Plasma and renal prorenin/renin, renin mRNA, and blood pressure in Dahl salt-sensitive and salt-resistant rats.

We measured plasma prorenin and renin levels, renal renin mRNA, renal anti-renin and anti-prorenin-prosequence immunoreactivity, and blood pressure in maturing Brookhaven Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats during 14 days of low (0%), medium (0.4%), or high 4%) NaCl diets. Blood pressure was higher in Dahl S rats and did not increase with high NaCl. Seven-week-old Dahl R rats had twofold and sixfold higher levels of plasma prorenin and renal prosequence immunoreactivity, respectively, which by 9 weeks were the same as in Dahl S rats. The anti-renin antiserum, BR1-5, was found to detect prorenin better than renin; Dahl S rats had suppressed renal anti-renin immunoreactivity relative to Dahl-R rats. Dahl R rats were unresponsive to high NaCl, whereas in Dahl S rats, plasma renin and renal prosequence immunoreactivity fell by 90% (P < .01), renal anti-renin immunoreactivity and renal renin MRNA fell by 35% (P < .05 for both), and plasma prorenin fell by 30% (P = NS). NaCl depletion increased prorenin/renin parameters similarly in both strains. There were direct relationships among all of the prorenin/renin parameters. Between low and high salt diets in Dahl S rats, plasma renin increased 20-fold, plasma total renin (renin plus prorenin) and renal renin mRNA both increased threefold, and plasma prorenin increased twofold. The results indicate that under steady-state conditions, plasma and renal renin/prorenin parameters change concordantly and that plasma total renin (renin plus prorenin) reflects changes in renal renin mRNA. The lower blood pressure of Dahl R rats is associated with later maturation-related declines in plasma and renal prorenin. Suppression of plasma renin may delay the salt-induced blood pressure rise in Dahl S rats. Finally, the renin system and blood pressure of Dahl R rats have remarkable disregard for a high salt diet.

Specific prorenin/renin binding (ProBP). Identification and characterization of a novel membrane site.

Renin can be detected in cardiovascular and other tissues but it disappears after bilateral nephrectomy indicating that tissues can take up or bind renal renin from the circulation. If renin uptake is the result of specific binding, plasma prorenin may be a natural antagonist of tissue directed renin-angiotensin systems. To investigate if specific prorenin/renin uptake occurs in rat tissues, binding studies were performed, with rat microsomal membrane preparations using recombinant rat prorenin metabolically labeled with 35S-methionine as a probe. A high affinity binding site for both renin and prorenin was identified. Affinities for prorenin and renin were approximately 200 and 900 pmol/L, respectively. Binding was reversible, saturable, and pH and temperature dependent. The relative binding capacities of membranes from various rat tissues were as follows (fmol/mg): renal cortex (55), liver (54), testis (63), lung (31), brain (18), renal medulla (15), adrenal (17), aorta (7), heart (4), and skeletal muscle (1). Bound prorenin was displaced by rat and human renin or prorenin but not by the prosequence of rat prorenin, angiotensin I or II, rat or human angiotensinogen, the renin inhibitor SQ30697, atrial natriuretic factor, amylase, insulin, bovine serum albumin, hemoglobin, heparin, lysozyme, ovalbumin, cytochrome C, pepsin, pepsinogen, ribonuclease A, mannose-6-phosphate, alpha-methyl mannoside, gonadotropin releasing hormone, or an antibody to hog renin binding protein. these results demonstrate specific binding of prorenin to a site in rat tissues, herein named ProBP, that also binds renin. It is possible that differences in prorenin/renin binding capacity determine the activity of tissue-directed renin-angiotensin systems and that prorenin is a natural antagonist. Alternatively, a prorenin/renin receptor may have been identified that may function by transducing an intracellular signal.

Angiotensinogen depletion by high renin levels in hypertensive rats: no evidence for tonic stimulation of angiotensinogen by angiotensin II.

OBJECTIVE: Data concerning the effect of angiotensin II (Ang II) on plasma angiotensinogen levels are conflicting. Although Ang II is reported to stimulate the biosynthesis of angiotensinogen, plasma angiotensinogen is often depleted by renin when the level of renin, and therefore Ang II, increases. In the present study we used the Ang II subtype 1 (AT1) receptor antagonist losartan to investigate whether rising plasma Ang II levels stimulate angiotensinogen production to counteract the falling plasma angiotensinogen levels caused by increasing renin activity in plasma. METHOD: Angiotensinogen was measured in plasma from two previously reported studies in which 6-week-old stroke-prone spontaneously hypertensive rats (SHRSP) or Dahl salt-sensitive (Dahl-S) rats were fed high-salt diets (4 and 8% sodium chloride, respectively) for 10-12 weeks with or without losartan. RESULTS: As reported previously, plasma renin was suppressed during the first 4 weeks of the high-salt diet but then paradoxically increased in both strains. When plasma renin increased, plasma angiotensinogen levels fell to 45 and 62% of the baseline value. The plasma renin concentration was negatively correlated with plasma angiotensinogen both in SHRSP and in Dahl-S rats (r = -0.76, P < 0.001 and r = -0.60, P < 0.001, respectively). In Dahl-S rats losartan treatment was associated with lower levels of plasma angiotensinogen but caused greater increases in plasma renin. When differences in renin were taken into account, plasma angiotensinogen levels were not different in losartan-treated and untreated Dahl-S rats. Similarly to Dahl-S rats, plasma angiotensinogen fell in SHRSP when renin increased, but SHRSP had higher plasma angiotensinogen levels during losartan treatment because plasma renin concentration was lower. CONCLUSION: The present study shows, in two strains of hypertensive rat, that an increase in plasma renin levels is associated with a fall in plasma angiotensinogen levels. Concurrent treatment with an Ang II AT1 receptor antagonist does not augment this fall, except to the extent that renin rises further. The results provide no evidence for a significant tonic stimulatory effect of Ang II on plasma angiotensinogen levels.

Angiotensinogen dependency of blood pressure in two high-renin hypertensive rat models.

In this analysis we investigated whether angiotensinogen (Aogn) levels were related to blood pressure (BP) in two hypertensive rat models when renin secretion was either under physiologic regulation or out of control. These relationships were investigated using BP data from previous reports in which SHRsp and Dahl S rats were studied for 10 to 12 weeks while ingesting a high-salt diet with and without the angiotensin II (AngII) antagonist losartan. During the first 4 weeks of high-salt diet, plasma renin concentration (PRC) was appropriately suppressed but it subsequently increased paradoxically in both strains. During the first 4 weeks, when renin secretion was under normal control, as indicated by its suppression by the high-salt diet and by an inverse relationship between PRC and BP (r = -0.69, P < .001 and r = -0.53, P < .01 in Dahl S and SHRsp, respectively), there was no relationship between BP and plasma Aogn. In contrast, when renin secretion increased paradoxically, the inverse relationship between BP and PRC was lost and a positive relationship was found between BP and plasma Aogn in both Dahl S rats (r = 0.70, P < .01) and SHRsp (r = 0.57, P < .01). There was no relationship between BP and Aogn in either strain during treatment with losartan either before or after 4 to 6 weeks of salt feeding. These results show Aogn dependency of BP, but only under conditions in which renin cannot feed back normally. The Aogn relationship to BP was most likely dependent on the vasoconstrictor effect of AngII since it was lost during AngII AT1 receptor antagonism.

Inhibition of human renin by rat plasma. Rat angiotensinogen is a competitive inhibitor of the human renin-substrate interaction.

Human renin can cleave rat angiotensinogen, yet infusion of human renin into rats causes only a modest increase in blood pressure. We therefore investigated whether there is a factor in rat plasma which inhibits human renin activity. The addition of 20% normal rat plasma to human plasma had a slight, but not significant, inhibitory effect on the rate of angiotensin formation, while nephrectomized rat plasma, which had a seven-fold higher concentration of angiotensinogen, caused a dose dependent inhibition (20 to 70%). The rat plasma inhibitor copurified with angiotensinogen. Analysis of the kinetics of the human renin-human substrate reaction and of the human renin-rat substrate reaction revealed that the rate of angiotensin I production in the presence of both substrates could be entirely accounted for by assuming that rat and human angiotensinogens are competitive inhibitors of each other. These results show that human renin can cleave rat substrate but the reaction rate is extremely slow relative to the cleavage of human angiotensinogen. They also indicate that rat angiotensinogen is an effective competitive inhibitor of the human renin-substrate reaction. These results may be relevant to the development of renin inhibitors and to transfection studies involving heterologous renin or substrate genes.

The greater renin system. Its prorenin-directed vasodilator limb. Relevance to diabetes mellitus, pregnancy, and hypertension.

A greater renin system is proposed. Evidence is presented that a greater renin system exists that has both vasodilator and vasoconstrictor properties. Vasodilator activity is induced by prorenin, vasoconstrictor activity by renin. Our model is based on evidence that both prorenin and renin have the capacity to generate angiotensin and that angiotensin causes vasodilation at high concentrations and vasoconstriction at low concentrations. In our model, prorenin acts only at particular target sites while renin of renal origin acts via the general circulation. Prorenin's designation as a biosynthetic precursor implies lack of intrinsic catalytic activity whereas in fact it can become reversibly active. Activation may occur in vivo at binding sites without cleavage of the prosequence. In this framework, prorenin should be more aptly called renin I and circulating active renin, renin II. In our model, the role of renin I (prorenin) is to generate localized high concentrations of angiotensin II, eg, in the afferent arteriole of the kidney and in other vital organs, causing regional dilation by rendering tissues insensitive (tachyphylactic) to the vasoconstrictor effect of circulating angiotensin II or by releasing vasodilator substances. The role of renin II (active renin) is to constrict resistance vessels and the efferent arteriole of the kidney, thereby raising blood pressure, maintaining glomerular filtration rate, and enabling more blood flow to those organs that selectively bind prorenin. This twin control system is ideally designed to maintain blood flow to vital organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypsin activation of human and cat prorenin: a comparative study.

Prorenin can be converted to renin by limited proteolysis with trypsin. In the current study we compared conditions for activation of human renal and ovarian prorenin and cat renal prorenin with either liquid-phase trypsin or trypsin bound to sepharose (solid phase). Higher concentrations of trypsin were required to activate cat prorenin than human prorenin. Human prorenin was destroyed by high concentrations of trypsin, while cat prorenin was not destroyed by up to 2 mg/mL solid-phase trypsin. For both human and cat prorenin, addition of the competitive serine protease inhibitor benzamidine--HCl increased the concentration of trypsin needed to activate prorenin, resulting in slightly higher levels of human prorenin but lower levels of cat prorenin. For human samples, activation with solid-phase trypsin resulted in slightly higher estimates of prorenin than liquid-phase trypsin. These results demonstrate species differences in the susceptibility of prorenin to trypsin cleavage. Cat prorenin requires more trypsin to be activated and is less susceptible to destruction than human prorenin.

Persistently low immunoreactive and normally bioactive plasma LH during male puberty.

Low basal and LRH-stimulated (50 mcg/m2 i.v.) levels of plasma immunoreactive (IR) LH were repeatedly found in 8 boys aged 13 to 16 years who had been referred because of delayed onset of puberty (basal IR-LH 0.19 +/- 0.1 and peak 0.48 +/- 0.2 vs. 0.24 +/- 0.09 and 1.71 +/- 0.9 mIU/ml in matched normal controls, respectively). Upon termination of puberty (no more than 3.5 years after referral) IR-LH levels were still low in all 8. Using a rat Leydig cell bioassay system with LER-907 (NPA) as standard, LH bioactivity in these patients was compared with that in 8 matched controls (basal LH was 0.32 +/- 0.33 and 0.11 +/- 0.07, and peak levels 0.8 +/- 0.47 and 1.22 +/- 0.44 mIU/ml respectively). The ratio of basal LH bioactivity to IR-LH was higher in the patients (1.24 +/- 0.95) than in controls (0.47 +/- 0.26, P less than .05) as was that of peak bioactivity (1.89 +/- 1.2 vs. 0.83 +/- 0.35, P less than .05). In the three patients tested during sleep IR-LH levels showed no significant change. Basal plasma testosterone levels were appropriate for pubertal stage (400 +/- 80 ng/dl) and in the four patients tested following prolonged LRH stimulation (500 mcg i.v. over 3 hours) increased to 530 +/- 60 ng/dl. It is concluded that in some boys there may be consistently low plasma levels of IR-LH in association with normal LH bioactivity.

Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture.

The in vitro effects of ovine PRL (oPRL) on testicular testosterone synthesis were determined using isolated, collagenase-dispersed, adult rat Leydig cells in culture. oPRL (50-1000 ng/ml) had no effect either on basal or on LH (50, 100 or 2000 pg/ml)-stimulated testosterone secretion by Leydig cells in short-term culture (4 h). 125I-oPRL binding studies revealed a single class of high affinity sites (Ka 8.7 nM) with a low capacity (Bmax 6.7 fmol/mg protein identical to approximately 980 sites/Leydig cell). Isolated Leydig cells were further purified on a continuous Percoll gradient and cultured in serum-free medium, at 34 degrees C, in 5% CO2 and 95% air. After 3 days of culture, the media were collected, the cells washed and then stimulated with hCG (3 ng/ml) for 3 h. oPRL (1-1000 ng/ml) added at plating, caused a log dose-dependent inhibition of testosterone accumulation during the 3-day culture period; the highest and most consistent inhibition (31%) was with 500 ng/ml oPRL. hCG increased the sensitivity to the inhibitory effect of PRL, 10 ng/ml oPRL causing 40% inhibition and 100 ng/ml causing a maximal inhibition of 50%. PRL in fact caused a reduction in the maximal effect (efficacy) of hCG on steroidogenesis, without significantly affecting the ED50 (sensitivity). The effects of an antiPRL receptor antibody raised by the antiidiotypic route and previously shown to bind to rat testis PRL receptors were tested. The antiPRL receptor IgG (13 micrograms/ml) mimicked the PRL inhibitory effect and acted synergistically with PRL (100 ng/ml) in inhibiting both testosterone accumulation in 3-day cultured Leydig cells and their subsequent response to hCG. In summary, a clear inhibitory effect of PRL and a synergistic effect of antiPRL receptor antibody were demonstrated on testosterone synthesis by rat Leydig cells in 3-day culture.