Bradykinin-mediated Relaxation Research Articles

Time-dependent activation of prostacyclin and nitric oxide pathways during continuous i.v. infusion of serelaxin (recombinant human H2 relaxin).

In the RELAX-AHF trial, a 48 h i.v. serelaxin infusion reduced systemic vascular resistance in patients with acute heart failure. Consistent with preclinical studies, serelaxin augments endothelial vasodilator function in rat mesenteric arteries. Little is known about the contribution of endothelium-derived relaxing factors after a longer duration of continuous serelaxin treatment. Here we have assessed vascular reactivity and mechanistic pathways in mesenteric arteries and veins and the aorta after 48 or 72 h continuous i.v. infusion of serelaxin. Male rats were infused with either placebo or serelaxin (13.3 μg·kg(-1) ·h(-1) ) via the jugular vein using osmotic minipumps. Vascular function was assessed using wire myography. Changes in gene and protein expression and 6-keto PGF1α levels were determined by quantitative PCR, Western blot and ELISA respectively. Continuous i.v. serelaxin infusion augmented endothelium-dependent relaxation in arteries (mesenteric and aorta) but not in mesenteric veins. In mesenteric arteries, 48 h i.v. serelaxin infusion increased basal NOS activity, associated with increased endothelial NOS (eNOS) expression. Interestingly, phosphorylated-eNOS(Ser1177) , eNOS and basal NOS activity were reduced in mesenteric arteries following 72 h serelaxin treatment. At 72 h, serelaxin treatment improved bradykinin-mediated relaxation through COX2-derived PGI2 production. Continuous i.v. serelaxin infusion enhanced endothelial vasodilator function in arteries but not in veins. The underlying mediator at 48 h was NO but there was a transition to PGI2 by 72 h. Activation of the PGI2 -dependent pathway is key to the prolonged vascular response to serelaxin treatment.

Exercise Training‐Induced Adaptations in Mediators of Sustained Endothelium‐Dependent Coronary Artery Relaxation in a Porcine Model of Ischemic Heart Disease

The aim of this study was to test the hypothesis that exercise training enhances sustained relaxation to persistent endothelium-dependent vasodilator exposure via increased nitric oxide contribution in small coronary arteries of control and ischemic hearts. Yucatan swine were designated to a control group or a group in which an ameroid constrictor was placed around the proximal LCX. Subsequently, pigs from both groups were assigned to exercise (five days/week; 16 weeks) or SED regimens. Coronary arteries (~100-350 μm) were isolated from control pigs and from both nonoccluded and collateral-dependent regions of chronically-occluded hearts. In arteries from control pigs, training significantly enhanced relaxation responses to increasing concentrations of bradykinin (10(-10) -10(-7) M) and sustained relaxation to a single bradykinin concentration (30 nM), which were abolished by NOS inhibition. Training also significantly prolonged bradykinin-mediated relaxation in collateral-dependent arteries of occluded pigs, which was associated with more persistent increases in endothelial cellular Ca(2+) levels, and reversed with NOS inhibition. Protein levels for eNOS and p-eNOS-(Ser1179), but not caveolin-1, Hsp90, or Akt, were significantly increased with occlusion, independent of training state. Exercise training enhances sustained relaxation to endothelium-dependent agonist stimulation in small arteries of control and ischemic hearts by enhanced nitric oxide contribution and endothelial Ca(2+) responses.

Exercise training enhances multiple mechanisms of relaxation in coronary arteries from ischemic hearts

Exercise training of coronary artery disease patients is of considerable interest, since it has been shown to improve vascular function and, thereby, enhance blood flow into compromised myocardial regions. However, the mechanisms underlying exercise-induced improvements in vascular function have not been fully elucidated. We tested the hypothesis that exercise training increases the contribution of multiple mediators to endothelium-dependent relaxation of coronary arteries in the underlying setting of chronic coronary artery occlusion. To induce gradual occlusion, an ameroid constrictor was placed around the proximal left circumflex coronary artery in Yucatan miniature swine. At 8 wk postoperatively, pigs were randomly assigned to sedentary or exercise (treadmill, 5 days/wk) regimens for 14 wk. Exercise training significantly enhanced the contribution of nitric oxide, prostanoids, and large-conductance Ca(2+)-dependent K(+) (BKCa) channels to endothelium-dependent, bradykinin-mediated relaxation in nonoccluded and collateral-dependent arteries. Combined nitric oxide synthase, prostanoid, and BKCa channel inhibition ablated the enhanced relaxation associated with exercise training. Exercise training significantly increased nitric oxide levels in response to bradykinin in endothelial cells isolated from nonoccluded and collateral-dependent arteries. Bradykinin treatment significantly increased PGI2 levels in all artery treatment groups and tended to be further enhanced after nitric oxide synthase inhibition in exercise-trained pigs. No differences were found in whole cell BKCa channel currents, BKCa channel protein levels, or arterial cyclic nucleotide levels. Although redundant, upregulation of parallel vasodilator pathways appears to contribute to enhanced endothelium-dependent relaxation, potentially providing a more refined control of blood flow after exercise training.

Abstract 5526: Maternal Smoking and Smoking Cessation in Early Pregnancy and Endothelium-Dependent Bradykinin-Mediated Relaxation of Uterine Small Arteries

Background - Impaired endothelium-dependent vasodilatation in pregnant smokers may reduce the uterine blood flow to the fetus and result in retarded fetal growth. The present study aimed at investigating endothelium-dependent bradykinin-mediated vasodilation in uterine small arteries from pregnant non-smokers, smokers, and ex-smokers who quitted smoking early in pregnancy. Methods and Results - Of 29 healthy, singleton pregnancies scheduled for elective cesarean section; 12 were non-smokers, 9 were smokers, and 8 were ex-smokers. Serum cotinine verified self-reported smoking. Newborns of smokers were shorter and had a smaller head circumference compared to those of non-smokers (P<0.05), but newborns of ex-smokers were similar to those of smokers and non-smokers. Uterine arteries were dissected from myometrial biopsies and vascular function was assessed on a small vessel myograph for isometric recordings. Preconstricted arteries from non-smokers had a better bradykinin-induced relaxation than arteries from smokers and ex-smokers (P<0.05). In the presence of L-N G -nitroarginine (nitric oxide (NO) synthase inhibitor), arteries from smokers and ex-smokers did not relax to bradykinin, while arteries from non-smokers relaxed at high bradykinin concentrations (P<0.05). In the presence of L-N G -nitroarginine and indomethacin (cyclooxygenase inhibitor) arteries from non-smokers no longer responded to bradykinin. Relaxations to sodium nitroprusside (NO donor) were similar in arteries from non-smokers and smokers, but were better in arteries from ex-smokers (P<0.05). Conclusions - The findings suggest that maternal smoking reduces endothelium-dependent bradykinin-mediated relaxation in uterine small arteries contributing to retarded fetal growth, and suggest that smoking cessation early in pregnancy does not abolish this effect, but improves smooth muscle sensitivity to NO.

Effects of ischemic heart disease and exercise training on mechanisms of nitric oxide production

Control and collateral‐dependent coronary arteries of exercise trained swine display enhanced nitric oxide‐dependent bradykinin‐mediated relaxation. For this study, we hypothesized that exercise training elicits cellular adaptations to enhance nitric oxide production in coronary arteries of ischemic hearts. Ameroid constrictors were placed around the proximal left circumflex coronary artery (LCX) of adult female Yucatan miniswine. Collateral‐dependent (LCX) and control (nonoccluded left anterior descending) arteries (∼1–2 mm diam) were isolated from sedentary and exercise trained (treadmill; 14 wks) pigs. Endothelial cells were dissociated and intracellular Ca2+ (fura‐2) responses to bradykinin were studied. Caveolin‐1 and eNOS protein levels were evaluated by immunoblot. Exercise training significantly enhanced bradykinin‐stimulated Ca2+ levels in cells of both control and collateral‐dependent arteries. Caveolin‐1 protein was significantly elevated in collateral‐dependent arteries of sedentary pigs; an increase that was reversed with exercise training. eNOS protein was not altered by occlusion or exercise training. Our studies suggest that increased bradykinin‐stimulated endothelial Ca2+ levels and reduced caveolin‐1 protein contribute to improved bradykinin‐mediated relaxation in both control and collateral‐dependent arteries of exercise trained swine. NIH HL64931

Adenosine instead of supranormal potassium in cardioplegic solution preserves endothelium-derived hyperpolarization factor-dependent vasodilation☆

We have recently shown that adenosine instead of supranormal potassium in cold crystalloid cardioplegia improves cardioprotection. Studies indicate that hyperkalemia has unfavorable effects on vascular endothelial function. Three pathways have been identified as major vasodilatory pathways: the nitric oxide (NO) pathway, the cyclooxygenase (COX) pathway, and the endothelium-derived hyperpolarization (EDHF) pathway, where the EDHF pathway, in particular, seems susceptible to hyperkalemia. We hypothesized that adenosine cardioplegia improves postcardioplegic endothelial function. Sixteen pigs were randomized to receive either cold (6 degrees C) hyperkalemic cardioplegia (n=8) or cardioplegia where hyperkalemia was substituted with 1.2 mM adenosine (n=8). After 1h of cold ischemic arrest, coronary blood flow was monitored for the following 2h. The LAD artery was then explanted, and cylindrical rings were mounted for isometric tension recordings in organ chambers. Vessels were preconstricted with U46610 (Thromboxane A(2) analog) and then bradykinin-mediated relaxation was investigated. To differentiate between the vasodilatory pathways the relaxation was assessed in the absence and presence of inhibitors of the COX (indomethacin), NO (L-NAME+carboxy-PTIO), and EDHF (apamin+charybdotoxin) pathways. Invivo: The adenosine group had, as distinct from the hyperkalemic group, a significantly increased coronary blood flow index 1h after cross-clamp release (from (ml/min/100 g, mean+/-SD) 50.9+/-13.9 to 72.8+/-21.9, p=0.010). The difference was, however, not statistically significant between groups. Invitro: Maximal relaxation without blockers was 27.4+/-10.1% of maximal tension in the adenosine group and 22.2+/-7.5% in the hyperkalemic group. To investigate EDHF-dependent vasodilation the vessel rings were simultaneously treated with indomethacin, L-NAME, and carboxy-PTIO. Maximal relaxation in the hyperkalemic group was then reduced to 47.4+/-17.4% of maximal tension, which was a significant reduction compared to the adenosine group with a maximal relaxation of 20.6+/-8.7% (p=0.028). Adenosine instead of supranormal potassium in cold crystalloid cardioplegia increases postcardioplegic myocardial blood flow and preserves EDHF-dependent vasodilation.

Isolated microparticles, but not whole plasma, from women with preeclampsia impair endothelium-dependent relaxation in isolated myometrial arteries from healthy pregnant women

Objective: This study was performed to establish whether microparticles from plasma of women with preeclampsia cause endothelial dysfunction, as described for isolated myometrial arteries in preeclampsia. Study Design: Myometrial arteries were isolated from biopsy specimens obtained at cesarean delivery from healthy pregnant women (n = 22) and mounted in a wire myograph. Bradykinin concentration-response curves were obtained before and after 1-hour incubation or after overnight incubation with one of the following preparations of plasma from individual women with preeclampsia (n = 16): Whole plasma, microparticle-free plasma, isolated microparticles resuspended in physiologic saline solution or physiologic saline solution. Overnight incubation was also performed with microparticles isolated from healthy pregnant women (n = 6). One-hour incubation was performed with 2% or 10% solution and overnight incubation with 5% solution. Results: No effect of preeclamptic plasma, with or without microparticles, on bradykinin-mediated relaxation was observed. Overnight, but not 1-hour, incubation with preeclamptic microparticles caused abolishment of bradykinin-mediated relaxation in contrast to healthy pregnant microparticles (P <.005). Conclusion: Preeclamptic microparticles, but not healthy pregnant microparticles cause endothelial dysfunction in isolated myometrial arteries from healthy pregnant women after overnight incubation, whereas other preeclamptic plasma constituents protect the endothelium from this effect. (Am J Obstet Gynecol 2002;187:1686-93.)

Functional compartmentation of endothelial P2Y receptor signaling.

The presence of multiple receptors for disparate nucleotides on endothelial cells makes it unclear how the endothelium differentiates among these signals. We propose that endothelial P2Y receptors are organized into cholesterol-rich signaling domains, such as caveolae and respond to nucleotide agonists by mobilizing intracellular calcium. Treatment of endothelial cells with 5 mmol/L beta-methyl-cyclodextrin prevents calcium release in response to the nucleotide receptor agonists 2-methylthio-ATP, ATP, ADP, and UTP, but not the kinin receptor agonist bradykinin, suggesting that depletion of membrane cholesterol disrupts signaling at P2Y receptors and that bradykinin receptors are not prelocalized to cholesterol microdomains in these cells. Direct measurement of cholesterol content after beta-methyl-cyclodextrin treatment of aortic rings reveals a concentration-dependent depletion of cholesterol that parallels functional antagonism of P2Y-mediated relaxation. Nucleotide- and bradykinin-mediated relaxation is disrupted by 5 to 15 mmol/L beta -methyl-cyclodextrin treatment or 1 to 10 microg/mL filipin III in a concentration-dependent fashion. Norepinephrine contracted aorta treated with A23187 relaxes in an endothelium-dependent fashion despite depletion of 84% of membrane-extractable cholesterol. These data indicate that in the basal state, P2Y receptors but not the kinin receptor may be compartmented to cholesterol-dependent signaling domains in guinea pig endothelium and that cholesterol-rich microdomains in these cells can respond to intracellular calcium in an agonist-specific manner. We suggest that the functional organization of cholesterol-rich signaling microdomains allows agonist-specific responses to increases in intracellular calcium and that this property may be a general phenomenon that permits cells to respond disparately to agonists that may signal through common calcium release pathways.

Endothelium-mediated relaxation of porcine collateral-dependent arterioles is improved by exercise training.

Endothelium-dependent modulation of coronary tone is impaired in the collateral-dependent coronary microcirculation. We used a porcine model of chronic coronary occlusion and collateral development to evaluate the hypothesis that exercise training enhances endothelium-mediated relaxation and increases endothelial nitric oxide synthase (ecNOS) mRNA levels of collateral-dependent microvasculature. Adult female miniature swine were subjected to chronic, progressive ameroid occlusion of the proximal left circumflex coronary artery (LCx); after 2 months, animals were randomly exposed to 16-week exercise-training (EX group; treadmill running) or sedentary (SED group; cage confinement) protocols. After completion of EX or SED programs, coronary arterioles ( approximately 100 microm in diameter) were isolated from collateral-dependent LCx (distal to occlusion) and nonoccluded left anterior descending coronary artery (LAD) regions of each heart. Arterioles were studied by in vitro videomicroscopy or frozen for ecNOS mRNA analysis (RT-PCR techniques). Relaxation to the endothelium-dependent vasodilator bradykinin was decreased (P<0.05) in arterioles isolated from collateral-dependent LCx versus nonoccluded LAD regions of SED animals. Bradykinin-mediated relaxation, however, was not different in LCx versus LAD arterioles isolated from EX animals. Nitroprusside-induced relaxation was unaffected by either chronic occlusion or exercise. Importantly, ecNOS mRNA expression was significantly decreased in arterioles isolated from LCx versus LAD regions of SED animals. After training, ecNOS mRNA expression was not different between LAD and LCx arterioles. These data indicate that exercise training enhances bradykinin-mediated relaxation of collateral-dependent LCx arterioles isolated after chronic coronary occlusion, most likely because of effects on ecNOS mRNA expression and increased production of NO.

Bradykinin-mediated relaxation of isolated maternal resistance arteries in normal pregnancy and preeclampsia

OBJECTIVE: The aim of the study was to investigate bradykinin-mediated vasodilator function in small arteries from normotensive pregnant and nonpregnant women and from women with preeclampsia. STUDY DESIGN: Small subcutaneous arteries (approximately 250 μm luminal diameter) were dissected from biopsy specimens obtained at cesarean section from 24 normotensive pregnant women and 6 women with preeclampsia and during abdominal surgery in 15 nonpregnant women. Vascular function was assessed after arteries were mounted on a small vessel myograph. RESULTS: Preconstricted arteries from normotensive pregnant women demonstrated enhanced relaxation to bradykinin compared with those from nonpregnant women ( p < 0.05), whereas arteries from women with preeclampsia showed blunted responses compared with those from normotensive pregnant women ( p < 0.01). Relaxation in all groups was attenuated in the presence of the nitric oxide synthase inhibitor N ω -nitro- l-arginine so that it became similar in the three groups. Indomethacin had a small but significant inhibitory effect on bradykinin-induced relaxation, but this component of relaxation was no different among groups. Sensitivity of arteries to norepinephrine and sodium nitroprusside showed no significant differences in the three groups of women. CONCLUSION: This study provides evidence for an increase in bradykinin-mediated nitric oxide synthesis from the vascular endothelium of small arteries from the peripheral circulation of normotensive pregnant women and a relative reduction in women with preeclampsia. In turn, these changes may contribute to vasodilation in normal pregnancy and elevation of the blood pressure in preeclampsia. (Am J Obstet Gynecol 1996;175:1668-74.)