RESUMO
1. To assess the involvement of endothelial alpha(2)-adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min(-1)). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA. 2. In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 microM L(omega)-nitro-L-arginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation. 3. In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123+/-13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, L(omega)-nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten microM ODQ obliterated the clonidine-induced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 - 100 nM sildenafil increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release. 4. alpha(2)-Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential alpha(2D)-adrenoceptor activation, the K(B)s for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively. 5. Rat pretreatment with 100 mg kg(-1) 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9+/-15.5 pmol NO. Perfusion with 1 microM guanethidine or 1 microM guanethidine plus 1 microM atropine did not modify the NO surge evoked by 100 nM clonidine. 6. Clonidine and congeners activate endothelial alpha(2D)-adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Clonidina/farmacologia , Óxido Nítrico/fisiologia , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , 3',5'-GMP Cíclico Fosfodiesterases , Acetilcolina/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Técnicas In Vitro , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Óxido Nítrico/metabolismo , Nitroarginina/farmacologia , Oxidiazóis/farmacologia , Oxidopamina/farmacologia , Fenilefrina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Piperazinas/farmacologia , Purinas , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 2/metabolismo , Saponinas/farmacologia , Citrato de Sildenafila , Solubilidade , Sulfonas , Simpatolíticos/farmacologia , Fatores de Tempo , Resistência Vascular , Vasodilatadores/farmacologia , Ioimbina/farmacologiaRESUMO
The purpose of this study was to assess the participation of the atrial natriuretic peptide (ANP)-cGMP system in electrolyte and volume handling of cholestatic rats submitted to an acute oral sodium load. Cholestasis was induced by ligation and section of the common bile duct (n = 51). Control rats were sham operated (n = 56). Three weeks after surgery, 24-hr urinary volume, sodium, potassium, cGMP and creatinine excretion were measured. Three days later, animals received 10 mmol/kg NaCl (1 M) by gavage, and urinary excretion was measured for 6 hr. In parallel groups of rats, plasma volume, electrolytes and ANP concentration, extracellular fluid volume (ECFV), and renal medullary ANP-induced cGMP production were determined in basal conditions or 1 hr after oral sodium overload. As compared with controls, cholestatic rats had a larger ECFV and higher plasma ANP (67.2 +/- 5.2 vs 39.7 +/- 3.5 pg/ml), but lower hematocrit and blood volume, and were hyponatremic. Cholestatic rats showed higher basal excretion of sodium, potassium, and volume than controls, but equal urinary cGMP. After the NaCl overload, cholestatic rats showed a reduced sodium excretion but equal urinary cGMP. One hr after sodium overload, both groups showed hypernatremia, but whereas in control rats ECFV and ANP increased (50.7 +/- 4.1 pg/ml), in cholestatic rats ECFV was unchanged, and plasma volume and ANP were reduced (37.5 +/- 5.8 pg/ml). ANP-induced cGMP production in renal medulla was similar in cholestatic and control nonloaded rats (14.2 +/- 5.2 vs 13.4 +/- 2.6 fmol/min/mg). One hr after the load, medullary cGMP production rose significantly in both groups, without difference between them (20.6 +/- 3.1 vs 22.7 +/- 1. 7 fmol/min/mg). We conclude that the blunted excretion of an acute oral sodium load in cholestatic rats is associated with lower plasma ANP due to differences in body fluid distribution and cannot be explained by renal refractoriness to ANP.
Assuntos
Fator Natriurético Atrial/fisiologia , Colestase/fisiopatologia , Natriurese , Sódio/administração & dosagem , Animais , Fator Natriurético Atrial/sangue , Ductos Biliares/cirurgia , Volume Sanguíneo , Creatinina/urina , GMP Cíclico/análise , GMP Cíclico/urina , Diurese , Feminino , Hematócrito , Medula Renal/química , Ligadura , Potássio/urina , Ratos , Ratos Sprague-Dawley , Sódio/sangue , UrinaRESUMO
We have shown previously that the kininogen-derived peptides bradykinin, prokinins, and PU-D1, given intravenously or into the duodenal lumen, block the atrial natriuretic peptide (ANP)-induced diuretic-natriuretic effect in fasting, anesthetized rats infused with isotonic glucose. HOE-140, an inhibitor of bradykinin B2 receptors, completely suppresses this ANP blockade. When intravenous glucose infusion is omitted, the above-described inhibition of ANP does not take place. Therefore, to clarify the role of glucose and/or feeding in this phenomenon, we used fasted, anesthetized rats to test how the ANP excretory response was affected by (1) short-term feeding before anesthesia, (2) 1 mL of isotonic glucose introduced into the stomach, and (3) the interaction of HOE-140 with these treatments. In addition, we tested the effects of 1 mL of intragastric glucose administration and HOE-140 on urinary excretion in awake rats. In anesthetized rats, both glucose administration and feeding significantly inhibited the diuretic-natriuretic effect of ANP for up to 90 minutes. Similarly, intragastric glucose delayed spontaneous sodium and water excretion for 90 minutes in awake rats. In all 3 cases, pretreatment with HOE-140 (2.5 microg IV) fully prevented the inhibition of ANP excretory action, ruling out osmotic effects as the cause of reduced diuresis. These results indicate that the presence of glucose in the digestive tract triggers an inhibitory effect on ANP renal actions that requires activation of kinin B2 receptors, providing strong support to our hypothesis that during the early prandial period, gastrointestinal signals elicit a transient blockade of renal excretion with a mechanism involving the kallikrein-kinin system.
Assuntos
Fator Natriurético Atrial/fisiologia , Glucose/farmacologia , Cininas/fisiologia , Natriurese/fisiologia , Receptores da Bradicinina/fisiologia , Animais , Ingestão de Alimentos , Jejum , Feminino , Masculino , Natriurese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor B2 da BradicininaRESUMO
To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 +/- 7 mmHg, accompanied by peaks of 23 +/- 3 pmol NE, 445 +/- 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 microM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 microM N(omega)-nitro-L-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 microM guanethidine or alpha1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. alpha2-Adrenoceptor blockade with 1 microM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.
Assuntos
Endotélio Vascular/fisiologia , Artérias Mesentéricas/fisiologia , Mesentério/irrigação sanguínea , Óxido Nítrico/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Artérias Mesentéricas/inervação , Mesentério/inervação , Mesentério/fisiologia , Nitroarginina/farmacologia , RatosRESUMO
We aimed at characterizing the receptor subtype and the signaling pathway involved in the inhibitory effect of neuropeptide Y on the release of endogenous noradrenaline from rat hypothalamus. Slices of hypothalamus were stimulated with two trains of electrical pulses, and the release of noradrenaline and nitric oxide was measured. The electrical stimulation of hypothalamic slices induced a consistent release of both endogenous noradrenaline and NO. Neuropeptide Y inhibited concentration dependently the stimulated noradrenaline release. Similarly, agonists for neuropeptide Y Y1, Y2 and Y5 receptors inhibited noradrenaline release, albeit with a potency lower than neuropeptide Y. GW1229, a selective neuropeptide Y Y1 receptor antagonist counteracted the effect of neuropeptide Y, but not that of PYY-(3-36), an agonist active at neuropeptide Y Y5 and Y2 receptors. These results indicate that the inhibitory effect of neuropeptide Y is likely mediated by several receptor subtypes, including neuropeptide Y Y1, Y5 and possibly Y2 receptors. One microM NPY significantly enhanced NO release induced by the electrical stimulation. NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, abolished NO release and blocked the inhibitory effect of neuropeptide Y on noradrenaline release. We conclude that nitric oxide participates in the signaling pathway of neuropeptide Y in the rat hypothalamus.
Assuntos
Hipotálamo/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Óxido Nítrico/metabolismo , Norepinefrina/metabolismo , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Animais , Estimulação Elétrica , Hipotálamo/metabolismo , Masculino , Oligopeptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismoRESUMO
This paper narrates Dr Héctor R Croxatto and collaborators' efforts over the past 50 years in search for peptidic hormones obtained by pepsin hydrolysis of blood plasma substrates. In the forties, Croxatto described three peptidic fractions characterized by their hypertensive, oxytocic and antidiuretic properties, designated as pepsitensin, pepsitocin and pepsanurin, respectively. While pepsitensin and pepsitocin were later identified as angiotensin I and metlys-bradykinin, pepsanurin was not identified and its research was halted for 35 years. During that time, Prof Croxatto and his group worked mostly on the renal kallikrein-kinin system, studying its physiological anti-hypertensive role, making significant contributions in the field of renovascular hypertension. After the discovery of atrial natriuretic peptide, Croxatto resumed his work with pepsanurin. In a series of papers from 1988 to 1998, it was shown that: 1) when injected intraperitoneally or in the intestinal lumen of anesthetized rats, or in the isolated perfused rat kidneys, pepsanurin is a potent inhibitor of the natriuretic effect of ANP; 2) plasma kininogens are identified as the substrates for pepsanurin formation; 3) bradykinin and prokinins exert the anti-ANP effect when injected either intravenously, intraperitoneally or intraduodenally, at small non-vasodilator doses; endogenous kinins also block ANP renal excretory effects; 4) a 20-amino acid peptide released by pepsin from domain 1 of purified LMW kininogen was isolated by Croxatto and collaborators, designed as PU-D1, and shown to exert similar anti-ANP effects as pepsanurin or kinins, but being more potent and longer lasting; 5) the anti-ANP effect of pepsanurin, kinins and PU-D1 is mediated by B2 kinin receptors, since it is blocked by a bradykinin receptor antagonist. Currently, Dr Croxatto is working on the hypothesis that intestinal-borne kinins and/or PU-D1 may reduce renal excretion during the prandial cycle.
Assuntos
Inibidores de Cisteína Proteinase , Cininogênios , Peptídeos , Animais , Fator Natriurético Atrial , Inibidores de Cisteína Proteinase/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal/metabolismo , Calicreínas , Rim/metabolismo , Cininogênios/farmacologia , Cininas , Pepsina A , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , RatosRESUMO
In anesthetized rats, the renal excretory actions of atrial natriuretic peptide (ANP) are inhibited by intravenous or intraperitoneal injections of bradykinin. To elucidate the mechanisms underlying this inhibition, we evaluated bradykinin effects on: i- ANP-induced natriuresis and diuresis in isolated perfused rat kidneys, and ii- ANP-induced cGMP production in rat renal medulla in vitro. In perfused kidneys, 1 microgram bradykinin completely inhibited the diuretic and natriuretic responses elicited by 0.5 microgram ANP, without changes in perfusion pressure. The inhibitory effects of bradykinin were abolished by HOE-140, a kinin-B2 receptor antagonist. Bradykinin alone had no effect on urinary excretion or perfusion pressure. Incubation with ANP (0.1 nM to 1 microM) increased renal medullary cGMP content up to 30-fold, in a concentration-dependent fashion. Medullary cGMP was moderately increased by the nitric oxide donor, sodium nitroprusside (1 microM), but it was unchanged by bradykinin (0.1 nM-0.1 microM). Despite this, ANP-induced cGMP production was significantly enhanced by co-incubation with low concentrations of bradykinin (up to 0.1 nM). In contrast, ANP-induced cGMP accumulation was unchanged by concentrations of 1 nM bradykinin or higher. In the presence of 100 nM HOE-140, bradykinin (0.1-1 nM) did not affect ANP-induced cGMP production. These results demonstrate that bradykinin counteracts ANP-stimulated sodium and water excretion, by acting directly on the kidney. The interaction between both peptides is complex; our data suggest that renal medullary ANP receptors are subjected to an on/off modulation by fluctuating bradykinin concentrations.
Assuntos
Fator Natriurético Atrial/efeitos dos fármacos , Bradicinina/farmacologia , GMP Cíclico , Rim/metabolismo , Natriurese/efeitos dos fármacos , Animais , Fator Natriurético Atrial/antagonistas & inibidores , Fator Natriurético Atrial/biossíntese , GMP Cíclico/biossíntese , Técnicas In Vitro , Medula Renal/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Héctor R. Croxatto, M.D., has recently celebrated his 90th anniversary, fully active in scientific research and in other academic activities. Along his productive life, he has contributed with original observations on the role of certain peptides in the pathogenesis of arterial hypertension. Furthermore, he has been a leader in the development of biological research in Chile and the mentor or teacher of several prominent disciples who have had, or are having, their own brilliant careers in medicine and in science in Chile and other Latinamerican countries. Although most of his scientific productivity has been published in top journals in the international field, several of his papers have been published in Revista Médica de Chile. In this issue, the journal pays a tribute to this outstanding scholar, his exceptional personality and fruitful academic career. The tribute includes this Editorial, a Letter to the Editor and its reply, and his most recent manuscript, all testifying the exceptional virtues of a great man.
Assuntos
Disciplinas das Ciências Biológicas/história , Chile , História do Século XX , Pesquisa/históriaRESUMO
This paper describes long term research efforts which have lead: 1) to the identification of peptides present in pepsanurin, a peptidic fraction obtained by pepsin hydrolysis of plasma globulins that inhibits the renal excretory action of atrial natriuretic peptide (ANP) and 2) to the discovery of an unexpected role of glucose, as a requisite for these inhibitory effects. The active peptides identified in pepsanurin are derived from plasma kininogens, substrates of the kallikrein-kinin system. Pro-kinins of 15, 16 and 18 amino acids, and bradykinin itself, block ANP-induced diuresis and natriuresis when injected i.v., i.p. or into the duodenal lumen of anesthetized rats in picomol doses. Furthermore, a novel 20 amino acids fragment derived from kininogen dominium-1, named PU-D1, is the most potent and longer lasting blocker of ANP renal effects. The anti-ANP effects of those peptides are prevented by B2-kinin receptor antagonists. The inhibition of ANP by kinins and PU-D1 was evident only in rats infused with isotonic glucose; whereas the excretory effect of ANP was not affected in fasted rats not infused, or infused with saline. These findings provide evidence that glucose facilitates liquid retention through a kinin-mediated inhibition of ANP excretory action that may be related to the prandial cycle.