Exercise training preserves endothelium-dependent relaxation in brachial arteries from hyperlipidemic pigs.

We tested the hypothesis that exercise training (Ex) attenuates the effects of hyperlipidemia on endothelial function by enhancing NO-mediated vasorelaxation in porcine brachial (Br) arteries. Adult female pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20 wk. Four weeks after initiation of the diet, pigs underwent Ex or remained sedentary (Sed) for 16 wk. Relaxation to ACh was impaired by HF (P = 0.03). The combination of HF and Sed impaired ACh-induced relaxation more than HF or Sed alone (P = 0.0002). Relaxation to high doses of bradykinin (BK) was impaired by HF (P = 0.0002). Ex significantly improved ACh-induced relaxation (P = 0.01) and tended to improve relaxation to BK (P = 0.38). To determine the mechanism(s) by which HF and Ex affected relaxation to ACh and BK, relaxation was assessed in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. In the presence of l-NAME, Indo, or l-NAME + Indo, ACh-induced relaxation was no longer different between HF and NF arteries; however, relaxation remained greater in Ex than in Sed arteries. In the presence of l-NAME or Indo, BK-induced relaxation was no longer altered by HF but was enhanced by Ex. In the presence of l-NAME + Indo, BK-induced relaxation was enhanced by HF and Ex. These data indicate that hyperlipidemia impairs ACh- and BK-induced relaxation by impairing NO- and PGI(2)-mediated relaxation. Ex attenuates the effects of HF by enhancing a vasodilator mechanism independent of NO and PGI(2).     

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.     

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.     

Chronic exercise training does not alter pulmonary vasorelaxation in normal pigs.

Exercise training increases acetylcholine-induced pulmonary vasorelaxation in pigs with coronary occlusion. The present study tested the hypothesis that chronic exercise training enhances endothelium-mediated vasorelaxation in pulmonary arteries from normal pigs. Yucatan miniswine exercised for 16 wk on a treadmill (Ex); control pigs (Sed) remained in pens. Pulmonary artery rings (2- to 3-mm OD) were studied using standard isometric techniques. Contractile responses to 80 mM KCl and norepinephrine (NE) were determined. Vessels were constricted with levels of NE that resulted in half-maximal contraction to examine endothelium-dependent relaxation to ACh and endothelium-independent relaxation to sodium nitroprusside in the presence and absence of nitric oxide synthase inhibition, cyclooxygenase inhibition, and endothelial denudation. Arteries from Ex pigs developed increased contraction to 80 mM KCl, but the response to NE did not differ between groups. Endothelium-dependent and endothelium-independent responses did not differ between Sed and Ex in the presence or absence of pharmacological inhibitors or denudation. We conclude that chronic exercise training does not alter endothelium-dependent or endothelium-independent vasorelaxation responses of pulmonary arteries from normal pigs.     

Exercise preserves endothelium-dependent relaxation in coronary arteries of hypercholesterolemic male pigs.

We tested the hypothesis that exercise training (Ex) attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in male porcine coronary arteries [left anterior descending coronary arteries (LAD)] by increasing nitric oxide (NO) release [due to increased endothelial NO synthase (NOS) expression] and/or increased bioactivity of NO. Adult male pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20-24 wk. Pigs were Ex or remained sedentary (Sed) for 16-20 wk, beginning after 4 wk on diet. Four groups of pigs were used: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. HF enhanced LAD contractions induced by KCl, aggregating platelets (AP), and serotonin (5-HT). AP and 5-HT produced EDR after blockade of cyclooxygenase with indomethacin (Indo) and smooth-muscle 5-HT(2) receptors with ketanserin. HF impaired EDR induced by AP, 5-HT, and bradykinin. Results indicate a decreased contribution of NO to EDR in HF-Sed LADs, because the percentage of bradykinin-induced EDR inhibited by N(G)-nitro-L-arginine methyl ester was 27% in NF-Sed and 34% in NF-Ex but only 17% in HF-Sed. Also, N(G)-nitro-L-arginine methyl ester + Indo results indicate that release of an Indo-sensitive vasoconstrictor contributes to blunted EDR in HF-Sed LAD. Immunoblot and immunohistochemistry results indicate the following: 1) LAD endothelial NOS protein content was similar among groups; 2) HF decreased LAD superoxide dismutase (SOD) but increased caveolin-1 content; and 3) Ex increased SOD content of HF LADs. We conclude that HF impairs EDR by impairing the contribution of NO released from NOS (due to decreased SOD and increased caveolin-1 protein content) and by production of an Indo-sensitive vasoconstrictor. Ex preserves EDR in HF LADs by decreasing the production of the constrictor and increasing NO-release by NOS and/or NO bioactivity and bioavailability.     

Chronic exercise training improves ACh-induced vasorelaxation in pulmonary arteries of pigs.

We hypothesized that exercise training would lead to enhanced endothelium-dependent vasodilation in porcine pulmonary arteries. Pulmonary artery rings (2- to 3-mm OD) were obtained from female Yucatan miniature swine with surgically induced coronary artery occlusion (ameroid occluder). Exercise training was performed for 16 wk, and vasomotor responses were studied by using standard isometric techniques. Contractile responses to 80 mM KCl, isosmotic KCl (10-100 mM), and norepinephrine (10(-8) to 10(-4) M) did not differ between sedentary (Sed) and exercise-trained (Ex) pigs. Relaxation was assessed to endothelium-dependent and endothelium-independent vasodilators after norepinephrine contraction. Pulmonary arteries of Ex pigs exhibited greater maximal relaxation to ACh (61.9 +/- 3.5%) than did those of Sed pigs (52.3 +/- 3.9%; P < 0.05). Endothelium-independent relaxation to sodium nitroprusside did not differ. Inhibition of nitric oxide synthase significantly decreased acetylcholine-induced relaxation, with greater inhibition in arteries from Ex pigs (P < 0.05). Inhibition of cyclooxygenase enhanced relaxation to acetylcholine in arteries from Sed pigs. We conclude that exercise training enhances endothelium-dependent (ACh-mediated) vasorelaxation in pulmonary arteries by mechanisms of increased reliance on nitric oxide and reduced production of a prostanoid constrictor.     

Impaired endothelium-mediated relaxation in isolated cerebral arteries from insulin-resistant rats

Insulin resistance (IR) impairs vascular responses in peripheral arteries. However, the effects of IR on cerebrovascular control mechanisms are completely unexplored. We examined the vascular function of isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats. Endothelium-dependent vasodilation elicited by bradykinin (BK) was reduced in IR compared with control MCAs. Maximal dilation to BK (10(-6) M) was 38 +/- 3% (n = 13) in control and 19 +/- 3% (n = 10) in IR arteries (P < 0.01). N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) decreased responses to BK in control arteries by approximately 65% and inhibited the already reduced responses completely in IR MCAs. Indomethacin (10 microM) reduced relaxation to BK in control MCAs by approximately 40% but was largely ineffective in IR arteries. Combined L-NAME and indomethacin treatments eliminated the BK-induced dilation in both groups. Similarly to BK, endothelium-mediated and mainly cyclooxygenase (COX)-dependent dilation to calcium ionophore A23187 was reduced in IR arteries compared with controls. In contrast, vascular relaxation to sodium nitroprusside was similar between the IR and control groups. These findings demonstrate that endothelium-dependent dilation in cerebral arteries is impaired in IR primarily because of a defect of the COX-mediated pathways. In contrast, nitric oxide-mediated dilation remains intact in IR arteries.     

Endothelial function in coronary arterioles from pigs with early-stage coronary disease induced by high-fat, high-cholesterol diet: effect of exercise.

Because hypercholesterolemia can attenuate endothelial function and exercise training can augment endothelial function, we hypothesized that exercise training would improve endothelial function of coronary arterioles from pigs in the early stages of cardiovascular disease induced by a high-fat, high-cholesterol (HF) diet. Yucatan miniature swine were fed a normal-fat (NF) diet or HF diet (2% cholesterol) for 20 wk in which 8 and 46% of their calories were derived from fat, respectively. Both groups were subdivided into sedentary (Sed) or exercise-trained (Ex) groups. This resulted in four experimental groups: NFSed, NFEx, HFSed, and HFEx. Endothelial function was assessed in coronary arterioles 75-100 microm in diameter dissected from the left ventricular apex. Responses to endothelial-dependent dilation induced by bradykinin (BK), ADP, and flow were similar in all four groups, whereas dilation to aggregating platelets in the presence of indomethacin and ketanserin was attenuated in HFSed arterioles (P = 0.01). The attenuated response to aggregating platelets was prevented or reversed in HFEx arterioles (P = 0.03). In HFSed arterioles, BK induced release of an indomethacin-sensitive prostanoid constrictor. In contrast, after exercise training, there was no evidence of this constrictor and BK-induced release of an indomethacin-sensitive prostanoid dilator in HFEx arterioles (P = 0.04). Endothelial nitric oxide synthase protein in arterioles was significantly reduced in HF groups (P < 0.05) and increased in Ex groups (P < 0.05). Interestingly, the relative contribution of nitric oxide to BK-induced dilation, as assessed with nitro-L-arginine methyl ester, was similar in arterioles in the NF, HF, Sed, and Ex groups. These results suggest that, in the early stages of cardiovascular disease, a high-fat, high-cholesterol diet has modest effects on endothelial-dependent dilation in coronary arterioles; nonetheless, these effects are prevented or reversed with exercise training.     

Acetylcholine causes endothelium-dependent contraction of mouse arteries

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).     

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.     

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.     

Connexin 43 mediates endothelium-derived hyperpolarizing factor-induced vasodilatation in subcutaneous resistance arteries from healthy pregnant women

The role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation of human arteries was assessed using connexin mimetic peptides (CMPs) designated (37,43)Gap27, (40)Gap27, and (43)Gap26 according to homology with the major vascular connexins (Cx37, Cx40, and Cx43). Resistance arteries were obtained from subcutaneous fat biopsies of healthy pregnant women undergoing elective cesarean section. Endothelium-dependent vasodilatation to bradykinin (BK) was assessed using wire myography. N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (nitric oxide synthase and cyclooxygenase inhibitors, respectively) attenuated maximal relaxation to BK (R(max)) by approximately 50%. Coincubation with l-NAME, indomethacin, and the combined CMPs ((37,43)Gap27, (40)Gap27, and (43)Gap26) almost abolished relaxation to BK (R(max) = 12.2 +/- 3.7%). In arteries incubated with l-NAME and indomethacin, the addition of either (37,43)Gap27 or (40)Gap27 had no significant effect on R(max), whereas (43)Gap26 caused marked inhibition (R(max) = 21 +/- 6.4%, P = 0.005 vs. l-NAME plus indomethacin alone) that was similar to that of the triple combination. Endothelium-independent vasorelaxation was unaffected by CMPs, l-NAME, or indomethacin. Immunohistochemistry demonstrated Cx37, Cx40, and Cx43 expression in the endothelium and vascular smooth muscle. In pregnant women, EDHF-mediated vasorelaxation of subcutaneous resistance arteries is dependent on Cx43 and gap junctions.     

Cyclic ADP ribose-mediated Ca2+ signaling in mediating endothelial nitric oxide production in bovine coronary arteries

The present study tested the hypothesis that cyclic ADP ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca2+ mobilization in coronary arterial endothelial cells (CAECs) and thereby contributes to endothelium-dependent vasodilation. In isolated and perfused small bovine coronary arteries, bradykinin (BK)-induced concentration-dependent vasodilation was significantly attenuated by 8-bromo-cADPR (a cell-permeable cADPR antagonist), ryanodine (an antagonist of ryanodine receptors), or nicotinamide (an ADP-ribosyl cyclase inhibitor). By in situ simultaneously fluorescent monitoring, Ca2+ transient and nitric oxide (NO) levels in the intact coronary arterial endothelium preparation, 8-bromo-cADPR (30 microM), ryanodine (50 microM), and nicotinamide (6 mM) substantially attenuated BK (1 microM)-induced increase in intracellular [Ca2+] by 78%, 80%, and 74%, respectively, whereas these compounds significantly blocked BK-induced NO increase by about 80%, and inositol 1,4,5-trisphosphate receptor blockade with 2-aminethoxydiphenyl borate (50 microM) only blunted BK-induced Ca2+-NO signaling by about 30%. With the use of cADPR-cycling assay, it was found that inhibition of ADP-ribosyl cyclase by nicotinamide substantially blocked BK-induced intracellular cADPR production. Furthermore, HPLC analysis showed that the conversion rate of beta-nicotinamide guanine dinucleotide into cyclic GDP ribose dramatically increased by stimulation with BK, which was blockable by nicotinamide. However, U-73122, a phospholipase C inhibitor, had no effect on this BK-induced increase in ADP-ribosyl cyclase activity for cADPR production. In conclusion, these results suggest that cADPR importantly contributes to BK- and A-23187-induced NO production and vasodilator response in coronary arteries through its Ca2+ signaling mechanism in CAECs.     

Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting and resistance vessels

Bradykinin (BK) is a potent dilator of the perinatal pulmonary circulation. We investigated segmental differences in BK-induced dilation in newborn pig large conducting pulmonary artery and vein rings and in pressurized pulmonary resistance arteries (PRA). In conducting pulmonary arteries and veins, BK-induced relaxation is abolished by endothelial disruption and by inhibition of nitric oxide (NO) synthase with nitro-L-arginine (L-NA). In PRA, two-thirds of the dilation response is L-NA insensitive. Charybdotoxin plus apamin and depolarization with KCl abolish the L-NA-insensitive dilations, findings that implicate the release of endothelium-derived hyperpolarizing factor (EDHF). However, endothelium-disrupted PRA retain the ability to dilate to BK but not to ACh or A-23187. In endothelium-disrupted PRA, dilation was inhibited by charybdotoxin. Thus in PRA, BK elicits dilation by multiple and duplicative signaling pathways. Release of NO and EDHF contributes to the response in endothelium-intact PRA; in endothelium-disrupted PRA, dilation occurs by direct activation of vascular smooth muscle calcium-dependent potassium channels. Redundant signaling pathways mediating pulmonary dilation to BK may be required to assure a smooth transition to extrauterine life.     

Oxidized low density lipoprotein inhibits bradykinin-induced phosphoinositide hydrolysis in cultured bovine aortic endothelial cells.

Vascular endothelial cells, in response to various neurohumoral and physical stimuli, produce an endothelium-derived relaxing factor, a substance which regulates vascular tone. We have demonstrated that oxidized low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. We studied the effect of oxidized LDL on inositol phosphates formation stimulated with bradykinin (BK) in cultured bovine aortic endothelial cells. BK elicited a rapid generation of inositol phosphates from inositol phospholipids. Accumulation of inositol 1,4,5-trisphosphate (IP3) stimulated with BK (0.1 microM) was markedly inhibited by oxidized LDL. However, native LDL had little effect on BK-induced accumulation of IP3. From these results, oxidized LDL inhibits receptor-mediated phosphoinositides hydrolysis and modulates the endothelial function.     

Functional characterization of the mechanisms underlying bradykinin-induced relaxation in the isolated rat carotid artery

This work aimed to functionally characterize the mechanisms underlying the relaxation induced by bradykinin (BK) in the rat carotid artery. Vascular reactivity experiments, using standard muscle bath procedures, showed that BK (0.1 nmol/L-3 mumol/L) induced relaxation of phenylephrine-pre-contracted rings in a concentration-dependent manner. Endothelial removal strongly attenuated BK-induced relaxation. HOE-140, the selective antagonist of bradykinin B(2) receptors concentration-dependently reduced the relaxation induced by BK. Pre-incubation of endothelium-intact rings with L-NAME (100 micromol/L), a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 micromol/L), a selective inhibitor of the eNOS or 7-nitroindazole (100 micromol/L), the selective inhibitor of nNOS, reduced BK-induced relaxation. Conversely, 1400 W (10 nmol/L), a selective inhibitor of iNOS, did not alter the relaxation induced by BK. Surprisingly, indomethacin (10 micromol/L) a non-selective inhibitor of cyclooxygenase (COX) increased BK-induced relaxation in endothelium-intact but not denuded rings. Neither SQ29548 (3 micromol/L), a competitive antagonist of PGH(2)/TXA(2) receptors nor AH6809 (10 micromol/L), an antagonist of PGF(2alpha) receptors significantly altered the relaxation induced by BK in endothelium-intact rings. The combination of SQ29548 and AH6809 increased BK-induced relaxation. The present study shows that the vasorelaxant action displayed by BK in the rat carotid is mediated by endothelial B(2) receptors and the activation of the NO pathway. The major finding of this work is that it demonstrated functionally that endothelial-derived vasoconstrictor prostanoids (probably PGH(2), TXA(2) and PGF(2alpha)) counteract the vasorelaxant action displayed by BK.     

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.     

Coronary microvascular dysfunction in a porcine model of early atherosclerosis and diabetes

Detailed evaluation of coronary function early in diabetes mellitus (DM)-associated coronary artery disease (CAD) development is difficult in patients. Therefore, we investigated coronary conduit and small artery function in a preatherosclerotic DM porcine model with type 2 characteristics. Streptozotocin-induced DM pigs on a saturated fat/cholesterol (SFC) diet (SFC + DM) were compared with control pigs on SFC and standard (control) diets. SFC + DM pigs showed DM-associated metabolic alterations and early atherosclerosis development in the aorta. Endothelium-dependent vasodilation to bradykinin (BK), with or without blockade of nitric oxide (NO) synthase, endothelium-independent vasodilation to an exogenous NO-donor (S-nitroso-N-acetylpenicillamine), and vasoconstriction to endothelin (ET)-1 with blockade of receptor subtypes, were assessed in vitro. Small coronary arteries, but not conduit vessels, showed functional alterations including impaired BK-induced vasodilatation due to loss of NO (P < 0.01 vs. SFC and control) and reduced vasoconstriction to ET-1 (P < 0.01 vs. SFC and control), due to a decreased ET(A) receptor dominance. Other vasomotor responses were unaltered. In conclusion, this model demonstrates specific coronary microvascular alterations with regard to NO and ET-1 systems in the process of early atherosclerosis in DM. In particular, the altered ET-1 system correlated with hyperglycemia in atherogenic conditions, emphasizing the importance of this system in DM-associated CAD development.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

Short-term training enhances endothelium-dependent dilation of coronary arteries, not arterioles.

Our objective was to test the hypothesis that short-term exercise training (STR) of pigs increases endothelium-dependent dilation (EDD) of coronary arteries but not coronary arterioles. Female Yucatan miniature swine ran on a treadmill for 1 h, at 3.5 mph, twice daily for 7 days (STR; n = 28). Skeletal muscle citrate synthase activity was increased in STR compared with sedentary controls (Sed; n = 26). Vasoreactivity was evaluated in isolated segments of conduit arteries (1-2 mm ID, 3-4 mm length) mounted on myographs and in arterioles (50-100 microm ID) isolated and cannulated with micropipettes with intraluminal pressure set at 60 cmH(2)O. EDD was assessed by examining responses to increasing concentrations of bradykinin (BK) (conduit arteries 10(-12)-10(-6) M and arterioles 10(-13)-10(-6) M). There were no differences in maximal EDD or BK sensitivity of coronary arterioles from Sed and STR hearts. In contrast, sensitivity of conduit arteries (precontracted with PGF(2alpha)) to BK was increased significantly (P < 0.05) in STR (EC(50), 2.33 +/- 0.62 nM, n = 12) compared with Sed animals (EC(50), 3.88 +/- 0.62 nM, n = 13). Immunoblot analysis revealed that coronary arteries from STR and Sed animals had similar levels of endothelial nitric oxide synthase (eNOS). In contrast, eNOS protein was increased in STR aortic endothelial cells. Neither protein nor mRNA levels of eNOS were different in coronary arterioles from STR compared with Sed animals. STR did not alter expression of superoxide dismutase (SOD-1) protein in any artery examined. We conclude that pigs exhibit increases in EDD of conduit arteries, but not in coronary arterioles, at the onset of exercise training. These adaptations in pigs do not appear to be mediated by alterations in eNOS or SOD-1 expression.