Effect of NOS inhibition on central response to atrial distension during pregnancy

Atrial distension increases c-fos expression in the paraventricular nucleus of virgin, but not pregnant, rats. We proposed that nitric oxide (NO), biosynthesis of which increases during pregnancy, blunts this reflex and that blocking NO biosynthesis would restore the response. Female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing either D- or N(G)-nitro-L-arginine methyl ester (L-NAME) (120 mg/2 ml at 10 microg/min) were implanted. On day 20, the rats were infused with saline (3 ml/h) with or without atrial balloon inflation (1 h). The brains were then processed for quantitation of c-fos expression. In the virgin rats, and in the pregnant rats treated with L-NAME, atrial distension significantly increased hypothalamic c-fos expression. In the pregnant animals treated with D-NAME, the response was greatly attenuated. NO had no effect on the increase in atrial receptor afferent discharge (single-fiber recordings) elicited by atrial distension. We conclude that, during pregnancy, NO attenuates central processing of the reflex response to atrial distension but does not alter the transducer properties of the volume receptors.     

Effect of pregnancy and 5alpha-pregnan-3alpha-ol-20-one on atrial receptor afferent discharge in rats

The atrial volume reflex is attenuated in pregnancy. This may be mimicked by chronic administration of 5alpha-pregnan-3alpha-ol-20-one (pregnan). We investigated whether afferent output from sensory receptors may be suppressed at this time. Vagal afferent nerve activity was measured during discrete localized stimulation of the atrial volume receptors by inflation of a balloon at the superior vena caval-right atrial junction. The receptors were classified as high- (HF) or low- (LF) frequency subtypes on the basis of their response to graded atrial distension. Although both HF (regression coefficient = 0.50 +/- 0.11 Hz/microl, r(2) = 0.47, P < 0.001) and LF (regression coefficient = 0.03 +/- 0.05 Hz/microl, r(2) = 0.009, P = 0.613) subtypes could be identified in virgin rats, only LF (regression coefficient = 0.09 +/- 0.05 Hz/microl, r(2) = 0.044, P = 0.099) receptors were found in late-pregnant animals. Similarly, in virgin rats treated chronically with pregnan (500 microg/24 h for 2 days), only LF receptors were identified (regression coefficient = -0.004 +/- 0.078 Hz/microl, r(2) = 0.000, P = 0.962), whereas both subtypes were present in the vehicle-treated animals (HF regression coefficient = 0.626 +/- 0.255 Hz/microl, r(2) = 0.317, P = 0.029; LF regression coefficient = -0.012 +/- 0.071 Hz/microl, r(2) = 0.002, P = 0.866). By contrast, acute intracardiac pregnan (2.6 microg/kg) did not alter vagal afferent nerve activity. In conclusion, stretch-induced discharge of high-frequency atrial receptors is suppressed during pregnancy, whereas that of low-frequency receptors is preserved. This effect may be mimicked by chronic, but not acute, pregnan. We propose that, during pregnancy, pregnan alters the transducer properties of the atrial volume receptors, thus allowing blood volume to increase.     

Effect of cardiac receptor stimulation on renal vascular resistance in the pregnant rat

Stimulation of cardiac receptors (CR) evokes blunted reflex reductions in mean arterial pressure (MAP) in pregnant compared with virgin rats. Because CR-mediated sympathoinhibition has preferential effects on the kidney, we tested whether, during pregnancy, renal vascular resistance (RVR) changes less in response to CR stimulation and investigated possible mechanisms. MAP, right atrial pressure, renal sympathetic nerve activity (RSNA), renal blood flow (RBF), and RVR were measured in anesthetized animals in response to CR stimulation by graded atrial injections of saline. Baseline MAP and RVR and reflex changes in these variables during CR stimulation were reduced in late-pregnant vs. virgin rats (P<0.05). Reflex changes in RSNA were attenuated in pregnant rats, but changes in RBF as a function of RSNA were similar in both groups. ANG II AT(1)-receptor blockade increased basal RBF more in virgin rats (P<0.05), but between-group differences in reflex changes in MAP, RSNA, and RVR were maintained after AT(1) blockade. Thus during CR simulation, reflex changes in RVR were reduced in pregnant versus virgin rats. This difference does not appear to involve differential effects of ANG II.     

Effect of nitric oxide synthase inhibition on cardiovascular and hormonal regulation during pregnancy in the rat

Recent studies have shown that nitric oxide (NO) biosynthesis increases in pregnancy and that inhibition of nitric oxide synthase (NOS) induces some pathological processes characteristic of preeclampsia. The current project sought to study the effect of the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 10 microg x min(-1), sc for 7 days) on plasma volume, plasma atrial natriuretic factor (ANF), plasma endothelin-1 (ET), and plasma renin activity (PRA) during gestation in conscious rats. NOS inhibition caused mean arterial pressure to increase in both virgin and 21-day pregnant rats. Plasma volume fell in the pregnant rats [L-NAME, 4.5 +/- 0.3 mL x 100 g(-1) body wt. (n = 7) vs. D-NAME, 6.8 +/- 0.2 mL x 100 g(-1) body wt. (n = 10); P < 0.05] but not in the virgin rats [L-NAME, 4.3 +/- 0.1 mL x 100 g(-1) body wt. (n = 6) vs. D-NAME, 4.8 +/- 0.2 mL x 100 g(-1) body wt. (n = 8)]. There was no effect of NOS inhibition on plasma ANF levels or PRA in either the virgin or pregnant rats. However, L-NAME did decrease plasma ET levels in the pregnant rats [L-NAME, 19.6 +/- 1.6 pg x mL(-1) (n = 8) vs. D-NAME, 11.6 +/- 2.5 pg x mL(-1) (n = 9); P < 0.05]. Our results confirm that NO is involved in cardiovascular homeostasis in pregnancy; NOS inhibition selectively reduces plasma volume in pregnant rats, thus mimicking a major pathophysiological perturbation of preeclampsia. However, it does not induce the hormonal changes characteristic of preeclampsia, namely the decrease in PRA and increase in plasma ET and ANF levels.     

The role of the hypothalamic nitric oxide in the pressor responses elicited by acute environmental stress in awake rats

We quantitatively investigated the change in nitric oxide (NO) in the hypothalamic paraventricular nucleus (PVN) and its effect on cardiovascular regulation during shaker stress (SS) using brain microdialysis in awake rats. Male Wistar rats were fed either N(G)-nitro-L-arginine methyl ester (L-NAME, 0.7 g/L) or tap water for 2 weeks. Two days after implantation of an arterial catheter and guide shaft, a microdialysis probe was placed to perfuse the PVN with degassed Ringer solution at 2 microl/min in awake normotensive Wistar (CONTROL) and chronic L-NAME-treated hypertensive rats. After the rat was placed in a plastic cage set on a shaker, the blood pressure and heart rate was monitored and 10-min SS was loaded at a frequency of 200 cycles/min. Dialysate samples were analyzed by NO analyzer (based on the Griess reaction) every 10 min, and NOx (NO(2)(-) + NO(3)(-)) was measured. Plasma NOx was also measured before and after SS. Pressor responses elicited by SS were significantly greater in L-NAME-treated rats than in the CONTROL. Although NOx in the PVN dialysate were increased by SS in the CONTROL, these responses were attenuated in chronic L-NAME-treated rats. Resting plasma NOx were higher in the CONTROL than in L-NAME-treated rats. SS elicited no difference between two groups in plasma NOx. These results indicated that NO within the PVN, but not in systemic circulation, may play a role on the attenuation of the pressor responses elicited by SS. The dysfunction of NO release within the PVN may, in part, play a role in the exaggerated pressor responses in acute environmental stress.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

Myogenic reactivity is reduced in small renal arteries isolated from relaxin-treated rats

Administration of the ovarian hormone relaxin to nonpregnant rats vasodilates the renal circulation comparable to pregnancy. This vasodilation is mediated by endothelin (ET), the ET(B) receptor, and nitric oxide. Furthermore, endogenous relaxin mediates the renal vasodilation and hyperfiltration that occur during gestation. The goal of this study was to investigate whether myogenic reactivity of small renal and mesenteric arteries is reduced in relaxin-treated rats comparable to the pregnant condition. Relaxin or vehicle was administered to virgin female Long-Evans rats for 5 days at 4 microg/h, thereby producing midgestational blood levels of the hormone. The myogenic responses of small renal arteries (200-300 microm in diameter) isolated from these animals were evaluated in an isobaric arteriograph system. Myogenic reactivity was significantly reduced in the small renal arteries from relaxin-treated compared with vehicle-treated rats. The reduced myogenic responses were mediated by the ET(B) receptor and nitric oxide since the selective ET(B) receptor antagonist RES-701-1 and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester restored myogenic reactivity to virgin levels. The influence of relaxin was not limited to the renal circulation because myogenic reactivity was also reduced in small mesenteric arteries isolated from relaxin-treated rats. Thus relaxin administration to nonpregnant rats mimics pregnancy, insofar as myogenic reactivity of small renal and mesenteric arteries is reduced in both conditions.     

Effect of 5 alpha-pregnan-3 alpha-ol-20-one on nitric oxide biosynthesis and plasma volume in rats

Plasma 5 alpha-pregnan-3 alpha-ol-20-one (pregnan) levels and nitric oxide (NO) biosynthesis increase during pregnancy. These factors have independently been implicated in the control of blood pressure and volume. We wished to determine whether pregnan might be responsible both for the increase in NO biosynthesis and for the increase in plasma volume observed during pregnancy. Virgin female Long-Evans rats were implanted with indwelling cannulas and maintained on a low nitrate/nitrite diet. After the rats recovered from surgery, 500 microg of pregnan or vehicle were given daily for 2 days. NO biosynthesis and plasma volume were measured in conscious animals before and after treatment. Pregnan caused a significant increase in NO biosynthesis (1.9 +/- 0.8 micromol/24 h, n = 10) compared with the vehicle-treated control group (0.3 +/- 0.4 micromol/24 h, n = 10, P < 0.05). Similarly, there was a significant increase in plasma volume in the pregnan-treated group (0.7 +/- 0.2 ml/100 g, n = 11) compared with the vehicle-treated control group (0.2 +/- 0.1 ml/100 g, n = 11, P < 0.05). These results confirm that pregnan can mimic pregnancy by its ability to increase both NO biosynthesis and plasma volume.     

Right atrial dimension-pressure relation during volume expansion is unaltered by pregnancy in the rat

Blood volume expands significantly during pregnancy, but afferent signals from cardiac receptors are reduced. In addition, during exogenous volume expansion, right atrial pressure (RAP) increases more for equivalent volumes in pregnant animals, implying reduced atrial compliance. To examine possible gestational alterations in atrial dimension during volume expansion, we compared the effects of volume expansion on RAP and right atrial dimension (RAD) in pregnant vs. virgin rats. Anesthetized animals were ventilated and catheterized for measurement of arterial pressure and RAP and for drug infusion. Through a parasternal incision, ultrasonic crystals were glued to the medial and lateral surfaces of the right atrium for measurement of RAD. Plasma volume and hematocrit were determined before experimentation. RAP, RAD, and arterial pressure were recorded at baseline and during progressive volume expansion (6% dextran, 60% of initial blood volume). Baseline RAP was similar in the two groups: 2.82 +/- 0.40 and 2.72 +/- 0.47 mmHg in pregnant and virgin rats, respectively. Basal RAD was significantly larger in pregnant than in virgin rats: 4.36 +/- 0.66 vs. 3.36 +/- 0.48 mm. Despite increased basal RAD in pregnant rats, the slope of the RAD-RAP relation during volume expansion was similar in the two groups. Results indicate that resting RAD is increased in pregnant rats and that the change in dimension during volume loads is similar to that in virgin rats. Thus, during pregnancy, the right atrium appears to accommodate the increased blood volume, and reduced afferent signaling most likely is due to mechanisms other than mechanical alterations of the atrium by expanded volume.     

Endothelin and nitric oxide mediate reduced myogenic reactivity of small renal arteries from pregnant rats

We tested the hypothesis that endothelin acting through the endothelial ET(B) receptor subtype and the nitric oxide (NO) pathway accounts for reduced myogenic reactivity of the renal resistance vasculature during pregnancy. Small renal arteries (100-200 microm) were isolated from virgin and midterm pregnant rats when gestational renal hyperfiltration and vasodilation are maximal in this species. Myogenic reactivity (the adjustment of arterial diameter in response to a change in transmural pressure) was assessed with a pressurized myograph system. A rapid increase in transmural pressure from 60 to 80 mmHg resulted in a 2.4% diameter increase in vessels from virgin compared with an 8.1% increase in arteries from midgestation rats (n = 8 each, P < 0.05). Thus myogenic reactivity is markedly reduced during pregnancy. Incubation with the NO synthase inhibitors, an ET(B) receptor subtype antagonist (RES-701-1), the nonselective ET(A/B) receptor blocker (SB-209670), or endothelial removal abrogated the reduced myogenic reactivity of vessels from gravid rats without affecting myogenic reactivity in arteries from virgin animals. Thus the endothelium mediates the reduced myogenic reactivity of small renal arteries of midgestation rats most likely through the ET(B) receptor subtype and NO pathway.     

Pregnancy-associated reduction in vascular protein kinase C activity rebounds during inhibition of NO synthesis

Vascular reactivity has been shown to be reduced during pregnancy and to be enhanced during chronic inhibition of nitric oxide (NO) synthesis in pregnant rats; however, the cellular mechanisms involved are unclear. The purpose of this study was to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of vascular protein kinase C (PKC). Active stress as well as the amount and activity of PKC was measured in deendothelialized thoracic aortic strips from virgin and pregnant rats untreated or treated with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In virgin rats, the PKC activator phorbol 12,13-dibutyrate (PDBu, 10(-6) M) and the alpha-adrenergic agonist phenylephrine (Phe, 10(-5) M) caused significant increases in active stress and PKC activity that were inhibited by the PKC inhibitors staurosporine and calphostin C. Western blot analysis in aortic strips of virgin rats showed significant amount of the alpha-PKC isoform. Both PDBu and Phe caused significant translocation of alpha-PKC from the cytosolic to the particulate fraction. Compared with virgin rats, the PDBu- and Phe-stimulated active stress and PKC activity as well as the amount and the PDBu- and Phe-induced translocation of alpha-PKC were significantly reduced in late pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. The PDBu- and Phe-induced changes in active stress and the amount, distribution, and activity of alpha-PKC in virgin rats treated with L-NAME were not significantly different from that in virgin rats, whereas the changes in pregnant rats treated with L-NAME + the NO synthase substrate L-arginine were not significantly different from that in pregnant rats. These results provide evidence that a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced during chronic inhibition of NO synthesis. The results suggest that one possible mechanism of the pregnancy-associated changes in vascular reactivity may involve changes in the amount and activity of the alpha-PKC isoform.     

Hemodynamic and renal effects of acute and progressive nitric oxide synthesis inhibition in anesthetized dogs

This study evaluated the effects of progressive nitric oxide (NO) inhibition in the regulation of systemic and regional hemodynamics and renal function in anesthetized dogs. The N(G)-nitro-L-arginine methyl ester group (n = 9) received progressive doses of 0.1, 1, 10, and 50 microg. kg(-1). min(-1). Renal (RBF), mesenteric (MBF), iliac (IBF) blood flows, mean arterial pressure (MAP), pulmonary pressures, cardiac output (CO), and systemic and pulmonary vascular resistances were measured. During N(G)-nitro-L-arginine methyl ester infusion, MAP and systemic vascular resistances increased in a dose-dependent manner. Mean pulmonary pressure and pulmonary vascular resistances increased in both the N(G)-nitro-L-arginine methyl ester and the control group, but the increase was more marked in the N(G)-nitro-L-arginine methyl ester group during the last two infusion periods. CO decreased progressively, before any significant change in blood pressure was noticeable in the N(G)-nitro-L-arginine methyl ester group. IBF decreased significantly from the first N(G)-nitro-L-arginine methyl ester dose, whereas RBF and MBF only decreased significantly during the highest N(G)-nitro-L-arginine methyl ester dose. Urinary volume and sodium excretion only increased significantly in the time control group during the two last time periods. The pulmonary vasculature was more sensitive than the systemic vasculature, whereas skeletal muscle and renal vasculatures showed a greater sensitivity to the inhibition of NO production than the mesenteric vasculature. NO synthesis inhibition induces a progressive antidiuretic and antinatriuretic effect, which is partially offset by the increase in blood pressure.     

NO and cGMP mediate angiotensin AT2 receptor-induced renal renin inhibition in young rats

We hypothesized that angiotensin subtype-2 receptor (AT(2)R) inhibits renal renin biosynthesis in young rats via nitric oxide (NO). We monitored changes in renal NO, cGMP, renal renin content (RRC), and ANG II in 4-wk-old rats in response to low sodium (LNa(+)) intake alone and combined with 8-h direct renal cortical administration of AT(1) receptor blocker valsartan (VAL), AT(2)R blocker PD123319 (PD), NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), NO donor S-nitroso-N-acetyl penicillamine (SNAP), or guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,2-alpha] quinoxaline-1-one (ODQ). In addition, we monitored renal endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in response to VAL or PD. LNa(+), VAL, PD, l-NAME, and ODQ increased RRC, ANG II, and renin mRNA. PD and l-NAME decreased NO and cGMP, while SNAP reduced RRC, ANG II, renin mRNA, and reversed the effects of PD. PD also reduced eNOS and nNOS protein and mRNA. Combined treatment with PD, l-NAME, or ODQ and VAL reversed the effects of VAL and caused further increase in RRC, ANG II, renin mRNA, and protein. ODQ reversed the effects of SNAP. These data demonstrate that the renal AT(2) receptor decreases renal renin biosynthesis and ANG II production in young rats. Reversal of the PD effects by SNAP and SNAP effects by ODQ confirms that NO and cGMP mediate the AT(2) receptor inhibition of renal renin production.     

Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension

A pharmacological inhibition of nitric oxide synthase (NOS) in rats for 4-6 weeks produces renal vasoconstriction, renal dysfunction, and severe hypertension. The present study was aimed at investigating whether Cudrania tricuspidata (C. tricuspidata) water extract ameliorates N(G)-Nitro-L-arginine methylester (L-NAME)-induced hypertension. Treatment of L-NAME (60 mg/L drinking water, 4 weeks) causes a sustained increase in systolic blood pressure (SBP). The concentration of plasma NO metabolites and NO/cGMP productions in the vascular tissues of the L-NAME-treated group were significantly reduced as compared with those in the control. C. tricuspidata water extract blocked increase of SBP in the L-NAME-treated group and restored SBP to normal level. Futhermore, C. tricuspidata water extract was able to preserve the vascular NO/cGMP production and plasma NO metabolites concentration. However, there are no changes in the expression of ecNOS and iNOS of thoracic aorta among the rats of control, L-NAME-treated group, and L-NAME and C. tricuspidata water extract co-treated group. The urinary sodium level, urine volume, and creatinine clearance were significantly higher in rats co-treated with C. tricuspidata water extract and L-NAME than in L-NAME-treated group. Taken together, these results suggest that C tricuspidata water extract prevents the increase of SBP in the L-NAME-induced hypertension that may have been caused by enhanced generation of vascular NO/cGMP.     

Gender-specific compensation for the lack of NO in the mediation of flow-induced arteriolar dilation

Flow-induced dilation of gracilis muscle arterioles was examined in both genders of control rats and rats chronically treated with N(omega)-nitro-L-arginine methyl ester (L-NAME). After L-NAME treatment (4 wk), systolic blood pressure was significantly increased compared with control, whereas the plasma concentration of nitrate/nitrite was significantly reduced. Isolated and pressurized arterioles dilated significantly in response to increases in flow (0-25 microl/min). Flow-induced dilation was comparable in arterioles of control and L-NAME-treated rats but was significantly greater in female than in male rats. L-NAME + indomethacin, which abolished flow-induced dilation in arterioles of male control rats, inhibited the dilation by only ~75% in female control rats. The residual portion of the response was eliminated by additional administration of miconazole, an inhibitor of cytochrome P-450. Indomethacin did not affect the dilation in female L-NAME-treated rats but completely inhibited the response in male L-NAME-treated rats. The indomethacin-insensitive, flow-induced dilation in female L-NAME-treated arterioles was abolished by miconazole, 6-(2-proparglyoxyphenyl)hexanoic acid, or charybdotoxin. Thus an augmented release of endothelial prostaglandins accounts for the preserved flow-induced dilation in arterioles of male rats, whereas a metabolite of cytochrome P-450 is responsible for the maintenance of flow-induced dilation in female rats, suggesting important differences in the adaptation of the endothelium of arterioles from male and female rats to the lack of nitric oxide (NO) synthesis.     

Chronic low-dose L-NAME treatment increases nitric oxide production and vasorelaxation in normotensive rats

N(G)-nitro-L-arginine methyl ester (L-NAME) is a non-specific nitric oxide (NO) synthase inhibitor, commonly used for the induction of NO-deficient hypertension. The aim of this study was to investigate the effect of chronic low-dose administration of L-NAME on NO production, vascular function and structure of the heart and selected arteries of rats. Adult male Wistar rats were treated with L-NAME in the dose of approximately 1.5 mg/kg/day in drinking water for 8 weeks. Basal blood pressure (BP) of rats (determined by tail-cuff) was 112+/-3 mm Hg. The low-dose administration of L-NAME significantly elevated BP measured on the third and sixth week of treatment vs. controls by approximately 9 % and 12 %, respectively. After this period, BP of L-NAME-treated rats returned to the control values. The relative left ventricular mass, heart fibrosis and collagen III/collagen I ratio were not affected by L-NAME. Similarly, there were no alterations in the cross-sectional area and wall thickness/diameter ratio of the aorta and the femoral artery of L-NAME-treated rats. NO synthase activity (determined by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline) was not altered in the hypothalamus of L-NAME-treated rats. Interestingly, chronic low-dose L-NAME treatment significantly elevated NO synthase activity in the left ventricle and aorta, increased endothelium-dependent acetylcholine-induced vasorelaxation and reduced serotonin-induced vasoconstriction of the femoral artery. The data suggest that chronic low-dose L-NAME treatment can increase NO production and vasorelaxation in normotensive rats without negative structural changes in the cardiovascular system.     

L-NAME- and ADMA-induced sympathetic neural activation in conscious rats

Although studies in anesthetized, sino-aortic denervated animals indicate that inhibition of central nitric oxide (NO) causes an excitatory influence on efferent sympathetic nerve activity (SNA) that is normally offset by baroreflex activation, studies in conscious animals have not provided clear-cut evidence for a sympathoexcitatory effect of N(omega)-nitro-l-arginine methyl ester (L-NAME) or the endogenous circulating NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus our goals were to 1) use surgical sino-aortic denervation to test for a sympathoexcititatory effect of intravenous l-NAME in conscious rats, and 2) to determine whether SNA responses to intravenous L-NAME can be extrapolated directly to intravenous ADMA. We recorded mean arterial blood pressure and renal SNA in both intact and sino-aortic-denervated conscious rats during 3 h of continuous intravenous infusion with either L-NAME or ADMA. When we eliminated the confounding influence of the sino-aortic baroreceptors, L-NAME produced a progressive increase in SNA with the peak response exceeding the baseline level of nerve firing by 150%. The same type of frank sympathetic activation was observed with intravenous ADMA. Taken together, these data offer straightforward evidence for l-NAME, as well as ADMA-induced sympathetic activation with direct recordings of SNA in conscious animals. These data confirm and extend the concept that circulating endogenous NOS inhibitors can constitute an excitatory signal to SNA.     

Attenuated glomerular arginine transport prevents hyperfiltration and induces HIF-1α in the pregnant uremic rat

Pregnancy worsens renal function in females with chronic renal failure (CRF) through an unknown mechanism. Reduced nitric oxide (NO) generation induces renal injury. Arginine transport by cationic amino acid transporter-1 (CAT-1), which governs endothelial NO generation, is reduced in both renal failure and pregnancy. We hypothesize that attenuated maternal glomerular arginine transport promotes renal damage in CRF pregnant rats. In uremic rats, pregnancy induced a significant decrease in glomerular arginine transport and cGMP generation (a measure of NO production) compared with CRF or pregnancy alone and these effects were prevented by l-arginine. While CAT-1 abundance was unchanged in all experimental groups, protein kinase C (PKC)-α, phosphorylated PKC-α (CAT-1 inhibitor), and phosphorylated CAT-1 were significantly augmented in CRF, pregnant, and pregnant CRF animals; phenomena that were prevented by coadministrating l-arginine. α-Tocopherol (PKC inhibitor) significantly increased arginine transport in both pregnant and CRF pregnant rats, effects that were attenuated by ex vivo incubation of glomeruli with PMA (a PKC stimulant). Renal histology revealed no differences between all experimental groups. Inulin and p-aminohippurate clearances failed to augment and renal cortical expression of hypoxia inducible factor-1α (HIF-1α) significantly increased in CRF pregnant rat, findings that were prevented by arginine. These studies suggest that in CRF rats, pregnancy induces a profound decrease in glomerular arginine transport, through posttranslational regulation of CAT-1 by PKC-α, resulting in attenuated NO generation. These events provoke renal damage manifested by upregulation of renal HIF-1α and loss of the ability to increase glomerular filtration rate during gestation.     

Gastrointestinal motility during pregnancy: role of nitrergic component of NANC nerves

This study evaluated whether increased release of nitric oxide (NO) from the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves may be partly responsible for the decrease in gastrointestinal motility observed during pregnancy. Segments of fundal strip, ileum, and colon were obtained from nonpregnant rats, rats in midpregnancy (days 9-11), and rats in late pregnancy (days 18-20). NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. The magnitude of change in cGMP levels in the tissues after application of EFS was also assessed. Our studies indicate that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon, after application of EFS to tissues obtained only from animals in late pregnancy. These results paralleled the changes in cGMP levels in tissues. NOS activity in the gastric fundus was significantly increased in animals in late pregnancy compared with nonpregnant controls. Our studies suggest that the delay in gastric emptying and increase in colonic transit time observed in rats during pregnancy may be caused in part by increased activity of the nitrergic component of the NANC nerves innervating these organs.     

A profound decrease in maternal arginine uptake provokes endothelial nitration in the pregnant rat

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. L-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (L-[3H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-1 mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-alpha, which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of alpha-tocopherol, a PKC-alpha inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-alpha. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like preeclampsia.