Effect of cyanide on nitrovasodilator-induced relaxation, cyclic GMP accumulation and guanylate cyclase activation in rat aorta

The effects of sodium cyanide on relaxation, increases in cyclic GMP accumulation and guanylate cyclase activation induced by sodium nitroprusside and other nitrovasodilators were examined in rat thoracic aorta. Cyanide abolished nitroprusside-induced relaxation and the associated increase in cyclic GMP levels. Basal levels of cyclic GMP and cyclic AMP were also depressed. Reversal of nitroprusside-induced relaxation by cyanide was independent of the tissue level of cyclic GMP prior to addition of cyanide. Incubation of nitroprusside with cyanide prior to addition to aortic strips did not alter the relaxant effect of nitroprusside. Sodium azide-, hydroxylamine-, N-methyl-N'-nitro-N-nitrosoguanide-, nitroglycerin- and acetylcholine-induced relaxations and increased levels of cyclic GMP were also inhibited by cyanide. Relaxations induced by nitric oxide were also inhibited by cyanide, although the relaxation with the low concentration of nitric oxide employed was not accompanied by detectable increases in cyclic GMP. Relaxation to 8-bromo-cyclic GMP was essentially unaltered by cyanide; however, isoproterenol-induced relaxation was inhibited. Guanylate cyclase in soluble and particulate fractions of aorta homogenates was activated by nitroprusside and the activation was prevented by cyanide. The present results suggest that cyanide inhibits nitrovasodilator-induced relaxation through inhibition of guanylate cyclase activation; however, cyanide may also have nonspecific effects which inhibit relaxation.     

Effects of glyceryl trinitrate on endothelium-dependent and -independent relaxation and cyclic GMP levels in rat aorta and human coronary artery

The effects of glyceryl trinitrate-induced desensitization on relaxations and/or elevated cyclic GMP levels due to the nitrogen oxide-containing vasodilators (glyceryl trinitrate and sodium nitroprusside), the endothelium-dependent vasodilators (acetylcholine and the Ca2+ ionophore A23187), and the atrial peptides (atriopeptin II) were investigated in the rat thoracic aorta and human coronary artery. Prior exposure of rat thoracic aorta to glyceryl trinitrate decreased relaxations to glyceryl trinitrate, sodium nitroprusside, and acetylcholine, whereas relaxations to atriopeptin II and 8-bromo cyclic GMP remained unaltered. In human coronary artery, glyceryl trinitrate pretreatment inhibited relaxations to glyceryl trinitrate, sodium nitroprusside, and the Ca2+ ionophore A23187. Relaxation to glyceryl trinitrate was inhibited more than that to sodium nitroprusside in both tissues. Acetylcholine-induced relaxation in rat thoracic aorta was slightly inhibited, whereas relaxation to the Ca2+ ionophore A23187 in human coronary artery was markedly depressed. Pretreatment with glyceryl trinitrate decreased the elevated cyclic GMP levels due to glyceryl trinitrate and acetylcholine in rat thoracic aorta and to glyceryl trinitrate and the Ca2+ ionophore A23187 in human coronary artery. Removal of the endothelium abolished the increased cyclic GMP levels and relaxation due to the Ca2+ ionophore A23187 and decreased basal cyclic GMP levels in the human coronary artery. In contrast, atriopeptin II-induced increased cyclic GMP levels were unaltered by glyceryl trinitrate pretreatment in rat thoracic aorta. The present results suggest that: glyceryl trinitrate-induced desensitization inhibits relaxation to the nitrogen oxide-containing vasodilators and endothelium-dependent vasodilators in both the rat thoracic aorta and human coronary artery: the inhibition of relaxation is associated with decreased formation of cyclic GMP;(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of atrial natriuretic factor, sodium nitroprusside, and acetylcholine on cyclic GMP levels and relaxation in rat aorta

The purpose of this study was to investigate the mechanisms whereby an endothelium-dependent vasodilator, acetylcholine, a nitrovasodilator, sodium nitroprusside and atrial natriuretic factor (atriopeptin II), elevate cyclic GMP levels and induce relaxation in rat thoracic aorta. Methylene blue inhibited the elevated cyclic GMP levels and relaxation due to sodium nitroprusside and acetylcholine, but not those to atriopeptin II. Cyanide inhibited relaxations to all three vasodilators, but inhibited the elevated cyclic GMP levels in response to only nitroprusside and acetylcholine. The reducing agents sodium borohydride, dithiothreitol, sucrose and isoproterenol all inhibited the elevated cyclic GMP levels due to nitroprusside and acetylcholine, while the increased cyclic GMP levels with atriopeptin II were unaffected by sodium borohydride, sucrose and isoproterenol. The effects of the reducing agents on relaxation induced by the vasodilators were difficult to interpret due to their nonspecific contractile and relaxant properties. Agents and procedures known to inhibit the Na+, K+-pump and relaxation to endothelium-dependent vasodilators and nitroprusside, including ouabain, K+-free, Mg2+-free and low Na+ Krebs-Ringer bicarbonate solution, all partially inhibited relaxations to atriopeptin II. Relaxations to atriopeptin II were also inhibited in tissues contracted with KCl. The present results suggest that the mechanism of atrial natriuretic factor-induced increased cyclic GMP levels, in contrast to that of nitroprusside and acetylcholine, does not involve the formation of free radicals, a reducible species or interaction with heme. Furthermore, the cyclic GMP formed in response to nitroprusside, acetylcholine and atrial natriuretic factor mediates relaxation through a common mechanism that may be functionally antagonized by agents and procedures which result in membrane depolarization.     

THE INHIBITION OF NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE BY DIPHENYLENE IODONIUM

1. The effects of diphenylene iodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate-dependent oxidases (which generate superoxide anions), were studied on nitric oxide (NO)-mediated responses in isolated preparations of the rat aorta and anococcygeus muscle. 2. In aortic rings, the endothelium-dependent relaxant action of acetylcholine was reduced by DPI (0.3–10 μmol/L) in a concentration-dependent manner and abolished by the NO synthase (NOS) inhibitor L-nitro-N^G-arginine methylester (l-NAME; 100 μmol/L). Relaxations induced by sodium nitroprusside (SNP) or NO were not affected by DPI or l-NAME. 3. In anococcygeus muscles, DPI (0.3–10 μmol/ L) as well as l-NAME (5–100 μmol/L) produced concentration-dependent reductions of relaxations produced by nitrergic nerve stimulation. Relaxations induced by NO and SNP were not affected by either DPI or l-NAME. l-Arginine (1 mmol/L) prevented the reduction of nitrergic relaxations by l-NAME but not by DPI. 4. Contractions of anococcygeus muscles elicited by exogenous noradrenaline (1 μmol/ L) were not affected or were inhibited by DPI (0.3–10 μmol/L), but the contractions elicited by noradrenergic nerve stimulation were significantly enhanced by DPI and l-NAME. When noradrenergic contractions had already been maximally enhanced by l-NAME (100 μmol/L), DPI produced no further enhancement. l-Arginine (1 mmol/L) prevented the enhancement of noradrenergic contractions by l-NAME but not by DPI. 5. The efflux of radioactivity induced by field stimulation from anococcygeus muscles previously incubated with [³H]-noradrenaline was not affected by either DPI or l-NAME. 6. Superoxide dismutase (SOD, 100 U/mL) had no significant effects on noradrenergic contractions, nitrergic relaxations, relaxations induced by NO or the actions of DPI in the rat anococcygeus muscle. 7. The results suggest that the effects of DPI in reducing the NO-mediated relaxations produced by acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle are due to the inhibition of NOS in these tissues. The effects of DPI were not sensitive to l-arginine, and thus the mechanism of inhibition of NOS differs from that of l-NAME.     

Role of selective cyclic GMP phosphodiesterase inhibition in the myorelaxant actions of M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine.

1. The mechanism by which M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated. 2. Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+-insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin-stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE). 3. M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 microM and 0.43 microM, respectively) and Ca2+ PDE (Ki = 9.9 microM and 0.55 microM, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 microM and 42 microM, respectively). MY-5445 selectivity inhibited cGMP PDE (Ki = 1.3 microM) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 microM). 4. M&B 22,948 and MIMAX induced dose-dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 microM) which blocks the actions of endothelium-derived relaxant factor. MY-5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation. 5. All four compounds caused dose-related relaxation of 5-hydroxytryptamine (10 microM) contracted, endothelium-intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 microM). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose-response curves of MY-5445 and vinpocetine, respectively. 6. The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY-5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis.     

Differential regulation by AT(1) and AT(2) receptors of angiotensin II-stimulated cyclic GMP production in rat uterine artery and aorta

1. In the present study we determined whether angiotensin II (Ang II) could increase cyclic GMP levels in two blood vessels that exhibit markedly different angiotensin II receptor subtype expression: rat uterine artery (UA; AT(2) receptor-predominant) and aorta (AT(1) receptor-predominant), and investigated the receptor subtype(s) and intracellular pathways involved. 2. UA and aorta were treated with Ang II in the absence and presence of losartan (AT(1) antagonist; 0.1 microm), PD 123319 (AT(2) antagonist; 1 microm), NOLA (NOS inhibitor; 30 microm), and HOE 140 (B(2) antagonist; 0.1 microm), or in combination. 3. Ang II (10 nm) induced a 60% increase in UA cyclic GMP content; an effect that was augmented with PD 123319 and HOE 140 pretreatment, and abolished by cotreatment with losartan, as well as by NOLA. 4. In aorta, Ang II produced concentration-dependent increases in cyclic GMP levels. Unlike effects in UA, these responses were abolished by PD 123319 and by NOLA, whereas losartan and HOE 140 caused partial inhibition. 5. Thus, in rat UA, Ang II stimulates cyclic GMP production through AT(1) and, to a less extent, AT(2) receptors. In rat aorta, the Ang II-mediated increase in cyclic GMP production is predominantly AT(2) receptor-mediated. In both preparations, NO plays a critical role in mediating the effect of Ang II, whereas bradykinin has differential roles in the two vessels. In UA, B(2) receptor blockade may result in a compensatory increase in cyclic GMP production, whilst in aorta, bradykinin accounts for approximately half of the cyclic GMP produced in response to Ang II.     

Autoregulation of nitric oxide-soluble guanylate cyclase-cyclic GMP signalling in mouse thoracic aorta

1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity.     

Role of gender and estrogen receptors in the rat aorta endothelium-dependent relaxation to red wine polyphenols

Regular intake of moderate amounts of beverages rich in polyphenols such as red wine is associated with a protective effect on the vascular system, in part, by increasing the endothelial formation of nitric oxide (NO), a major vasoprotective factor. Since estrogens are potent inducers of NO formation and polyphenols have been shown to have phytoestrogen properties, we determined whether estrogen receptors mediate the stimulatory effect of red wine polyphenols (RWPs) on the endothelial formation of NO using isolated rat aortic rings and cultured endothelial cells.RWPs caused endothelium-dependent relaxations, which were more pronounced in the aorta of female than male rats. Increased relaxations were also observed to acetylcholine but not to sodium nitroprusside. Relaxations to RWPs were abolished by nitro l-arginine and MnTMPyP, markedly reduced by polyethyleneglycol-catalase and wortmannin, and not affected by the estrogen antagonist ICI 182,780 in aortic rings from males and females. eNOS expression was higher in aortic sections of female than male rats. RWPs caused the phosphorylation of Akt and eNOS in endothelial cells, which was unaffected by ICI 182,780.Thus, RWPs cause redox-sensitive PI3-kinase/Akt-dependent NO-mediated relaxations, which are more pronounced in the aorta of female than male rats; an effect most likely due to the increased expression level of eNOS rather than activation of estrogen receptors.     

Pharmacological comparison between the nitrergic responses produced by intramural nerve stimulation and exogenous NO-donors in rat gastric fundus.

To investigate whether the nitrergic nerve-mediated smooth muscle relaxation is caused by authentic nitric oxide (NO) and is mediated via guanosine 3':5'-cyclic monophosphate (cyclic GMP), we compared the response to electrical field stimulation of nitrergic nerve (EFS) with other NO-related responses in rat gastric fundus strips. EFS, sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and acidified NaNO2 and inducible NO synthase (iNOS)-mediated NO all produced relaxation and elevated cyclic GMP level in rat fundus strips. However, the basal and stimulated cyclic GMP levels were significantly lower than the basal level in aorta (40+/-4 pmol/g wet tissue). Methylene blue and 6-anilino-5,8-quinolinedione (LY83583), both known as soluble guanylyl cyclase inhibitors and O2- generators that scavenge NO, reduced the elevation of cyclic GMP level by all stimuli and inhibited the relaxations only in response to NaNO2 and iNOS-mediated NO but not to the other stimuli. These results suggest that in the rat gastric fundus strips the relaxations induced by not only nitrergic nerve but also SNP and SNAP are not associated with cyclic GMP production, in contrast to the relaxations mediated by authentic NO.     

Impairment of endothelium-dependent relaxation and changes in levels of cyclic GMP in aorta from streptozotocin-induced diabetic rats.

1. Acetylcholine (ACh)-induced relaxation of aortic strips with endothelium and production of cyclic GMP between streptozotocin-induced diabetic and age-matched control rats were compared. 2. The concentration-response curve for ACh-induced relaxation was shifted to the right in diabetic rats. IC50 values for ACh were 4.57 +/- 0.67 x 10(-8) M and 1.00 +/- 0.87 x 10(-7) M in aortic strips from age-matched control and diabetic rats, respectively (n = 6, P less than 0.05). 3. Relaxations produced by atrial natriuretic peptide (ANP) in diabetic aortae were similar to those in age-matched vessels. 4. Relaxations produced by sodium nitroprusside (SNP) in diabetic aortae were similar to those in age-matched vessels. 5. Basal levels of cyclic GMP and ACh-induced production of cyclic GMP were significantly decreased in diabetic rats. 6. These results suggest that functional changes in endothelium but not in guanylate cyclase activity in the aorta may occur in diabetes, and thus, spontaneous and ACh-induced formation of cyclic GMP may be decreased. This decrease in production of cyclic GMP may be responsible for the decreased response of the aorta to the relaxant effect of ACh.     

Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice

We examined the direct involvement of endothelial nitric oxide (eNOS) in nitrate tolerance using eNOS knockout (eNOS (-/-)) and wild-type (eNOS (+/+)) mice. Animals were treated with either nitroglycerin (NTG, 20 mg kg(-1)s.c. 3 x daily for 3 days) or vehicle (5% dextrose, D5W), and nitrate tolerance was assessed ex vivo in isolated aorta by vascular relaxation studies and cyclic GMP accumulation. Western blot was performed to determine NOS expression after NTG treatment. In both the eNOS (-/-) and (+/+) mice, the EC(50) from NTG concentration-response curve was increased by approximately 3 fold, and vascular cyclic GMP accumulation was similarly decreased after NTG pretreatment. Vascular tolerance did not lead to changes in eNOS protein expression in eNOS (+/+) mice. These results indicate that vascular nitrate tolerance was similarly induced in eNOS (-/-) and (+/+) mice, suggesting that eNOS may not be critically involved in nitrate tolerance development in mice.     

Correlation between airway epithelium-induced relaxation of rat aorta in the co-axial bioassay and cyclic nucleotide levels.

1. In co-axial bioassays, in the presence of indomethacin, addition of histamine (100 microM) or methacholine (100 microM) to guinea-pig trachea produced an epithelium-dependent relaxation of precontracted rat aorta which was associated with an approximately 2 fold elevation in tissue levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Removal of the airway epithelium abolished the histamine-induced relaxation of rat aorta and the associated increase in intracellular cyclic GMP. 2. Epithelium-dependent relaxation was not associated with altered adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in rat aorta. Unstimulated intact or denuded guinea-pig trachea also did not affect the levels of cyclic AMP or cyclic GMP in rat aorta. 3. Methylene blue (10 microM) abolished the methacholine-induced, endothelium-derived relaxing factor (EDRF)-mediated rise in intracellular cyclic GMP in rat endothelium-intact aorta alone. In contrast, methylene blue (10 microM) did not affect the methacholine-induced epithelium-dependent rise in intracellular cyclic GMP in rat endothelium-denuded aorta in the co-axial bioassay. 4. Relaxation of the rat aorta without endothelium was associated with increased levels of cyclic GMP (but not cyclic AMP) in response to sodium nitroprusside (5 nM) and of cyclic AMP (but not cyclic GMP) in response to isoprenaline (1 microM). 5. These results provide evidence that the postulated epithelium-derived inhibitory factor (EpDIF) may produce relaxation of vascular tissue via elevation in cyclic GMP levels. Furthermore, some data suggest that EpDIF may act by stimulation of the particulate, rather than the soluble form of guanylate cyclase.     

Effects of nonsteroidal phospholipase inhibitors and glucocorticoids on endothelium-dependent relaxations of rabbit aorta induced by different agents

The present study investigates the effect of described nonsteroidal phospholipase inhibitors [mepacrine, papaverine, trifluoperazine (TFP), p-bromophenacyl bromide (pBPB), compound CB 874] and of glucocorticoids on endothelium-dependent and -independent relaxations of rabbit aorta. Endothelium-dependent relaxations were elicited by acetylcholine, Ca2+ ionophore A23187, melittin, and thimerosal. These four agents also stimulated vascular prostacyclin formation. Prostacyclin does not relax rabbit aorta; it was measured as an indicator of phospholipid hydrolysis. All putative nonsteroidal phospholipase inhibitors blocked acetylcholine-induced relaxations. Since papaverine did not inhibit prostacyclin production, it cannot be considered a phospholipase inhibitor in this tissue. Relaxations in response to A23187 were blocked only by pBPB and CB 874, which can interact with phospholipases directly. Melittin-induced relaxations were suppressed by mepacrine, TFP, pBPB, and CB 874. Relaxations elicited by thimerosal were not affected by mepacrine, but were abolished by the other three inhibitors. All inhibitors were ineffective against endothelium-independent relaxations induced by glyceryl trinitrate. Effective blockade of endothelial relaxations correlated with inhibition of prostacyclin formation. In the presence of glucocorticoids, no inhibition of endothelium-mediated relaxations and no inhibition of prostacyclin formation occurred, indicating unimpaired phospholipase activity. These findings suggest that cleavage of phospholipids may be important in the triggering of the production of endothelium-derived relaxing factor.     

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.     

Mechanisms of relaxation by carbon monoxide-releasing molecule-2 in murine gastric fundus and jejunum

This study investigated the effects and mechanisms of action of carbon monoxide-releasing molecule-2 (CORM-2), compared to those of carbon monoxide (CO), in murine gastric fundus and jejunal circular smooth muscle. Functional in vitro experiments and cGMP measurements were conducted. In both tissues, CO and CORM-2 induced concentration-dependent relaxations. CO-induced relaxations were abolished by the soluble guanylyl cyclase (sGC) inhibitor ODQ, while CORM-2-evoked inhibitory responses were only partly prevented by ODQ. Relaxations elicited by CO (300 microM) were associated with a significant increase in cGMP levels, whereas for CORM-2 (300 microM) no significant increase in cGMP levels could be measured. The sGC sensitizer YC-1 was able to accelerate and potentiate both CO- and CORM-2-induced relaxations. Furthermore, the intermediate- and large-conductance Ca2+-activated K+ (IKCa-BKCa) channel blocker charybdotoxin significantly reduced CO- and CORM-2-induced relaxations in jejunal tissue; this same effect was observed with the BKCa channel blocker iberiotoxin. The combination of apamin plus charybdotoxin significantly reduced relaxations in gastric fundus and had synergistic inhibitory effects in jejunum. The NOS inhibitor L-NAME had no effect on the induced relaxations in gastric fundus, but significantly reduced CO- and CORM-2-evoked relaxations in jejunum. In conclusion, these results demonstrate that CO and CORM-2 produce relaxation in gastric fundus and jejunum via sGC and activation of KCa channels, and a nitric oxide (NO)-mediated amplification of CO signaling in jejunum is suggested.     

Effects of phosphodiesterase inhibition on cortical spreading depression and associated changes in extracellular cyclic GMP

Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME, 1mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.     

Genistein prevents myocardial hypertrophy in 2-kidney 1-clip renal hypertensive rats by restoring eNOS pathway

Genistein has been shown to increase nitric oxide (NO) production derived from endothelial nitric oxide synthase (eNOS). This study was to investigate whether genistein could prevent myocardial hypertrophy in the 2-kidney 1-clip (2K1C) renohypertensive rat through the NO pathway and to clarify the underlying mechanisms. After the 2K1C operation, plasma angiotensin II increased, and the rats developed significant left ventricular hypertrophy (LVH) and increased collagen I expression. Phosphorylated eNOS, NOS activity, NO production and cGMP contents were markedly decreased in ventricular tissues of 2K1C rats. Chronic administration of genistein to 2K1C rats restored NO, NOS activity, phosphorylated eNOS expression, cGMP in ventricular tissues, and the restoration was parallel with the improvement of LVH and attenuated the excessive ventricular collagen I expression. Genistein also elevated angiotensin II type 2 receptor (AT2) expression, and the effects of genistein on LVH could be completely abolished by an AT2 antagonist, PD123319. The antagonist also reversed the increase in eNOS activity, NO and cGMP restored by genistein in hypertensive rats. We further explored the mechanisms by which genistein restored NO in hypertension and found that genistein significantly enhanced phosphorylated eNOS but left relatively unchanged total eNOS and the eNOS dimer/monomer ratio. In addition, genistein decreased the binding of eNOS with caveolin 3 and simultaneously promoted its binding with calmodulin and heat shock protein 90. We conclude that the preventive effects of genistein on cardiac remodeling induced by 2K1C hypertension are mediated by AT2-dependent NO production.     

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.     

EFFECTS OF ARGININOSUCCINIC ACID ON NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE

1. Argininosuccinic acid (ASA), a naturally occurring NG derivative of arginine, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were compared for their ability to reduce responses to nitric oxide (NO) derived from endothelial cells (aorta) and nitrergic nerves (anococcygeus muscle). 2. In isolated rings of rat aorta, endothelium-dependent relaxation responses to acetylcholine were abolished by L-NAME (0.1 mmol/L) and were reduced by ASA (0.1 and 0.3 mmol/L). Relaxations induced by sodium nitroprusside (SNP) were not affected by L-NAME but were reduced by ASA. 3. In rat isolated anococcygeus muscles, relaxations elicited by nitrergic nerve stimulation at 1 Hz were abolished by L-NAME (0.1 mmol/L) but were only slightly reduced by ASA (1 mmol/L). The effect of ASA was not sustained. L-Arginine (1 mmol/L) prevented the effect of L-NAME but not that of ASA. Neither ASA or L-NAME inhibited SNP-induced relaxation in the anococcygeus muscle. 4. The results suggest that ASA inhibits NOS but this does not totally account for its effects in reducing NO-mediated relaxations produced by the endothelium-dependent vasodilator acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle.     

Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors.

1. The effects of selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterases (PDEs) were investigated on PDEs isolated from the rat aorta and on relaxation of noradrenaline (1 microM) precontracted rat aortic rings, with and without functional endothelium. 2. Four PDE forms were isolated by DEAE-sephacel chromatography from endothelium-denuded rat aorta: a calmodulin-activated PDE (PDE I) which hydrolyzed preferentially cyclic GMP, two cyclic AMP PDEs (PDE III and PDE IV) and one cyclic GMP-specific PDE (PDE V). The latter was selectively and potently inhibited by zaprinast. The two cyclic AMP PDEs were discriminated by specific inhibitors: one was inhibited by cyclic GMP (PDE III) and by new cardiotonic agents (milrinone, CI 930, LY 195115 and SK&F 94120); the other was inhibited by denbufylline and rolipram (PDE IV). None of these drugs significantly inhibited PDE I. 3. The PDE III inhibitors caused endothelium-independent relaxations of rat aortic rings with the following EC50 values (microM concentration producing 50% relaxation): LY 195115: 3.4, milrinone: 5.7, CI 930; 7.8, SK&F 94120: 14.7. Neither NG-monomethyl-L-arginine (L-NMMA, 300 microM), an inhibitor of the L-arginine-NO pathway, nor L-arginine (1 mM) modified the effect of PDE III inhibitors. However, methylene blue (10 microM) an inhibitor of soluble guanylate cyclase abolished relaxation induced by PDE III inhibitors except in the case of compound CI 930. 4. The specific PDE IV and PDE V inhibitors both produced endothelium-dependent relaxations which were inhibited by L-NMMA and by methylene blue (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)