Increase In Cyclic GMP Content Research Articles

KMUP-1 relaxes rabbit corpus cavernosum smooth muscle in vitro and in vivo: involvement of cyclic GMP and K(+) channels.

1. In isolated endothelium-intact or denuded rabbit corpus cavernosum preconstricted with phenylephrine, KMUP-1 (0.001 - 10 microM) caused a concentration-dependent relaxation. 2. This relaxation of KMUP-1 was attenuated by endothelium removed, high K(+) and pretreatments with a soluble guanylyl cyclase (sGC) inhibitor ODQ (1 microM), a NOS inhibitor L-NAME (100 microM), a K(+) channel blocker TEA (10 mM), a K(ATP) channel blocker glibenclamide (1 microM), a voltage-dependent K(+) channel blocker 4-AP (100 microM) and Ca(2+)-dependent K(+) channel blockers apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). 3. The relaxant responses of KMUP-1 (0.01, 0.05, 0.1 microM) together with a PDE inhibitor IBMX (0.5 microM) had additive actions on rabbit corpus cavernosum smooth muscle (CCSM). 4. KMUP-1 (0.01 - 10 microM) induced increase of intracellular cyclic GMP level in the primary cell culture of rabbit CCSM. This increase in cyclic GMP content was abolished in the presence of ODQ (10 microM). 5. Both KMUP-1 and sildenafil at 0.2, 0.4, 0.6 mg kg(-1) caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose-dependent manner. These in vivo activities of ICP for sildenafil and KMUP-1 are consistent with those of in vitro effects of cyclic GMP. 6. KMUP-1 has the following merits: (1) inhibition of PDE or cyclic GMP breakdown, (2) stimulation of NO/sGC/cyclic GMP pathway, and (3) subsequent stimulation of K(+) channels, in rabbit CCSM. We suggest that these merits play prominent roles in KMUP-1-induced CCSM relaxation-associated increases of ICP and penile erection.

A xanthine-based KMUP-1 with cyclic GMP enhancing and K(+) channels opening activities in rat aortic smooth muscle.

1. KMUP-1 (1, 3, 5 mg kg(-1), i.v.), a xanthine derivative, produced dose-dependent sustained hypotensive and short-acting bradycardiac effects in anaesthetized rats. This hypotensive effect was inhibited by pretreatment with glibenclamide (5 mg kg(-1), i.v.). 2. In endothelium-intact or denuded aortic rings preconstricted with phenylephrine, KMUP-1 caused a concentration-dependent relaxation. This relaxation was reduced by endothelium removal, the presence of NOS inhibitor L-NAME (100 microM) and sGC inhibitors methylene blue (10 microM) and ODQ (1 microM). 3. The vasorelaxant effects of KMUP-1 was attenuated by pretreatment with various K(+) channel blockers TEA (10 mM), glibenclamide (1 microM), 4-AP (100 microM), apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). 4. Increased extracellular potassium levels (30 - 80 mM) caused a concentration-related reduction of KMUP-1-induced vasorelaxations. Preincubation with KMUP-1 (1, 10, 100 nM) increased the ACh-induced maximal vasorelaxations mediated by endogenous NO release, and enhanced the potency of exogenous NO-donor SNP. 5. The vasorelaxant responses of KMUP-1 (0.01, 0.05, 0.1 microM) together with a PDE inhibitor IBMX (0.5 microM) had an additive action. Additionally, KMUP-1 (100 microM) affected cyclic GMP metabolism since it inhibited the activity of PDE in human platelets. 6. KMUP-1 induced a dose-related increase in intracellular cyclic GMP levels in rat A10 vascular smooth muscle (VSM) cells, but not cyclic AMP. The increase in cyclic GMP content of KMUP-1 (0.1 - 100 microM) was almost completely abolished in the presence of methylene blue (10 microM), ODQ (10 microM), and L-NAME (100 microM). 7. In conclusion, these results indicate that KMUP-1 possesses the following merits: (1) stimulation of NO/sGC/cyclic GMP pathway and subsequent elevation of cyclic GMP, (2) K(+) channels opening, and (3) inhibition of PDE or cyclic GMP breakdown. Increased cyclic GMP display a prominent role in KMUP-1-induced VSM relaxations.

Evidence for cocaine and methylecgonidine stimulation of M(2) muscarinic receptors in cultured human embryonic lung cells.

1. Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M(2) muscarinic receptors. 2. A concentration-dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 microM carbachol (P < 0.01), 3.1 fold for 1 microM cocaine (P < 0.01), and 7.8 fold for 1 microM MEG (P < 0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M(2) blocker methoctramine. 3. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 microM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 microM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 microM significantly attenuated or abolished the cocaine-induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine-induced reduction of cyclic AMP production. 4. Western blot analysis showed that HEL299 cells specifically express M(2) muscarinic receptors without detectable M(1) and M(3). Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M(2) protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. 5. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug-induced stimulation of M(2) muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.

Long-lasting cyclic guanosine-3',5'-monophosphate accumulation in the medium of cultured smooth muscle cells from atherosclerotic rabbit aortas in response to exogenous or endogenous nitric oxide.

Although atherosclerosis causes a marked inhibition of the endothelium-dependent vasorelaxation it also leads to expression of inducible nitric oxide synthase (iNOS), accompanied by an increase in cyclic GMP content, in the arterial wall. The aim of our present study as to evaluate the influence of atherosclerosis on the soluble guanylyl cyclase pathway in viable cultured smooth muscle cells (SMC) from rabbit atherosclerotic rabbit aortas (atherosclerotic SMC) and from control rabbit aortas (control SMC). In response to 100 microM sodium nitroprusside (SNP), the intracellular production of cyclic GMP was similar in both types of cells, reaching a maximum after 5 min of incubation. In the culture medium, SNP evokes an increased cyclic GMP concentration lasting 6 h in control SMC and 24 h in atherosclerotic SMC. Interleukin-1beta (100 IU/mL), which induces iNOS in SMC from both control and atherosclerotic aortas causes accumulation of cyclic GMIP in the extracellular medium between 3 and 6 h for control SMC and between 3 and 24 h with atherosclerotic SMC. These results demonstrate a long-lasting egression of cyclic GMP in the extracellular medium of cultured SMC from rabbit aortas in response to endogenous or exogenous NO. Since this egression of cyclic GMP lasts longer in atherosclerotic than in control SMC, we suggest that atherosclerosis dysregulates the long-term soluble guanylyl cyclase response to NO in SMC.

Action of a NO donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery.

The aim of the present study was to investigate the actions of NO donors in ratsuperior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (Vm), cytosolic calcium concentration ([Ca2+]cyt) and accumulation of inositol phosphates. In unstimulated arteries, SNAP (S-nitroso-N-acetylpenicillamine, 10 microM) hyperpolarised Vm by 3.0 +/- 0.5 mV (n = 9). In KCl-stimulated arteries, SNAP relaxed contraction without changing Vm and [Ca2+]cyt. In noradrenaline-stimulated arteries, SNAP relaxed tension, repolarised Vm and decreased [Ca2+]cyt with the same potency. Responses to SNAP were unaffected by the following K+ channel blockers: glibenclamide, 4-aminopyridine, apamin and charybdotoxin, and by increasing the KCl concentration to 25 mM. In SNAP-pretreated arteries, the production of inositol phosphates and the contraction stimulated by noradrenaline were inhibited similarly. The guanylate cyclase inhibitor ODQ abolished the increase in cyclic GMP content evoked by SNAP and inhibited the effects of SNAP on contraction, Vm and accumulation of inositol phosphates in noradrenaline-stimulated artery. These results indicate that, in rat superior mesenteric arteries activated by noradrenaline, inhibition of production of inositol phosphates is responsible for the effects of the NO donor SNAP on membrane potential, [Ca2+]cyt and contraction through a cyclic GMP-dependent mechanism.

Norepinephrine increases cyclic GMP levels in cerebellar cells from neuronal nitric oxide synthase knockout mice.

Cyclic GMP is an important intracellular messenger in the nervous system that may mediate cellular forms of neuronal plasticity. Previous studies show that most neurotransmitters stimulate cyclic GMP levels by the activation of nitric oxide synthase (NOS). In this study, we report that in primary cell cultures from the cerebellum of neuronal NOS knockout mice, norepinephrine stimulates an increase in cyclic GMP content. This increase is seen in both granule cell and astrocyte cultures and is not blocked by inhibitors of NOS or by inhibition of soluble guanylyl cyclase. These results suggest a novel pathway by which norepinephrine enhances cyclic GMP levels in the nervous system.

Cyclosporine A-induced increase in glomerular cyclic GMP in rats and the involvement of the endothelinB receptor.

1. A transient two fold increase in the cyclic GMP content was observed in rat freshly isolated glomeruli 6 to 9 h after a single subcutaneous injection of 20 mg kg-1 cyclosporine A (CsA) in conscious animals. 2.In vitro stimulation with endothelin 3 (ET-3) of isolated glomeruli obtained from CsA-untreated rats resulted in a dose-dependent increase in cyclic GMP content. The increase observed with 10 nM ET-3 was similar to that observed in glomeruli isolated 9 h after in vivo CsA administration. 3. The rise in glomerular cyclic GMP content after in vivo CsA injection was prevented by in vivo treatment with L-NAME (10 mg kg-1) or by in vitro calcium deprivation of the incubation medium. 4. The stimulating effects of CsA on glomerular cyclic GMP content were inhibited by in vivo administration of the ETB receptor antagonist BQ-788 (2 mg kg-1) but not by the ETA receptor antagonist BQ-123 (2 mg kg-1). 5. The maximum increase in glomerular cyclic GMP content induced in vitro by acetylcholine (100 microM) and by ET-3 (100 nM) was slightly lower (approximately by 20-25%; P < 0.05) in glomeruli from CsA-treated rats than in glomeruli from untreated rats. In contrast, the maximum increase achieved with 1 microM sodium nitroprusside was similar in both groups. 6. A single subcutaneous injection of CsA did not significantly alter the glomerular mRNA expression of constitutive endothelial NO synthase (eNOS), as evaluated by RT-PCR, whereas the mRNA expression of the inducible NO synthase (iNOS), which follows pretreatment with lipopolysaccharide, was prevented. 7. These results indicate that in vivo administration of a single dose of cyclosporine A transiently increases the cyclic GMP content of freshly isolated glomeruli, and that activation of ETB receptors and stimulation of the NO pathway are involved in this process. Furthermore, a single administration of CsA does not impair eNOS mRNA expression and only slightly reduces NO-dependent glomerular cyclic GMP production.

Alteration by lipopolysaccharide of the relationship between intracellular calcium levels and contraction in rat mesenteric artery.

1. The aim of this work was to investigate the effect of lipopolysaccharide (LPS) treatment on the relationship between the cytosolic Ca2+ ion concentration ([Ca2+]i) and contraction in rat resistance arteries, and the involvement of the L-arginine-nitric oxide (NO)-guanosine 3'-5' cyclic monophosphate (cyclic GMP) pathway in these effects. 2. [Ca2+]i and tension were simultaneously recorded in small mesenteric arteries removed from rats 4 h after intraperitoneal injection of E. coli LPS (30 mg kg-1) or solvent. Cyclic GMP was assayed in vessels submitted to identical treatments. 3. Basal [Ca2+]i was higher in vessels from LPS-treated rats compared to controls. LPS did not modify the concentration-contraction curve of noradrenaline. However, the increase in basal [Ca2+]i produced by LPS resulted in a shift of the noradrenaline [Ca2+]i-contraction curve to higher [Ca2+]i concentrations. 4. L-Arginine (300 microM) relaxed noradrenaline (10 microM) pre-contracted arteries from LPS-treated but not from control rats. This effect of L-arginine was reversed by two inhibitors of NO synthase: N omega-nitro-L-arginine-methyl-ester (L-NAME, 1 mM) and S-methyl-isothiourea (SMT, 0.1 mM). Both the relaxing effect of L-arginine and its reversal by L-NAME or SMT occurred without any change in [Ca2+]i. 5. LPS treatment did not modify the cyclic GMP content of the small mesenteric arteries. In arteries removed from LPS-treated rats but not from controls, addition of L-arginine (300 microM) was associated with a significant increase in cyclic GMP content, an effect which was prevented by both L-NAME (1 mM) and SMT (0.1 mM). 6. L-NAME (1 mM) produced a greater reduction in cyclic GMP content than SMT (0.1 mM) in control vessels exposed to L-arginine (300 microM). Under the same conditions, SMT produced a larger decrease in cyclic GMP level than L-NAME in arteries taken from LPS-treated rats, consistent with selective inhibition by SMT of the inducible NO-synthase after LPS. 7. These results show that LPS produced two effects in small mesenteric arteries: (i) alterations in Ca2+ handling and a decreased sensitivity of myofilaments to Ca2+, (ii) induction of NO-synthase activity resulting in exogenous L-arginine-dependent production of NO and cyclic GMP accumulation. Both effects are likely to be involved in the hyporeactivity induced by LPS in resistance arteries.

BIOCHEMICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF NITRIC OXIDE SYNTHASE IN THE RAT RETINA

The increase in cyclic GMP contents in phototransduction is likely to be mediated with release of nitric oxide (NO). In this study we have characterized a constitutive NO synthase (NOS) isolated from rat retina supernatant. The activity of NOS was determined by monitoring L-citrulline formation from L-arginine. Soluble NOS from retina used L-arginine as substrate, with NADPH, tetrahydrobiopterin and FAD as cofactors. The enzyme activity was raised with additional calcium and was reduced with high concentrations of calmodulin inhibitors. Protein immunoblot and immunohistochemical analysis using a specific antibody against Type I NOS indicated that the same type of NOS is mainly localized in amacrine cells in the retina. However, the enzyme activity in freshly prepared sample was not completely abolished in the absence of calcium or with calmodulin inhibitors. In addition, NADPH diaphorase staining in retina was wider and more intensive than staining with the antibody against Type I NOS. These results indicate that rat retina contains more than one type of NOS.

Study of NO and VIP as non‐adrenergic non‐cholinergic neurotransmitters in the pig gastric fundus

1. The contribution of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) to non-adrenergic non-cholinergic (NANC) relaxations in the pig gastric fundus was investigated. 2. Circular and longitudinal muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine; tone was raised with 5-hydroxytryptamine. Electrical field stimulation with 10 s trains at 5 min intervals induced responses were abolished by tetrodotoxin. 3. The short-lasting as well as the sustained electrically induced NANC relaxations were significantly reduced by NG-nitro-L-arginine methyl ester (L-NAME). Pretreatment with L-arginine but not D-arginine, prevented the inhibitory effect of L-NAME except for sustained relaxations in the longitudinal muscle strips. 4. Sodium nitroprusside, forskolin, zaprinast and 3-isobutyl-l-methylxanthine induced concentration-dependent relaxations. Exogenous NO mimicked the short-lasting electrically induced relaxations, while endogenous VIP evoked sustained relaxations. The responses to exogenous NO and VIP were not influenced by tetrodotoxin and L-NAME. 5. alpha-Chymotrypsin abolished the responses to exogenous VIP but only moderately reduced NANC relaxations induced by continuous electrical stimulation. Zaprinast potentiated the relaxant responses to sodium nitroprusside and increased the duration of the NANC relaxations induced by electrical stimulation with 10 s trains in circular muscle strips but not in longitudinal muscle strips. 6. The cyclic GMP and cyclic AMP response to electrical stimulation, NO and VIP was measured in circular muscle strips. Short-lasting as well as sustained electrical field stimulation induced an approximately 1.5 fold increase in cyclic GMP content, while NO induced nearly a 40 fold increase. An increase in cyclic AMP content was obtained only with sustained electrical field stimulation. 7. Immunocytochemistry for NO synthase (NOS) revealed immunoreactive neuronal cell bodies in the submucous and myenteric plexuses and nerve fibres in both the circular and longitudinal muscle layer; double-labelling for NOS and VIP showed that VIP coexists in a major part of the intrinsic neurones. NADPH diaphorase-histochemistry showed the same pattern of nitrergic neurones and nerves as NOS-immunocytochemistry. 8. It is concluded that a cyclic GMP- and a cyclic AMP-dependent pathway for relaxation is present in both the circular and longitudinal muscle layer of the pig gastric fundus. NO appears to contribute to short-lasting as well as sustained NANC relaxations. A peptide, possibly VIP, may be involved during sustained stimulation at lower frequencies of stimulation.

Factors involved in the relaxation of female pig urethra evoked by electrical field stimulation.

1. Non-adrenergic, non-cholinergic (NANC) relaxations induced by electrical field stimulation (EFS) were studied in pig isolated urethra. The mechanism for relaxation was characterized by measurement of cyclic nucleotides and by study of involvement of different subsets of voltage-operated calcium channels (VOCCs). 2. EFS evoked frequency-dependent and tetrodotoxin-sensitive relaxations in the presence of propranolol (1 microM), phentolamine (1 microM) and scopolamine (1 microM). At low frequencies (< 12 Hz), relaxations were rapid, whereas at high (> 12 Hz) frequencies distinct biphasic relaxations were evoked. The latter consisted of a rapidly developing first phase followed by a more long-lasting second phase. 3. Treatment with the NO-synthesis inhibitor NG-nitro-L-arginine (L-NOARG; 0.3 mM) inhibited relaxations at low frequencies of stimulation. At high frequencies (> 12 Hz) only the first relaxation phase was affected. 4. Measurement of cyclic nucleotides in preparations subjected to continuous nerve-stimulation, revealed an increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels from 1.3 +/- 0.3 to 3.0 +/- 0.4 pmol mg-1 protein (P < 0.01). In the presence of L-NOARG, there was a significant decrease in cyclic GMP content to control. However, there was no increase in cyclic GMP content in response to EFS. Levels of cyclic AMP remained unchanged following EFS. 5. Treatment with the N-type VOCC-inhibitor, omega-conotoxin GVIA (0.1 microM) reduced NO-dependent relaxations, the effect being most pronounced at low frequencies (1-4 Hz) of stimulation. The NO independent second phase of the relaxation, studied in the presence of L-NOARG (0.3 mM) at 16-30 Hz, was however markedly reduced or abolished by omega-conotoxin GVIA. omega-Conotoxin MVIIC (1 microM)or omega-agatoxin IVA (30 nM) had no effect on electrically evoked relaxations.6. These results suggest that NANC-nerve derived urethral relaxation in the pig consists of two apparently independent components. One is mediated by NO and associated with an increase in cyclic GMP content. The other mediator is unknown and produces relaxations not associated with changes in levels of cyclic nucleotides. The release of this mediator seems to involve the N-type VOCC, since the relaxation was markedly reduced or abolished by omega-conotoxin GVIA.

Rolipram increases cyclic GMP content in L-arginine-treated cultured bovine aortic endothelial cells

Cultured bovine aortic endothelial cells only contain two cyclic nucleotide phosphodiesterases isoforms: PDE II (cyclic GMP stimulated) and PDE IV (rolipram sensitive). The effects of cilostamide or rolipram alone or together, on cyclic AMP and cyclic GMP levels, were measured in indomethacin-treated endothelial cells alone or in the presence of nitric oxide (NO) modulators. In all conditions, cyclic AMP levels were potently increased (8–13-fold) only when PDE II and PDE IV inhibitors were given together. Cyclic GMP levels were not modified by these PDE inhibitors in control and N G-nitro- L-arginine-methyl ester-treated cells. But surprisingly, in L-arginine-treated cells, cyclic GMP content was increased by 42% by rolipram alone, and combination of rolipram with cilostamide resulted in a further increase in cyclic GMP content (to 153% compared to control cells). These results suggest that in presence of the NO synthase substrate ( L-arginine), an increase in cyclic AMP level may upregulate the L-arginine/NO/cyclic GMP pathway.

Nitric oxide and cyclic GMP formation following relaxant nerve stimulation in isolated human corpus cavernosum.

To investigate further the role of the nitric oxide (NO)-cyclic GMP pathway as the mediator of relaxant neurotransmission in human corpus cavernosum and to establish whether impaired activity of this pathway contributes to the pathophysiology of impotence. Samples of cavernosal tissue were obtained from 59 men undergoing penile operations. The controls comprised four men with penile carcinoma and 17 with Peyronie's disease. Of the impotent men, 35 had clinical evidence of penile vascular disease on pre-operative investigation, whilst three had non-vascular impotence. Each biopsy was divided into two strips which were then suspended under tension in organ bath chambers. The relaxant innervation of one strip of each pair was stimulated electrically whilst the other strip was left unstimulated. The formation of NO and cyclic GMP was calculated by comparing their respective tissue content in the stimulated and unstimulated strips. Overall, stimulation of the relaxant innervation produced significant increases in the tissue content of both NO and cyclic GMP. Incubation with an inhibitor of NO biosynthesis abolished the mechanical relaxant response and the formation of both NO and cyclic GMP. The magnitude of relaxant response and the formation of NO was diminished in tissue from men with vascular impotence compared to controls. The increase in cyclic GMP content was similar in both these groups. Relaxant response, NO formation and cyclic GMP formation in tissue from men with non-vascular impotence was similar to controls. This study provides further evidence that the NO-cyclic GMP pathway acts as the mediator of nerve-evoked smooth muscle relaxation in human corpus cavernosum. Diminished NO formation following relaxant nerve stimulation may account for impaired relaxant responses found in tissue from men with vascular impotence and may contribute to the cause of their erectile dysfunction.

DQ-2511, having an antiulcer action, elicits vasodilation through an increase in cyclic GMP contents in rat arteries.

It has been reported that 3-(([2-(3,4-dimethoxyphenyl)ethyl]carbamoyl)methyl)amino-N-methylbenz amide (DQ-2511: ecabapide) effectively increases gastric mucosal blood flow in rats, suggesting that this property may contribute to the antiulcer activity. To clarify the mechanisms underlying the increase in gastric mucosal blood flow, we investigated the dilator response of rat isolated thoracic aorta, mesenteric artery and celiac artery smooth muscle preparations to DQ-2511. This compound prevented noradrenaline-induced contraction in both the presence and absence of endothelium in the arterial specimen, and it (0.01-1 mM) inhibited these contractions induced by noradrenaline in all tissues in a concentration-dependent manner. This inhibitory effect of DQ-2511 was most evident in the celiac artery. The dilator response to DQ-2511 (0.1 mM) was abolished after pretreatment with methylene blue (3 microM), a guanylate cyclase inhibitor. Under the same conditions, methylene blue inhibited the dilator response to acetylcholine (1 microM), but not that to papaverine (10 microM). Furthermore, when DQ-2511 (0.01-1 mM) was incubated with the arterial preparations, this compound increased cyclic GMP content in segments in a concentration-dependent manner. These findings demonstrate that the vasodilation induced by DQ-2511 is independent of the endothelium and is related to the augmentation of intracellular cyclic GMP content, which may consequently contribute to the increased gastric mucosal blood flow.

Contribution of cyclic GMP generation to the relaxation by nipradilol in the rabbit aorta

1. 1. Contribution of cyclic GMP generation to the relaxation by nipradilol of vascular smooth muscle was studied in the isolated rabbit aorta contracted by phenylephrine (10 −7 M) or prostaglandin F 2α (PGF 2α, 3 × 10 −6 M). 2. 2. Nipradilol-induced relaxation in both contractions and increase in cyclic GMP content were inhibited by methylene blue (10 −5 M). 3. 3. The relations between the increase in cyclic GMP and the relaxation produced by nipradilol and nitroglycerin were fitted to sigmoid curves. The increase in cyclic GMP at 50% relaxation by nipradilol was smaller than that by nitroglycerin (1.2-fold increase as against 3-fold increase). 4. 4. These results suggest a smaller contribution of cyclic GMP generation through activation of soluble guanylate cyclase to nipradilol-induced relaxation in the rabbit aorta.

Role of phosphodiesterases III and IV in the modulation of vascular cyclic AMP content by the NO/cyclic GMP pathway

1. The effect on cyclic nucleotide contents of selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) isoforms III and IV (respectively SK&F 94120 and rolipram) and their interactions with endothelium and NO have been studied in rat aorta in the presence of indomethacin (10 microM). The participation of NO was assessed by using either NG-nitro-L-arginine methyl ester (L-NAME) (NO synthase inhibitor: 30 microM) or 3-morpholinosydnonimine (SIN-1, NO donor: 10 microM with SOD 100 units ml-1). 2. The presence of endothelium significantly increased both adenosine 3':5'-cyclic monophosphate (cyclic AMP, 1.7 fold) and guanosine 3':5'-cyclic monophosphate (cyclic GMP, 2.2 fold) contents. Cyclic GMP was largely affected by L-NAME or SIN-1 treatment, this was not the case for cyclic AMP suggesting that the presence of endothelium modified cyclic AMP content in aorta independently of the NO production. 3. In the presence or absence of endothelium, neither SK&F 94120 nor rolipram, alone or combined, significantly modified cyclic GMP content. 4. The PDE III inhibitor significantly affected cyclic AMP content only in non treated aorta without endothelium. In contrast, the PDE IV inhibitor increased cyclic AMP in all conditions. These increases were generally about 2 fold but markedly higher in aorta treated with SIN-1 and superoxide dismutase (SOD, 6 fold). Association of a low concentration of the PDE III inhibitor (5 microM) with the PDE IV inhibitor (30 microM) potentiated the effect of the PDE IV inhibitor on cyclic AMP content, except for aorta without endothelium treated with SIN-1 plus SOD. 5. These data indicate that the presence of the endothelium could increase cyclic AMP content independently of NO and prostacyclin (PGI2) production. Furthermore, an increase in cyclic GMP content (modulated by NO production) could enhance the cyclic AMP accumulation induced by the PDE IV inhibitor. This result supports the hypothesis that PDE III inhibition by endogenous cyclic GMP may potentiate the effect of PDE IV inhibition on cyclic AMP content. Taken together with our previous studies on relaxation, these results suggest that the NO/cyclic GMP pathway could induce PDE IV-dependent regulation of cyclic AMP via PDE III inhibition.

Segmental differences in the effects of guanylin and Escherichia coli heat‐stable enterotoxin on Cl‐ secretion in human gut.

1. Mucosally added synthetic guanylin and Escherichia coli heat-stable enterotoxin (STa) increased short-circuit current (ISC) across isolated muscle-stripped human intestine in vitro. 2. Serosal bumetanide inhibited ISC responses indicating that guanylin and STa stimulate electrogenic chloride secretion. 3. ISC responses were markedly greater in the colon than in the jejunum. 4. Pretreatment with indomethacin did not significantly alter the effects of guanylin and STa. 5. Both peptides induced concentration-dependent increases in the cyclic GMP content of human intestinal mucosa in vitro; cyclic AMP levels remained unchanged. 6. In contrast to ISC responses, increases in cyclic GMP content induced by guanylin and STa were markedly greater in the jejunum than in the colon. 7. Sodium nitroprusside (SNP) but not human alpha-atrial natriuratic peptide (CDD/ANP(99-126)) increased chloride secretion in human intestine; both agents induced small increases in intestinal cyclic GMP content. 8. Guanylin, STa and the nitric oxide (NO) donor SNP increased electrogenic chloride secretion across human intestinal mucosa in vitro by stimulation of cyclic GMP. The discrepancy between the effects on chloride secretion and intracellular cyclic GMP content suggest different cellular action sites of guanylin and STa in human small and large intestine.

Vasorelaxant mechanism of the new vasodilator, FK409

To define the vasorelaxation mechanism of FK409, we examined the effect of the compound on vascular tension and cyclic nucleotide levels in isolated rat thoracic aorta contracted with norepinephrine, and on activities of guanylate cyclase and cyclic GMP phosphodiesterase prepared from rat or rabbit thoracic aorta. FK409 (1 × 10 −9 to 1 × 10 −6 M), like nitroglycerin (1 × 10 −9 to 1 × 10 −6 M), produced a potent vasorelaxant effect associated with an increase in cyclic GMP content of the tissue. There was no change in cyclic AMP levels. The vasorelaxant effect of FK409 was independent of the integrity of the endothelium, and was unaffected by L-N G-monomethylarginine (0.1 mM) or oxyhemoglobin (1 μM). On the other hand, FK409 (3.2 × 10 −7 M) activated soluble guanylate cyclase, and the activating effect was completely inhibited by oxyhemoglobin (10 nM). Cyclic GMP phosphodiesterase was unaffected by FK409 (1 × 10 −7 to 1 × 10 −5 M). Furthermore, in rat aortic soluble fraction FK409 (3 mM) was found to liberate nitric oxide (NO) which was evaluated spectrophotometrically after diazotization of sulfanilic acid and coupling with N-(1-naphtyl)-ethylenediamine. The liberation occurred even in the absence of L-cysteine (5 mM), in contrast to the case with nitroglycerin (3 mM). These results suggest that the vasorelaxant effect of FK409 is associated with an increase in intracellular cyclic GMP, and that the cyclic GMP accumulation is due to activation of soluble guanylate cyclase. The enzyme activation is probably due to NO released from the compound molecule in the vascular smooth muscle cells.

Leukotriene D4 receptors are not negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma.

1. The possibility that receptors for the peptide-containing leukotrienes may be negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma was investigated by comparing the effect of leukotriene D4 (LTD4) on the intracellular cyclic nucleotide (cyclic AMP and cyclic GMP) content and on the activity of cyclic AMP-dependent protein kinase (PKA). In addition, the potential association between changes in the cyclic nucleotide content and the ability of LTD4 to increase lung parenchymal tone was also evaluated. 2. Non-cumulative challenge of parenchymal lung strips with LTD4 elicited concentration-dependent contractions (pD2 = 8.23) that were paralleled by concentration-related increases in the intracellular level of cyclic AMP and cyclic GMP, and in the activation state of PKA (Kact = 33 nM). Temporally, these biochemical effects of LTD4 were transient, peaking after approximately 5 min drug contact thereafter decaying, despite the continued generation of tone. Both the biochemical and mechanical effects of LTD4 were antagonized by the LTD4-receptor blocking drug, ICI 198,615 (1 microM for 60 min), indicating that they were receptor-mediated events. 3. Challenge of guinea-pig lung with LTD4 (200 nM; EC100 for tension generation) stimulated a 150 and 70 fold increase in the elaboration of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) respectively, relative to that generated spontaneously. 4. Pretreatment of lung strips for 60 min with an irreversible inhibitor of cyclo-oxygenase, flurbiprofen,at a concentration (8 microM) that abolished both basal and LTD4 (200 nM)-induced TXB2 and 6-keto-PGF1alpha release, relaxed rapidly the spontaneous tone of the tissues, reduced the cyclic AMP content by ~50%and lowered the PKA activity ratio from 29% to 17%. In addition, flurbiprofen abolished the ability of LTD4 (200 nM) to increase the cyclic AMP content and to activate PKA. Functionally, the magnitude of LTD4 (200 nM)-induced tone and the increase in cyclic GMP content were attenuated by approximately 20% and 50% respectively in flurbiprofen-treated tissues.5. In flurbiprofen-treated tissues, isoprenaline (10 microM for 10 min) increased the cyclic AMP content(from 4 to 27 pmol mg-1 protein) and activated PKA (from 15% to 26%). Preincubation (30 s or 5 min)of lung with LTD4 (200 nM) did not inhibit (or enhance) these isoprenaline-induced effects.6. Pretreatment of lung strips for 60 min with the thromboxane synthetase inhibitor, dazmegrel (10 microM),relaxed the spontaneous tone of the tissues, abolished the LTD4 (200 nM)-stimulated release of TXB2 and significantly enhanced (~two fold) the elaboration of 6-keto-PGF1alpha. In addition, dazmegrel attenuated (by ~50%) LTD4 (200 nM)-induced cyclic GMP accumulation but approximately doubled both the cyclic AMP content and PKA activity ratio. LTD4-induced contractions, in contrast, were not affected by dazmegrel.7. EP 092 (1 microM for 60 min), a selective TP-receptor blocking drug, had no effect on spontaneous tone,eicosanoid formation or on the cyclic GMP content of guinea-pig lung parenchymal strips. Likewise,EP 092 exerted no significant mechancial effect in lung challenged with LTD4 (200 nM) although it did potentiate, to a small extent, the ability of LTD4 (200 nM) to increase the cyclic AMP content.8. It is concluded that LTD4 can increase the intracellular level of cyclic AMP in guinea-pig parenchyma and activate PKA by a leukotriene-receptor-mediated mechanism sensitive to ICI 198,615. However,these biochemical actions of LTD4 are induced indirectly by an arachidonic acid-derived cyclo-oxygenase product(s) other than TXA2. Thus, contrary to reports of other investigators, no evidence was found to corroborate the finding that stimulation of leukotriene receptors on guinea-pig lung parenchyma results in a rapid lowering of the cyclic AMP content even in cyclo-oxygenase-blocked tissues. These data,therefore, do not support the hypothesis that leukotriene-induced tension generation is dependent upon a prior reduction in the cyclic AMP content.

Prostacyclin and nitric oxide contribute to the vasodilator action of acetylcholine and bradykinin in the intact rabbit coronary bed.

The relative contribution of nitric oxide (NO) and cyclo-oxygenase products in the dilator response to equieffective doses of acetylcholine (ACh) and bradykinin (Bk) was studied in the isolated, saline-perfused rabbit heart under constant flow conditions. ACh (1 microM) and Bk (10 nM) induced a similar vasodilation, with a maximum reduction in coronary perfusion pressure (CPP) of 27 +/- 2%. The vasodilation induced by both agonists was associated with an enhanced release of 6-keto-PGF1 alpha from the coronary bed, with the Bk-induced increase in 6-keto-PGF1 alpha being threefold greater than that induced by ACh. The angiotensin converting enzyme (ACE) inhibitor ramiprilat (0.3 microM) selectively enhanced both the 6-keto-PGF1 alpha outflow and the dilator response to Bk. The B2-receptor antagonist Hoe 140 (0.1 microM) blocked both Bk effects. The cyclo-oxygenase inhibitor diclofenac (1 microM) halved the dilator response to Bk, but did not affect the vasodilation to ACh. Both agonists induced the release of NO, as assessed by the increase in cyclic GMP content of platelets passing through the vascular bed. However, ACh induced a 2.5-fold greater increase in platelet cyclic GMP content, compared to Bk. Treatment of hearts with NG-nitro-L-arginine (L-NNA, 30 microM) halved the ACh- and Bk-induced maximum reduction in CPP. Combined infusion of L-NNA and diclofenac completely blocked the dilator response to Bk, and inhibited the vasodilation to ACh more efficiently than L-NNA alone. We conclude that both NO and PGI2 contribute to the coronary dilator response to Bk and ACh in the rabbit Langendorff heart.(ABSTRACT TRUNCATED AT 250 WORDS)