Effects of blood viscosity on renin secretion.

The effects of alterations in blood and plasma viscosities on plasma renin activity (PRA) were studied in dogs anesthetized with pentobarbital. Blood viscosity was altered by changing the hematocrit (Hct) level by isovolemic exchange using packed red blood cells or plasma. Plasma viscosity was elevated by isovolemic exchange using Hct-matched blood with high molecular weight dextran (Dx, mean m.w. approximately 450,000) dissolved in plasma. Following control measurements of plasma and blood viscosities, plasma [Dx], PRA, Hct and hemodynamic functions, the dog was subjected to isovolemic exchange transfusions to either alter the Hct or administer the Dx. Various measurements were repeated 40-60 min after each exchange. Arterial pressure and renal blood flow remained relatively constant after exchanges; increases in plasma and blood viscosities were accompanied by a decrease in renal vascular hindrance (vasodilation) to keep the renal flow resistance at control level. PRA rose with increases in plasma [Dx] and viscosity, and the rise in PRA was best correlated with the decrease in renal hindrance. The changes in PRA and renal hindrance have the same regression line whether blood viscosity was altered by Hct variation or Dx administration. The results indicate that increases in viscosity cause a compensatory vasodilation of renal vessels to cause renin secretion.     

Effects of right atrial stretch on plasma renin activity.

In anaesthetized dog, right atrial stretch leads in the first five minutes to a decrease in plasma renin activity, when measured in inferior vena cava just above the renal veins. Bilateral cervical vagotomy increases plasma renin activity. After vagotomy, atrial stretch no longer has any effect on plasma renin activity. The results support the hypothesis of a control of renin secretion originating from atrial volume receptors.     

Effects of long-term administration of clonidine on plasma renin activity.

Plasma renin activity (PRA) was studied before and during long-term treatment with moderate oral doses (0.2 or 0.3 mg/d) of clonidine. Nine outpatients with essential hypertension received clonidine for 12 weeks; a significant decrease in blood pressure was evident in all patients. Except for a nonsignificant increase after 12 weeks of treatment, PRA values were not notably changed by clonidine therapy. No correlation was found between individual blood pressure changes and PRA variation during the study. The absence of a net effect on PRA in this study does not exclude more complex interactions of clonidine with the renin-angiotensin system. Nonetheless, clonidine cannot generally be classified as a "renin-inhibiting" drug.     

Conversion between renin and high-molecular-weight renin in the dog.

Two forms of renin, one of mol.wt. 43,000 and the other 60,000, were found in the dog kidney. Conversion between the two forms of renin was reversible at neutral pH. Though the molecular weight of renin in kidney-cortex homogenate was 43,000, it was completely converted into high-molecular-weight renin in the presence of substances that react with thiol groups. On the contrary, stored renin in the granules was the form of normal size (mol. wt. 43,000) regardless of the absence or presence of such substances. The present experiments indicated that renin is stored in the granules as the form of normal size and might be converted into high-molecular-weight renin when it is released from the granules and attached to some substance in the soluble fraction of renal-cortical tissue.     

Effects of substance P on renin release and renal function in anesthetized dogs.

Substance P (SP), a naturally occuring undecapeptide with hypotensive, vasodilatory and smooth muscle stimulating properties, was infused intravenously or intrarenally into anesthetized dogs. Infusions of SP intravenously suppressed renin secretion rate (RSR) from 204±45 to 52±18 ng/min (p < 0.02) at an infusion rate of 0.5 ng/kg/min, and to 50±22 ng/min (p < 0.05) at 5 ng/kg/min. When the concentration of SP was further increased to 50 ng/kg/min, RSR increased to a level above the control value (728±81, p < 0.01). Intrarenal infusion of SP produced similar changes in renin release. At infusion rates of 0.5 ng/kg/min and 5 ng/kg/min, RSR was suppressed from 145±18 to 56±18 ng/min (p < 0.05) and to 26±8 ng/min (p < 0.01) respectively. At 50 ng/kg/min, RSR increased to 251±59 (p > 0.1). Both intravenous and intrarenal administration of the peptide significantly lowered arterial blood pressure at the highest two doses. Intrarenal infusion of SP resulted in a significant dose-related increase in urine volume, sodium and potassium excretion, and renal blood flow. In contrast, intravenous infusions did not alter these parameters. Thus SP suppresses renin release in the presence and in the absence of diuresis, natriuresis, and vasodilation.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Purification of human renin substrate.

Renin substrate, angiotensinogen, has been purified from human plasma by methods which permit the processing of large amounts of outdated bank blood. The purified protein is homogeneous by disc gel electrophoresis at pH 9.5. The specific activity of 18 nmol/mg corresponds to a molecular weight of 56,000, while a higher value, 90,000, is found by gel filtration. Chromatography of partially purified renin substrate on DEAE-cellulose in a descending pH gradient shows evidence for the existence of multiple forms. However, some of these forms appear to be lost after chromatography on hydroxylapatite.     

Plasma renin, renin substrate and angiotensin II changes following experimental endotoxinaemia.

The effect of a single dose of endotoxin (B. coli, 1.5 mg/kg intravenously) on plasma renin concentration (PRC), renin substrate (PRSC), and angiotensin II (AT II) was studied in rats over a period of 48 hours. All determinations were performed by specific radioimmunoassay. Six and nine hours following endotoxin administration, renin secretion was decreased, whereas at 48 hours a slight increase in the PRC was found. In contrast, a three-fold elevation of the PRSC occurred during the first 24 hour period, attributable to a stimulation of the hepatic biosynthesis as result of corticosterone oversecretion. According to the observed changes in PRC and PRSC, AT II remains unchanged after six and nine hours, whereas a significant increase was detected after 24 and 48 hours. Based on the actual AT II level, the findings emphasize that in the rat the RAS does participate in the later stages of endotoxin stress only.     

Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP)

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.     

New renin inhibitors homologous with pepstatin.

Four homologues of pepstatin, the potent but poorly soluble inhibitor of aspartic proteinases, were synthesized by coupling to the C-terminus of the natural pentapeptide the following amino acid residues: L-arginine methyl ester, L-aspartic acid, L-glutamic acid and the dipeptide L-aspartyl-L-arginine. The peptide-coupling reagent we used, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, allowed us to obtain readily pure pepstatin homologues with high yields (60-83%). Pepstatylarginine methyl ester and pepstatylglutamic acid were about one order of magnitude more water-soluble than pepstatin. The four homologues and pepstatin were tested in vitro as inhibitors for highly purified pig and human renins acting on the N-acetyltetradecapeptide substrate. The 50% inhibitory concentrations (IC50) of the homologues were ranged from 0.01 to 1 microM against porcine renin at pH 6.0 (pepstatin IC50 approximately 0.32 microM) and from 5.8 to 41 microM against human renin at pH 6.5 (pepstatin IC 50 approximately 17 microM). By three different graphical methods we showed that pepstatin and the four homologues behaved as competitive inhibitors for porcine renin. The most potent inhibitors were pepstatylaspartic acid and pepstatylglutamic acid, with inhibitory constants respectively 2- and 10-fold smaller than that of pepstatin. By coupling glutamic acid to pepstatin, the ratio solubility/Ki was increased by two orders of magnitude.