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.     

Maturation enhances fluid shear-induced activation of eNOS in perfused ovine carotid arteries

The present study tests the hypothesis that age-dependent increases in endothelial vasodilator capacity are due to maturational increases in endothelial nitric oxide (NO) synthesis and release. Intact 4-cm carotid artery segments taken from term fetal lambs and nonpregnant adult sheep were perfused by using a closed system that enabled independent control of flow and inflow pressure and facilitated complete recovery of all NO released. Fluid shear stress induced a graded release of NO (in nmol NO x min x cm(-2) of luminal surface area) that was significantly greater in adult (890 +/- 140) than in fetal (300 +/- 40) carotid arteries at corresponding values of shear stress (5.9 +/- 0.3 dyn/cm2) but was independent of inflow pressure in both age groups. These age-related differences in NO release were not attributable to corresponding differences in endothelial NO synthase (eNOS) abundance, as eNOS protein levels (in ng of eNOS/cm2 of luminal surface area) were similar in adult (14 +/- 2) and fetal (12 +/- 1) arteries. Adult (80 +/- 15) and fetal (89 +/- 32) levels of eNOS mRNA (in 10(6) copies/cm2 of luminal surface area) were also similar. However, when NO release was normalized relative to the associated mass of eNOS protein to estimate eNOS-specific activity in situ, this value (in nmol NO x microg of eNOS(-1) x min(-1)) was significantly greater in adult (177 +/- 44) than in fetal (97 +/- 36) arteries when the endothelium was maximally activated by A-23187. Similarly, the slope of the relation between fluid shear stress and estimated eNOS-specific activity (in nmol NO x microg of eNOS(-1) x min(-1) per dyn/cm2) was also significantly greater in adult (6.8 +/- 0.1) than in fetal (2.9 +/- 0.1) arteries, which suggests that eNOS may be more sensitive to or more efficiently coupled to activating stimuli in adult compared with fetal arteries. We conclude that maturational increases in endothelial vasodilator capacity are attributable to age-dependent increases in NO release secondary to elevated eNOS-specific activity and involve more efficient coupling between endothelial activation and enhancement of eNOS activity in adult compared with fetal arteries.     

Endothelial dysfunction and arterial pressure regulation during early diabetes in mice: roles for nitric oxide and endothelium-derived hyperpolarizing factor

We determined whether nitric oxide (NO) counters the development of hypertension at the onset of diabetes in mice, whether this is dependent on endothelial NO synthase (eNOS), and whether non-NO endothelium-dependent vasodilator mechanisms are altered in diabetes in mice. Male mice were instrumented for chronic measurement of mean arterial pressure (MAP). In wild-type mice, MAP was greater after 5 wk of N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 mg x kg(-1) x day(-1) in drinking water; 97 +/- 3 mmHg) than after vehicle treatment (88 +/- 3 mmHg). MAP was also elevated in eNOS null mice (113 +/- 4 mmHg). Seven days after streptozotocin treatment (200 mg/kg iv) MAP was further increased in L-NAME-treated mice (108 +/- 5 mmHg) but not in vehicle-treated mice (88 +/- 3 mmHg) nor eNOS null mice (104 +/- 3 mmHg). In wild-type mice, maximal vasorelaxation of mesenteric arteries to acetylcholine was not altered by chronic L-NAME or induction of diabetes but was reduced by 42 +/- 6% in L-NAME-treated diabetic mice. Furthermore, the relative roles of NO and endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced vasorelaxation were altered; the EDHF component was enhanced by L-NAME and blunted by diabetes. These data suggest that NO protects against the development of hypertension during early-stage diabetes in mice, even in the absence of eNOS. Furthermore, in mesenteric arteries, diabetes is associated with reduced EDHF function, with an apparent compensatory increase in NO function. Thus, prior inhibition of NOS results in endothelial dysfunction in early diabetes, since the diabetes-induced reduction in EDHF function cannot be compensated by increases in NO production.     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties

Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (−)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation.     

Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3+/-3.8 to 467+/-22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4+/-0.3 x 10(-6) to 8.5+/-0.4 x 10(-6) cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation.     

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.     

Chronic nitric oxide exposure alters the balance between endothelium-derived relaxing factors released from rat renal arteries: prevention by treatment with NOX-100, a NO scavenger

We investigated whether nitric oxide (NO) exposure alters the balance between NO and endothelium-derived hyperpolarizing factor (EDHF) released from rat renal arteries. To produce states of acutely or chronically excessive NO, lipopolysaccharide (LPS) was administered intraperitoneally to rats in a single dose of 4 mg/kg (LPS-single group) or in stepwise doses of 0.5, 1.0 and 2.0 mg/kg every other day (LPS-repeated group). On the day after LPS treatment, the protein levels of inducible NO synthase (iNOS) and endothelial NOS (eNOS) were measured, and the relaxation responses were determined in the renal arteries. The protein levels of iNOS markedly increased in both LPS-treated groups, while those of eNOS significantly increased in the LPS-repeated group compared with those in the respective control groups. In both LPS-treated groups, the relaxations in response to acetylcholine (ACh) and sodium nitroprusside remained unchanged. The ACh-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, or by 1H-[1, 2, 4-] oxadiazole [4, 3-a] quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, i.e. EDHF-mediated relaxations were significantly impaired in the LPS-repeated group but not in the LPS-single group, indicating increase in NO-mediated relaxation in the LPS-repeated group. These changes in the protein levels and EDHF-mediated relaxations induced by ACh observed in the LPS-repeated group were restored by treatment with NOX-100, a NO scavenger. These results suggest that persistent but not acute excessive NO exposure in rats impairs EDHF-mediated relaxation in renal arteries, leading to a compensatory upregulation of the eNOS/NO pathway.     

Endothelial NO formation does not control myocardial O2 consumption in mouse heart

To test whether endothelium-derived nitric oxide (NO) regulates mitochondrial respiration, NO was pharmacologically modulated in isolated mouse hearts, which were perfused at constant flow to sensitively detect small changes in myocardial O2 consumption (MVO2). Stimulation of NO formation by 10 microM bradykinin (BK) increased coronary venous nitrite release fivefold to 58 +/- 33 nM (n = 17). Vasodilatation by BK, adenosine (1 microM), or papaverine (10 microM) decreased perfusion pressure, left ventricular developed pressure (LVDP), and MVO2. In the presence of adenosine-induced vasodilatation, stimulation of endothelial NO synthesis by BK had no effect on LVDP and MVO2. Also, inhibition of NO formation by NG-monomethyl-l-arginine (l-NMMA, 100 microM) did not significantly alter LVDP and MVO2. Similarly, intracoronary infusion of authentic NO 2 microM were contractile dysfunction and MVO2 reduction observed. Because BK-induced stimulation of endothelial NO formation and basal NO are not sufficient to impair MVO2 in the saline-perfused mouse heart, a tonic control of the respiratory chain by endothelial NO is difficult to conceive.     

Mechanisms of histamine-induced relaxation in bovine small adrenal cortical arteries

Adrenal steroidogenesis is closely correlated with increases in adrenal blood flow. Many reports have studied the regulation of adrenal blood flow in vivo and in perfused glands, but until recently few studies have been conducted on isolated adrenal arteries. The present study examined vasomotor responses of isolated bovine small adrenal cortical arteries to histamine, an endogenous vasoactive compound, and its mechanism of action. In U-46619-precontracted arteries, histamine (10(-9)-5 x 10(-6) M) elicited concentration-dependent relaxations. The relaxations were blocked by the H(1) receptor antagonists diphenhydramine (10 microM) or mepyramine (1 microM) (maximal relaxations of 18 +/- 6 and 22 +/- 6%, respectively, vs. 55 +/- 5% of control) but only partially inhibited by the H(2) receptor antagonist cimetidine (10 microM) and the H(3) receptor antagonist thioperamide (1 microM). Histamine-induced relaxations were also blocked by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microM; maximal relaxation of 13 +/- 7%) and eliminated by endothelial removal or L-NA combined with the cyclooxgenase inhibitor indomethacin (10 microM). In the presence of adrenal zona glomerulosa (ZG) cells, histamine did not induce further relaxations compared with histamine alone. Histamine (10(-7)-10(-5) M) concentration-dependently increased aldosterone production by adrenal ZG cells. Compound 48/80 (10 microg/ml), a mast cell degranulator, induced significant relaxations (93 +/- 0.6%), which were blocked by L-NA plus indomethacin or endothelium removal, partially inhibited by the combination of the H(1), H(2), and H(3) receptor antagonists, but not affected by the mast cell stabilizer sodium cromoglycate (1 mM). These results demonstrate that histamine causes direct relaxation of small adrenal cortical arteries, which is largely mediated by endothelial NO and prostaglandins via H(1) receptors. The potential role of histamine in linking adrenal vascular events and steroid secretion requires further investigation.     

Effects of pulsatile shear stress on nitric oxide production and endothelial cell nitric oxide synthase expression by ovine fetoplacental artery endothelial cells

Placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression increase during pregnancy. Shear stress, the frictional force exerted on endothelial cells by blood flow, stimulates vessel dilation, endothelial NO production, and eNOS expression. In order to study the effects of pulsatile flow/shear stress, we adapted Cellco CELLMAX artificial capillary modules to study ovine fetoplacental artery endothelial (OFPAE) cells for NO production and eNOS expression. OFPAE cells were grown in the artificial capillary modules at 3 dynes/cm2. Confluent cells were then exposed to 10, 15, or 25 dynes/cm2 for up to 24 h. NO production by OFPAE cells exposed to pulsatile shear stress was inhibited to nondetectable levels by the NOS inhibitor l-NMMA and reversed by excess NOS substrate l-arginine. NO production and expression of eNOS mRNA and protein by OFPAE cells were elevated by shear stress in a graded fashion (P < 0.05). The rise in NO production with 25 dynes/cm2 shear stress (8-fold) was greater (P < 0.05) than that observed for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). The acute shear stress-induced rise in NO production by OFPAE cells was via eNOS activation, whereas the prolonged NO rise occurred by elevations in both eNOS expression and enzyme activation. Thus, elevations of placental blood flow and physiologic shear stress may be partly responsible for the increases in placental arterial endothelial eNOS expression and NO production during pregnancy.     

Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension

Patients with left-to-right shunt congenital heart disease may develop pulmonary hypertension. Perioperative mortality of these patients is high due to abnormal vasoreactivity of the pulmonary artery (PA). We studied the changes in the PA induced by high pulmonary blood flow in rats with aortocaval fistula. Eight weeks after surgery, morphological changes of the PA were studied and vasomotor function was assessed by isometric force recording. Expression of endothelial nitric oxide (NO) synthase (eNOS), VEGF, and cyclooxygenase-2 (COX-2) proteins and levels of cGMP in the PA were analyzed. Rats with high pulmonary blood flow developed pulmonary hypertension, medial thickening, and increasing of internal elastic lamina and basement membrane in the PA. When compared with sham-operated animals, rats with fistula had significantly increased contractions in the PA, whereas relaxations to acetylcholine and NO donor were reduced. Concentrations of cGMP were reduced in the PA of rats with pulmonary hypertension (18.4 +/- 3.3 vs. 9.4 +/- 1.7 pmol/mg protein; P = 0.04). The altered vasomotor function was normalized by treatment with indomethacin. The PA of rats with fistula expressed higher levels of eNOS, phosphorylated eNOS, and COX-2. Sustained high PA blood flow in rats causes pulmonary hypertension that is morphologically and functionally identical with patients with flow-induced pulmonary hypertension. Abnormal vasomotor function of the PA in these animals appears to be mediated by reduced availability and the biological effect of endogenous NO and the high production of vasoconstrictor prostanoids. Increased eNOS and phosphorylated eNOS are most likely the adaptive changes in response to an increase in PA pressure secondary to high blood flow.     

Endothelium-derived microparticles inhibit angiogenesis in the heart and enhance the inhibitory effects of hypercholesterolemia on angiogenesis

Therapeutic angiogenesis remains unsuccessful in coronary artery disease. It is known that plasma endothelium-derived microparticles (EMPs) are increased in coronary artery disease and that hypercholesterolemia can inhibit angiogenesis. We evaluated the relationship between EMPs and hypercholesterolemia in the impairment of angiogenesis. EMPs isolated from human umbilical vein endothelial cells were injected into low-density lipoprotein receptor-null (LDLr(-/-)) mice fed a Western diet for 2 wk and C57BL6 mice for 6 h or were directly added to the tissue culture media. Hearts isolated from mice were sectioned and cultured, and endothelial tube formation was measured. The expression and phosphorylation of endothelial NO synthase (eNOS) and the generation of NO in the hearts were determined. Angiogenesis was inhibited by pathophysiological concentrations of EMPs but not physiological concentrations of EMPs in hearts from C57BL6 mice. However, angiogenesis was inhibited by EMPs at both physiological and pathophysiological concentrations of EMPs in hearts from hypercholesterolemic LDLr(-/-) mice. Pathophysiological concentrations of EMPs decreased eNOS phosphorylation at Ser(1177) and NO generation without altering eNOS expression in hearts from C57BL6 mice. Both physiological and pathophysiological concentrations of EMPs decreased not only eNOS phosphorylation at Ser(1177) and NO generation, but eNOS expression in hypercholesterolemic hearts from LDLr(-/-) mice. These data demonstrated that pathophysiological concentrations of EMPs could inhibit angiogenesis in hearts by decreasing eNOS activity. EMPs and hypercholesterolemia mutually enhanced their inhibitory effect of angiogenesis by inducing eNOS dysfunction. Our findings suggest a novel mechanism by which hypercholesterolemia impairs angiogenesis.     

Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NO(x)

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110-130 days gestation; term = 145 +/- 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n = 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO(2)-NO(3) (NO(x)). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT < or = FOL < PREG (P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG (P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls (P < 0.01). Circulating NO(x) levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 +/- 1.1 microM in LUT to 12 +/- 4, 22 +/- 3, and 41 +/- 3 microM at 110, 120, and 130 days gestation (P < 0.01). Systemic NO(x) levels in singleton (12.5 +/- 1.6 microM) were less (P < 0.01) than in multiple (twin 27.6 +/- 6.5 microM; triplet = 46 +/- 10 microM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NO(x) levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.     

Ethanol inhibits L-arginine uptake and enhances NO formation in human placenta.

The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.     

Acute effects of 17beta -estradiol on femoral veins from adult gonadally intact and ovariectomized female pigs

Our experiments were designed to determine the acute effects of 17beta-estradiol on femoral veins from intact and ovariectomized female pigs. Rings of femoral veins with or without endothelium were suspended in organ chambers for measurement of isometric force. Concentration-response curves to 17beta-estradiol (10(-9) to 10(-5) M) were obtained in veins contracted with prostaglandin F(2alpha) in the absence and presence of inhibitors of either estrogen receptors (ICI-182780; 10(-5) M), nitric oxide synthase [N(G)-monomethyl-l-arginine (l-NMMA); 10(-4) M], soluble guanylate cyclase (1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10(-5) M), or potassium channels (tetraethylammonium; 10(-2) M). Estrogen receptors were identified with the use of Western blotting and immunostaining in veins of both groups. 17beta-Estradiol caused acute endothelium-dependent relaxations in both groups. Relaxations to 17beta-estradiol were inhibited by l-NMMA and 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but not ICI-182780. Tetraethylammonium inhibited relaxations only in veins with endothelium from intact females. Results indicate that 17beta-estradiol causes acute endothelium-dependent relaxations in femoral veins. The relative contribution of nitric oxide and K(+) channels as mechanisms involved in relaxations to 17beta-estradiol in femoral veins is modulated by ovarian status.     

ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K+

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K+, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10(-10) to 10(-6) M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca(2+)-activated K+ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 microM) and apamin but not barium plus charybdotoxin. Addition of K+ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K+ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K+ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10(-7) M) stimulated voltage-dependent outward K+ current from endothelial cells, which was inhibited by charybdotoxin, indicating K+ efflux. Arachidonic acid (10(-7) to 10(-4) M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K+ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K+ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell K+ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.     

Effects of 1400W, a potent selective inducible NOS inhibitor, on histamine- and leukotriene D4-induced relaxation of isolated guinea pig nasal mucosa.

To clarify the role of inducible nitric oxide synthase (iNOS) in the relaxation of nasal vasculature, the effects of a potent selective iNOS inhibitor, N-[(3-aminomethyl)benzyl]acetamidine (1400W), on histamine- and leukotriene D4 (LTD4)-induced relaxations of isolated nasal septal mucosae were examined in naive guinea pigs. In addition to eNOS and nNOS, Western blots demonstrated a distinct expression of iNOS in nasal mucosal tissues of naive guinea pigs. In isolated nasal septal mucosae precontracted with norepinephrine (3 x 10(-5)M), both histamine (10(-7)-10(-3)M) and LTD4 (10(-10)-10(-7)M) exhibited relaxations, which were inhibited by a NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 10(-4)M). The inhibitory effect of L-NMMA was reversed by L-arginine (10(-3)M), indicating that the relaxations induced by histamine and LTD4 are mediated by NO. Furthermore, both the histamine- and LTD4-induced relaxations were also significantly attenuated by 1400W (10(-5)M). These findings suggest an involvement of NO generated by iNOS in agonist-induced relaxation of nasal mucosal vasculature in naive guinea pigs.