Andrea L. Fellet

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.

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.

Role of nitric oxide as a key mediator on cardiovascular actions of atrial natriuretic peptide in spontaneously hypertensive rats

The objective was to study atrial natriuretic peptide (ANP) effects on mean arterial pressure (MAP) and cardiovascular nitric oxide (NO) system in spontaneously hypertensive rats (SHRs), investigating the receptors and signaling pathways involved. In vivo, SHRs and Wistar-Kyoto (WKY) rats were infused with saline (0.05 ml/min) or ANP (0.2 microg.kg(-1).min(-1)) for 1 h. MAP and nitrites and nitrates excretion (NOx) were determined. NO synthase (NOS) activity and endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) NOS expression were measured in the heart and aorta. In vitro, heart and aortic NOS activity induced by ANP was determined in the presence of iNOS and nNOS inhibitors, natriuretic peptide receptor (NPR)-A/B blocker, G(i) protein, and calmodulin inhibitors. As a result, ANP diminished MAP and increased NOx in both groups. Cardiovascular NOS activity was higher in SHRs than in WKY rats. ANP increased NOS activity, but the activation was lower in SHRs than in WKY rats. ANP had no effect on NOS isoform expression. NOS activity induced by ANP was not modified by iNOS and nNOS inhibitors. NPR-A/B blockade blunted NOS stimulation via ANP in ventricle and aorta but not in atria. Cardiovascular NOS response to ANP was reduced by G(i) protein and calmodulin inhibitors in both groups. In conclusion, in atria, ventricle, and aorta, ANP interacts with NPR-C receptors, activating Ca(2+)-calmodulin eNOS through G(i) protein. In ventricle and aorta, NOS activation also involves NPR-A/B. The NOS response to ANP was impaired in heart and aorta of SHRs. The impaired NO-system response to ANP in hypertensive animals, involving alterations in the signaling pathway, could participate in the maintenance of high blood pressure in this model of hypertension.

Hypothyroidism: age-related influence on cardiovascular nitric oxide system in rats

This study investigates whether changes in nitric oxide (NO) production participate in the cardiovascular manifestations of hypothyroidism and whether these changes are age-related. Sprague-Dawley rats aged 2 and 18 months old were treated with 0.02% methimazole (wt/vol) during 28 days. Left ventricular function was evaluated by echocardiography. Measurements of arterial blood pressure, heart rate, nitric oxide synthase (NOS) activity and NOS/caveolin-1 and -3 protein levels were performed. Hypothyroidism enhanced the age-related changes in heart function. Hypothyroid state decreased atrial NOS activity in both young and adult rats, associated with a reduction in protein levels of the three NOS isoforms in young animals and increased caveolin (cav) 1 expression in adult rats. Ventricle and aorta NOS activity increased in young and adult hypothyroid animals. In ventricle, changes in NOS activity were accompanied by an increase in inducible NOS isoform in young rats and by an increase in caveolins expression in adult rats. Greater aorta NOS activity level in young and in adult Hypo rats would derive from the inducible and the endothelial NOS isoform, respectively. Thyroid hormones would be one of the factors involved in the modulation of cardiovascular NO production and caveolin-1 and -3 tissue-specific abundance, regardless of age. Hypothyroidism appears to contribute in a differential way to aging-induced changes in the myocardium and aorta tissues. Low thyroid hormones levels would enhance the aging effect on the heart. Age-related changes in NO production participate in the cardiovascular manifestations of hypothyroidism.

Endothelin-1 and -3 induce choleresis in the rat through ETB receptors coupled to nitric oxide and vagovagal reflexes

We have reported previously that centrally applied ET (endothelin)-1 and ET-3 induce either choleresis or cholestasis depending on the dose. In the present study, we sought to establish the role of these endothelins in the short-term peripheral regulation of bile secretion in the rat. Intravenously infused endothelins induced significant choleresis in a dose-dependent fashion, ET-1 being more potent than ET-3. Endothelins (with the exception of a higher dose of ET-1) did not affect BP (blood pressure), portal venous pressure or portal blood flow. ET-1 and ET-3 augmented the biliary excretion of bile salts, glutathione and electrolytes, suggesting enhanced bile acid-dependent and -independent bile flows. ET-induced choleresis was mediated by ET(B) receptors coupled to NO and inhibited by truncal vagotomy, atropine administration and capsaicin perivagal application, supporting the participation of vagovagal reflexes. RT (reverse transcription)-PCR and Western blot analysis revealed ETA and ET(B) receptor expression in the vagus nerve. Endothelins, through ET(B) receptors, augmented the hepatocyte plasma membrane expression of Ntcp (Na⁺/taurocholate co-transporting polypeptide; Slc10a1), Bsep (bile-salt export pump; Abcb11), Mrp2 (multidrug resistance protein-2; Abcc2) and Aqp8 (aquaporin 8). Endothelins also increased the mRNAs of these transporters. ET-1 and ET-3 induced choleresis mediated by ET(B) receptors coupled to NO release and vagovagal reflexes without involving haemodynamic changes. Endothelin-induced choleresis seems to be caused by increased plasma membrane translocation and transcriptional expression of key bile transporters. These findings indicate that endothelins are able to elicit haemodynamic-independent biological effects in the liver and suggest that these peptides may play a beneficial role in pathophysiological situations where bile secretion is impaired.

Nitric oxide synthase blockade and body fluid volumes

The influence of chronic nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on body fluid distribution was studied in male Wistar rats weighing 260-340 g. Extracellular, interstitial and intracellular spaces, as well as plasma volume were measured after a three-week treatment with L-NAME (approximately 70 mg/kg per 24 h in drinking water). An increase in extracellular space (16.1 +/- 1.1 vs 13.7 +/- 0.6 ml/100 g in control group, N = 12, P<0.01), interstitial space (14.0 +/- 0.9 vs 9.7 +/- 0.6 ml/100 g in control group, P<0.001) and total water (68.7 +/- 3.9 vs 59.0 +/- 2.9 ml/100 g, P<0.001) was observed in the L-NAME group (N = 8). Plasma volume was lower in L-NAME-treated rats (2.8 +/- 0.2 ml/100 g) than in the control group (3.6 +/- 0.1 ml/100 g, P<0.001). Blood volume was also lower in L-NAME-treated rats (5.2 +/- 0.3 ml/100 g) than in the control group (7.2 +/- 0.3 ml/100 g, P<0.001). The increase in total ratio of kidney wet weight to body weight in the L-NAME group (903 +/- 31 vs 773 +/- 45 mg/100 g in control group, P<0.01) but not in total kidney water suggests that this experimental hypertension occurs with an increase in renal mass. The fact that the heart weight to body weight ratio and the total heart water remained constant indicates that, despite the presence of high blood pressure, no modification in cardiac mass occurred. These data show that L-NAME-induced hypertension causes alterations in body fluid distribution and in renal mass.

Hypovolemic state: Involvement of nitric oxide in the aged related alterations of aquaporins-2 abundance in rat kidney

AIM To examine the effect of nitric oxide (NO) on the expression and/or localization of inner medulla collecting duct aquaporin-2 water channel (AQP2) in young and adult hemorrhaged anesthetized rats. METHODS Rats of 2 (young) and 12 mo (adult) old (n=15) were divided into: Sham animals with and without NG-nitro-l-arginine methyl ester (L-NAME) treatment (S L-NAME and S); hemorrhaged animals (20% blood loss) with and without L-NAME (H L-NAME and H). Mean arterial pressure (MAP) was continuously monitored and AQP2 expression and inmunolocalization were evaluated at 120 min after bleeding. RESULTS L-NAME blunted the hypotension induced by hemorrhage at 120 min in young (106+/-2 mm Hg) and adult (103+/-4 mm Hg) rats. AQP2 expression increased after bleeding in young (from 22 to 50 densitometric units) and adult rats (from 15 to 30 densitometric units). Pretreatment with L-NAME enhanced this effect, being this rise lower in adult than young animals (young: 318%, adult: 233%). Electron microscopy showed that AQP2 labeling increased after withdrawal, being the number of gold particles smaller in adult than young animals in the inner medulla. L-NAME enhanced this effect. CONCLUSION NOS activity decreases AQP2 expression/traffick in the inner collecting duct principal cells in response to hemorrhage and this effect is lower with aging.

Cardiac Mitochondrial Nitric Oxide: A Regulator of Heart Rate?

Alterations in autonomic control and myocardial nitric-oxide (NO) production are likely linked to the development and progression of heart dysfunction. By focusing on heart rate, the complexity of the actions of NO at distinct levels throughout the autonomic nervous system and its relationship with other regulators can be demonstrated. Given the multiple and opposing actions of NO on cardiac control, it is difficult to interpret a response after a global intervention in the NO system. The diversity of intracellular pathways activated by NO, and their differing sensitivities to different levels of NO, might account for some aspects of reported specific but opposite effects. We discuss factors that might contribute to this diversity of actions. A proper elucidation of the effects of NO on metabolic pathways and on energy generation could lead to novel therapeutic strategies aimed at the early treatment of heart dysfunction.

Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg) elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade) and complete autonomic blockade (38 +/- 3, 55 +/- 6, 54 +/- 5, 45 +/- 5 mmHg, respectively; N = 9, P = NS). L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 +/- 8, 38 +/- 5, 46 +/- 6 bpm, respectively; N = 9, P = NS). Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 3 bpm, P<0.05 vs intact animals, N = 9). Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9) caused a similar increase in blood pressure (45 +/- 5, 38 +/- 3, 44 +/- 9 mmHg, respectively; P = NS) and heart rate (31 +/- 4, 34 +/- 3, 35 +/- 4 bpm, respectively; P = NS). Addition of L-NAME (500 micro M) to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9). In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.