Masao Kakoki

Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via a cyclic GMP-independent mechanism.

Adrenomedullin, which was discovered as a vasodilating peptide, has been reported to be produced in various organs, in which adrenomedullin regulates not only vascular tone but also cell proliferation and differentiation in an autocrine/paracrine manner. We evaluated the effect of adrenomedullin on endothelial cell apoptosis. Human umbilical vein endothelial cells underwent apoptosis when cultured in serum-free medium. Treatment with adrenomedullin reduced the number of cells with pyknotic nuclei (Hoechst 33258 staining) and inhibited cell death (dimethylthiazol-diphenyltetrazolium bromide assay) in a dose-dependent manner. The administration of adrenomedullin did not alter the expression levels of Bcl-2 family proteins. Experiments with analogs of cAMP or a cAMP-elevating agonist demonstrated that elevation of the intracellular cAMP concentration does not mediate the antiapoptotic effect of adrenomedullin. The coadministration of N-nitro-L-arginine methyl ester (2 mmol/L), an inhibitor of nitric oxide synthase, abrogated the effect of adrenomedullin. Lower doses of sodium nitroprusside (1 to 10 micromol/L), a nitric oxide donor, mimicked the antiapoptotic effect of adrenomedullin. The antiapoptotic effect of sodium nitroprusside was not attenuated by the inhibition of soluble guanylyl cyclase with 1 micromol/L oxadiazolo-quinoxalin-1-one nor could apoptosis be inhibited by the incubation of human umbilical vein endothelial cells with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analog. These results indicate that adrenomedullin and nitric oxide inhibit endothelial cell apoptosis via a cGMP-independent mechanism.

Role of Nitric Oxide–cGMP Pathway in Adrenomedullin-Induced Vasodilation in the Rat

We previously reported that adrenomedullin (AM), a potent vasodilator peptide discovered in pheochromocytoma cells, stimulates nitric oxide (NO) release in the rat kidney. To further investigate whether the NO-cGMP pathway is involved in the mechanisms of AM-induced vasodilation, we examined the effects of E-4021, a cGMP-specific phosphodiesterase inhibitor, on AM-induced vasorelaxation in aortic rings and perfused kidneys isolated from Wistar rats. We also measured NO release from the kidneys using a chemiluminescence assay. AM (10(-10) to 10(-7) mol/L) relaxed the aorta precontracted with phenylephrine in a dose-dependent manner. Denudation of endothelium (E) attenuated the vasodilatory action of AM (10(-7) mol/L AM: intact (E+) -25.7+/-5.2% versus denuded (E-) -7. 8+/-0.6%, P<0.05). On the other hand, pretreatment with 10(-8) mol/L E-4021 augmented AM-induced vasorelaxation in the intact aorta (-49. 0+/-7.9%, P<0.05) but not in the denuded one. E-4021 also enhanced acetylcholine (ACh)-induced vasorelaxation in the rat intact aorta (10(-7) mol/L ACh -36.6+/-8.4% versus 10(-8) mol/L E-4021+10(-7) mol/L ACh -62.7+/-3.1%, P<0.05). In perfused kidneys, AM-induced vasorelaxation was also augmented by preincubation with E-4021 (10(-9) mol/L AM -15.4+/-0.6% versus 10(-8) mol/L E-4021+10(-9) mol/L AM -23.6+/-1.2%, P<0.01). AM significantly increased NO release from rat kidneys (DeltaNO: +11.3+/-0.8 fmol. min-1. g-1 kidney at 10(-9) mol/L AM), which was not affected by E-4021. E-4021 enhanced ACh-induced vasorelaxation (10(-9) mol/L ACh -9.7+/-1.7% versus 10(-8) mol/L E-4021+10(-9) mol/L ACh -18.8+/-2.9%, P<0.01) but did not affect ACh-induced NO release from the kidneys. In the aorta and the kidney, 10(-4) mol/L of NG-nitro-L-arginine methyl ester, an NO synthase inhibitor, and 10(-5) mol/L of methylene blue, a guanylate cyclase inhibitor, reduced the vasodilatory effect of AM. These results suggest that the NO-cGMP pathway is involved in the mechanism of AM-induced vasorelaxation, at least in the rat aorta and kidney.

Increased Nitric Oxide in the Exhaled Air of Patients with Decompensated Liver Cirrhosis

Patients with liver cirrhosis often present with several systemic hemodynamic disturbances, including hypotension, low systemic vascular resistance, and a reduced sensitivity to vasoconstrictors [1]. As cirrhosis progresses, vascular resistance continues to decrease, and the low arterial pressure may lead to secondary disturbances in renal and hepatic blood flow and to ascites [1]. The precise mechanisms of these hemodynamic disorders have not yet been clearly elucidated. Excessive production of vasodilators, such as prostacyclin, bradykinin, substance P, and atrial natriuretic peptide, has been proposed, but there is no clear evidence to show that vasodilators are involved. Vallance and Moncada [2] hypothesized that nitric oxide, originally discovered as an endothelium-derived relaxing factor [3], may be a causative factor in hemodynamic disorders in patients with liver cirrhosis. High concentrations of circulating endotoxin are frequently found in patients with cirrhosis who have no clinical evidence of infection [4]. Thus, the endotoxemia of liver cirrhosis may induce nitric oxide synthase directly in blood vessels or indirectly through cytokines, leading to an increased synthesis and release of nitric oxide that may account for the hemodynamic abnormalities. Recent studies show that nitric oxide concentration in exhaled air can be measured [5-7] and that it is increased in patients with bronchial asthma [5, 6]. To test the hypothesis that an increased synthesis and release of nitric oxide accounts for hemodynamic abnormalities in patients with liver cirrhosis, we investigated whether nitric oxide output in exhaled air is increased in these patients. Methods Patients Fifty-six patients were consecutively selected from those hospitalized in our department. All had biopsy-proven chronic hepatitis or liver cirrhosis; none had primary lung disease, hypertension, or infection. They could walk in the ward unaided and did not need intensive care. Physical examination findings and blood data were analyzed to classify hepatocellular function in liver cirrhosis according to the Child criteria. The clinical background of these patients is summarized in Table 1. Healthy volunteers served as controls (15 men; 34 2 years of age; body surface area, 1.84 0.03 m2). All medications were discontinued 24 hours before each study began. No antihypertensives or vasodilators, including nitrates and angiotensin-converting enzyme inhibitors, were used in these patients. The study was approved by the hospital ethics committee, and informed consent was obtained from each study participant. Table 1. Clinical Background of Patients with Chronic Hepatitis and Liver Cirrhosis Nitric Oxide Measurement The nitric oxide concentration in exhaled air was determined at least 3 hours after meals while each participant was at rest in the sitting position, as previously described [7]. Each participant was asked to inhale synthetic air (Taiyo Sanso Co., Osaka, Japan) free of nitric oxide (< 3 parts per billion [ppb]) through a mask and a T-valve, and to exhale the air into a wide-bore Teflon tube (internal diameter, 25 mm; length, 600 mm). Exhaled air was continuously drawn from this tube with a vacuum pump and was introduced into a chemiluminescence analyzer (APNE-350E, Horiba Co., Kyoto, Japan). Measurement of nitric oxide concentration was based on the reaction of nitric oxide with ozone. The sensitivity of the analyzer to nitric oxide ranged from 2 to 1000 ppb. The system was calibrated with dilutions of certified nitric oxide gas (450 ppb in nitrogen; Taiyo Sanso Co.) using mass flowmeters (Estec Co., Kyoto, Japan). Expired volume was measured with a hot-wire flow meter connected to the T-valve on the expiratory side, and minute ventilation was calculated using a breath-by-breath respirometer (RM-280, Minato Medical Science Co., Tokyo, Japan). The nitric oxide concentration and minute ventilation were recorded with a computer-assisted data recorder (DS1100, Fukuda Denshi Co., Tokyo, Japan), and the output of nitric oxide was calculated as follows: nitric oxide output = (nitric oxideex nitric oxidein) x minute ventilation/body surface area, where nitric oxideex was the nitric oxide concentration in exhaled air, and nitric oxidein was the nitric oxide concentration in inhaled air. Nitric oxide concentration and minute ventilation were monitored simultaneously for 10 minutes, and the data obtained during the last 3 minutes was averaged. During the study period, the ambient levels of nitric oxide concentration were less than 5 ppb. Nitric oxide output was reproducible in patients with cirrhosis and in controls (coefficient of variation, 10.8% [n = 5] for patients and 9.3% [n = 5] for controls) on separate days, and there was no significant time-course change in nitric oxide output at rest. Echocardiographic Measurement To examine the relation between systemic hemodynamics and nitric oxide production, we measured cardiac output using transthoracic two-dimensional echocardiography (SSD-2200, Aloka Co., Tokyo, Japan) in 19 patients with liver cirrhosis and in 6 controls. This was done on the same day that nitric oxide concentrations were measured. A physician, who was blinded to the patient characteristics and the exhaled nitric oxide output values, obtained echocardiographic views and recorded them on videotape. Another physician, who was also blinded to these data, measured cardiac output using the echocardiographic images. Left ventricular dimension was measured in the long-axis view of the left ventricle while the patient was in the left lateral decubitus position. Left ventricular volume and cardiac index were obtained by the following formulae according to the Teichholz equation [8]: left ventricular volume = 7.0 x dimension3/(2.4 + dimension); cardiac index = (left ventricular end-diastolic volume -end-systolic volume) x heart rate/body surface area. Blood pressure was measured with a sphygmomanometer. Total peripheral resistance index was calculated as mean blood pressure 80/cardiac index. The cardiac index obtained by this method on separate days was reproducible in patients with cirrhosis and in controls (coefficient of variation, 9.1% [n = 6] in patients and 8.6% [n = 5] in controls). Statistical Analysis Values are expressed as the mean SE. Differences between patients and controls were compared using one-way analysis of variance (ANOVA) followed by the Fisher test. The correlation coefficient was calculated using the least-squares method. Statistical significance was set at P < 0.05. Results Patients with decompensated liver cirrhosis had markedly depressed liver function but normal serum creatinine levels (Table 1). There were no intergroup differences in minute ventilation per m2 body surface area (patients with chronic hepatitis, 5.1 0.3 L/min; Child A patients, 5.6 0.3 L/min; Child B patients, 5.4 0.2 L/min; Child C patients, 6.2 0.3 L/min; and controls, 5.4 0.2 L/min; P = 0.12). The level of exhaled nitric oxide output per m2 body surface area was significantly greater in patients with Child C (190 11 nL/min; P < 0.001) or Child B liver cirrhosis (166 12 nL/min; P < 0.001) than in controls (97 8 nL/min) (Figure 1). In patients with Child A liver cirrhosis (119 10 nL/min; P = 0.17) or chronic hepatitis (129 19 nL/min; P = 0.13), the level of nitric oxide output per m2 body surface area was similar to that in controls. Figure 1. Nitric oxide (NO) output in exhaled air in controls, patients with chronic hepatitis (CH), and patients with liver cirrhosis. The results of hemodynamic measurements showed that patients with Child C liver cirrhosis had a greater cardiac index per m2 body surface area (4.3 0.3 L/min compared with 2.9 0.2 L/min; P < 0.001) and a smaller total peripheral resistance per m2 body surface area (1732 125 dyne/s x cm5 compared with 2680 235 dyne/s x cm5; P = 0.004) than controls. There was a positive correlation between the level of nitric oxide output and cardiac index (r = 0.621; P < 0.001) (Figure 2). Figure 2. Relation between nitric oxide (NO) output and cardiac index in patients with liver cirrhosis and controls. Discussion We have shown that nitric oxide output is increased in the air exhaled by patients with cirrhosis, especially patients with decompensated cirrhosis. Although we did not identify the origin of the increased synthesis of nitric oxide, several potential sources can be considered. Patients with liver cirrhosis often have endotoxemia even when they have no signs of infection [4], and elevated concentrations of cytokines, such as tumor necrosis factor-, have been shown in patients with liver diseases [9, 10]. The liver may produce large amounts of nitric oxide in these patients: Hepatocytes and Kupffer cells are known to produce nitric oxide in vitro in response to lipopolysaccharide and several cytokines [11, 12]. The plasma levels of cytokines, including tumor necrosis factor, are much lower in patients with liver cirrhosis than in these in vitro studies [10-15]. However, in vitro studies have also shown that endotoxin and cytokines also induce nitric oxide synthase in other tissues, including vascular endothelium, smooth muscle, and bronchial epithelium [13-15]. Thus, it is possible that vascular and bronchial tissues in the lungs of patients with liver cirrhosis produce nitric oxide as a result of continuous stimulation by the lower concentrations of cytokines, because the plasma levels of cytokines in patients with cirrhosis are similar to those in normal persons who have become hypotensive through the administration of endotoxin [10, 16]. Because most nitric oxide is inactivated by hemoglobin or rapidly metabolized to nitrite and nitrate [3], nitric oxide in exhaled air may be the residual of excessive local production of nitric oxide by the lung rather than a product of the liver. Because plasma nitrite and nitrate levels reflect the sum of nitric oxide production in the entire body, includ

Effects of Vasodilatory β-Adrenoceptor Antagonists on Endothelium-Derived Nitric Oxide Release in Rat Kidney

The mechanisms for the vascular actions of vasodilatory beta-blockers remain undetermined. For some kinds of beta-blockers, the involvement of nitric oxide (NO) has been suggested. We studied the effects of vasodilatory beta-blockers on renal perfusion pressure (RPP) and NO release in the rat kidney. Infusion of bopindolol, celiprolol, and nebivolol caused a dose-dependent reduction in RPP and an increase in NO release (RPP: bopindolol 10(-6) mol/L, -23+/-2%; celiprolol 10(-4) mol/L, -27+/-2%; nebivolol 10(-5) mol/L, -35+/-3%; NO: bopindolol 10(-6) mol/L, +33+/-2; celiprolol 10(-4) mol/L, +41+/-2; nebivolol 10(-5) mol/L, +45+/-5 fmol. min-1. g kidney-1, mean+/-SEM). Metergoline (10(-6) mol/L), a 5-hydroxytryptamine (5-HT)1/2 antagonist, or NAN-190 (10(-6) mol/L), a 5-HT1A antagonist, almost completely abolished the vasorelaxation and NO release caused by bopindolol, celiprolol, and nebivolol. However, neither propranolol nor bisoprolol decreased RPP. Celiprolol and nebivolol caused vasodilation in the rat thoracic aorta, and it was markedly reduced by endothelial denudation, Nomega-nitro-L-arginine methyl ester (10(-4) mol/L), or NAN-190 (10(-6) mol/L). In deoxycorticosterone acetate-salt hypertensive rats, 4-week administration of celiprolol (50 mg. kg-1. d-1 IV) restored the responses regarding RPP and NO release to acetylcholine. These results suggest that several beta-blockers exert their vasodilatory action through the 5-HT1A receptor/NO pathway and that treatment with these beta-blockers may protect against endothelial injury in hypertension.

Role of Endogenous Adrenomedullin in the Regulation of Vascular Tone and Ischemic Renal Injury: Studies on Transgenic/Knockout Mice of Adrenomedullin Gene

Adrenomedullin (AM) is a potent depressor peptide whose vascular action is suggested to involve nitric oxide (NO) release. To explore the role of endogenous AM in vascular and renal function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists AM(22-52) and CGRP(8-37) on the renal perfusion pressure (RPP) of kidneys isolated from AM transgenic (TG)/heterozygote knockout (KO) mice and wild-type littermates (WT). Furthermore, we evaluated the renal function and histology 24 hours after bilateral renal artery clamp for 45 minutes in TG, KO, and WT mice. Baseline RPP was significantly lower in TG than in KO and WT mice (KO 93.4±4.6, WT 85.8±4.2, TG 72.4±2.4 mm Hg [mean±SE], P <0.01). ACh and AM caused a dose-related reduction in RPP, but the degree of vasodilatation was smaller in TG than that in KO and WT (%&Dgr;RPP 10−7 mol/L ACh: KO −48.1±3.9%, WT −57.5±5.6%, TG −22.8±4.8%, P <0.01), whereas NG-nitro-l-arginine methyl ester (L-NAME) caused greater vasoconstriction in TG (%&Dgr;RPP 10−4 mol/L: KO 33.1±3.3%, WT 55.5±7.2%, TG 152.6±21.2%, P <0.01). Both AM antagonists increased RPP in TG to a greater extent compared with KO and WT mice (%&Dgr;RPP 10−6 mol/L CGRP(8-37): KO 12.8±2.6%, WT 19.4±3.6%, TG 41.8±8.7%, P <0.01). In mice with ischemic kidneys, serum levels of urea nitrogen and renal damage scores showed smaller values in TG and greater values in KO mice (urea nitrogen: KO 104±5>WT 98±15>TG 38±7 mg/dL, P <0.05 each). Renal NO synthase activity was also greater in TG mice. However, the differences in serum urea nitrogen and renal damage scores among the 3 groups of mice were not observed in mice pretreated with L-NAME. In conclusion, AM antagonists increased renal vascular tone in WT as well as in TG, suggesting that endogenous AM plays a role in the physiological regulation of the vascular tone. AM is likely to protect renal tissues from ischemia/reperfusion injury through its NO releasing activity.

Effects of hypertension, diabetes mellitus, and hypercholesterolemia on endothelin type B receptor-mediated nitric oxide release from rat kidney.

BACKGROUND Although endothelin-1 is a potent vasoconstrictor peptide, stimulation of endothelin type B receptor (ETBR) causes bidirectional changes in vascular tone, ie, vasodilation and vasoconstriction. Roles of ETBR in pathological conditions are largely unknown. METHODS AND RESULTS We studied the effect of BQ-3020, a highly selective ETBR agonist, on renal vascular resistance and nitric oxide (NO) release in the isolated, perfused kidney of rats with hypertension, diabetes mellitus, and hypercholesterolemia. Immunohistochemistry of endothelial NO synthase and ETBR was also examined. Infusion of BQ-3020 at concentrations of </=10(-10) mol/L reduced renal perfusion pressure in Dahl salt-resistant (R) rats but increased renal perfusion pressure in Dahl salt-sensitive (S) rats (10(-10) mol/L: -10.3+/-0. 6% versus 11.2+/-1.5%, R versus S; P<0.01). BQ-3020 caused a dose-dependent release of NO in both R and S rats, although the level of NO release in S rats was lower, as detected by chemiluminescence (10(-10) mol/L: 10.7+/-0.7 versus 3.1+/-0.4 fmol/min per gram of kidney, R versus S; P<0.01). Similar effects of BQ-3020 were observed in streptozotocin-induced diabetic rats and diet-induced hypercholesterolemic rats. Expression of endothelial NO synthase decreased in S rats but not in diabetic or hypercholesterolemic rats. In contrast, expression of ETBR in the endothelium was decreased in all 3 disease models compared with that in the vascular smooth muscle cell. CONCLUSIONS These results suggest that impaired NO release in response to stimulation of ETBR is due, at least in part, to a decrease in endothelial ETBR and may play a role in vascular dysfunction usually associated with arteriosclerosis-related diseases.

Increased excretion of nitric oxide in exhaled air of patients with chronic renal failure

Nitric oxide exerts multiple effects on renal function. It remains unclear whether endogenous nitric oxide production is increased or decreased in patients with chronic renal failure. To evaluate endogenous nitric oxide production in these patients we studied exhaled nitric oxide output by an ozone chemiluminescence method and plasma NO2(-)/NO3(-) levels by the Griess method in 40 patients with end-stage chronic renal failure who underwent regular continuous ambulatory peritoneal dialysis (n=30) or haemodialysis (n=10), and in 28 healthy subjects. Patients with chronic renal failure had a higher exhaled nitric oxide concentration [39+/-3 versus 19+/-1 parts per billion, (mean+/-S.E.M.), P<0.0001], a greater nitric oxide output (177+/-11 versus 96+/-7 nl.min-1.m-2, P<0.001) and a higher plasma NO2(-)/NO3(-) concentration (96+/-14 versus 33+/-4 micromol, P<0.01) than controls. These values did not differ between patients on haemodialysis and those on continuous ambulatory peritoneal dialysis. Patients with chronic renal failure had significantly higher plasma concentrations of both interleukin-1beta and interferon-gamma than controls. The exhaled nitric oxide output did not correlate with plasma NO2(-)/NO3(-) or with peritoneal dialysate NO2(-)/NO3(-), but plasma NO2(-)/NO3(-) correlated with dialysate NO2(-)/NO3(-) in patients who underwent continuous ambulatory peritoneal dialysis (r=0.77, P<0.01). Haemodialysis for 4 h acutely decreased plasma NO2(-)/NO3(-) (92+/-17 versus 50+/-8 micromol, P<0.05) and cGMP concentration (16.5+/-4.3 versus 5.1+/-1. 7 pmol/ml, P<0.01), but did not decrease exhaled nitric oxide output. The increase in exhaled nitric oxide with the simultaneous increase in circulating cytokines suggests that nitric oxide synthase seems to be induced significantly in patients with chronic renal failure. Increased endogenous nitric oxide production may have a pathophysiological role in patients with uraemia.

Molecular Mechanisms of Endothelin-1–Induced Cell-Cycle Progression Involvement of Extracellular Signal-Regulated Kinase, Protein Kinase C, and Phosphatidylinositol 3-Kinase at Distinct Points

Although it is well established that endothelin-1 (ET-1) has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this study, we examined the effects of ET-1 on the cell-cycle regulatory machinery, including cyclins, cyclin-dependent kinase (cdk), and cdk inhibitors in NIH3T3 cells. ET-1 increased cyclin D1 protein (5.1+/-1.9-fold increase, 8 hours after stimulation, P<0.05), cdk4 kinase activity (2.8+/-0. 5-fold increase, 12 hours after stimulation, P<0.01), and cdk2 kinase activity (2.1+/-0.4-fold increase, 16 hours after stimulation, P<0.05) in a time- and dose-dependent manner. ET-1-induced increase in cyclin D1 protein, and cdk4 kinase activity was not significantly inhibited by an inhibitor of the mitogen-activated protein kinase kinase 1/2, PD98059, nor by the protein kinase C inhibitor calphostin C, whereas ET-1-induced upregulation of cyclin D1 protein and cdk4 kinase activity was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. In contrast, ET-1-induced activation of cdk2 kinase was significantly inhibited by PD98059, calphostin C, and LY294002. ET-1 increased 3H-thymidine uptake in a time-dependent fashion (0 hours, 4216+/-264 cpm per well; 8 hours, 5025+/-197 cpm per well; 16 hours, 9239+/-79 cpm per well, P<0.001 versus 0 hours). ET-1-induced increase in 3H-thymidine uptake was significantly inhibited by PD98059, calphostin C, and LY294002. These results suggest that ET-1-induced cell-cycle progression is, at least in part, mediated by the extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase and that those pathways may be involved in the progression of the cell cycle at distinct points.

Nitric Oxide Release From Kidneys of Hypertensive Rats Treated With Imidapril

To examine whether endothelial dysfunction in hypertension is reversible or not, we studied the effects of imidapril, an angiotensin-converting enzyme inhibitor, on nitric oxide release in stroke-prone spontaneously hypertensive rats (SHR) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. After a 4-week treatment with imidapril (1 or 10 mg/d SC) or vehicle, acetylcholine-induced vasodilation and nitric oxide release in the isolated kidneys were determined. Nitric oxide release was measured by a chemiluminescense assay. Imidapril lowered blood pressure in stroke-prone SHR in a dose-dependent manner. Untreated stroke-prone SHR exhibited significantly attenuated responses to acetylcholine (10(-8) mol/L) of both renal perfusion pressure (stroke-prone SHE 42 +/- 4% versus Wistar-Kyoto rats [WKY] 58 +/- 4% [mean +/- SE], P < .01) and nitric oxide release (stroke-prone SHR +7.6 +/- 2.1 versus WKY +29.7 +/- 9.7 fmol/min per gram of kidney wt, P < .01). Imidapril at 10 mg/d significantly increased acetylcholine-induced renal vasodilation and nitric oxide release in stroke-prone SHR (renal perfusion pressure, 56 +/- 3%; nitric oxide release, +27.1 +/- 6.4 fmol/min per gram of kidney wt; both P < .01 versus stroke-prone SHR treated with vehicle). On the other hand, imidapril neither decreased blood pressure nor changed nitric oxide release induced by acetylcholine in DOCA-salt hypertensive rats. Staining for endothelial nitric oxide synthase and brain nitric oxide synthase was clearly detected in the kidneys of both stroke-prone SHR and WKY, whereas staining intensity was weaker in DOCA-salt hypertensive rats. Inducible nitric oxide synthase immunoreactivity was barely noticeable in any type of rat. Thus, imidapril restored endothelial damage by pressure-dependent mechanisms. Most of the nitric oxide detected in the perfusate seemed to be derived from constitutive nitric oxide synthase.

Cyclin A downregulation and p21(cip1) upregulation correlate with GATA-6-induced growth arrest in glomerular mesangial cells.

The GATA-6 transcription factor is reported to be expressed in vascular myocytes. Because glomerular mesangial cells (GMCs) and vascular myocytes have similar properties, we examined whether GATA-6 was expressed in cultured GMCs and whether overexpression of GATA-6 induced cell cycle arrest in GMCs, using a recombinant adenovirus that expresses GATA-6 (Ad GATA-6). GATA-6 expression in GMCs was downregulated when quiescent GMCs were stimulated by serum to reenter the cell cycle. [3H]thymidine uptake was inhibited in GMCs infected with Ad GATA-6 in a dose- and time-dependent manner. The expression of cyclin A protein was decreased and that of the cyclin-dependent kinase inhibitor p21cip1 was increased in GMCs infected with Ad GATA-6. Although the expression of p21cip1 transcripts did not change remarkably, p21cip1 protein was stabilized in GMCs infected with Ad GATA-6, suggesting a post-transcriptional regulation of p21cip1 expression. Northern blot analysis showed that expression of the cyclin A transcript was decreased in Ad GATA-6–infected cells, whereas this decrease of cyclin A was not observed in GMCs derived from p21cip1 null mice. Our results demonstrate that GATA-6 is endogenously expressed in GMCs and that overexpression of GATA-6 can induce cell cycle arrest. Our results also show that GATA-6–induced cell cycle arrest is associated with inhibition of cyclin A expression and p21cip1 upregulation. Finally, our results indicate that the GATA-6–induced suppression of cyclin A expression depends on the presence of p21cip1.