The mechanism of EDHF-mediated responses in subcutaneous small arteries from healthy pregnant women

We studied the importance of endothelium-derived hyperpolarizing factor (EDHF) vs. nitric oxide (NO) and prostacyclin (PGI(2)) in bradykinin (BK)-induced relaxation in isolated small subcutaneous arteries from normal pregnant women. We also explored the contribution of cytochrome P-450 (CYP450) product of arachidonic acid (AA) metabolism, hydrogen peroxide (H(2)O(2)), and gap junctions that have been suggested to be involved in EDHF-mediated responses. Isolated arteries obtained from subcutaneous fat biopsies of normal pregnant women (n = 30) undergoing planned cesarean section were mounted in a wire-myography system. In norepinephrine-constricted vessels, incubation with N(G)-nitro-L-arginine methyl ester (L-NAME) resulted in a significant reduction in relaxation to BK. Simultaneous incubation with L-NAME and indomethacin failed to modify this response further. BK-mediated dilatation in the presence of K(+)-modified solution was decreased to similar level as obtained after incubation with L-NAME. Incubation with L-NAME abolished BK-induced responses in K(+)-modified solution. Sulfaphenazole, a specific inhibitor of CYP450 epoxygenase, and catalase (an enzyme that decomposes H(2)O(2)) did not affect the EDHF-mediated relaxation because concentration-response curves to BK were similar in arteries after incubation with L-NAME vs. L-NAME + sulfaphenazole and L-NAME + catalase. The inhibitor of gap junctions, 18 alpha-glycyrrhetinic acid, significantly reduced BK-mediated relaxation both without and with incubation with L-NAME. We found that both NO and EDHF, but not PGI(2), are involved in the endothelium-dependent dilatation to BK. BK-induced relaxation is almost equally mediated by NO and EDHF. CYP450 epoxygenase metabolites of AA or H(2)O(2) do not account for EDHF-mediated response; however, gap junctions are involved in the EDHF-mediated responses to BK in subcutaneous small arteries in normal pregnancy.     

In vivo mechanisms of acetylcholine-induced vasodilation in rat sciatic nerve

We examined the importance of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and neurogenic activity in agonist-induced vasodilation and baseline blood flow [i.e., nerve microvascular conductance (NMVC)] in rat sciatic nerve using laser Doppler flowmetry. Agonists were acetylcholine (ACh) and 3-morpholinosydnonimine (SIN-1). Vasodilation occurring despite NO synthase (NOS) and cyclooxygenase inhibition and showing dependence on K(+) channel activity was taken as being mediated by EDHF. NOS and cyclooxygenase inhibition with N(omega)-nitro-L-arginine (L-NNA) + indomethacin (Indo) revealed two phases of ACh-induced vasodilation: an initial, transient L-NNA + Indo-resistant vasodilation, peaking at 23 +/- 6 s and lasting 145 +/- 69 s, followed by sustained L-NNA + Indo-sensitive vasodilation. L-NNA alone did not affect sustained ACh-induced vasodilation but decreased baseline NMVC by 55%. In the presence of L-NNA + Indo, the K(+) channel blocker tetraethylammonium (TEA) inhibited transient ACh-induced vasodilation by 58% and reduced baseline NMVC by 25%. SIN-1-induced vasodilation increased fourfold in the presence of L-NNA, whereas the specific guanylyl cyclase inhibitor 1H-(1, 2, 4)oxadiazolo(4,3-alpha)quinoxalin-1-one abolished it. However, in homogenates of rat sciatic nerve, SIN-1-stimulated soluble guanylyl cyclase (sGC) activity was unaffected by L-NNA. TTX affected neither SIN-1- nor ACh-induced vasodilation. In conclusion, ACh-induced vasodilation consisted of two components, the first partially mediated by EDHF and the second by a vasodilatory prostanoid + NO. Baseline NMVC was dependent on NO and EDHF. Although L-NNA enhanced SIN-1-induced vasodilation, it had no effect on sGC-activity.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Nitric oxide modulates the cardiovascular effects elicited by acetylcholine in the NTS of awake rats

Microinjection of acetylcholine chloride (ACh) in the nucleus of the solitary tract (NTS) of awake rats caused a transient and dose-dependent hypotension and bradycardia. Because it is known that cardiovascular reflexes are affected by nitric oxide (NO) produced in the NTS, we investigated whether these ACh-induced responses depend on NO in the NTS. Responses to ACh (500 pmol in 100 nl) were strongly reduced by ipsilateral microinjection of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 nmol in 100 nl) in the NTS: mean arterial pressure (MAP) fell by 50 +/- 5 mmHg before L-NAME to 9 +/- 4 mmHg, 10 min after L-NAME, and HR fell by 100 +/- 26 bpm before L-NAME to 20 +/- 10 bpm, 10 min after L-NAME (both P < 0.05). Microinjection of the selective inhibitor of neuronal nitric oxide synthase (nNOS), 1-(2-trifluoromethylphenyl) imidazole (TRIM; 13.3 nmol in 100 nl), in the NTS also reduced responses to ACh: MAP fell from 42 +/- 3 mmHg before TRIM to 27 +/- 6 mmHg, 10 min after TRIM (P < 0.05). TRIM also tended to reduce ACh-induced bradycardia, but this effect was not statistically significant. ACh-induced hypotension and bradycardia returned to control levels 30-45 min after NOS inhibition. Control injections with D-NAME and saline did not affect resting values or the response to ACh. In conclusion, injection of ACh into the NTS of conscious rats induces hypotension and bradycardia, and these effects may be mediated at least partly by NO produced in NTS neurons.     

Modulation by NO of acetylcholine release in the ileum of wild-type and NOS gene knockout mice

Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.     

Endothelial dysfunction and arterial pressure regulation during early diabetes in mice: roles for nitric oxide and endothelium-derived hyperpolarizing factor

We determined whether nitric oxide (NO) counters the development of hypertension at the onset of diabetes in mice, whether this is dependent on endothelial NO synthase (eNOS), and whether non-NO endothelium-dependent vasodilator mechanisms are altered in diabetes in mice. Male mice were instrumented for chronic measurement of mean arterial pressure (MAP). In wild-type mice, MAP was greater after 5 wk of N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 mg x kg(-1) x day(-1) in drinking water; 97 +/- 3 mmHg) than after vehicle treatment (88 +/- 3 mmHg). MAP was also elevated in eNOS null mice (113 +/- 4 mmHg). Seven days after streptozotocin treatment (200 mg/kg iv) MAP was further increased in L-NAME-treated mice (108 +/- 5 mmHg) but not in vehicle-treated mice (88 +/- 3 mmHg) nor eNOS null mice (104 +/- 3 mmHg). In wild-type mice, maximal vasorelaxation of mesenteric arteries to acetylcholine was not altered by chronic L-NAME or induction of diabetes but was reduced by 42 +/- 6% in L-NAME-treated diabetic mice. Furthermore, the relative roles of NO and endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced vasorelaxation were altered; the EDHF component was enhanced by L-NAME and blunted by diabetes. These data suggest that NO protects against the development of hypertension during early-stage diabetes in mice, even in the absence of eNOS. Furthermore, in mesenteric arteries, diabetes is associated with reduced EDHF function, with an apparent compensatory increase in NO function. Thus, prior inhibition of NOS results in endothelial dysfunction in early diabetes, since the diabetes-induced reduction in EDHF function cannot be compensated by increases in NO production.     

Role of nitric oxide in the nicotine-induced pituitary-adrenocortical response.

Nitric oxide (NO) is a major signaling molecule and biological mediator of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the role of NO formed by endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) in the stimulatory effect of nicotine on the HPA axis in rats under basal conditions. Also possible interaction of NOS systems with endogenous prostaglandins (PG) in that stimulation was assessed. NOS and cyclooxygenase inhibitors were administered i.p. 15 min prior to nicotine (2, 5 mg/kg i.p.). Plasma ACTH and serum corticosterone levels were measured 1 h after nicotine injection. NOS blockers given alone did not markedly affect the resting ACTH and corticosterone levels. L-NAME (2-10 mg/kg), a broad spectrum NOS inhibitor considerably and dose dependently enhanced the nicotine-induced ACTH and corticosterone secretion. L-NNA (2 mg/kg) and 7-nitroindazole (7-NI 20 mg/kg), neuronal NOS inhibitors in vivo also significantly augmented the nicotine-induced ACTH and corticosterone levels. L-arginine greatly impaired the nicotine-induced hormone responses and reversed the L-NNA elicited enhancement of the nicotine-evoked ACTH and corticosterone response. In contrast to the constitutive eNOS and nNOS antagonists, an inducible NOS antagonist guanethidine (50-100 mg/kg i.p.) did not substantially affect the nicotine-elicited pituitary-adrenocortical responses. Indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase blocker abolished the L-NAME and L-NNA-induced enhancement of the nicotine-evoked ACTH and corticosterone response. These results indicate that NO is an inhibitory mediator in the HPA axis activity. Inhibition of its generation by eNOS and nNOS significantly enhances the nicotine-induced HPA response. Under basal conditions iNOS is not involved in the nicotine-induced ACTH and corticosterone secretion. Prostaglandins play an obligatory role in the response of HPA axis to systemic nicotine administration.     

Disruption of endothelial caveolae is associated with impairment of both NO- as well as EDHF in acetylcholine-induced relaxation depending on their relative contribution in different vascular beds

Caveolae represent an important structural element involved in endothelial signal-transduction. The present study was designed to investigate the role of caveolae in endothelium-dependent relaxation of different vascular beds. Caveolae were disrupted by cholesterol depletion with filipin (4x10(-6) g L(-1)) or methyl-beta-cyclodextrin (MCD; 1x10(-3) mol L(-1)) and the effect on endothelium-dependent relaxation was studied in rat aorta, small renal arteries and mesenteric arteries in the absence and presence of L-NMMA. The contribution of NO and EDHF, respectively, to total relaxation in response to acetylcholine (ACh) gradually changed from aorta (71.2+/-6.1% and 28.8+/-6.1%), to renal arteries (48.6+/-6.4% and 51.4+/-6.4%) and to mesenteric arteries (9.1+/-4.0% and 90.9+/-4.1%). Electron microscopy confirmed filipin to decrease the number of endothelial caveolae in all vessels studied. Incubation with filipin inhibited endothelium-dependent relaxation induced by cumulative doses of ACh (3x10(-9)-10(-4) mol L(-1)) in all three vascular beds. In aorta, treatment with either filipin or MCD only inhibited the NO component, whereas in renal artery both NO and EDHF formation were affected. In contrast, in mesenteric arteries, filipin treatment only reduced EDHF formation. Disruption of endothelial caveolae is associated with the impairment of both NO and EDHF in acetylcholine-induced relaxation.     

Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.     

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.     

Selected Contribution: Aging impairs nitric oxide and prostacyclin mediation of endothelium-dependent dilation in soleus feed arteries.

We tested the hypothesis that endothelium-dependent dilation in soleus muscle feed arteries (SFA) is impaired by aging due to attenuated nitric oxide (NO)-mediated vasodilation. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two glass micropipettes for examination of endothelium-dependent [flow or acetylcholine (ACh)] and endothelium-independent [sodium nitroprusside (SNP)] vasodilator function. Flow- and ACh-induced dilation was significantly attenuated by age, whereas dilation to SNP was not compromised. To determine the mechanism(s) by which aging affected dilator responses to flow and ACh, dilation was assessed in the presence of Nomega-nitro-L-arginine (L-NNA; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), and L-NNA + Indo. In the presence of L-NNA, Indo, or L-NNA + Indo, flow-induced dilation was inhibited in young SFA, resulting in a response to flow that was no longer greater than old SFA. In the presence of L-NNA or Indo, ACh-induced dilation was not significantly inhibited in young or old SFA; however, double blockade with L-NNA + Indo inhibited ACh-induced dilation in young SFA such that the response to ACh was no longer greater than old SFA. Collectively, these data indicate that aging impairs vasodilator responses in SFA by attenuating NO- and prostacyclin-mediated, endothelium-dependent, dilation.     

Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release

Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(-/-)], and nNOS-deficient [nNOS(-/-)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC(50) and maximal pressor responses to angiotensin II were greater in eNOS(-/-) than in nNOS(-/-) and smaller in wild-type mice. The nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. L-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3', 5'-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.     

Secretagogue-stimulated pancreatic secretion is differentially regulated by constitutive NOS isoforms in mice

Nitric oxide (NO) and NO synthase (NOS) play controversial roles in pancreatic secretion. NOS inhibition reduces CCK-stimulated in vivo pancreatic secretion, but it is unclear which NOS isoform is responsible, because NOS inhibitors lack specificity and three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Mice having individual NOS gene deletions were used to clarify the NOS species and cellular interactions influencing pancreatic secretion. In vivo secretion was performed in anesthetized mice by collecting extraduodenal pancreatic duct juice and measuring protein output. Nonselective NOS blockade was induced with N(omega)-nitro-L-arginine (L-NNA; 10 mg/kg). In vivo pancreatic secretion was maximal at 160 pmol.kg(-1).h(-1) CCK octapeptide (CCK-8) and was reduced by NOS blockade (45%) and eNOS deletion (44%). Secretion was unaffected by iNOS deletion but was increased by nNOS deletion (91%). To determine whether the influence of NOS on secretion involved nonacinar events, in vitro CCK-8-stimulated secretion of amylase from isolated acini was studied and found to be unaltered by NOS blockade and eNOS deletion. Influence of NOS on in vivo secretion was further examined with carbachol. Protein secretion, which was maximal at 100 nmol.kg(-1).h(-1) carbachol, was reduced by NOS blockade and eNOS deletion but unaffected by nNOS deletion. NOS blockade by L-NNA had no effect on carbachol-stimulated amylase secretion in vitro. Thus constitutive NOS isoforms can exert opposite effects on in vivo pancreatic secretion. eNOS likely plays a dominant role, because eNOS deletion mimics NOS blockade by inhibiting CCK-8 and carbachol-stimulated secretion, whereas nNOS deletion augments CCK-8 but not carbachol-stimulated secretion.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.     

Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms

ADP mediates platelet-induced relaxation of blood vessels and may function as an important intercellular signaling molecule in the brain. We used pharmacological and genetic approaches to examine mechanisms that mediate responses of cerebral arterioles to ADP, including the role of endothelial nitric oxide synthase (eNOS). We examined responses of cerebral arterioles (control diameter approximately 30 microm) in anesthetized wild-type (WT, eNOS+/+) and eNOS-deficient (eNOS-/-) mice using a cranial window. In WT mice, local application of ADP produced vasodilation that was not altered by indomethacin but was reduced by approximately 50% by NG-nitro-L-arginine (L-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (inhibitors of NOS and soluble guanylate cyclase, respectively). In eNOS-/- mice, responses to ADP were largely preserved, and a significant component of the response was resistant to L-NNA (a finding similar to that in WT mice treated with L-NNA). In the absence of L-NNA, responses to ADP were markedly reduced by charybdotoxin plus apamin [inhibitors of Ca2+-dependent K+ channels and responses mediated by endothelium-derived hyperpolarizing factor (EDHF)] in both WT and eNOS-/- mice. Thus pharmacological and genetic evidence suggests that a significant portion of the response to ADP in cerebral microvessels is mediated by a mechanism independent of eNOS. The eNOS-independent mechanism is functional in the absence of inhibited eNOS and most likely is mediated by an EDHF.     

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.     

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.     

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.     

Cytochrome P-450 metabolites but not NO,PGI2, and H2O2 contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels

The endothelium-dependent hyperpolarization of cells has a crucial role in regulating vascular tone, especially in microvessels. Nitric oxide (NO) and prostacyclin (PGI2), in addition to endothelium-derived hyperpolarizing factor (EDHF), have been reported to hyperpolarize vascular smooth muscle in several organs. Studies have reported the hyperpolarizing effects of these factors are increased by a stretch in large coronary arteries. EDHF has not yet been identified and cytochrome P-450 metabolites and H2O2 are candidates for EDHF. With the use of the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethione oxonol [DiBAC4(3)], we examined whether NO, PGI2, cytochrome P-450 metabolites, and H2O2 contribute to ACh-induced hyperpolarization in pressurized coronary microvessels. Canine coronary arterial microvessels (60-356 mum internal diameter) were cannulated and pressurized at 60 cmH2O in a vessel chamber perfused with physiological salt solution containing DiBAC4(3). Fluorescence intensity and diameter were measured on a computer. There was a linear correlation between changes in the fluorescence intensity and membrane potential. ACh significantly decreased the fluorescence intensity (hyperpolarization) of the microvessels without any inhibitors. Endothelial damage caused by air perfusion abolished the ACh-induced decrease in fluorescence intensity. The inhibitors of NO synthase and cyclooxygenase did not affect the ACh-induced decreases in the fluorescence intensity. The addition of 17-octadecynoic acid, a cytochrome P-450 monooxygenase inhibitor, to those inhibitors significantly attenuated the ACh-induced decreases in fluorescence intensity, whereas catalase, an enzyme that dismutates H2O2 to form water and oxygen, did not. Furthermore, catalase did not affect the vasodilation produced by ACh. These results indicate that NO and PGI2 do not contribute to the ACh-induced hyperpolarization and that the cytochrome P-450 metabolites but not H2O2 are involved in EDHF-mediated hyperpolarization in canine coronary arterial microvessels.     

Nascent EDHF-mediated cerebral vasodilation in ovariectomized rats is not induced by eNOS dysfunction

In estrogen-depleted [i.e., ovariectomized (Ovx)] animals, an endothelium-derived hyperpolarizing factor (EDHF)-like mechanism may arise to, at least partially, replace endothelial nitric oxide (NO) synthase (eNOS)-derived NO in modulating cerebral arteriolar tone. Additional findings show that eNOS expression and function is restored in estrogen-treated Ovx female rats, while the nascent EDHF-like activity disappears. Because NO has been linked to repression of EDHF activity in the periphery, the current study was undertaken to examine whether the nascent EDHF role in cerebral vessels of Ovx females relates to a chronically repressed eNOS-derived NO-generating function. We compared the effects of chronic NOS inhibition with Nomega-nitro-L-arginine-methyl ester (L-NAME; 100 mg. kg-1. day-1 for 3 wk) on EDHF-mediated pial arteriolar vasodilation in anesthetized intact, Ovx, and 17beta-estradiol-treated (0.1 mg. kg-1. day-1 ip, 1 wk) Ovx (OVE) female rats as well as in male rats that were prepared with closed cranial windows. In the chronic NOS inhibition groups, pial arteriolar responses were monitored in the absence (all groups) and presence (females only) of indomethacin (Indo; 10 mg/kg iv). Finally, the gap junction inhibitory peptide Gap 27 (300 muM) was applied to block EDHF-related vasodilation. NO donor (S-nitroso-N-acetyl-penicillamine) responses were similar in all rats studied. Acetylcholine (ACh) reactivity was virtually absent in control Ovx rats and chronically NOS-inhibited intact female, OVE, and male rats. However, a partial recovery of ACh reactivity was seen in L-NAME-treated Ovx females. In addition, in the presence of L-NAME, a normal CO2 reactivity was observed in all females, whereas a 50% reduction in CO2 reactivity was seen in males. In intact and OVE rats, both chronic and acute (NG-nitro-L-arginine suffusion) NOS inhibition, combined with Indo, depressed ADP-induced dilation by > or =50%, and subsequent application of Gap 27 had no further effect on ADP-induced vasodilation. ADP reactivity was retained in Ovx rats after combined chronic NOS inhibition and acute Indo, but was attenuated significantly by Gap 27. In males, Gap 27 had no effect on arteriolar reactivity. Taken together, our data demonstrate that in the cerebral microcirculation, NO does not have an inhibitory effect on EDHF production or action. The increased EDHF-like function in chronic estrogen-depleted animals is not due to eNOS deficiency, suggesting a more direct effect of estrogen in modulating EDHF-mediated cerebral vasodilation.