Cristina Arranz

Moderate zinc restriction during fetal and postnatal growth of rats: effects on adult arterial blood pressure and kidney

Intrauterine and postnatal zinc restriction may result in an adverse environment for the development of cardiovascular and renal systems. This study evaluated the effects of moderate zinc deficiency during fetal life, lactation, and/or postweaning growth on systolic blood pressure, renal function, and morphology in adult life. Female Wistar rats received low (8 ppm) or control (30 ppm) zinc diets from the beginning of pregnancy up to weaning. After weaning, male offspring of each group of mothers were fed low or control zinc diet. Systolic blood pressure, creatinine clearance, proteinuria, renal morphology, renal apoptosis. and renal oxidative stress state were evaluated after 60 days. Zinc deficiency during pre- and postweaning growth induced an increase in systolic blood pressure and a decrease in the glomerular filtration rate associated with a reduction in the number and size of nephrons. Activation of renal apoptosis, reduction in catalase activity, glutathione peroxidase activity, and glutathione levels and increase in lipid peroxidation end products could explain these morphometric changes. Zinc deficiency through pre- and postweaning growth induced more pronounced renal alteration than postweaning zinc deficiency. These animals showed signs of renal fibrosis, proteinuria, increased renal apoptosis, and higher lipid peroxidation end products. A control diet during postweaning growth did not totally overcome renal oxidative stress damage, apoptosis, and fibrosis induced by zinc deficiency before weaning. In conclusion, zinc deficiency during a critical period of renal development and maturation could induce functional and morphological alterations that result in elevated blood pressure and renal dysfunction in adult life.

Angiotensin-(1-7) improves cardiac remodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats.

The present study examined whether chronic treatment with angiotensin (ANG)-(1-7) reduces cardiac remodeling and inhibits growth-promoting signaling pathways in the heart of fructose-fed rats (FFR), an animal model of insulin resistance. Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of the corresponding diet, control and FFR were implanted with osmotic pumps that delivered ANG-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We determined heart weight, myocyte diameter, interstitial fibrosis, and perivascular collagen type III deposition as well as the phosphorylation degree of ERK1/2, JNK1/2, and p38MAPK. FFR showed a mild hypertension that was significantly reduced after ANG-(1-7) treatment. Also, FFR displayed higher ANG II circulating and local levels in the heart that remained unaltered after chronic ANG-(1-7) infusion. An increased heart-to-body weight ratio, myocyte diameter, as well as left ventricular fibrosis and perivascular collagen type III deposition were detected in the heart of FFR. Interestingly, significant improvements in these cardiac alterations were obtained after ANG-(1-7) treatment. Finally, FFR that received ANG-(1-7) chronically displayed significantly lower phosphorylation levels of ERK1/2, JNK1/2, and p38MAPK. The beneficial effects obtained by ANG-(1-7) were associated with normal values of Src-homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity in the heart. In conclusion, chronic ANG-(1-7) treatment ameliorated cardiac hypertrophy and fibrosis and attenuated the growth-promoting pathways in the heart. These findings show an important protective role of ANG-(1-7) in the heart of insulin-resistant rats.

Angiotensin-(1–7) upregulates cardiac nitric oxide synthase in spontaneously hypertensive rats

It has been shown that angiotensin (ANG)-(1-7) activates nitric oxide synthase (NOS) in isolated ventricular myocytes from normotensive rats. Since many ANG-(1-7) actions are enhanced in situations of increased ANG II activity, as in hypertension, in this study we investigated the in vivo effect of ANG-(1-7) on NOS activity and expression of endothelial (eNOS), neuronal (nNOS), and inducible NOS (iNOS) in ventricles from spontaneously hypertensive rats (SHR). Rats were subjected to a 60-min ANG-(1-7) infusion (0.35 nmol/min); controls received saline. NOS activity was measured using the NADPH diaphorase histochemical method and by the conversion of L-[(14)C]arginine to citrulline, and NOS phosphorylation and expression were determined using Western blotting. In SHR, ANG-(1-7) infusion diminished mean arterial pressure from 180 ± 9 to 146 ± 9 mmHg (P < 0.05), and this effect was prevented by nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor. In addition, NOS activity and eNOS phosphorylation were increased by ANG-(1-7) infusion. Ventricular eNOS and nNOS expression were increased 67.4 ± 6.4 and 51 ± 10%, respectively, by ANG-(1-7), whereas iNOS was not changed. In another set of experiments, we evaluated the mechanism by which ANG-(1-7) modifies NOS activity. Isolated ventricle slices preincubated with ANG-(1-7) showed an increase in NOS activity and eNOS phosphorylation, which was blocked by an AT(2) and a bradykinin B(2) receptor antagonist, but not by the Mas receptor antagonist. Our results show that in rats in a hypertensive state, ANG-(1-7) infusion upregulates cardiac NOS expression and activity through an AT(2)- and bradykinin-dependent mechanism. In this way ANG-(1-7) may elicit its cardioprotective action and contribute to some of the counterregulatory AT(2) receptor effects that oppose the AT(1) receptor-mediated effects.

Atrial natriuretic peptide influence on nitric oxide system in kidney and heart.

Atrial natriuretic peptide (ANP) and nitric oxide (NO) induce diuresis, natriuresis and diminish vascular tone. Our previous studies showed NO system is involved in ANP hypotensive effect. The aim was to investigate ANP effects on renal and cardiac NO-synthase (NOS) activity. Rats were divided into two groups: group I, infused with saline (1 h, 0.05 ml/min); group II, received ANP bolus (5 microg/kg)+ANP infusion (1 h, 0.2 microg/kg x min). NADPH-diaphorase activity (NADPH-d) was determined in kidney and heart. NOS catalytic activity was determined in renal medulla and cortex and cardiac atria and ventricle by measuring the conversion of l-[U(14)C]-arginine to l-[U(14)C]-citrulline. In group I, NOS activity was determined in basal conditions and plus 1 microM ANP and in group II, NOS activity was determined in basal conditions. NADPH-d was higher in group II than in group I in glomeruli, proximal tubule, cortical and medullar collecting duct, right atria and left ventricle. NOS activity was increased by in vitro ANP addition and, in vivo, ANP infusion in all the studied tissues. ANP treatment increases renal and cardiac NO synthesis. This effect would be independent on the hemodynamic changes induced by ANP. The activation of NO pathway would be one of the mechanisms involved in diuretic, natriuretic and hypotensive effects of ANP.

Exposure to zinc deficiency in fetal and postnatal life determines nitric oxide system activity and arterial blood pressure levels in adult rats

We had previously shown that prenatal exposure to Zn-deficient diets induces an increase in blood pressure and impairs renal function in adult rats. The aim of the present study was to investigate if moderate Zn restriction during early growth periods, fetal life and lactation would induce impairment in the vascular and renal NO system and alterations in plasma lipid profile. We also investigated if these effects persisted into adult life, even when a Zn-replete diet was provided after weaning. Pregnant rats were fed control (30 parts per million (ppm)) or low (8 ppm) Zn diets throughout gestation up to weaning. Afterwards, male offspring from low-Zn mothers were assigned to low- or control-Zn diets during 60 d. Male offspring from control mothers were fed a control diet. Animals exposed to Zn restriction showed low birth weight, increased systolic blood pressure and serum TAG levels, and decreased glomerular filtration rate in adulthood. Zn restriction induced a decrease in vascular and renal NO synthase activity and a reduced expression of the endothelial NO synthase isoform in aorta. A control-Zn diet during post-weaning growth returned TAG levels to normal but was unsuccessful in normalising systolic blood pressure, glomerular filtration rate or NO system activity in Zn-deficient offspring. Zn restriction during fetal life, lactation and/or post-weaning growth induced alterations in the vascular and renal NO system and in lipid metabolism that could contribute to the programming of hypertension and renal dysfunction in adulthood.

Signaling cascade that mediates endothelial nitric oxide synthase activation induced by atrial natriuretic peptide.

UNLABELLED Atrial natriuretic peptide (ANP) induces activation of nitric oxide-synthase (NOS). AIMS to identify the isoform of NOS involved in ANP effects, to study whether ANP modifies NOS expression and to investigate the signaling pathways and receptors involved in NOS stimulation. NOS activation induced by ANP would be mediated by endothelial NOS (eNOS) since neuronal or inducible NOS inhibition did not alter ANP effect. The peptide induced no changes in eNOS protein expression. NOS activity stimulated by ANP, in the kidney, aorta and left ventricle, was partially abolished by the NPR-A/B antagonist, as well as PKG inhibition, but no difference in atria was observed. 8-Br-cGMP partially mimicked the effect of ANP on NOS in all tissues. NOS stimulation by ANP in atria disappeared when G protein was inhibited, but this effect was partial in the other tissues. Calmodulin antagonist abolished NOS stimulation via ANP. Inhibition of the PLC, PKC or PI3 kinase/Akt pathway failed to alter NOS activation induced by ANP. ANP would activate eNOS in the aorta, heart and kidney without modifying the expression of the enzyme. ANP would interact with NPR-C coupled via G proteins leading to the activation of Ca(2+)-calmodulin-dependent NOS in atria; while in ventricle, aorta and kidney, ANP could also interact with NPR-A/B, increasing cGMP, which in turns activates PKG to stimulate eNOS.

Role of NPR-C natriuretic receptor in nitric oxide system activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.

Moderate zinc deficiency influences arterial blood pressure and vascular nitric oxide pathway in growing rats.

There is an increasing interest in the involvement of trace elements such as zinc in the pathogenesis of cardiovascular diseases. This study was designed to examine whether moderate zinc deficiency during growth influences blood pressure (BP) and vascular nitric oxide (NO) pathway. Three-week-old weaned male Wistar rats were randomly divided into two dietary groups and fed either a moderately zinc-deficient diet (zinc content 9 mg/kg; n = 12) or a control diet (zinc content 30 mg/kg; n = 12) for 60 d. The following were measured: systolic BP, nitrates and nitrites urinary excretion, urinary chemiluminescence intensity, NADPH-diaphorase activity in the thoracic aorta and intestinal arterioles, and NO synthase (NOS) catalytic activity using l-[U14C]-arginine as substrate in the thoracic aorta. Zinc deficiency during growth induced an increase in BP from day 30 of the experimental period, leading to hypertension on day 60. Animals that were fed the zinc-deficient diet had lower urinary excretion levels of nitrates and nitrites and higher intensity of spontaneous luminescence on day 60. At the end of the experiment, zinc-deficient rats showed decreased NADPH diaphorase activity in endothelium and smooth muscle of the thoracic aorta and intestinal arterioles and decreased activity of NOS in thoracic aortic tissue. An imbalance in zinc bioavailability during postnatal and growing periods may be may be a risk factor in development of cardiovascular alterations in adult life. The mechanisms involved may include an impaired vascular NO system as a result of decreased NOS activity and higher systemic oxidative stress.

C-type natriuretic peptide effects on cardiovascular nitric oxide system in spontaneously hypertensive rats.

The aim was to study the effects of C-type natriuretic peptide (CNP) on mean arterial pressure (MAP) and the cardiovascular nitric oxide (NO) system in spontaneously hypertensive rats (SHR), and to investigate the signaling pathways involved in this interaction. SHR and WKY rats were infused with saline or CNP. MAP and nitrites and nitrates excretion (NO(x)) were determined. Catalytic NO synthase (NOS) activity and endothelial (eNOS), neuronal (nNOS) and inducible NOS (iNOS) were measured in the heart and aorta artery. NOS activity induced by CNP was determined in presence of: iNOS or nNOS inhibitors, NPR-A/B natriuretic peptide receptors blocker and Gi protein and calmodulin inhibitors. CNP diminished MAP and increased NO(x) in both groups. Cardiovascular NOS activity was higher in SHR than in WKY. CNP increased NOS activity, but this activation was lower in SHR. CNP had no effect on NOS isoforms expression. iNOS and nNOS inhibitors did not modify CNP-induced NOS activity. NPR-A/B blockade induced no changes in NOS stimulation via CNP in both tissues. Cardiovascular NOS response to CNP was reduced by Gi protein and calmodulin inhibitors in both groups. CNP interacts with NPR-C receptors, activating Ca-calmodulin eNOS via Gi protein. NOS response to CNP is impaired in the heart and aorta of SHR. Alterations in the interaction between CNP and NO would be involved in the maintenance of high blood pressure in this model of hypertension.

Nitric oxide and thyroid gland: modulation of cardiovascular function in autonomic-blocked anaesthetized rats.

We have previously reported that acute administration of NG‐nitro‐l‐arginine methyl ester (l‐NAME) increases the mean arterial pressure (MAP) and heart rate (HR) in autonomic‐blocked (CAB) anaesthetized rats. In the present study we examined whether thyroid and adrenal glands are involved in these pressor and chronotropic responses. Sprague‐Dawley rats were studied after bilateral vagotomy and ganglionic blockade with hexamethonium (10 mg kg−1), and stabilization of MAP with infusion of phenylephrine (PE) (6 μg kg−1 min−1). The rats were divided into groups: L, CAB; PE, CAB + PE bolus (6 μg kg−1); L‐TX, thyroidectomy + CAB; L‐AX, adrenalectomy + CAB; TX, only thyroidectomy; C, CAB. L, L‐AX and L‐TX groups received a bolus of l‐NAME (7.5 mg kg−1). Triiodothyronine (T3), thyroxin (T4) and thyrotropin (TSH) levels were measured in L and L‐TX rats before and after l‐NAME administration. Reduced nicotamide adenine dinucleotide (NADPH) diaphorase activity was determined in heart and aorta of the TX group. The pressor response induced by l‐NAME was similar in all groups. l‐NAME‐induced‐tachycardia was associated with this rise in MAP. Adrenalectomy did not modify this chronotropic response, but it was attenuated by thyroidectomy. Thyroidectomy by itself decreased the circulating levels of T3 but it had no effect on the plasma levels of T4 and TSH. L and L‐TX groups showed similar levels of circulating T4 and TSH, meanwhile the plasma level of T3 decreased in the L group. Nitric oxide synthase (NOS) activity in atria as well as in aorta was greater in the TX group compared with C. When autonomic influences are removed, the thyroid gland modulates intrinsic heart rate via a mechanism that involves, at least in part, the nitric oxide pathway.