Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload.

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.

Regulation of Dopamine Uptake by Vasoactive Peptides in the Kidney.

Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na(+), K(+)-ATPase inhibition. Present results show that CNP did not affect either (3)H-dopamine uptake in renal tissue or Na(+), K(+)-ATPase activity; meanwhile, Ang-(1-7) was able to increase (3)H-dopamine uptake and decreased Na(+), K(+)-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na(+), K(+)-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on (3)H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on (3)H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on (3)H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na(+), K(+)-ATPase activity inhibition, contributing to its natriuretic and diuretic effects.

Role of angiotensin II and oxidative stress in renal inflammation by hypernatremia: benefits of atrial natriuretic peptide, losartan, and tempol.

The body regulates plasma sodium levels within a small physiologic range, despite large variations in daily sodium and water intake. It is known that sodium transport in the kidneys plays an important role in hypoxia, being the major determinant of renal oxygen consumption. Tubular epithelial cell hypoxia is an important contributor to the development of renal inflammation, and the damage may progress to structural injury, ending in acute renal failure. In this review, we will summarize the renal inflammatory effects of high acute plasma sodium (acute hypernatremia), and the molecular mechanisms involved. We will also discuss recent findings related to the role of oxidative stress and angiotensin II (Ang II) in the pathogenesis of renal injury. We will comment on the effects of agents used to prevent or attenuate the inflammatory response, such as the atrial natriuretic peptide, the superoxide dismutase mimetic - tempol, and losartan.

Signaling pathways involved in renal oxidative injury: role of the vasoactive peptides and the renal dopaminergic system.

The physiological hydroelectrolytic balance and the redox steady state in the kidney are accomplished by an intricate interaction between signals from extrarenal and intrarenal sources and between antinatriuretic and natriuretic factors. Angiotensin II, atrial natriuretic peptide and intrarenal dopamine play a pivotal role in this interactive network. The balance between endogenous antioxidant agents like the renal dopaminergic system and atrial natriuretic peptide, by one side, and the prooxidant effect of the renin angiotensin system, by the other side, contributes to ensuring the normal function of the kidney. Different pathological scenarios, as nephrotic syndrome and hypertension, where renal sodium excretion is altered, are associated with an impaired interaction between two natriuretic systems as the renal dopaminergic system and atrial natriuretic peptide that may be involved in the pathogenesis of renal diseases. The aim of this review is to update and comment the most recent evidences about the intracellular pathways involved in the relationship between endogenous antioxidant agents like the renal dopaminergic system and atrial natriuretic peptide and the prooxidant effect of the renin angiotensin system in the pathogenesis of renal inflammation.

Central insulin-angiotensin II interaction in blood pressure regulation in fructose overloaded rats.

The aim of the present study was to determine if insulin is able to modulate the pressor response to intracerebroventricularly administered angiotensin II in insulin resistant fructose overloaded rats. Male Sprague-Dawley rats were divided into two groups: 1) Control group (C) with tap water to drink for 6 weeks (n=36); and 2) fructose treated (F), with fructose solution (10% w/v) to drink for 6 weeks (n=36). On the day of the experiment, anesthetized male C and F rats were intracerebroventricularly infused with insulin (12 mU/h, n=15) or Ringer's solution as vehicle (n=15) for 2h. Immediately, changes in mean arterial pressure (MAP) in response to an intracerebroventricular subpressor dose of angiotensin II (5 pmol, n=10) or vehicle (n=5) were measured for 10 min. Then, hypothalami were removed and Akt and ERK1/2 phosphorylation levels were determined. In a subset of C (n=10) and F (n=20) animals, PD98059 (p44/42 MAPK inhibitor) or vehicle was administered intracerebroventricularly at a flow rate of 5 µl/min for 1 min. Ten minutes later, insulin (12 mU/h, n=5 for each group) or vehicle (Ringer's solution, only in the F group, n=5) was perfused for 2h at a flow rate of 4 µl/h, and cardiovascular parameters were measured every 15 min. Immediately, changes in MAP and HR in response to a subpressor dose of Ang II (5 pmol/2 µl) were evaluated for 10 min (n=5 for each group). In other subset of animals (n=6 for each group), AT1 and AT2 hypothalamic receptor levels were measured by Western blotting. Intracerebroventricular insulin pre-treatment increased the pressor response to angiotensin II in C rats. In F rats (with or without insulin pretreatment), the pressor response to angiotensin II was higher than that in vehicle pre-treated C animals, but similar to that observed in C after insulin infusion. In C rats phospho-ERK 1/2 hypothalamic levels significantly increased after angiotensin II injection in insulin pretreated animals compared to vehicle pre-treated rats, suggesting that MAPK activation might be involved in insulin potentiation of blood pressure response to angiotensin II in the brain. Phospho-ERK 1/2 hypothalamic levels were significantly increased in vehicle treated F rats compared to C, suggesting that basal MAPK activation might play a role in the enhanced response to angiotensin II observed in these animals. Finally, in F rats, either after vehicle or insulin infusion, angiotensin II injection was associated with a similar increase in phospho-ERK 1/2 hypothalamic levels, comparable to that observed after angiotensin II injection in insulin pre-treated C animals. ERK 1/2 blockade significantly reduced MAP in F rats compared to C. Moreover, ERK 1/2 inhibition completely abolished the Ang II pressor response in F rats and in insulin pre-treated C animals. All these findings suggest that insulin-angiotensin II interaction at hypothalamic level might be involved in the increase in blood pressure observed in the insulin resistant state.

Brain derived neurotrophic factor and neurotrophin-4 employ different intracellular pathways to modulate norepinephrine uptake and release in rat hypothalamus.

Classical actions of the neurotrophin family are related to cellular survival and differentiation. Moreover, acute effects of neurotrophins have been reported. Although neurotrophins effects on synaptic transmission at central nervous system level have been largely studied, acute effects of neurotrophins on hypothalamic noradrenergic transmission are still poorly understood. Thus, we have studied the effects of the neurotrophin family members nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) on norepinephrine (NE) neuronal uptake and its evoked release, as well as the receptor and the intracellular pathways involved in these processes in rat hypothalamus. Present results indicate that BDNF increased NE uptake and decreased its evoked release through a mechanism that involve Trk B receptor and phospholipase C. Moreover, NT-4, also through the Trk B receptor, decreased NE uptake and its evoked release by activating phosphatidylinositol 3-OH-kinase. These effects were observed in whole hypothalamus as well as in the anterior hypothalamic zone. On the other hand, NGF did not modify noradrenergic transmission. In conclusion, we showed for the first time that BDNF and NT-4 activate two different intracellular signalling pathways through a Trk B receptor dependent mechanism. Furthermore, present findings support the hypothesis that BDNF and NT-4 acutely applied, could be considered as modulators of noradrenergic transmission and thus may regulate hypothalamic physiological as well as pathophysiological responses.

Acute sodium overload produces renal tubulointerstitial inflammation in normal rats.

The aim of the present study was to determine whether acute sodium overload could trigger an inflammatory reaction in the tubulointerstitial (TI) compartment in normal rats. Four groups of Sprague-Dawley rats received increasing NaCl concentrations by intravenous infusion. Control (C): Na+ 0.15 M; G1: Na+ 0.5 M; G2: Na+ 1.0 M; and G3: Na+ 1.5 M. Creatinine clearance, mean arterial pressure (MAP), renal blood flow (RBF), and sodium fractional excretion were determined. Transforming growth factor beta1 (TGF-beta1), alpha-smooth muscle actin (alpha-SMA), RANTES, transcription factor nuclear factor-kappa B (NF-kappaB), and angiotensin II (ANG II) were evaluated in kidneys by immunohistochemistry. Animals with NaCl overload showed normal glomerular function without MAP and RBF modifications and exhibited a concentration-dependent natriuretic response. Plasmatic sodium increased in G2 (P < 0.01) and G3 (P < 0.001). Light microscopy did not show renal morphological damage. Immunohistochemistry revealed an increased number of ANG II-positive tubular cells in G2 and G3, and positive immunostaining for NF-kappaB only in G3 (P < 0.01). Increased staining of alpha-SMA in the interstitium (P < 0.01), TGF-beta1 in tubular cells (P < 0.01), and a significant percentage (P < 0.01) of positive immunostaining for RANTES in tubular epithelium and in glomerular and peritubular endothelium were detected in G3 > G2 > C group. These results suggest that an acute sodium overload is able 'per se' to initiate TI endothelial inflammatory reaction (glomerular and peritubular) and incipient fibrosis in normal rats, independently of hemodynamic modifications. Furthermore, these findings are consistent with the possibility that activation of NF-kappaB and local ANG II may be involved in the pathway of this inflammatory process.

Salivary glands and noradrenergic transmission in diabetic rats.

1 Type 2 diabetes is associated with diverse oral pathologies in which salivary flow reduction is one of the causes of these oral abnormalities. Scarce literature exists regarding noradrenergic transmission and adrenergic-induced salivary flow in submaxillary and parotid glands of type 2 diabetic rats. 2 We studied noradrenergic transmission as well as the secretory response to alpha1- and beta-adrenoceptor stimulation in the parotid and submaxillary glands of type 2 diabetic rats. 3 Diabetic rats exhibited diminished neuronal uptake, release and endogenous content of noradrenaline (NE) in both salivary glands. Further, NE synthesis was also diminished accompanied by decreased tyrosine hydroxylase activity. Salivary flow responses to alpha1-(methoxamine) and beta-(isoprenaline) adrenoceptor stimulation were reduced in the submaxillary as well as the parotid glands of diabetic rats. 4 Our results suggest that the reduction of noradrenergic transmission in the salivary glands of type 2 diabetic rats is in part responsible for the diminished salivary flow evoked by alpha1- and beta-adrenergic stimulation. Reduced noradrenergic activity may contribute to the pathophysiology of oral abnormalities in diabetic patients.

Centrally applied atrial natriuretic factor diminishes bile secretion in the rat.

Little is known about the role of centrally applied peptides in the regulation of bile secretion. We previously reported that the intravenous injection of atrial natriuretic factor (ANF) reduces bile acid dependent flow without affecting portal venous pressure in the rat. In the present work, we studied the effects of centrally applied ANF on bile secretion and the possible pathways involved. Rats were cannulated in the brain lateral ventricle for the administration of 1, 10 and 100 ng/microl ANF. After 1 week, the common bile duct was cannulated and bile samples were collected every 15 min for 60 min after the administration of ANF. The excretion rate of various biliary components was assessed. Bile secretion experiments were also performed after bilateral truncal vagotomy or atropine administration to evaluate the participation of a vagal pathway. In addition, the role of the sympathetic system was addressed by combined administration of propranolol and phentolamine. Centrally applied ANF did not modify blood pressure but diminished bile flow and bile acid output. It also reduced sodium and potassium secretion but did not modify protein or phospholipid excretion. Neither bilateral truncal vagotomy nor atropine administration abolished ANF response. Furthermore, combined administration of adrenergic antagonists did not alter ANF inhibitory effect on bile flow. In conclusion, centrally applied ANF reduced bile acid dependent flow not through a vagal or adrenergic pathway in the rat, suggesting the involvement of a peptidergic pathway.

Behavior of atrial natriuretic factor in an experimental model of Trypanosoma cruzi infection in rats.

Enhanced atrial natriuretic factor (ANF) production by the heart is related to hemodynamic overload, cardiac growth, and hypertrophy. The heart is one of the most affected organs during Trypanosoma cruzi infection. We tested the hypothesis that myocarditis produced by parasite infection alters the natriuretic peptide system by investigating the behavior of plasma ANF during the acute and chronic stages of T. cruzi infection in rats. Sprague-Dawley rats were infected with T. cruzi clone Sylvio-X10/7. Cardiac morphology showed damage to myocardial cells and lymphocyte infiltration in the acute phase; and fibrosis and cell atrophy in the chronic period. Plasma ANF levels (radioimmunoassay) were significantly higher in acute (348 +/- 40 vs. 195 +/- 36 pg/ml, P < 0.05, n = 17) and chronic T. cruzi myocarditis (545 +/- 81 vs. 229 +/- 38 pg/ml, P < 0.001, n = 11) than in their respective controls (n = 10 and 14). Rats in the chronic phase also showed higher levels than rats in the acute phase (P < 0.01). The damage of myocardial cells produced by the parasite and the subsequent inflammatory response could be responsible for the elevation of plasma ANF during the acute period of T. cruzi infection. The highest plasma ANF levels found in chronically infected rats could be derived from the progressive failure of cardiac function.

B-Type and C-type natriuretic peptides modify norepinephrine uptake in discrete encephalic nuclei of the rat.

1. We previously demonstrated that atrial natriuretic factor and B- and C-type natriuretic peptides (ANF, BNP, and CNP, respectively) modified catecholamine metabolism by increasing the neuronal uptake and decreasing the neuronal release of norepinephrine in the rat hypothalamus. The aim of the present work was to study the effects of natriuretic peptides BNP and CNP on norepinephrine uptake as an index of the amine metabolism in discrete areas and nuclei of the central nervous system (CNS) of the rat. 2. Experiments were carried out in vitro using the punchout technique in diverse areas and nuclei of rat CNS. Results showed that 100 nM BNP and 1 nM CNP increased norepinephrine (NE) uptake in all brain areas and nuclei studied. 3. Present results permit us to conclude that BNP and CNP regulate NE metabolism independently of the encephalic area or nucleus involved. In fact, NE uptake increased in nuclei related to the regulation of cardiovascular activity as well as nuclei associated with endocrine metabolism and hydrosaline homeostasis. These observations suggest that BNP and CNP may be involved in the regulation of these physiological processes in an indirect manner through modifications of noradrenergic neurotransmission. Present findings provide further support to the hypothesis that CNP would be the main natriuretic peptide in brain. Furthermore, previous as well as present results support the role of the natriureic peptides as neuromodulators of noradrenergic transmission at the presynaptic level.

Angiotensin-(1-7) does not affect norepinephrine neuronal uptake or catabolism in rat hypothalamus and atria.

1. Since we previously reported that angiotensin-(1-7) [Ang-(1-7)] increases or inhibits norepinephrine (NE) release in rat atria or hypothalamus, respectively, the present work was undertaken to investigate the effect of the heptapeptide on NE neuronal uptake and metabolism in atria and hypothalamus isolated from rats. 2. Ang II (1-10 microM) caused a decrease in neuronal NE uptake in both atria and hypothalami isolated from rats. On the contrary, tissues incubated with [3H]NE in the presence of 0.1-10 microM Ang-(1-7) showed no modification in [3H]NE content with respect to the control group, suggesting that the heptapeptide did not modify [3H]NE neuronal uptake. 3. To study the effect of the heptapeptide on NE catabolism, monoamine-oxidase (MAO) and catechol-O-methyltransferase (COMT) activities were determined. Pretreatment of the tissue with Ang-(1-7) (0.1-1.0 microM) showed a tendency to diminish MAO activity in rat atria, while no significant changes were observed in hypothalamic MAO activity. Moreover, the heptapeptide (0.1-1.0 microM) did not affect central COMT activity with respect to the control group. 4. Present results allow us to conclude that Ang-(1-7) interacts with noradrenergic neurotransmission by increasing or inhibiting NE release at the peripheral and central levels, respectively, without affecting either the neurotransmitter neuronal uptake or catabolism.

Central natriuretic peptides regulation of peripheral atrial natriuretic factor release.

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP) receptors have been described in encephalic areas and nuclei related to the regulation of cardiovascular as well as sodium and water homeostasis. Stimulation of the anterior ventral third ventricular region of the brain modifies plasma ANF concentration, suggesting the participation of the central nervous system in the regulation of circulating ANF. The aim of this work was to study the effect of centrally applied ANF or CNP on plasma ANF. Normal and blood volume expanded rats (0.8 ml isotonic saline/100 g body weight) were intra cerebralventricularly injected with 1, 10 or 100 ng/microl/min ANF. Blood volume expanded animals were also centrally injected with the same doses of CNP. Blood samples were collected at 5 and 15 min. after intracerebralventricular administration of either ANF or CNP. Centrally applied ANF did not affect circulating ANF in normal blood volume rats. In blood volume expanded animals both ANF (1, 10 or 100 ng/microl/min) and CNP (1 ng/microl/min) decreased plasma ANF concentration after 15 min. Moreover, CNP (10 and 100 ng/microl/min) lowered circulating ANF levels not only at 15 min but also at 5 min. Neither ANF nor CNP elicited any change in mean arterial pressure and heart rate in normal and blood volume expanded rats. These results suggest the existence of a central regulation exerted by natriuretic peptides on circulating ANF levels. Furthermore, this is the first study reporting an effect on plasma ANF induced by centrally applied CNP.

Angiotensin-(1-7) reduces norepinephrine release through a nitric oxide mechanism in rat hypothalamus.

Angiotensin (Ang)-(1-7) elicits a facilitatory presynaptic effect on peripheral noradrenergic neurotransmission, and because biological responses to the heptapeptide on occasion are tissue specific, the present investigation was undertaken to study its action on noradrenergic neurotransmission at the central level. In rat hypothalamus labeled with [(3)H]-norepinephrine, 100 to 600 nmol/L Ang-(1-7) diminished norepinephrine released by 25 mmol/L KCl. This effect was blocked by the selective angiotensin type 2 receptor antagonist PD 123319 (1 micromol/L) and by the specific Ang-(1-7) receptor antagonist ([D-Ala(7)]Ang-(1-7) (1 micromol/L) but not by losartan (10 nmol/L to 1 micromol/L), a selective angiotensin type 1 receptor antagonist. The inhibitory effect on noradrenergic neurotransmission caused by Ang-(1-7) was prevented by 10 micromol/L N(omega)-nitro-L-arginine methylester, an inhibitor of nitric oxide synthase activity, and was restored by 100 micromol/L L-arginine, precursor of nitric oxide synthesis. Methylene blue (10 micromol/L), an inhibitor of guanylate cyclase considered as the target of nitric oxide action, as well as Hoe 140 (10 micromol/L), a bradykinin B(2)-receptor antagonist, prevented the inhibitory effect of the heptapeptide on neuronal norepinephrine release, whereas no modification was observed in the presence of 0.1 to 10 micromol/L indomethacin, a cyclooxygenase inhibitor. Our results indicate that Ang-(1-7) has a tissue-specific neuromodulatory effect on noradrenergic neurotransmission, being inhibitory at the central nervous system by a nitric oxide-dependent mechanism that involves angiotensin type 2 receptors and local bradykinin production.

Increased plasma atrial natriuretic factor in catecholamine-producing tumor patients.

The aim of this study was to evaluate plasma levels of ANF in patients with catecholamine-secreting tumors with and without hypertension and to relate ANF secretion to levels of plasma and urinary catecholamines and blood pressure. Twenty-one pheochromocytoma (15 with sustained, 6 with paroxysmal hypertension), 6 neuroblastoma (1 hypertensive) patients and 28 aged-matched controls were studied in basal conditions. Plasma and urinary norepinephrine (NE),epinephrine (E), dopamine (DA) and DOPA were determined by HPLC-ED and plasma ANF by RIA. Both neuroblastoma and pheochromocytoma patients had significantly higher plasma ANF levels than controls. Neuroblastomas showed higher ANF concentration than pheochromocytomas. No differences were found in plasma ANF between hypertensive and normotensive patients. Pheochromocytomas with ANF levels within the normal range had plasma and urinary NE and urinary DA and DOPA levels significantly higher than patients with high ANF. Plasma ANF levels were unrelated to systolic or diastolic blood pressure or heart rate. A negative correlation between plasma ANF and urinary DA was found only in the patients groups. In conclusion, plasma ANF was increased in pheochromocytoma and neuroblastoma patients. Our data suggest that the excessive catecholamine secretion is not responsible for the increased ANF secretion in these patients. The significance of the relationships among plasma ANF and urinary and plasma catecholamines requires further investigation.

Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus.

We have previously reported that atrial natriuretic factor (ANF) increased neuronal norepinephrine (NE) uptake and reduced basal and evoked neuronal NE release. Changes in NE uptake and release are generally associated to modifications in the synthesis and/or turnover of the amine. On this basis, the aim of the present work was to study ANF effects in the rat hypothalamus on the following processes: endogenous content, utilization and turn-over of NE; tyrosine hydroxylase (TH) activity; cAMP and cGMP accumulation and phosphatidylinositol hydrolysis. Results showed that centrally applied ANF (100 ng/microl/min) increased the endogenous content of NE (45%) and diminished NE utilization. Ten nM ANF reduced the turnover of NE (53%). In addition, ANF (10 nM) inhibited basal and evoked (with 25 mM KCl) TH activity (30 and 64%, respectively). Cyclic GMP levels were increased by 10 nM ANF (100%). However, neither cAMP accumulation nor phosphatidylinositol breakdown were affected in the presence of 10 nM ANF. The results further support the role of ANF in the regulation of NE metabolism in the rat hypothalamus. ANF is likely to act as a negative putative neuromodulator inhibiting noradrenergic neurotransmission by signaling through the activation of guanylate cyclase. Thus, ANF may be involved in the regulation of several central as well as peripheral physiological processes such as cardiovascular function, electrolyte and fluid homeostasis, endocrine and neuroendocrine synthesis and secretion, behavior, thirst, appetite and anxiety that are mediated by central noradrenergic activity.

Atrial natriuretic factor does not affect norepinephrine catabolism in rat hypothalamus and adrenal medulla.

We have previously reported that atrial natriuretic factor (ANF) increases neuronal uptake and endogenous content of norepinephrine (NE) and diminishes neuronal release, synthesis and turn-over of NE in rat hypothalamus and adrenal medulla. The aim of the present work was to study another aspect of NE metabolism and therefore investigate the possible effects of ANF on NE catabolism. The determination of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) activity and deaminates metabolites formation were studied in vitro in rat hypothalamus and adrenal medulla slices. Results showed that, in the hypothalamus, 100 nM ANF diminished MAO activity while 10 nM ANF did not modify the enzyme activity. Conversely, 10 and 100 nM ANF reduced MAO activity in adrenal medulla. On the other hand, the atrial factor modified neither COMT activity nor the formation of deaminates metabolites in the hypothalamus and adrenal medulla. Present results as well as previous findings support a putative role for ANF in the modulation of NE metabolism not only in the hypothalamus but also in the adrenal medulla of the rat, affecting the storage, release and uptake of NE but not its catabolism.

Atrial natriuretic factor in two kidney--two clip renovascular hypertension in the rat.

Hig levels of circulating atrial natriuretic factor (ANF) have been reported in several physiopathologic conditions like hypertension, heart and renal failure, pregnancy and high sodium intake. Nevertheless, neither relationships with water-sodium space regulation nor the role of an ANF vascular relaxant effect have been yet defined. The aim of present experiments was to characterize the contribution of circulating ANF and its vascular relaxing effects in the two kidney-two clip (2K2C) experimental model of renovascular hypertension. Complementary, plasma metabolites nitrite/nitrate of nitric oxide (NO) was examined because of mediation for both (NO an ANF) through cGMP. Three results showed (two-four weeks after surgery): indirect systolic blood pressure (mmHg), 186 +/- 4 in HT and 122 +/- 1 in SH (p < 0.001); a significant increase of plasma ANF (fmol/ml) in HT (n = 7, 1221 +/- 253) vs. SH (n = 9, 476 +/- 82; p < 0.02). Nitrate/nitrite plasma concentrations (mumol/l) were mpt different between SH and. The relaxant effect of ANF (10(-9), 10(-8) and 10(-7) M) on phenylephrine (3,5 x 10(-6) M) contracted rings from HT rats was smaller than SH rats (10(-8) M, p < 0.05). Contractions to phorbol 12, 13-dibutyrate (seven weeks after surgery) were significantly higher in rings from HT rats (p < 0.001). We conclude: 1) in addition to decreased granularity in atrial myocardiocytes, high circulating values of ANF here described suggest an increased turnover of the peptide in 2K2C hypertensive rats; 2) lower significant vascular relaxant effects in HT rats would indicate down regulation of ANF receptors in this model; the latter would derive from high plasma ANF concentration and, tentatively, because of greater activity of protein kinase C in the vascular wall; 39 similar values of plasma nitrite/nitrate in SH and HT rats would indicate a comparable NO circulating availability in both groups.

Atrial natriuretic factor-induced amylase output in the rat parotid gland appears to be mediated by the inositol phosphate pathway.

In previous in vivo studies we have reported that atrial natriuretic factor enhanced induced salivary secretion and increased isoproterenol-induced amylase release in the rat suggesting that, ANF effect could be mediated by phosphatidylinositol hydrolysis. In the present work, the effect of ANF on rat parotid tissue incubated in vitro was investigated with the aim to assess whether the phosphoinositol pathway was involved in ANF intracellular signaling in the parotid gland. Results showed that ANF induced a dose dependent increase in amylase fractional release, which was lower than that evoked by any concentration of isoproterenol. Furthermore 100 nM ANF enhanced isoproterenol-evoked amylase release. The effect of ANF was not affected in the presence of propranolol suggesting the noninvolvement of the beta adrenergic receptor, which is the main stimulus for the output of the enzyme in the parotid gland. However, ANF increased phosphatidylinositol hydrolysis, which implies an increase in intracellular calcium, which is necessary for the achievement of maximal response in amylase release. This effect was abolished in the presence of neomycin supporting ANF direct stimulation of phospholipase C. These results suggest the involvement of the C type natriuretic peptide receptor coupled to phospholipase C in ANF evoked amylase release and ANF enhancement of the isoproterenol-induced output of the enzyme.