Regulation of hepatic nuclear guanylate cyclase.

In immunohistochemical studies of rat liver tissue slices and purified nuclei, adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) immunofluorescence on the nuclear membrane are sequentially increased after glucagon administration. An explanation for the increased cGMP immunofluorescence was sought in experiments in which guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]activity of hepatic subcellular fractions was determined. The results showed that a nuclear guanylate cyclase exists which can be distinguished from the soluble and crude particulate guanylate cyclases. The activity of the nuclear enzyme was increased by 35% in nuclei isolated from rats 30 min after glucagon injection, the time at which maximal nuclear membrane cGMP immunofluorescence is observed. Because glucagon altered both cAMP location and levels prior to the observed changes in nuclear cGMP metabolism, the hypothesis that cAMP acted as the second messenger was tested. In vitro incubation of nuclei isolated from control rats with 10(-5) M cAMP produced a 25% increase in nuclear guanylate cyclase activity. With nuclei isolated from glucagon-treated rats, no significant increase in enzyme activity was observed; this indicates that maximal stimulation of nuclear guanylate cyclase by cAMP occurred at levels that are obtained in vivo after glucagon administration. These findings suggest that hepatic nuclear cGMP content may be regulated by a specific organelle guanylate cyclase and that cAMP may be one of the determinants of this enzyme's activity.     

Role of cyclic nucleotides in rapid platelet adhesion to collagen

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4- fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2- fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.     

cGMP-induced differentiation of the promyelocytic cell line HL-60.

cGMP is a second messenger that mediates numerous metabolic events; in the present work a role in myeloid cell differentiation was demonstrated. Nitroprusside and NaNO2, which activate cytosolic guanylate cyclase and increase the intracellular cGMP concentration, induced granulocytic differentiation of the human promyelocytic cell line HL-60; differentiation was measured by acquisition of the OKM1 antigen, morphological changes, and nitroblue tetrazolium reduction. When theophylline, a phosphodiesterase inhibitor, which by itself induced modest differentiation, was added to nitroprusside or NaNO2, differentiation increased in an additive fashion. The degree of differentiation correlated with the increase in the intracellular cGMP concentration. 8-Bromoguanosine 3',5'-cyclic monophosphate, a membrane-permeable cGMP analogue, also induced differentiation of HL-60 cells but was much more effective in the presence of theophylline, with the two agents interacting synergistically. The effect of theophylline in these studies could not be attributed to increasing the intracellular cAMP concentration. Dimethyl sulfoxide, and established inducer of differentiation of HL-60 cells, markedly enhanced the differentiation induced by nitroprusside and NaNO2.     

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

AIMS: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). METHODS: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrulline production from l-[3H]-arginine and cyclic nucleotides by radioimmunoassay. RESULTS: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 micro mol/l calcium ionophore ionomycin (from 0.1094+/-0.0229 to 0.2685+/-0.0560 pmol/min per mg cell protein, P=0.011) and to 2 nmol/l insulin (from 0.1214+/-0.0149 to 0.2045+/-0.0290 pmol/min per mg cell protein, P=0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71+/-0.10 to 6.80+/-0.40 pmol/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=0.0001). NOS inhibitor N(G)-monomethyl-l-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. CONCLUSION: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.     

Influence of insulin and of insulin resistance on platelet and vascular smooth muscle cell function

In this short review, we present the main results obtained in our laboratory in the last 15 years concerning the influence exerted by insulin on platelets and human vascular smooth muscle cells (VSMCs). In particular, we discuss: (i) the insulin ability to rapidly activate a constitutive nitric oxide synthase (NOS) in both cell types, with a consequent increase of the two nucleotides guanosine-3',5'-cyclic monophosphate (cGMP) and adenosine-3',5'-cyclic monophosphate (cAMP), well-known mediators of antiaggregation and vasodilation; (ii) the interplay of insulin with substances able to activate adenylate cyclase in both cell types; (iii) the impairment of the antiaggregating insulin effects in insulin-resistant subjects; (iv) the insulin-induced increase on endothelin in the VSMCs; (v) the insulin ability to slightly stimulate VSMC proliferation.     

Endothelium-dependent modulation of cGMP levels and intrinsic smooth muscle tone in isolated bovine intrapulmonary artery and vein

The role of the endothelium in modulating cyclic nucleotide levels and intrinsic smooth muscle tone was studied in isolated rings of bovine intrapulmonary artery and vein. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were threefold to fourfold higher in unrubbed artery and vein than in vessels that had been denuded of endothelium. Cyclic 3',5'-adenosine monophosphate (cAMP) levels were twofold higher in unrubbed than in endothelium-denuded artery, but no differences were observed in veins. Methylene blue, an inhibitor of guanylate cyclase, decreased cGMP but not cAMP levels, and this was accompanied by increases in smooth muscle tone. M&B 22,948, an inhibitor of cGMP-phosphodiesterase, increased cGMP but not cAMP levels, and this was accompanied by decreases in smooth muscle tone. Unrubbed vessels were more sensitive than endothelium-denuded vessels to the actions of both methylene blue and M&B 22,948, and this may be attributed to endothelium-dependent increases in cGMP turnover. Moreover, unrubbed vessels were more sensitive than endothelium-denuded vessels to contractile responses to phenylephrine and potassium, and these responses were potentiated by methylene blue and attenuated by M&B 22,948. Although indomethacin lowered cAMP levels in unrubbed artery, no changes in tone or contractile responsiveness were observed. A consistent observation was that the smaller branches of unrubbed but not endothelium-denuded intrapulmonary artery and vein had higher levels of cGMP but not cAMP, were sensitive to endothelium-dependent vasodilators, were more sensitive to methylene blue, and would not maintain a steady level of submaximal tone to phenylephrine when compared with larger branches from a common vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle.

A 26 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) relaxed isolated rabbit aortic segments in which the endothelium was either intact or functionally destroyed. The relaxations were temporally associated with increases in levels of cGMP with no change in the levels of cAMP. The ANF-induced increases in cGMP were also observed in aortic segments pretreated with calcium-free buffer or the cGMP phosphodiesterase inhibitor M&B 22,948. Qualitatively similar results were obtained for sodium nitroprusside. ANF selectively activated particulate guanylate cyclase, having no effect on the soluble form of the enzyme. Thus, the direct (endothelium-independent) vasodilator effect of ANF may be mediated via increased tissue levels of cGMP. ANF appears to increase vascular cGMP levels by activation of particulate guanylate cyclase.     

Hormone and neurotransmitter receptors in an established vascular endothelial cell line.

A cell line from the intima of the rabbit aorta has been established. This cell line exhibits strict contact inhibition, and morphologically resembles intimal endothelial cells. B-type blood group antigens and the presence of fibrinolytic activity also distinguish these cells from smooth muscle cells and from fibroblasts of the aortic wall. Endothelial cells were assayed for changes in levels of adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) in response to a series of vasoactive drugs. Control levels for cAMP and cGMP are 7.01 +/- 0.82 and 1.50 +/- 0.06, respectively (mean +/- SEM). Norepinephrine, acetylcholine, 5-hydroxytryptamine, and phenylephrine increased the levels of both nucleotides significantly. Propranolol (10-5 M) and phentolamine (10-5M) inhibited, respectively, the cAMP and cGMP response to norepinephrine. Angiotensin II and histamine significantly increased cGMP levels but not cAMP levels of the endothelial cells. The cGMP increases with acetylcholine were inhibited by atropine. These results indicate that the established cell line is endothelial in nature and contains cellular receptors to a variety of vasoactive agents.     

Atrial natriuretic peptide and sodium nitroprusside stimulate cyclic GMP accumulation by avian skin fibroblasts and epiphyseal growth-plate chondroprogenitor cells

Chondroprogenitor cells derived from avian tibia epiphyseal growth plate, and skin fibroblasts were cultured in vitro. In the fibroblasts, human (1-28) and rat (5-28) atrial natriuretic peptide (ANP) stimulated cyclic GMP (cGMP) production in a dose-dependent manner without affecting cAMP. Sodium nitroprusside also stimulated cGMP accumulation by chondroprogenitor cells and fibroblasts, but the maximum cGMP accumulation elicited by sodium nitroprusside was much lower than that obtained with ANP. The effects of ANP and sodium nitroprusside on chondroprogenitor cells and skin fibroblasts were additive. Human ANP increased cGMP production by the particulate fraction prepared either from chondroprogenitor cells or fibroblasts. Sodium nitroprusside, at concentrations of up to 1 mmol/l, did not affect cGMP production by the particulate fraction prepared from either cell type. The present study provides additional evidence that avian growth-plate chondroprogenitor cells and skin fibroblasts are targets for ANP. ANP and nitroprusside activate different guanylate cyclase isoenzymes--the particulate and soluble forms of the enzyme respectively. The data suggest that most of the guanylate cyclase activity in these cells is localized in the particulate fraction.     

Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator

To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.     

Immunocytology on microwave-fixed cells reveals rapid and agonist-specific changes in subcellular accumulation patterns for cAMP or cGMP.

We developed a method for cAMP and cGMP immunocytology based upon fixation by microwave irradiation. Fixation by microwave irradiation prevented three problems found with other fixation methods: nucleotide loss from cells, nucleotide diffusion within cells, and chemical modification of immunologic epitopes. Six agonists (four that stimulate adenylate cyclase and two that stimulate guanylate cyclase) produced cAMP or cGMP accumulation patterns that were agonist-specific, dose-dependent, detectable at physiologic concentrations of hormone, and time-dependent within 15 sec to 30 min. cAMP accumulation after 1 mM forskolin was greatest in the nucleus. Isoproterenol, prostaglandin E2, or calcitonin caused initial accumulation of cAMP along the plasma membrane, but later accumulation was greater in the cytoplasm. With calcitonin the later accumulation of cAMP was selectively perinuclear and along the nuclear membrane. Sodium nitroprusside stimulated cGMP accumulation diffusely throughout the cytoplasm. Atrial natriuretic peptide initiated cGMP accumulation near the plasma membrane, and cGMP accumulation moved from there into the cytoplasm. In conclusion, microwave irradiation preserved cell structure and allowed visualization of expected as well as unsuspected changes in intracellular accumulation patterns of cAMP and cGMP.     

Atrial natriuretic peptide inhibits spontaneous rat oocyte maturation

We report results of experiments demonstrating a dose-dependent inhibition of spontaneous maturation (resumption of meiosis) in rat oocyte-cumulus complexes by atrial natriuretic peptide (ANP). The inhibition was persistent over the time period studied. The ANP analog Tyr8-ANP, which mediates smooth muscle relaxation in other organs without elevating cGMP levels, did not inhibit the spontaneous maturation. ANP, but not Tyr8-ANP, dose-dependently stimulated cGMP accumulation in oocyte-cumulus complexes. Furthermore, sodium nitroprusside (SNP), that stimulates a soluble form of guanylate cyclase, inhibited spontaneous maturation in oocyte-cumulus complexes and stimulated cGMP accumulation in oocyte-cumulus complexes. Neither ANP nor SNP stimulated cAMP accumulation. In oocytes where the surrounding cumulus cells had been removed neither ANP nor SNP inhibited the spontaneous maturation. These results demonstrate that cumulus cells, but not the oocyte itself, have ANP receptors and guanylate cyclases. Furthermore, ANP, via cGMP, can influence oocyte meiosis, suggesting a possible involvement of ANP and cGMP in the control of the meiotic process in rat oocytes.     

cGMP-dependent protein kinase expression restores contractile function in cultured vascular smooth muscle cells

Vascular diseases, such as atherosclerosis and restenosis following angioplasty or transplantation, are due to abnormal vascular smooth muscle growth and gene expression. The smooth muscle cells (SMC) in response to injury lose their contractile function, become highly proliferative and synthesize and secrete extracellular matrix proteins. Similar changes in the phenotypic properties of vascular SMC occur during in vitro culture. In this report, we examined whether restoration of the expression of the major receptor protein for nitric oxide (NO) signaling in smooth muscle, the guanosine 3':5' cyclic monophosphate (cGMP)-dependent protein kinase (PKG), reestablished contractile function to cultured rat aortic SMC. Contractile function was monitored using the silicone polymer wrinkle assay used previously to determine contractility in cultured mesangial cells. Noncontractile rat aortic smooth muscle cells transfected with the cDNA encoding the type I isoform of PKG, but not those transfected with empty vector, formed discreet wrinkles on the substratum in response to serum indicative of contraction. Treatment of the PKG-expressing SMC with sodium nitroprusside (SNP), an NO donor, and with cGMP analogs, or with the adenylyl cyclase activator, forskolin, and with adenosine 3':5' cyclic monophosphate (cAMP) analogs reduced wrinkling. The expression of a major PKG substrate protein involved in smooth muscle relaxation, heat shock-related protein-20 (HSP20), was also reestablished in PKG-expressing SMC. Treatment of the PKG-expressing SMC with nitroprusside resulted in phosphorylation of HSP20. Collectively, these results indicate that PKG expression is important to establish contractility to SMC in culture.     

Cyclic nucleotide metabolism in compensatory renal hypertrophy and neonatal kidney growth.

Cyclic nucleotide metabolism was investigated in growing kidneys of rats during compensatory hypertrophy and during neonatal development. After unilateral nephrectomy a mild and short-lasting decrease in cyclic 3':5" adenosine monophosphate (cAMP) was observed in the hypertrophying kidney. In contrast, cyclic 3':5' guanosine monophosphate (cGMP) showed a sharp decline to 20% of control at 15 min and a rapid rise to 200-300% above base-line at 1-72 hr. The alterations in renal tissue levels of cGMP were associated with parallel changes in the soluble, 100,000 X g supernatant guanylate cyclase activity [GTP pyrophosphate-lyase (cyclizing): EC 4.6.1.2]. No change was observed in total cGMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase; EC 3.1.4.17). In the rapidly growing kidney of newborn rats cAMP levels were 983 +/- 65 and 833 +/- 42 pmol/g of kidney at 4 and 7 days after birth, and increased to adult levels (1518 +/- 57 pmol/g) at 21 days whereas cGMP levels were 59.8 +/- 6.8 and 92.5 +/- 13.9 pmol/g at 4 and 7 days and decreased to adult levels (36 +/- 1.5) at 21 days. The results indicate that compensatory renal hypertrophy and neonatal kidney growth are associated with changes in cAMP and cGMP metabolism.     

Sodium nitroprusside inhibition of parathyroid hormone release is not mediated through cyclic GMP

Sodium nitroprusside effected a rapid, dose-dependent increase in intracellular cGMP accumulation in freshly dispersed bovine parathyroid cells. The effect was half-maximal between 10(-4) and 3 X 10(-4)M, maximal at 3 X 10(-3)M nitroprusside and could be amplified (approximately 50%) by the addition of methylisobutylxanthine (4 X 10(-4)M). The dose-response characteristics were similar to those previously described for the inhibition of cAMP accumulation and PTH release by this agent. Neither dibutyryl cGMP (10(-3)M) nor 8'-bromo-cGMP (10(-3)M) mimicked the inhibitory effect of nitroprusside on cAMP accumulation or PTH release. Dose-dependent stimulation of guanylate cyclase was found in a particulate preparation of parathyroid cells; activity was maximal at 10(-4)M nitroprusside while higher concentrations appeared to inhibit the enzyme. Nitroprusside significantly reduced both (-)isoproterenol and guanine nucleotide-stimulated adenylate cyclase activity in the particulate preparation with maximum inhibition between 10(-3)-10(-2)M. cGMP concentrations as high as 10(-4)M did not affect agonist-stimulated cAMP synthesis. Thus, although the kinetic and dose-response characteristics of the nitroprusside effect on cGMP suggest a linkage to its previously described effects on cAMP and PTH secretion, no direct evidence was found to indicate a causal relationship between the two. Rather it would appear that the effects on the adenylate and guanylate cyclase enzymes occur in parallel, possibly the result of some common primary perturbation of cellular physiology.     

Catecholamine-stimulated cyclic GMP accumulation in the rat pineal: apparent presynaptic site of action.

Guanosine 3':5'-cyclic monophosphate (cGMP) increased 7-fold in rat pineal glands incubated in the presence of l-norepinephrine. This response consisted of two components-one was stereospecific and inhibited by alpha-adrenergic antagonists while the other was not stereospecific and not readily inhibited by antagonists. Although l-isoproterenol was more potent than l-norepinephrine it had less intrinsic activity and its action was not stereospecifc. The increase in cGMP caused by these catecholamines, unlike that of adenosine 3':5'-cyclic monophosphate (cAMP), was dependent upon extracellular calcium. Ouabain and high levels of potassium produced a marked, calcium-dependent increase in pineal cGMP, without affecting cAMP. There was no effect of cholinergic agonists on cGMP. Surgical denervation markedly reduced the cGMP response to stimulation by l-norepinephrine, potassium, or ouabain. This was in contrast to the enhanced response of cAMP in denervated glands. The nonspecific increase in cGMP caused by l-isoproterenol, however, was not affected by denervation. These data demonstrate the existence of a calcium-dependent presynaptic mechanism for the generation of cGMP which may be mediated by an alpha-adrenergic-like receptor. In addition, the mechanisms regulating pineal cGMP appear to be physiologically distinct from those regulating cAMP.     

Epinephrine-induced elevation of guanosine 3′:5′-cyclic monophosphate in isolated fat cells of rat

The effects of epinephrine (as low as 0.1 muM) on guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) in isolated fat cells were examined. Epinephrine increased both cGMP and cAMP levels, with the elevation of cAMP preceding the rise of cGMP. Maximal elevation was obtained with 1 muM epinephrine for each nucleotide. The increase in content of cGMP and cAMP due to epinephrine was completely blocked by a beta-adrenergic antagonist (5 muM propranolol). Phentolamine (10-100 muM), an alpha-adrenergic antagonist, enhanced the response to epinephrine resulting in elevation of cAMP levels, whereas a high concentration (100 muM) of phentolamine suppressed the elevation of cGMP. The ability of epinephrine to increase cGMP and cAMP levels was markedly diminished by "feedback regulator" partially purified from the incubation mixtures of isolated fat cells exposed to epinephrine [Ho, R.J. & Sutherland, E. W. (1971) J. Biol. Chem. 246, 6822-6827], whereas an increase in cGMP, but not cAMP, levels was observed in isolated fat cells incubated with "feedback regulator" alone (without epinephrine). These observations suggest the possibility that the epinephrine-induced elevation of cGMP levels in isolated fat cells might be mediated by an increase in formation of intracellular "feedback regulator" due to an elevation of cAMP by epinephrine.     

Pulmonary hypertension alters soluble guanylate cyclase activity and expression in pulmonary arteries isolated from fetal lambs

The nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate (cGMP) signaling pathway plays an important role in the pulmonary vascular transition at birth. We studied pulmonary arteries and veins isolated from normal late-gestation fetal lambs and from fetal lambs with persistent pulmonary hypertension (PPHN) following prenatal ligation of the ductus arteriosus. We additionally used double immunolabeling and immunoblot analysis to determine relative vascular contents of endothelial nitric oxide synthase (NOS-III) and soluble guanylate cyclase (sGC). Cyclic GMP content and sGC activity were significantly lower in arteries from hypertensive lambs than controls. A rank order for contents of both soluble guanylate cyclase and NOS-III was observed by both immunolabeling and immunoblotting: Control vein = Hypertensive vein > Control artery > Hypertensive artery. Our data demonstrate that the relative expression of sGC correlates well with the relative expression of NOS-III, and indicate the potential importance of soluble guanylate cyclase in the regulation of the perinatal pulmonary circulation. These data may help us understand vascular mechanisms producing PPHN, as well as patterns of response to exogenous NO.     

YC-1 prevents sodium nitroprusside-mediated apoptosis in vascular smooth muscle cells

OBJECTIVE: Nitric oxide signaling pathways are of central importance in both the maintenance of vascular homeostasis and the progression of vascular disease. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, the authors investigated whether YC-1, a soluble guanylyl cyclase (sGC) activator, regulates apoptosis in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: Sodium nitroprusside (SNP) (1 mM) induced cGMP (guanosine 3':5'-cyclic monophosphate)-independent apoptosis in rat vascular smooth muscle cells using MTT assay and TUNEL-reaction techniques. Furthermore, sodium nitroprusside induced apoptosis via Bcl-2 down-regulation, cytochrome c release reaction, and caspase-3 activation by Western blotting analysis and enzymatic assay methods. YC-1 abolished these apoptotic signaling cascades and prevented apoptosis through a cGMP-involved pathway, and phosphatidylinositol (PI) 3-kinase behaved a downstream event in this pathway. CONCLUSIONS: These results suggest that YC-1 inhibits sodium nitroprusside-induced vascular smooth muscle cells apoptosis via a cGMP- and phosphatidylinositol 3-kinase-involved inhibition on Bcl-2 down-regulation/cytochrome c release/caspase-3 activation cascades. The ability of YC-1 to prevent smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.     

Nitric oxide/cGMP signaling inhibits TSH-stimulated iodide uptake and expression of thyroid peroxidase and thyroglobulin mRNA in FRTL-5 thyroid cells.

OBJECTIVE: Nitric oxide (NO) induces morphological and functional alterations in primary cultured thyroid cells. The aim of this paper was to analyze the direct influence of a long-term exposition to NO on parameters of thyroid hormone biosynthesis in FRTL-5 cells. DESIGN: Cells were treated with the NO donor sodium nitroprusside (SNP) for 24-72 h. MAIN OUTCOME: SNP (50-500 micromol/L) reduced iodide uptake in a concentration-dependent manner. The inhibition of iodide uptake increased progressively with time and matched nitrite accumulation. SNP inhibited thyroperoxidase (TPO) and thyroglobulin (TG) mRNA expression in a concentration-dependent manner. SNP enhanced 3',5'-cyclic guanosine monophosphate (cGMP) production. 3',5'-cyclic adenosine phosphate (cAMP) generation was reduced by a high SNP concentration after 48 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog, inhibited iodide uptake as well as TPO and TG mRNA expression. The cGMP-dependent protein kinase (cGK) inhibitor KT-5823 reversed SNP or 8-Br-cGMP-inhibited iodide uptake. Thyroid-stimulating hormone pretreatment for 24-48 h prevented SNP-reduced iodide uptake although nitrite levels remained unaffected. CONCLUSION: These findings favor a long-term inhibitory role of the NO/cGMP pathway on parameters of thyroid hormone biosynthesis. A novel property of NO to inhibit TPO and TG mRNA expression is supported. The NO action on iodide uptake could involve cGK mediation. The long-term inhibition of steps of thyroid hormonogenesis by NO could be of interest in thyroid pathophysiology.