Flow regulation of ecNOS and Cu/Zn SOD mRNA expression in porcine coronary arterioles.
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The purpose of this study was to test the hypothesis that increased flow through coronary arterioles increases endothelial cell nitric oxide synthase (ecNOS) and Cu/Zn superoxide dismutase (SOD) mRNA expression. Single porcine coronary arterioles (ID 100-160 micrometers; pressurized) were cannulated, perfused, and exposed to intraluminal flow sufficient to produce maximal flow-induced dilation of coronary arterioles (high flow; 7.52 +/- 0.22 microliter/min), low flow (0.84 +/- 0.05 microliter/min), or no flow for 2 or 4 h. Mean shear stress was calculated to be 5.7 +/- 1.0 dyn/cm2 for high-flow arterioles and 1. 6 +/- 1.0 dyn/cm2 for low-flow arterioles. At the end of the treatment period, mRNA was isolated from each vessel, and ecNOS and SOD mRNA expression was assessed using a semiquantitative RT-PCR. All data were standardized by coamplifying ecNOS or SOD with glyceraldehyde-3-phosphate dehydrogenase. The results indicate that ecNOS mRNA expression is increased in arterioles exposed to 2 or 4 h of high flow. In contrast, SOD mRNA expression was increased only after 4 h of high flow. Neither gene is induced by exposure to low flow. On the basis of these data, we concluded that ecNOS and SOD mRNA expression is regulated by flow in porcine coronary arterioles. In addition, we concluded that a threshold level of flow and shear stress must be sustained to elicit the upregulation of ecNOS and SOD mRNA expression. (+info)
Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.
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Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion. (+info)
Inducible NO synthase inhibition attenuates shear stress-induced pulmonary vasodilation in the ovine fetus.
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Recent studies have suggested that type II (inducible) nitric oxide (NO) synthase (NOS II) is present in the fetal lung, but its physiological roles are uncertain. Whether NOS II activity contributes to the NO-mediated fall in pulmonary vascular resistance (PVR) during shear stress-induced pulmonary vasodilation is unknown. We studied the hemodynamic effects of two selective NOS II antagonists [aminoguanidine (AG) and S-ethylisothiourea (EIT)], a nonselective NOS antagonist [nitro-L-arginine (L-NNA)], and a nonselective vasoconstrictor (U-46619) on PVR during partial compression of the ductus arteriosus (DA) in 20 chronically prepared fetal lambs (mean age 132 +/- 2 days, term 147 days). At surgery, catheters were placed in the left pulmonary artery (LPA) for selective drug infusion, an ultrasonic flow transducer was placed on the LPA to measure blood flow, and an inflatable vascular occluder was placed loosely around the DA for compression. On alternate days, a brief intrapulmonary infusion of normal saline (control), AG, EIT, L-NNA, or U-46619 was infused in random order into the LPA. The DA was compressed to increase mean pulmonary arterial pressure (MPAP) 12-15 mmHg above baseline values and held constant for 30 min. In control studies, DA compression reduced PVR by 42% from baseline values (P < 0.01). L-NNA treatment completely blocked the fall in PVR during DA compression. AG and EIT attenuated the decrease in PVR by 30 and 19%, respectively (P < 0.05). Nonspecific elevation in PVR by U-46619 did not affect the fall in PVR during DA compression. Immunostaining for NOS II identified this isoform in airway epithelium and vascular smooth muscle in the late-gestation ovine fetal lung. We conclude that selective NOS II antagonists attenuate but do not block shear stress-induced vasodilation in the fetal lung. We speculate that stimulation of NOS II activity, perhaps from smooth muscle cells, contributes in part to the NO-mediated fall in PVR during shear stress-induced pulmonary vasodilation. (+info)
Impact of development and chronic hypoxia on NE release from adrenergic nerves in sheep arteries.
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To examine effects of development and chronic high-altitude hypoxia on sympathetic nerve function in sheep, norepinephrine release was measured in vitro from middle cerebral and facial arteries. Capsaicin was used to test the role of capsaicin-sensitive sensory nerves; norepinephrine release was not altered by capsaicin treatment. Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, decreased stimulation-evoked norepinephrine release in middle cerebral arteries from normoxic sheep with no effect in hypoxic arteries or facial arteries. Thus NO-releasing nerves augmented norepinephrine release. Furthermore, the function of NO-releasing nerves declined after chronic hypoxia. Despite loss of the augmenting effects of NO, stimulation-evoked fractional norepinephrine release was unchanged after chronic hypoxia, suggesting that middle cerebral arteries adapt to hypoxia by increasing stimulation-evoked norepinephrine release. In fetal facial arteries, chronic hypoxia resulted in a decline in stimulation-evoked norepinephrine release, but there was an increase in the adult facial artery. In the adult, adaptation to chronic hypoxia is similar in both cerebral and facial arteries. However, differential adaptation in fetal adrenergic nerves may reflect differences in fetal redistribution of blood flow in the face of chronic hypoxia but could also possibly contribute to increased incidence of fetal morbidity. (+info)
Genetic control of renal thiazide receptor response to dietary NaCl and hypertension.
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Excess NaCl increases blood pressure in some strains of animals but not others. An 8% NaCl diet did not change renal thiazide receptor (TZR) density in two salt-resistant normotensive rat strains (Wistar-Kyoto and Sprague-Dawley) [Fanestil, D. D., D. A. Vaughn, and P. Blakely. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R1241-R1245, 1997]. However, the renal response to salt differs in normal and hypertensive kidneys [Rettig, R., N. Bandelow, O. Patschan, B. Kuttler, B. Frey, and A. Uber. J. Hum. Hypertens. 10: 641-644, 1996]. Therefore, we examined two strains with salt-aggravated hypertension. Renal TZR did not change when Dahl-S (salt sensitive) animals became hypertensive with 8% dietary NaCl. In contrast, renal TZR decreased 34%, whereas blood pressure increased further, in SHR with 8% dietary NaCl. Blood pressure increased after NG-nitro-L-arginine in SHR, but renal TZR did not change, indicating the salt-induced decrease in TZR in SHR cannot be attributed nonspecifically to elevated arterial pressure. We conclude that the renal response to NaCl-induced increases in blood pressure can be genetically modulated independently of the genes that mediate either the primary hypertension or the salt sensitivity of the hypertension. This finding may be of use in future studies directed at identifying genotypes associated with salt-dependent hypertension. (+info)
AVP inhibits LPS- and IL-1beta-stimulated NO and cGMP via V1 receptor in cultured rat mesangial cells.
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The present study examined how arginine vasopressin (AVP) affects nitric oxide (NO) metabolism in cultured rat glomerular mesangial cells (GMC). GMC were incubated with test agents and nitrite, and intracellular cGMP content, inducible nitric oxide synthase (iNOS) mRNA, and iNOS protein were analyzed by the Griess method, enzyme immunoassay, and Northern and Western blotting, respectively. AVP inhibited lipopolysaccharide (LPS)- and interleukin-1beta (IL-1beta)-induced nitrite production in a dose- and time-dependent manner, with concomitant changes in cGMP content, iNOS mRNA, and iNOS protein. This inhibition by AVP was reversed by V1- but not by oxytocin-receptor antagonist. Inhibition by AVP was also reproduced on LPS and interferon-gamma (IFN-gamma). Protein kinase C (PKC) inhibitors reversed AVP inhibition, whereas PKC activator inhibited nitrite production. Although dexamethasone and pyrrolidinedithiocarbamate (PDTC), inhibitors of nuclear factor-kappaB, inhibited nitrite production, further inhibition by AVP was not observed. AVP did not show further inhibition of nitrite production with actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of protein synthesis. In conclusion, AVP inhibits LPS- and IL-1beta-induced NO production through a V1 receptor. The inhibitory action of AVP involves both the activation of PKC and the transcription of iNOS mRNA in cultured rat GMC. (+info)
An inhibitor of nitric oxide synthase does not increase contraction or beta-adrenoceptor sensitivity of ventricular myocytes from failing human heart.
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OBJECTIVE: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in an attempt to restore contractile function. METHODS AND RESULTS: Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32 degrees C, 1-2 mmol/l Ca2+) and electrical stimulation (0.1-2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70 +/- 0.53% cell shortening (mean +/- SEM, n = 13 subjects), compared with 2.18 +/- 0.27% (P < 0.01, 11 patients) from patients with ischaemic heart disease (IHD) or 2.56 +/- 0.74% (P < 0.02, six patients) with dilated cardiomyopathy (DCM). Superfusion with 0.1 mmol/l L-NMMA did not increase contraction amplitude in myocytes from failing heart at either 0.2 Hz (n = 11) or 1 Hz (n = 7). Responses to beta-adrenoceptor stimulation were reduced in myocytes from failing human heart, with contraction amplitude in maximum isoprenaline 0.47 +/- 0.11 of that in high Ca2+ in the same cell (n = 6), compared to 0.99 +/- 0.07 in non-failing heart (n = 14, P < 0.01). The presence of 0.1 mmol/l L-NMMA did not increase the isoprenaline/Ca2+ ratio in myocytes from failing heart (0.40 +/- 0.09, P = NS). CONCLUSION: These results do not suggest a functional role for tonic NO production in the frequency-dependent depression of contraction or beta-adrenoceptor desensitisation in myocytes from failing human ventricle. (+info)
Role of innate and adaptive immunity in the outcome of primary infection with Chlamydia pneumoniae, as analyzed in genetically modified mice.
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Infection with Chlamydia pneumoniae is a common cause of acute respiratory disease in man and is also associated with atherosclerotic cardiovascular disorder. Herein, we have compared bacterial load and immune parameters of C. pneumoniae-infected mice genomically lacking T cell coreceptors, cytokine receptors, or cytotoxic effector molecules. A protective role for CD8+ cells is shown by the enhanced severity of infection of CD8-/- or TAP-1-/-/beta2-microglobulin -/- mice. CD8+ cells hindered a parasite growth-promoting role of CD4+ T cells, as indicated by the higher sensitivity to early infection of CD8-/- than CD4-/-/CD8-/- mice, which was further confirmed in experiments in which SCID mice were reconstituted with either CD4+ or CD4+ plus CD8+ T cells. Interestingly, CD4+ T cells played a dual role, detrimental early (14 and 24 days) after infection but protective at later time points (60 days after infection). The CD8+ T cell protection was perforin independent. The early deleterious role of CD4+ in the absence of CD8+ T cells was associated with enhanced IL-4 and IL-10 mRNA levels and delayed IFN-gamma mRNA accumulation in lungs. In line with this, IFN-gammaR-/- (but not TNFRp55 -/-) mice showed dramatically increased susceptibility to C. pneumoniae, linked to reduced inducible nitric oxide synthase (iNOS) mRNA accumulation, but not to diminished levels of specific Abs. The increased susceptibility of iNOS-/- mice indicates a protective role for iNOS activity during infection with C. pneumoniae. The higher sensitivity of IFN-gammaR-/- mice to C. pneumoniae compared with that of SCID or recombination-activating gene-1-/- mice suggested a relevant protective role of IFN-gamma-dependent innate mechanisms of protection. (+info)