Blockade Of Nitric Oxide Synthesis Research Articles

Role for endothelium‐derived hyperpolarizing factor in vascular tone in rat mesenteric and hindlimb circulations in vivo

The role of endothelium-derived hyperpolarizing factor (EDHF) in the regulation of blood flow in vivo was examined in the mesenteric and hindlimb circulations of anaesthetized rats. Basal mesenteric conductance decreased from 57 +/- 5 to 20 +/- 6 microl min(-1) mmHg(-1) when nitric oxide (NO) production was inhibited, and combined blockade of intermediate- and small-conductance Ca(2+)-activated K(+) (K(Ca)) channels with charybdotoxin (ChTx) and apamin had no further effect. Basal hindlimb conductance was reduced from 39 +/- 3 to 22 +/- 2 microl min(-1) mmHg(-1) by NO synthesis inhibition, with no effect of the K(Ca) channel blockers. Endothelial stimulation with acetylcholine (ACh) infusion directly into the mesenteric bed increased conductance by 20 +/- 2 microl min(-1) mmHg(-1). Blockade of NO synthesis decreased this conductance to 15 +/- 1 microl min(-1) mmHg(-1), leaving the response attributable to EDHF. This was reduced to 2 +/- 1 microl min(-1) mmHg(-1) by ChTx plus apamin but not by iberiotoxin, which selectively blocks large-conductance K(Ca) channels. Similar results were obtained when bradykinin (BK) was used to stimulate the endothelium. Nitroprusside, which directly relaxes smooth muscle, evoked an increase in conductance that was resistant to all blockers tested. ACh-induced increases in hindlimb conductance were reduced from 19 +/- 1 to 12 +/- 1 microl min(-1) mmHg(-1) by NO synthesis inhibition and further reduced to 2 +/- 2 microl min(-1) mmHg(-1) by ChTx plus apamin. In contrast to NO, ChTx- and apamin-sensitive EDHF appears to contribute little to basal conductance in rat mesenteric and hindlimb beds. However, EDHF accounts for a significant component of the conductance increase during endothelial stimulation by ACh and BK. In these beds, intermediate- and small-conductance K(Ca) channels underpin EDHF-mediated vasodilatation.

Nitric Oxide Modulates Vascular Disease in the Remnant Kidney Model

A loss of the microvascular endothelium occurs in the remnant kidney model of renal disease and may play an important role in progression (Kang et al, J Am Soc Nephrol, 12:1434, 2001). Given that nitric oxide (NO) is a potent endothelial cell survival factor, we hypothesized that stimulating (with L-arginine) or blocking (with nitro-L-arginine methyl ester, (L-NAME)) NO synthesis could modulate the integrity of the microvasculature and hence affect progression of renal disease. Rats underwent 5/6 nephrectomy (RK) and then were randomized at 4 weeks to receive vehicle, L-NAME, or L-arginine for 4 weeks. Systolic blood pressure and renal function was measured, and tissues were collected at 8 weeks for histological and molecular analyses. The effect of modulation of NO on vascular endothelial growth factor (VEGF) expression in rat aortic vascular smooth muscle cells (SMC) and mouse medullary thick ascending limb tubular epithelial cells (mTAL) was also studied. Inhibition of NO with L-NAME was associated with more rapid progression compared to RK alone, with worse blood pressure, proteinuria, renal function, glomerulosclerosis, and tubulointerstitial fibrosis. The injury was also associated with more glomerular and peritubular capillary endothelial cell loss in association with an impaired endothelial proliferative response. Interestingly, the preglomerular endothelium remained intact or was occasionally hyperplastic, and this was associated with a pronounced proliferation of the vascular SMCs with de novo expression of VEGF. Cell culture studies confirmed a divergent effect of NO inhibition on VEGF expression, with inhibition of VEGF synthesis in mTAL cells and stimulation of VEGF in vascular SMC. In contrast to the effects of NO inhibition, stimulation of NO with L-arginine had minimal effects in this rat model of progressive renal disease. These studies confirm that blockade of NO synthesis accelerates progression of renal disease in the remnant kidney model, and support the hypothesis that one of the pathogenic mechanisms may involve accelerated capillary loss and impaired angiogenesis of the renal microvasculature. Interestingly, inhibition of NO synthesis did not lead to a loss of the preglomerular endothelium, which may relate to the effect of NO blockade to stimulate VEGF synthesis in the adjacent vascular smooth muscle cell.

Attenuation of hypertension, cardiomyocyte hypertrophy, and myocardial fibrosis by beta-adrenoceptor blockers in rats under long-term blockade of nitric oxide synthesis.

The effects of propranolol and atenolol were investigated on arterial hypertension, cardiomyocyte hypertrophy, and ventricular ischaemic lesions induced by an 8-week treatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 20 mg/rat per day) in Wistar rats. Propranolol and atenolol (30 mg/rat per day each) were given in the drinking water concomitantly to L-NAME. Treatment with L-NAME induced marked arterial hypertension and cardiomyocyte hypertrophy, both of which were significantly reduced by propranolol and atenolol. A marked repairing fibrosis was also observed in L-NAME-treated rats and this was significantly attenuated in animals receiving the beta-blockers. In L-NAME group, 33% mortality was observed, whereas all the animals from the other groups survived. Our study demonstrates that propranolol and atenolol reduce arterial hypertension, cardiomyocyte hypertrophy and myocardial fibrosis induced by L-NAME, suggesting that beta-blockers are of beneficial value in treatment of vascular and cardiac alterations caused by chronic nitric oxide deficiency.

Important role of Rho-kinase in the pathogenesis of cardiovascular inflammation and remodeling induced by long-term blockade of nitric oxide synthesis in rats.

Chronic inhibition of endothelial NO synthesis by the administration of N(G)-nitro-L-arginine methyl ester (L-NAME) to rats induces early vascular inflammation (monocyte infiltration into coronary vessels and monocyte chemoattractant protein-1 expression) as well as subsequent arteriosclerosis. The small GTPase Rho controls cell adhesion, motility, and proliferation and is activated by several growth factors such as angiotensin II. We investigated the effect of a specific inhibitor of Rho-kinase, Y-27632, in rats treated with L-NAME to determine the role of the Rho/Rho-kinase pathway in the development of arteriosclerosis. We found here increased activity of Rho/Rho-kinase after L-NAME administration and its prevention by angiotensin II type 1 receptor blockade. Hydralazine or lecithinized superoxide dismutase (l-SOD) did not affect Rho/Rho-kinase activity. Co-treatment with Y-27632 did not affect the L-NAME-induced increase in cardiovascular tissue ACE activity or L-NAME-induced decrease in plasma NO concentrations, but did prevent the L-NAME-induced early inflammation and late coronary arteriosclerosis. In addition, Y-27632 prevented the increased gene expression of monocyte chemoattractant protein-1 and transforming growth factor-beta1 as well as cardiac fibrosis and glomerulosclerosis. These findings suggest that increased activity of Rho/Rho-kinase pathway mediated via the angiotensin II type 1 receptor may thus be important in the pathogenesis of early vascular inflammation and late remodeling induced by chronic inhibition of NO synthesis. The beneficial effects of Rho-kinase inhibition are not mediated by restoration of NO production. The Rho-kinase pathway could be a new therapeutic target for treatment of vascular diseases.

Role of nitric oxide in catalepsy and hyperthermia in morphine-dependent rats

The possible involvement of nitric oxide (NO) in morphine-induced catalepsy and hyperthermia was studied in morphine-dependent rats. Four days repeated injection regimen was used to induce morphine dependence, which was assessed by naloxone challenge (0.5 mg kg −1, s.c.). Pretreatment of rats with the NO synthase inhibitor, NG-nitro-l-arginine (l-NA, 8 mg kg −1twice daily, i.p.) potentiated the cataleptic response of morphine as shown by a rightward shift in the morphine-log dose–response curve. Prior treatment of rats with the NO precursor, l-arginine (200 mg kg −1, twice daily, i.p.) abolished the potent effect of l-NA and restored the cataleptic scores to levels similar to those of morphine-dependent rats. The same dose of l-NA significantly blocked morphine-induced hyperthermia at the dose levels of morphine (15–105 mg kg −1) and this effect was reversed by l-arginine. These data provide the first experimental evidence that NO is involved in morphine induced catalepsy and hyperthermia and demonstrated that blockade of NO synthesis may suggest a dangerous interaction with opioids in the control of motor function.

Vasomotor effects of arg-gly-asp (RGD) peptides are limited and not related to endothelium-derived hyperpolarizing factor-mediated relaxation in rat mesenteric arteries.

1. In the present study we tested the effect of arg-gly-asp (RGD) peptides on vasomotor responses in rat isolated mesenteric arteries. More specifically, the hypothesis was tested that RGD interaction with integrins mediates relaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). 2. The presence of the beta3 integrin subunit was shown by western blot analysis. To study its functional role, arteries (355 +/- 11 microm; n = 50) were mounted in a wire myograph set-up to measure isometric force generation. After blockade of nitric oxide synthesis with N(G)-nitro-L-arginine (0.1 mmol/L) and prostaglandin synthesis with indomethacin (10 micromol/L), methacholine (10 micromol/L) induced a transient relaxation within 1 min of 72 +/- 4.0% (as percentage of precontraction with phenylephrine; n = 27). 3. These responses were inhibited by a 60 mmol/L potassium buffer (18 +/- 6.0%; n = 6) or endothelium denudation (12 +/- 3.2%; n = 7), consistent with EDHF. 4. A function-blocking monoclonal antibody against the integrin beta3 chain did not affect relaxation. 5. The RGD peptides gly-arg-gly-asp-thr-pro (GRGDTP), gly-arg-gly-asp-ser (GRGDS) and cyclic RGD, ligands for the RGD binding site of integrins, also did not affect relaxation induced by methacholine. 6. Cyclic RGD increased contraction from 91 +/- 3 to 98 +/- 3% (as percentage of 120 mmol/L potassium). 7. In conclusion, these data show that vasomotor responses related to integrins are small and not involved in hyperpolarization attributed to EDHF in rat mesenteric artery.

Blockade of nitric oxide synthesis reduces responding for cocaine self-administration during extinction and reinstatement

Nitric oxide is a gaseous neurotransmitter that plays a significant role in various forms of synaptic plasticity and may play a role in the behavioral effects of psychostimulant drugs and in cocaine addiction. The course of drug addiction consists of different phases. Relapse into drug-seeking behavior following a period of abstinence is believed to represent one of the major factors leading to the perpetuation of the addictive cycle. In this respect, experimental extinction procedures provide a measure of the motivational properties of drugs as reflected by the persistence of drug-seeking behavior in the absence of the drug and by the reinstatement of responding by non-contingent drug administration. Pretreatment with the nitric oxide synthase inhibitor l-NG-nitroarginine methyl ester ( l-NAME, 50 mg/kg IP twice daily for 4 days) impaired responding for cocaine self-administration when the drug was available and the increase of drug-seeking behavior upon abrupt cessation of cocaine availability observed in control rats was significantly reduced after treatment with l-NAME. In addition, the priming effect of a non-contingent injection of cocaine on extinguished cocaine self-administration was also diminished by the same treatment. The acquisition of cocaine self-administration, in contrast, was not affected by treatment with l-NAME. These observations lend further support to the hypothesis of the involvement of nitric oxide in cocaine addiction and extend previous findings to components of the cocaine addictive cycle associated with relapse.

Involvement of Rho-kinase in vascular remodeling caused by long-term inhibition of nitric oxide synthesis in rats

Long-term inhibition of nitric oxide (NO) synthesis with N ω-nitro- l-arginine methyl ester ( l-NAME) induces coronary vascular remodeling in rats. To determine the pathogenic mechanism involved in vascular remodeling, we examined the effects of fasudil, a Rho-kinase inhibitor, on vascular lesion formation. In rats treated with l-NAME at 10 mg/kg/day, vascular remodeling was evident in both large and small coronary arteries at the fourth week. Fasudil (3 mg/kg, p.o., twice daily) markedly prevented the development of vascular remodeling in small coronary arteries. Coronary flow was measured in Langendorff perfused isolated heart preparations. Long-term treatment with l-NAME caused a significant decrease in coronary flow, which was significantly inhibited by fasudil. Fasudil suppressed the structural and functional changes in coronary arteries by chronic blockade of NO synthesis. Thus, the Rho-kinase pathway may be substantially involved in the pathogenesis of vascular remodeling in this rat model.

Endogenous nitric oxide modulates myocardial oxygen consumption in canine right ventricle.

The role of endogenous nitric oxide (NO) in modulating myocardial oxygen consumption (MVO2) is unclear, in part because of systemic and coronary hemodynamic effects of blocking NO release. This study evaluated the effect of NO on right ventricular MVO2 under controlled hemodynamic conditions. In 12 open-chest dogs, N(omega)-nitro-L-arginine methyl ester (L-NAME, 150 microg/min), a NO synthase (NOS) blocker, was infused into the right coronary artery. Heart rate and mean aortic pressure were constant. Right coronary blood flow and right ventricular MVO2 were measured at normal and elevated right coronary perfusion pressures (RCP) before and after L-NAME. To avoid effects of NO synthesis blockade on right coronary blood flow, which might have altered right ventricular MVO2, experiments, were conducted during adenosine-induced maximal coronary vasodilation. L-NAME did not affect right coronary blood flow (P = 0.51). However, L-NAME significantly increased right ventricular MVO2 (6% at RCP 100 mmHg, and 21% at RCP 180 mmHg). Right coronary blood flow varied with perfusion pressure (P < 0.02), and the elevation of MVO2 produced by L-NAME increased at higher flows (P < 0.04), consistent with the greater shear stress-mediated release of NO. These findings indicate that endogenous NO limits right ventricular MVO2.

The role of iNOS in wound healing

Background. We have previously shown that the blockade of nitric oxide (NO) synthesis impairs wound healing, in particular collagen synthesis. Conversely, impaired wound healing is accompanied by decreased wound NO synthesis. Fibroblast collagen synthesis, proliferation, and fibroblast-mediated matrix contraction are critical to wound healing. We examined the wound healing–related phenotypic changes that are induced by the loss of inducible nitric oxide synthase (iNOS) gene function in fibroblasts. Methods. Dermal fibroblasts were obtained from 8- to 12-week-old iNOS–knock out (KO; C57BL/Ai-[KO] Nos2 N5) and wild type mice by an explant technique and used after 1 to 3 passages. Proliferation ([3H]-thymidine incorporation) and collagen synthesis ([3H]-proline incorporation into collagenase-sensitive protein) were studied after stimulation with 10% fetal bovine serum. Matrix remodeling was assessed by the measurement of the contraction of fibroblast-populated collagen lattices. Results. iNOS-KO fibroblasts proliferated more slowly, synthesized less collagen, and contracted fibroblast-populated collagen lattices more slowly than wild-type fibroblast. Collagen synthesis was restored to normal in KO fibroblasts in response to NO donors (s-nitroso-N-acetylpenicillamine). Conclusions. iNOS deficiency causes significant impairment in wound healing–related properties of fibroblasts, which suggests that NO plays an important role in wound healing. (Surgery 2001;130:225-9.)

Cirrhosis serum induces a nitric oxide-associated vascular hyporeactivity of aortic segments from healthy rats in vitro.

Arterial vasodilation with concomitant hyperdynamic circulation are common findings in liver cirrhosis. Nitric oxide acting at a local level has been suggested to be pathophysiologically relevant in this context. Several systemic factors in conjunction with nitric oxide might interfere with the observed phenomena. The study has been designed to demonstrate the influence of cirrhotic serum on the nitric oxide system and vascular contractility. The contractile response of aortic segments from healthy rats was studied in vitro after incubation with serum of healthy and cirrhosis-induced rats (1 week, 2 weeks, 3 weeks and 4 weeks after bile duct ligation). A cumulative dose response curve to phenylephrine (10--10-4 mol) was established before and after incubation with nitric oxide synthesis blocker N(omega)-nitro-L-arginine, the more selective aminoguanidine (nitric oxide synthase [NOS]-2 inhibitor) and W7 (NOS-3 inhibitor). NOS-2 expression in incubated aortic rings was evaluated by Western blot analysis. A 4-hour incubation with serum of cirrhosis-induced rats reduced the maximum contractile response to phenylephrine to 66.8 +/- 9.1% after 1 week, 50.4 +/- 7.8% after 2 weeks, 43.2 +/- 2.8% after 3 weeks and 35 +/- 5.2% after 4 weeks of bile duct ligation. This reduction in the contractility response to phenylephrine was completely reversed by blocking nitric oxide synthesis with N(omega)-nitro-L-arginine and aminoguanidine, but not after W7. Incubation with cirrhotic serum induced NOS-2 expression in aortic rings. In Western blot analysis, the most intensive signal for NOS-2 protein was obtained in rings incubated with serum from rats 3 weeks and 4 weeks after induction of cirrhosis. Cirrhotic serum decreases the contractile response to phenylephrine even in an early stage of secondary cirrhosis. Reversibility of this effect after nitric oxide synthesis blockade suggests an induction of nitric oxide synthesis by systemic factors as a major point in vascular hyporeactivity to vasoconstrictors in cirrhosis.

Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxidedelivery

Nitric oxide (NO) may be stabilized by binding to hemoglobin, by nitrosating thiol-containing plasma molecules, or by conversion to nitrite, all reactions potentially preserving its bioactivity in blood. Here we examined the contribution of blood-transported NO to regional vascular tone in humans before and during NO inhalation. While breathing room air and then room air with NO at 80 parts per million, forearm blood flow was measured in 16 subjects at rest and after blockade of forearm NO synthesis with NG-monomethyl-L-arginine (L-NMMA) followed by forearm exercise stress. L-NMMA reduced blood flow by 25% and increased resistance by 50%, an effect that was blocked by NO inhalation. With NO inhalation, resistance was significantly lower during L-NMMA infusion, both at rest and during repetitive hand-grip exercise. S-nitrosohemoglobin and plasma S-nitrosothiols did not change with NO inhalation. Arterial nitrite levels increased by 11% and arterial nitrosyl(heme)hemoglobin levels increased tenfold to the micromolar range, and both measures were consistently higher in the arterial than in venous blood. S-nitrosohemoglobin levels were in the nanomolar range, with no significant artery-to-vein gradients. These results indicate that inhaled NO during blockade of regional NO synthesis can supply intravascular NO to maintain normal vascular function. This effect may have application for the treatment of diseases characterized by endothelial dysfunction.

Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition.

Recent studies have suggested that subtle microvascular and tubulointerstitial injury in the kidney can cause salt-sensitive hypertension. To test this hypothesis, we determined whether the mild renal disease induced by transient blockade of nitric oxide (NO) synthesis would result in salt-sensitive hypertension and whether prevention of the renal injury by coadministration of the immunosuppressive agent mycophenolate mofetil (MMF) would block the development of salt sensitivity. N(omega)-nitro-L-arginine-methyl ester (L-NAME; 70 mg/100 ml in the drinking water) was administered for 3 wk to rats with or without MMF (30 mg x kg(-1) x day(-1) by gastric gavage), followed by a 1-wk "washout" period in which the MMF was continued, which was followed in turn by placement on a high-salt (4% NaCl) diet for an additional 4 wk. Renal histology was examined at 3 and 8 wk, and blood pressure was measured serially. L-NAME treatment resulted in acute hypertension and the development of mild renal injury. During the washout period, blood pressure returned to normal, only to return to the hypertensive range on exposure of the animals to a high-salt diet. MMF treatment prevented the development of hypertension in response to a high-salt diet. This correlated with the ability of MMF to inhibit specific aspects of the renal injury, including the development of segmental glomerulosclerosis, the infiltration of T cells and ANG II-positive cells, and the thickening of afferent arterioles.

Renal cortical remodelling by NO-synthesis blockers in rats is prevented by angiotensin-converting enzyme inhibitor and calcium channel blocker.

The cortical remodelling was studied when chronically nitric oxide synthesis (NOs) blockade (L-NAME-induced) hypertensive rats are simultaneously treated, or not, with angiotensin-converting enzyme inhibitor or calcium channel blocker. Four groups of eight rats each were studied as follows: Control (C), L-NAME (L), L-NAME+Enalapril (L+E) and L-NAME+Verapamil (L+V). The systolic blood pressure (SBP) was weekly recorded. The cortex of the left kidneys was analysed according to the vertical section design. The volume-weighted mean glomerular volume (VWGV) was made through the "point-sampled intercepts" method. Enalapril and verapamil were efficient in reducing the SBP in rats submitted to NOs blockade. Glomeruli had considerable alterations in L group rats (glomerular hypertrophy or sclerosis) and tubular atrophy. The VWGV was 100% greater in L group rats than in the C group rats, while it was 30% smaller in L+E and L+V groups than in L group. The tubular volume was 30-50% greater, while the tubular length was 20-30% smaller in the L group than in the other groups. The renal cortical region showed glomerular sclerosis/hypertrophy and tubular remodelling in rats with NOs blockade that was efficiently prevented with the simultaneous treatment with enalapril or verapamil.

The Role of Nitric Oxide in Development of Topographic Precision in the Retinotectal Projection of Chick

The axonal projection from the retina to the tectum exhibits a precise topographic order in the mature chick such that neighboring ganglion cells send axons to neighboring termination zones in the contralateral tectum. The initial pattern formed during development is much less organized and is refined to the adult pattern during a discrete period of development. Refinement includes elimination of radically aberrant projections, such as those from the temporal side of the retina to posterior regions of the tectum, as well as a more subtle improvement in the topographic precision of the projection. The enzyme that synthesizes nitric oxide is expressed at high levels in the tectum during the developmental period in which the topography improves. Pharmacological blockade of nitric oxide synthesis during this period prevented elimination of topographically inappropriate retinotectal projections in a dose-dependent manner. This effect could not be duplicated by treatment of embryos with a vasoconstrictor, indicating that vascular changes were not a factor. These results show that nitric oxide is involved in refinement of the topography of the retinotectal projection as well as in other aspects of refinement of this projection in developing chick.

Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries.

Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2alpha (3 microM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC(50)=9.64+/-0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC(50)=9.23+/-0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 microM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC(50)=0.043 microg ml(-1)), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC(50)=0.14 microg ml(-1)) of the cathepsin G effect, whereas neither indomethacin (3 microM) nor the thrombin inhibitor hirudin (5 ATU ml(-1)) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC(50)=0.11 microg ml(-1)), heparin (IC(50)=0.48 microg ml(-1)) and suramin (IC(50)=1.85 microg ml(-1)) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like thrombin and trypsin, cathepsin G is able to induce endothelium-dependent vascular relaxation. It can be released from activated leukocytes at sites of vascular injury and inflammation and, therefore, sufficiently high concentrations might be reached locally in the vascular space to induce vasodilatation.

Countervailing influence of tumor necrosis factor-alpha and nitric oxide in endotoxemia.

Tumor necrosis factor-alpha (TNF-alpha), a crucial mediator in sepsis, elicits multiple biologic effects, including intravascular thrombosis and circulatory shock. TNF-alpha exerts its biologic effects through two distinct cell surface receptors, TNF-R1 and TNF-R2. The pathophysiologic interaction between TNF-alpha and nitric oxide (NO) in glomerular thrombosis caused by endotoxemia in rats and wild-type mice (C57BL6) as well as in knockout mice that are deficient in TNF-R1 (R1 -/-), TNF-R2 (R2 -/-), or both receptors (R1R2 -/-) was studied. Administration of lipopolysaccharide (LPS; Escherichia coli endotoxin) resulted in increased NO and TNF-alpha production but failed to induce glomerular thrombosis. Concomitant administration of LPS + NG-nitro-L-arginine methyl ester (L-NAME; an NO synthesis inhibitor) resulted in glomerular thrombosis in rats and in wild-type mice. Intraperitoneal administration of pentoxifylline before LPS inhibited TNF-alpha synthesis and prevented glomerular thrombosis in rats given LPS + L-NAME. In contrast to the results observed in rats and wild-type mice, administration of LPS + L-NAME did not result in glomerular thrombosis in knockout mice with either single or double TNF-alpha receptor deletion. Thus, during endotoxemia, (1) TNF-alpha fosters glomerular thrombosis if there is deficiency of NO synthesis and (2) both TNF-alpha receptors are necessary for TNF-alpha's prothrombogenic action. Clinically, these novel studies suggest that in gram-negative endotoxemia, inhibition of NO synthesis and selective blockade of TNF-alpha receptors may provide unique therapeutic approaches for mitigation of glomerular thrombosis and restitution of vascular tone.

Regression by ACE inhibition of arteriosclerotic changes induced by chronic blockade of NO synthesis in rats

We previously reported that chronic inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces vascular inflammation at week 1 and produces subsequent arteriosclerosis at week 4 and that cotreatment with an angiotensin-converting enzyme (ACE) inhibitor prevents such changes. In the present study, we tested the hypothesis that treatment with an ACE inhibitor after development of vascular inflammation could inhibit arteriosclerosis in rats. Wistar-Kyoto rats were randomized to four groups: the control group received no drugs, the 4wL-NAME group received L-NAME (100 mg x kg(-1) x day(-1)) for 4 wk, the 1wL + 3wNT group received L-NAME for 1 wk and no treatment for the subsequent 3 wk, and the 1wL + 3wACEI group received L-NAME for 1 wk and the ACE inhibitor imidapril (20 mg x kg(-1) x day(-1)) for the subsequent 3 wk. After 4 wk, we observed significant arteriosclerosis of the coronary artery (medial thickening and fibrosis) and increased cardiac ACE activity in the 1wL + 3wNT group as well as in the 4wL-NAME group, but not in the 1wL + 3wACEI group. In a separate study, we examined apoptosis formation and found that posttreatment with imidapril (20 mg x kg(-1) x day(-1)) or an ANG II AT1-receptor antagonist, CS-866 (5 mg x kg(-1) x day(-1)), induced apoptosis (TdT-mediated nick end-labeling) in monocytes and myofibroblasts appearing in the inflammatory lesions associated with a clear degradation in the heart (DNA electrophoresis). In conclusion, treatment with the ACE inhibitor after 1 wk of L-NAME administration inhibited arteriosclerosis by inducing apoptosis in the cells with inflammatory lesions in this study, suggesting that increased ANG II activity inhibited apoptosis of the cells with inflammatory lesions and thus contributed to the development of arteriosclerosis.

Decreased influence of nitric oxide on deoxycorticosterone acetate (DOCA)–salt hypertension

The response to exogenous administration of acetylcholine and the effects of nitric oxide (NO) synthesis blockade were assessed for small arterioles in the cremaster muscle of deoxycorticosterone acetate (DOCA)–salt hypertensive and normotensive rats using intravital microscopy. The NO synthesis inhibitor Nω- L-nitro-arginine methyl ester (L-NAME) produced significantly less constriction in the arterioles of DOCA–salt hypertensive rats. Exposure to increasing concentrations of acetylcholine (10 −10 to 10 −5 mol/L) or sodium nitroprusside (10 −10 to 10 −5 mol/L) produced similar arteriolar dilation in both groups. These results suggest that attenuated basal release of NO by arterioles may play a role in the development of increased peripheral resistance observed in DOCA–salt hypertension.

Volume-weighted mean nuclear volume and numerical nuclear density in the cardiomyocyte following enalapril and verapamil treatment.

The estimation of the volume-weighted mean nuclear volume, nu(nu), and the numerical nuclear density in the plane, N(A), was used to analyze cardiomyocyte nuclei in the condition of cardiac hypertrophy caused by nitric oxide (NO) synthesis blockade and simultaneous antihypertensive treatment for 40 days (four groups of ten rats each: control, L-NAME, L-NAME+enalapril, L-NAME+verapamil). The blood pressure (BP) increased 71% in the L-NAME group. In the L-NAME+enalapril and L-NAME+verapamil groups, the BP did not show any alteration when compared with the respective controls. In comparison with the control group, nu(nu) was 250% greater, and the N(A) was 25% smaller in the L-NAME animals, while no difference occurred in the other two groups. With respect to cardiomyocyte nuclear size, the present results suggest a beneficial effect of the angiotensin-converting enzyme inhibitor enalapril and the calcium channel blocker verapamil when NO synthesis is blockaded.