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.     

Repeated pregnancies (multiparity) increases venous tone and reduces compliance

In humans, multiparity (repeated pregnancy) is associated with increased risk of cardiovascular disease. In rats, multiparity increases the pressor response to phenylephrine and to acute stress, due in part to changes in tone of the splanchnic arterial vasculature. Given that the venous system also changes during pregnancy, we studied the effects of multiparity on venous tone and compliance. Cardiovascular responses to volume loading (2 ml/100 g body wt), and mean circulatory filling pressure (MCFP, an index of venomotor tone) were measured in conscious, repeatedly bred (RB), and age-matched virgin rats. In addition, passive compliance and venous reactivity of isolated mesenteric veins were measured by pressure myography. There was a greater increase in mean arterial pressure after volume loading in RB rats (+7.2 +/- 2.5 mmHg, n = 8) than virgin rats (-1.4 +/- 1.7 mmHg, n = 7) (P < 0.05). The increase in MCFP in response to norepinephrine (NE) was also greater in RB rats [half maximal effective dose (ED(50)) 3.1 +/- 0.5 nmol.kg(-1).min(-1), n = 6] than virgins (ED(50): 12.1 +/- 2.7 nmol.kg(-1).min(-1), n = 6) (P < 0.05). Pressure-induced changes in passive diameter were lower in isolated mesenteric veins from RB rats (29.3 +/- 1.8 microm/mmHg, n = 6) than from virgins (36.9 +/- 1.3 microm/mmHg, n = 6) (P < 0.05). Venous reactivity to NE in isolated veins was also greater in RB rats (EC(50): 2.68 +/- 0.37x10(-8) M, n = 5) than virgins (EC(50): 4.67 +/- 0.93 x 10(-8) M, n = 8). We conclude that repeated pregnancy induces a long-term reduction in splanchnic venous compliance and augments splanchnic venous reactivity and sympathetic tonic control of total body venous tone. This compromises the ability of the capacitance (venous) system to accommodate volume overloads and to buffer changes in cardiac preload.     

Endogenous estrogen mediates vascular reactivity and distensibility in pregnant rat mesenteric arteries

The role of estrogen in the maternal systemic cardiovascular adaptations during pregnancy is still controversial. Female Sprague-Dawley rats were implanted at day 14 of pregnancy with either a 50-mg tamoxifen pellet (estrogen receptor blocker, n = 10) or placebo pellet (n = 10). Virgin female rats were a nonpregnant control (n = 7). At days 20-22 of pregnancy, resistance-sized mesenteric arteries were mounted onto a dual-chamber arteriograph system. Pregnancy significantly blunted the pressor response to phenylephrine [measurement of the effective concentration that yielded 50% maximum response (EC(50)) values were 1.5 +/- 0.22 vs. 0.69 +/- 0.16 microM (P < 0.05)] and enhanced vasodilation to ACh [EC(50) = 1.13 +/- 2.53 vs. 3.13 +/- 6.04 nM (P < 0.05)] compared with nonpregnant rats. However, tamoxifen treatment during pregnancy reversed these effects. Inhibition of nitric oxide (NO) synthase with N(G)-monomethyl-L-arginine (250 microM) shifted only the responses of the placebo-treated pregnant group to both phenylephrine and ACh. Arterial distensibility in the placebo-treated pregnant group was also significantly increased (P < 0.05) compared with nonpregnant and tamoxifen-treated pregnant animals. In summary, endogenous estrogen during pregnancy increases NO-dependent modulation of vessel tone and arterial distensibility.     

IL-10 deficiency increases superoxide and endothelial dysfunction during inflammation

Little is known about the role of interleukin-10 (IL-10), an anti-inflammatory cytokine, in blood vessels. We used IL-10-deficient mice (IL-10 -/-) to examine the hypothesis that IL-10 protects endothelial function after lipopolysaccharide (LPS) treatment. The responses of carotid arteries were studied in vitro 6 h after injection of a relatively low dose of LPS (10 microgram ip). In IL-10 -/- mice, the maximum relaxation to ACh (3 microM) was 56 +/- 6% (means +/- SE) after LPS injection and 84 +/- 4% after vehicle injection (P < 0.05). Thus endothelium-dependent relaxation was impaired in carotid arteries from IL-10 -/- mice after LPS injection. In contrast, this dose of LPS did not alter relaxation to ACh in vessels from wild-type (IL-10 +/+) mice. Relaxation to nitroprusside and papaverine was similar in arteries from both IL-10 -/- and IL-10 +/+ mice after vehicle or LPS injection. Because inflammation is associated with increased levels of reactive oxygen species, we also tested the hypothesis that superoxide contributes to the impairment of endothelial function by LPS in the absence of IL-10. Results using confocal microscopy and hydroethidine indicated that levels of superoxide are elevated in carotid arteries from IL-10 -/- mice compared with IL-10 +/+ mice after LPS injection. The impaired relaxation of arteries from IL-10 -/- mice after LPS injection was restored to normal by polyethylene glycol-suspended superoxide dismutase (50 U/ml) or allopurinol (1 mM), an inhibitor of xanthine oxidase. These data provide direct evidence that IL-10 protects endothelial function after an acute inflammatory stimulus by limiting local increases in superoxide. The source of superoxide in this model may be xanthine oxidase.     

Temperature effects on morphological integrity and Ca²⁺ signaling in freshly isolated murine feed artery endothelial cell tubes

To study Ca(2+) signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5-9 mo, 25-35 g). We tested the hypothesis that intracellular Ca(2+) concentration ([Ca(2+)](i)) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1-2 mm, width: 65-80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca(2+)](i) was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca(2+) K(d) values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca(2+)](i) remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ～220 nM at 24°C to ～500 nM at 32°C (P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca(2+)](i) increased by ～30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca(2+) signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca(2+) responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.     

Reduced molecular expression of K(+) channel proteins in vascular smooth muscle from rats made hypertensive with N{omega}-nitro-L-arginine

Smooth muscle membrane potential (E(m)) depends on K(+) channels, and arteries from rats made hypertensive with N(omega)-nitro-l-arginine (LHR) are depolarized compared with control. We hypothesized that decreased K(+) channel function, due to decreased K(+) channel protein expression, underlies E(m) depolarization. Furthermore, K(+) channel blockers should move control E(m) (-46 +/- 1 mV) toward that in LHR (-37 +/- 2 mV) and normalize contraction. The E(m) vs. K(+) relationship was less steep in LHR (23 +/- 2 vs. 28 +/- 1 mV/log K(+) concentration), and contractile sensitivity to K(+) was increased (EC(50) = 37 +/- 1 vs. 23 +/- 1 mM). Iberiotoxin (10 nM), an inhibitor of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, depolarized control and LHR E(m) to -35 +/- 1 and -30 +/- 2 mV, respectively; however, effects on K(+) sensitivity were more profound in LHR (EC(50) = 25 +/- 2 vs. 15 +/- 3 mM). The voltage-dependent K(+) (K(V)) channel blocker 4-aminopyridine (3 mM) depolarized control E(m) to the level of LHR (-28 +/- 1 vs. -28 +/- 1 mV); however, effects on K(+) sensitivity were greater in LHR (EC(50) = 17 +/- 4 vs. 4 +/- 4 mM). Western blots revealed reduced BK(Ca) and K(V)1.5 channel expression in LHR arteries. The findings suggest that diminished expression of K(+) channels contributes to depolarization and enhanced contractile sensitivity. These conclusions are supported by direct electrophysiological assessment of BK(Ca) and K(V) channel function in control and LHR smooth muscle cells.     

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.     

Multiparity leads to obesity and inflammation in mothers and obesity in male offspring

Multiparity is an independent risk factor for obesity in parous females. In addition to being a health issue for the mother, offspring of multiparous females may also be at risk for obesity later in life. The aim of the current study was to establish a mouse model that mimics the human pathology of multiparity and determine the effects of multiparity-induced obesity (MIO) on offspring in adulthood. C57BL/6 mice were mated and studied when primiparous (1st pregnancy) or multiparous (4th pregnancy). Dams became obese with multiparity, an effect that was independent of the age of the dam. Multiparous dams also had increased markers of inflammation (JNK activation, cytokine expression) in adipose tissue and liver that was greater than inflammation in nulliparous females made obese with a high-fat diet. Placental inflammation was prevalent in multiparous vs. primiparous dams as well. Male offspring of the multiparous dams developed increased adiposity by 24 wk of age relative to the progeny of primiparous dams, although food consumption was similar in both groups. Lipid metabolism was altered in liver and fat in that mRNA levels of regulatory genes (PGC-1α) as well as metabolic genes (CPT I) and Akt phosphorylation were decreased in offspring of multiparous dams. Thus, in mice, as in humans, multiparity increases adiposity and is associated with hepatic and placental inflammation and abnormal glucose tolerance. Importantly, MIO leads to increased body fat and metabolic dysfunction in the offspring, suggesting a role in the propagation of obesity.     

Microvascular versus macrovascular dysfunction in type 2 diabetes: differences in contractile responses to endothelin-1

Vascular dysfunction characterized by a hyperreactivity to vasoconstrictors and/or impaired vascular relaxation contributes to increased incidence of cardiovascular disease in diabetes. Endothelin (ET)-1, a potent vasoconstrictor, is chronically elevated in diabetes. However, the role of ET-1 in resistance versus larger vessel function in mild diabetes remains unknown. Accordingly, this study investigated vascular function of third-order mesenteric arteries and basilar arteries in control Wistar and Goto-Kakizaki (GK) rats, a model of mild Type 2 diabetes. Six weeks after the onset of diabetes, contractile responses to 0.1-100 nM ET-1 and relaxation responses to 1 nM-10 microM acetylcholine (ACh) in vessels preconstricted (baseline + 60%) with serotonin (5-HT) were assessed by myograph studies in the presence or absence of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (L-NNA). Maximum contractile response to ET-1 was augmented in mesenteric vessels (155 +/- 18% in GK vs. 81 +/- 6% in control; n = 5-7) but not in the basilar artery (134 +/- 29% in GK vs. 107 +/- 17% in control; n = 4 per group). However, vascular relaxation was impaired in the basilar arteries (22 +/- 4% in GK vs. 53 +/- 7% in control; n = 4 per group) but not in mesenteric arteries of GK rats. Inhibition of NOS decreased the relaxation response of basilar arteries to 15 +/- 8% and 42 +/- 5% in GK and control rats, respectively; whereas, in resistance vessels, corresponding values were 56 +/- 7% and 89 +/- 3% (vs. 109 +/- 2% and 112 +/- 3% without NOS blockade), indicating the involvement of different vasorelaxation-promoting pathways in these vascular beds. These findings provide evidence that the ET system is activated even under mild hyperglycemia and that it contributes to the hyperreactivity of resistance vessels, therefore, the ET system may play an important role in elevated blood pressure in Type 2 diabetes.     

Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis.

Equine laminitis is a crippling condition associated with a variety of systemic diseases. Although it is apparent that the prodromal stages of laminitis involve microvascular dysfunction, little is known regarding the physiology of this vasculature. The aim of the present study was to determine the relative responses of equine laminar arteries and veins to the vasoconstrictor agonists phenylephrine (1 nM-10 microM), 5-HT (1 nM-10 microM), PGF2alpha (1 nM-100 microM), and endothelin-1 (1 pM-1 microM). We have determined that laminar veins were more sensitive, with respect to the concentration of agonist required to initiate a contractile response and to achieve EC(50), for all agonists tested. EC50 values, for veins and arteries, respectively, were 84+/-7 vs. 688+/-42 nM for phenylephrine, 35+/-6 vs. 224+/-13 nM for 5-HT, 496+/-43 nM vs. 3.0+/-0.6 microM for PGF2alpha, and 467+/-38 pM vs. 70.6+/-6.4 nM for endothelin-1. Moreover, when expressed as a percentage of the response to a depolarizing stimulus (80 mM potassium), the maximal contractile response of laminar veins exceeded that for the laminar arteries for each agonist. These results indicate that there may be a predisposition for venoconstriction within the vasculature of the equine digit. While this physiological predisposition for venoconstriction may be important in the regulation of blood flow during exercise, it also may help to explain why laminitis can result from a variety of pathological systemic conditions.     

Inhibition of NOS enhances pulmonary vascular changes in stroke-prone spontaneously hypertensive rats

To determine the effects of chronic nitric oxide (NO) blockade on the pulmonary vasculature, 58-day-old spontaneously hypertensive rats of the stroke-prone substrain (SHRSP) and Wistar-Kyoto rats (WKY) received N(omega)-nitro-L-arginine (L-NNA; 15 mg. kg(-1). day(-1) orally for 8 days). Relaxation to acetylcholine (ACh) in hilar pulmonary arteries (PAs), the ratio of right ventricular (RV) to body weight (RV/BW) to assess RV hypertrophy (RVH), and the percent medial wall thickness (WT) of resistance PAs were examined. L-NNA did not alter the PA relaxation, RV/BW, or WT in WKY. Although the PA relaxation and RV/BW in control SHRSP were comparable to those in WKY, the WT was increased (31 +/- 2 vs. 19 +/- 1%). L-NNA-treated SHRSP showed two patterns: in one group, the relaxation, RV/BW, and WT were comparable to those in the control SHRSP; in the other, impaired relaxation (36 +/- 7 vs. 88 +/- 4% for WKY) was associated with an increase in WT (37 +/- 1%) and RV/BW (0. 76 +/- 0.05). Thus the abnormal pulmonary vasculature in SHRSP at <10 wk of age is not accompanied by impaired relaxation in PAs or RVH; however, impaired relaxation is associated with increased WT and RVH.     

Glutathione depletion in vivo enhances contraction and attenuates endothelium-dependent relaxation of isolated rat aorta

Ten-day administration of the glutamate-cysteine ligase inhibitor L-buthionine-[S,R]-sulfoximine (BSO; 20 or 30 mM in drinking water) to adult male Sprague-Dawley rats induced 50-60% glutathione depletion (p<0.001) and elevated aortic ring reactive oxygen species release and tissue and plasma H2O2 concentrations (p<0.001) compared to control animals (CON) that consumed normal drinking water. In contrast to previous studies using tail cuff plethysmography methods, BSO had no significant effect on systolic blood pressure assessed by indwelling femoral artery catheters in conscious animals (10-day values, 119+/-3 mn Hg vs 122+/-4 mm Hg in CON vs BSO, respectively). Thoracic aorta rings were excised for in vitro assessment of vasomotor function. BSO shifted the phenylephrine (PE) dose-response curve to the left (p=0.003), lowering the EC50 for PE contraction (from -6.752+/-0.056 to -7.056+/-0.055 log units; p=0.001). Endothelium-dependent relaxation to acetylcholine (ACh) was significantly blunted (p=0.019) and the EC50 for ACh relaxation was significantly increased (from -7.428+/-0.117 to -7.129+/-0.048 log units; p=0.02) in BSO vs CON. Endothelium-independent vasorelaxation to sodium nitroprusside was similar in BSO and CON groups. Thoracic aorta immunoblot analyses revealed increases in endothelial nitric oxide synthase, superoxide dismutase 1 and 2, and soluble guanylate cyclase in BSO vs CON (all p<0.01). Thus, enhanced PE contraction, blunted endothelium-dependent relaxation, and adaptations in nitric oxide bioavailability pathways provide the first evidence of chronic, in vivo BSO-induced, oxidative stress-mediated direct effects on the vasomotor function of arteries.     

Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart

Bradykinin and acetylcholine (ACh) trigger preconditioning by ATP-sensitive K(+) (K(ATP)) channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cGMP-dependent protein kinase (PKG). This study utilized DT-2 and DT-3 peptides, highly specific membrane-permeable blockers of PKG. Rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 microM) caused a 49.9 +/- 5.9% and 46.8 +/- 1.7% increase in ROS production, respectively (P < 0.005 vs. untreated cells). Coincubation with DT-3 (250 nM) abolished both the ACh- and bradykinin-induced ROS signal, whereas a nonpermeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100 microM), a selective opener of mitochondrial K(ATP) channels, suggesting that these channels are downstream of PKG. DT-2 (125 nM) also prevented ACh from triggering ROS production. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100 microM), a cGMP analog and potent direct activator of PKG, increased ROS production of cardiomyocytes by 44.7 +/- 7.1% (P < 0.001 vs. untreated cells). This increase was blocked by DT-2. Neither DT-2 nor DT-3 could block the anti-infarct effect of bradykinin in isolated rabbit hearts. Studies with fluorescent-tagged DT-3 revealed that it was confined to endothelial cells and never reached the myocytes. We conclude that both bradykinin and ACh trigger ROS generation by a pathway that includes PKG. Although the peptides may be inappropriate for a whole heart model, they are likely to become important tool drugs for elucidation of signal transduction pathways in cell preparations.     

Intracoronary beta-irradiation enhances balloon-injury-induced tissue factor expression in the porcine injury model

Intracoronary brachytherapy (ICBT) effectively reduces restenosis but is associated with late thrombosis. Since tissue factor (TF) is an important mediator of arterial thrombosis, we tested the hypothesis that ICBT results in persistently augmented TF expression. Coronary arteries from 12 pigs were randomized to: control (C; no injury), oversized balloon injury (BI), or BI followed by ICBT. Animals were sacrificed at 1, 7, 14, or 60 days postprocedure, and coronary arteries collected for expression analyses and immunostaining. ICBT-treated arteries had higher TF antigen and activity at all time-points compared to BI arteries (Western blot: 16 571 +/- 2090 vs 10 135 +/- 2939 densitometric units, p = 0.001; ELISA: 0.42 +/- 0.13 nM vs 0.25 +/- 0.14 nM, p = 0.001; TF activity assay: 0.303 +/- 0.11 nM vs 0.18 +/- 0.07 nM, p = 0.01; immunohistochemical staining: 30.6 +/- 6.6% vs 11.5% +/- 3.2%, p = 0.01). TF expression increased following BI, increased further following ICBT, and persisted for the duration of the study. We conclude that TF expression increases after BI, but is further increased and persists for a longer duration following ICBT, suggesting that a TF-mediated mechanism may play a role in late thrombosis following ICBT.     

Interleukin-10 counteracts impaired endothelium-dependent relaxation induced by ANG II in murine aortic rings

ANG II stimulates the production of reactive oxygen species and activates proinflammatory cytokines leading to endothelial dysfunction. We hypothesized that the anti-inflammatory cytokine IL-10 counteracts the impairment in endothelium-dependent ACh relaxation caused by ANG II. Aortic rings of C57BL/6 mice were incubated in DMEM in the presence of vehicle (deionized H(2)O), ANG II (100 nmol/l), recombinant mouse IL-10 (300 ng/ml), or both ANG II and IL-10 for 22 h at 37 degrees C. After incubation, rings were mounted in a wire myograph to assess endothelium-dependent vasorelaxation to cumulative concentrations of ACh. Overnight exposure of aortic rings to ANG II resulted in blunted ACh-induced vasorelaxation compared with that shown in untreated rings (maximal response = 44 +/- 3% vs. 64 +/- 3%, respectively; P<0.05). IL-10 treatment significantly restored this impairment in relaxation (63 +/- 2%). In addition, the NADPH oxidase inhibitor apocynin restored the impairment in relaxation (maximal response = 76 +/- 3%). Western blotting showed increased gp91(phox) expression (a subunit of NADPH oxidase) in response to ANG II. Vessels treated with a combination of ANG II and IL-10 showed decreased expression of gp91(phox). Immunohistochemical analysis showed increased gp91(phox) expression in ANG II-treated vessels compared with those treated with combined ANG II and IL-10. We found that the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation in response to long-term incubation with ANG II via decreasing NADPH oxidase expression.     

Similarity of vasorelaxant effects of natriuretic peptides in isolated blood vessels of salmonids

Natriuretic peptides (NPs) have been implicated in cardiovascular regulation in rainbow trout (Oncorhyncus mykiss), and it has been observed that the vasorelaxant activity of distinct trout and human NPs is similar in isolated trout arteries. This study characterizes the response of a variety of vessels from rainbow trout and other salmonids to different NPs. The effects of heterologous (rat atrial and human atrial) and homologous (rainbow trout atrial and rainbow trout ventricular) NPs were examined in precontracted efferent branchial arteries from rainbow trout (O. mykiss, Kamloops strain), lake whitefish (Coregonus clupeaformis), and in rainbow trout celiacomesenteric arteries and anterior cardinal veins. The response to mammalian NPs was also examined in efferent branchial arteries from the steelhead (O. mykiss, Skamania strain), coho salmon (Oncorhyncus kisutch), brook trout (Salvelinus fontinalis), and brown trout (Salmo trutta). In general, there were relatively few differences that were species, peptide, or vessel specific. There was no difference in the sensitivity (concentration producing a half-maximal response EC(50)) or efficacy (percent relaxation, i.e., E(max)) of trout or whitefish efferent branchial arteries to any NP, except human NP, which was significantly less effective (greater EC(50) and lower E(max)) in whitefish arteries. There were no differences in E(max) of mammalian NPs in efferent branchial arteries from any species, and only coho and brook trout had significantly different EC(50)'s (coho, 1.0+/-0.2 nM; brook trout, 4. 2+/-0.6 nM; and other species, from 1.9 to 3.5 nM). Rainbow and coho anterior cardinal veins were less sensitive than arteries to mammalian NPs (EC(50)'s; 8.8+/-2.0, 2.0+/-0.1 vs. 3.0+/-0.9, 1.0+/-0. 2, respectively), whereas brown trout veins were more sensitive (1. 0+/-0.2, 3.5+/-1.3, respectively). Sodium nitroprusside (SNP), which activates soluble guanylate cyclase, was vasodilatory, albeit significantly less potent than all NPs, in efferent branchial arteries of all species. SNP was significantly more potent in trout than whitefish efferent branchial arteries, whereas it was equally efficacious in these vessels. These results demonstrate that multiple vessels from various salmonids are similarly responsive to the vasorelaxant effects of a variety of NPs and that the salmonid NP receptor has relatively little ability to discriminate between homologous and heterologous peptides. We conclude that the vascular NP receptor complex is highly conserved among salmonids. Further, salmonids utilize cyclic guanosine monophosphate (cGMP) elevations for reductions of vascular tonus by both particulate and soluble guanylate cyclase pathways.     

The binding of 3H-acetylcholine to cholinergic receptors in bovine cerebral arteries

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, 3H-acetylcholine (ACh), as the ligand. Specific binding of 3H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (KD) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of 3H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC50 values of cholinergic drugs for 3H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC50 values of nicotinic cholinergic agents such as nicotine, cytisine and alpha-bungarotoxin exceeded 50 microM. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic receptors in these arteries.     

Higher sensitivity of cerebral arteries isolated from premature and newborn baboons to adrenergic and cholinergic stimulation

To find whether effects of adrenergic and cholinergic agents on cerebral artery were dependent on maturity, we examined responses of isolated cerebral artery strips harvested from premature, term newborn and adult baboons. Although cerebral arteries from many species are only mildly sensitive to norepinephrine, we found the perinatal cerebral arteries to be quite responsive to the amine. Cerebral arteries from premature and newborn baboons were significantly (P less than 0.001) more sensitive to norepinephrine than were arteries from adults; medium effective concentration (EC50) for norepinephrine were 3 X 10(-8), 6 X 10(-8) and 32 X 10(-8)M for prematures, newborns and adults, respectively. Arteries showed a similar age-dependence in the sensitivity of the response to phenylephrine, an alpha 1-adrenoceptor agonist. EC50 values for KC1 did not differ among groups, nor did the maximum response to norepinephrine. Arteries from premature and newborn baboons showed marked contractile response to acetylcholine (maximum tensions 5.9 +/- 0.6 and 6.4 +/- 0.8 g/mm2, respectively), whereas arteries from adult baboons showed little response (0.6 +/- 0.1 g/mm2). Arteries from premature and newborn animals showed a more marked relaxation response to isoproterenol than did arteries from adult animals; the degree of relaxation from an induced contraction was 63% (premature), 72% (newborn) and 10% (adult). There was no age-dependence in the relaxation response to sodium nitrite. We conclude that the events coupling alpha 1, beta or muscarinic receptor activation with cerebral arterial contraction or relaxation are more effective in perinatal than in adult baboons.     

High-fat diet-induced reduction in nitric oxide-dependent arteriolar dilation in rats: role of xanthine oxidase-derived superoxide anion

Obesity frequently leads to the development of hypertension. We hypothesized that high-fat diet (HFD)-induced obesity impairs the endothelium-dependent dilation of arterioles. Male Wistar rats were fed with normal (control) or HFD (60% of saturated fat, for 10 wk). In rats with HFD, body weight, mean arterial blood pressure, and serum insulin, cholesterol, and glucose were elevated. In isolated gracilis muscle arterioles (diameter: approximately 160 microm) of HFD, rat dilations to ACh (at 1 microM, maximum: 83 +/- 3%) and histamine (at 10 microM, maximum: 16 +/- 4%) were significantly (P < 0.05) decreased compared with those of control responses (maximum: 90 +/- 2 and 46 +/- 4%, respectively). Dilations to the NO donor sodium nitroprusside were similar in the two groups. Inhibition of NO synthesis by N(omega)-nitro-l-arginine methyl ester reduced ACh- and histamine-induced dilations in control arterioles but had no effect on microvessels of HFD rats. The superoxide dismutase mimetic Tiron or xanthine oxidase inhibitor allopurinol enhanced ACh (maximum: 90 +/- 2 and 93 +/- 2%, respectively)- and histamine (maximum: 30 +/- 7 and 37 +/- 8%, respectively)-induced dilations in HFD arterioles, whereas the NAD(P)H oxidase inhibitor apocynin had no significant effect. Correspondingly, in carotid arteries of HFD rats, an enhanced superoxide production was shown by lucigenin-enhanced chemiluminescence, in association with an increased xanthine oxidase, but not NAD(P)H oxidase activity. In addition, a marked xanthine oxidase immunostaining was detected in the endothelial layer of the gracilis arterioles of HFD, but not in control rats. These findings suggest that, in obese rats, NO mediation of endothelium-dependent dilation of skeletal muscle arterioles is reduced because of an enhanced xanthine oxidase-derived superoxide production. These alterations demonstrate substantial dysregulation of arteriolar tone by the endothelium in HFD-induced obesity, which may contribute to disturbed tissue blood flow and development of increased peripheral resistance.     

Beneficial effects of L-arginine supplementation in experimental hyperlipemia-hyperglycemia in the hamster

The objective of this study was to evaluate whether administration of L-arginine, the substrate for nitric oxide synthesis, was able to ameliorate the endothelial dysfunction and the morphological changes induced by the combined insult of hyperlipemia and hyperglycemia. To this purpose, golden Syrian hamsters were rendered simultaneously hyperlipemic and diabetic (HD group) for 24 weeks, and then orally treated with 622.14 mg/kg per day L-arginine, for 12 weeks (HD + L-arg group). The following assays were carried out: (1) spectrophotometric: concentrations of circulating glucose, cholesterol, and creatinine, the activity of angiotensin-converting enzyme (ACE), and the osmotic fragility of erythrocyte plasmalemma; (2) myographic: the endothelium-dependent and -independent relaxation of the resistance arteries (i.d. 210-250 microm) to 10(-8) to 10(-4) M acetylcholine (ACh) or sodium nitroprusside (SNP); and (3) electron-microscopic: the ultrastructure of the resistance arteries, myocardium, and kidney glomeruli, which are main targets of hypertensive complications.The results showed that oral supplementation with L-arginine in simultaneous hyperlipemia-hyperglycemia induced in hamsters had favorable effects on: (1) homeostasis, i.e., diminished the concentration of circulating glucose (by ~63%) and cholesterol (by approximately 10%), reduced the ACE activity (by approximately 45%), and lowered the osmotic fragility of erythrocyte plasmalemma (as marker for the oxidative stress in plasma); (2) mesenteric resistance arteries, which showed (in 10(-4) M ACh) an improved endothelium-dependent relaxation (72.40+/-4.6% in the HD + L-arg group vs 61.90+/-1.45% in the HD group) and a reduced thickness (approximately 1.32-fold) of the smooth muscle cells' extracellular matrix; and (3) the heart, which displayed approximately 16% diminishing of the thickness of the left ventricular wall, and an apparently normal structure of the myocardium; the restoration of the thickness of the pericapillary extracellular matrix to almost normal dimensions was also observed. Administration of L-arginine did not modify the high level of plasma creatinine determined for the HD group (approximately 48% increased vs control group) and had no effect on the thickened, nodular basal lamina of the kidney capillaries. The results indicate that endothelial dysfunction established in combined hyperlipemia-diabetes is distinctive for each vascular bed (mesenteric arterioles, heart capillaries, kidney glomerular capillaries), and there is a reversible stage of the dysfunction in which L-arginine oral supplementation induced beneficial effects.