ADP-induced pial arteriolar dilation in ovariectomized rats involves gap junctional communication

It was previously shown that, despite the loss of nitric oxide (NO) dependence, ADP-induced pial arteriolar dilation was not attenuated in estrogen-depleted [i.e., ovariectomized (Ovx)] rats. Additional evidence suggested that the NO was replaced by an endothelium-dependent hyperpolarizing factor (EDHF)-like mechanism. To further characterize the nascent EDHF role in Ovx females, the current study was undertaken to test whether, in Ovx rats, ADP-induced pial arteriolar dilation retained its endothelial dependence and whether gap junctions are involved in that response. A closed cranial window and intravital microscopy system was used to monitor pial arteriolar diameter changes in anesthetized rats. The endothelial portion of the ADP-induced dilation was evaluated using light dye endothelial injury (L/D). The study was organized around three experimental approaches. First, the responses of pial arterioles to ADP before and after L/D exposure in intact and Ovx female rats were tested. L/D reduced the ADP response by 50-70% in both groups, thereby indicating that the endothelium dependence of ADP-induced vasodilation is not altered by chronic estrogen depletion. Second, the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) and the prostanoid synthesis inhibitor indomethacin (Indo) were coapplied. In intact females, L-NNA-Indo attenuated the response to ADP by 50%, with no further changes upon the addition of L/D. On the other hand, L-NNA-Indo did not affect ADP reactivity in Ovx rats, but subsequent L/D exposure reduced the ADP response by >50%. The NO-prostanoid-independent, but endothelium-dependent, nature of the response in Ovx females is a hallmark of EDHF participation. Third, gap junctional inhibition strategies were applied. A selective inhibitor of gap junctional function, Gap 27, did not affect ADP reactivity in intact females but reduced the the ADP response by 50% in Ovx females. A similar result was obtained following application of a connexin43 antisense oligonucleotide. These findings suggest that the nascent EDHF dependency of ADP-induced pial arteriolar dilation in Ovx females involves connexin43-related gap junctional communication.     

Chronic estrogen depletion alters adenosine diphosphate-induced pial arteriolar dilation in female rats

We examined pial arteriolar reactivity to a partially endothelial nitric oxide synthase (eNOS)-dependent vasodilator ADP as a function of chronic estrogen status. The eNOS-dependent portion of the ADP response was ascertained by comparing ADP-induced pial arteriolar dilations before and after suffusion of a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA; 1 mM) in intact, ovariectomized (Ovx), and 17beta-estradiol (E2)-treated Ovx females. We also examined whether ovariectomy altered the participation of other factors in the ADP response. Those factors were the following: 1) the prostanoid indomethacin (Indo); 2) the Ca2+-dependent K+ (K(Ca)) channel, iberiotoxin (IbTX); 3) the ATP-regulated K+ (K(ATP)) channel glibenclamide (Glib); 4) the K(Ca)-regulating epoxygenase pathway miconazole (Mic); and 5) the adenosine receptor 8-sulfophenyltheophylline (8-SPT). In intact females, the eNOS-dependent (L-NNA sensitive) portion of the ADP response represented approximately 50% of the total. The ADP response was retained in the Ovx rats but L-NNA sensitivity disappeared. On E2 replacement, the initial pattern was restored. ADP reactivity was unaffected by Indo, Glib, Mic, and 8-SPT. IbTX was associated with 50-80% reductions in the response to ADP in the intact group that was nonadditive with L-NNA, and 60-100% reductions in the Ovx group. The present findings suggest that estrogen influences the mechanisms responsible for ADP-induced vasodilation. The continued sensitivity to IbTX in Ovx rats, despite the loss of a NO contribution, is suggestive of a conversion to a hyperpolarizing factor dependency in the absence of E2.     

The role of the glia limitans in ADP-induced pial arteriolar relaxation in intact and ovariectomized female rats

We examined whether the glia limitans (GL) influences pial arteriolar relaxation elicited in vivo by the purinergic (P(2)Y(1) receptor) agonist ADP in female rats, and whether that influence is altered in ovariectomized (Ovx) females. A validated model for GL injury was used, topical application of the gliotoxin L-alpha-aminoadipic acid (L-alphaAAA), 24 h before the study. In both intact and Ovx females, L-alphaAAA had no effect on responses to the NO donor, S-nitroso-N-acetyl penicillamine, but ADP-induced pial arteriolar dilations were significantly reduced (by 33-90%), compared with vehicle-treated controls. When N(G)-nitro-L-arginine (L-NNA) was administered to L-alphaAAA-treated rats, the ADP response was virtually lost in intact females, but no further reductions were observed in the Ovx rats. On the other hand, in L-alphaAAA-treated Ovx females, when the gap junction blocker, Gap 27, was subsequently added to the suffusate, ADP reactivity fell to very low levels. In vehicle-treated control rats, L-NNA and Gap 27 reduced ADP reactivity by approximately 50% in intact and Ovx females, respectively. An earlier study indicated that the endothelium was a key site of influence for L-NNA (intact) and Gap 27 (Ovx). Thus present and previous results imply that the ADP response in pial arterioles represents the additive actions of an endothelial and a GL component. That supposition was confirmed in the present study by the finding that combining endothelial and GL injury produced an essentially complete loss of ADP reactivity in both intact and Ovx females. Finally, topical application of the selective P(2)Y(1) antagonist, MRS-2179, was associated with a nearly complete suppression of the ADP response in both intact and Ovx females. These results suggest that 1) ADP-induced pial arteriolar dilation involves additive contributions from P(2)Y(1) receptors present in both vascular endothelium and the GL; 2) the influence of the GL component is not altered by ovariectomy; and 3) the gap junction-dependent component of the ADP response in Ovx females is unlikely to include the GL and probably resides in the vessels themselves.     

Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

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.     

Chronic nitric oxide exposure alters the balance between endothelium-derived relaxing factors released from rat renal arteries: prevention by treatment with NOX-100, a NO scavenger

We investigated whether nitric oxide (NO) exposure alters the balance between NO and endothelium-derived hyperpolarizing factor (EDHF) released from rat renal arteries. To produce states of acutely or chronically excessive NO, lipopolysaccharide (LPS) was administered intraperitoneally to rats in a single dose of 4 mg/kg (LPS-single group) or in stepwise doses of 0.5, 1.0 and 2.0 mg/kg every other day (LPS-repeated group). On the day after LPS treatment, the protein levels of inducible NO synthase (iNOS) and endothelial NOS (eNOS) were measured, and the relaxation responses were determined in the renal arteries. The protein levels of iNOS markedly increased in both LPS-treated groups, while those of eNOS significantly increased in the LPS-repeated group compared with those in the respective control groups. In both LPS-treated groups, the relaxations in response to acetylcholine (ACh) and sodium nitroprusside remained unchanged. The ACh-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, or by 1H-[1, 2, 4-] oxadiazole [4, 3-a] quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, i.e. EDHF-mediated relaxations were significantly impaired in the LPS-repeated group but not in the LPS-single group, indicating increase in NO-mediated relaxation in the LPS-repeated group. These changes in the protein levels and EDHF-mediated relaxations induced by ACh observed in the LPS-repeated group were restored by treatment with NOX-100, a NO scavenger. These results suggest that persistent but not acute excessive NO exposure in rats impairs EDHF-mediated relaxation in renal arteries, leading to a compensatory upregulation of the eNOS/NO pathway.     

Interaction of nitric oxide, 20-HETE, and EETs during functional hyperemia in whisker barrel cortex

Nitric oxide (NO) modulates vasodilation in cerebral cortex during sensory activation. NO is known to inhibit the synthesis of 20-HETE, which has been implicated in arteriolar constriction during astrocyte activation in brain slices. We tested the hypothesis that the attenuated cerebral blood flow (CBF) response to whisker stimulation seen after NO synthase (NOS) inhibition requires 20-HETE synthesis and that the ability of an epoxyeicosatrienoic acids (EETs) antagonist to reduce the CBF response is blunted after NOS inhibition but restored with simultaneous blockade of 20-HETE synthesis. In anesthetized rats, the increase in CBF during whisker stimulation was attenuated after the blockade of neuronal NOS with 7-nitroindazole. Subsequent administration of the 20-HETE synthesis inhibitor N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) restored the CBF response to control levels. After the administration of 7-nitroindazole, the inhibitory effect of an EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on the CBF response was lost, whereas the simultaneous administration of 7-nitroindazole and HET0016 restored the inhibitory effect of 14,15-EEZE. The administration of HET0016 alone had only a small effect on the evoked CBF response in rats. Furthermore, in neuronal NOS(+/+) and NOS(-/-) mice, HET0016 administration did not increase the CBF response to whisker stimulation. In neuronal NOS(+/+) mice, HET0016 also blocked the reduction in the response seen with acute NOS inhibition. These results indicate that 20-HETE synthesis normally does not substantially restrict functional hyperemia. Increased NO production during functional activation may act dynamically to suppress 20-HETE synthesis or downstream signaling and permit EETs-dependent vasodilation. With the chronic loss of neuronal NOS in mice, other mechanisms apparently suppress 20-HETE synthesis or signaling.     

Nitric-oxide-dependent pial arteriolar dilation in the female rat: effects of chronic estrogen depletion and repletion

In this study, we compared endothelial nitric oxide synthase (eNOS)-mediated cerebral vasodilating responses in intact female rats, chronically ovariectomized (OVX) rats, and OVX rats treated for 2 weeks with 17beta-estradiol (E(2)). Under anesthesia, using intravital microscopy and a closed cranial window system, pial arteriolar diameter changes were monitored during sequential cortical suffusions of an eNOS-dependent dilator [acetylcholine (ACh)] and a direct NO donor [S-nitrosoacetylpenicillamine (SNAP)]. In separate rats from the same groups, we compared eNOS and caveolin-1 (CAV-1) protein abundance in pial arterioles (via immunofluorescence analyses). In untreated and low-dose E(2)-treated (1.0 microg x kg(-1) x day(-1)) OVX rats, ACh-induced vasodilations were virtually absent. High-dose E(2) treatment (100 microg x kg(-1) x day(-1)) restored ACh-induced pial arteriolar dilations to levels seen in intact females. The vasodilations elicited by SNAP and ADO were unaffected by chronic estrogen changes, indicating no direct estrogen influence on vascular smooth muscle (VSM) reactivity. Pial arteriolar eNOS protein abundance was diminished by ovariectomy and restored by high-dose E(2) treatment. Pial arteriolar CAV-1 expression was higher in OVX versus intact and E(2)-treated OVX females. These results suggest that long-term changes in estrogen directly influence brain eNOS functional activity. The estrogen-related changes in eNOS-dependent vasodilating function appear to be related, in part, to a capacity for E(2) to increase eNOS protein expression and, in part, to an E(2)-associated diminution in endothelial CAV-1 expression.     

Impaired NO-mediated vasodilation with increased superoxide but robust EDHF function in right ventricular arterial microvessels of pulmonary hypertensive rats

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n=126) to induce PH or with saline as controls (n=114). After 3 wk, coronary arterioles (diameter = 30-100 microm) and small arteries (diameter = 100-200 microm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small arteries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.     

EDHF-mediated vasodilation involves different mechanisms in normotensive and hypertensive rat lungs

The role of endothelium-derived hyperpolarizing factor (EDHF) in regulating the pulmonary circulation and the participation of cytochrome P-450 (CYP450) activity and gap junction intercellular communication in EDHF-mediated pulmonary vasodilation are unclear. We tested whether tonic EDHF activity regulated pulmonary vascular tone and examined the mechanism of EDHF-mediated pulmonary vasodilation induced by thapsigargin in salt solution-perfused normotensive and hypoxia-induced hypertensive rat lungs. After blockade of both cyclooxygenase and nitric oxide synthase, inhibition of EDHF with charybdotoxin plus apamin did not affect either normotensive or hypertensive vascular tone or acute hypoxic vasoconstriction but abolished thapsigargin vasodilation in both groups of lungs. The CYP450 inhibitors 7-ethoxyresorufin and sulfaphenazole and the gap junction inhibitor palmitoleic acid, but not 18alpha-glycyrrhetinic acid, inhibited thapsigargin vasodilation in normotensive lungs. None of these agents inhibited the vasodilation in hypertensive lungs. Thus tonic EDHF activity does not regulate either normotensive or hypertensive pulmonary vascular tone or acute hypoxic vasoconstriction. Whereas thapsigargin-induced EDHF-mediated vasodilation in normotensive rat lungs involves CYP450 activity and might act through gap junctions, the mechanism of vasodilation is apparently different in hypertensive lungs.     

Influence of the glia limitans on pial arteriolar relaxation in the rat

We examined whether damage to the glia limitans (GL), via exposure to the gliotoxin l-alpha-aminoadipic acid (l-alphaAAA), alters hypercapnia-induced pial arteriolar dilation in vivo. Anesthetized female rats were prepared with closed cranial windows. Pial arteriolar diameters were measured using intravital microscopy. l-alphaAAA (2 mM) was injected into the space under the cranial windows 24 h before the study, and injury to the GL was confirmed by light microscopy. l-alphaAAA was associated with a reduction in pial arteriolar CO(2) reactivity to 40-50% of the level seen in vehicle-treated controls, with no further reduction in the CO(2) response after nitric oxide (NO) synthase (NOS) inhibition via N(omega)-nitro-l-arginine (l-NNA). Subsequent blockade of prostanoid synthesis, via indomethacin (Indo), reduced CO(2) reactivity to 10-15% of normal. In vehicle-treated controls, l-NNA, followed by Indo, reduced the response to approximately 50% and then to 15-20% of the normocapnic value, respectively. On the other hand, l-alphaAAA had no effect on vascular responses to the endothelium-dependent vasodilator acetylcholine or the NO donor SNAP and did not alter cortical somatosensory evoked responses. This indicates an absence of any direct l-alphaAAA actions on pial arterioles or influence on neuronal transmission. Furthermore, l-alphaAAA did not alter the vasodilation elicited by topical application of an acidic artificial cerebrospinal fluid solution, suggesting that the GL influences the pial arteriolar relaxation elicited by hypercapnic, but not local extracellular (EC), acidosis. That differences exist in the mechanisms mediating hypercapnia- versus EC acidosis-induced pial arteriolar dilations was further exemplified by the finding that topical application of a neuronal NOS (nNOS)-selective blocker (ARR-17477) reduced the response to hypercapnia (by approximately 65%) but not the response to EC acidosis. Disruption of GL gap junctional communication, using an antisense oligodeoxynucleotide (ODN) connexin43 knockdown approach, was accompanied by a 33% lower CO(2) reactivity versus missense ODN-treated controls. These results suggest that the GL contribution to the hypercapnic vascular response appears to involve the NO-dependent component rather than the prostanoid-dependent component and may involve gap junctional communication. We speculate that the GL may act to facilitate the spread, to pial vessels, of hypercapnia-induced vasodilating signals arising in the comparatively few scattered nNOS neurons that lie well beneath the GL.     

Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts

Previous studies have shown that estrogen modulation of endothelial nitric oxide (NO) synthase (eNOS) may confer protection against heart disease. Here, we demonstrate an association between reductions in baroreflex-mediated bradycardia and in cardiac NOS activity in ovariectomized (Ovx) rats compared with controls. The latter resulted, at least in part, from a reduction in cardiac eNOS protein. eNOS-derived NO and its biological effects are determined by the levels of eNOS protein and by eNOS catalytic activity; the latter is regulated partly through the dynamic interaction with an inhibitory protein (caveolin) and a stimulatory protein (calmodulin). The association of eNOS immunoprecipitated with caveolin-3 and calmodulin was examined. Caveolin-3 and calmodulin binding with eNOS was increased and decreased, respectively, in Ovx rats. 17 beta-Estradiol replacement restored, to within normal levels, the baroreflex-mediated bradycardic responses along with eNOS activity, eNOS expression, and the association of eNOS with caveolin-3 and calmodulin. Our findings may help to elucidate the molecular mechanism underlying the favorable effects of estrogen on cardiac responses to baroreflex activation.     

Effects of Trientine,a Metal Chelator,on Defective Endothelium-dependent Relaxation in the Mesenteric Vasculature of Diabetic Rats

Diabetes mellitus compromises endothelium-dependent relaxation of blood vessels. This has been linked to the generation of reactive oxygen species (ROS), which neutralise nitric oxide (NO) and interfere with vasodilator function. Experiments using chelators have emphasised the importance of ROS produced by transition metal catalysed reactions. However, particularly for the small arteries and arterioles that control microcirculatory blood flow, NO is not the only endothelium-derived mediator; endothelium-derived hyperpolarizing factor (EDHF) has a major role. EDHF-mediated vasodilation is severely curtailed by diabetes; however, little information exists on the underlying pathophysiology. Deficits in the EDHF system, alone or in combination with the NO system, are crucial for the development of diabetic microvascular complications. To further elucidate the mechanisms involved, the aim was to examine the effects of diabetes and preventive and intervention chelator therapy with trientine on a preparation that has well-defined NO and EDHF-mediated responses, the rat mesenteric vascular bed. In phenylephrine-preconstricted preparations, maximum vasodilation to acetylcholine was reduced by 35 and 44% after 4 and 8 weeks of streptozotocin-induced diabetes, respectively. Trientine treatment over the first 4 weeks gave 72% protection; intervention therapy over the final 4 weeks prevented deterioration and corrected the initial deficit by 68%. These responses depend on both NO and EDHF. When the latter mechanism was isolated by NO synthase inhibition, diabetic deficits of 53.4 (4 weeks) and 65.4% (8 weeks) were revealed, that were 65% prevented and 50% corrected by trientine treatment. Neither diabetes nor trientine altered vascular smooth muscle responses to the NO donor, sodium nitroprusside (SNP). Thus, the data suggest that metal catalysed ROS production makes a substantial contribution to defects in both the EDHF and NO endothelial mechanisms in diabetes, which has therapeutic implications for microvascular complications.     

Effects of trientine,a metal chelator,on defective endothelium-dependent relaxation in the mesenteric vasculature of diabetic rats

Diabetes mellitus compromises endothelium-dependent relaxation of blood vessels. This has been linked to the generation of reactive oxygen species (ROS), which neutralise nitric oxide (NO) and interfere with vasodilator function. Experiments using chelators have emphasised the importance of ROS produced by transition metal catalysed reactions. However, particularly for the small arteries and arterioles that control microcirculatory blood flow, NO is not the only endothelium-derived mediator; endothelium-derived hyperpolarizing factor (EDHF) has a major role. EDHF-mediated vasodilation is severely curtailed by diabetes; however, little information exists on the underlying pathophysiology. Deficits in the EDHF system, alone or in combination with the NO system, are crucial for the development of diabetic microvascular complications. To further elucidate the mechanisms involved, the aim was to examine the effects of diabetes and preventive and intervention chelator therapy with trientine on a preparation that has well-defined NO and EDHF-mediated responses, the rat mesenteric vascular bed. In phenylephrine-preconstricted preparations, maximum vasodilation to acetylcholine was reduced by 35 and 44% after 4 and 8 weeks of streptozotocin-induced diabetes, respectively. Trientine treatment over the first 4 weeks gave 72% protection; intervention therapy over the final 4 weeks prevented deterioration and corrected the initial deficit by 68%. These responses depend on both NO and EDHF. When the latter mechanism was isolated by NO synthase inhibition, diabetic deficits of 53.4 (4 weeks) and 65.4% (8 weeks) were revealed, that were 65% prevented and 50% corrected by trientine treatment. Neither diabetes nor trientine altered vascular smooth muscle responses to the NO donor, sodium nitroprusside (SNP). Thus, the data suggest that metal catalysed ROS production makes a substantial contribution to defects in both the EDHF and NO endothelial mechanisms in diabetes, which has therapeutic implications for microvascular complications.     

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.     

Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

Role of EDHF in type 2 diabetes-induced endothelial dysfunction

Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in modulating vasomotor tone, especially in microvessels when nitric oxide-dependent control is compromised such as in diabetes. Epoxyeicosatrienoic acids (EETs), potassium ions (K+), and hydrogen peroxide (H2O2) are proposed as EDHFs. However, the identity (or identities) of EDHF-dependent endothelial dilators has not been clearly elucidated in diabetes. We assessed the mechanisms of EDHF-induced vasodilation in wild-type (WT, normal), db/db (advanced type 2 diabetic) mice, and db/db mice null for TNF (dbTNF-/dbTNF-). In db/db mice, EDHF-induced vasodilation [ACh-induced vasodilation in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 10 micromol/l) and prostaglandin synthase inhibitor indomethacin (Indo, 10 mumol/l)] was diminished after the administration of catalase (an enzyme that selectively dismutates H2O2 to water and oxygen, 1,000 U/ml); administration of the combination of charybdotoxin (a nonselective blocker of intermediate-conductance Ca2+-activated K+ channels, 10 micromol/l) and apamin (a selective blocker of small-conductance Ca2+-activated K+ channels, 50 micromol/l) also attenuated EDHF-induced vasodilation, but the inhibition of EETs synthesis [14,15-epoxyeicosa-5(Z)-enoic acid; 10 mumol/l] did not alter EDHF-induced vasodilation. In WT controls, EDHF-dependent vasodilation was significantly diminished after an inhibition of K+ channel, EETs synthesis, or H2O2 production. Our molecular results indicate that mRNA and protein expression of interleukin-6 (IL-6) were greater in db/db versus WT and dbTNF-/dbTNF- mice, but neutralizing antibody to IL-6 (anti-IL-6; 0.28 mg.ml(-1).kg(-1) ip for 3 days) attenuated IL-6 expression in db/db mice. The incubation of the microvessels with IL-6 (5 ng/ml) induced endothelial dysfunction in the presence of l-NAME and Indo in WT mice, but anti-IL-6 restored ACh-induced vasodilation in the presence of L-NAME and Indo in db/db mice. In db(TNF-)/db(TNF-) mice, EDHF-induced vasodilation was greater and comparable with controls, but IL-6 decreased EDHF-mediated vasodilation. Our results indicate that EDHF compensates for diminished NO-dependent dilation in IL-6-induced endothelial dysfunction by the activation of H2O2 or a K+ channel in type 2 diabetes.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Chronic administration of indomethacin increases role of nitric oxide in hypercapnic cerebrovasodilation in piglets.

Hypercapnia-induced cerebral vasodilation is associated with prostanoids in the piglet, but is a primarily nitric oxide (NO) associated response in many adult models. Hypercapnia-induced cerebral vasodilation is both NO and prostanoid associated in the juvenile pig. We hypothesized that with chronic administration of indomethacin the piglet would advance the role of the NO system in cerebrovascular responses. The closed cranial window technique was used in piglets to determine pial arteriolar response. Chronically indomethacin treated newborn animals dilated in response to CO2 similarly to control newborns (40.9+/-4.4% vs 48.4+/-4.1%). Topical n-nitro L-arginine (L-NA, 10(-3) M), attenuated CO2 induced dilation in the chronically indomethacin treated animals (11.7+/-3.3% vs 40.9+/-4.4%; p < 0.001), but had no effect on the response to hypercapnia of piglets not treated with indomethacin. Neither indomethacin nor L-NA altered response to topical isoproterenol (10(-6) M). We conclude that with chronic indomethacin administration there develops a significant hypercapnia-induced cerebral vasodilation in which NO has an important role. The chronic inhibition of the newborn's principal dilator system appears to increase the role of NO in newborn cerebral hemodynamics.