Mohinder P. Sambhi

Estrogen replacement therapy by transdermal estradiol administration

To determine whether the nonoral administration of estradiol (E2) might provide physiologic replacement without alteration of hepatic function, 20 postmenopausal women were studied before and after 3 weeks of treatment with either E2-containing transdermal therapeutic systems or placebo. Twenty premenopausal women were also studied. With E2-containing systems, serum E2 and estrone levels were restored to the premenopausal range. Variable responses of the different biochemical and biologic markers of the actions of E2 were observed. The most sensitive marker was vaginal cytology, with the E2 dosage reverting the maturation index to premenopausal values. Hot flashes, measured objectively, were reduced in frequency but not abolished. Serum levels of follicle-stimulating hormone and luteinizing hormone were lowered but remained higher than the premenopausal range. No significant changes were noted in urinary calcium/creatinine and hydroxyproline/creatinine ratios, which were used as markers of bone resorption. With active systems, no significant changes were noted in the concentrations of the hepatic proteins renin substrate and thyroxine-binding globulin or in the binding capacities of cortisol-binding globulin and sex hormone-binding globulin. These results indicate that transdermal E2 administration may be used to provide estrogen replacement while exerting limited effects on hepatic function.

Hyporeninemic Hypoaldosteronism in Diabetes Mellitus: Studies of the Autonomic Nervous System's Control of Renin Release

The proposal that neurogenic mechanisms may contribute to suppressed plasma renin activity in diabetic patients with hyporeninemic hypoaldosteronism was examined. Five hyperkalemic, diabetic patients with mild to moderate renal insufficiency and evidence of autonomic or peripheral neuropathy were studied. Mean plasma renin (1.1 ± 0.2 ng/ml/h)andaldosterone(2.8 ± 0.8 ng/dl) values were low and showed minimal increase after a low-sodium diet. Sequential renin responses during 4 h upright posture were significantly diminished in all patients compared with control subjects. The normal increment in plasma norepinephrine after upright posture and exercise was also blunted in the patients. Infusion of the beta-adrenergic agonist isoproterenol at a dose to augment pulse rate by 20% of baseline produced a rapid twofold increase in renin in control subjects. The patients had no renin response to isoproterenol infusion at doses that elicited similar cardiovascular responses in both groups. These findings suggest that in hyporeninemic hypoaldosteronism associated with diabetes mellitus and neuropathy, an alteration in sympathetic nervous system activity may contribute to the decreased plasma renin levels. The diminished catecholamine response to upright posture could lead to a blunting of renin response during orthostasis and other maneuvers. Secondly, loss of renin response to isoproterenol infusion in the face of normal cardiovascular responses is consistent with a localized intrarenal defect at the beta-adrenergic receptor level or at more distal sites in patients with hyporeninemic hypoaldosteronism.

Multiple forms of human plasma renin substrate.

The objective of this investigation was to determine whether heterogeneity of plasma renin substrate could be observed in states of steroid excess and various forms of hypertensive disease. In states of stimulated renin substrate production by estrogens or glucocorticoids, multiple forms of renin substrate were apparent when stimulation was excessive. Stimulation of substrate production caused by uremia associated with hypertension showed similar results. None, or only trace quantities of the additional forms of renin substrate were evident in subjects with normal or suppressed levels of plasma renin substrate. The additional forms of renin substrate could be distinguished from the normal form on the basis of cross-reactivity with a specific antiserum to the normal form, electrophoretic mobility, and kinetic rate constants. Differences in rate constants of the various forms of plasma renin substrate may account for the altered rate of the renin reaction associated with several states of hypertension. In plasma of patients with renovascular hypertension, significant quantities of a protein which cross-reacted with the antiserum but could not generate angiotensin I were observed.

Increased EGF binding and EGFR mRNA expression in rat aorta with chronic administration of pressor angiotensin II

This study examines the changes in the mRNA expression of epidermal growth factor (EGF), EGF receptor (EGFR), platelet derived growth factor (PDGF-B), and transforming growth factor beta (TGF-beta 1) before and after sustained pressor infusion of angiotensin II (Ang II) for 4 weeks. A threefold increase occurred in the levels of EGFR mRNA (17,240 +/- 827 vs 6403 +/- 1372 units, P less than 0.01) and TGF-beta 1 mRNA (1644 +/- 584 vs 475 +/- 30 units, P less than 0.01) only in the aorta and not in the heart and kidney tissues. This increase in both of the above mRNA transcripts highly correlated (r = 0.96 and 0.92, P less than 0.01) with the elevation of blood pressure. The specific binding of 125I-labeled EGF to aortic membranes also increased (11,429 +/- 728 vs 8630 +/- 420 cpm/mg protein, P less than 0.05) with a parallel increase in the protein tyrosine kinase activity of the membranes indicating that the enhanced EGFR mRNA expression resulted in increased activity of a functional receptor. No significant changes were observed in either EGF mRNA or PDGF-B mRNA levels. These findings suggest that EGFR and TGF-beta 1 participate in the long-term progressive pressor response to Ang II and thus potentially in the progression and the maintenance of chronic hypertension.

Influence of angiotensinase inhibitors on the enzymatic activity of renin

Abstract Asp 1 -Ileu 5 -angiotensin I, added to pooled normal human plasma at pH 5.5 or at 7.4, was shown to be fully protected from enzymatic degradation and conversion to angiotensin II by addition to plasma of a combination of angiotensinase inhibitors including ethylenediaminetetraacetic acid, diisopropylfluorphosphate, dimercaptopropanol, and 8-hydroxyquinoline hemisulfate. Addition of Dowex resin to the incubation media (pH 5.5) did not increase the protection of angiotensin I afforded by this combination of inhibitors. For these studies, both bioassay and radioimmunoassay were employed, which allowed for evaluation of N-terminal and C-terminal alteration of the peptide. In the presence of Dowex resin (pH 5.5), plasma renin activity was shown to be depressed. By the use of an angiotensinase-free isolated system of human angiotensinogen and homologous renin, this decrease in activity was found to be due to binding of renin, and not of the renin substrate, to the resin. At pH 5.5, although 3.4 m m 8-hydroxyquinoline was shown to exert maximal inhibition of angiotensin degradation, yielding an increase in measured plasma renin activity, at higher concentrations of 8-hydroxyquinoline an overall inhibition of angiotensin generation was observed. At pH 7.4, on the other hand, 8-hydroxyquinoline inhibited renin activity at all concentrations studied. The inhibition of renin activity at pH 7.4 was minimized by reduction of the 8-hydroxyquinoline concentration to 1.7 m m . This latter concentration was of equal effectiveness in preventing angiotensin degradation. Although protection of angiotensin I was complete at pH 5.5 and 7.4, it was found that plasma renin activity was increased at pH 7.4. It was concluded that maximization of angiotensin I generation in plasma, by endogenous renin, was dependent not only on the pH of incubation, but also upon the type and concentration of angiotensinase inhibitors employed which affect the renin reaction itself.

An increase in high-molecular weight renin substrate associated with estrogenic hypertension.

We have previously reported that estrogens have the potential to induce new forms of renin substrate in addition to elevating the major circulating form of this protein. One of these estrogen-induced forms had a molecular weight in excess of 150,000. In this study we have compared the plasma concentration of the high-molecular-weight renin substrate in normotensive women receiving estrogen therapy and women with estrogenic hypertension. A statistically significant elevation of this protein was associated with estrogenic hypertension and normotensive pregnant women at term. This form of renin substrate differed from the major form with respect to electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. In addition, kinetic analysis indicated that this high-molecular-weight substrate has a significantly higher affinity for the enzyme renin than the major circulating form (Km = 1800 +/- 290 versus 3520 +/- 260 ng angiotensin I equivalents/ml). These results suggest that in addition to renin substrate concentration, substrate composition may play an important role in blood pressure regulation.

Renin activation in the venous plasma from the involved kidney in the patient with renal hypertension.

Measurements of plasma renin activity (PRA) in renal vein blood from the ischemic kidney are reported to be generally higher than from the contralateral kidney. Importance of factors other than renin content of renal venous plasma has not been investigated. Initial rate measurements of angiotensin generation with added excess of homologous renin (Plasma Renin Substrate Activity [PRSA]-20 min) were made in bilateral renal venous plasma from 31 patients suspected of suffering from unilateral renal hypertension. The mean values from the involved vs. the contralateral kidney were 551 vs. 331 ng respectively of angiotensin II equivalents generated per milliliter of plasma per 20 min of incubation. The measurement of maximal angiotensin generation under the same conditions with incubation prolonged to 3 hr (PRSA-180 min), however, were bilaterally equal in renal venous plasma from selected patients with renal hypertension who showed distinct differences in PRSA-20 min and PRA measurements. Prior extraction of plasma lipids did not significantly change the bilateral renal venous PRSA-20 min determinations. Stimulation of endogenous renin release in normal dogs did not change the PRSA determinations. The data suggest strongly the presence of a renin activating mechanism in the renal venous plasma from the involved kidney of patients with renal hypertension.

Heterogeneity of renin substrate released from hepatocytes and in brain extracts.

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17 beta estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000-65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.

Renin substrate and the renin-angiotensin system in hog tissues.

The individual components of the renin-angiotensin system has been identified in numerous tissues. In this study we have examined whether a functional renin-angiotensin system is operative in several hog tissues including brain, aorta, and liver. The contribution of tissue renin substrate to the rate of local angiotensin generation was also assessed. Electrophoretic differences in plasma and tissue renin substrates, indicating structural differences, were employed as an index of independence of the tissue system from that of the peripheral circulation. Our results indicate that all tissues studied had the potential to locally generate angiotensin and that renin substrate limited to rate of the renin reaction in these tissues. Electrophoretic parameters, polyacrylamide gel electrophoresis, and isoelectric focusing suggest that the tissue renin systems are of local origin. The potential magnitude of local angiotensin production is such that tissue renin-angiotensin systems may significantly contribute to the control and regulation of blood pressure and other regulatory mechanisms influenced by angiotensin.

The influence of plasma lipoproteins on the renin reaction in normal human plasma.

Abstract Several studies have indicated that lipids isolated from plasma and kidney may be potential inhibitors of the renin reaction and that the increased renin reactivity observed in hypertensive subjects may be due to the lack of a plasma lipid found in normotensive subjects. In the present study lipoproteins were removed from normal human plasma by using density gradient centrifugation ( d = 1.195 g/ml, 360,000 g × 45 hr). This experimental design more closely resembles physiological conditions. Following dialysis against 0.15 m NaCl, the kinetic parameters of plasma renin in delipidated plasma and fresh plasma which was enriched with lipoproteins were compared. The renin reactivity and Michaelis-Menten kinetics of normal human plasma were not altered by the removal of lipoproteins ( K m = 2464 ng AI/ml, V max = 6.3 ng AI/ml/min). Only at abnormally high concentrations of added lipoproteins was inhibition of the renin reaction evident ( K m = 886 ng AI/ml, V max = 2.8 ng AI/ml/min). These results indicate that lipids of normal human plasma bound to apolipoproteins at normal physiological concentrations do not have an inhibitory effect on the renin reaction.