Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney

To evaluate the role of a potential interaction between superoxide anion (O(2)(-)) and nitric oxide (NO) in regulating kidney function, we examined the renal responses to intra-arterial infusion of a superoxide dismutase mimetic, tempol (0.5 mg.kg(-1).min(-1)), in anesthetized dogs treated with or without NO synthase inhibitor, N(omega)-nitro-l-arginine (NLA; 50 microg.kg(-1).min(-1)). In one group of dogs (n = 10), tempol infusion alone for 30 min before NLA infusion did not cause any significant changes in renal blood flow (RBF; 5.2 +/- 0.4 to 5.0 +/- 0.4 ml.min(-1).g(-1)), glomerular filtration rate (GFR; 0.79 +/- 0.04 to 0.77 +/- 0.04 ml.min(-1).g(-1)), urine flow (V; 13.6 +/- 2.1 to 13.9 +/- 2.5 microl.min(-1).g(-1)), or sodium excretion (U(Na)V; 2.4 +/- 0.3 to 2.2 +/- 0.3 micromol.min(-1).g(-1)). Interestingly, when tempol was infused in another group of dogs (n = 12) pretreated with NLA, it caused increases in V (4.4 +/- 0.4 to 9.7 +/- 1.4 microl.min(-1).g(-1)) and in U(Na)V (0.7 +/- 0.1 to 1.3 +/- 0.2 micromol.min(-1).g(-1)) without affecting RBF or GFR. Although NO inhibition caused usual qualitative responses in both groups of dogs, the antidiuretic (47 +/- 5 vs. 26 +/- 4%) and antinatriuretic (67 +/- 4 vs. 45 +/- 11%) responses to NLA were seen much less in dogs pretreated with tempol. NLA infusion alone increased urinary excretion of 8-isoprostane (13.9 +/- 2.7 to 22.8 +/- 3.6 pg.min(-1).g(-1); n = 7), which returned to the control levels (11.6 +/- 3.4 pg.min(-1).g(-1)) during coadministration of tempol. These data suggest that NO synthase inhibition causes enhancement of endogenous O(2)(-) levels and support the hypothesis that NO plays a protective role against the actions of O(2)(-) in the kidney.     

Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries

Communication between vascular smooth muscle (VSM) cells via low-resistance gap junctions may facilitate vascular function by synchronizing the contractile state of individual cells within the vessel wall. We hypothesized that inhibition of gap junctional communication would impair constrictor responses of mesenteric resistance arteries. Immunohistochemical experiments revealed positive staining for connexin 37 (Cx37) in both endothelium and smooth muscle of rat mesenteric arterioles, whereas connexin 43 (Cx43) immunoreactivity was not detected in the mesenteric vasculature. Administration of the gap junction inhibitory peptide Gap27, which targets Cx37 and Cx43, significantly diminished myogenic vasoconstriction (8.6 +/- 3.8% of passive diameter at 100 Torr) and changes in vessel wall intracellular [Ca2+] of mesenteric resistance arteries compared with vessels treated with either vehicle (physiological saline solution) (33.5 +/- 6.1%) or a control peptide (32.1 +/- 6.5%). Administration of 18alpha-glycyrrhetinic acid, structurally distinct from Gap27, also significantly attenuated myogenic constriction compared with its vehicle control (DMSO) (9.6 +/- 3.2% vs. 23.8 +/- 4.6%). In contrast, phenylephrine-induced vasoconstriction was not altered by gap junction blockers. Attenuated myogenic vasoconstriction resulting from inhibition of gap junctions persisted after disruption of the endothelium. In additional experiments, VSM cell membrane potential was recorded in mesenteric resistance arteries pressurized to 20 or 100 Torr. VSM membrane potential was depolarized at 100 Torr compared with 20 Torr. However, VSM cells in arteries treated with Gap27 were significantly hyperpolarized (-48.6 +/- 1.4 mV) at the higher pressure compared with vehicle (-41.4 +/- 1.5 mV) and Gap20-treated (-38.4 +/- 0.7 mV) vessels. Our findings suggest that inhibition of smooth muscle gap junctions attenuates pressure-induced VSM cell depolarization and myogenic vasoconstriction.     

Increased H(2)O(2) counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla

A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2 counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+ excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2 microassay, it was found that interstitial H2O2 levels in the renal cortex and medulla in anesthetized rats averaged 55.91 +/- 3.66 and 102.18 +/- 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 micromol x min-1x kg-1) significantly increased medullary H2O2 levels by 46%, and coinfusion of catalase (10 mg x min-1x kg-1) completely abolished this increase. Functionally, removal of H2O2 by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2 by renal medullary interstitial infusion (7.5-30 nmol x min-1x kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2 may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.     

Superoxide formation and interaction with nitric oxide modulate systemic arterial pressure and renal function in salt-depleted dogs

To determine the role of superoxide (O(2)(-)) formation in the kidney during alterations in the renin-angiotensin system, we evaluated responses to the intra-arterial infusion of an O(2)(-) - scavenging agent, tempol, in the denervated kidney of anesthetized salt-depleted (SD, n=6) dogs and salt-replete (SR, n=6) dogs. As expected, basal plasma renin activity was higher in SD than in SR dogs (8.4 +/- 1.0 vs. 2.3 +/- 0.6 ng angiotensin 1/ml/hr). Interestingly, the basal level of urinary F(2)-isoprostanes excretion (marker for endogenous O(2)(-) activity) relative to creatinine (Cr) excretion was also significantly higher in SD compared to SR dogs (9.1 +/- 2.8 vs. 1.6 +/- 0.4 ng F(2)-isoprostanes/mg of Cr). There was a significant increase in renal blood flow (4.3 +/- 0.5 to 4.9 +/- 0.6 ml/min/g) and decreases in renal vascular resistance (38.2 +/- 5.8 to 33.2 +/- 4.7 mm Hg/ml/min/g) and mean systemic arterial pressure (148 +/- 6 to 112 +/- 10 mm Hg) in SD dogs but not in SR dogs during infusion of tempol at 1 mg/kg/min for 30 mins. Glomerular filtration rate and urinary sodium excretion (U(Na)V) did not change significantly during tempol infusion in both groups of dogs. Administration of the nitric oxide synthase inhibitor nitro-L-arginine (50 mug/kg/min) during tempol infusion caused a reduction in U(Na)V in SR dogs (47% +/- 12%) but did not cause a decrease in SD dogs. These data show that low salt intake enhances O(2)(-) activity that influences renal and systemic hemodynamics and thus may contribute to the regulation of arterial pressure in the salt-restricted state.     

Constriction to hypoxia-reoxygenation in isolated mouse coronary arteries: role of endothelium and superoxide.

The aim of the present study was to determine the role of endothelium and superoxide in the responses of isolated mouse coronary arteries to hypoxia-reoxygenation. Isolated mouse coronary artery was cannulated, pressurized at 60 mmHg, and constantly superfused with recirculating Krebs-Ringer bicarbonate solution for continuous measurement of intraluminal diameter (ID) by video microscopy. Under a no-flow condition, hypoxia (0% O(2), 30 min) caused vasoconstriction. Reoxygenation caused a further vasoconstriction (ID change from 111.4 +/- 11.1 to 91 +/- 16.5 microm) that was significantly reduced by removal of endothelium (ID change from 105.4 +/- 27 to 109.9 +/- 23.4 microm). Cu/Zn superoxide dismutase (150 U/ml) did not alter the hypoxic vasoconstriction but abolished the reoxygenation-caused endothelium-dependent vasoconstriction. Hypoxia-reoxygenation markedly enhanced the generation of superoxide that was significantly reduced by either removing the endothelium or treated these endothelium-intact vessels with superoxide dismutase. These results suggest that, in isolated mouse coronary arteries, hypoxia causes vasoconstriction that is independent of endothelium, whereas reoxygenation causes vasoconstriction that is mediated by enhanced generation of superoxide from endothelium.     

Simulated microgravity alters rat mesenteric artery vasoconstrictor dynamics through an intracellular Ca(2+) release mechanism

Previous work has shown that orthostatic hypotension associated with cardiovascular deconditioning results from inadequate peripheral vasoconstriction. We used the hindlimb-unloaded (HU) rat in this study as a model to induce cardiovascular deconditioning. The purpose of this study was to test the hypothesis that 14 days of HU diminishes vasoconstrictor responsiveness of mesenteric resistance arteries. Mesenteric resistance arteries from control (n = 43) and HU (n = 44) rats were isolated, cannulated, and pressurized to 108 cm H(2)O for in vitro experimentation. Myogenic (intralumenal pressure ranging from 30 to 180 cm H(2)O), KCl (2-100 mM), norepinephrine (NE, 10(-9)-10(-4) M) and caffeine (1-20 mM) induced vasoconstriction, as well as the temporal dynamics of vasoconstriction to NE, were determined. The active myogenic and passive pressure responses were unaltered by HU when pressures remained within physiological range. However, vasoconstrictor responses to KCl, NE, and caffeine were diminished by HU, as well as the rate of constriction to NE (C, 14.8 +/- 3.6 microm/s vs. HU 7.6 +/- 1.8 microm/s). Expression of sarcoplasmic reticulum Ca(2+)ATPase 2 and ryanodine 3 receptor mRNA was unaffected by HU, while ryanodine 2 receptor mRNA and protein expression were diminished in mesenteric arteries from HU rats. These data suggest that HU-induced and microgravity-associated orthostatic intolerance may be due, in part, to an attenuated vasoconstrictor responsiveness of mesenteric resistance arteries resulting from a diminished ryanodine 2 receptor Ca(2+) release mechanism.     

Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.     

Hypoxia-induced reactive oxygen species downregulate ETB receptor-mediated contraction of rat pulmonary arteries

Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (approximately 0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po2) compared with normoxic control (140 mmHg Po2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.     

Cyclooxygenase- and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries

We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA (10(-9)-10(-4) M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 +/- 0.5% and EC(50) of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 microM), NS-398 (10 microM, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 muM), nordihydroguaiaretic acid (NDGA; 1 microM), and ebselen (1 microM). HPLC analysis showed that [(14)C]AA was metabolized by mesenteric arteries to PGI(2), PGE(2), THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI(2) and PGE(2) was blocked by indomethacin, and the production of THETAs, HEETAs, and 15-HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15-THETA. PGI(2), PGE(2), and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high K(+) (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways.     

Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically via increasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have been developed that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavenging activities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity, and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation with paraquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities of superoxide anions and hydroxyl radicals. Both EUK-134 (10-300 microM) and tempol (0.3-1.0 mM) were able to improve cell viability and reduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radical generation. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoid the pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies for paraquat nephrotoxicity.     

Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS-/- mice

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.     

Age-related changes in adenosine 5'-triphosphate-induced constriction of isolated, perfused mesenteric arteries of rats

In isolated mesenteric arteries of rats, dose-dependent increase in perfusion pressure by adenosine 5'-triphosphate (ATP, 0.1 approximately 3000 nmole) diminished with age. ATP responses of both 4- and 32-week-old rats were enhanced by indomethacin (5 microM), and further by the combination of indomethacin and N(G)-nitro-L-arginine methyl ester (L-NAME, 5 microM). The enhancement with each of the treatments was less in 32-week-old rats than that in 4-week-old rats, and there was no enhancement in 75-week-old rats. The ATP response was enhanced by removing the endothelium only in 4-week-old rats. The constrictions in response to ATP (1000 nmole) in both 4- and 32-week-old rats were equally enhanced by reactive blue 2 (30 micromole) and were inhibited by pyridoxal-phosphate-6-azophenyl-2',4-disulphonic acid (PPADS, 30 microM) and alpha, beta-methylene ATP (alpha, beta-mATP, 100 nmole) to a similar extent. The increased tone which was produced by the perfusion with physiological solution containing 100 mM potassium chloride was greater in older animals. This age-related change in the vascular tone disappeared when the responses were potentiated by L-NAME. These results demonstrate that in rat mesenteric arteries, ATP-induced constriction decreases with age. The age-related decline of vasoconstriction is not likely to arise from the changes in the contractility of smooth muscle, from the counterbalancing regulation by the endothelium, or from the cooperation of P2 purinoceptor subtypes. The density of purinoceptors and some post-receptor signal transduction mechanisms in the vascular smooth muscle cells may change with age. The enhanced ATP response might have special physiological significance in rats during development.     

Statins as coronary vasodilators in isolated bovine coronary arteries--involvement of PGI2 and NO

Here we studied direct vasodilation induced by statins in isolated bovine coronary arteries. In rings of coronary bovine arteries preconstricted with prostaglandin F(2 alpha) (3 x 10(-8) - 10(-5)), lovastatin, simvastatin, atorvastatin and cerivastatin (3-30 microM) but not pravastatin induced concentration-dependent vasodilation. Removal of endothelium diminished response to simvastatin, cerivastatin and atorvastatin (30 microM) (67.4+/-4.56 vs. 22.7+/-8.14%, 96.9+/-2.27% vs. 54.5+/-6.86%, 67.4+/-4.01% vs. 34.6+/-5.66%, respectively). In presence of L-NAME (300 microM) or indomethacin (5 microM) responses to simvastatin, atorvastatin and cerivastatin, were also partially diminished. In contrast, lovastatin-induced vasorelaxation was not significantly affected by removal of endothelium (35.6+/-4.19% vs. 28.8+/-5.24%) or by pretreatment with L-NAME or indomethacin. In summary, with the exception of pravastatin, statins act as coronary vasodilators. Simvastatin, cerivastatin and atorvastatin but not lovastatin induced vasodilation displayed endothelium dependent- and endothelium-independent components. The endothelium-dependent effect of statins was mediated by NO and PGI(2), while the mechanism of smooth muscle cells-dependent component remains to be determined.     

In vitro pharmacological actions of sinomenine on the smooth muscle and the endothelial cell activity in rat aorta

Vasodilating actions of sinomenine were examined using rat aorta ring strips. Sinomenine (0.1 to 100 microM) dilated norepinephrine (NE, 5 microM)-induced vasoconstriction in a concentration-dependent manner reaching 68.8+/-5.1% (n=6, P<0.01) at a concentration of 100 microM. Sinomenine (100 microM) also attenuated KCl (60 mM) and phorbol 12, 13-dibutyrate (PDB, a protein kinase C, PK-C, activator, 300 nM)-induced vasoconstriction by 86.9+/-8.5% (n=6, P<0.01) and 49.9+/-9.8% (n=6, P<0.01), respectively. Pretreatment with nicardipine (a Ca2+ channel antagonist), staurosporine (a PK-C inhibitor), NG-monomethyl-L-arginine acetate (L-NMMA, a nitric oxide, NO, synthesis inhibitor), and indomethacin (a cyclooxygenase inhibitor) were carried out. Nicardipine (0.1 microM) led to a significant decrease in the vasodilating potential of sinomenine (at 100 microM, 68.8+/-5.1% vs. 35.5+/-6.9%; n=5, P<0.001). Pretreatment with staurosporine (30 nM) reduced sinomenine-associated vasodilation from 68.8+/-5.1% to 49.5+/-7.7% (n=5, P<0.001), and L-NMMA (100 microM) and indomethacin (10 microM), to 25.3+/-2.3% (n=5, P<0.001) and to 37.1+/-9.3% (n=5, P<0.001), respectively. The responses were almost similar to the results without endothelium. Therefore, these results indicate that sinomenine causes the vasorelaxation by the mechanisms involved with the inhibitions of Ca2+ channel and PK-C activity, and also with the activations of NO and prostaglandin (PG) I2 syntheses in endothelium.     

Differences in acute hypoxic pulmonary vasoresponsiveness between rat strains: role of endothelium.

Intact Madison (M) rats have greater pulmonary pressor responses to acute hypoxia than Hilltop (H) rats. We tested the hypothesis that the difference in pressor response is intrinsic to pulmonary arteries and that endothelium contributes to the difference. Pulmonary arteries precontracted with phenylephrine (10(-7) M) from M rats had greater constrictor responses [hypoxic pulmonary vasoconstriction (HPV)] to acute hypoxia (0% O(2)) than those from H rats: 473 +/- 30 vs. 394 +/- 29 mg (P < 0.05). Removal of the endothelium or inhibition of nitric oxide (NO) synthase by N(omega)-nitro-L-arginine (L-NA, 10(-3) M) significantly blunted HPV in both strains. Inhibition of cyclooxygenase by meclofenamate (10(-5) M) or blockade of endothelin type A and B receptors by BQ-610 (10(-5) M) + BQ-788 (10(-5) M), respectively, had no effect on HPV. Constrictor responses to phenylephrine, endothelin-1, and prostaglandin F(2alpha) were similar in pulmonary arteries from both strains. The relaxation response to ACh, an NO synthase stimulator, was significantly greater in M than in H rats (80 +/- 3 vs. 62 +/- 4%, P < 0.01), but there was no difference in response to sodium nitroprusside, an NO donor. L-NA potentiated phenylephrine-induced contraction to a greater extent in pulmonary arteries from M than from H rats. These findings indicate that at least part of the strain-related difference in acute HPV is attributable to differences in endothelial function, possibly related to differences in NO production.     

CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 +/- 0.06 mN/mm) in pulmonary vascular rings treated with 15 micromol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has no effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 micromol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 micromol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 micromol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity.     

Streptozotocin-induced Diabetes Decreases Conducted Vasoconstrictor Response in Mouse Cremaster Arterioles

A conducted vasomotor response (CVR) is characterized by the spread of vasoconstriction or vasodilatation both up- and downstream from a local stimulation site in the microcirculation. It is believed to coordinate vasomotor responses within the microcirculation, and to contribute to the control of the major feed arteries to a given organ or tissue. Microvascular disease is a common and severe complication in diabetes, and we therefore studied CVR in streptozotocin (STZ) diabetic mice to examine whether changes in CVR might have a role in the pathophysiology of microvascular dysfunction in diabetes. The mouse cremasteric arterioles were stimulated locally with KCl and the resulting local response as well as conducted responses at 500 mum and 1000 mum were measured in control and STZ treated mice. Diabetes (n=8) induced by intraperitoneal injection of STZ in a dose of 100 mg/kg (mean blood glucose 16.8+/-2.1 mmol/l) decreased the conduction of vasoconstriction from 27.3+/-1.1% to 21.4+/-1.6% at 500 mum (p<0.01) and from 17.4+/-1.0% to 9.8+/-1.1% at 1000 mum (p<0.01) as compared with control (n=9). Treatment with either the protein kinase C beta II inhibitor (LY341684) or the oxygen radical scavenger tempol, did not improve the decreased conduction of vasoconstriction, but when administered together, the conduction of vasoconstriction was improved from 21.4+/-1.6% to 26.5+/-0.8% at 500 mum and 9.8+/-1.1% to 16.5+/-0.7% at 1000 mum (p<0.01). We conclude that STZ induced diabetes reduces conducted vasoconstriction to KCl in mouse cremasteric arterioles, and combined treatment with both an oxygen radical scavenger and a protein kinase C beta II inhibitor improves the reduced conducted vasoconstriction.     

Low nanomolar concentration of mercury chloride increases vascular reactivity to phenylephrine and local angiotensin production in rats

Exposure to mercury at nanomolar level affects cardiac function but its effects on vascular reactivity have yet to be investigated. Pressor responses to phenylephrine (PHE) were investigated in perfused rat tail arteries before and after treatment with 6 nM HgCl2 during 1 h, in the presence (E+) and absence (E-) of endothelium, after L-NAME (10(-4) M), indomethacin (10(-5 )M), enalaprilate (1 microM), tempol (1 microM) and deferoxamine (300 microM) treatments. HgCl2 increased sensitivity (pD2) without modifying the maximum response (Emax) to PHE, but the pD2 increase was abolished after endothelial damage. L-NAME treatment increased pD2 and Emax. However, in the presence of HgCl2, this increase was smaller, and it did not modify Emax. After indomethacin treatment, the increase of pD2 induced by HgCl2 was maintained. Enalaprilate, tempol and deferoxamine reversed the increase of pD2 evoked by HgCl2. HgCl2 increased the angiotensin converting enzyme (ACE) activity explaining the result obtained with enalaprilate. Results suggest that at nanomolar concentrations HgCl2 increase the vascular reactivity to PHE. This response is endothelium mediated and involves the reduction of NO bioavailability and the action of reactive oxygen species. The local ACE participates in mercury actions and depends on the angiotensin II generation.