Intermittent hypoxia-induced increases in reactive oxygen species activate NFATc3 increasing endothelin-1 vasoconstrictor reactivity.

Sleep apnea (SA), defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension, peripheral vascular disease, stroke, and sudden cardiac death. We have shown that intermittent hypoxia with CO2 supplementation (IH), a model for SA, increases blood pressure and circulating ET-1 levels, upregulates lung pre-pro ET-1 mRNA, increases vasoconstrictor reactivity to ET-1 in rat small mesenteric arteries (MA) and increases vascular reactive oxygen species (ROS). NFAT activity is increased in the aorta (AO) and MA of mice exposed to IH in an ET-1-dependent manner, and the genetic ablation of the isoform NFATc3 prevents IH-induced hypertension. We hypothesized that IH causes an increase in arterial ROS generation, which activates NFATc3 to increase vasoconstrictor reactivity to ET-1. In support of our hypothesis, we found that IH increases ROS in AO and MA. In vivo administration of the SOD mimetic tempol during IH exposure prevents IH-induced increases in NFAT activity in mouse MA and AO. We found that IH causes an NFATc3-dependent increase in vasoconstrictor reactivity to ET-1, accompanied by an increase in vessel wall [Ca²âº]. Our results indicate that IH exposure causes an increase in arterial ROS to activate NFATc3, which then increases vasoconstrictor reactivity and Ca²âº response to ET-1. These studies highlight a novel regulatory pathway, and demonstrate the potential clinical relevance of NFAT inhibition to prevent hypertension in SA patients.

NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice.

Pulmonary hypertension occurs with prolonged exposure to chronic hypoxia in both adults and neonates. The Ca(2+)-dependent transcription factor, nuclear factor of activated T cells isoform c3 (NFATc3), has been implicated in chronic hypoxia-induced pulmonary arterial remodeling in adult mice. Therefore, we hypothesized that NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice. The aim of this study was to determine whether 1) NFATc3 mediates chronic hypoxia-induced increases in right ventricular systolic pressure in adult mice; 2) NFATc3 is activated in neonatal mice exposed to chronic hypoxia; and 3) NFATc3 is involved in chronic hypoxia-induced right ventricular hypertrophy and pulmonary vascular remodeling in neonatal mice. Adult mice were exposed to hypobaric hypoxia for 2, 7, and 21 days. Neonatal mouse pups were exposed for 7 days to hypobaric chronic hypoxia within 2 days after delivery. Hypoxia-induced increases in right ventricular systolic pressure were absent in NFATc3 knockout adult mice. In neonatal mice, chronic hypoxia caused NFAT activation in whole lung and nuclear accumulation of NFATc3 in both pulmonary vascular smooth muscle and endothelial cells. In addition, heterozygous NFATc3 neonates showed less right ventricular hypertrophy and pulmonary artery wall thickness in response to chronic hypoxia than did wild-type neonates. Our results suggest that NFATc3 mediates pulmonary hypertension and vascular remodeling in both adult and neonatal mice.

Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter.

Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491-5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069-75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na+ and D-glucose. Using alpha-14C-methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant (K(0.5) approximately 2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.

Effect of chronic angiotensin II inhibition on the nitric oxide synthase in the normal rat during aging.

OBJECTIVE: To assess the effect on the cardiovascular system, of enalapril (E) or losartan (L) given since weaning during 6 or 18 months to normal rats. METHODS: Animals were divided in three groups: control (C), E-treated and L-treated; treated rats received 10 mg/ kg per day of drug. Systolic blood pressure (SBP), body weight, water and food intake (WI, FI), cardiac, left ventricular and aortic weight as well as the length of the tail were recorded. NADPH-diaphorase activity was determined as a marker of nitric oxide synthase (NOS) activity in aorta, arterioles of small intestine, heart and kidney of normal rats. NOS activity was measured as optical density (OD) in the stained tissue. Nitrate + nitrite urinary excretion was measured in 24 h urine. Only significant differences (P < 0.05) are reported. RESULTS: SBP, absolute cardiac, left ventricular and aortic weight increased with age. Both treatments delayed these increments. At 6 and 18 months, NOS activity was higher in aortic endothelium (Em) of L- and E-treated animals. Losartan treatment during 6 months also increased NOS activity in aortic smooth muscle (SM). Aortic Em NOS activity fell in the 18 months-treated and untreated animals. E increased NOS activity in the SM of intestinal arterioles at 6 months but reduced it at 18 months. CONCLUSIONS: The fact that both E and L delayed cardiac hypertrophy/hyperplasia and aortic growth and raised aortic endothelium NOS activity indicates a protective effect on cardiovascular damage due to aging, exerted through inhibition of angiotensin II.

Effects of endothelin-3 on intestinal ion transport.

We investigated the effects of endothelin 3 (ET-3) on electrolyte transport in rat small intestine using a voltage clamp technique in Ussing's chamber. ET-3 diminished potential difference (PD) and short circuit current (Isc). ET-3 did not affect PD or Isc in low Na(+) and/or D-glucose-free medium. Phloridzine (an inhibitor of sodium-glucose cotransporter [SGLT1]) pretreatment abolished the effect of ET-3 on Isc. Methylene blue (a soluble guanylate cyclase inhibitor) or N-nitro-L-arginine methyl ester (a NOS inhibitor) pretreatment delayed the effect of ET-3 on PD and Isc. ET-3 enhanced NOS activity on enterocytes and systemic NO production. Then, ET-3 could inhibit SGLT1 with the participation of NO.

Effects of atrial natriuretic peptide in the gut.

The intestinal tract is a target organ for atrial natriuretic peptide (ANP), characterized by various biologic activities, immunoreactivity, as well as specific binding sites for ANP. A review of previous studies reveals that ANP is an important regulator of water and nutrient intake, which acts via multiple signaling pathways including activation of guanylyl cyclase to produce its biologic responses. As a regulator, the peptide locally controls hydrosaline balance and acute systemic effects. Therefore, ANP could also act as a local mediator or paracrine effector of intestinal function.

Atrial natriuretic peptide modifies arterial blood pressure through nitric oxide pathway in rats.

The aim of the present study was to determine the relationship between the hypotensive effect of the atrial natriuretic peptide (ANP) and the nitric oxide (NO) pathway. N(G)-nitro-L-arginine methyl ester bolus (L-NAME, 1 mg/kg) reverted the decrease in mean arterial pressure induced by ANP administration (5 microg/kg bolus and 0.2 microg x kg(-1) x min(-1) infusion), and the injection of L-NAME before peptide administration suppressed the ANP hypotensive response. To confirm these findings, a histochemical reaction was used to determine NADPH-diaphorase activity (a NO synthase marker) in the endothelium and smooth muscle of aorta and arterioles of the small and large intestine. ANP increased aorta and arteriole endothelium staining after both in vivo administration and in vitro tissue incubation. In both cases, L-NAME prevented the ANP effect on NADPH-diaphorase activity. Tissues incubated with 8-bromoguanosine 3',5'-cyclic monophosphate mimicked ANP action. In addition, ANP administration increased urinary excretion of NO(x) end products. These findings indicate that ANP increases NO synthesis capability and NO production and suggest that the cGMP pathway may be involved. In conclusion, the NO pathway could be an intercellular messenger in the ANP endothelium-dependent vasorelaxation mechanism.

Atrial natriuretic peptide effect on NADPH-diaphorase in rat intestinal tract.

Histochemical reaction of NADPH-diaphorase (NOS-NADPH-d) was used to identify NO synthesis. A 30-min 0.1 microg microg/kg/min ANP infusion led to about a 10% and 35% increase in small and large intestine enterocytes stain respectively. This increase was abolished by a bolus of 1 mg/kg L-NAME before ANP infusion in small intestine, and partially abolished it in colon. Incubation of small and large intestine with 0.5 microM ANP increased stain at about 20%. In both tissues the preincubation with 0.1 mM L-NAME abolished the ANP effect. Incubation with 0.1 mM 8-Br-cGMP enhanced staining about 70% and 30% in small and large intestine respectively. Our results show that ANP enhances NOS-NADPH-d activity, suggesting that ANP stimulates NO synthase in enterocytes by L-arginine-NO pathway. 8-Br-cGMP mimicked the effect of ANP described above. Therefore, the guanylate cyclase-coupled natriuretic receptors, NPR-A and NPR-B, probably mediate this ANP effect.

Effect of atrial natriuretic peptide on alpha-methyl-D-glucoside intestinal active uptake in rats.

In vivo, atrial natriuretic peptide (ANP) inhibits water and sodium absorption by the intestine. In addition, ANP inhibits glucose (re)absorption at the level of both the intestine and kidney. ANP also decreases sodium absorption in the rat small intestine in vitro, but only if glucose is present on the luminal side of the tissue. These findings suggest that ANP inhibits the sodium-glucose cotransporter (SGLT) of enterocytes. In the present study the inhibitory effect of 1 microM ANP on SGLT1 in rat small intestine and colon was tested. For this purpose, the apparent kinetic constants of SGLT1 were determined using radioactive alpha-methyl-D-glucoside (alpha-MG), a non-metabolizable glucose analogue that selectively serves the luminal Na+-dependent intestinal uptake, but not the serosal-facilitated diffusion sugar carrier. In both tissues, incubation with ANP increased Km without modifying the Vmax. In addition, Vmax in the small intestine was found to be higher than in the colon. The evidence presented here suggests that ANP, through its second messenger, may be a competitive inhibitor of SGLT1. Since SGLT1 is also expressed in the brush-border membrane of the renal proximal tubule, we suggest that this peptide might regulate the hydro-saline balance at intestinal and proximal tubular nephron levels.

Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine.

Atrial natriuretic peptide (ANP) decreases sodium absorption in small intestine of rats in vitro under sodium concentration-gradient conditions (SCG) and this effect may be mediated by the inhibition of the sodium/glucose cotransporter (SGLT). In order to assess this hypothesis, the effects of ANP, phloridzine (Phlz) and methylene blue (MB), added alone or together, using a voltage clamp technique in Ussing's chamber with SCG were studied. ANP and Phlz significantly decreased potential difference and short circuit current. Effects of Phlz and ANP were not additive. The addition of MB alone did not affect ion transport, whereas it abolished ANP effects. These data suggest that ANP blocks the SGLT through mechanisms mediated by cGMP and/or NO.