Reduction in soluble guanylyl cyclase-specific activity following prolonged treatment of porcine pulmonary artery with nitric oxide

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (-log EC(50) 6.55 +/- 0.12 and 5.02 +/- 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCalpha(1) and sGCbeta(1). sGCbeta(1) protein expression was 42 +/- 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCbeta(1) protein expression (36 +/- 3 and 31 +/- 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)(i)] was significantly increased, and NO-induced (cGMP)(i) was significantly decreased by 24-h DETA-NO treatment. (cGMP)(i) normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.     

Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.     

氯化钠对培养的大鼠主动脉平滑肌细胞的心房钠尿肽受体的调节

培养的卒中型自发性高血压大鼠(SHR_(sp))及其对照 WKY 大鼠主动脉平滑肌细胞(VSMC)上存在心房钠尿肽(ANP)的特异性受体,它们与~(125)I-ANP 的最大结合量(B_(max))是:SHR_(sp)3.65±0.13和 WKY 1.89±0.09 pmol/mg pr(P<0.01);解离平衡常数(Kd)值分别是72.6±10.2和42.0±4.8×10~(-12)mol/L(P<0.01)。 两种细胞内介导舒血管作用的第二信使、环磷酸乌苷(cGMP)的基础浓度无显著差异,对相同剂量 ANP 刺激引起 cGMP 分别增加139(SHRsp)和271(WKY)倍。可见 SHRsp 的 VSMC ANP 受体数量虽比 WKY大鼠增多,但对相同剂量 ANP 引起的 cGMP 增加反应及 ANP 受体的亲和力均显著降低。高盐培养液孵育24h 后,细胞表面 ANP 受体的亲和力改变不明显,但受体数量下调,SHRsp 和 WKY 大鼠分别降至对照的34.8±8.2%和38.6±9.4%,细胞对 ANP 引起的 cGMP增加反应明显降低,且均以 SHR_(sp)较显著。提示后两种变化可能在高盐促进血压升高的机制中起作用。     

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.     

Functional characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.     

Cyclic guanosine monophosphate-dependent protein kinase I promotes adhesion of primary vascular smooth muscle cells

The cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates many cellular functions. The current study shows that 8-Br-cGMP stimulates the number of attached primary but not that of subcultured murine vascular smooth muscle cells (VSMCs). These effects of 8-Br-cGMP require the presence of cGKI. In agreement with previous studies, cGKI inhibited the number of cells in repeatedly passaged murine VSMCs. Activation of the cGMP/cGKI pathway in freshly isolated primary VSMCs slightly decreased apoptosis and strongly increased cell adhesion. The stimulation of cell adhesion by cGKI involves an inhibition of the RhoA/Rho kinase pathway and increased exposure of β₁ and β₃ integrins on the cell surface. Together, these results identify a novel proadhesive function of cGMP/cGKI signaling in primary VSMCs and suggest that the opposing effects of this pathway on VSMC number depend on the phenotypic context of the cells.     

Increased urinary excretion of uroguanylin in patients with congestive heart failure

Uroguanylin is a small-molecular-weight peptide that activates membrane-bound receptor-guanylate cyclases in the intestine, kidney, and other epithelia. Uroguanylin has been shown to participate in the regulation of salt and water homeostasis in mammals via cGMP-mediated processes, bearing a distinct similarity to the action of the atriopeptins, which play a defined role in natriuresis and act as prognostic indicators of severe congestive heart failure (CHF). The objectives of this study were to measure the urinary levels of uroguanylin and the circulating plasma levels of atrial natriuretic peptide (ANP) in healthy individuals (n = 53) and patients with CHF (n = 16). Urinary excretion of uroguanylin was assessed by a cGMP accumulation bioassay employing human T84 intestinal cells. In individuals without CHF, the concentration of uroguanylin bioactivity was 1.31 +/- 0.27 nmol cGMP/ml urine and 1.73 +/- 0.25 micromol cGMP/24-h urine collection. The urinary bioactivity of uroguanylin in males (1.74 +/- 0.55 nmol cGMP/ml urine; n = 27) tended to be higher than the excretion levels in females (0.94 +/- 0.16 nmol cGMP/ml urine; n = 26) over a 24-h period but did not achieve statistical significance. Both male and female groups showed 24-h temporal diurnal variations with the highest uroguanylin levels observed between the hours of 8:00 AM and 2:00 PM. The circulating level of ANP was 12.1 +/- 1.6 pg/ml plasma and did not significantly vary with respect to male/female population or diurnal variation. In patients with CHF, the concentration of plasma ANP and urinary uroguanylin bioactivity increased substantially (7.5-fold and 70-fold, respectively, both P 相似文献   

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.     

Cyclic GMP down-regulates atrial natriuretic peptide receptors on cultured vascular endothelial cells.

Down-regulation of atrial natriuretic peptide (ANP) receptors was investigated using a cultured bovine pulmonary artery endothelial (CPAE) cell line. Endothelial cells have been shown to possess two subtypes of ANP receptors, a guanylate cyclase-coupled receptor (B-receptor) and a clearance receptor (C-receptor). The treatment with APIII, rat ANP (103-126), at concentrations of 10(-8) to 10(-6) M for 24 h, resulted in a significantly (p less than 0.01) greater decrease in maximum 125I-APIII binding to CPAE cells than the identical concentration of API, rat ANP (103-123). APIII at concentrations of 10(-8) to 10(-6) M stimulated cyclic GMP (cGMP) production 3.3-17.5-fold greater than similar concentrations of API. From these findings, we hypothesized that cGMP produced following ANP binding to the B-receptor participates in ANP receptor regulation. M&B 22948, a selective inhibitor of cGMP-specific phosphodiesterase, significantly (p less than 0.01) potentiated the effect of both API and APIII on 125I-APIII binding, while M&B 22948 itself had no significant effect on 125I-APIII binding. Treatment of the cells with 1 mM 8-bromo-cGMP also significantly (p less than 0.01) decreased 125I-APIII binding to the cells, and a potentiation of this effect was observed by M&B 22948. Scatchard analysis of binding data from 8-bromo-cGMP-treated cells showed a significant decrease in Bmax (1.79 +/- 0.15 to 1.20 +/- 0.07 fmol/mg protein, p less than 0.05) without a significant change in Kd. Affinity cross-linking of 125I-APIII to 8-bromo-cGMP-treated cells showed a decrease in the labeling of 60- and 70-kDa bands corresponding to the C-receptor. In addition, the APIII-stimulated cGMP response remained unchanged in the 8-bromo-cGMP-treated cells, indicating that the B-receptor was not down-regulated. We conclude that cGMP regulates ANP-binding sites on the endothelial cell and that the evidence indicates that the C-receptor may preferentially be down-regulated by cGMP in CPAE cells.     

Hormonal, renal, hemodynamic responses to acute neutral endopeptidase inhibition in heart transplant patients.

We investigated the hemodynamic, renal, and hormonal responses to neutral endopeptidase (NEP) inhibition during a 6-h, double-blind, randomized, placebo-controlled study in seven chronic, stable heart transplant patients. Baseline characteristics were similar during both experiments, and no significant changes were observed after placebo. NEP inhibition increased circulating endothelin-1 (from 2.01 +/- 0.1 to 2.90 +/- 0.2 pmol/l; P < 0.01), atrial natriuretic peptide (ANP; from 21.5 +/- 2.7 to 29.6 +/- 3.7 pmol/l; P < 0.01), and the ANP second messenger cGMP. Noteworthy, systemic blood pressure did not increase. Renal plasma flow and glomerular filtration rate remained unmodified after NEP inhibition. Filtration fraction (33 +/- 13%), diuresis (196 +/- 62%), and natriuresis (315 +/- 105%) increased significantly in relation to ANP and cGMP. A strong inverse relationship was observed between excreted cGMP and sodium reabsorption (r = -0.71, P < 0.0001). Thus, despite significantly increasing endothelin-1, NEP inhibition did not adversely influence systemic or renal hemodynamics in transplant patients. ANP, possibly through a tubular action, enhances the natriuresis observed after NEP inhibition.     

Atrial natriuretic peptide reduces cyclosporin toxicity in renal cells: role of cGMP and heme oxygenase-1.

Using cultured proximal renal tubular epithelial cells (LLC-PK1), the present study investigates the effect of atrial natriuretic peptide (ANP) on cytotoxicity induced by cyclosporin A (CsA). Preincubation with ANP (1-100 nM) protected LLC-PK1 cells from CsA-induced toxicity in a concentration-dependent manner. A cytoprotective effect comparable to ANP was observed when preincubating the cells with 8-bromo cGMP (1-100 microM) or the antioxidant heme oxygenase (HO) metabolite bilirubin (0.1-10 microM). ANP or cGMP produced increases in HO-1 protein levels at concentrations that were also effective in cellular protection. Moreover, incubation with ANP or 8-bromo cGMP led to increased HO activity, i.e., formation of bilirubin in the cell lysate (up to 3-fold over basal). Tin protoporphyrin-IX (SnPP; 19 microM), an inhibitor of HO activity, completely abolished ANP-induced cytoprotection. Our results demonstrate that HO-1 is a cellular target of ANP and cGMP in renal cells. HO-1 induction and ensuing formation of antioxidant metabolites may be a novel pathway by which ANP protects from CsA-dependent nephrotoxicity and preserves renal function.     

Atrial natriuretic peptide inhibits iodide uptake and thyroglobulin messenger ribonucleic acid expression in cultured bovine thyroid follicles

The involvement of atrial natriuretic peptide (ANP) in the regulation of thyroid gland is supported by the presence of high-affinity ANP receptors and the identification of the peptide in thyroid follicular cells. The aim of this work was to study the action of ANP on parameters of thyroid hormone biosynthesis and analyze the intracellular mechanism of the ANP action in cultured bovine thyroid follicles. The addition of ANP (0.1-10 nM) to the culture medium for 24 h inhibited the TSH (thyroid-stimulating hormone)-stimulated iodide uptake with a maximal inhibition at 1 nM ANP. When thyrocytes were incubated with 10 nM ANP the inhibitory effect slightly increased from 24 to 72 h. Thyroglobulin (Tg) mRNA expression was reduced by 1 and 10 nM ANP. After 24 h of treatment with the cGMP analogue, N(2),2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate [(Bu)(2)cGMP] (0.1 and 1 mM), an inhibition of iodide uptake and Tg mRNA expression was obtained, evidencing a cGMP-mediated inhibitory signal in the thyroid cell. A reduction of the cAMP production was induced by incubation of thyroid follicles with 1 and 10 nM ANP for 24 h. Under a similar treatment the cGMP accumulation was increased only by 10 nM ANP. The inhibitory effect of ANP on Tg mRNA level was reverted in the presence of pertussis toxin, an inhibitor of the G(i)-protein-mediated reduction of the adenylate cyclase activity. These results indicate an inhibitory action of ANP on parameters of thyroid hormone biosynthesis. A G(i)-protein-mediated reduction of the cAMP production seems to be the main factor involved in the ANP action although a role of the cGMP pathway should not be discarded specially at high ANP levels.     

Effect of atrial natriuretic peptide on adrenal renin and aldosterone

The effect of atrial natriuretic peptide (ANP) on adrenal renin and aldosterone was investigated in anesthetized rats. Under pentobarbital anesthesia 40 mg/kg), intravenous infusion of ANP (0.25 micrograms/kg/min) for 45 min failed to alter the adrenal renin, adrenal aldosterone, and plasma aldosterone (PA). In this condition, intraperitoneal injection of ACTH (10 micrograms/kg) significantly increased the adrenal renin (from 2.4 +/- 0.1 to 5.0 +/- 0.08 ng/mg protein/h, P less than 0.05), adrenal aldosterone (from 13.6 +/- 1.3 to 22.7 +/- 2.3 ng/mg protein, P less than 0.01) and PA (from 59.8 +/- 5.8 to 75.5 +/- 7.4 ng/dl, P less than 0.05), respectively. Under ACTH stimulation, ANP infusion induced significant decreases in adrenal renin (from 5.0 +/- 0.08 to 2.8 +/- 0.2 ng/mg protein/h, P less than 0.05), adrenal aldosterone (from 22.7 +/- 2.3 to 16.2 +/- 1.8 ng/mg protein, P less than 0.05) and PA (from 75.5 +/- 7.4 to 61.6 +/- 4.9 ng/dl). These results suggest a possible role for adrenal renin in the mechanism underlying the inhibitory effect of ANP on aldosterone production in vivo.     

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 μM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 μM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.     

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO-mediated pathways. Fluorescently labeled mitochondria were studied using time-lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re-oxygenation or to diethyleneamine/nitric oxide adduct, DETA-NO (100-500 microm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 +/- 1.3% at 0 h and 7.4 +/- 1.7% at 1 h of re-oxygenation, versus 25.6 +/- 1.4% in controls (p < 0.05). Mean mitochondrial velocity (microm s(-1)) decreased from 0.374 +/- 0.01 in controls to 0.146 +/- 0.01 at 0 h and 0.177 +/- 0.02 at 1 h of re-oxygenation (p < 0.001). Exposure to DETA-NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG-nitro-L-arginine methyl ester (L-NAME) prevented the hypoxia-induced decrease in mitochondrial movement at 0 h (30.1 +/- 1.6%) and at 1 h (26.1 +/- 9%) of re-oxygenation. Exposure to either hypoxia/re-oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO-mediated pathway is at least partially involved.     

Biphasic 24-Hour Variations in Cyclic GMP Accumulation in the Rat Pineal Gland Are Due to Corresponding Changes in the Activity of Cytosolic and Particulate Guanylate Cyclase

Various parameters of the rat pineal gland display a 24-h rhythm. However, nothing is known about possible 24-h variations in cyclic GMP (cGMP) metabolism. In the present study, 24-h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate-saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24-h variations with two peaks--one approximately 7 h after lights "on" and the other approximately 7 h after lights "off." The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine, indicating that 24-h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.     

Lymphocyte cytochrome c oxidase, cyclic GMP and cholinergic muscarinic receptors as peripheral indicators of carbon monoxide neurotoxicity after acute and repeated exposure in the rat

Changes in cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) have been reported in rodents acutely exposed to carbon monoxide (CO). These endpoints measurable in lymphocytes may serve as peripheral markers of CO neurotoxicity. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was similarly affected both centrally and peripherally. Male Wistar rats either inhaled 500 ppm CO, 6 h/day, 5 days/week, 4 weeks (repeated exposure) or 2,400 ppm, 1 h (single exposure). Neither treatment altered brain or lymphocyte COX activity 1 and 7 days post-treatment. Also ineffective were repeated and acute CO treatments towards (3)H-quinuclidinyl benzilate (QNB) binding to MRs in cerebral cortex, hippocampus, striatum, cerebellum (respective controls, mean+/-S.D.: 171 +/- 45, 245 +/- 53, 263 +/- 14 and 77 +/- 7 fmol/mg protein) and lymphocytes (24 +/- 10 fmol/million cells) at the same time points. In lymphocytes control cGMP levels averaged 1.98 +/- 0.99 pmol/mg protein under basal conditions, and 3.94 +/- 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the CO-treated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after, compared to air-exposed rats. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NO-cGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain [Hernández-Viadel, M., Castoldi, A.F., Coccini, T., Manzo, L., Erceg, S., Felipo, V., 2004. In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum. Journal of Neurochemistry 89, 1,157-1,165], and supports the use of this peripheral endpoint as a biomarker of CO central effects.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.     

Reduced natriuresis after oral sodium load in cholestatic rats: role of compartment volumes and ANP

The purpose of this study was to assess the participation of the atrial natriuretic peptide (ANP)-cGMP system in electrolyte and volume handling of cholestatic rats submitted to an acute oral sodium load. Cholestasis was induced by ligation and section of the common bile duct (n = 51). Control rats were sham operated (n = 56). Three weeks after surgery, 24-hr urinary volume, sodium, potassium, cGMP and creatinine excretion were measured. Three days later, animals received 10 mmol/kg NaCl (1 M) by gavage, and urinary excretion was measured for 6 hr. In parallel groups of rats, plasma volume, electrolytes and ANP concentration, extracellular fluid volume (ECFV), and renal medullary ANP-induced cGMP production were determined in basal conditions or 1 hr after oral sodium overload. As compared with controls, cholestatic rats had a larger ECFV and higher plasma ANP (67.2 +/- 5.2 vs 39.7 +/- 3.5 pg/ml), but lower hematocrit and blood volume, and were hyponatremic. Cholestatic rats showed higher basal excretion of sodium, potassium, and volume than controls, but equal urinary cGMP. After the NaCl overload, cholestatic rats showed a reduced sodium excretion but equal urinary cGMP. One hr after sodium overload, both groups showed hypernatremia, but whereas in control rats ECFV and ANP increased (50.7 +/- 4.1 pg/ml), in cholestatic rats ECFV was unchanged, and plasma volume and ANP were reduced (37.5 +/- 5.8 pg/ml). ANP-induced cGMP production in renal medulla was similar in cholestatic and control nonloaded rats (14.2 +/- 5.2 vs 13.4 +/- 2.6 fmol/min/mg). One hr after the load, medullary cGMP production rose significantly in both groups, without difference between them (20.6 +/- 3.1 vs 22.7 +/- 1. 7 fmol/min/mg). We conclude that the blunted excretion of an acute oral sodium load in cholestatic rats is associated with lower plasma ANP due to differences in body fluid distribution and cannot be explained by renal refractoriness to ANP.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.