Clonidine-induced nitric oxide-dependent vasorelaxation mediated by endothelial alpha(2)-adrenoceptor activation.

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.

In vivo assessment of microvascular nitric oxide production and its relation with blood flow.

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.

Reduced natriuresis after oral sodium load in cholestatic rats: role of compartment volumes and ANP.

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.

Stimulation of NO production and of eNOS phosphorylation in the microcirculation in vivo.

The role of nitric oxide (NO) in microvascular permeability is controversial, in part because the regulation of its endothelial constitutive synthase, eNOS, has been studied in vitro but not in vivo. Our study was designed to detect the morphologic and functional presence of eNOS and to test whether eNOS could be phosphorylated by platelet-activating factor (PAF), an agent that induces hyperpermeability. Immunocytochemistry was applied using human anti-eNOS antibodies in the hamster cheek pouch (hcp). The hcp microvessels demonstrated positive reaction products in the endothelium. The functional presence of eNOS in hcp was investigated by topical application of 10(-7) M PAF to the hcp and by measuring NO production by chemiluminescence. The mean baseline value of NO release was 63.3 +/- 6.9 pmol/ml (mean +/- SE). Application of PAF led to an increase in mean NO release to 120.8 +/- 31.2 pmol/ml (P < 0.05). In another series of experiments, 10(-7) M PAF was applied topically to hcp preincubated with [(32)P]orthophosphoric acid. Immunoprecipitation and Western blots detected (32)P-labeled bands that migrated with the mobility of positive eNOS indicating phosphorylated eNOS protein. The intensity of the radioactive bands was evaluated by computer-assisted image analysis. Comparison of the net band intensities yielded a mean PAF-treated/control ratio of 1.6 +/- 0.1. Our data demonstrate the morphologic and functional presence of eNOS in the microcirculation. The data also provide evidence that the function of microvascular eNOS is subject to regulation by phosphorylation.

Kinin B2 receptors mediate blockade of atrial natriuretic peptide natriuresis induced by glucose or feeding in fasted rats.

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.

Rise in endothelium-derived NO after stimulation of rat perivascular sympathetic mesenteric nerves.

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.

Neuropeptide Y induced inhibition of noradrenaline release in rat hypothalamus: role of receptor subtype and nitric oxide.

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.

Croxatto's fifty-year pursuit: from pepsanurin to the discovery of a new kininogen-derived peptide (PU-D1).

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.

Interactions between bradykinin and ANP in rat kidney in vitro: inhibition of natriuresis and modulation of medullary cyclic GMP.

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.

[A tribute to professor Héctor Croxatto Rezzio: setting up science in Chile. "Nature is always more amazing than our imagination can conceive"]. / Homenaje al Doctor Héctor Croxatto Rezzio: instalando la ciencia en Chile. "La naturaleza siempre es más maravillosa que lo que imaginamos".

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.

[Glucose in conjunction with peptides derived from kininogens might act from digestive tract as blockers of atrial natriuretic peptide mediated diuresis-natriuresis]. / La glucosa en conjunto con péptidos derivados de cininógenos podrían actuar desde el aparato digestivo como bloqueadores de la acción diurética-natriurética del péptido natriurético auricular.

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.

Kinins mediate the inhibition of atrial natriuretic peptide diuretic effect induced by pepsanurin.

Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B2 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 microgram i.v.) given before and after i.p. injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/kg), and bradykinin (5-20 micrograms/kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 micrograms, i.v.) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 micrograms/kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.

A peptide released by pepsin from kininogen domain 1 is a potent blocker of ANP-mediated diuresis-natriuresis in the rat.

A 20-amino acid peptide, KYEIKEGDCPVQSGKTWQDC (PU-D1), released by pepsin hydrolysis of LMW kininogen domain 1 was tested for its ability to antagonize the diuretic and natriuretic effect of ANP(103-125) in anesthetized rats. A single dose of 10.8 or 21.6 pmol (25 or 50 ng) PU-D1 given intravenously or into the duodenal lumen suppressed the diuresis-natriuresis induced by 209 pmol (500 ng) ANP by 43% to 59% and 69% to 96%, respectively. None of the doses tested (2.16 to 432 pmol, 5 ng to 1 microg) modified systemic blood pressure. Strikingly, a single IV dose of 10.8 pmol PU-D1 blocked the action of ANP for more than 3 hours. ANP blockade by PU-D1 was annulled completely by the bradykinin (BK) B2 receptor inhibitor Hoe 140. On a molar basis, PU-D1 is more effective than BK and kinins of 15, 16, and 18 amino acids for blocking the ANP-mediated diuresis-natriuresis. As with BK and other kinins, the inhibitory effect of Pu-D1 on ANP is obtained only within a small range of picomol doses. A single dose of 2.16 or 4.32 pmol PU-D1 or 47 pmol (50 ng) BK is ineffective against ANP if injected alone. However, when both substances are administered concomitantly at these subthreshold doses, they totally suppress ANP-induced diuresis-natriuresis. These results raise the question of whether PU-D1, released from kininogen domain 1, either alone or associated with BK, may interact with ANP in the regulation of urinary water and electrolyte excretion in physiological and pathological conditions.

GW1229, a novel neuropeptide Y Y1 receptor antagonist, inhibits the vasoconstrictor effect on neuropeptide Y in the hamster microcirculation.

We studied the effect of GW1229, a novel neuropeptide Y Y1 receptor antagonists, on the vasoconstriction induced by neuropeptide Y and structurally related analogs in the hamster cheek pouch microcirculation. Changes in arteriolar diameter and microvascular conductance were assessed by intravital microscopy and measurement of sodium22 clearance. GW1229 did not affect basal vascular conductance but inhibited, concentration dependently, the reduction in arteriolar diameter and vascular conductance induced by 100 nM neuropeptide Y. GW1229 also counteracted the vasoconstrictor effect of 100 nM [Leu31,Pro34]neuropeptide Y, and that of 300 nM neuropeptide Y-[(13-36). In contrast, GW1229 had no effect on the vasoconstriction induced by noradrenaline. We conclude that the vasoconstrictor effect on neuropeptide Y in the hamster cheek pouch is mediated by neuropeptide Y Y1 receptors. The maintenance of physiological tone in this vascular bed does not involve the participation of endogenous neuropeptide Y.

A fragment of human kininogen containing bradykinin blunts the diuretic effect of atrial natriuretic peptide.

A synthetic 15 aminoacids kinin, named PU-15, is able to block the diuretic natriuretic action of Atrial Natriuretic Peptide (ANP). The structure of PU-15 is Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Iso, having the aminoacid sequence of a fragment of human kininogens. The increase in the urinary excretion of sodium, potassium, and water, elicited by a bolus of 0.5 microg of ANP in anesthetized rats, is blocked by PU-15 (100-150 ng) given either intravenously 3 min before ANP injection, or injected intraperitoneally or in the duodenal lumen, 40 min before ANP. This ANP blockade, which mimics the action of pepsanurin, is only obtained with doses of PU-15 in a narrow range around 100 picomol/rat, and do not modify blood pressure. Larger doses, 2- to 8-fold the effective dose, either do not change the response to ANP or raise the excretion of sodium and water. The administration of HOE-140, a bradykinin B2 receptor blocker, prior to PU-15, completely abolishes the anti-ANP action of PU-15. These findings lend support to the proposal that kinins released from the intestinal tract during prandial period can modulate renal excretory function.

Neuropeptide Y is a vasoconstrictor and adrenergic modulator in the hamster microcirculation by acting on neuropeptide Y1 and Y2 receptors.

The microvascular effects of neuropeptide Y, and two analogs with preferential affinity for different neuropeptide Y receptor subtypes, were assessed by intravital microscopy on the hamster cheek pouch. The interaction of neuropeptide Y and its analogs with noradrenaline was also studied. Superfusion with 0.1-300 nM neuropeptide Y caused a concentration-dependent reduction in microvascular conductance that was paralleled by reductions in arteriolar and venular diameters. These effects of neuropeptide Y were equipotent with noradrenaline, but slower to develop and longer-lasting than that of noradrenaline. Neuropeptide Y did not affect permeability to macromolecules, as measured by extravasation of fluorescent dextran. The neuropeptide Y Y1 receptor agonist, [Leu31,Pro34]neuropeptide Y, mimicked neuropeptide Y with similar potency but shorter duration, while neuropeptide Y-(13-36), a neuropeptide Y Y2 receptor agonist, was at least 10-fold less potent than neuropeptide Y to induce a delayed and prolonged reduction in microvascular conductance. The joint superfusion of 1 nM neuropeptide Y plus 0.1 mu M noradrenaline did not cause synergism, nor even summation of effects, but reduced the contractile effect of noradrenaline. No synergism was observed after a 10 min priming with 1 nM neuropeptide Y, followed by its joint application with 0.1 mu M noradrenaline, but a significant vasodilation and hyperemia ensued upon stopping noradrenaline application. Priming with 1 nM [Leu31,Pro34]neuropeptide Y prolonged noradrenaline vasoconstriction without evidence of hyperemia. In contrast, priming with 1 nM neuropeptide Y-(13-36) significantly antagonized noradrenaline vasoconstriction. These findings indicate that both neuropeptide Y receptor subtypes are present in arterioles and venules of the hamster, and suggest that their activation with neuropeptide Y induces a rapid (Y1 receptor subtype activation) and a delayed (Y2 receptor subtype activation) vasocontractile response. The interaction with noradrenaline is complex, without evidence for synergism, but neuropeptide Y Y2 receptor activation seems to antagonize noradrenaline and/or to facilitate auto-regulatory vasodilation after the catecholamine-induced vasoconstriction.

Inhibition of atrial natriuretic peptide excretory action by bradykinin.

We examined whether the excretory effect of atrial natriuretic peptide could be antagonized by intravenously administered bradykinin or by elevated endogenous kinin levels attained during converting enzyme inhibition. Urinary volume and sodium and potassium excretion were determined every 20 minutes in female, anesthetized Sprague-Dawley rats (weight, 0.19 to 0.22 kg) infused with 10 microL/min isotonic glucose. In some experiments, urinary cGMP content was measured by radioimmunoassay. Two intravenous boluses of 209 pmol (0.5 micrograms) atrial natriuretic peptide were given before and after the injection of test substances, and the response ratio was used to quantify inhibition. Single injections of 94.3 or 142 pmol (100 or 150 ng) bradykinin, 3 minutes prior to atrial natriuretic peptide, inhibited the excretion of water, sodium, and potassium by 70%, 75%, and 50%, respectively. Larger (236 to 472 pmol) or smaller (23.6 to 47.2 pmol) bradykinin doses were ineffective. None of the bradykinin doses tested affected basal urinary output, systemic pressure, or the modest depressor effect of atrial natriuretic peptide. The anti-atrial natriuretic peptide effect of bradykinin was completely prevented by the kinin receptor antagonist Hoe 140. Converting enzyme inhibition with ramipril (96 nmol IV) also blunted atrial natriuretic peptide diuresis and natriuresis by 70% and reduced urinary cGMP excretion by 50%. These effects of ramipril were mediated by endogenous kinin accumulation, since they were abolished by pretreatment with Hoe 140. It is concluded that intrarenal kinins modulate the renal actions of atrial natriuretic peptide, and at a precise concentration bradykinin strongly antagonizes atrial natriuretic peptide by preventing its transduction mechanism.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

Blunting effect of pepsanurin introduced in the duodenum on the atrial natriuretic peptide diuretic action in rats.

Pepsanurin (PU) is a peptide(s) obtained by pepsin hydrolysis of human plasma or its globulin fraction. We have reported that the accelerated renal excretory rate induced by atrial natriuretic peptide (ANP) can be considerably blunted by PU either in the intact rat or in the isolated perfused rat kidney. We explored whether or not PU can be part of a signaling mechanism originated in the digestive tract, involved in the regulation of water and electrolyte homeostasis. PU obtained either from human (0.5 ml) or rat plasma (0.25-0.5 ml) administered into the duodenal lumen of rats, counteracted significantly the diuretic-saluretic action of a 0.5- microgram bolus of ANP, reproducing qualitatively the effect of its intraperitoneal administration. Human PU reduced the ANP-stimulated renal excretion by 67-90% for Na (P < 0.001) and by 35-54% for water (P < 0.02-P < 0.001); the inhibition induced by rat PU was 45-96% for Na (P < 0.05-P < 0.01) and 35-65% for water (P < 0.05-P < 0.01). Rat PU (0.5 ml) abolished the rise of glomerular filtration rate induced by ANP without affecting fractional Na excretion. All the samples tested decreased K excretion, but in some experiments, the difference did not reach statistical significance. Contrary to the effect of PU, the introduction in the duodenum of (i) isotonic glucose solution, (ii) hydrolysate of bovine serum albumin, or (iii) hydrolysate of casein prepared after the same procedure yielding PU from plasma failed to produce an inhibition of the ANP stimulation of renal excretory rate. In addition, human plasma incubated at 37 degrees C for 24 to 48 hr, prior to pepsin digestion, did not yield PU, which indicates that PU is generated from a substrate sensitive to endogenous enzymes and/or that its stability is vulnerable to endogenous enzymes.