Lufei Hu

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.

Appropriate Regulation of Renin and Blood Pressure in 45-kb Human Renin/Human Angiotensinogen Transgenic Mice

The renin-angiotensin system is normally subject to servo control mechanisms that suppress plasma renin levels in response to increased blood pressure and increase plasma renin levels when blood pressure falls. In most species, renin is rate limiting, and angiotensinogen circulates at a concentration close to the Km, so varying the concentration of either can affect the rate of angiotensin formation. However, only the plasma renin level responds to changes in blood pressure and sodium balance to maintain blood pressure homeostasis. Therefore, the high plasma human renin levels and the hypertension of mice and rats containing both human renin and angiotensinogen transgenes indicate inappropriate regulation of renin and blood pressure. These anomalies led us to develop new lines of transgenic mice with a longer human renin gene fragment (45 kb) than earlier lines (13 to 15 kb). Unlike their predecessors, the 45-kb hREN mice secrete human renin only from the kidneys, and both the human and mouse renins respond appropriately to physiological stimuli. To determine whether blood pressure is also regulated appropriately, we crossed these new 45-kb hREN mice with mice containing the human angiotensinogen gene. All doubly transgenic mice were normotensive like their singly transgenic and nontransgenic littermates. Moreover, among doubly transgenic mice, both human and mouse plasma renin concentrations were suppressed relative to the singly transgenic 45-kb hREN mice. These findings demonstrate the importance of appropriate cell and tissue specificity of gene expression in constructing transgenic models and affirm the pivotal role played by renal renin secretion in blood pressure control.

Identical hemodynamic and hormonal responses to 14-day infusions of renin or angiotensin II in conscious rats

Objective To investigate whether plasma angiotensin II (Ang II) determines the effects of the renin-angiotensin system or whether tissue uptake of renin and localized production of Ang II might account for any cardiovascular, renal, hormonal or drinking effect of circulating renin. Design Intravenous infusions of renin (0.6 ng/min; n = 10) and Ang II (3.5 ng/min; n = 10) that produce similar plasma Ang II levels were compared for 2 weeks with vehicle (n = 7) in conscious rats after a 1-week control period. Mean arterial pressure (MAP) and the heart rate were measured continuously. Hormones and renal function were measured twice weekly. Plasma Ang II and recovery data were measured in seven additional rats. Results In renin and Ang II-infused rats, respectively, plasma Ang II increased similarly from 4.5 ± 0.8 and 4.4 ± 0.9 to 10.8 ± 0.7 and 10.6 ± 0.7 pg/ml and declined similarly in the second week to 7.0 ± 1.1 and 7.0 ± 1.5 pg/ml. Plasma renin increased from 4.2 ± 0.7 to 21.7 ± 1.3 and fell from 5.9 ± 0.5 to 0.6 ± 0.2 ng/ml per h respectively. Plasma prorenin fell similarly (> 70%); angiotensinogen was unchanged. MAP rose initially by 25.6 ± 1.2 and 23.3 ± 0.9 mmHg and by an additional 21.1 ± 2.4 and 27.4 ± 1.8 mmHg on days 5–8. The heart rate fell gradually but transiently by -11% in both. Although the initial MAP rise was slower in renin-infused rats (P < 0.05) MAP returned to baseline within 2 h after both infusions were stopped. Changes in renal vascular resistance, renal blood flow, glomerular filtration rate, urinary sodium, potassium and water excretion and water intake were not significantly different between renin-and Ang II-infused rats. Conclusions Intravenous infusions of low doses of renin or Ang II into conscious rats increase MAP identically. MAP increases in two phases 5–8 days apart, in coordination with transient falls in the heart rate. Reninand Ang II-induced chronic hypertension are identically sustained by very small increases in plasma Ang II. Blood pressure increases more slowly with renin infusions, consistent with tissue binding. Notwithstanding, no evidence was obtained for a physiological role of tissue-bound renin in causing the cardiovascular, renal, hormonal and drinking responses measured in this study.

Delayed recovery of hypertension after single dose losartan in angiotensin II-infused conscious rats.

OBJECTIVE In a conscious unrestrained rat model, it takes approximately 1 week for angiotensin II to increase blood pressure to maximum levels. We investigated the time required for hypertension to fully recover after acute angiotensin II receptor blockade in this angiotensin II dependent hypertensive model. DESIGN Conscious unrestrained rats (n = 8) infused with 10 ng/kg per min angiotensin II for 21 days received losartan (10 mg/kg) on day 17 of angiotensin II infusion. Mean arterial pressure (MAP) and heart rate were monitored continuously. The acute pressor response to 50 ng/kg per min angiotensin II was monitored for 2 h on days 15, 17, 18, 19 and 20 of angiotensin II infusion. Plasma renin concentration (PRC) was measured daily. RESULTS Angiotensin II increased MAP acutely by 26 +/- 2 mmHg and by a further 23 +/- 4 mmHg between days 4 and 8. Losartan acutely reduced MAP by 75 +/- 2 mmHg; 24 h later MAP had partially recovered but remained suppressed by 47 +/- 3 mmHg. MAP had not fully recovered 4 days later. Some 2 h after losartan, the acute pressor response to angiotensin II had fallen from 24 +/- 2 mmHg to zero. This recovered to 13 +/- 5 and 28 +/- 2 mmHg 24 and 48 h post losartan. After losartan PRC rose from 0.1 +/- 0.05 to above 1 ng/ml per h for less than 24 h. CONCLUSION A single dose of losartan reverses both the fast and slow pressor effects of continuous angiotensin II infusions. While losartan is metabolized, the fast vasoconstrictor effect recovers quickly but the slow pressor effect takes almost a week to build up again to maximum levels. Since the slow pressor effect is mediated via the AT1 receptor, any means of blocking the renin-angiotensin system is likely to keep blood pressure below maximum hypertensive levels for several days after the drug has disappeared from the circulation.

Increased arterial pressure is required for myocardial infarction to occur in L-NAME + ang II-treated rats

Abstract P-570 Key Words: Renin Angiotensin System, Myocardial Infarction, Arterial Pressure