Alterations in KATP and KCa channel function in cerebral arteries of insulin-resistant rats

We examined whether insulin resistance alters the function of ATP-dependent and Ca(2+)-activated K(+) channels (K(ATP) and K(Ca) channels, respectively) in pressurized isolated middle cerebral arteries (MCAs) from fructose-fed insulin-resistant (IR) and control rats. Blockade of K(Ca) channels with tetraethylammonium chloride (TEA, 2.5 mM) or iberiotoxin (IBTX, 0.1 microM) increased the spontaneously developed tone in control MCAs by 10.5 +/- 1.3% (n = 10) and 13.3 +/- 2.3% (n = 6), respectively. In the IR arteries, TEA induced similar constrictions (8.0 +/- 1.1%, n = 10), but IBTX constricted the IR arteries by only 3.1 +/- 0.9% (n = 8; P < 0.01). Bradykinin (BK)-induced endothelium-mediated relaxation was reduced in IR MCAs. Maximum relaxation to BK (10(-6) M) was 42 +/- 4% in control (n = 9) and 19 +/- 2% in IR (n = 10; P < 0.01) arteries. Pretreatment with TEA, IBTX, or the K(ATP) channel blocker glibenclamide (10 microM) inhibited relaxation to BK in control MCAs but did not alter dilation in IR arteries. Relaxation to the K(ATP) channel opener cromakalim was also diminished in IR MCAs. Maximum relaxation to cromakalim (10(-5) M) was 48 +/- 3% in control (n = 6) and 19 +/- 2% in IR arteries (n = 6; P < 0.01). These findings demonstrate that insulin resistance alters the function of K(ATP) and K(Ca) channels in isolated MCAs and affects the control of resting vascular tone and the mediation of dilator stimuli.     

Insulin resistance does not impair contractile responses of cerebral arteries

Insulin resistance (IR) impairs endothelium-mediated vasodilation in cerebral arteries as well as K+ channel function in vascular smooth muscle. Peripheral arteries also show an impaired endothelium-dependent vasodilation in IR and concomitantly show an enhanced contractile response to endothelin-1 (ET-1). However, the contractile responses of the cerebral arteries in IR have not been examined systematically. This study examined the contractile responses of pressurized isolated middle cerebral arteries (MCAs) in fructose-fed IR and control rats. IR MCAs showed no difference in pressure-mediated (80 mmHg) vasoconstriction compared to controls, either in time to develop spontaneous tone (control: 61+/-3 min, n=30; IR: 63+/-2 min, n=26) or in the degree of that tone (control: 60 min: 33+/-2%, n=22 vs. IR 60 min: 34+/-3%, n=17). MCAs treated with ET-1 (10(-8.5) M) constrict similarly in control (53+/-3%, n=14) and IR (53+/-3%, n=14) arteries. Constrictor responses to U46619 (10(-6) M) are also similar in control (48+/-9%, n=8) and IR (42+/-5%, n=6) MCAs as are responses to extraluminal uridine 5'-triphosphate (UTP; 10(-4.5) M) (control: 35+/-7%, n=11 vs. IR: 38+/-3%, n=10). These findings demonstrate that constrictor responses remain intact in IR despite a selective impairment of dilator responses and endothelial and vascular smooth muscle K+ channel function in cerebral arteries. Thus, it appears that the increased susceptibility to cerebrovascular abnormalities associated with IR and diabetes (including cerebral ischemia, stroke, vertebrobasilar transient ischemic attacks) is not due to an enhanced vasoreactivity to constrictor agents.     

Chronic nitric oxide synthase inhibition blunts endothelium-dependent function of conduit coronary arteries, not arterioles

Current literature suggests that chronic nitric oxide synthase (NOS) inhibition has differential effects on endothelium-dependent dilation (EDD) of conduit arteries vs. arterioles. Therefore, we hypothesized that chronic inhibition of NOS would impair EDD of porcine left anterior descending (LAD) coronary arteries but not coronary arterioles. Thirty-nine female Yucatan miniature swine were included in the study. Animals drank either tap water or water with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 mg/l), resulting in control and chronic NOS inhibition (CNI) groups, respectively. Treatment was continued for 1-3 mo (8.3 +/- 0.6 mg x kg(-1) x day(-1)). In vitro EDD of coronary LADs and arterioles was assessed via responses to ADP (LADs only) and bradykinin (BK), and endothelium-independent function was assessed via responses to sodium nitroprusside (SNP). Chronic NOS inhibition diminished coronary artery EDD to ADP and BK. Incubating LAD rings with L-NAME decreased relaxation responses of LADs from control pigs but not from CNI pigs such that between-group differences were abolished. Neither indomethacin (Indo) nor sulfaphenazole incubation significantly affected relaxation responses of LAD rings to ADP or BK. Coronary arteries from CNI pigs showed enhanced relaxation responses to SNP. In contrast to coronary arteries, coronary arterioles from CNI pigs demonstrated preserved EDD to BK and no increase in dilation responses to SNP. L-NAME, Indo, and L-NAME + Indo incubation did not result in significant between-group differences in arteriole dilation responses to BK. These results suggest that although chronic NOS inhibition diminishes EDD of LAD rings, most likely via a NOS-dependent mechanism, it does not affect EDD of coronary arterioles.     

Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release

The present study was performed to determine the importance of nitric oxide in eliciting epicardial coronary artery dilation during sustained increases in shear stress in the absence of pulsatile flow. Isolated first-order porcine epicardial coronary conduit arteries (approximately 500 microm) were preconstricted (U-46619) and subjected to steady-state changes in flow in vitro. Nonpulsatile flow (shear stress range from 0 to approximately 100 dyn/cm2) produced a graded dilation of epicardial arteries. Inhibiting nitric oxide synthase with 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) blocked bradykinin-induced vasodilation but did not affect the flow-diameter relation or the maximum change in diameter from static conditions (67 +/- 10 microm in control vs. 71 +/- 8 microm after L-NAME, P = not significant). The addition of indomethacin (10(-5) M) had no effect on flow-mediated vasodilation. Depolarizing vascular smooth muscle with KCl (60 mM) or removing the endothelium blocked bradykinin vasodilation and completely abolished flow-mediated responses. The K+ channel blocker tetraethylammonium chloride (TEA; 10(-4)M) attenuated flow-mediated vasodilation (maximum diameter change was 110 +/- 18 microm under control conditions vs. 58 +/- 10 microm after TEA, P < 0.001). These data indicate that epicardial coronary arteries dilate to steady-state changes in nonpulsatile flow via a mechanism that is independent of nitric oxide production. The ability to completely block this with KCl and attenuate it with TEA supports the hypothesis that epicardial coronary arteries dilate to steady levels of shear stress through hyperpolarization of vascular smooth muscle. This may be secondary to the release of an endothelium-dependent hyperpolarizing factor.     

Endothelium-dependent and -independent relaxation in the forelimb and hindlimb vasculatures of swine

Limb differences in endothelial function exist between arm and leg vasculatures of humans. The current investigation tested the hypothesis that forelimb and hindlimb vasorelaxation are similar in the absence of limb differences in blood pressure. Conduit arteries (brachials/femorals) and second order arterioles were harvested from 22 miniature Yucatan swine. In vitro assessment of vasorelaxation was determined by administering increasing doses of bradykinin (BK), acetylcholine (ACh), and sodium nitroprusside (SNP). The role of the nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways was assessed in conduit arteries but not resistance arterioles through L-NAME (300 microM) and INDO (5 microM) incubation, respectively. The relaxation responses to BK and ACh were similar in brachial and femoral arteries. SNP relaxation response was greater in the brachial compared to femoral arteries. There were also no significant differences in the relaxation responses of second order arterioles of the forelimb and hindlimb to BK, ACh, and SNP. Incubation of conduit arterial rings in L-NAME produced a greater reduction in BK and ACh relaxation in the brachial (approximately 25%) compared to femoral (approximately 13%) arterial rings. The current results of this investigation suggest that the forelimb and hindlimb vasculatures of swine have relatively similar vasorelaxation responses to both endothelium-dependent and -independent vasodilators.     

Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery

We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant role in the female cerebrovasculature. The contribution of EDHF to the ATP-mediated dilation was determined in middle cerebral arteries (MCAs) isolated from male and female rats. Four groups of rats were tested: intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20) rats. Maximal dilation to ATP was similar in all groups. However, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) and indomethacin (10(-5) M), the maximal dilation to ATP was significantly reduced in intact female (24 +/- 9%) and estrogen-treated ovariectomized female (29 +/- 9%) rats compared with intact male (95 +/- 4%) and vehicle-treated ovariectomized female (96 +/- 2%) rats. The ATP-mediated dilation in L-NAME- and indomethacin-treated MCAs isolated from male and ovariectomized female rats was inhibited by charybdotoxin (10(-7) M), an inhibitor of large-conductance Ca2+-sensitive K+ channels. We have defined EDHF as the L-NAME- and indomethacin-insensitive component of the ATP-mediated dilation. Our findings indicate that EDHF-mediated dilations are negligible in the female rat MCA; these dilations can be significantly enhanced after ovariectomy, suggesting that this effect is mediated by estrogen.     

Induction of iNOS restricts functional activity of both eNOS and nNOS in pig cerebral artery

The aim of the study was to investigate the effect of iNOS expression on eNOS and nNOS functional activity in porcine cerebral arteries. iNOS was induced in pig basilar arteries using lipopolysaccharide (LPS). Arteries expressing iNOS generated NO and relaxed when challenged with L-arginine (30 microM), an effect that was reduced by treatment with dexamethasone (coincubated with LPS) and prevented by the iNOS inhibitor 1400 W (administered 10 min prior to precontraction). eNOS was activated by A23187 and was found to be impaired in arteries that had iNOS induced (A23187 1 microM relaxation: control 110+/-8%, LPS-treated 50+/-16% ; p<0.05, N=5-6). This was due mainly to reduced formation of NO by A23187 (NO concentration in response to A23187 1 microM: control 25+/-6 nM, LPS-treated 0.8+/-1.2 nM; p<0.001, N=5-6), in addition to a small reduction in the vasodilator response to the NO-donors NOC-22 and SIN-1. Cerebral vasodilation produced by stimulation of intramural nitrergic nerves was impaired in arteries that had iNOS induced, and this was reversed by 1400 W (control 23+/-4% relaxation, LPS-treated 11+/-1% relaxation, LPS plus 1400 W 10 microM treated 25+/-2% relaxation; p<0.01 for control versus LPS, N=6). It is concluded that the induction of iNOS in cerebral arteries reduces NO-mediated vasodilation initiated by eNOS and by nNOS, primarily by modulation of NO formation.     

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.     

Exercise attenuates the effects of hypercholesterolemia on endothelium-dependent relaxation in coronary arteries from adult female pigs.

We tested the hypothesis that exercise training (Ex) attenuates the effects of hypercholesterolemia on endothelium-dependent relaxation in left anterior descending coronary arteries. Adult female pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were trained or remained sedentary (Sed) for 16 wk, yielding four groups of pigs: 1) NF-Sed, 2) NF-Ex, 3) HF-Sed, and 4) HF-Ex. Sensitivity (EC(50)) to bradykinin (BK) was impaired in HF-Sed arteries. Ex improved BK-induced relaxation such that the EC(50) and maximal response to BK in HF-Ex arteries was not different from that in NF-Sed and NF-Ex. ACh-induced constriction was less in HF-Ex arteries than in HF-Sed, NF-Sed, and NF-Ex. To determine the mechanism(s) by which HF and Ex affected responses to BK and ACh, vasoactive responses were assessed in the presence of N(G)-nitro-L-arginine methyl ester [L-NAME; to inhibit nitric oxide (NO) synthase], indomethacin (Indo; to inhibit cyclooxygenase), and L-NAME + Indo. L-NAME inhibited BK-induced relaxation in NF (not HF) arteries. Indo did not significantly alter relaxation to BK in NF arteries; however, relaxation was enhanced in HF-Sed arteries. Double blockade with L-NAME + Indo attenuated BK-induced relaxation in NF arteries and eliminated relaxation in HF arteries. Neither L-NAME nor Indo altered constrictor responses to ACh in NF or HF arteries; however, double blockade with L-NAME + Indo attenuated constriction to ACh in NF-Ex arteries. Endothelium-independent relaxation to sodium nitroprusside was enhanced in HF-Sed and HF-Ex arteries. Collectively, these results indicate that HF impaired endothelial function in coronary arteries by impairing production of NO and by enhancing production of a constrictor that was inhibited by Indo. Ex attenuated the effects of hypercholesterolemia by improving NO-mediated, endothelium-dependent relaxation and by reducing the influence of the Indo-sensitive constrictor.     

Effect of gender on endothelium-dependent dilation to bradykinin in human adipose microvessels

We examined the influence of gender and climacteric status, two coronary risk factors, on bradykinin (BK)-induced dilation in adipose arterioles from men and women of different ages [premenopausal women (Pre-W), postmenopausal women (Post-W), and similar aged men (Y-M and O-M), respectively]. We examined the responses from both omental (more closely associated with coronary disease) and subcutaneous fat. Tissues were obtained at surgery and cannulated (60 mmHg) for measurement of internal diameter. In vessels from omental tissue, dilation to BK was more sensitive in Pre-W than other groups, whereas in vessels from subcutaneous tissue, sensitivity to BK was greater in both Pre-W and Post-W compared with Y-M and O-M. Maximal dilation was similar among groups. Indomethacin (Indo; 10(-5) M) alone had no effect on dilation to BK in any groups, but Indo and N(omega)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M) reduced dilation to BK in Pre-W more than in Y-M. L-NAME increased dilation to BK in subcutaneous fat from Y-M but had no effect in Post-W and O-M. Indo- and L-NAME-resistant dilation in all vessels was markedly reduced by 30 mM KCl. There was no difference in sodium nitroprusside-induced dilation among groups. We conclude that gender and climacteric state contribute to mechanisms of microvascular regulation in humans. Functional vascular differences in visceral and subcutaneous fat may underlie the proposed differential influence of these tissues on cardiovascular risk.     

Maturational modulation of endothelium-dependent vasodilatation in ovine cerebral arteries

To address the hypothesis that maturation enhances endothelial vasodilator function in cerebral arteries, relaxant responses to ADP and A-23187 were determined in ovine carotid and cerebral arteries harvested from 25 newborn lambs (3-7 days) and 23 adult sheep. Maturation significantly increased pD(2) values for A-23187 (newborn range: 4.9 +/- 0.3 to 5.4 +/- 0.3; adult range: 6.0 +/- 0.2 to 7.1 +/- 0.2) and the maximal vasodilator response to A-23187 by 10-18%. In contrast, maturation decreased maximum responses to ADP by 5-25% with no change in pD(2). The magnitudes of endothelium-dependent relaxation were not affected by 10 microM indomethacin but were virtually abolished by 100 microM N(G)-nitro-L-arginine methyl ester/L-nitro arginine, indicating that nitric oxide (NO) is the primary endothelium-dependent vasodilator in these arteries. Maturation also modestly decreased endothelial NO synthase (eNOS) abundance in both carotid (32%) and cerebral (26%) arteries. Together, these findings reinforce the view that receptor coupling to endothelial activation is tightly regulated and may offset underlying changes in maximal endothelial vasodilator capacity. This capacity, in turn, appears to increase with postnatal age despite major growth and expansion of endothelial cell size and vascular wall volume. In ovine cerebral arteries, endothelial vasodilator capacity appears completely dependent on eNOS activity but not on cyclooxygenase activity. In turn, eNOS activity appears to be postnatally regulated by mechanisms independent of changes in eNOS abundance alone.     

Nitric oxide-mediated nonadrenergic noncholinergic relaxation of piglet pulmonary arteries decreases with postnatal age.

Nonadrenergic noncholinergic (NANC) vasodilator mechanisms may contribute to the maintenance of adult pulmonary and systemic vascular tone. However, their actions in the neonatal circulation have not been studied. We aimed to investigate NANC vasorelaxation in neonatal and 2-week-old piglet pulmonary and mesenteric arteries and to examine the potential role of nitric oxide (NO) in this phenomenon. Responses to electric field stimulation (EFS, 50V, 0.25-32 Hz) were investigated in pulmonary and mesenteric artery rings (external diameter 150-200 microm) precontracted with the thromboxane A2 mimetic U46619, in the presence of guanethidine (10 microM) and atropine (10 microM). Under these conditions, EFS resulted in a frequency dependent relaxation of newborn pulmonary (maximal relaxation of 53+/-9.1%), mesenteric (68.8.2+/-7.1%) and 2-wk-old mesenteric (46 6.3%) arteries but this relaxation was significantly reduced (4.5+/-2.2%) in 2-week-old pulmonary arteries. In neonatal pulmonary arteries, the neurotoxin tetrodotoxin (0.3 muM), the NO synthase inhibitor L-NAME (0.1 mM), and the guanylyl cyclase inhibitor ODQ (10 microM) abolished EFS-induced relaxations, suggesting that NANC relaxation of porcine neonatal pulmonary arteries is mediated by NO, which is probably neuronal in origin. However, The expression in pulmonary arteries of the neuronal NO synthase (nNOS), as determined by Western-blot analysis, increased with postnatal age whereas the expression of the endothelial NOS (eNOS) did not change. In conclusion, NANC relaxation is present in neonatal pulmonary and mesenteric arteries and it is, at least partially, mediated through NO. NANC relaxation of porcine pulmonary and mesenteric arteries decreases with postnatal maturation.     

Urotensin-II is a nitric oxide-dependent vasodilator in the pial arteries of the newborn pig

Urotensin-II (UT-II) is a small circular peptide and is described as the most potent endogenous vasoconstrictor in various vascular beds. However, the in vivo effects of UT-II can be either vasoconstriction or vasodilation depending on the species and the tissue investigated. The present study sought to characterize the vasoactive effect of UT-II in the piglet cerebral circulation in vivo. Pial arteries of 99 +/- 6 microm were visualized with intravital microscopy through a closed cranial window in anesthetized newborn piglets. Topical application of UT-II elicited a weak dose-dependent vasodilation of the arteries (0.001 microM: 3 +/- 3 microm, 0.1 microM: 10 +/- 5 microm, 10 microM: 14 +/- 7 microm). Smaller arteries with an initial diameter below 100 microm showed minimal or no vasodilation, while larger arteries between 100 and 120 microm had a pronounced dose-dependent effect. Systemic application of 15 mg/kg Nomega-nitro-L-arginine-methyl ester (L-NAME) completely inhibited the vasodilation. We conclude that UT-II, in contrast to most other vascular beds, is a weak NO-dependent vasodilator in the piglet pial vasculature.     

Effects of carbon monoxide and heme oxygenase inhibitors in cerebral vessels of rats and mice

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10(-6) to 10(-4) M). Mouse MCAs, however, dilated by 21 +/- 10% at 10(-4) M CO. Authentic nitric oxide (NO., 10(-10) to 10(-7) M) dilated both rat and mouse MCAs. At 10(-8) M NO., rat vessels dilated by 84 +/- 4%, and at 10(-7) M NO., mouse vessels dilated by 59 +/- 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor delta-aminolevulinic acid (10(-10) to 10(-4) M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 +/- 2% at 3 x 10(-5) M). Importantly, this constriction was unaltered by exogenous CO (10(-4) M) or CO plus 10(-5) M biliverdine (both HO products). In contrast, exogenous CO (10(-4) M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 +/- 1% in response to 10(-5) M CrMP. Magnesium protoporphyrin IX (10(-5) M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries.     

Potassium (BK(Ca)) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats

Insulin resistance (IR) syndrome is associated with impaired vascular relaxation; however, the underlying pathophysiology is unknown. Potassium channel activation causes vascular smooth muscle hyperpolarization and relaxation. The present study determined whether a reduction in large conductance calcium- and voltage-activated potassium (BK(Ca)) channel activity contributes to impaired vascular relaxation in IR rats. BK(Ca) channels were characterized in mesenteric microvessels from IR and control rats. Macroscopic current density was reduced in myocytes from IR animals compared with controls. In addition, inhibition of BK(Ca) channels with tetraethylammonium (1 mM) or iberiotoxin (100 nM) was greater in myocytes from control (70%) compared with IR animals (approximately 20%). Furthermore, activation of BK(Ca) channels with NS-1619 was three times more effective at increasing outward current in cells from control versus IR animals. Single channel and Western blot analysis of BK(Ca) channels revealed similar conductance, amplitude, voltage sensitivity, Ca2+ sensitivity, and expression density between the two groups. These data provide the first direct evidence that microvascular potassium currents are reduced in IR and suggest a molecular mechanism that could account for impaired vascular relaxation in IR.     

Vasoconstrictor mechanisms in the cerebral circulation are unaffected by insulin resistance

Insulin-resistance (IR) impairs agonist-induced relaxation in cerebral arteries, but little is known about its effect on constrictor mechanisms. We examined the vascular responses of the basilar artery (BA) and its side branches in anesthetized Zucker lean (ZL) and IR Zucker obese (ZO) rats using a cranial window technique. Endothelin-1 (ET-1) constricted the BAs in both the ZL and ZO rats, but there was no significant difference between the two groups (ZL: 36 +/- 8%; ZO: 33 +/- 3% at 10(-8) M). Inhibition of the ET(A) receptors by BQ-123 slightly increased the diameters of the BAs, with no difference shown between the ZL (6 +/- 1%) and ZO (5 +/- 3%) rats. Expressions of the ET(A) receptors and ET-1 mRNA examined by immunoblot analysis and RT-PCR, respectively, were also similar in the ZL and ZO groups. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), and the thromboxane A(2) (TxA(2)) mimetic U-46619 constricted the BAs, but similarly to ET-1, there was no significant difference between the ZL and ZO groups (10(-6) M PDBu: ZL: 33 +/- 2%; ZO: 32 +/- 4%; and 10(-7) M U-46619: ZL: 23 +/- 1%; ZO: 19 +/- 2%). Inhibition of Rho-kinase with Y-27632 induced dilation of the BAs, and these responses were also comparable in the ZL and ZO rats (ZL: 39 +/- 4%; ZO: 38 +/- 2% at 10(-5) M). In contrast, nitric oxide-dependent relaxation to bradykinin was significantly reduced in the ZO rats (10(-6) M: 10 +/- 3%) compared with ZLs (29 +/- 7%, P < 0.01). These findings indicate that vasoconstrictor responses of the BA mediated by ET-1, TxA(2), PKC, and Rho-kinase are not affected by IR.     

Hydrogen peroxide acts as an EDHF in the piglet pial vasculature in response to bradykinin

We investigated the mechanism of EDHF-mediated dilation to bradykinin (BK) in piglet pial arteries. Topically applied BK (3 micromol/l) induced vasodilation (62 +/- 12%) after the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which was inhibited by endothelial impairment or by the BK(2) receptor antagonist HOE-140 (0.3 micromol/l). Western blotting showed the presence of BK(2) receptors in brain cortex and pial vascular tissue samples. The cytochrome P-450 antagonist miconazole (20 micromol/l) and the lipoxygenase inhibitors baicalein (10 micromol/l) and cinnamyl-3,4-dyhydroxy-alpha-cyanocinnamate (1 micromol/l) failed to reduce the BK-induced dilation. However, the H(2)O(2) scavenger catalase (400 U/ml) abolished the response (from 54 +/- 11 to 0 +/- 2 microm; P < 0.01). The ATP-dependent K(+) (K(ATP)) channel inhibitor glibenclamide (10 micromol/l) had a similar effect as well (from 54 +/- 11 to 16 +/- 5 microm; P < 0.05). Coapplication of the Ca(2+)-dependent K(+) channel inhibitors charybdotoxin (0.1 micromol/l) and apamin (0.5 micromol/l) failed to reduce the response. We conclude that H(2)O(2) mediates the non-nitric oxide-, non-prostanoid-dependent vasorelaxation to BK in the piglet pial vasculature. The response is mediated via BK(2) receptors and the opening of K(ATP) channels.     

Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance

We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca(2+)-activated K(+) (BK(Ca)) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BK(Ca) channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, N(omega)-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BK(Ca) activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BK(Ca) channel activity in IR myocytes, whereas in the I/O configuration, BK(Ca) channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through K(Ca) channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BK(Ca) channel.     

The vasorelaxation of cerebral arteries by carbon monoxide

Carbon monoxide (CO) is known to increase cerebral blood flow, but the effect of CO on the vascular tone of large cerebral arteries is uncertain. We tested whether CO affects cerebral artery tone by measuring tension generated by ex vivo segments of dog basilar artery upon exposure to CO. In cerebral artery segments contracted with either KCl or prostaglandin F(2alpha), CO caused a concentration-related relaxation beginning with a concentration of 57 microM. Relaxation did not occur if CO was administered in the presence of bubbling carboxygen (95% O(2):5% CO(2)), which reduces greater than 99% of CO from the solution. Furthermore, the CO-induced relaxation of cerebral artery segments was reduced in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM)or the potassium channel blocker tetraethylammonium (TEA, 1 mM). Neither ODQ nor TEA completely eliminated the relaxation caused by CO and there was no additive effect if ODQ and TEA were administered together. These results suggest that cerebral arteries are directly relaxed by CO and that this relaxation depends upon the activation of guanylyl cyclase and the opening of potassium channels.