP level in the presence of IBMX. Irsogladine inhibited concentration-dependently the superoxide (O2−) production induced by various stimuli including formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, guanosine 5′-[gamma-thio] triphosphate, A23187 and phorbol 12-myristate 13-acetate. These effects of irsogladine were mimicked by rolipram, IBMX and dibutyryl cAMP. The inhibitory effects of irsogladine and rolipram on the O2− production were reversed by a protein kinase A inhibitor H-89. These results indicate that irsogladine inhibits the superoxide production in human neutrophils by the increase of cAMP content by PDE 4 inhibition, which in turn contributing to the anti-ulcer effects of irsogladine on gastric mucosal lesions associated with oxidative stress.

Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation.

We have compared the mechanisms that contribute to endothelium-derived hyperpolarizing factor (EDHF)-type responses induced by ACh and the Ca(2+) ionophore A-23187 in the rabbit iliac artery. Relaxations to both agents were associated with ~1.5-fold elevations in smooth muscle cAMP levels and were attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (DDA) and potentiated by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Mechanical responses were inhibited by coadministration of the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin, both in the absence and presence of IBMX, but were unaffected by blockade of ATP-sensitive K(+) channels with the sulphonylurea glibenclamide. Relaxations and elevations in cAMP evoked by ACh were abolished by 18alpha-glycyrrhetinic acid, which disrupts gap junction plaques, whereas the corresponding responses to A-23187 were unaffected by this agent. Consistently, in "sandwich" bioassay experiments, A-23187, but not ACh, elicited extracellular release of a factor that evoked relaxations that were inhibited by DDA and potentiated by IBMX. These findings provide evidence that EDHF-type relaxations of rabbit iliac arteries evoked by ACh and A-23187 depend on cAMP accumulation in smooth muscle, but involve signaling via myoendothelial gap junctions and the extracellular space, respectively.

A Novel Adenylyl Cyclase Detected in Rapidly Developing Mutants of Dictyostelium

Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium. The RDEA gene product displays homology to certain H2-type phosphotransferases, while REGA encodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multistep phosphorelay. To test this proposal, we examined cAMP accumulation in rdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyostelium adenylyl cyclases, aggregation stage adenylyl cyclase (ACA) or germination stage adenylyl cyclase (ACG), since it occurred in an acaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn2+ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel adenylyl cyclase activity in lysates of an acaA/acgA double mutant. The fact that disruption of the RDEA gene as well as inhibition of the REGA-phosphodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA.

Increased cAMP levels in stimulated neutrophils inhibit their adhesion to human bronchial epithelial cells.

Bronchial epithelial cells express the intercellular adhesion molecule-1 that mediates binding of activated neutrophils via interaction with Mac-1 and/or leukocyte function-associated antigen-1. In this study, we examined whether increased intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) affected neutrophil adhesion to the human bronchial epithelial cells. It was found that the N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated neutrophil adhesion was concentration dependently inhibited when the cAMP analogs dibutyryl adenosine 3',5'-cyclic monophosphate or 8-bromoadenosine 3',5'-cyclic monophosphate were present. The beta-adrenergic receptor agonists isoprenaline and salmeterol, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), were also able to inhibit the fMLP-stimulated adhesion of neutrophils to bronchial epithelial cells. These agonists in combination with IBMX significantly increased the intracellular cAMP level in both neutrophils and epithelial cells. Preincubation of neutrophils with the long-acting beta2-adrenergic receptor agonist salmeterol (in the presence of IBMX) inhibited their fMLP-stimulated adhesion to epithelial cells, whereas pretreatment of epithelial cells did not influence the adhesion process. When ethanol-fixed epithelium was used, salmeterol pretreatment also diminished the adhesion of stimulated neutrophils. Moreover, combinations of salmeterol or isoprenaline with IBMX inhibited fMLP-upregulated Mac-1 expression. Therefore, we conclude from these data that elevation of intracellular cAMP in the neutrophil inhibits stimulated neutrophil adhesion to bronchial epithelial cells via Mac-1.

Pituitary adenylate cyclase-activating polypeptide stimulates cholecystokinin secretion in STC-1 cells.

Secretion of cholecystokinin (CCK) from the endocrine cells of small intestinal mucosa and the murine intestinal tumor cell line STC-1 is known to involve both adenosine 3',5'-cyclic monophosphate (cAMP)-and Ca(2+)-dependent signal transduction pathways. However, the endogenous stimulant(s) that acts through the cAMP-dependent cascade has not been identified. We determined the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on CCK secretion and cAMP production and its interaction with other CCK secretagogues in STC-1 cells. At concentrations > 10 nM, PACAP-27 stimulated the release of large intestinal CCK from STC-1 cells in a time- and dose-dependent manner. The stimulatory effect of PACAP-27 was enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). PACAP-27, PACAP-38, and vasoactive intestinal polypeptide (VIP), with or without IBMX, were equally effective and potent to elicit CCK release with similar half-maximal doses and maximal levels of stimulation. Both forms of PACAP and VIP stimulated a transient but not significant increase in the cellular cAMP level. In the presence of IBMX, all three peptides increased significantly the cellular cAMP level between 2 and 5 min, but PACAP produced a two times higher level than VIP. The stimulatory effect of PACAP-27 on CCK release was also potentiated by bombesin and KCl but without a synergistic production of cAMP. With or without IBMX, PACAP-27-stimulated CCK secretion was not affected by the Ca2+ channel blocker diltiazem (1 microM), the cell-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 25 microM), or by downregulation of protein kinase C. The stimulatory effects of KCl and bombesin were either reduced or abolished by these treatments. The synergistic effect of bombesin with PACAP was abolished by diltiazem and BAPTA-AM but not by downregulation of protein kinase C, whereas KCl remained synergistic with PACAP after these treatments. Taken together, these results indicate that PACAP may be a neuromodulator of CCK secretion that acts through activation of adenylate cyclase and may function as a coregulator with other CCK secretagogues that are known to increase intracellular Ca2+ concentration.

Negative chronotropic effect of endothelin 1 mediated through ETA receptors in guinea pig atria.

Endothelins exert potent excitatory cardiac effects by acting on specific receptors on myocytes. In this study, we have examined the signal transduction mechanism for the chronotropic effect of endothelins in guinea pig atria. A competition binding of [125I]endothelin 1 ([125I]ET-1) using the recently developed ETA receptor-selective antagonist BQ123 showed the presence of almost equal populations of ETA (44%) and ETB (56%) receptors in the guinea pig right atria. In a concentration-response study, endothelin 3 (ET-3), an agonist with higher affinity to ETB receptors than to ETA receptors, and sarafotoxin S6c (STXS6c), an ETB receptor-selective agonist, increased the rate of spontaneous beating at all concentrations tested (10 pmol/L to 100 nmol/L). In contrast, ET-1, a nonselective agonist, increased the heart rate at lower concentrations (10 pmol/L to 10 nmol/L) but decreased it at higher concentrations (30 to 100 nmol/L). When ET-1 (100 nmol/L) was applied in a single amount, heart rate was strongly increased; however, this increase was followed by a rapid decline in the response. ET-1 (100 nmol/L) but not ET-3 or STXS6c significantly reduced the heart rate when it was raised by isoproterenol (ISO, 300 nmol/L) either in the absence or presence of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Correspondingly, ET-1 significantly reduced the ISO-induced elevation of cAMP accumulation (19.1 +/- 1.7 pmol/mg protein [n = 8] and 12.6 +/- 1.2 pmol/mg protein [n = 7] in the absence and presence of ET-1, respectively; P < .01), which was also observed even in the presence of IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation by forskolin of both SNP- and ANP-stimulated cyclic GMP accumulation in porcine isolated palmar lateral vein.

1. The aim of this study was to examine the effect of modulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels (by using forskolin, a direct activator of adenylyl cyclase, or rolipram, a cyclic AMP selective phosphodiesterase inhibitor) on basal and stimulated guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in the porcine isolated palmer lateral vein by use of a [3H]-guanine prelabelling technique. 2. Sodium nitroprusside (SNP; 10(-5) - 10(-3) M) and atrial natriuretic peptide (ANP; 10(-8) - 10(-6) M), produced concentration-dependent increases in [3H]-cyclic GMP levels via stimulation of soluble and particulate guanylyl cyclase respectively. The SNP-stimulated [3H]-cyclic GMP response peaked after 5 min in the presence and absence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). 3. In the absence of IBMX, forskolin (3 x 10(-5) M) significantly increased [3H]-cyclic GMP levels to 118.5 +/- 8.7% of basal values (P < 0.05, n = 8), and significantly increased both the SNP- and ANP-stimulated [3H]-cyclic GMP accumulation at all concentrations of SNP and ANP used. For example, effects at the maximal SNP (10(-3) M) and ANP (10(-6) M) concentrations were: SNP: 154.7 +/- 15.4% of basal; SNP+forskolin: 191.3 +/- 14.8% of basal (P < 0.05, n = 4); ANP: 161.4 +/- 17.4% of basal; ANP+forskolin: 220.0 +/- 20.0% of basal (P < 0.05, n = 4). 4. The cyclic AMP-selective phosphodiesterase inhibitor, rolipram (10-5 M), had no effect on basal or SNP-stimulated [3H]-cyclic GMP levels; however, the combination of forskolin and rolipram produced an increase in the basal (158.7 +/- 27.1% of basal) and SNP-stimulated [3H]-cyclic GMP accumulation(SNP (10-3 M): 165.3 +/- 8.7% of basal; SNP + forskolin + rolipram: 510.7 +/- 64.8% of basal; P<0.05,n = 5), greater than either forskolin or rolipram alone. The phosphodiesterase inhibitor, IBMX (10-3 M)significantly raised [3H]-cyclic GMP levels, and forskolin (3 x 10- M) in the presence of IBMX had no significant effect on either basal or SNP-stimulated [3H]-cyclic GMP levels (e.g. in the presence of IBMX: SNP (10-3 M): 660 +/- 90% of basal; SNP + forskolin: 790 +/- 86% of basal, n = 3).5. The data indicate the presence of both soluble and particulate guanylyl cyclase in the porcine isolated palmar lateral vein. The ability of forskolin to potentiate SNP- and ANP-stimulated [3H]-cyclic GMP accumulation may suggest a cyclic AMP-cyclic GMP interaction at the level of the phosphodiesterases.Further, the ability of cyclic AMP to influence cyclic GMP levels may indicate that the two nucleotides, as well as having independent mechanisms to induce smooth muscle relaxation, could produce vasodilatation via a common mechanism.

Influence of isoproterenol and phosphodiesterase inhibitors on platelet-activating factor biosynthesis in the human neutrophil.

Increasing cellular levels of cAMP inhibit eicosanoid production in the human neutrophil; however, little is known about the effects of cAMP on platelet-activating factor (PAF) biosynthesis in this cell. In the current study, the beta-adrenergic receptor agonist isoproterenol, alone or in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), was used to increase cAMP in neutrophils. The incorporation of [3H]acetate into PAF and the synthesis of leukotrienes in response to ionophore A23187 were significantly inhibited by 10 microM isoproterenol. The inhibitory effect on PAF was potentiated by the addition of 10 microM IBMX. The effects of IBMX were mimicked by rolipram, an inhibitor of the cAMP-specific phosphodiesterase IV. Mass spectrometric analysis of the PAF molecular species in stimulated neutrophils indicated that the combination of isoproterenol and IBMX inhibited (> 50%) ionophore- and fMLP-induced production of PAF. To better understand the mechanism involved in the inhibition of PAF formation, the major biosynthetic steps were examined in the presence and absence of a maximally effective concentration of isoproterenol and IBMX. Isoproterenol alone or in the presence of IBMX had no measurable effect on the ionomycin-induced increase in cytosolic calcium concentration, as assessed by fura-2 fluorescence. Treating intact neutrophils with a combination of isoproterenol and IBMX did not inhibit acetyltransferase activity when assayed in a subsequent broken cell preparation. Finally, increasing cellular cAMP with these drugs did not influence the ability of the neutrophil to catabolize PAF. Phospholipase A2-like activity was assayed in the whole cell by measuring the mobilization of phospholipase A2 products, PAF, lyso PAF, and arachidonic acid, from cellular phosphoglycerides. Treatment of neutrophils with isoproterenol and IBMX significantly reduced the production of lyso PAF and PAF from 1-alkyl-2-arachidonoyl sn-glycero-3-phosphocholine. Similarly, increasing cellular levels of cAMP inhibited the cell's ability to mobilize arachidonic acid upon cell activation. These data suggest that increasing cellular levels of cAMP leads to the inhibition of PAF and leukotriene biosynthesis, at least in part, by regulation of phospholipase A2 activity.

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

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

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

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

Atriopeptin-induced increases in endothelial cell permeability are associated with elevated cGMP levels.

To investigate the mechanism of nonrenal capillary hyperfiltration, we studied the effect of atriopeptin (AP) III and AP I on permeability and intracellular cyclic nucleotide levels in cultured bovine pulmonary artery endothelial cell monolayers. Permeability to albumin was assessed by the albumin transfer rate across endothelial cell monolayers, following a 4-h incubation with atriopeptins. AP III (0.01, 0.1, and 1 microM) caused a concentration-dependent increase in the albumin transfer rate. AP III induced a threefold increase in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels during the incubation period. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), enhanced the AP III-induced increase in permeability and cGMP accumulation by 16-fold at maximum. 8-Bromoguanosine 3',5'-cyclic monophosphate, a hydrolysis-resistant cGMP analogue, caused a slight but significant increase in permeability. In contrast, AP I, a weak agonist of the cGMP-coupled ANP receptor, did not elicit an increase in permeability at concentrations of 0.1 and 1 microM. Although AP I (1 microM) caused a significant increase in cGMP by 33 and 60% in the absence and presence of IBMX, the increase was markedly less compared with AP III. AP III did not cause a change in intracellular cAMP levels during the incubation period. These observations suggest that in our system AP III increases the permeability of endothelial cell monolayers in association with an elevated cGMP level. Thus an increase in permeability might be involved in the mechanism of ANP-induced capillary hyperfiltration.

Effects of Sperm-Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water.

The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K+ ] sea water were examined. In high [K+ ] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K+ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K+ sea water. However, in high [K+ ] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca2+ concentration of spermatozoa in normal sea water but not in 100mM K+ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K+ ] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K+ channels.

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

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

Regulation of Cyclic AMP by the μ‐Opioid Receptor in Human Neuroblastoma SH‐SY5Y Cells

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive hormones and cAMP affect pericyte contraction and stress fibresin vitro

Pericytes are contractile cells of the microvascular wall that may influence capillary haemodynamics and permeability. We examined the contractile responses of cultured pericytes to selected vasoactive agents and cAMP agonists. Morphological and biochemical changes associated with these responses were also studied. Pericytes seeded onto silicone rubber contracted when stimulated with histamine or serotonin, relaxed in response to the beta-adrenergic agonist isoproterenol and did not respond to epinephrine. Since hormonal-induced relaxation of vascular smooth muscle involves cAMP, we investigated the ability of cAMP, to modulate pericyte contraction. Dibutyryl cAMP and forskolin (an adenylate cyclase activator) both induced pericyte relaxation and elevated intracellular cAMP levels. Isoproterenol increased cAMP levels but epinephrine had no effect. However, when epinephrine and isoproterenol were co-incubated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), cAMP was increased to levels above those elicited by these agonists alone. Serotonin and histamine in the presence of IBMX did not affect cAMP levels. These results suggest that certain vasoactive agents may relax pericytes by cAMP-dependent processes. We have shown previously that stress fibres are also involved in pericyte contraction. Hence, changes in the staining patterns of stress fibres in response to these selected agonists were studied. Histamine, serotonin and epinephrine had no apparent effect on stress fibre staining. Dibutyryl cAMP, forskolin, and isoproterenol, which relax pericytes and increase cAMP, disassembled fibres. In summary, the results demonstrate that the contractile activity of cultured pericytes in vitro can be regulated by vasoactive agonists and that changes in cAMP and stress fibres may mediate the regulation.