Jack D. Barrett

Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA.

The observation that nuclei from hepatic tissue exhibit specific angiotensin II (Ang II) binding led us to explore whether Ang II modulates mRNA in general, mRNA specific for renin system components, or both. Nuclei from hepatic tissue exhibited a single high-affinity (Kd = 0.4 nmol/L) Ang II-specific binding site, which was associated with increased RNA transcription. Whereas total RNA extracted from nuclei increased 1.5-fold in response to Ang II (10(-9) mol/L), specific mRNA for renin and angiotensinogen increased 7.8- and 2.5-fold, respectively. Ang II binding and induced transcription showed parallel Ang II dose responses that were both inhibited by 10(-5) mol/L DuP 753 or saralasin. Maximum Ang II binding and RNA transcription occurred at the same Ang II concentration (10(-9) mol/L). Higher doses of Ang II resulted in a progressive decrease in RNA transcription. Together, these results demonstrate that hepatic nuclei have functional Ang II-specific receptors. It is concluded that Ang II may elicit responses at nuclear receptors, which heretofore were associated only with Ang II receptors located on plasma membranes. However, the individual contribution of plasma and nuclear membrane Ang II receptors to the overall cellular Ang II transcriptional response and their possible interactions remain to be determined.

Hepatic angiotensin II nuclear receptors and transcription of growth-related factors

Objectives To investigate the influence of angiotensin II (All) receptors in isolated hepatic nuclei on other genes regulated by All and to determine whether the function of these intracellular receptors is influenced by alterations in the endocrine renin system. Methods Nuclei were isolated from hepatic tissue of normal and bilaterally nephrectomized or adrenalectomized Wistar rats. Following nuclear run-off, in the presence of varying All concentrations, specific messenger RNAs (mRNA) were determined by slot blot hybridization. Tissue levels of renin system components were measured by radioimmunoassay and nuclear receptors characterized by displacement of radiolabeled All with specific All receptor antagonists. Results All binding in the presence of DUP 753 and PD 123177 confirmed that nuclear All receptors can be classified as AT1 receptors and that as much as 10% of the specific binding is attributable to nuclear chromatin. All stimulated not only the production of mRNA for renin system components such as renin and angiotensinogen, but also that of mRNA for growth-related factors such as platelet-derived growth factor and the oncogene c-myc. Maximal stimulation occurred at 10−9 mol/l All; higher concentrations reduced this response. After stimulation or suppression of the plasma renin system by adrenalectomy or bilateral nephrectomy, nuclei isolated from rat hepatic tissue contained elevated endogenous levels of growth-related and renin system mRNA including AT1 and AT2 All receptors. However, despite the level of receptor mRNA having been elevated, the total All receptor density of isolated nuclei decreased. In addition, after both maneuvers, isolated nuclei were refractory to All-induced gene transcription. Conclusion The existence of mechanisms producing intracellular All and regulating its level, which in turn exert local regulatory responses via nuclear All receptors, lends significance to the presence of a functional intracrine renin system that could act in concert with or independently of the endocrine renin system.

Renal Effects of Nitrendipine Monotherapy in Essential Hypertension

Summary Ten male patients with essential hypertension were studied on a constant diet (100 mEq of sodium daily) while on placebo, immediately following a 20-mg oral dose of nitrendipine, and during 1 week of nitrendipine therapy (10 mg twice daily). After 2 weeks of outpatient follow-up on 20–40 mg nitrendipine daily, the patients were readmitted and restudied with the same protocol. Glomerular filtration rate, effective renal blood flow, blood volume, and cardiac output were determined isotopically. Free water clearance and osmolar clearance following a water load were also measured. Twenty-four-hour urinary creatinine and electrolyte excretion were measured during the hospitalization periods. Blood pressure decreased significantly during the acute and chronic periods when compared to placebo. During the first 7 days of nitrendipine treatment, urinary sodium excretion increased significantly from control, and a mean deficit of 148 ± 7 mEq (p < 0.001) was calculated. There was a mean weight loss of 1.0 ± 0.1 kg (p < 0.05) after a week of treatment. Glomerular filtration rate, renal blood flow, blood volume, cardiac output, plasma renin activity, plasma catecholamines, and urinary aldosterone remained unchanged from control in both the acute and chronic periods. Compared to the placebo period, free water clearance (1.8 ± 0.6 versus 2.9 ± 0.5 ml/min; p < 0.05) and osmolar clearance (1.9 ±0.3 versus 2.9 ± 0.5 ml/min; p < 0.05)increased significantly in the chronic period. The natriuresis and diuresis associated with nitrendipine therapy without any changes in renal function may represent a direct renal tubular effect of the drug.

Insulin-Mediated Growth in Aortic Smooth Muscle and the Vascular Renin-Angiotensin System

Insulin has been shown to directly affect blood vessel tone and to promote vascular hypertrophy, but the mechanism of these actions remains uncertain. Because angiotensin I (Ang I)-converting enzyme inhibitors have been shown to improve insulin action and to impede the progression of vascular hypertrophy in hypertensive animal models, it is possible that the vascular properties of insulin may be mediated through the tissue renin-angiotensin system (RAS). To evaluate this relationship, we first investigated the effect of insulin on components of the RAS using cultured rat vascular smooth muscle cells (VSMCs). Insulin treatment (1000 microU/mL) markedly increased angiotensinogen mRNA expression and angiotensinogen production. We next investigated the role of the RAS in insulin-mediated cell proliferation, using [3H]thymidine uptake. Studies were done both with insulin alone and in the presence of captopril (1x10(-7) to 10(-5) mol/L) and losartan (1x10(-9) to 10(-7) mol/L). [3H]Thymidine uptake was increased significantly by 1000 microU/mL insulin, and this stimulation was reduced by 1x10(-6) mol/L captopril (-38.8%, P<0.05) and by 1x10(-8) mol/L losartan (-37. 5%, P<0.05). Further studies showed that the degree of insulin-mediated [3H]thymidine uptake in VSMCs could be duplicated by 4x10(-10) mol/L Ang II. Losartan reduced the effects of both Ang II and insulin on [3H]thymidine uptake by about 40% to 45% of baseline (P<0.05). Captopril reduced insulin-mediated [3H]thymidine uptake but did not affect Ang II-mediated [3H]thymidine uptake. In summary, insulin induced significant stimulation of angiotensinogen expression and production and stimulated growth similar to that seen with Ang II in cultured rat VSMCs. Inhibition of Ang II production or its binding to the Ang II type 1 (AT1) receptor inhibited insulin-mediated growth in a fashion similar to that seen with inhibition of Ang II-mediated growth. Thus, insulin can modulate the vascular RAS, and the effect of insulin on vascular growth may be via direct effects on angiotensinogen expression and translation operative through both the AT1 receptor and the conversion of Ang I to Ang II.

Angiotensin-converting enzyme inhibition in the treatment of renal transplant erythrocytosis. Clinical experience and observation of mechanism.

Recent observations indicate that angiotensin-converting enzyme (ACE) inhibition corrects renal transplant erythrocytosis (RTE). The mechanism for this association is not known. We examined the effect of ACE inhibition on hematocrit, erythropoietin (EPO), and renin substrate. ACE inhibition has been reported to suppress renin substrate, which is known to stimulate EPO and erythropoiesis. In 15 patients with RTE, hematocrit dropped from 52.8 +/- 0.6 (SEM) to 45.8 +/- 1.4% after 8 weeks of treatment with Enalapril, 2.5-20 mg/day. Serum EPO (normal range: 9-30 mU/ml) was high in one, normal in seven, and low in seven patients. ACE inhibition reduced EPO in patients with initial high or normal levels but induced no change in patients with initial low levels. ACE inhibition had no significant effect on renin substrate. In one patient who rejected his first graft, erythrocytosis recurred following a second, successful transplant. Treatment was discontinued because of cough in two patients and symptomatic drop in blood pressure in one patient. We conclude RTE is not caused by hypererythropoietinemia. In patients with normal circulating EPO, erythrocytosis may result from an increase sensitivity to EPO, and ACE inhibition lowered hematocrit by further reduction of this hormone. However, the finding of erythrocytosis in half our patients with suppressed EPO, suggests the participation of non-EPO-mediated mechanism(s). The recurrence of RTE in a patient after a second transplant raises the additional possibility of patient-specific factors in the pathogenesis of this disorder. In contrast to other reports, we documented side-effects (cough, hypotension) in three (20%) of our patients. Our clinical experience, coupled with prior reports of spontaneous resolution of RTE in some patients, suggests that intermittent courses of ACE-inhibition may be the optimal strategy in the use of this form of therapy for RTE.

Insulin and insulin-like growth factor-I promotes angiotensinogen production and growth in vascular smooth muscle cells.

Background Circulating insulin and insulin-like growth factor-I (IGF-I) levels are increased in patients with hypertension and insulin resistance. Since both hormones are known to have cell growth-promoting effects, they may contribute to the progression of vascular hypertrophy in patients with insulin resistance. Insulin-mediated activation of the vascular renin–angiotensin system (RAS) stimulates growth in cultured rat vascular smooth muscle cells (VSMC). Objective In order to evaluate the role of IGF-I-mediated activation of components of the tissue RAS, we examined the effect of IGF-I receptor stimulation on cell proliferation, and production of angiotensinogen in cultured VSMC. Study Design Aortic VSMC were derived from male Sprague-Dawley rats. IGF-I and insulin-mediated DNA synthesis were estimated by 3 H-thymidine uptake (3H-TdR) with or without the angiotensin I converting enzyme inhibitor, captopril. Moreover, angiotensinogen released by the cells to the culture medium was determined by radioimmunoassay with or without the anti-IGF-I receptor antibody αIR3 or captopril. Results Both IGF-I and insulin increased 3H-TdR uptake by cultured rat VSMC (P < 0.05). Captopril blocked IGF-I and insulin-mediated 3H-TdR uptake (−34.4 ± 1.9% and −32.7 ± 1.8%, P < 0.05, respectively). IGF-I increased the angiotensinogen level in the medium by 30.6 ± 2.9% (P < 0.01). Insulin also stimulated angiotensinogen synthesis by 26.3 ± 2.2% (P < 0.01). Captopril and αIR3 significantly suppressed angiotensinogen production stimulated by both IGF-I and insulin. Conclusions These results indicate that IGF-I as well as insulin stimulates angiotensinogen production and growth in VSMC. Thus, both hormones may independently play a role in progression of the vascular hypertrophy and atherosclerosis in patients with hypertension and insulin resistance through activation of the tissue RAS.

Erythropoietin upregulates angiotensin receptors in cultured rat vascular smooth muscle cells

Objective Plasma renin is not elevated in recombinant human erythropoietin (rhEPO)-induced hypertension but angiotensin converting enzyme inhibitors reduce blood pressure in both human and animal studies. Since rhEPO elevates renin and angiotensinogen messenger RNAs in angiotensin II target tissues such as the aorta, we explored the actions of rhEPO on renin–angiotensin system-related gene transcription of cultured rat vascular smooth muscle cells. Design and methods To separate direct actions of rhEPO from those mediated secondarily by potential activation of the renin–angiotensin system, vascular smooth muscle cells were cultured with rhEPO and enalapril to inhibit the angiotensin converting enzyme and losartan to inhibit angiotensin II type 1 receptors. Results Vascular smooth muscle cells cultured with rhEPO (6–8 units/ml) demonstrated elevations (40–120%) in messenger RNAs of the renin-angiotensin system (renin, angiotensinogen, angiotensin receptor types 1 and 2) and increased levels of several messenger RNAs known to respond to angiotensin II (transforming growth factor-β, insulin-like growth factor-II, epidermal growth factor, c-fos and platelet-derived growth factor). In contrast, cells cultured in the presence of rhEPO and enalapril or losartan showed elevations of messenger RNA for only the two types of angiotensin II receptor. This increase was higher than that obtained when cells were cultured with rhEPO or either antagonist alone. The increase in specific binding of angiotensin II to cells cultured in the presence of rhEPO and enalapril or rhEPO and losartan paralleled the changes in receptor messenger RNA. Conclusions rhEPO exerts its primary action on vascular smooth muscle cells via an increase in angiotensin receptor messenger RNA, resulting in a parallel increase in angiotensin II receptor expression. We suggest that increased receptor expression secondarily mediates the expression of other renin-angiotensin system messenger RNAs, which leads to angiotensin II-responsive gene transcription. The elevation in angiotensin II receptors, as observed in response to rhEPO, may provide a mechanism by which other forms of renin-dependent hypertension are initiated.

A direct radioimmunoassay for human renin substrate and identification of multiple substrate types in plasma.

SUMMARY Plasma renin substrate, a widely measured parameter of the renin reaction, is quantitated indirectly by the measurement of liberated angiotensin I upon exhaustive incubation of plasma with added renin. To overcome methodological problems of this assay system, we have developed a direct radioimmunoassay for this plasma protein using renin substrate purified from pooled plasma of normotensive subjects as the antigen. Comparison of substrate quantitated by the two assay systems (direct and indirect) indicates a 1:1 correlation with the exception of certain subjects with elevated substrate levels induced by estrogen therapy. To study the possibility of multiple substrate forms, we have made a comparison of substrate quantitated by both radioimmunoassays in conjunction with electrophoresis of plasma on polyacrylamide gel. One major form of substrate with a retardation factor (Rf) = 0.60 was found in normotensive and essential hypertensive subjects which gave a 1:1 correspondence on quantitation by the two methods. In contrast, six of 16 women on oral contraceptives demonstrated three forms of substrate (Rf = 0.16, 0.35, and 0.60) on electrophoresis. Substrate with Rr = 0.16 and 0.35 did not cross-react with the antiserum prepared against substrate from normotensive subjects, implying structural differences in these proteins.

EXPRESSION OF RENIN AND ANGIOTENSINOGEN GENES IN PREECLAMPTIC AND NORMAL HUMAN PLACENTAL TISSUE

Objective: We have quantitated renin activity and angiotensinogen and measured the expression of renin and angiotensinogen genes in placentas from normal and preeclamptic subjects to determine if tissue renin system parameters are elevated in preeclampsia.Methods: Tissue renin activity and tissue levels of angiotensinogen were measured by enzymatic techniques, and renin and angiotensinogen mRNA were quantitated by blot hybrodization in placentae from young nulliparous black women with preeclampsia and normal controls.Main Outcome: Placental ischemia is considered one of the etiological factors of preeclampsia. All components of the renin system are present in human placenta, and enhanced expression of this system may contribute to placental ischemia.Results: Tissue active renin concentration was slightly elevated in preeclamptics (130 ± 20 ng Al/mg protein/h) versus controls (80 ± 20), but angiotensinogen levels did not differ. In preeclamptic placentae renin mRNA was also elevated (249 ± 35 versus 207 ± ...

Effects of trypsin on the measurement of inactive renin in rat plasma by radio-immunoassay: decrease in inactive renin following nephrectomy.

We have examined the effect of trypsin treatment of rat plasma on the rate of angiotensin (Ang) I generation and measurement of this peptide by radio-immunoassay. Trypsin increased the renin incubation blank but did not alter the kinetics of the renin reaction with exogenous renin. The quantity of immunoreactive material detected in trypsin-treated plasma was not proportional to the volume of plasma assayed. Consequently, the level of inactive renin was dependent upon the volume of plasma subjected to the assay. This discrepancy occurred with two independent radio-immunoassay systems. The rate of Ang I generation was linear and significantly elevated following the addition of renin substrate to trypsin-treated plasma. However, if trypsin degradation of endogenous renin substrate was extensive and additional renin substrate was not provided, non-linear rates of Ang I generation occurred. Multiple additions of trypsin were necessary to activate maximally inactive rat plasma renin. Inactive renin accounted for 79 +/- 2% of the total enzyme activity in normal rats. Although active renin declined following bilateral nephrectomy, the ratio of active to inactive renin did not change. The data suggest that the kidney is the primary source of inactive renin in the normal rat.