Robert R. Smeby

Measurement of Renin Activity in Human Plasma

A method is described for estimating plasma renin activity by using renin substrate present in plasma. This method differs from other indirect renin assay methods by (1) incubation in the absence of ions thus establishing conditions for zero order kinetics for the reaction between endogeneous renin and substrate and (2) the use of angiotensinase inhibitors di-sodium ethylenediamine tetraacetic acid (EDTA) and d-isopropylfluorophosphate (DFP). Recoveries of renin added to plasma in levels similar to those occurring in plasma are 85% SD±7%. The incubation was done at pH 5.5 which was shown to be the optimum for human renin reacting with human substrate. By incubating human plasma samples with known quantities of human renin, evidence was obtained suggesting that factors other than enzyme or total substrate concentrations affect the velocity of angiotensin formation. This variability of reaction rate may be explained by the existence of an inhibitor or activator in this system or by a variation in the type of substrate.

Renin in Rats with Spontaneous Hypertension

An age-dependent study of plasma renin activity (PRA), kidney renin activity (KRA), and plasma renin substrate was carried out in rats with spontaneous hypertension during the prehypertensive, the early hypertensive, and the established hypertensive phases of their disease. PRA and KRA were both significantly elevated before and during the initial phase of hypertension and normal or subnormal during the established phase. In normal controls, neither KRA nor PRA was significantly different during the entire growth period. Plasma renin substrate was elevated throughout the growth period in rats with hypertension. This relationship between renin and blood pressure suggests that renin may play a primary role, possibly along with other factors, in the initiation of hypertension in rats with spontaneous hypertension.

Angiotensinase with a high degree of specificity in plasma and red cells.

A peptidase with a high degree of specificity for angiotensin II occurs in normal human plasma and red cells. Preparations from both sources have the same pH optimum, require calcium ions, and hydrolyze valyl5- or isoleucyl5-angiotensin II, but do not hydrolyze β-aspartyl1-angiotensin II, arginyl1-angiotensin II or deaminoangiotensin II. This enzyme, given the name angiotensinase A, requires α-L-aspartic acid or α-L-asparagine as the N-terminal amino acid in its angiotensin substrate, and thus differs from kidney leucine aminopeptidase. Other peptidases known to hydrolyze angiotensin also hydrolyze at least one of the other angiotensin analogs with substitution in the one position.

The relationship of structure to pressor and oxytocic actions of isoleucine5 angiotensin octapeptide and various analogues

Abstract Isolated natural angiotensin I and synthetic angiotensin II have similar pressor activity but the latter has a much higher oxytocic activity. On a weight basis, the oxytocic activity of angiotensin II is about 1 8 that of oxytocin. Examination of analogues of angiotensin II demonstrate the following conditions must be met to have pressor and oxytocic activity: (a) the C-terminal amino acid must be l -phenylalanine, (b) the terminal carboxyl group must be free, (c) tyrosine must be present, and (d) the peptide must contain at least amino acids numbered 3 to 8. It is suggested that the spatial configuration of the molecule is an important aspect of the biological specificity of peptides in addition to the amino acid composition and sequence.

A proposed conformation of isoleucyl5-angiotensin II

Abstract Optical rotatory dispersion studies on isoleucyl 5 -angiotensin II show the peptide has a definite degree of order which is decreased by urea. Ultraviolet spectral studies on the phenolic hydroxyl group indicate it does not interact with any other groups in the molecule. A three-dimensional model of angiotensin II is suggested which is consistent with physical and biological data available.

Measurement of immunoreactive angiotensin peptides in rat tissues: Some pitfalls in angiotensin II analysis

Angiotensin II, the major effector peptide of the renin-angiotensin system, is an endocrine and paracrine regulator of tissue function. To determine its physiological role, it is important to quantify angiotensin II and related fragment peptides in tissues and plasma as a first step toward understanding angiotensin II metabolism within tissues. A fully characterized, sensitive, and reproducible immunochemical assay has been developed for quantitating angiotensin II immunoreactivity in tissues and plasma. We identified two methodological events of critical importance, incompletely addressed in previously reported studies. First, the nonspecific interference resulting from Sep-Pak processing was found to be due to hydrophobic impurities in the octade-casilane absorbent which were eliminated by washing the Sep-Pak with tetrahydrofuran and hexane before use. Second, a significant discrepancy was observed in the recoveries of angiotensin II and 125I-angiotensin II added to tissue extracts following high-pressure liquid chromatography. Angiotensin II immunoreactivity extracted from decapitated rat adrenal gland, brain, and kidney (target organs for angiotensin II), ovary and uterus (potential target organs for angiotensin II), and plasma has been characterized. The predominant component of the angiotensin II immunoreactivity was the biologically active octapeptide angiotensin II. However, in the brain, the ratio of angiotensin II to C-terminal angiotensin II immunoreactive fragments was lower than observed in other tissues studied. Other angiotensin II C-terminal immunoreactive peptide fragments-the biologically active heptapeptide and the biologically inactive angiotensin(3-8) and angiotensin(4-8)--were also detected in variable quantities in the various tissues.

Angiotensin II: its metabolic fate.

Randomly labeled tritiated angiotensin has been prepared with a specific activity of 300 �c/mg and with undiminished pressor and oxytocic activity. After infusion, angiotensin accumulated in the kidneys, adrenal glands, and uterus. Thirty minutes after infusion high levels of radioactivity appeared in brain, but the electrophoretic mobility differed from that of angiotensin II. Incubation of angiotensin with hemolyzed human red blood cells or diluted human plasma rapidly inactivated the pressor activity with production of metabolic products separable by paper chromatography. But if undiluted plasma is used with incubation up to 6 hours, no loss of activity occurs.

Role of renin-angiotensin system in chronic renal hypertensive rats.

SUMMARY The role of renin-angiotensin system has been examined in the maintenance of hypertension in acute and chronic two-kidney (36 weeks) and chronic one-kidney (12 weeks) Goldblatt hypertensive rats using three inhibitors of this system. The inhibitors used were URI-73A, a synthetic analog of lysopbosphatidylethanolamine, which inhibits renin both in vivo and in vitro, SQ14.225, a potent conrerting enzyme inhibitor, and [Sar1, Thr*] angiotensin II, an angiotensin II antagonist. When the inhibitors were administered in acute (high renin) hypertensive rats, they all lowered blood pressure significantly. However, in the chronic (low renin) hypertensive phase, both renin and converting enzyme inhibitors lowered blood pressure, whereas, Sar, Thr* failed to lower Mood pressure. The renin inhibitor lowered plasma renin activity (PRA), and SQ 14,225 and [Sar1, Thr*] Ang II increased PRA. Further studies on water and electrolyte balance with one-kidney model hypertensive and uninephrectomized control rats showed no change in plasma volume. However, there was increased 24-bour urinary output and increased sodium excretion. This study indicates that in chronic renal hypertensive rats, blood pressure reduction is possible by either renin or converting enzyme inhibitor, but not by angiotensin antagonists. Since volume did not change either during the development or reversal of hypertension, volume did not appear to play a major role in the maintenance of hypertension.

Production of Renin by Human Juxtaglomerular Cells in Vitro

New organ culture methods permit growth of human renal cortical cells fulfilling all known criteria of juxtaglomerular cells. Two types of granules were observed with the electron microscope. These granules showed an increase in size and number during the growth of these cultures. Their presence in cells was demonstrated by Bowies, basic fuchsin-crystal violet, and fluorochrome staining methods. Puromycin or actinomycin D caused a reduction in the number of cytoplasmic granules, while 1% oxygen in the gas phase of the culture caused an increase. Specific reaction of these granules with rabbit antihuman renin antibody was demonstrated. Other cellular structures did not react with this antibody. Similar granules were observed in the media of hyperplastic, interlobular arterioles but not in normal arterioles or arterioles of other vascular beds. Bundles of myofilaments could be observed between the cytoplasmic granules in cultured cells. Bioassays of renin activity of the supernatant culture medium showed that it contained renin if granulated cells were present in the culture. All juxtaglomerular cells in culture produced granules, but only those cells which grew from kidneys with pathological changes released renin, suggesting that the stimulus for renin release existed in the intact kidney. This stimulus for renin release seemed to be rapidly lost during culture growth.

Cerebrospinal fluid angiotensin II immunoreactivity is not derived from the plasma.

To elucidate whether the presence of angiotensin II immunoreactivity (ANG II-ir) in the cerebrospinal fluid (CSF) of the dog is in part due to passage of the peptide across the CSF-blood-brain barrier, [Ile5] angiotensin II (ANG II) was infused intravenously for 7 days in conscious, trained dogs at a rate of 10 micrograms/kg/day. Mean arterial pressure (MAP) and heart rate were monitored each day, and samples of arterial blood and CSF (with a catheter secured into the cisterna magna) were drawn at regular intervals for determination of catecholamine levels, ANG II-ir, and electrolyte levels. Within 2 days after ANG II infusion, MAP stabilized at 35 +/- 1 mm Hg (mean +/- SE, p less than 0.001) above control values. The hypertension was associated with bradycardia, suppressed plasma renin activity, and a fall in both plasma and CSF Na+ concentrations. These changes coincided with a considerable and sustained decrease in the levels of plasma and CSF norepinephrine. On the other hand, levels of epinephrine and K+ in the two compartments remained unchanged. Although concentration of ANG II-ir in plasma was augmented markedly (368% above control values, p less than 0.001), ANG II-ir in the CSF remained within the low values measured in the control period.(ABSTRACT TRUNCATED AT 250 WORDS)