Anita Racz

ADMA Impairs Nitric Oxide–Mediated Arteriolar Function Due to Increased Superoxide Production by Angiotensin II–NAD(P)H Oxidase Pathway

Asymmetrical dimethylarginine (ADMA) is thought to be an endogenous regulator of arteriolar tone by inhibiting NO synthase. However, our previous studies showed that, in isolated arterioles, ADMA induced superoxide production as well. Thus, the mechanisms by which ADMA affects arteriolar tone remain obscure. We hypothesized that ADMA, by activating NAD(P)H oxidase, increases superoxide production, interfering with NO mediation of flow-induced dilation. In the presence of indomethacin, isolated arterioles from rat gracilis muscle (≈160 &mgr;m at 80 mm Hg) were incubated with ADMA (10−4 mol/L), which elicited significant constriction (from 162±4 to 143±4 &mgr;m) and eliminated the dilations to increases in intraluminal flow (from a maximum 31±2% to 3±1%; P<0.05). In the presence of ADMA, superoxide dismutase plus catalase restored dilations to flow (from a maximum 3±1% to 28±2%). Endothelial denudation or incubation of arterioles with the NAD(P)H oxidase inhibitor apocynin or the angiotensin-converting enzyme inhibitor quinapril inhibited ADMA-induced constriction. In addition, apocynin, quinapril, or the angiotensin type 1 receptor blocker losartan restored flow-induced dilations reduced by ADMA. Furthermore, inhibition of NO synthase abolished the “superoxide dismutase/catalase-restored” flow-induced dilation in the presence of ADMA. ADMA-induced increased production of superoxide, assessed by dihydroethidium fluorescence, was inhibited by apocynin, quinapril, or losartan. We suggest that ADMA activates the local renin-angiotensin system, and the angiotensin II released activates NAD(P)H oxidase; superoxide produced interferes with the bioavailability of NO, resulting in diminished flow-induced dilation, a mechanism that may contribute to the development of arteriolar dysfunction and increased tone associated with elevated ADMA levels.

Asymmetrical Dimethylarginine Inhibits Shear Stress–Induced Nitric Oxide Release and Dilation and Elicits Superoxide-Mediated Increase in Arteriolar Tone

l-arginine is the substrate used by NO synthase to produce the vasodilator NO. However, in several human diseases, such as hyperhomocysteinemia, diabetes mellitus, and hypertension, there is an increase in serum levels of methylated l-arginines, such as asymmetrical dimethylarginine (ADMA), which cannot be used by NO synthase to produce NO. Yet, the functional consequence of increased levels of ADMA on the vasomotor function of resistance vessels has not been delineated. We hypothesized that elevated levels of exogenous ADMA inhibit NO mediation of flow/shear stress–dependent dilation of isolated arterioles. In the presence of indomethacin, isolated arterioles from rat gracilis muscle (≈165 &mgr;m at 80 mm Hg) were incubated with ADMA (10−4 mol/L), which eliminated the dilations to increases in intraluminal flow (control: from 164±5.4 to 188±3.8 &mgr;m versus ADMA: from 171±6.1 to 173±6.3 &mgr;m at 20 &mgr;L/min). ADMA did not affect dilations to nifedipine (10−6 mol/L; control: 63.4±2%, ADMA: 65.8±3%) or 8-bromo cGMP (10−4 mol/L; control: 51.2±2.1%, ADMA: 49.3±3.4%). In addition, ADMA elicited significant constriction of arterioles (from 173±17 &mgr;m to 138±16 &mgr;m at 80 mm Hg), which was prevented by previous incubation of arterioles with polyethylene-glycol (PEG) superoxide dismutase (SOD; 120 U/mL, control: 155±11 &mgr;m versus ADMA: 150±14 &mgr;m). Correspondingly, ADMA increased PEG-SOD reversible manner the production of vascular superoxide assessed by lucigenin-enhanced chemiluminescence and ethidium bromide fluorescence. Thus, increased levels of ADMA in various diseases could inhibit the regulation of arteriolar resistance by shear stress–induced release of NO and elicit superoxide-mediated increase in basal tone, both of which favor the development of hypertension.

Cyclooxygenase-2 derived thromboxane A2 and reactive oxygen species mediate flow-induced constrictions of venules in hyperhomocysteinemia

OBJECTIVE Hyperhomocysteinemia (HHcy) has been shown to impair the endothelial function of arterial vessels and promote thrombosis. There are no studies, however, assessing the effects of HHcy on the vasomotor function of venules. We hypothesized that HHcy activates pathophysiological mechanisms impairing flow/shear stress-dependent responses of venules. METHODS AND RESULTS Changes in diameter of isolated gracilis muscle venules (diameter: approximately 250 microm at 10 mmHg) of control and HHcy rats (induced by methionine diet for 5 weeks) to increases in intraluminal flow were measured. Increases in flow elicited dilations in control (at max.: 14+/-1%), but induced constrictions in HHcy venules (at max.: -24+/-4%). Flow-induced constrictions in HHcy venules were converted to dilations in the presence of the thromboxane A(2) (TxA(2)) receptor (TP) antagonist SQ 29,548, which were then abolished by the simultaneous administration of nitric oxide (NO) synthase inhibitor, L-NAME and non-selective cyclooxygenase (COX) blocker, indomethacin. In addition, the selective COX-2 inhibitor NS 398 reversed flow-induced constrictions to dilations, which were significantly decreased by additional COX-1 inhibitor, SC 560. Also, as compared to controls, a SOD/CAT sensitive increased ethidium bromide fluorescence was detected in HHcy small veins, indicating substantial production of reactive oxygen species (ROS) in HHcy. Correspondingly, SOD/CAT diminished flow-induced constrictions in venules of HHcy rats. CONCLUSIONS In hyperhomocysteinemia increases in flow/shear stress increases the production of COX-2-derived TxA(2), and reactive oxygen species--that overcome the dilator effects of NO and prostaglandins--eliciting constrictions in skeletal muscle venules; changes which can increase vascular resistance and favor thrombus formation in the venular circulation.

Thromboxane A2 Contributes to the Mediation of Flow-Induced Responses of Skeletal Muscle Venules: Role of Cyclooxygenases 1 and 2

Background: It has been shown that increases in intraluminal flow elicit dilation in venules, but the mediation of response is not yet clarified. We hypothesized that – in addition to nitric oxide (NO) and dilator prostaglandins (PGI2/ PGE2) – thromboxane A2 (TxA2) contributes to the mediation of flow-induced responses of venules. Methods and Results: Isolated rat gracilis muscle venules (259 ± 11 μm at 10 mm Hg) dilated as a function of intraluminal flow, which was augmented in the presence of the TxA2 receptor antagonist SQ 29,548 or the TxA2 synthase inhibitor ozagrel. In the presence of SQ 29,548, indomethacin or Nω-nitro-L-arginine methyl-ester decreased flow-induced dilations, whereas in their simultaneous presence dilations were abolished. The selective cyclooxygenase (COX) 1 inhibitor SC 560 reduced, whereas the selective COX-2 inhibitor NS 398 enhanced flow-induced dilations. Immunohistochemistry showed that both COX-1 and COX-2 are present in the wall of venules. Conclusion: In skeletal muscle venules, increases in intraluminal flow elicit production of constrictor TxA2, in addition to the dilator NO and PGI2/PGE2, with an overall effect of limited dilation. These mediators are likely to have important roles in the multiple feedback regulation of wall shear stress in venules during changes in blood flow velocity and/or viscosity.

Hydrogen peroxide via thromboxane A2 receptors mediates myogenic response of small skeletal muscle veins in rats.

UNLABELLED We aimed to test two hypotheses: (1) isolated small veins develop substantial myogenic tone in response to elevation of intraluminal pressure, (2) H2O2 contributes to the mediation of myogenic response via activation of arachidonic acid (AA) cascade and release constrictor prostaglandins. METHODS Small veins were isolated from gracilis muscle of male rats, then cannulated. Changes of diameter to increases in intraluminal pressure, H2O2 and arachidonic acid in the presence and absence of various inhibitors were measured by videomicroscope and microangiometer. At the end of experiments the passive diameter were obtained in Ca2+ -free PSS solution. RESULTS Isolated rat gracilis muscle small veins developed a substantial myogenic tone in response to increases in intraluminal pressure (1-12 mmHg). Calculated maximum myogenic tone was 70 ± 5% at 10 mmHg. Presence of catalase or indomethacin or SQ 29,548 reduced significantly the pressure-induced myogenic response. Also, H2O2 (10-9-10-5 M) and arachidonic acid (10-7-10-4 M) elicited concentration dependent constrictions, which were inhibited by the presence of indomethacin or SQ 29,548. CONCLUSION We propose that both myogenic response and pressure-induced release of H2O2 play important roles in regulating the vasomotor function of venules both in physiological and pathological conditions.

Vázizomkisvénák vazomotortónusának intrinszik szabályozómechanizmusai

In many developed countries the prevalence of venous disorders and its consequences are higher than that of arterial diseases. Thus it is very important to understand the exact physiological and pathophysiological function of small veins and their control mechanisms. Small veins and venules have an important role in the regulation of capillary fluid exchange, as well as return of the venous blood into the heart. However, there is only limited knowledge available regarding the role of local mechanisms controlling the vasomotor tone and diameter of small veins. In the last decade the authors focused on the elucidation of these mechanisms in isolated skeletal muscle venules of rats. Their results suggest that the tone of small veins is controlled by the integration of several mechanisms, activated by the intraluminal pressure and flow/wall shear stress, in addition to numerous local mediators synthesized and released from the smooth muscle and endothelium. These mechanisms are involved - in a complex manner - in the control of postcapillary resistance, thus regulation of tissue blood supply, venous return and consequently in the modulation of the cardiac output, as well.