Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation

1. The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) in the effects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca²⁺]_i) was studied in rat aorta. Cyclic AMP-elevating agents used were a β-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram).
2. In rat intact aorta, the relaxant effect induced by isoprenaline (0.01–0.3 μM) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 μM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca²⁺]_i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01–0.3 μM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved.
3. The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no significant effect of H-89. Neither SK&F 94120, nor rolipram at 30 μM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca²⁺]_i. This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant effect. These results suggest that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta.
4. The combination of 5 μM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H-89 and not significantly modified by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell.
5. These findings support a role for both PKA and PKG in cyclic AMP-mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

     

Critical dependence of the NO-mediated component of cyclic AMP-induced vasorelaxation on extracellular L-arginine in pulmonary arteries of the rat

A component of isoprenaline-mediated vasorelaxation in pulmonary arteries is mediated by nitric oxide (NO). We examined the effects of physiological concentrations (150 microM.     

Potentiation by neuropeptide Y of vasoconstriction in rat resistance arteries.

1. The effects of neuropeptide Y (NPY) on resistance arteries were investigated on 3rd generation mesenteric arterioles of the rat. 2. Contractions were elicited by noradrenaline (NA), 5-hydroxytryptamine (5-HT), prostaglandin F2 alpha (PGF2 alpha), depolarization (KCl substituted for NaCl) and by the calcium agonist Bay K 8644, in the absence and in the presence of NPY (100 nM), a concentration which by itself did not induce vasoconstriction. 3. NPY produced a leftward shift of the concentration-response curves to the agonists and to KCl, without any alteration of maximal contractions. 4. NPY also potentiated contractions elicited by addition of CaCl2 to KCl-depolarized vessels, but its effect on calcium-induced contractions decreased with increasing KCl concentrations (from 20 to 100 mM). 5. Calcium-induced contractions were inhibited by the calcium channel blocker nitrendipine, both in the presence and absence of NPY (100 nM). NPY increased slightly (but significantly) the sensitivity to nitrendipine (the apparent KB increased from 2.9 x 10(-10) M to 1.6 x 10(-10) M). 6. The KCl concentration necessary for the maximal effect of Bay K 8644 was decreased in the presence of NPY, and the sensitivity to the calcium channel agonist was increased. 7. Elevating the KCl concentration in the bath from 5 to 20 mM (which gives the same displacement to the left of the KCl concentration-effect curve seen in the presence of NPY) induced a parallel leftward shift of NA and 5-HT concentration-response curves. This shift was identical to the one induced by NPY on 5-HT-evoked contractions, but it was significantly smaller (P less than 0.001) than the shift of the NA concentration-response curve observed in the presence of NPY. In the latter case, NPY enhanced more markedly the contractions induced by low NA concentrations (between 10(-9) and 3 x 10(-8 M) than those induced by high concentrations (up to 3 x 10(-7) M), thus giving a shallow concentration-response curve. 8. The results strongly suggest that NPY partially depolarizes the arterioles and induces an increase in calcium entry through voltage-dependent channels, thus enhancing contractions elicited by agonists or by KCl-depolarization. In addition, they support the view that another mechanism also plays a part in the potentiation by NPY of the effects of low concentrations of NA.     

Potentiation of cyclic AMP-mediated proopiomelanocortin gene promoter activity by calcium channel blockers in a pituitary cell line

Although it is known that the expression of proopiomelanocortin, a precursor protein of adrenocorticotropic hormone, can be affected by a variety of drugs, the effects of calcium channel blockers have not been studied. This study examined the effect of calcium channel blockers on proopiomelanocortin gene expression. Mouse pituitary tumor cells stably transfected with approximately 0.7 kb of the rat proopiomelanocortin 5' promoter-luciferase fusion gene were stimulated by potassium chloride, corticotropin-releasing hormone (CRH) or forskolin, in the presence or absence of calcium channel blockers (nifedipine, verapamil and diltiazem). Assessments were made of proopiomelanocortin gene promoter activity and cyclic adenosine 3',5'-monophosphate (cyclic AMP) efflux. A dose-dependent enhancement of CRH- or forskolin-stimulated proopiomelanocortin promoter activity was observed with nifedipine and verapamil, but not diltiazem. Cyclic AMP efflux induced by CRH or forskolin was also enhanced by nifedipine and verapamil. In the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, enhancement of proopiomelanocortin promoter activity and cyclic AMP efflux by nifedipine and verapamil was not observed. It was concluded that the inhibition of phosphodiesterase is a probable mechanism for the effect of nifedipine and verapamil on CRH or forskolin induction of proopiomelanocortin gene expression.     

Mechanisms of noradrenaline-induced vasorelaxation in isolated femoral arteries of the neonatal rat.

Isolated arteries from the femoral circulation of Wistar rats mounted on a small vessel myograph demonstrated age related tension development to noradrenaline (NA, 1 x 10(-8) - 5 x 10(-5) M) day 20 greater than day 10 (P<0.005); day 100 greater than day 20 (P<0.001) and depolarizing potassium (125 mM) buffer day 20 greater than day 10 (P<0.001). NA evoked dilatation in femoral arteries from neonatal rats (10 days) when added to unstimulated vessels or to those preconstricted with the thromboxane mimetic, U46619. Relaxation to NA was inhibited by L-NAME (0.1 mM) (P<0.001), endothelial removal (P<0.001) and the alpha2-adrenoceptor antagonist, yohimbine (0.1 microM) (P<0.001). Alpha1- or beta-adrenoceptor antagonism was without effect. Relaxation was evoked in femoral arteries of the 10-day-old rats by the alpha2-adrenoceptor agonist UK14304 (1 x 10(-8) - 5 x 10(-5) M). This relaxation was also abolished by L-NAME (0.1 mM) (P<0.001) or endothelial removal (P<0.001). Alpha2-adrenoceptor-mediated vasorelaxation was the predominant response to NA stimulation in femoral arteries of the neonatal rat. These responses were endothelium-dependent and were NO-mediated.     

Mechanisms of anandamide-induced vasorelaxation in rat isolated coronary arteries

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.     

Mitochondrial uncoupler triclosan induces vasorelaxation of rat arteries

Our previous studies found that mitochondrial uncouplers induced vasodilation. Triclosan, the broad spectrum antibacterial agent, is the active ingredient in soaps and toothpastes. It was reported that triclosan induced mitochondrial uncoupling, so we aim to investigate the effects of triclosan on vascular function of rat mesenteric arteries and aorta. The isometric tension of rat mesenteric artery and thoracic aorta was recorded by multi-wire myograph system. The cytosolic [Ca²⁺]_i, mitochondrial reactive oxygen species (mitoROS), and mitochondrial membrane potential of smooth muscle cells (A10 cells) were measured using laser scanning confocal microscopy. Triclosan treatment relaxed phenylephrine (PE)- and high K⁺ (KPSS)-induced constriction, and pre-treatment with triclosan inhibited PE- and KPSS-induced constriction of rat mesenteric arteries. In rat thoracic aorta, triclosan also relaxed PE- and KPSS-induced constriction. Triclosan induces vasorelaxation without involving K_ATP channel activation in smooth muscle cells of arteries. Triclosan treatment increased cytosolic [Ca²⁺]_i, mitochondrial ROS production and depolarized mitochondrial membrane potential in A10 cells. In conclusion, triclosan induces mitochondrial uncoupling in vascular smooth muscle cells and relaxes the constricted rat mesenteric arteries and aorta of rats. The present results suggest that triclosan would indicate vasodilation effect if absorbed excessively in vivo.     

Mechanisms of vasorelaxation to 17beta-oestradiol in rat arteries

We have investigated the involvement of the endothelium, K+ channels, oestradiol receptors, and Ca2+ influx in 17beta-oestradiol-induced vasorelaxation in rat mesenteric arterial beds and aortae. 17beta-Oestradiol (10 pM-1 mM) caused acute vasorelaxations in mesenteric arterial beds and aortae from male and female rats. In male rat mesenteric vessels and aortae, the vasorelaxations were mostly independent of the endothelium and nitric oxide (NO). However, indomethacin (10 microM) enhanced the relaxant responses to 17beta-oestradiol. In male rat mesenteric beds, 60 mM KCl, tetrabutylammonium chloride (300 microM), 4-aminopyridine (1 mM), and barium chloride (30 microM), charybdotoxin (100 nM), but not glibenclamide (10 microM) and tamoxifen (10 microM), inhibited vasorelaxation to 17beta-oestradiol. In male rat aortae, 60 mM KCl did not affect vasorelaxation to 17beta-oestradiol. However, in the presence of indomethacin, vasorelaxation to 17beta-oestradiol was enhanced but this was sensitive to 60 mM KCl. Pre-treatment with 17beta-oestradiol (100 microM) inhibited CaCl2-induced contraction. The present findings indicate that, in rat mesenteric beds and aortae, 17beta-oestradiol causes acute and potent vasorelaxation which may be enhanced in the presence of a cyclooxygenase inhibitor. In mesenteric arterial bed, 17beta-oestradiol-induced vasorelaxation occurs primarily via activation of K+ channels. In the aorta, vasorelaxations involved activation of K+ efflux when the cyclooxygenase pathway was inhibited, and also inhibition of Ca2+ influx.     

Essential role of L-arginine uptake and protein tyrosine kinase activity for NO-dependent vasorelaxation induced by stretch, isometric tension and cyclic AMP in rat pulmonary arteries

1. The NO-dependent component of cyclic AMP-induced vasorelaxation in rat pulmonary arteries is critically dependent on extracellular L-arginine but independent of endothelial cell intracellular [Ca(2+)]. We examined whether L-arginine uptake was also essential for NO production induced by passive stretch or isometric tension, processes also reported to be Ca(2+)-independent. 2. The passive length-tension curve was depressed by physiological concentrations of L-arginine (400 microM; P<0.05). Inhibition of the y(+) transporter with 10 mM L-lysine, NO synthase with L-NAME (100 microM), or protein tyrosine kinase with erbstatin A (30 microM) caused identical upward shifts (P<0.001), alone or in combination. Tyrphostin 23 was similar to erbstatin A, whilst the inactive analogue tyrphostin A1 and genistein were without effect. 3. L-arginine (400 microM) shifted the PGF(2 alpha) concentration-response curve under isometric conditions to the right (P<0.05), whereas L-NAME or L-lysine caused a leftward shift (P<0.001). Tyrphostin 23 (30 microM) more than reversed the L-arginine-induced suppression of PGF(2 alpha)-induced tension; subsequent addition of L-NAME had no effect. The L-lysine-sensitive component of CPT cyclic AMP-induced vasorelaxation was abolished by erbstatin A. 4. ACh-induced vasorelaxation was approximately 80% inhibited by L-NAME, but was not affected by L-lysine or 400 microM L-arginine. Erbstatin A reduced the vasorelaxation by only approximately 25%. 5. We conclude that activation of NO production by stretch, isometric tension, or cyclic AMP in rat pulmonary arteries is critically dependent on the presence and uptake of physiological concentrations of extracellular L-arginine, and protein tyrosine kinase activity. This directly contrasts with ACh-induced vasorelaxation, which was independent of extracellular L-arginine, and relatively unaffected by tyrosine kinase inhibition.     

Potentiation of vasoconstrictor response and inhibition of endothelium-dependent vasorelaxation by gallic acid in rat aorta

In the isolated rat thoracic aorta, gallic acid potentiated the vasoconstrictor response to phenylephrine. The potentiation produced by gallic acid was absent in endothelium-denuded arteries. The potentiation was abolished by N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis, and slightly attenuated by an addition of L-arginine, while indomethacin or BQ610 had no effect. The potentiation of response to phenylephrine was not found for structural modifications of gallic acid, except for caffeic acid. Gallic acid also inhibited vasorelaxation induced by acetylcholine, sodium nitroprusside or prostacyclin, especially that by acetylcholine. The effect on vasorelaxation induced by acetylcholine was decreased by esterification of the carboxy group of gallic acid, and in the absence or by the methylation of the o-dihydroxy group. Caffeic acid inhibited the vasorelaxation, though the effect was smaller than that of gallic acid. These findings indicate that gallic acid produces a potentiation of contractile response and inhibition of vasorelaxant responses, probably through inactivation of nitric oxide (NO), in which endothelially produced NO is principally involved, and that the modification of functional groups of the gallic acid molecule abolishes the potentiation of contractile response and attenuates the inhibition of vasorelaxant responses.     

An investigation of the mechanisms of hydrogen sulfide-induced vasorelaxation in rat middle cerebral arteries

Hydrogen sulfide (H(2)S) is an endogenous mediator with peripheral vasorelaxant effects; however, the mechanism of H(2)S-induced vasorelaxation in cerebral blood vessels has not been extensively studied. Vasorelaxation studies were performed on middle cerebral arteries from male Sprague Dawley rats using wire myography. Immunofluorescence staining was used to detect the presence of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE). CSE was present in the endothelium and smooth muscle of middle cerebral arteries. The CSE substrate, L-cysteine, induced vasorelaxation that was sensitive to the CSE inhibitor DL-propargylglycine. This relaxation was independent of endothelium, suggesting that H(2)S was produced in the vascular smooth muscle. The H(2)S donor, sodium hydrogen sulfide (NaHS; 0.1-3.0?mM) produced concentration-dependent relaxation, which was unaffected by endothelium removal. Nifedipine (3?μM) significantly reduced the maximum relaxation elicited by NaHS. Inhibiting potassium (K(+)) conductance with 50?mM K(+) significantly attenuated NaHS-induced relaxation, however, selective blockers of ATP sensitive (K(ATP)), calcium sensitive (K(Ca)), voltage dependent (K(V)), or inward rectifier (K(ir)) channels alone or in combination did not affect the response to NaHS. 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 300?μM) caused a significant rightward shift of the NaHS concentration-response curve, but this effect could not be explained by inhibition of Cl(-) channels or Cl(-)/ HCO (3) (-) exchange, as selective blockade of these mechanisms had no effect. These findings suggest endogenous H(2)S can regulate cerebral vascular function. The H(2)S-mediated relaxation of middle cerebral arteries is DIDS sensitive and partly mediated by inhibition of L-type calcium channels, with an additional contribution by K channels but not K(ATP), K(Ca), K(V), or K(ir) subtypes.     

Prior vasorelaxation enhances diadenosine polyphosphate-induced contractility of rat mesenteric resistance arteries

Low-threshold concentrations of diadenosine polyphosphates (ApnA: Ap3A, Ap4A, Ap5A, Ap6A) or ATP, which at basal vessel tone induce just measurable vasoconstrictions, induce up to ten times enhanced vasoconstrictions of previously relaxed (by acetylcholine or sodium nitroprusside or 8Br₂ cGMP or isoproterenol or levcromakalim) pre-contracted rat mesenteric resistance arteries (MrA) in a microvessel–myograph. These enhanced vasoconstrictions were of similar magnitude for threshold concentrations of all ApnA.Possibly, the low concentrations of ApnA reverse the prior vasorelaxation by inhibiting a common vasorelaxation pathway, but obviously this is not due to inhibition of guanylate cyclase, which has been previously described to be inhibited by ApnA, because the enhanced vasoconstrictions can be observed with guanylate cyclase-independent vasorelaxants (8Br₂ cGMP, isoproterenol or levcromakalim), too. The enhanced vasoconstrictions are endothelium-independent because after mechanical vascular de-endothelialization the results were identical. De-endothelialized vessels, which fail to relax by acetylcholine, showed no enhanced ApnA-induced vasoconstrictions, demonstrating that the mere prior vasorelaxation of the vessel is required to provide the enhanced vasoconstriction by ApnA. Furthermore, the enhanced contractility is not based on a potentiation of the phenylephrine contraction because it equally occurs with other agents used for arterial pre-contraction. Systemically applied ApnA considerably decrease arteriovascular resistance, resulting in hypotension. But here it is demonstrated that a preceding vasorelaxation enables the resistance arteries to generate a strong and persistent ApnA-induced vasoconstriction. Thus, in vivo at very low concentrations ApnA may serve to counteract severe conditions of hypotension (e.g., shock syndrome or anaphylaxis) by the constriction of resistance arteries.     

Kaempferol-induces vasorelaxation via endothelium-independent pathways in rat isolated pulmonary artery

Background

Kaempferol, a flavonoid, is the essential part of human diet. Flavonoids have different pharmacological activities like cardioprotective, anti-inflammatory and anti-oxidant. The aim of current study was to investigate vasorelaxant potential of kaempferol on rat isolated pulmonary artery and to assess the underling mechanisms.

Heterogeneity in the mechanisms of vasorelaxation to anandamide in resistance and conduit rat mesenteric arteries

1 In order to address mechanistic differences between arterial vessel types, we have compared the vasorelaxant actions of anandamide in resistance (G3) and conduit (G0) mesenteric arteries. 2 Anandamide produced concentration-dependent relaxations of pre-constricted G3 arteries with a maximal response that was significantly greater than seen in G0. 3 The CB1 receptor selective antagonists SR141716A (100 nm) and AM251 (100 nm) caused reductions in the vasorelaxant responses to anandamide in both arteries. Maximal vasorelaxant responses to anandamide were reduced in both arteries after treatment with capsaicin to deplete sensory neurotransmitters (10 microm for 1 h). 4 Vasorelaxation to anandamide was not affected by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 300 microm) in either artery. Only responses in G3 arteries were sensitive to removal of the endothelium. In G3 vessels only, vasorelaxation to anandamide was reduced by inhibition of EDHF activity with a combination of charybdotoxin (100 nm) and apamin (500 nm) in the presence of L-NAME (300 microm) and indomethacin (10 microm). 5 Antagonism of the novel endothelial cannabinoid receptor (O-1918, 1 microm) caused a reduction in the sensitivity to anandamide in G3 but not G0. 6 G3, but not G0, vessels showed a small reduction in vasorelaxant responses to anandamide after inhibition of gap junctional communication with 18alpha-GA (100 microm). 7 These results demonstrate that there are differences in the mechanisms of vasorelaxation to anandamide between conduit and resistance mesenteric arteries. In small resistance vessels, vasorelaxation occurs through stimulation of vanilloid receptors, CB1 receptors, and an endothelial receptor coupled to EDHF release. By contrast, in the larger mesenteric artery, vasorelaxation is almost entirely due to stimulation of vanilloid receptors and CB1 receptors, and is endothelium-independent.     

Potentiation of the hypoxic contraction of guinea-pig isolated pulmonary arteries by two inhibitors of superoxide dismutase

• 1.1. Isolated proximal and distal extralobar branches of the pulmonary artery of the guinea-pig develop slow and well-sustained contractions in response to hypoxia (P_O2 11–15 mm Hg) without prior stimulation with an agonist. These contractions are readily reversible by readministration of oxygen.
• 2.2. Incubation of these preparations with diethyldithiocarbamic acid (DETCA, 5 mM for 30 min), an inhibitor of superoxide dismutase, significantly increased the hypoxic contractions whether DETCA was added before the challenge with hypoxia or after the hypoxic contraction had reached a plateau. This treatment also reduced the oxygen-induced relaxation.
• 3.3. Similarly, incubation with triethylenetetramine (TETA, 5 mM for 30 min), another inhibitor of superoxide dismutase, produced larger potentiation of the hypoxic contraction in the two preparations and reduced the oxygen-induced relaxation.
• 4.4. Furthermore, addition of H₂O₂ (10⁻⁵ M −3 × 10⁻⁴M) caused concentration-dependent relaxation of the hypoxic contraction while larger concentrations (10⁻³M and 3 × 10⁻³M) caused contraction that did not respond to readministration of oxygen.
• 5.5. These observations suggest that during hypoxic stress, the accumulation of superoxide anions may participate in the hypoxia-induced contraction and that the metabolism of these radicals into H₂O₂ by superoxide dismutase maintains the relaxed state during normoxia.

     

Potentiation of atrial natriuretic peptide-stimulated cyclic guanosine monophosphate formation by glucocorticoids in cultured rat renal cells.

1. The effect of steroid hormones on atrial natriuretic peptide (ANP)-stimulated cyclic guanosine monophosphate (cyclic GMP) formation was studied in cultured rat renal cells. 2. ANP increased cyclic GMP formation in a dose-dependent manner, while cyclic AMP was not changed by ANP. 3. Steroid hormones did not affect basal cyclic GMP levels in cultured rat renal cells. 4. Dexamethasone at 10(-8) M increased ANP (human and rat ANP)-stimulated cyclic GMP dose-dependently in cultured rat renal cells. Cortisol, corticosterone and aldosterone at a concentration of 10(-7) M also potentiated ANP-stimulated cyclic GMP formation, although triiodothyronine, oestradiol and testosterone were ineffective. Potentiation of ANP action by these steroids seems to parallel glucocorticoid activity. 5. Dexamethasone did not affect cyclic GMP formation stimulated by sodium nitroprusside which stimulates soluble guanylate cyclase in the cytosol. Therefore, the potentiating action of dexamethasone may be mediated through the action on particulate guanylate cyclase at the plasma membrane. 6. It is suggested that the diuretic action of glucocorticoids may, at least in part, be mediated through the potentiating effect of glucocorticoids on cyclic GMP response to ANP.     

Sulphonylureas induced vasorelaxation of mouse arteries

Sulphonylureas, such as glybenclamide and gliclazide, are classical blockers of ATP-dependent potassium channels (K(ATP)). Closure of K(ATP) channels in vascular smooth muscles by sulphonylureas would lead to membrane depolarization and vasoconstriction but this hypothesis has not been consistently proved in different types of vascular tissues. Our present study examined mouse vascular contractility changes induced by sulphonylureas using a wire myograph. The phenylephrine-precontracted aorta (n=6), super mesenteric arteries (n=10) and third-order mesenteric artery (n=10) from C57BL mice were relaxed by glybenclamide with IC(50) of 116+/-13.0, 61.8+/-5.5, and 41.4+/-1.3 microM, respectively. Gliclazide or U37883A (a vascular K(ATP) blocker structurally different from that of sulphonylureas) had similar vasorelaxant effects on mesenteric arteries with IC(50) of 14.8+/-1.5 microM (n=5) or 15.6+/-1.3 microM (n=9), respectively. The presence of Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME, 300 microM, n=8), apamin+charybdotoxin (n=4), or 1H-[1, 2, 4]-oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ) (10 microM, n=10) partially reduced vasorelaxant effect of glybenclamide on mesenteric arteries. Removal of endothelium (n=7) or precontraction with 100 mM [K(+)](o) (n=10) also significantly reduced vasorelaxant effect of glybenclamide on mesenteric arteries. The relaxation of mouse resistance arteries by K(ATP) channel blockers calls for further investigation into the selectivity and suitability of these drugs in treatment of different vascular disease.     

Hydrogen peroxide as a mediator of vasorelaxation evoked by N-oleoylethanolamine and anandamide in rat small mesenteric arteries

Hydrogen peroxide (H(2)O(2)) has been shown to participate in endothelium-derived hyperpolarising factor (EDHF)-mediated mechanisms. Vasorelaxation to the endocannabinoid-like N-oleoylethanolamine (OEA) and anandamide has been shown to be endothelium-dependent. Therefore, the principal aim was to investigate whether H(2)O(2) plays a role in vasorelaxation to endocannabinoids in rat mesenteric arteries. We have also investigated the effects of catalase on endothelium-dependent relaxations and vascular responses to H(2)O(2). First- (G1) and third- (G3) order branches of the superior mesenteric artery from male, Wistar rats were mounted in a wire myograph, contracted with methoxamine, and concentration-response curves to anandamide, OEA carbachol or H(2)O(2), were constructed. The influence of nitric oxide production and H(2)O(2) breakdown on these responses were then investigated using L-NAME (300 μM), and catalase (1000 Uml(-1)) respectively. In G1 mesenteric arteries, vasorelaxations to carbachol and H(2)O(2) were inhibited by L-NAME, but not by catalase. Responses to both anandamide and OEA were also unaffected by catalase. In G3 mesenteric arteries, endothelium-dependent relaxations to carbachol were modestly affected by L-NAME, unaffected by catalase alone, but their combination greatly inhibited vasorelaxation. Similarly, catalase inhibited vasorelaxation to anandamide and OEA, and combined treatment with L-NAME further reduced this response. In G1 mesenteric arteries, vasorelaxation to H(2)O(2) is predominantly mediated by nitric oxide. We conclude that in G3 arteries H(2)O(2) activity contributes towards EDHF-type responses and vasorelaxation to endocannabinoids, either directly or indirectly. Given the association between vascular pathophysiology and H(2)O(2), these findings may provide a mechanism whereby disease states may influence responses to endocannabinoid and related mediators.     

Effects of L-NG-monomethyl arginine on the cyclic GMP formations in rat mesenteric arteries.

To evaluate the contribution of L-arginine as a precursor of the endothelium-derived relaxing factor (EDRF) on vascular cyclic GMP formation, we examined the effects of L-NG-monomethyl arginine (L-NMMA), and analog of L-arginine, on basal and acetylcholine (ACh)-, sodium nitroprusside (SNP)- and atrial natriuretic peptide (ANP)-induced cyclic GMP formations in rat mesenteric arteries. The mesenteric arteries were perfused with Krebs-Henseleit solution containing 0.2 mM isobutyl methyl xanthine. The effluents from the arteries were collected before and after infusions of graded doses of ACh, SNP or ANP in the absence or presence of 100 microM L-NMMA, and the levels of cyclic GMP were measured. Basal and ACh-induced cyclic GMP formations in the mesenteric arteries were significantly inhibited in the presence of L-NMMA, whereas a concomitant infusion of 300 microM L-arginine restored the inhibition of basal as well as ACh-induced cyclic GMP formations. L-NMMA did not affect SNP- and ANP-stimulated cyclic GMP formations, respectively. These results suggest that L-arginine is necessary for not only the stimulated cyclic GMP formation but also the basal cyclic GMP formation in the mesenteric arteries, whereas the SNP- and ANP-stimulated cyclic GMP formations in the arteries are independent of L-arginine.     

Beta-adrenoceptor stimulating effects of phenylephrine and noradrenaline in the rat pulmonary vascular bed

The effects of phenylephrine and noradrenaline have been investigated on the perfusion pressure of the rat isolated lung. Both drugs (0.3-30 micrograms) produced a dose-dependent decrease in perfusion pressure elevated by 20 mM KCl, which was reversed to a dose-dependent increase after addition of propranolol (1 x 10(-7) M) to the perfusion fluid. Increments due to both agonists in the presence of propranolol were antagonized by prazosin (1 x 10(-6) M). Propranolol, but not prazosin, elevated the basal perfusion pressure. The results indicate that phenylephrine and noradrenaline are more effective in stimulating beta-adrenoceptors than alpha-adrenoceptors in the rat pulmonary vascular bed and that beta-adrenoceptors may regulate the vascular tone of the rat pulmonary circulation.