Sodium nitroprusside potentiates the depressor response to the phosphodiesterase inhibitor zaprinast in rats

To determine if the presence of an activator of guanylate cyclase alters the depressor response to a selective inhibitor of low Km cyclic GMP (cGMP) phosphodiesterase (PDE), zaprinast (3-30 mg/kg) was given i.v. to conscious, spontaneously hypertensive rats during a steady state of i.v. infusion of sodium nitroprusside (15 micrograms/kg per min). Sodium nitroprusside significantly increased the magnitude of the depressor response to zaprinast. In contrast, fenoldopam (20 micrograms/kg per min), an activator of adenylate cyclase, did not affect the depressor response to zaprinast. Zaprinast (10 mg/kg) significantly decreased mean arterial pressure (MAP) in rats given an infusion of sodium nitroprusside, an activator of soluble guanylate cyclase, at doses of 15 and 25 micrograms/kg per min but not at a dose of 5 micrograms/kg per min. However, in rats given atrial natriuretic peptide (ANP; 0.5, 1 and 2 micrograms/kg per min), an activator of particulate guanylate cyclase, zaprinast (10 mg/kg) did not affect MAP. In contrast to the potentiation of the depressor response to zaprinast, sodium nitroprusside (15 micrograms/kg per min) significantly attenuated the reductions in MAP produced by CI-930, a selective inhibitor of low Km cAMP PDE. It is concluded that sodium nitroprusside, but not ANP or fenoldopam, potentiates the depressor response to zaprinast. Furthermore, the potentiation of the depressor response to zaprinast is dependent upon the dose of sodium nitroprusside and is selective for zaprinast; the depressor response to CI-930 is attenuated by sodium nitroprusside.     

Impaired effect of salt loading on nitric oxide-mediated relaxation in aortas from stroke-prone spontaneously hypertensive rats

1. The present study was designed to characterize the effects of salt on vasorelaxation via the nitric oxide (NO)/cGMP pathway in stroke-prone spontaneously hypertensive rats (SHRSP), which are highly salt sensitive. 2. Male 8-week-old SHRSP were given 1% NaCl solution as drinking water for 4 weeks, whereas control animals were given water only. 3. In aortic rings from salt-loaded SHRSP, relaxations in response to acetylcholine and sodium nitroprusside were significantly impaired compared with those in the control. In the presence of zaprinast, a cGMP-specific cyclic nucleotide phosphodiesterase (PDE)-5 inhibitor, the cGMP levels induced by these drugs were significantly reduced by salt loading, but remained unchanged in the absence of zaprinast. The protein levels of endothelial NO synthase, soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) remained unchanged with salt loading, but those of PDE-5 decreased significantly and those of phosphorylated PKG tended to decrease, although the change was not statistically significant. Salt loading significantly impaired the relaxation in response to 8-bromo-cGMP. 4. These results indicate that, in aortas from SHRSP, salt loading causes impairment of relaxation in response to NO, which may be due to a decrease in cGMP production by sGC and impairment of the relaxation pathway downstream of cGMP, which, in turn, probably causes a decrease in PKG activity. Reduced PDE-5 protein expression may act, in part, as a compensatory response to impairment of cGMP-mediated relaxation.     

The effect of tributyltin chloride on vascular responses to atrial natriuretic peptide.

The effects of tributylin chloride (TBT) on vascular smooth muscle responses to norepinephrine, nitroprusside (SNP) and atrial natriuretic peptide (ANP) were studied in isolated aortic rings of rats. TBT did not interfere with norepinephrine-induced contraction or SNP-induced vasorelaxation. However, TBT produced a dose-dependent inhibition of ANP-induced vasorelaxation. Inhibition was not observed with inorganic tin chloride, SnCl2. The inhibition of vasorelaxation was accompanied by a parallel inhibition of ANP-induced cGMP generation. SNP-induced generation of cGMP was not affected by TBT. TBT did not interfere with binding of ANP to its receptor in bovine adrenal glands suggesting that the effects of TBT were mediated by direct interaction with membrane-bound guanylate cyclase.     

Desensitization to nitroglycerin in vascular smooth muscle from rat and human

Guanylate cyclase in high speed supernatant fractions obtained from rat thoracic aorta or human coronary arteries pretreated with nitroglycerin exhibited a marked desensitization to activation by nitroglycerin, nitroprusside, and nitric oxide. However, activation of soluble guanylate cyclase by arachidonic acid was unaffected by pretreatment of vessels with nitroglycerin. Furthermore, activation of soluble guanylate cyclase by protoporphyrin IX was increased 4-fold when vessels were pretreated with nitroglycerin. Soluble guanylate cyclase partially purified from nitroglycerin-pretreated rat thoracic aorta by immunoprecipitation with a specific monoclonal antibody exhibited persistent desensitization to nitrate-induced activation. These data suggest that nitroglycerin-induced desensitization of guanylate cyclase to activation by nitrovasodilators represents a stable alteration of the enzyme. In contrast, activation by protoporphyrin IX of guanylate cyclase immunoprecipitated from nitroglycerin-pretreated or control vessels was not significantly different. This suggests that the mechanism of protoporphyrin activation of guanylate cyclase is different than the mechanism with nitrovasodilators. Activation of particulate guanylate cyclase by Lubrol-PX, hemin, or atrial natriuretic factor was not significantly different with enzyme prepared from nitroglycerin-pretreated or control vessels from rat and human. Thus, nitroglycerin-induced desensitization of rat thoracic aorta or human coronary artery results in a relatively stable molecular alteration of soluble guanylate cyclase such that the enzyme is specifically less sensitive to activation by nitrovasodilators whereas the effects of other activators of the enzyme are either unchanged or increased.     

Inhibition of low Km cyclic GMP phosphodiesterases and potentiation of guanylate cyclase activators by cicletanine.

Cicletanine is an antihypertensive/vasorelaxant/natriuretic agent of unknown mechanism. We wished (a) to determine if cicletanine interacts with guanylate cyclase activators that modulate vasomotor tone and sodium balance [i.e., atriopeptin II (AP II), endothelium-derived relaxing factor (EDRF), and sodium nitroprusside (SNP)], and (b) to define the subcellular basis for this interaction by quantitating the effects of cicletanine on low Km cyclic GMP phosphodiesterase (PDE) activity. In phenylephrine-contracted rat aortic smooth muscle, the vasorelaxant potency of cicletanine was increased twofold in the presence of a threshold-relaxant concentration of AP II, and functional cyclic GMP PDE inhibition was also evident from the three- to sixfold potentiation by cicletanine of AP II- or SNP-induced vasorelaxation. Vasorelaxation produced by cicletanine was not endothelium dependent, however. In further studies, intravenous (i.v.) administration of cicletanine or the low Km cyclic GMP PDE inhibitor, zaprinast, decreased blood pressure (BP) greater than or equal to 20% in conscious spontaneously hypertensive rats (SHR). These results are consistent with the additional finding that cicletanine inhibited Ca2(+)-calmodulin (CaM) cyclic GMP PDE and zaprinast-sensitive cyclic GMP specific PDE over a concentration range (10-600 microM) similar to that for vasorelaxation. Thus, inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant effect of cicletanine as well as the potentiation by cicletanine of the vasorelaxant actions of guanylate cyclase activators. The extent to which this mechanism contributes to the antihypertensive efficacy of cicletanine has not yet been fully determined.     

N omega-nitro-L-arginine attenuates the accumulation of aortic cyclic GMP and the hypotension produced by zaprinast.

To determine if N omega-nitro-L-arginine (NNA), an inhibitor of the synthesis and/or release of endothelium-derived relaxing factor (EDRF), alters the response to zaprinast, a selective inhibitor of cyclic GMP (cGMP) phosphodiesterase, zaprinast (3-30 mg/kg) or vehicle (1 ml/kg) was given to conscious, spontaneously hypertensive rats (SHR) in a cumulative i.v. dose-response manner 30 min after pretreatment with NNA (1 or 3 mg/kg) or saline (1 ml/kg). Mean arterial pressure (MAP) was measured 5 min after each dose of zaprinast. Five minutes after the last dose of zaprinast (30 mg/kg), the rats were anesthetized with pentobarbital (25 mg i.v.). A segment of the abdominal aorta was freeze-clamped in situ and removed for the determination of cGMP levels. NNA (3 mg/kg) decreased basal aortic cGMP levels by 54% and increased MAP by 37 +/- 2 mm Hg. Zaprinast (30 mg/kg) increased aortic cGMP by 187% and decreased MAP by 49 +/- 4 mm Hg. NNA (3 mg/kg) reduced the accumulation of cGMP in aortic tissue (from 4.1 +/- 0.4 to 1.3 +/- 0.1 fmol/microgram protein) and attenuated the depressor response (from -49 +/- 4 to -31 +/- 4 mm Hg) produced by zaprinast. These data are consistent with the hypothesis that NNA inhibits the tonic release of EDRF and that the depressor effects of zaprinast are due, at least in part, to the potentiation of the vasodilator effects of EDRF in vivo. Moreover, since the changes in MAP produced by NNA and zaprinast were significantly correlated with cGMP levels in aortic tissue, the concentration of cGMP in vascular tissue may be a determinant of blood pressure in SHR.     

The role of cGMP hydrolysing phosphodiesterases 1 and 5 in cerebral artery dilatation.

The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (cGMP) pathway. Immunoreactivity to phosphodiesterases 1A, 1B and 5, but not phosphodiesterase 1C was found in fractions of homogenised cerebral arteries eluted by high-pressure liquid chromatography (HPLC). Both the phosphodiesterase 1 inhibitor 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine (8-MM-IBMX) and the phosphodiesterase 5 inhibitors zaprinast and dipyridamole induced dilatation of cerebral arteries. The dilatory response to 8-MM-IBMX was reduced by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microM) and endothelial removal and restored by sodium nitroprusside (0.1 microM) pretreatment, indicating a close relation to the nitric oxide-cGMP pathway. The responses to zaprinast and dipyridamole, however, were not only moderately affected, but also restored by sodium nitroprusside (0.1 microM) pretreatment. At high concentrations, the dilatory effects of zaprinast and dipyridamole were partly caused by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease.     

Endothelium- and sydnonimine-induced responses of native and cultured aortic smooth muscle cells are not impaired by nitroglycerin tolerance

Summary Tolerance to the cyclic GMP-mediated vasodilator action of nitroglycerin develops with prolonged exposure and may be mediated either by formation of less nitric oxide from nitroglycerin or by desensitization of soluble guanylate cyclase to activation with nitric oxide. In the latter case, smooth muscle cells tolerant to nitroglycerin should show cross-tolerance to nitric oxide released from sydnonimines and endothelial cells (endothelium-derived relaxing factor).Therefore cultured smooth muscle cells from rabbit aorta were pretreated for 1 h with vehicle or high concentrations (0.55 mM) of nitroglycerin or the sydnonimine SIN-1. The formation of cyclic GMP induced by subsequent small doses of nitroglycerin, sydnonimine SIN-1 and endothelium-derived relaxing factor (released from cultured endothelial cells) was compared with the changes in activation of soluble guanylate cyclase, cyclic GMP formation and vasodilation in response to the same stimuli in similarly pretreated segments from rabbit thoracic aortae.Both cultured and native smooth muscle cells remained responsive to stimulation with sydnonimine SIN-1 and endothelium-derived relaxing factor after pretreatment with nitroglycerin, vehicle, or sydnonimine SIN-1, even though they were tolerant to nitroglycerin after pretreatment with nitroglycerin. In contrast, activation of soluble guanylate cyclase by nitroglycerin and sydnonimine SIN-1 was attenuated in homogenates of nitrate-tolerant aortae. The findings suggest that nitroglycerin tolerance in intact cells does not involve desensitization of soluble guanylate cyclase, because in intact cells nitrate tolerance can be overcome by direct activators of soluble guanylate cyclase. Send offprint requests to A. Mülsch at the above address     

Desensitization of guanylate cyclase in nitrate tolerance does not impair endothelium-dependent responses

Tolerance of vascular smooth muscle to nitroglycerin could be induced by an impaired biotransformation of nitroglycerin to nitric oxide, the activator of soluble guanylate cyclase, or by desensitization of guanylate cyclase to activation with nitric oxide. The latter would imply that there would also be tolerance to nitric oxide delivered from sodium nitroprusside or endothelial cells. Therefore, endothelium-denuded segments of rabbit aorta were treated with nitroglycerin to induce tolerance, and were then assessed for mechanical response, cyclic GMP content, and activity of soluble guanylate cyclase after addition of nitrovasodilators. Nitrate tolerance decreased the vasodilation and the increase in cyclic GMP elicited by nitroglycerin, but not that elicited by sodium nitroprusside or endothelium-derived relaxing factor, in norepinephrine-contracted segments. However, soluble guanylate cyclase in the supernatants of homogenates of nitrate-tolerant aortas was desensitized to activation with nitroglycerin and sodium nitroprusside. As the guanylate cyclase was still responsive to activation by nitric oxide in the intact, tolerant smooth muscle, an impaired biotransformation of nitroglycerin rather than desensitization of soluble guanylate cyclase may be the mechanism by which nitrate tolerance develops.     

Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover

Rats were made tolerant towards nitroglycerin (GTN) by subcutaneous injections of 50 mg/kg GTN, 3 times daily for 3 consecutive days. The effects of a test dose of GTN on tension and on the cGMP response were studied in aortic preparations. The activities of the enzymes regulating cGMP turnover were also investigated. In noradrenaline (2.5 μM)-contracted tissue from GTN-treated animals, the relaxant response towards the test dose of GTN (44 μM) was reduced by about 75% as compared to the ethanol-treated control rats. The cGMP-elevating action of GTN was reduced by 55%, while no significant effect on the cAMP level could be detected. The cyclic GMP-phosphodiesterase (G-PDE) activity was increased from 770 pmol/min × mg prot (ethanol-pretreated rats) to 1340 pmol/min × mg prot (GTN-pretreated animals). The guanylate cyclase activity, stimulated with 1 mM nitroprusside, was determined with both MnGTP and MgGTP as substrate and found to be reduced by about 75% in aortic homogenates from the GTN-pretreated animals. A slight depression in cGMP-dependent protein kinase activity was detected in aortas from GTN-treated animals. However, this depression seemed to be due to an increased breakdown of the activator (cGMP) since the inclusion of 2.5 mM theophylline in the assay solution abolished the difference. These results strongly support the suggestion that cGMP acts as a mediator of GTN-induced vascular smooth muscle relaxation. The tolerance towards the pharmacological action of GTN after repeated exposure could well be explained by the reduced formation and increased rate of breakdown of cGMP as demonstrated in this study.     

Contribution of phosphodiesterase isoenzymes and cyclic nucleotide efflux to the regulation of cyclic GMP levels in aortic smooth muscle cells.

Involvement of phosphodiesterase isoenzymes (PDEs) in guanosine-3',5'-cyclic monophosphate (cGMP) hydrolysis was analyzed in aortic smooth muscle cells. Four families of PDEs were separated from pig aorta: PDE1 (calcium-calmodulin-activated), PDE3 (cGMP-inhibited), PDE4 (adenosine 3',5'-cyclic monophosphate [cAMP]-specific), and PDE5 (cGMP-specific). Within this PDE complement, PDE1 and PDE5 mostly contributed to the hydrolysis of cGMP both in the presence and absence of calcium-calmodulin. The role of these isoenzymes in cGMP degradation was analyzed in primary cultures of porcine aortic smooth muscle cells after stimulation with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF). Pretreatment with 10 microM zaprinast, a concentration that selectively inhibits PDE5, did not potentiate the SNP- or ANF-induced rise of cGMP, questioning the widespread opinion that only PDE5 accounts for cGMP hydrolysis in this tissue. Further evidence came from experiments assessing the effect of zaprinast or 3-isobutyl-1-methylxanthine at concentrations inhibiting both type 1 and type 5 isoenzymes, in which this potentiation was clearly seen. Contribution of cGMP egression to the control of intracellular cGMP levels after SNP or ANF stimulation was also investigated. Shortly after guanylate cyclase activation, extracellular cGMP levels surpassed intracellular levels. However, comparison of the amounts of cGMP extruded to the extracellular medium with those degraded by PDEs leads to the conclusion that efflux is of relatively minor importance in regulating intracellular cGMP levels. In cells made tolerant to SNP, selective PDE5 inhibition synergistically increased intra- and extracellular cGMP amounts after SNP stimulation. These results indicate a previously undescribed greater relevance of PDE5 after tolerance development in aortic smooth muscle cells.     

Effects of nucleotide cyclase activators on the depressor response to selective low Km cyclic AMP phosphodiesterase inhibitors in rats

The potential interaction between agents which promote vasodilation through cyclic AMP (cAMP)- and cyclic GMP (cGMP)-dependent mechanisms was examined in conscious, spontaneously hypertensive rats (SHR). Selective inhibitors of low Km cAMP phosphodiesterase (PDE), CI-930 (0.1?10 mg/kg) and milrinone (0.03?3 mg/kg), were administered i.v. to SHR during a steady-state i.v. infusion of either the guanylate cyclase activator sodium nitroprusside (SNP, 5 or 15 μg/kg/min) or the adenylate cyclase activator fenoldopam (20 μg/kg/min). The depressor responses to Cl-930 and milrinone were significantly attenuated in the presence of SNP. This attenuation of the depressor response to Cl-930 was apparently not due to a hemodynamic floor effect since an equihypotensive dose of the calcium channel blocker verapamil (20 μg/kg/min infusion preceeded by a loading dose of 1.2 mg/kg i.v.) did not alter the response to Cl-930. The depressor response to Cl-930 was also attenuated during an infusion of fenoldopam, although this interaction was dependent upon the order of administration of these agents since pretreatment with Cl-930 (0.3 mg/kg, i.v.) potentiated the depressor response to infusions of fenoldopam (10 and 20 μg/kg/min). These data suggest that activation of either adenylate or guanylate cyclase may alter the intrinsic activity of low Km cAMP PDE and functionally impair the ability of inhibitors of this PDE isozyme to lower blood pressure. Furthemore, teh interactive effects of fenoldoparm and Cl-930 on blood pressure are dependent upon the order of administration.     

Effect of calcium on endothelium-derived relaxing factor formation and cGMP levels in endothelial cells

Various stimulants of the release of EDRF (endothelium-derived relaxing factor) increased intracellular cGMP levels in bovine aortic endothelial cells. ATP was the most effective compound tested, increasing cGMP 7-fold, followed by the calcium ionophore, A23187 (4.8-fold), and bradykinin (4.0-fold). The EC₅₀ values were similar to those obtained when EDRF release was measured with the bioassay technique, which suggests a stimulation of endothelial guanylate cyclase by EDRF. The direct acting stimulants of soluble guanylate cyclase, sodium nitroprusside and SIN-1 (3-morpholino-sydnonimine), also increased the cGMP content of endothelial cells by 9.4 and 7.2 times, respectively. The effects of both groups of stimulants of cGMP levels were antagonized by the lipoxygenase inhibitor, nordihydroguaiaretic acid, and by the radical scavenger, phenylbutylnitrone, whereas gossypol or canavanine only antagonized the EDRF-induced effect on endothelial cGMP levels. Bradykinin, ATP and A23187 also increased the uptake of ⁴⁵CaCl₂ into endothelial cells but since the complete removal of extracellular Ca²⁺ or blockade of Ca²⁺ transport by LaCl₃ did not affect the ability of these compounds to elevate cGMP levels, the formation of EDRF appears not to be triggered by an influx of extracellular calcium. This study provides evidence that EDRF stimulators enhance cGMP levels in endothelial cells, probably due to a direct activation of guanylate cyclase by EDRF.     

Cyclic GMP as the mediator of molsidomine-induced vasodilatation

The mode of action of the in vitro active metabolites SIN-1 and SIN-1A of the vasodilator prodrug molsidomine was studied in bovine coronary artery strips. Both compounds increased cyclic GMP levels in close association with, but prior to their relaxing action. Relaxation and rises in cyclic GMP by SIN-1 were potentiated by M & B 22,948, an inhibitor of cyclic GMP phosphodiesterase and attenuated by methylene blue, a dye that inhibits activation of guanylate cyclase by SIN-1 and various nitrovasodilators. A single significant correlation between rises in cGMP and relaxation was obtained for both SIN compounds and various nitrovasodilators. Relaxation by SIN-1A was independent of the presence of endothelium and was not affected by various inhibitors of arachidonic acid metabolism. In contrast to nitroglycerin, SIN-1 did not induce substantial tolerance nor were its actions reduced in artery strips that were tolerant to nitroglycerin. The results indicate that SIN-1A relaxes coronary smooth muscle by a direct stimulant effect on soluble guanylate cyclase in vascular smooth muscle cells.     

Role of cyclic nucleotides in vasodilations of the rat thoracic aorta induced by adenosine analogues.

Although adenosine analogues such as 5'-N-ethylcarboxamidoadenosine (NECA) relax the rat thoracic aorta in a partially endothelium-dependent manner via adenosine A(2A) receptors, others such as N(6)-R-phenylisopropyladenosine (R-PIA) act via an endothelium-independent, antagonist-insensitive mechanism. The role of cyclic nucleotides in these relaxations was investigated in isolated aortic rings using inhibitors of adenylate and guanylate cyclases as well as subtype-selective phosphodiesterase inhibitors. The adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536; 100 microM) significantly inhibited responses to NECA, but not responses to R-PIA. The type IV (cyclic AMP-selective) phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 30 microM) significantly enhanced responses to NECA and to a lesser extent those to R-PIA. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ; 100 microM) significantly inhibited responses to NECA and acetylcholine but not responses to R-PIA. The selective phosphodiesterase V (cyclic GMP-selective) inhibitors, zaprinast (10 microM) and 4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline (MMQ; 1 microM), had no significant effect on responses to either NECA or R-PIA, but enhanced responses to acetylcholine. These results are consistent with the effects of NECA being via activation of endothelial receptors to release NO which stimulates guanylate cyclase, as well as smooth muscle receptors coupled to stimulation of adenylate cyclase. The lack of effect of zaprinast and MMQ on responses to NECA are likely to be due to simultaneous activation of both adenylate and guanylate cyclases in the smooth muscle, as cyclic AMP reduces the sensitivity of phosphodiesterase V to inhibitors. These results also suggest that the effects of R-PIA are via neither of these mechanisms.     

Tolerance to relaxation in rat aorta: comparison of an S-nitrosothiol with nitroglycerin

Tolerance and cross-tolerance to the relaxant effects of S-nitroso-N-acetylpenicillamine (SNAP) and nitroglycerin were examined in rat aortic rings. Nitroglycerin-tolerant rings remained fully responsive to SNAP and sodium nitroprusside. Thus, reduced metabolic activation of nitroglycerin, rather than impaired guanylate cyclase activity, appeared to be the operating mechanism for nitrate tolerance in this preparation. Under similar conditions, SNAP incubation induced less tolerance than nitroglycerin. S-nitrosothiols, therefore, appear to be less tolerance-producing than nitroglycerin.     

Effect of zaprinast on nitric oxide levels in serum and aortic tissue

Nitric oxide (NO), which is synthesized from the guanidino nitrogen of l-arginine by nitric oxide synthase (NOS), plays an important role in many physiological and pathological processes. Most of the effects of NO are mediated by cyclic guanosine 3'5 monophosphate (cGMP), which is synthesized by soluble guanylate cyclase (sGC) and degraded by phosphodiesterases (PDEs). Although the NO/cGMP pathway has been extensively studied, remarkably little is known about the regulation of NO release. Furthermore, controversial studies have indicated that intervention of the sGC/cGMP pathway modulates the release of NO. The purpose of this study was to evaluate the hypothesis that drugs that affect the sGC/cGMP pathway may modulate NO release and, if so, is there a correlation between NO levels and blood pressure effect? To this end, we investigated the effects of the PDE 5 inhibitor zaprinast on mean arterial pressure (MAP), nitrite/nitrate levels and cGMP in normotensive male Sprague Dawley rats. The results of the current study indicated that zaprinast dose-dependently increased plasma cGMP levels at 18, 24 and 36 mg/kg and decreased MAP at 24 and 36 mg/kg. However, zaprinast at 18, 24 and 36 mg/kg did not affect NO levels either in serum or aortic tissue. We have concluded that the PDE 5 inhibitor zaprinast has no regulatory effect on NO release in serum and aortic tissue, and NO was not involved in the hypotensive effect of zaprinast.     

Role of cyclic nucleotide phosphodiesterase isozymes in intact canine trachealis.

The role of individual cyclic nucleotide phosphodiesterase (PDE) isozymes in regulating cAMP and cGMP content in intact canine trachealis was examined using isozyme-selective and nonselective PDE inhibitors. The inhibitors used in this study were characterized previously [Mol. Pharmacol. 37:206-214 (1990)] and included: 1) zaprinast, an inhibitor (Ki = 0.1 microM) of the cGMP-specific PDE (cAMP Km = 135 microM; cGMP Km = 4 microM); 2) SK&F 94120, an inhibitor (Ki = 7 microM) of the cGMP-inhibited PDE (cAMP Km = 0.3 microM; cGMP Km = 8 microM); 3) Ro 20-1724, an inhibitor (Ki = 5 microM) of the cAMP-specific PDE (cAMP Km = 4 microM; cGMP Km = 40 microM); and 4) 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor (IC50 = 1-30 microM). In addition to the aforementioned isozymes, canine trachealis contains a Ca2+/calmodulin-stimulated PDE (cAMP Km = 1 microM; cGMP Km = 2 microM) and a GMP-stimulated PDE (cAMP Km = 93 microM; cGMP Km = 60 microM), for which selective inhibitors are not available. Isolated canine trachealis strips were contracted with methacholine and exposed to various concentrations of PDE inhibitors, before being relaxed by the cumulative addition of isoproterenol, an adenylate cyclase activator, or sodium nitroprusside, a guanylate cyclase activator. At the completion of the concentration-response studies, tissues were flash-frozen and assayed for cyclic nucleotide content. Neither isoproterenol-induced relaxation nor cAMP accumulation was altered by zaprinast, but both of these responses were potentiated by pretreatment of tissues with either SK&F 94120 or Ro 20-1724. The effects of SK&F 94120 and Ro 20-1724 were additive, and the combination of SK&F 94120, Ro-1724, and IBMX had no greater effect on the responses to isoproperenol than did either IBMX alone or the combination of SK&F 94120 plus Ro 20-1724. In contrast, zaprinast potentiated sodium nitroprusside-induced relaxation and cGMP accumulation, whereas neither SK&F 94120 nor Ro 20-1724 altered these responses. IBMX produced a greater potentiation than did zaprinast, and the combination of zaprinast and IBMX had a greater effect than either agent alone. The results of this study suggest that the cGMP-inhibited and cAMP-specific PDEs are responsible for cAMP hydrolysis in intact canine trachealis, whereas cGMP hydrolysis is mediated by the cGMP-specific PDE as well as the Ca2+/calmodulin-stimulated PDE and/or the cGMP-stimulated PDE.     

Differential hemodynamic responses to selective inhibitors of cyclic nucleotide phosphodiesterases in conscious rats.

Selective inhibition of either the low Km cyclic AMP (cAMP) or low Km cyclic GMP (cGMP) phosphodiesterase (PDE) promotes vasorelaxation and, consequently, produces depressor effects. To evaluate the systemic and regional hemodynamic effects of selective inhibitors of these PDE isozymes, CI-930 (0.1-10 mg/kg), an inhibitor of low Km cAMP PDE, or zaprinast (3-30 mg/kg), an inhibitor of low Km cGMP PDE, was given i.v. to conscious, normotensive rats. The rats were chronically instrumented with vascular catheters and either an ultrasonic transit-time flow probe around the ascending aorta or miniaturized pulsed Doppler flow probes around the superior mesenteric and left renal arteries and the abdominal aorta. CI-930 and zaprinast, at cumulative doses of 3 and 30 mg/kg, respectively, produced comparable reductions in mean arterial pressure (-22 +/- 3 and -19 +/- 4 mm Hg, respectively) and total peripheral resistance (-0.41 +/- 0.07 and -0.42 +/- 0.06 mm Hg/ml/min, respectively) but affected other hemodynamic variables differently. CI-930 at 3 mg/kg increased the heart rate (HR), maximal aortic flow acceleration (dF/dt), and peak aortic flow and decreased the stroke volume (SV). Cardiac output (CO) was not affected by CI-930. Zaprinast at 30 mg/kg increased the CO, dF/dt, and peak aortic blood flow. The HR and SV were unaffected by zaprinast. Although both CI-930 and zaprinast increased the dF/dt and peak aortic flow, these parameters were affected more by CI-930 than by zaprinast. CI-930 decreased hindquarter, mesenteric, and renal vascular resistances in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide-dependent reduction in soluble guanylate cyclase functionality accounts for early lipopolysaccharide-induced changes in vascular reactivity

We investigated the role of soluble guanylate cyclase in lipopolysaccharide-induced hyporesponsiveness to phenylephrine. The effects of phenylephrine on the blood pressure of female Wistar rats were evaluated at 2, 8, and 24 h after lipopolysaccharide injection (12.5 mg/kg i.p.). Vasoconstrictive responses to phenylephrine were reduced 40 to 50% in all time periods. Methylene blue, a soluble guanylate cyclase inhibitor (15 micromol/kg i.v.) restored the reactivity to phenylephrine in animals injected with lipopolysaccharide 2 and 24 h earlier. However, it failed to do so in animals injected with lipopolysaccharide 8 h earlier. Incubation with sodium nitroprusside (SNP) increased lung and aorta cGMP levels in control animals and in tissues of rats treated with lipopolysaccharide 24 h earlier. However, SNP failed to increase tissue cGMP in rats injected 8 h earlier. Lipopolysaccharide reduced the vasodilatory response to NO donors 8 h after injection. This effect and the decreased lung cGMP accumulation in response to SNP were reversed by an NO synthase blocker. Guanylate cyclase protein levels were lower than controls in lungs harvested from rats injected 8 h earlier and were back to normal values in lungs of rats injected 24 h earlier with lipopolysaccharide. Thus, data indicate that there is a temporal window of 8 h after lipopolysaccharide injection in which soluble guanylate cyclase is not functional and that this loss of function is NO-dependent. Thus, the putative use of soluble guanylate cyclase inhibitors in the treatment of endotoxemia may be beneficial mainly at early stages of this condition.