Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

Effects of manipulating the nitric oxide/cyclic GMP pathway on bovine oocyte meiotic resumption in vitro

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture. The iNOS inhibitor, aminoguanidine (AG, 10 and 50 mM) and the NO donor sodium nitroprusside (SNP, 100 and 500 microM) significantly inhibited GVBD after 7h of culture. However, a lower concentration of SNP (0.01 microM) stimulated GVBD. The inhibitory effects of AG and SNP were reversible, indicating that they were not toxic effects. Although SNP (500 microM) increased cGMP levels in cumulus-oocyte complexes after 3 h of culture, the inhibitor of soluble guanylate cyclase ODQ and the protein kinase G (PKG) inhibitor KT5823 did not reverse the inhibitory effect of SNP on meiosis, suggesting that SNP does not inhibit meiosis through the cGMP/PKG pathway. Similarly, an analogue of cGMP (8-Bromo-cGMP 0.5, 1, 3, and 6 mM), as well as activation of guanylate cyclase with Protoporphyrin IX or atrial natriuretic peptide, or inhibition of the enzyme with ODQ, did not have any significant effect on GVBD after 7 h of culture, supporting the idea that the effects of AG and SNP were not due to altered cGMP levels. Atrial natriuretic peptide, Protoporphyrin IX and SNP 500 microM increased cGMP levels after 3 h but not 6 h of culture. In conclusion, soluble and particulate guanylate cyclases could be activated in bovine cumulus-oocyte complexes, but accumulation of cGMP was probably not responsible for the effects of NO on meiosis.     

Exogenous nitric oxide stimulates cell proliferation via activation of a mitogen-activated protein kinase pathway in ovine fetoplacental artery endothelial cells

Sodium nitroprusside (SNP), a nitric oxide (NO) donor and a nitrovasodilator drug used for patients with hypertensive crisis, has been shown to promote angiogenesis. However, direct evidence showing the involvement of NO in the SNP-induced angiogenesis is not available. Accordingly, we assessed whether NO generated from SNP-stimulated ovine fetoplacental artery endothelial (OFPAE) cell proliferation via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also termed ERK1/2). We observed that SNP dose dependently stimulated (P < 0.05) cell proliferation with a maximal effect at 1 microM and that SNP rapidly (相似文献   

Insulin and nitric oxide stimulates glucose transport in human placenta

The present work examines whether insulin and NO can act as regulators of glucose transport in placenta. Glucose uptake (2-deoxy D-[(3)H]glucose) was measured in the absence (control or basal values) and in the presence of insulin (1200 microU/ml) or SNP (20 microM) in isolated perfused cotyledons and tissue slices preparations of human placenta. Both insulin and NO significantly increased glucose uptake by 20 and 27 per cent, respectively. Insulin decreased the Km of glucose transport from 42.5 +/- 2.69 to 35.1 +/- 2.58 mM. The stimulatory effect of SNP was mimicked by 8-CPT-cGMP and was completely blocked by the guanylate cyclase inhibitor, ODQ (10 microM). ODQ and the NOS inhibitor, L-NAME (100 microM), decreased basal glucose uptake but did not affect insulin-stimulated glucose transport. Taken together, these findings indicate that insulin and NO stimulate glucose uptake in human placenta and suggest that both potential regulators of glucose transport use different signaling pathways.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by polyamines

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.     

Signal pathway responsible for hepatocyte preconditioning by nitric oxide

Nitric oxide (NO) improves liver resistance to hypoxia/reperfusion injury acting as a mediator of hepatic preconditioning. However, the mechanisms involved are still poorly understood. In this study, we have investigated the mechanisms by which short-term exposure to the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium-1,2-diolate (NOC-9) increases hepatocyte tolerance to hypoxic injury. Isolated rat hepatocytes preincubated 15 min with NOC-9 (0.250 mM) became resistant to the killing caused by hypoxia. NOC-9 cytoprotection did not involve the activation of protein kinase C, but was instead blocked by inhibiting soluble guanylate cyclase with 1H-(1,2,4)-oxadiazolo-(4,3) quinoxalin-1-one (ODQ) (50 microM) or cGMP-dependent kinase (cGK) with KT 5823 (5 microM). Conversely, cGMP analogue, 8Br-cGMP (50 microM) mimicked the effect of NOC-9. Western blot analysis revealed that hepatocyte treatment with NOC-9 or 8Br-cGMP significantly increased dual phosphorylation of p38 MAPK. The activation of p38 MAPK was abolished by inhibiting guanylate cyclase or cGK. Pretreatment with NO significantly reduced intracellular Na(+) accumulation in hypoxic hepatocytes. This effect was reverted by KT 5823 as well as by the p38 MAPK inhibitor SB203580. SB203580 also reverted NOC-9 protection against hypoxic injury. Altogether, these results demonstrated that NO can induce hepatic preconditioning by activating p38 MAPK through a guanylate cyclase/cGK-mediated pathway.     

Cyclic GMP-dependent and -independent regulation of MAP kinases by sodium nitroprusside in isolated cardiomyocytes

Sodium nitroprusside (SNP) elicits various physiological effects, in part through generation of the membrane permeable mediator nitric oxide (NO). In the heart, besides its role in regulating contractility, NO is involved in both protection from and induction of cellular damage. The present study investigated the role of SNP in the regulation of the mitogen-activated protein kinases (MAPKs) in isolated adult rat cardiomyocytes. SNP maximally activated Erk1, Erk2, p38 MAPK and MAPKAPK2 in 5-10 min. The activation of MAPKAPK2 by SNP was blocked by the soluble guanylyl cyclase inhibitor, 1H-[1, 2,4]oxadiazolol[4,3-a]quinoxalin-1-one (ODQ) and the p38 MAPK inhibitor, SB203580. The activation of Erk1 was insensitive to ODQ but completely blocked by the Mek1 inhibitor PD98059. The membrane-permeable homologue of cGMP, 8-Br-cGMP, also activated p38 MAPK (A(0.5) approximately 50 microM) but not Erk1 and Erk2. These results indicate that p38 MAPK and MAPKAPK2 are activated by SNP in cGMP-dependent pathways, while the Erk1 activation by SNP is independent of cGMP levels.     

17β-Estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells

Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17β-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17β-estradiol decreases cGMP levels in PC12 cells. 17β-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17β-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17β-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17β-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17β-estradiol inhibits soluble guanylate cyclase activity through SHP-1.     

Regulation of NHE3 by nitric oxide in Caco-2 cells

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.     

Carnosine as a regulator of soluble guanylate cyclase

The molecular mechanism of the participation of carnosine in the functioning of soluble guanylate cyclase is discussed. It is shown that carnosine inhibits the activation of soluble guanylate cyclase by sodium nitroprusside and a derivative of furoxan--1,2,5-oxadiazolo-trioxide (an NO donor). However, carnosine has no effect on stimulation of the enzyme by a structural analog of the latter compound, a furazan derivative (1,2,5-oxadiazolo-dioxide) that is not an NO donor; nor was carnosine involved in the enzyme activation by protoporphyrin IX, whose stimulatory effect is not associated with the guanylate cyclase heme. The inhibition by carnosine of guanylate cyclase activation by an NO donor is due to the interaction of carnosine with heme iron with subsequent formation of a chelate complex. It was first demonstrated that carnosine is a selective inhibitor of NO-dependent activation of guanylate cyclase and may be used for suppression of activity of the intracellular signaling system NO-soluble guanylate cyclase-cGMP, whose sharp increase is observed in malignant tumors, sepsis, septic shock, asthma, and migraine.     

Differential effects of nitric oxide on rod and cone pathways in carp retina

The effects of nitric oxide (NO) on electroretinograms and light responses of horizontal cells intracellularly recorded from isolated, superfused carp retinas were studied. Sodium nitroprusside (SNP), an NO donor, suppressed scotopic b wave, while enhancing photopic b wave, and the effects could be blocked by hemoglobin, an NO chelator. Furthermore, following SNP application, light responses of rod horizontal cells were reduced in size and those of cone horizontal cells were increased. These results suggest that NO suppresses the activity of rod pathway, but enhances that of cone pathway in the outer retina. Moreover, the effects of methylene blue, an inhibitor of soluble guanylate cyclase, on rod and cone horizontal cells were just opposite to those of SNP, implying that the effects of NO may be mediated by cGMP. Project supported by the “Climbing Project” of the State Commission of Science and Technology (China) and the National Natural Science Foundation of China.     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

Ginsenoside Re promotes human sperm capacitation through nitric oxide-dependent pathway

The regulation of sperm capacitation is important for successful fertilization. Ginsenosides, the biologically effective components of ginseng, have been found to enhance intracellular nitric oxide (NO) production and the latter has recently been indicated to play a significant role in modulation of sperm functions. We investigated the effect of Ginsenoside Re on human sperm capacitation in vitro and the mechanism by which the Ginsenosides play their roles. Spermatozoa were separated by Percoll and incubated with 0, 1, 10, or 100 microM of Ginsenoside Re. The percentages of spontaneous and lysophosphatidylcholine (LPC)-induced acrosome reaction (AR), as a measure of sperm capacitation, were assayed with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA). The intracellular cGMP level was measured by [(3)H] cGMP radioimmunoassay system. The results showed that the percentages of both spontaneous and LPC-induced AR and intracellular cGMP level were significantly enhanced by Ginsenoside Re with a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside Re. And pretreatment with a NOS inhibitor N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-acetyl-l-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside Re. Furthermore, the AR-inducing effect of Ginsenoside Re was significantly reduced in the presence of the soluble guanylate cyclase inhibitor LY83583 or cGMP-dependent protein kinase (PCK) inhibitor KT5823, whereas addition of the cGMP analogue 8-Br-cGMP significantly increased the AR of human spermatozoa. Data suggested that Ginsenoside Re is beneficial to sperm capacitation and AR, and that the effect is accomplished through NO/cGMP/PKG pathway.     

Effect of centrally administered nitric oxide modulators in Brewer's yeast-induced nociception in rats

Possible modulation of Brewer's yeast-induced nociception by centrally (icv) administered nitric oxide (NO) modulators, viz., NO synthase (NOS) inhibitors, NO precursor, donors, scavengers and co-administration of NO donor (SIN-1) with NOS inhibitor (L-NAME) and NO scavenger (Hb) was investigated in rats. Administration of NOS inhibitors and NO scavenger Hb increased the pain threshold capacity significantly, whereas NO donors SIN-1, SNP and NO precursor L-arginine were found to be hyperalgesic. D-arginine, the inactive isomer of L-arginine and methylene blue, inhibitor of soluble guanylate cyclase failed to alter the nociceptive behaviour in rats. Co-administration of SIN-1 with L-NAME and Hb found to increase the nociceptive threshold. The results indicate, that centrally administered NO modulators alter the nociceptive transmission induced by Brewer's yeast in rats.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms

Inhibition of platelet activation by nitric oxide (NO) is not exclusively cGMP-dependent. Here, we tested whether inhibition of platelet aggregation by structurally distinct NO donors is mediated by different mechanisms, partly determined by the site of NO release. Glyceryl trinitrate (GTN), sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), diethylamine diazeniumdiolate (DEA/NO), and a novel S-nitrosothiol, RIG200, were examined in ADP (8 microM)- and collagen (2.5 microgram/ml)-activated human platelet rich plasma. GTN was a poor inhibitor of aggregation whilst the other NO donors inhibited aggregation, irrespective of agonist. These effects were abolished by the NO scavenger, hemoglobin (Hb; 10 microM, P < 0.05, n = 6), except with high concentrations of DEA/NO, when NO concentrations exceeded the capacity of Hb. However, experiments with the soluble guanylate cyclase inhibitor, ODQ (100 microM), indicated that only SNP-mediated inhibition was exclusively cGMP-dependent. Furthermore, the cGMP-independent effects of S-nitrosothiols were distinct from those of DEA/NO, suggesting that different NO-related mediators (e.g., nitrosonium and peroxynitrite, respectively) are responsible for their actions.     

Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase.

The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized. Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner. S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration. Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO. In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively. GSNO did not cause cell death. Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect. LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2. GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2. Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect. In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS. Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.     

Inositol uptake in rat aorta.

The purpose of this study was to investigate the mechanism of inositol uptake into rat thoracic aorta. 3H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na(+)-dependent, and a nonsaturable, Na(+)-independent component. The Na(+)-dependent component of inositol uptake had a Km of 50 microM and a Vmax of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca2(+)-free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, an activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake into both the endothelial and smooth muscle cells. These results suggest that the uptake of inositol into vascular smooth muscle is: (1) dependent upon an inward Na(+)-gradient; (2) carrier mediated, and (3) inhibited by alpha 1 adrenoceptor agonists.