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.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.     

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.     

Activation of cerebral guanylate cyclase by nitric oxide.

Mouse cerebral guanylate cyclase was activated by catalase in the presence of sodium azide (NaN₃), which is known to form catalase-NO complex, while nitrosamines and nitric oxide (NO gas) were capable of activating cerebral guanylate cyclase in the absence of catalase. The activation of guanylate cyclase by NaN₃-catalase or nitrosamines was markedly inhibited by ferrohemoglobin which has a high affinity for NO, but not by ferrihemoglobin. These data suggest that NO or NO containing compounds may activate guanylate cyclase, whereas ferrohemoglobin may exhibit an inhibitory effect on the activation of guanylate cyclase, possibly by interacting with NO or NO containing compounds.     

Synthesis of a nitric oxide-like factor from L-arginine by rat serosal mast cells: stimulation of guanylate cyclase and inhibition of platelet aggregation

Rat serosal mast cells were tested for their ability to generate a nitric oxide-like factor by two bioassay systems: inhibition of platelet aggregation and stimulation of mast cell guanylate cyclase. Incubation of rat serosal mast cells with human washed platelets resulted in an inhibition of thrombin-induced platelet aggregation proportional to the number of cells. The inhibition was potentiated by superoxide dismutase (SOD) and reversed by oxyhaemoglobin (oxyHb). The inhibitory activity of mast cells was also prevented by NG-monomethyl-L-arginine (MeArg), an effect reversed by co-incubation with L-Arg but not D-Arg. When mast cells alone were stirred at 1,000 rpm, a time-dependent increase in the levels of their cGMP but not cAMP was observed. This increase was reduced by pretreatment with MeArg. The inhibitory effect of MeArg was reversed by L-Arg but not D-Arg. These results demonstrate that rat mast cells release a factor with the same pharmacological profile as NO, and that this NO-like factor is derived from L-arginine.     

Suppression of Proliferation of Human Coronary Artery Smooth Muscle Cells by the Nitric Oxide Donor,S-Nitrosoglutathione,Is cGMP-Independent

Nitric oxide (NO), delivered by a single addition of S-nitrosoglutathione (GSNO, IC₅₀ = 60–75 μM), causes the prolonged, multi-day suppression of proliferation of asynchronous, logarithmically growing human (hCASMC, two cell strains), and porcine (porCASMC) coronary artery smooth muscle cells. The inhibition is not cytotoxic, but cytostatic and reversible. Transient exposure (>4–12 h) to GSNO is sufficient to elicit prolonged suppression, but a less than 4 h exposure produces little or no inhibition. Unlike porCASMC and rat and rabbit aortic SMC, hCASMC synthesize little cGMP in response to GSNO stimulation, suggesting loss of NO responsive guanylate cyclase in vitro. The guanylate cyclase inhibitor, ODQ, blocks the slight cGMP synthesis induced by GSNO in hCASMC, but does not prevent GSNO suppression of proliferation. These data support a cGMP independent mechanism for NO induced suppression of hCASMC proliferation which may be significant in the treatment of proliferative coronary artery diseases.     

Effects of combined inhibition of ATP-sensitive potassium channels, nitric oxide, and prostaglandins on hyperemia during moderate exercise.

ATP-sensitive potassium (KATP) channels have been suggested to contribute to coronary and skeletal muscle vasodilation during exercise, either alone or interacting in a parallel or redundant process with nitric oxide (NO), prostaglandins (PGs), and adenosine. We tested the hypothesis that KATP channels, alone or in combination with NO and PGs, regulate exercise hyperemia in forearm muscle. Eighteen healthy young adults performed 20 min of moderate dynamic forearm exercise, with forearm blood flow (FBF) measured via Doppler ultrasound. After steady-state FBF was achieved for 5 min (saline control), the KATP inhibitor glibenclamide (Glib) was infused into the brachial artery for 5 min (10 microg.dl(-1).min(-1)), followed by saline infusion during the final 10 min of exercise (n = 9). Exercise increased FBF from 71 +/- 11 to 239 +/- 24 ml/min, and FBF was not altered by 5 min of Glib. Systemic plasma Glib levels were above the therapeutic range, and Glib increased insulin levels by approximately 50%, whereas blood glucose was unchanged (88 +/- 2 vs. 90 +/- 2 mg/dl). In nine additional subjects, Glib was followed by combined infusion of NG-nitro-L-arginine methyl ester (L-NAME) plus ketorolac (to inhibit NO and PGs, respectively). As above, Glib had no effect on FBF but addition of L-NAME + ketorolac (i.e., triple blockade) reduced FBF by approximately 15% below steady-state exercise levels in seven of nine subjects. Interestingly, triple blockade in two subjects caused FBF to transiently and dramatically decrease. This was followed by an acute recovery of flow above steady-state exercise values. We conclude 1) opening of KATP channels is not obligatory for forearm exercise hyperemia, and 2) triple blockade of NO, PGs, and KATP channels does not reduce hyperemia more than the inhibition of NO and PGs in most subjects. However, some subjects are sensitive to triple blockade, but they are able to restore FBF acutely during exercise. Future studies are required to determine the nature of these compensatory mechanisms in the affected individuals.     

Nitric Oxide Coordinates Cell Proliferation and Cell Movements During Early Development of Xenopus

The establishment of a vertebrate body plan during embryogenesis is achieved through precise coordination of cell proliferation and morphogenetic cell movements. Here we show that nitric oxide (NO) suppresses cell division and facilitates cell movements during early development of Xenopus, such that inhibition of NO synthase (NOS) increases proliferation in the neuroectoderm and suppresses convergent extension in the axial mesoderm and neuroectoderm. NO controls cell division and cell movement through two separate signaling pathways. Both rely on RhoA-ROCK signaling but can be distinguished by the involvement of either guanylate cyclase or the planar cell polarity regulator Dishevelled. Through the cGMP-dependent pathway, NO suppresses cell division by negatively regulating RhoA and controlling the nuclear distribution of ROCK and p21WAF1. Through the cGMP-independent pathway, NO facilitates cell movement by regulating the intracellular distribution and level of Dishevelled and the activity of RhoA, thereby controlling the activity of ROCK and regulating actin cytoskeleton remodeling and cell polarization. Concurrent control by NO helps ensure that the crucial processes of cell proliferation and morphogenetic movements are coordinated during early development.     

C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations

C-reactive protein (CRP), an acute-phase protein and newly recognized indicator of cardiovascular risk, may have direct actions on the vascular wall. Previous studies suggest that CRP is a vasodilator that activates smooth muscle K(+) channels. We examined the reported vasoactive properties of CRP and further explored its mechanisms of action. CRP decreased blood pressure in rats and increased coronary flow in open-chest dogs at a constant coronary perfusion pressure. CRP relaxed rat aortic rings and mesenteric small arteries that were contracted with phenylephrine. Relaxation was not affected by endothelial denudation or inhibition of nitric oxide (NO) synthase but was blocked by inhibition of soluble guanylate cyclase or K(+) channels. CRP solutions remained effective, i.e., elicited vasodilation, even after boiling or enzymatic digestion, which suggests the presence of a nonprotein contaminant. Sodium azide (NaN(3), 0.1%) is the preservative used for commercially available CRP and a potential source of NO. NaN(3) elicited the same cardiovascular effects as CRP preparations at equal concentrations, and its actions were blocked by inhibition of guanylate cyclase and K(+) channels. NaN(3)-free CRP, prepared by gel-filtration centrifugation and confirmed by electrophoresis, had no effect on vascular tone. Inhibition of vascular smooth muscle catalase with 3-amino-1,2,4-triazole completely prevented the effects of NaN(3) and NaN(3)-containing CRP solutions. We demonstrate that the acute vasoactive properties of commercially available CRP preparations are attributable to NaN(3) (and subsequent production of NO by catalase); therefore, this study suggests a reappraisal of the acute role of CRP in regulating vascular tone.     

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of I(to) and augmentation of K(ATP) channels

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.     

The influence of 8-prenylnaringenin on the activity of voltage-gated kv1.3 potassium channels in human jurkat t cells

Using the whole-cell patch-clamp technique, we investigated the influence of 8-prenylnaringenin on the activity of the voltage-gated Kv1.3 potassium channels in the human leukemic T lymphocyte cell line Jurkat. 8-prenylnaringenin is a potent plant-derived phytoestrogen that has been found to inhibit cancer cell proliferation. The results show that it inhibited the Kv1.3 channels in a concentration-dependent manner. Complete inhibition occurred at concentrations higher than 10 ??M. The inhibitory effect of 8-prenylnaringenin was reversible. It was accompanied by a significant acceleration of channel inactivation without any pronounced change in the activation rate. Of the naringenin derivatives tested to date, 8-prenylnaringenin is the most potent inhibitor of the Kv1.3 channels. The potency of the inhibition may be due to the presence of a prenyl group in the molecule of this flavonoid. The inhibition of the Kv1.3 channels might be involved in the antiproliferative and pro-apoptotic effects of 8-prenylnaringenin that have been observed in cancer cell lines expressing these channels.     

Nitric oxide and cardiac function

Nitric oxide (NO) participates in the control of contractility and heart rate, limits cardiac remodeling after an infarction and contributes to the protective effect of ischemic pre- and postconditioning. Low concentrations of NO, with production of small amounts of cGMP, inhibit phosphodiesterase III, thus preventing the hydrolysis of cAMP. The subsequent activation of a protein-kinase A causes the opening of sarcolemmal voltage-operated and sarcoplasmic ryanodin receptor Ca(2+) channels, thus increasing myocardial contractility. High concentrations of NO induce the production of larger amounts of cGMP which are responsible for a cardiodepression in response to an activation of protein kinase G (PKG) with blockade of sarcolemmal Ca(2+) channels. NO is also involved in reduced contractile response to adrenergic stimulation in heart failure. A reduction of heart rate is an evident effect of NO-synthase (NOS) inhibition. It is noteworthy that the direct effect of NOS inhibition can be altered if baroreceptors are stimulated by increases in blood pressure. Finally, NO can limit the deleterious effects of cardiac remodeling after myocardial infarction possibly via the cGMP pathway. The protective effect of NO is mainly mediated by the guanylyl cyclase-cGMP pathway resulting in activation of PKG with opening of mitochondrial ATP-sensitive potassium channels and inhibition of the mitochondrial permeability transition pores. NO acting on heart is produced by vascular and endocardial endothelial NOS, as well as neuronal and inducible synthases. In particular, while in the basal control of contractility, endothelial synthase has a predominant role, the inducible isoform is mainly responsible for the cardiodepression in septic shock.     

Endothelial cell nitric oxide inhibits aldosterone synthesis in zona glomerulosa cells: modulation by oxygen

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [(3)H]L-arginine to [(3)H]L-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.     

Mechanisms of acetylcholine-mediated vasodilation in systemic arteries from mourning doves (Zenaida macroura)

For mammals, acetylcholine (ACh) promotes endothelium-dependent vasodilation primarily through nitric oxide (NO) and prostaglandin-mediated pathways, with varying reliance on endothelial-derived hyperpolarizing factors. Currently, no studies have been conducted on small systemic arteries from wild birds. We hypothesized that ACh-mediated vasodilation of isolated small arteries from mourning doves (Zenaida macroura) would likewise depend on endothelial-derived factors. Small resistance mesenteric and cranial tibial (c. tibial) arteries (80–150 μm, inner diameter) were cannulated and pre-constricted to 50 % of resting inner diameter with phenylephrine then exposed to increasing concentrations of ACh (10^?9–10^?5 M) or the NO donor, sodium nitroprusside (SNP; 10^?12–10^?3 M). For mesenteric arteries, ACh-mediated vasodilation was significantly blunted with the potassium channel antagonist tetraethylammonium chloride (TEA, 10 mM); whereas responses were only moderately impaired with endothelial disruption or inhibition of prostaglandins (indomethacin, 10 μM). In contrast, endothelial disruption as well as exposure to TEA largely abolished vasodilatory responses to ACh in c. tibial arteries while no effect of prostaglandin inhibition was observed. For both vascular beds, responses to ACh were moderately dependent on the NO signaling pathway. Inhibition of NO synthase had no impact, despite complete reversal of phenylephrine-mediated tone with SNP, whereas inhibition of soluble guanylate cyclase (sGC) caused minor impairments. Endothelium-independent vasodilation also relied on potassium channels. In summary, ACh-mediated vasodilation of mesenteric and c. tibial arteries occurs through the activation of potassium channels to induce hyperpolarization with moderate reliance on sGC. Prostaglandins likewise play a small role in the vasodilatory response to ACh in mesenteric arteries.     

Nitric oxide deficit in chronic intermittent hypoxia impairs large conductance calcium-activated potassium channel activity in rat hippocampal neurons

Sleep apnea associated with chronic intermittent hypoxia (IH) impairs hippocampal functions but the pathogenic mechanisms involving dysfunction of nitric oxide (NO) and ionic channels remain unclear. We examined the hypothesis that hippocampal NO deficit impairs the activity of large conductance calcium-activated potassium (BK) channels in rats with chronic IH, mimicking conditions in patients with sleep apnea. A patch-clamp study was performed on hippocampal CA1 neurons acutely dissociated from IH and control rats. The levels of endogenous NO and intracellular calcium in the CA1 region of the hippocampal slices were measured respectively by electrochemical microsensors and spectrofluorometry. We found that the open probability of BK channels remarkably decreased in the CA1 pyramidal neurons in a time-dependent manner with the IH treatment, without changes in the unitary conductance and reversal potential. NO donors, SNP or DETA/NO, significantly restored the activity of BK channels in the IH neurons, which was prevented by blockade of S-nitrosylation with NEM or MTSES but not by inhibition of the cGMP pathway with ODQ or 8-bromo-cGMP. Endogenous NO levels were substantially lowered in the IH hippocampus during resting and hypoxia. Also, the level of protein expression of neuronal NO synthase was markedly lessened in the IH neurons with decreased intracellular calcium response to hypoxia. Collectively, the results suggest that the IH-induced NO deficit mediated by a down-regulation of the expression of neuronal NO synthase plays a causative role in the impaired activity of BK channels, which could account for the hippocampal injury in patients with sleep apnea.     

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.