Increase In cGMP Content Research Articles

Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes.

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate growth of cultured cardiac myocytes. First, we investigated the effects of exogenous NO and cGMP analog on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor 3-morpholino-sydnonimine-hydrochloride (SIN-1) and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine-stimulated incorporation of 3H-leucine into cardiac myocytes. We then transferred endothelial constitutive NO synthase (ecNOS) gene into cultured neonatal rat cardiac myocytes using adeno-associated virus (AAV) vectors. ecNOS gene transfer into cardiac myocytes induced 140 kD ecNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. ecNOS gene transfer inhibited 3H-leucine incorporation into cardiac myocytes in response to phenylephrine, which was significantly recovered in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine acetate. These results indicate that endogenously generated NO by ecNOS gene transfer using AAV vectors inhibits the alpha-adrenergic agonist-induced cardiac protein synthesis at least partially via cGMP production.

NO donor sodium nitroprusside inhibits excitation-contraction coupling in guinea pig taenia coli.

In guinea pig taenia coli, the nitric oxide (NO) donor sodium nitroprusside (SNP, 1 microM) reduced the carbachol-stimulated increases in muscle force in parallel with a decrease in intracellular Ca(2+) concentration ([Ca(2+)](i)). A decrease in the myosin light chain phosphorylation was also observed that was closely correlated with the decrease in [Ca(2+)](i). With the patch-clamp technique, 10 microM SNP decreased the peak Ba(2+) current, and this effect was blocked by an inhibitor of soluble guanylate cyclase. Carbachol (10 microM) induced an inward current, and this effect was markedly inhibited by SNP. SNP markedly increased the depolarization-activated outward K(+) currents, and this current was completely blocked by 0.3 micorM iberiotoxin. SNP (1 microM) significantly increased cGMP content without changing cAMP content. Decreased Ca(2+) sensitivity by SNP of contractile elements was not prominent in the permeabilized taenia, which was consistent with the [Ca(2+)](i)-force relationship in the intact tissue. These results suggest that SNP inhibits myosin light chain phosphorylation and smooth muscle contraction stimulated by carbachol, mainly by decreasing [Ca(2+)](i), which resulted from the combination of the inhibition of voltage-dependent Ca(2+) channels, the inhibition of nonselective cation currents, and the activation of Ca(2+)-activated K(+) currents.

Maturational Changes in Endothelium-Derived Nitric Oxide-Mediated Relaxation of Ovine Pulmonary Arteries

Endothelium-derived nitric oxide (EDNO) modulates the responses of pulmonary vasculature. The present study was designed to determine EDNO-mediated responses of pulmonary arteries (PA) of term fetal, newborn, and adult sheep. Vessel rings were suspended in organ chambers and their isometric tension was recorded. In vessels preconstricted with endothelin-1, acetylcholine had no effect on fetal PA but caused a greater endothelium-dependent relaxation in adult PA than in newborn PA. In vessels without endothelium, nitric oxide and 8-bromo-cGMP caused greater relaxation in adult PA than in newborn PA while causing the least relaxation in fetal PA. Acetylcholine had no effect on cGMP content in fetal PA but caused a greater endothelium-dependent increase in cGMP content in adult PA than in newborn PA. In vessels without endothelium, nitric oxide caused a smaller increase in cGMP content of fetal PA than of newborn PA while causing the greatest increase in cGMP of adult PA. These results demonstrate an age-dependent increase in EDNO-mediated responses of ovine pulmonary arteries. A change in receptor and/or receptor coupling, in soluble guanylate cyclase activity, and in cGMP responsiveness of vascular smooth muscle may contribute to the phenomenon.

NNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle

Nitric oxide (NO) from Ca(2+)-dependent neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers may modulate vascular tone by a cGMP-dependent pathway similar to NO derived from NOS in endothelial cells (eNOS). In isolated fast-twitch extensor digitorum longus (EDL) muscles from control mice, cGMP formation increased approximately 166% with electrical stimulation (30 Hz, 15 s). cGMP levels were not altered in slow-twitch soleus muscles. The NOS inhibitor N(omega)-nitro-l-arginine abolished the contraction-induced increase in cGMP content in EDL muscles, and the NO donor sodium nitroprusside (SNP) increased cGMP content approximately 167% in noncontracting EDL muscles. SNP treatment but not electrical stimulation increased cGMP formation in muscles from nNOS(-/-) mice. cGMP formation in control and stimulated EDL muscles from eNOS(-/-) mice was less than that obtained with similarly treated muscles from control mice. Arteriolar relaxation in contracting fast-twitch mouse cremaster muscle was attenuated in muscles from mice lacking either nNOS or eNOS. These findings suggest that increases in cGMP and NO-dependent vascular relaxation in contracting fast-twitch skeletal muscle may require both nNOS and eNOS.

Urodilatin limits acute reperfusion injury in the isolated rat heart.

Hypercontracture is an important mechanism of myocyte death during reperfusion. cGMP modulates the sensitivity of contractile myofilaments to Ca2+, and increasing cGMP concentration during the last minutes of anoxia prevents reoxygenation-induced hypercontracture in isolated cardiomyocytes. The purpose of this study was to determine whether stimulation of particulate guanylyl cyclase with the natriuretic peptide urodilatin, given at the time of reperfusion, reduces myocardial necrosis in the rat heart submitted to transient ischemia. Isolated rat hearts (n = 38) were submitted to either 40 or 60 min of no-flow ischemia and 2 h of reperfusion, and were allocated to receive or not receive 0.05 microM urodilatin during the first 15 min of reperfusion or non-reperfusion treatment. A marked reduction in myocardial cGMP concentration was observed in control hearts during reperfusion after 40 or 60 min of ischemia. Urodilatin significantly attenuated cGMP depletion during initial reperfusion, markedly improved contractile recovery after 40 min of ischemia (P < 0.0309), and reduced reperfusion-induced increase in left ventricular end-diastolic pressure (P = 0.0139), LDH release (P = 0.0263), and contraction band necrosis (P = 0.0179) after 60 min of ischemia. The beneficial effect of urodilatin was reproduced by the membrane permeable cGMP analog 8-Bromo-cGMP. These results indicate that reduced cGMP concentration may impair myocyte survival during reperfusion. Stimulation of particulate guanylyl cyclase may appear as a new strategy to prevent immediate lethal reperfusion injury.

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells

Purpose. Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair. Methods. The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or other agents. At prescribed time intervals, the cells were stained by Giemsa and the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured by radioimmunoassay. Results. Addition of EGF (1–100 ng/ml) to RCEC stimulated cell proliferation which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 µM) or SNP (10 µM to 1 mM), in the presence of EGF, dose-dependently inhibited the growth factor-stimulated wound closure in RCEC. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 µM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibited the wound closure with a corresponding increase in cGMP contents. Treatment of the cells with ODQ (10 nM to 10 µM), a soluble guanylate cyclase inhibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, and reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Addition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited the EGF-stimulated wound closure in a dose-dependent manner. Treatment of RCEC with mitomycin C (5 µM) exerted a marked inhibitory effect on wound closure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated cells. Conclusions. The results demonstrate that ANP and SNP inhibit the EGF-stimulated wound repair in RCEC. The effect of these agents is mediated via activation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exert its inhibitory effect at the level of cell proliferation and not cell migration. The data suggest an important role for cGMP-dependent protein kinase in proliferation of RCEC stimulated by EGF.

Inhibitory mechanism of papaverine on the smooth muscle of guinea pig urinary bladder.

In guinea pig urinary bladder, the hyperosmotic 65 mM KCI (H-65K+)- or carbachol (CCh)-induced contraction was inhibited by an addition of papaverine in a concentration-dependent manner. The cAMP content of the muscle in the presence of H-65K+ or CCh was increased by papaverine only at the higher concentration of 100 microM, but cGMP content was not affected by papaverine. Forskolin, compared with papaverine, increased cAMP content in a concentration-dependent manner, and nitroprusside did not significantly increase cGMP content. In a fura 2 loaded muscle, papaverine did not affect an increase of [Ca2+]i level by high K+ or CCh. The increase of oxidized flavoprotein (FPox) fluorescence and muscle contraction in the presence of H-65K+ or CCh was decreased by papaverine (1 - 100 microM), and the increase of pyridine nucleotide (PNred) fluorescence was not affected by papaverine. In summary, it was concluded that papaverine induced relaxation by inhibiting mitochondrial respiration in guinea pig urinary bladder as well as ileum. Moreover, it is proposed that the mechanism of papaverine-induced relaxation in the smooth muscle, which shows predominantly a metabolic dependency on its contraction, is an inhibition of mitochondrial respiration.

SILDENAFIL INHIBITS PHOSPHODIESTERASE TYPE 5 IN HUMAN CLITORAL CORPUS CAVERNOSUM SMOOTH MUSCLE

Abstract Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-cyclic GMP phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.

Sildenafil citrate and blood-pressure–lowering drugs: results of drug interaction studies with an organic nitrate and a calcium antagonist

Sildenafil, a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), is a well-tolerated and highly effective treatment for erectile dysfunction. The mechanism of action of sildenafil depends on activation of the nitric oxide (NO)–cGMP pathway during sexual stimulation, which results in corpus cavernosal smooth muscle relaxation and penile erection. Endogenously derived NO is also involved in blood pressure regulation through its effect on basal vascular tone, which is mediated by cGMP levels. Organic nitrates and NO donors exert their therapeutic effects on blood pressure and vascular smooth muscle by the same mechanism as endogenous NO. Since both sildenafil and organic nitrates exert their pharmacologic effects via increases in cGMP concentrations, a double-blind, placebo-controlled, crossover study was undertaken to investigate the effects of sildenafil coadministered with glyceryl trinitrate on blood pressure and heart rate in healthy male subjects. The hemodynamic effects of sildenafil were also evaluated in a second placebo-controlled crossover study in men with hypertension who were taking the calcium antagonist amlodipine, which has a mechanism of action that does not involve the cGMP pathway. In the first crossover study, subjects were treated with oral sildenafil (25 mg, 3 times a day for 4 days) or placebo and then challenged on day 4 with a 40-minute, stepwise, intravenous infusion of glyceryl trinitrate (0.5 mg/mL in 5% dextrose at an initial infusion rate of 2.5 μg/min and doubling every 5 minutes to a maximum rate of 40 μg/min) 1 hour after taking sildenafil or placebo. On day 5, subjects received a sublingual glyceryl trinitrate tablet (500 μg) 1 hour after taking 25 mg of sildenafil or placebo. During sildenafil treatment, the subjects were significantly less tolerant of intravenously administered glyceryl trinitrate than during placebo treatment, based on the occurrence of a >25 mm Hg decrease in blood pressure or the incidence of symptomatic hypotension (p <0.01). When a sublingual glyceryl trinitrate tablet was administered on day 5, a 4-fold greater decrease in systolic blood pressure was observed for the subjects during the sildenafil treatment period than during the placebo treatment period. The changes in heart rate were negligible during both glyceryl trinitrate challenges. In conclusion, sildenafil potentiated the hypotensive effects of glyceryl trinitrate, an organic nitrate. Thus, sildenafil administration to patients who are using organic nitrates, either regularly and/or intermittently, in any form is contraindicated. In the second crossover study, men with hypertension, who were taking 5 or 10 mg/day of amlodipine, received a single oral dose of 100 mg sildenafil or placebo. Coadministration of sildenafil did not significantly affect the pharmacokinetics of amlodipine. In the 4 hours after dosing, differences in the mean maximum change from baseline in supine systolic and diastolic blood pressures between the sildenafil plus amlodipine and the placebo plus amlodipine treatment periods were −8 mm Hg and −7 mm Hg, respectively (p ≤0.002). The mean maximum supine heart rate increased 2.1 beats/min during sildenafil plus amlodipine treatment and decreased 1.5 beats/min during placebo plus amlodipine treatment (p <0.02). The adverse events in this study were predominantly mild or moderate and did not cause discontinuation of treatment. Adverse events considered to be related to sildenafil treatment included headache, nausea, and dyspepsia. In patients with hypertension who were taking amlodipine therapy, sildenafil produced additive, but not synergistic, reductions in blood pressure. The difference in the mean maximum change from baseline in blood pressure between sildenafil plus amlodipine and placebo plus amlodipine was comparable to the decrease in blood pressure reported for healthy men taking sildenafil alone. Thus, no synergistic interaction was observed between sildenafil and the calcium antagonist amlodipine in patients with hypertension.

Pharmacological profiles of sildenafil (VIAGRA) in the treatment of erectile dysfunction: efficacy and drug interaction with nitrate

Penile erection follows relaxation of the corpus cavernosum in which nitric oxide (NO) released during sexual stimulation from non-adrenergic non-cholinergic nerve endings and from endothelial cells of the corpus cavernosum plays a crucial role. Sildenafil (VIAGRA) selectively inhibited phosphodiesterase type 5 (PDE5) activity in the human corpus cavernosum and increased cGMP concentrations in the rabbit cavernosum in the presence of NO. Sildenafil enhanced the NO-dependent relaxation of the isolated human corpus cavernosum and the intracavernosal pressure in the anesthetized dog without affecting systemic blood pressure and heart rate. In the patients with erectile dysfunction, an orally administered sildenafil enhanced the penile rigidity during visual sexual stimulation. Sildenafil did not affect the phenylephrine-induced contraction of the isolated rabbit aorta, but enhanced the relaxant effect of glyceryl trinitrate. The pharmacodynamic interaction with glyceryl trinitrate was also observed in human studies where sildenafil potentiated the hypotensive effect of the nitrate. These results indicate that sildenafil, which enhances the physiological process of penile erection during sexual arousal, is a novel orally effective treatment for erectile dysfunction. It should be noted, however, that sildenafil enhanced the hypotensive effect of glyceryl trinitrate, as a result of inhibition of PDE5 in vascular smooth muscle. Therefore, administration of sildenafil to patients who are using nitrates and NO donors is contraindicated.

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs.

Endothelium-derived nitric oxide (EDNO) plays a pivotal role in regulating pulmonary circulation. To determine whether there is a heterogeneity in EDNO-mediated responses of different sized pulmonary vessels, we studied small and large isolated pulmonary arteries of newborn lambs (diameter, 0.4-0.7 and 1.5-2.5 mm, respectively). The isometric tension of vessel rings were recorded while suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2-5% CO2, 37 degrees C). In vessels preconstricted with norepinephrine, acetylcholine and bradykinin induced a greater relaxation of small pulmonary arteries than of large pulmonary arteries. Acetylcholine, bradykinin, and nitric oxide also induced a greater increase in cGMP content in small arteries than in large ones. The responses to acetylcholine and bradykinin were endothelium-dependent and inhibited by nitro-L-arginine, an inhibitor of nitric oxide synthase. In vessels without endothelium, the response to nitric oxide was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. The activity of soluble guanylyl cyclase of small arteries was greater than that of large arteries under basal conditions and after stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor. These results demonstrate that heterogeneity exists in EDNO-mediated relaxation of small and large pulmonary arteries in newborn lambs. A difference in the soluble guanylate cyclase activity of vascular smooth muscle may have contributed to this phenomenon.

Propofol-induced relaxation of rat mesenteric arteries: evidence for a cyclic GMP-mediated mechanism.

Experiments were designed to evaluate whether guanosine 3',5'-cyclic monophosphate (cGMP)-mediated mechanisms contribute to vasodilation via propofol in rat mesenteric resistance arteries. Ring segments were suspended in the myograph system for isometric tension recording, and responses to propofol were tested in the presence and absence of methylene blue (MB), an inhibitor of guanylate cyclase. At concentrations > or = 1 microM, propofol caused concentration-dependent relaxation of vessel rings precontracted with U46619 (a thromboxane analog). The effect was not affected by N-monomethyl-L-arginine (L-NMMA; 50 microM). MB (5 microM) reversed propofol-induced vasodilation by 30% (p < 0.001). In contrast, MB has no effect on nifedipine-inhibited vasocontraction. The propofol-induced relaxation was further tested in rings incubated in Ca2+-free solution. U46619-induced contractions were significantly reduced by propofol (40 microM) but not by nifedipine (1 microM). Propofol reduced to a similar degree the contractions obtained to exogenously added calcium chloride in the absence and the presence of MB. Furthermore, propofol (10-100 microM) increased cGMP content in cultured bovine vascular smooth-muscle cells. Soluble guanylate cyclase inhibitors, such as MB and LY83583, attenuated this effect. This investigation suggests that propofol-induced relaxations in small arteries, in addition to inhibition of calcium influx, are mediated by increases of cGMP in the smooth muscle cells.

Estrogenic effect of estradiol-sulfamate on the male rat anterior pituitry

Estrogen sulfamates (ES) are used for a new treatment strategy to avoid liver-hormone and hormone-liver interactions. ES represent new synthetic steroids having an increased systemic and reduced hepatic estrogenicity when given orally [1, 2]. In the present study effects of ES and estradiol-benzoate (EB) on adenohypophyseal (AP) and serum concentrations of prolactin (PRL), luteinizing hormone (LH), and pituitary contents of cAMP and cGMP in the male rat are demonstrated. The weight gain of experimental animals treated by ES, EB or both hormones simultaneously was significantly lower compared to controls. EB but not ES significantly increased the weight of the AP. The amounts of PRL in the AP and serum were significantly increased after EB administration. ES significantly increased only AP content of PRL. EB administered simultaneously with ES exhibited an additive effect on the AP plasma concentrations of PRL. The EB or ES significantly decreased AP and serum concentrations of LH. ES given simultaneously with EB further decreased AP and serum concentrations of LH. After administration of either ES or EB, AP contents of cAMP and cGMP were significantly increased. An additive effect of these estrogens on the cGMP content was found. ES given simultaneously with EB further increased cGMP content in the AP but partially inhibited the effect of EB on the AP cAMP content. The present results demonstrate that the effects of ES on the AP content of PRL, LH, cAMP, and cGMP differ from the effects of EB. Whether this is due to lower levels of estradiol after the administration of ES secondary to its different absorption when compared to EB is unknown. Thus, our data support the concept that the ES has a lesser estrogenic effect on the AP function.

Estrogen increases cGMP in selected brain regions and in cerebral microvessels.

We have previously reported that exogenous and endogenous estrogen can amplify residual cerebral blood flow during experimental cerebral ischemia. Because estrogen has been linked to nitric oxide and cyclic guanosine monophosphate (cGMP) signaling in noncerebral tissue, we tested the hypothesis that long-term 17beta-estradiol treatment increases basal cGMP in brain homogenates and cerebral microvessels in female rabbits. We also determined whether there are important baseline gender-specific differences in regional cGMP. Adult female rabbits were implanted with 17beta-estradiol pellets, 10 mg (F10, n = 10) or 50 mg (F50, n = 13), and compared with untreated females (F, n = 19) and males with negligible estrogen (M, n = 19) (plasma 17beta-estradiol levels of 4+/-4 pg/mL in M, 7+/-5 pg/mL in F, 141+/-74 pg/mL in F10, and 289 +/-10 pg/mL in F50). Cyclic GMP was determined by radioimmunoassay in cerebellum, hypothalamus, caudate nucleus, hippocampus, and cortex. Cerebral microvessels were harvested from additional cohorts of untreated males and females or estradiol-implanted females (n = 6 per group). Basal cGMP was higher in F versus M only in cerebellum. Estrogen-induced increases in regional cGMP were prominent in hippocampus at all doses (M = 43+/-26, F = 43+/-21, F10 = 84+/-24, F50 = 117+/-55 fmol/mg protein) and in cortex at the high dose (M = 78+/-55, F = 88+/-51, F10 = 69+/-34, F50 = 143+/-52 fmol/mg protein). Similarly, microvascular cGMP increased only in females treated with the 50 mg dose (M = 77+/-13, F = 86+/-25, F10O = 106+/-35, F50 = 192+/-88 fmol/mg protein). Therefore, 17beta-estradiol increases cGMP content in parenchymal regions that are known physiologic targets for reproductive steroids but are also areas of selective vulnerability to ischemic insult. Further, high doses of estrogenic steroids could amplify cGMP signaling within the cerebral microvasculature.

24R,25-Dihydroxyvitamin D3Increases Cyclic GMP Contents, Leading to an Enhancement of Osteocalcin Synthesis by 1,25-Dihydroxyvitamin D3in Cultured Human Osteoblastic Cells

The effect of the physiological vitamin D metabolite 24R,25-dihydroxyvitamin D3[24R,25(OH)2D3] on human osteoblastic cells was assessed. Physiological concentrations (10−9–10−8M) of 24R,25(OH)2D3significantly increased the cyclic guanosine 5′-monophosphate (cGMP) content in the human osteoblastic cells by approximately 200% in 5 to 15 min. In contrast, 24S,25-dihydroxyvitamin D3had only a weak effect on the cGMP content, and 1,25-dihydroxyvitamin D3[1,25(OH)2D3] did not affect the content. The production of osteocalcin was not induced by 10−9–10−8M of 24R,25(OH)2D3in the absence of 1,25(OH)2D3. However, the same concentration of 24R,25(OH)2D3showed stimulatory effects on osteocalcin synthesis in the presence of 10−9M 1,25(OH)2D3. Rp-8Br-cyclic GMP, a specific inhibitor of cyclic GMP-dependent protein kinase, significantly inhibited the cooperative effect of 24R,25(OH)2D3with 1,25(OH)2D3on the osteocalcin synthesis, although Rp-8Br-cyclic AMP, a specific inhibitor of cyclic AMP-dependent protein kinase, did not affect the cooperative effect. In addition, okadaic acid enhanced the osteocalcin synthesis induced by 1,25(OH)2D3. These observations suggest that 24R,25(OH)2D3has a unique activity of increasing cGMP contents in osteoblastic cells, and that the increase in cGMP contents may lead to the cooperative effect of 24R,25(OH)2D3with 1,25(OH)2D3on osteocalcin synthesis. These data support the hypothesis that 24R,25(OH)2D3has a physiological role in human bone and mineral metabolism.

Sildenafil Inhibits Phosphodiesterase Type 5 in Human Clitoral Corpus Cavernosum Smooth Muscle

Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-cyclic GMP phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.

Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide

Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP-mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with N G-nitro- l-arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine-induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.

Cyclic 3 ′, 5 ′-Guanosine Monophosphate Synthesis Induced by Atrial Natriuretic Peptide, C-Type Natriuretic Peptide, and Nitric Oxide in the Rat Retina

This study was undertaken to determine whether pathways exist in the rat retina for atrial natriuretic peptide (ANP)-, C-type natriuretic peptide (CNP)-, and nitric oxide (NO)- cyclic 3′, 5′-guanosine monophosphate (cGMP). Exposure of the retina to ANP (10 −7 mol/L), CNP (10 −7 mol/L), S-nitroso-N-acetylpenicillamine (10 −5 mol/L, SNAP; a NO donor), A23187 (10 −5 mol/L; a Ca 2+ ionophore), and carbachol (10 −3 mol/L) caused 1.45 ∼ 1.67-fold increases in cGMP content ( P < .01). The increase in cGMP content induced by A23187 was blocked by 2-4-carboxyphenyl · 4455-tetramethyl imidazoline 1-oxyl 3-oxide (10 −3 mol/L, carboxy-PTIO; a NO scavenger). Both carboxy-PTIO (10 −3 mol/L) and N G-nitro- l-arginine (10 −3 mol/L, l-NNA; a NO synthase inhibitor) blocked the increase in cGMP content induced by carbachol. Atropine (10 −5 mol/L; a muscarinic receptor antagonist) also blocked the cGMP increase induced by carbachol. These data demonstrate that ANP-, CNP-, and NO-cGMP pathways exist in the rat retina and that the NO-cGMP pathway may be linked to the activation of the muscarinic receptor.

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase.

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes.

Nitric oxide increases the activity of the apical 70-pS K+ channel in TAL of rat kidney.

We have previously shown that nitric oxide (NO) mediates the stimulatory effect of angiotensin II on the apical 70-pS K+ channel in the thick ascending limb (TAL) of Henle's loop of the rat kidney (12). In the present study, we used the patch-clamp technique to examine the effects of NO on the 70-pS K+ channel. Addition of 10 microM S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, increased the channel activity in cell-attached patches. In contrast, application of 100 microM N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), reduced the channel activity by 75 +/- 7%. The effect of L-NAME was the result of inhibiting NOS, since D-NAME, which does not block NOS activity, had no effect on the channel activity. In addition, the effect of L-NAME was abolished in the presence of 1 mM L-arginine or by addition of 10 microM SNAP, further supporting the role of NO. Finally, the L-NAME-induced inhibition was also reversed by adding 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). That the effect of NO is mediated by the cGMP-dependent pathway is also suggested by experiments in which inhibition of guanylate cyclase abolished the effect of SNAP. Finally, 10 microM SNAP significantly increased cGMP concentration of the medullary TAL from 12.4 fM/microgram protein to 38.9 fM/microgram protein, as measured with ELISA. We conclude that NO is involved in regulating the activity of the apical 70-pS K+ channel in the TAL of the rat kidney.