Membrane depolarization and NADPH oxidase activation in aortic endothelium during ischemia reflect altered mechanotransduction

Vascular endothelial growth factor (VEGF) is considered to be important in promotion of capillary growth in skeletal muscles exposed to increased activity. We studied its interactions with nitric oxide (NO) by examining the expression of endothelial NO synthase (NOS), VEGF, and VEGF receptor-2 (VEGFR-2) proteins in relation to capillary growth in rat extensor digitorum longus muscles electrically stimulated for 2, 4, or 7 days with and without NOS inhibition by N(omega)-nitro-L-arginine (L-NNA, 3 mg/day). Stimulation increased all proteins from 2 days onward, concomitantly with capillary proliferation (labeling for proliferating cell nuclear antigen). Capillary-to-fiber ratio was elevated by 25% after 7 days. Concurrent oral administration of L-NNA did not affect the increase in endothelial NOS but depressed its activity, as shown by increased blood pressure and decreased arteriolar diameters in 2-day-stimulated muscles. NOS inhibition eliminated the increased expression of VEGFR-2 and VEGF proteins in muscles stimulated for 2 and 4 days but not for 7 days. However, it depressed capillary proliferation and the increase in C/F at all time points. We conclude that, in stimulated muscles, NO, generated by activation of neuronal NOS by muscle activity or endothelial NOS by increased blood flow and capillary shear stress, may increase capillary proliferation in the early stages of stimulation through upregulation of VEGFR-2 and VEGF. With longer stimulation, capillary growth appears to require NO, and high levels of VEGF and VEGFR-2 may be contributing to maintenance of the increased capillary bed.     

The flavan-3-ol fraction of cocoa powder suppressed changes associated with early-stage metabolic syndrome in high-fat diet-fed rats

Aims

Previous epidemiological studies have suggested that ingestion of chocolate reduces the risk of cardiovascular disease. In the present study, we examined the effects of flavan-3-ols derived from cocoa on blood pressure, lipolysis, and thermogenesis in rats fed a high-fat diet and that showed early signs of metabolic syndrome.

Main methods

The rats were divided into three groups, and fed either normal diet (normal), 60% fat high-fat diet (HFD), or HFD containing 0.2% flavan-3-ols (HFD-flavan) for 4 weeks. At the end of the feeding period, blood pressure was measured and animals were sacrificed under anesthesia. Lipolysis and thermogenesis-related protein levels were measured in several tissues by Western blotting, and mitochondrial DNA copy number was measured by RT-PCR.

Key findings

Mean blood pressure and epididymal adipose tissue weight of HFD-flavan were significantly lower compared with those of HFD. Uncoupling protein (UCP)1 in brown adipose tissue and UCP3 in gastrocnemius of HFD-flavan were significantly increased compared with those of HFD group. Carnitine palmitoyltransferase (CPT) 2 levels in liver and medium-chain acyl-CoA dehydrogenase (MCAD) levels in gastrocnemius and liver were significantly increased by the supplementation of flavan-3-ols.

Significance

In addition to having hypotensive effects, flavan-3-ols enhance thermogenesis and lipolysis and consequently reduce white adipose tissue weight gain in response to high-fat diet feeding.     

Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm(2))-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.     

L-arginine protects from ischemia-reperfusion-induced endothelial dysfunction in humans in vivo.

It has been shown that nitric oxide (NO) protects from myocardial ischemia-reperfusion injury in animal models. The present study investigated whether administration of the NO substrate l-arginine protects against ischemia-reperfusion-induced endothelial dysfunction in humans. Forearm blood flow was measured with venous occlusion plethysmography in 16 healthy male subjects who were investigated on two occasions. Forearm ischemia was induced for 20 min followed by 60-min reperfusion. With the use of a crossover protocol, the subject received a 15-min intrabrachial artery infusion of l-arginine (20 mg/min) and vehicle (saline, n = 12 or d-arginine, n = 4) starting at 15 min of ischemia on two separate occasions. Compared with preischemia, endothelium-dependent increase in forearm blood flow induced by intra-arterial acetylcholine (3-30 microg/min) was significantly impaired at 15 and 30 min of reperfusion when the subjects received saline (P < 0.001). When the subjects received l-arginine, the acetylcholine-induced increase in forearm blood flow was not significantly affected by ischemia-reperfusion. The recovery of endothelium-dependent vasodilatation at 15- and 30-min reperfusion was significantly greater after administration of l-arginine than after saline (P < 0.05). d-Arginine did not affect the response to acetylcholine. Endothelium-independent vasodilatation to nitroprusside was not affected during reperfusion. These results demonstrate that the NO substrate l-arginine significantly attenuates ischemia-reperfusion-induced endothelial dysfunction in humans in vivo. This suggests that l-arginine may be useful as a therapeutic agent in the treatment of ischemia-reperfusion injury in humans.     

Flow-mediated release of nitric oxide in isolated, perfused rabbit lungs.

The effects of changing perfusate flow on lung nitric oxide (NO) production and pulmonary arterial pressure (Ppa) were tested during normoxia and hypoxia and after N(G)-monomethyl-L-arginine (L-NMMA) treatment during normoxia in both blood- and buffer-perfused rabbit lungs. Exhaled NO (eNO) was unaltered by changing perfusate flow in blood-perfused lungs. In buffer-perfused lungs, bolus injections of ACh into the pulmonary artery evoked a transient increase in eNO from 67 +/- 3 (SE) to 83 +/- 7 parts/billion with decrease in Ppa, whereas perfusate NO metabolites (pNOx) remained unchanged. Stepwise increments in flow from 25 to 150 ml/min caused corresponding stepwise elevations in eNO production (46 +/- 2 to 73 +/- 3 nl/min) without changes in pNOx during normoxia. Despite a reduction in the baseline level of eNO, flow-dependent increases in eNO were still observed during hypoxia. L-NMMA caused declines in both eNO and pNOx with a rise in Ppa. Pulmonary vascular conductance progressively increased with increasing flow during normoxia and hypoxia. However, L-NMMA blocked the flow-dependent increase in conductance over the range of 50-150 ml/min of flow. In the more physiological conditions of blood perfusion, eNO does not reflect endothelial NO production. However, from the buffer perfusion study, we suggest that endothelial NO production secondary to increasing flow, may contribute to capillary recruitment and/or shear stress-induced vasodilation.     

Impaired nitric oxide-mediated vasodilation in transgenic sickle mouse

Transgenic sickle mice expressing human beta(S)- and beta(S-Antilles)-globins show intravascular sickling, red blood cell adhesion, and attenuated arteriolar constriction in response to oxygen. We hypothesize that these abnormalities and the likely endothelial damage, also reported in sickle cell anemia, alter nitric oxide (NO)-mediated microvascular responses and hemodynamics in this mouse model. Transgenic mice showed a lower mean arterial pressure (MAP) compared with control groups (90 +/- 7 vs. 113 +/- 8 mmHg, P < 0.00001), accompanied by increased endothelial nitric oxide synthase (eNOS) expression. N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of NOS, caused an approximately 30% increase in MAP and approximately 40% decrease in the diameters of cremaster muscle arterioles (branching orders: A2 and A3) in both control and transgenic mice, confirming NOS activity; these changes were reversible after L-arginine administration. Aminoguanidine, an inhibitor of inducible NOS, had no effect. Transgenic mice showed a decreased (P < 0.02-0.01) arteriolar dilation in response to NO-mediated vasodilators, i.e., ACh and sodium nitroprusside (SNP). Indomethacin did not alter the responses to ACh and SNP. Forskolin, a cAMP-activating agent, caused a comparable dilation of A2 and A3 vessels ( approximately 44 and 70%) in both groups of mice. Thus in transgenic mice, an increased eNOS/NO activity results in lower blood pressure and diminished arteriolar responses to NO-mediated vasodilators. Although the increased NOS/NO activity may compensate for flow abnormalities, it may also cause pathophysiological alterations in vascular tone.     

Vascular wall energetics in arterioles during nitric oxide-dependent and -independent vasodilation.

The objective of this study was to evaluate whether the nitric oxide (NO) released from vascular endothelial cells would decrease vessel wall oxygen consumption by decreasing the energy expenditure of mechanical work by vascular smooth muscle. The oxygen consumption rate of arteriolar walls in rat cremaster muscle was determined in vivo during NO-dependent and -independent vasodilation on the basis of the intra- and perivascular oxygen tension (Po2) measured by phosphorescence quenching laser microscopy. NO-dependent vasodilation was induced by increased NO production due to increased blood flow, whereas NO-independent vasodilation was induced by topical administration of papaverine. The energy efficiency of vessel walls was evaluated by the variable ratio of circumferential wall stress (amount of mechanical work) to vessel wall oxygen consumption rate (energy cost) in the arteriole between normal and vasodilated conditions. NO-dependent and -independent dilation increased arteriolar diameters by 13 and 17%, respectively, relative to the values under normal condition. Vessel wall oxygen consumption decreased significantly during both NO-dependent and -independent vasodilation compared with that under normal condition. However, vessel wall oxygen consumption during NO-independent vasodilation was significantly lower than that during NO-dependent vasodilation. On the other hand, there was no significant difference between the energy efficiency of vessel walls during NO-dependent and -independent vasodilation, suggesting the decrease in vessel wall oxygen consumption produced by NO to be related to reduced mechanical work of vascular smooth muscle.     

Oral administration of l-citrulline alters the vascular delivery of substances to rat skeletal muscles

Vascular endothelial function deteriorates with age and disease, and the production of vasodilator factors like nitric oxide (NO) decreases. The free amino acid l-citrulline increases vasodilation and blood flow through increased NO production. We examined the effects of oral l-citrulline administration on vascular delivery of substances to skeletal muscles. In Experiment 1, following oral l-citrulline administration and subsequent intravenous Evans blue dye (EBD) administration to rats, EBD levels delivered to skeletal muscles were measured after 60 min. In Experiment 2, plasma concentrations of amino acids and NOx, an indicator of vasodilation, were measured over time after oral l-citrulline administration. In Experiment 3, we measured EBD levels in skeletal muscles of streptozotocin-induced type 1 diabetic rats following l-citrulline administration. In these experiments, EBD levels in the soleus muscle were higher in the l-citrulline group than in the control group (19.9 ± 0.7 vs. 22.5 ± 1.9 μg/g tissue, p < 0.05). Plasma l-arginine, l-citrulline, and NOx levels were increased within 30 min after l-citrulline administration. EBD levels in the soleus and gastrocnemius muscles were higher in diabetic rats with l-citrulline administration (18.7 ± 2.2 vs. 25.0 ± 4.3 μg/g tissue, p < 0.05 and 8.0 ± 0.5 vs. 9.2 ± 0.8 μg/g tissue, p = 0.05, respectively). These data suggest that oral l-citrulline administration may increase the level of substances delivered to skeletal muscles by increasing the NO production in both normal and vascular endothelial dysfunction models.     

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.     

Plasma from conscious hypoxic rats stimulates leukocyte-endothelial interactions in normoxic cremaster venules.

Systemic hypoxia results in rapid increases in leukocyte-endothelial adherence (LEA) and emigration, vascular permeability, and mast cell activation in several microcirculations. Observations in cremaster muscle suggest that this response is initiated by a mediator released from a distant site (Dix R, Orth T, Allen JA, Wood JG, and Gonzalez NC. J Appl Physiol 95: 2495-2502, 2003). The present experiments in rat cremaster muscle tested the hypothesis that, if a circulating mediator triggers hypoxia-induced inflammation, then plasma from hypoxic rats should elicit LEA in normoxic cremaster venules. Plasma from conscious donor rats breathing 10% O2-90% N2 for 5 min was applied topically to the cremaster of normoxic anesthetized rats. In this and all other groups described below, the donor plasma had attained normoxic PO2 when applied to the cremaster. LEA (leukocytes/100-microm venule) increased from 2.7 +/- 0.8 to 12.3 +/- 2.4, and venular shear rate and arteriolar diameter decreased to 79 +/- 9% (P < 0.05, n = 6) and 77 +/- 5% of control (P < 0.05, n = 5), respectively, 10 min after application of plasma from hypoxic donors. The decrease in venular shear rate was exclusively due to a reduction of venular blood flow, secondary to the upstream arteriolar vasoconstriction. Plasma from normoxic donors had no effects. Plasma from blood equilibrated in vitro for 5 min with 5% CO2-95% N2 did not alter LEA or shear rate of normoxic cremasters, suggesting that the putative mediator does not originate in blood cells. The effects of plasma from hypoxic rats persisted when the donors were pretreated with the mast cell stabilizer cromolyn, which prevents hypoxia-induced LEA. This suggests that the effects of hypoxic plasma are not due to inflammatory mediators released by adherent leukocytes in the donor rat. There was a positive correlation between LEA and mast cell degranulation observed histologically. These results support the idea that systemic hypoxia produces the release of a substance transported by the circulation that initiates the microvascular inflammation.     

Effect of rhythmic tetanic skeletal muscle contractions on peak muscle perfusion.

The purpose of this investigation was to examine the effect of rhythmic tetanic skeletal muscle contractions on peak muscle perfusion by using spontaneously perfused canine gastrocnemii in situ. Simultaneous pulsatile blood pressures were measured by means of transducers placed in the popliteal artery and vein, and pulsatile flow was measured with a flow-through-type transit-time ultrasound probe placed in the venous return line. Two series of experiments were performed. In series 1, maximal vasodilation of the muscles' vascular beds was elicited by infusing a normal saline solution containing adenosine (29.3 mg/min) and sodium nitroprusside (180 microg/min) for 15 s and then simultaneously occluding both the popliteal artery and vein for 5 min. The release of occlusion initiated a maximal hyperemic response, during which time four tetanic contractions were induced with supramaximal voltage (6-8 V, 0.2-ms stimuli for 200-ms duration at 50 Hz, 1/s). In series 2, the muscles were stimulated for 3 min before the muscle contractions were stopped for a period of 3 s; stimulation was then resumed. The results of series 1 indicate that, although contractions lowered venous pressure, muscle blood flow was significantly reduced from 2,056 +/- 246 to 1,738 +/- 225 ml x kg(-1) x min(-1) when contractions were initiated and then increased significantly to 1,925 +/- 225 ml x kg(-1) x min(-1) during the first 5 s after contractions were stopped. In series 2, blood flow after 3 min of contractions averaged 1,454 +/- 149 ml x kg(-1) x min(-1). Stopping the contractions for 3 s caused blood flow to increase significantly to 1,874 +/- 172 ml x kg(-1) x min(-1); blood flow declined significantly to 1,458 +/- 139 ml x kg(-1) x min(-1) when contractions were resumed. We conclude that the mechanical action of rhythmic, synchronous, maximal isometric tetanic skeletal muscle contractions inhibits peak muscle perfusion during maximal and near-maximal vasodilation of the muscle's vascular bed. This argues against a primary role for the muscle pump in achieving peak skeletal muscle blood flow.     

Mechanisms of shear stress-induced endothelial nitric-oxide synthase phosphorylation and expression in ovine fetoplacental artery endothelial cells

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.     

Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase

Estrogen increases nitric oxide (NO) production by inducing the activity of endothelial NO synthase (eNOS) (Simoncini et al. Nature 407: 538, 2000). Ischemia (30 min) and reperfusion (I/R) increased the number of adherent leukocytes and decreased their rolling velocities in mouse cremaster muscle venules with a strong dependence on wall shear rate. Minimum rolling velocity at approximately 5 min after the onset of reperfusion was accompanied by increased P-selectin expression. This preceded the peak in leukocyte adhesion (at 10-15 min). In untreated wild-type mice, I/R caused a decrease of leukocyte rolling velocity from 37 to 26 microm/s and a 2.0-fold increase in leukocyte adhesion. Both were completely abolished by 0.25 mg ip estrogen 1 h before surgery. In eNOS(-/-) mice, the decrease of leukocyte rolling velocity and increase in adhesion were similar but were only marginally improved by estrogen. We conclude that the protective effect of estrogen, as measured by leukocyte rolling and adhesion, is significantly reduced in eNOS(-/-) mice, suggesting that induction of eNOS activity is the major mechanism of vasoprotection by estrogen in this model.     

内源性一氧化氮在内毒素引起的肺动脉高压和肺损伤中的作用

本实验观察了家兔静脉内注入内毒素的主要成分脂多糖(LPS)后平均动脉血压(MAP)、肺动脉压(PAP)及入、出肺血NO含量的变化,并观察了静脉内预注入NO生成抑制剂Nω-硝基-L-精氨酸(L-NNA)及诱生型NO生成抑制剂氨基胍(AG)后PAP和肺损伤的变化.结果观察到:家兔LPS注入后,MAP均明显下降,LPS注入后0.5、1、1.5、2h PAP明显增高(P<0.05).LPS注入后PAP的高峰期(1h)入肺血NO含量明显降低,出肺血NO无明显变化.与对照组相比,LPS注入后3h出肺血NO含量和5h入、出肺血NO含量均明显增多.相关分析表明,兔LPS注入前和LPS注入后1h PAP与入肺血NO含量呈明显的负相关,而LPS注入后 3h和5h两者相关不明显.静脉预注入L-NNA后,LPS处理组的动物PAP明显增高,入、出肺血丙二醛(MDA)含量也明显增高,动物生存率明显降低.肺组织光镜下可见肺萎陷和小血管淤血加重,白细胞明显增加.静脉预注入AG后,LPS处理组的动物MAP在3～5h明显增高,此时PAP无明显改变,但5h时血中MDA含量明显减低,5h时与LPS组相比肺萎陷和小血管淤血减轻,白细胞也明显减少.以上结果提示,内毒素入血后较早期阶段可出现PAP的升高,此时入肺血NO的减少是参与肺动脉压增高(PAH)的机制之一.家兔内毒素进入血后较早期阶段NO对减轻内毒素引起的PAH和肺损伤起重要作用,而较晚的时期当诱生型NO合酶(iNOS)诱生后释放的NO则参与内毒素引起的肺组织炎症反应和肺损伤.     

Systemic alpha-adrenergic and nitric oxide inhibition on basal limb blood flow: effects of endurance training in middle-aged and older adults

Endurance training improves endothelium-dependent vasodilation, yet it does not increase basal blood flow in the legs. We determined the effects of a 3-mo aerobic exercise intervention on basal leg blood flow and alpha-adrenergic vasoconstriction and nitric oxide (NO) release in seven apparently healthy middle-aged and older adults (60 +/- 3 yr). Basal femoral artery blood flow (via Doppler ultrasound) (pretraining: 354 +/- 29; posttraining: 335 +/- 34 ml/min) and vascular conductance did not change significantly with the exercise training. Before the exercise intervention, femoral artery blood flow increased 32 +/- 16% with systemic alpha-adrenergic blockade (with phentolamine) (P < 0.05), and the addition of nitric oxide synthase (NOS) inhibition using N(G)-monomethyl-L-arginine (L-NMMA) did not affect femoral artery blood flow. After training was completed, femoral artery blood flow increased 47 +/- 7% with alpha-adrenergic blockade (P < 0.01) and then decreased 18 +/- 7% with the subsequent administration of L-NMMA (P < 0.05). Leg vascular conductance showed a greater alpha-adrenergic blockade-induced vasodilation (+1.7 +/- 0.5 to +3.0 +/- 0.5 units, P < 0.05) as well as NOS inhibition-induced vasoconstriction (-0.8 +/- 0.4 to -2.7 +/- 0.7 units, P < 0.05) after the exercise intervention. Resting plasma norepinephrine concentration significantly increased after the training. These results suggest that regular aerobic exercise training enhances NO bioavailability in middle-aged and older adults and that basal limb blood flow does not change with exercise training because of the contrasting influences of sympathetic nervous system activity and endothelium-derived vasodilation on the vasculature.     

Effect of uterine blood flow occlusion on shear stress-mediated nitric oxide production and endothelial nitric oxide synthase expression during ovine pregnancy

During normal pregnancy, uterine blood flow (UBF) is increased in association with elevations of endothelial nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Shear stress increases endothelial-derived NO production to reduce vasomotor tone. We hypothesized that decreasing in vivo UBF, and thus shear stress, will decrease NO and/or eNOS levels. In this experiment, one of the main uterine arteries of chronically instrumented late pregnant sheep (125 +/- 1 days' gestation [mean +/- SEM]; n = 15) was occluded for 24 h. Cardiovascular parameters (systemic and uterine arterial pressure, heart rate [HR], and ipsilateral and contralateral UBF) and NO(2)/NO(3) (NO(x)) levels were evaluated. Although UBF measured using Transonic flow probes was reduced unilaterally 41.5% +/- 2.1%, uterine perfusion pressure only fell 12.2% +/- 4.5%. Systemic arterial blood pressure and HR were unaltered. Using radioactive microspheres, ipsilateral UBF was reduced approximately 28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NO(x) levels were reduced 22.1% +/- 6.7% and 22.6% +/- 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial ( approximately 2.5 h) increase in systemic arterial and uterine venous NO(x) levels by 116% +/- 30% and 97% +/- 49%, respectively. Despite a decline in NO(x) levels after 6 h, no significant differences versus preocclusion NO(x) levels were detected by 24 h of occlusion in this experimental group. In contrast, NO(x), UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NO(x).     

Evidence for a basal release of a cytochrome-related endothelium-derived hyperpolarizing factor in the radial artery in humans

Whether a cytochrome P-450 (CYP)-related endothelium-derived hyperpolarizing factor (EDHF), acting through calcium-activated potassium (K(Ca)) channels, interacts with nitric oxide (NO) to regulate the basal diameter of human peripheral conduit arteries is unexplored in vivo. Radial artery diameter (echo tracking) and blood flow (Doppler) were measured, after oral aspirin (500 mg), in eight healthy volunteers during local infusion for 8 min of tetraethylammonium chloride (TEA; 9 micromol/min), as K(Ca) channel inhibitor, and fluconazole (0.4 micromol/min), as CYP inhibitor, alone and in combination with N(G)-monomethyl-L-arginine (L-NMMA; 8 micromol/min), as endothelial NO synthase inhibitor. Endothelium-independent dilatation was assessed by using sodium nitroprusside (SNP). Radial diameter was unaffected by L-NMMA (0.4 +/- 0.9%) and fluconazole (-1.6 +/- 0.8%) but was decreased by TEA (-5.0 +/- 1.0%), L-NMMA + fluconazole (-5.3 +/- 0.5%), and L-NMMA + TEA (-9.9 +/- 1.3%). These effects are still significant even when the concomitant decreases in blood flow induced by L-NMMA (-24 +/- 4%), TEA (-21 +/- 3%), L-NMMA + fluconazole (-26 +/- 5%), and L-NMMA + TEA (-35 +/- 4%) were taken as covariate into analysis. Conversely, fluconazole alone slightly but not significantly increased radial flow (13 +/- 6%). L-NMMA alone or with TEA and with fluconazole enhanced radial artery dilatation to SNP, whereas TEA and fluconazole alone did not modify this response. These results confirm in humans the involvement of NO and K(Ca) channels in the regulation of basal conduit artery diameter. Moreover, the synergistic effect of combined inhibition of NO synthesis and CYP on the decrease in radial diameter in the absence of such effect after L-NMMA and fluconazole alone unmasks the role of CYP in this regulation and shows the presence of an interaction between NO and a CYP-related EDHF to maintain peripheral conduit artery diameter in vivo. Furthermore, the higher vasoconstrictor effect of TEA compared with fluconazole suggests that different K(Ca) channel-dependent hyperpolarizing mechanisms could exist in conduit arteries.     

Permanently increased conductance of the murine uterine arcade after the first pregnancy

Mechanical forces due to increased blood flow during the first pregnancy might induce a permanently higher conductance of the uterine arcade. Number of endothelial and smooth muscle cells, cross-sectional area and wall thickness of the uterine artery were measured in nulliparous mice (n = 11) and until the 93rd day after parturition in primiparous mice (n = 44). Inner diameter and wall area were calculated. Three months after the first pregnancy, uterine artery wall thickness was not altered compared to nulliparous mice. In contrast, inner diameter increased 1.6-fold, wall area 1.5-fold and the numbers of endothelial and smooth muscle cells increased 1.5 times. These changes were completely stable during the whole observation period. The increased blood flow during the first pregnancy might be a mandatory mechanical stimulus for uterine arcade maturation. This physiological maturation process could result in abortion explaining the higher prevalence of unexplained pregnancy losses in primiparous women.     

Reperfusion injury is reduced in skeletal muscle by inhibition of inducible nitric oxide synthase.

This study evaluated the effects of the selective inducible nitric oxide synthase (iNOS) inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W) on the microcirculation in reperfused skeletal muscle. The cremaster muscles from 32 rats underwent 5 h of ischemia followed by 90 min of reperfusion. Rats received either 3 mg/kg 1400W or PBS subcutaneously before reperfusion. We found that blood flow in reperfused muscles was <45% of baseline in controls but sharply recovered to near baseline levels in 1400W-treated animals. There was a significant (P < 0.01 to P < 0.001) difference between the two groups at each time point throughout the 90 min of reperfusion. Vessel diameters remained <80% of baseline in controls during reperfusion, but recovered to the baseline level in the 1400W group by 20 min, and reached a maximum of 121 +/- 14% (mean +/- SD) of baseline in 10- to 20-micro m arterioles, 121 +/- 6% in 21- to 40-micro m arterioles, and 115 +/- 8% in 41- to 70-micro m arteries (P < 0.01 to P < 0.001). The muscle weight ratio between ischemia-reperfused (left) and non-ischemia-reperfused (right) cremaster muscles was 193 +/- 42% of normal in controls and 124 +/- 12% in the 1400W group (P < 0.001). Histology showed that neutrophil extravasation and edema were markedly reduced in 1400W-treated muscles compared with controls. We conclude that ischemia-reperfusion leads to increased generation of NO from iNOS in skeletal muscle and that the selective iNOS inhibitor 1400W reduces the negative effects of ischemia-reperfusion on vessel diameter and muscle blood flow. Thus 1400W may have therapeutic potential in treatment of ischemia-reperfusion injury.     

Antihypertensive effects of tannins isolated from traditional Chinese herbs as non-specific inhibitors of angiontensin converting enzyme

The tannins are natural polyphenols, able to precipitate water-soluble alkaloids and possess an inhibitory action on the angiotensin converting enzyme (ACE). We identified 18 polyphenolic compounds (tannins) from Chinese herbs and examined the in vitro effects of these tannins on ACE activity, including determination of the 50% inhibitory concentrations (IC50), specificity and mode of inhibition. We also assessed the in vivo inhibitory effect of the tannins on angiotensin I-induced blood pressure elevation in spontaneously hypertensive rats (SHR). Nine tannins with an IC50 <200 microM for ACE inhibitors were identified belonging to three tannin classes: caffeoylquinates, flavan-3-ols and gallotannins. In vitro, we found caffeoylquinates chelate the ACE zinc cofactor. Two of the flavan-3-ols: epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, and one of gallotannin: 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose were non-specific inhibitors because also reduced the activity of trypsin and chymotrypsin. The ACE inhibition of 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose was also reduced after addition of bovine serum albumin, suggesting a non-specific mode of action. In vivo, 1,2,3,6-tetra-O-galloyl-beta-D-glucose and epigallocatechin-3-O-methylgallate had a strong dose-dependent hypotensive effect reducing the blood pressure significantly in the SHR with infusion of the angiotensin I. These findings indicate that some of the tannins isolated from herbs inhibit ACE activity non-specifically. The ACE inhibitory effect of these tannins may explain the hypotensive effects of some traditional Chinese herbs.