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.

Jorge Mardones: oration at his funeral service.

Oratios at the funeral service of Prof Jorge Mardones, MD. A very successful and influential pharmacologist, he was Chairman of the Department of Pharmacology, Faculty of Medicine, University of Chile; President of the Academy of Sciences, Institute of Chile; National Prize of Sciences, 1977; and Minister of Public Health. He also served as Chief Editor of "Archivos de Biología y Medicina Experimentales" (former name of this journal) between 1964 and 1978.

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.

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.

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.

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.

Inhibition of atrial natriuretic peptide-induced natriuresis by plasma hydrolysates containing pepsanurin.

The specificity of antidiuretic actions of pepsanurin, a peptidic fraction obtained by pepsin hydrolysis of plasma, was studied in anesthetized rats and in isolated perfused rat kidneys. Pepsanurin was obtained from fresh dialyzed human plasma digested with pepsin (2,400 units/ml, 18 hours at 37 degrees C, pH 2.5), deproteinized (10 minutes at 80 degrees C), and centrifuged. In the rat, intraperitoneal injections of pepsanurin (0.5 ml/100 g body wt) significantly inhibited the effects of an intravenous bolus of atrial natriuretic peptide (ANP) (0.5 micrograms) on water, sodium, and potassium excretion without altering systemic blood pressure. In addition, pepsanurin abolished the peak in glomerular filtration rate and reduced the ANP-induced rise in fractional sodium excretion. Pepsanurin also inhibited the natriuretic effects of amiloride (10 micrograms/100 g body wt i.v.) without changing glomerular filtration rate, but it did not inhibit the potassium-retaining effect of amiloride. In contrast, pepsanurin had no effect on basal urinary excretion, and it did not affect the diuretic response induced by furosemide (doses of 25, 50, or 100 micrograms i.v.). Control peptidic hydrolysates prepared from human plasma preincubated 48 hours at 37 degrees C (PIPH), bovine albumin (BSAH), or human albumin did not inhibit ANP, amiloride, or furosemide. In perfused kidneys, pepsanurin significantly and reversibly reduced sodium and water excretion. Furthermore, pepsanurin, but not PIPH or BSAH, blocked the natriuretic and diuretic effects of ANP. These results support the existence of a specific plasma substrate able to release a peptide or peptides that counteract distal tubule diuresis and natriuresis by an intrarenal mechanism.

Atrial natriuretic factors (ANF) and antidiuretic agents.

The effects of lysine-vasopressin (VP) and pepsanurin (PU) on the diuretic-saluretic action of ANF were investigated. Anaesthetized female rats under constant intravenous perfusion with isotonic glucose solution (0.6 ml/h/100 g body weight) were used. Blood pressure was recorded continuously and in the urine collected every 20 min, volume, NA and K excretion were measured. In each rat two intravenous boluses of either 2.5 ug synthetic rat atriopeptin II or an equivalent amount of a semipurified rat atrial extract were assayed. Thirty min before the ANF second bolus, an i.v. injection of lysine-vasopressin was given. Doses of 0.1, 1, 5, 10 and 50 mU were used. PU in the dose of 0.5 ml, obtained from 20 ml of human plasma was administered intraperitoneally, 40-60 min before the second bolus of ANF. The smaller doses of VP did not inhibit the urinary response to ANF, and the higher one (50 mU) produced a significant facilitation of ANF effect. Contrariwise, PU produced a considerable inhibition on water, NA and K excretion promoted by ANF.

Identification of kallikrein in cultures of adult renal cells.

Cortical tubular cultures enriched with distal segments were prepared from the kidney of adult Fisher 344 rats bearing thyroid tumors. After two years of cultivation (20 passages) cell monolayers contained immunocytochemically detectable cytokeratin and kallikrein material in their cytoplasm. Furthermore cell pellets showed a true renal type of kininogenase activity corresponding to active kallikrein which ranged from 22.3 to 1.5, and total Kallikrein from 29.9 to 2.8 pg kinins/min per mg of protein.

Potentiating effect of aldosterone on the diuretic action of atrial extract.

The typical stimulatory effect of a rat heart atrial extract on urinary water, sodium, potassium and kallikrein excretion is significantly increased by a previous administration of aldosterone (0.5 microgram/100 g b. wt) in the rat.

Urinary kallikrein activity during rat pregnancy.

Changes in urinary kallikrein activity and its possible correlation with changes in blood pressure and renal excretory function during pregnancy were studied in the rat. To establish a possible physiological role of kallikrein in this condition aprotinin, which inhibits kallikrein as well as other serine protease was administered to pregnant rats. Urinary kallikrein activity was markedly increased during pregnancy and correlated positively with urine volume and electrolytes excretion, but not with blood pressure. Aprotinin administration almost completely inhibited kallikrein activity, however, blood pressure levels, urine volume and electrolytes were not changed after one day of aprotinin treatment. In conclusion, although renal kallikrein is highly enhanced during pregnancy, its physiologic role in this condition remains elusive.

Effect of amiloride on urinary and renal kallikrein in the rat.

A single intraperitoneal injection of amiloride in the range of 2.7, 5.4, 10.9, and 21 mumol/100 g body wt in female adult rats produced, in the two successive periods of 4 h following its administration, a significant decrease in the urinary excretory rate of kallikrein. Amiloride, 10.9 mumol/100 g body wt, which significantly reduced active kallikrein, also decreased, but less significantly, the trypsin-activated kallikrein in the urine. The fall in the excretory rate of kallikrein cannot be explained by its enzymatic inhibition by amiloride, since the inhibition was only present at higher concentrations. In hyperhydrated rats amiloride did not change the kallikrein excretory rate in the urine collected within 4 h after the injection. Rats simultaneously injected with 7.6 mumol/100 g body wt furosemide and 10.9 mumol/100 g body wt amiloride excreted levels of kallikrein similar to those found in rats injected with furosemide alone. The kidneys of rats removed after 4 h of administration of 10.9 mumol/100 g body wt amiloride showed a significant lowering of the kallikrein activity compared with the respective controls. The decrease of renal kallikrein tended to be similarly pronounced in those rats that received amiloride and furosemide simultaneously. These results confirm the depressive effect of amiloride on kallikrein excretion, which may be explained by an inhibitory action on kallikrein release, activation, and synthesis by the renal cells.

Urinary kallikrein excretion in pregnant adrenalectomized rats.

The mechanisms responsible for the increase of the urinary kallikrein activity (UKA) during pregnancy is still unknown. Since aldosterone has been described as one of the stimulatory factor of UKA, and this hormone is considerably elevated in normal pregnancy it is feasible to postulate that it might be involved in the process. A preliminary approach to solve the role of the mineralocorticoid was to investigate the effect of the adrenalectomy upon UKA, of pregnant rats. UKA activity and blood pressure (BP) were measured at weekly intervals in four groups of rats: pregnant-adrenalectomized (P-ADX); pregnant-sham operated (P-C); non pregnant-adrenalectomized (NP-ADX) and non pregnant-sham operated (NP-C). In addition serum aldosterone levels in pregnant rats were determined. All the groups were maintained in similar conditions drinking 1% NaCl solution. The increase in UKA was not detected in P-C, as observed in normal pregnant rats drinking tap water, indicating that a high NaCl intake prevents the rise in UKA during gestation. However, P-C rats had higher UKA than P-ADX on day 14 of pregnancy (p < 0,05). The lowest level of UKA was found in NP-ADX. In NP-C and NP-ADX BP rose progressively throughout the experiment. BP in P-ADX rats during pregnancy and on day 7 of post partum was significantly lower than in P-C rats (p < 0,01-0,02) but in both pregnant groups it decreased significantly before parturition (p < 0,02) increasing at post-partum. Serum aldosterone levels on days 14 ans 21 of gestation, showed higher values in P-C than in P-ADX (p < 0,02-0,005). The results provide support to the assumption that adrenal gland has a regulatory action on UKA during pregnancy and that aldosterone can be one of the main factors involved.

Isolation and characterization of rat plasma glandular kallikrein.

A method has been developed to purify glandular kallikrein present in rat plasma by using Sepharose-Aprotinin affinity chromatography and elution of the enzyme with p-aminobenzamidine. The isolated enzyme liberated kinins from kininogen II of low molecular weight (sp. act. 14 ng kinins/min X mg) and p-nitroaniline (pNA) from the substrate S-2266 (sp. act. 1.23 nmoles pNA/min X mg); it was inhibited by aprotinin, benzamidine and rat urinary antikallikrein antibody but not by ovomucoid. In polyacrylamide gel electrophoresis, the enzymatic activities of the preparation were associated with two light protein bands of molecular weights equal to that of urinary kallikrein (35,000 daltons). Using this method, the recovery of [125I]kallikrein added to the plasma was 82-88%. The concentration of the enzyme in normal rat plasma was equivalent to 6.1 +/- 2.1 (S.D.) ng kallikrein/ml. The mean value found in nephrectomized rats was 20.0 +/- 6.3 (S.D.) ng kallikrein/ml. This increment was highly significant (P less than 0.001). Our results confirm the presence of glandular kallikrein in plasma which had been detected by other methods; they also demonstrate that the material purified from plasma is enzymatically active, suggesting that kallikrein may play a biological role in the control of blood circulation.

Fisiopatologia de la hipertension arterial. Sistema calicreina-cininas-prostaglandinas. / Physiopathology of the arterial hypertension.Kallikrein-kinins-prostaglandins system

Se ha demostrado que el rinon es uno de los organos productores de prostaglandinas (PGs) entre las cuales se cuentan la PGE2, PGE2a y la PGI2 (prostaciclina). Sus principales acciones renales incluyen la induccion de vasodilatacion, mayor excrecion de sodio y agua y accion reguladora sobre la secrecion de renina y eritropoyetina.Por otro lado, se ha demostrado tambien que el rinon dispone de un sistema vasodilatador y natriuretico distinto del de las PG, constituido por una glicoproteina que es una enzima proteolitica, similar a la renina, la calicreina, un substrato, el cininogeno y pepticos vasodilatadores. Se discuten en este trabajo las interrelaciones de los factores mencionados, en la regulacion de la presion arterial