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 3H-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 alphaIR3 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 alphaIR3 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-beta, 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.

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.

Cigarette smoke increases gastric ulcer size in part by an angiotensin II-mediated mechanism in rats.

To assess the mechanism of the effect of cigarette smoke on ulcer disease we employed a rat model in which cigarette smoke increases the size of acetic acid-induced gastric ulcer and decreases the hyperemia at the ulcer margin. We postulate that cigarette smoke increases angiotensin II (a vasoconstrictor) in ulcer tissue. Since direct measurement of angiotensin II in small tissue samples is problematic, we compared the messenger ribonucleic acid (mRNA) for its precursors (angiotensinogen and renin) in ulcer and normal gastric tissue. We also evaluated the effect of enalapril, which blocks the conversion of angiotensin I to angiotensin II on ulcer size. In the ulcer tissue, cigarette smoke produced a significant increase in mRNA for angiotensinogen but not for renin. Enalapril decreased the size of the gastric ulcer in rats exposed to cigarette smoke. The data support the possibility that in ulcer tissue cigarette smoke stimulates an angiotensin II-mediated mechanism, which may in part be responsible for the impairment of ulcer margin hyperemia and aggravation of ulcer size.

The effectiveness of various nitrogen sources in white spruce [Picea glauca (Moench) Voss] somatic embryogenesis.

The effects of glutamine-based dipeptides, glutamine and casein hydrolysate, as well as the deletion of organic nitrogen, were investigated during white spruce [Picea glauca (Moench) Voss] somatic embryogenesis. There were no differences in the fresh weight increase of the tissue masses grown on initiation medium with different combinations of organic nitrogen. This was also the case for subsequent growth on kinetin medium, except that glutamine alone produced a significantly lower fresh weight increase than the other organic nitrogen combinations. Without organic (i.e. with only inorganic) nitrogen in the medium, the fresh weight increase was significantly less than with organic nitrogen on both initiation and kinetin medium. No differences were found between the dry/fresh weight ratios obtained with the various nitrogen treatments. The number of mature embryos produced per gram fresh weight when cultured in the absence of organic nitrogen was significantly higher than that obtained in its presence. There were no differences in the total number of mature embryos produced in cultures grown with various organic nitrogen combinations or without organic nitrogen. There were large clone differences with respect to the number of mature somatic embryos per gram tissue and the total number of somatic embryos produced. Hence, nitrogen type influences culture growth rate but not the number of mature somatic embryos produced. The latter was clone dependent.

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.

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.

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.

Inactivation of renin substrate by soybean trypsin inhibitors: implications for measurement of circulating inactive renin.

Semipurified soybean trypsin inhibitor added to rat and human plasma leads to a concentration dependent decrease in the rate of angiotensin I generation. This inhibition is due to binding of renin substrate to the inhibitor. Renin substrate present in nephrectomized rat plasma was more susceptible to binding than substrate of the normal rat suggesting structural differences in the substrate generated following nephrectomy. Because trypsin inhibition is necessary for measurement of active and inactive renin, we examined several alternate trypsin inhibitors. The Bowman-Birk inhibitor from soybean had similar actions as purified soybean trypsin inhibitor while trypsin inhibitors from lima bean and chicken did not depress renin substrate, but did have variable effects on the measured levels of active and total plasma renin. Surprisingly, crude soybean trypsin inhibitor did not suppress renin substrate and actually increased angiotensin I generation during PRA and PRC measurements. Since the crude preparation did not suppress renin substrate, changes in the specificity of the inhibitor may occur during its purification. The augmentation of PRA and PRC may be related to angiotensinase inhibitory actions.

Influence of recombinant human erythropoietin on blood pressure and tissue renin-angiotensin systems.

In humans, blockade of the renin-angiotensin system with angiotensin converting-enzyme inhibitors (ANG CEI) prevents the rise in blood pressure associated with the administration of recombinant human erythropoietin (rhEPO). This study was conducted to determine whether rhEPO elevates blood pressure in normal Wistar rats and whether the renin-ANG system is affected. Groups of 10 rats each were given rhEPO, ANG CEI (enalapril), rhEPO + ANG CEI, or vehicle. Renin and/or renin substrate mRNA was measured in aortas, kidney, and heart; renin activity (PRA), inactive renin, and renin substrate were measured in plasma. rhEPO raised blood pressure in the normal rat without changing the plasma renin system. ANG CEI prevented this blood pressure rise. Renin-specific mRNA was increased by rhEPO in renal tissue, and renin substrate mRNA was significantly elevated in the kidney and aorta. mRNA for renin and renin substrate were not altered in the heart. In both aorta and kidney, a significant correlation was observed between renin substrate mRNA and blood pressure. The data indicate that rhEPO modulates specific tissue renin-ANG systems, which may contribute to blood pressure elevation.

Trypsin activation of inactive renin: plasma blanks and angiotensin I radioimmunoassay.

Divergent conclusions exist as to whether inactive renin is present in nephrectomized rat plasma. A major factor contributing to this conflict may be related to significant changes in the "plasma blank" when trypsin-treated plasma is subjected to angiotensin I (AI) radioimmunoassay (RIA). In normal, but not nephrectomized rat plasma, AI-like substances are present in direct proportion to active renin. These substances are destroyed by trypsin. However, trypsin generates additional AI-like material, in both normal and nephrectomized rat plasma. This material, which is present in proportion to the renin substrate concentration, does not appear to be tetradecapeptide (TDP). In normal plasma, however, exogenous TDP is converted to AI in proportion to the active renin concentration and AI generation from TDP is increased by activation of inactive renin. However, in nephrectomized rat plasma, no AI generation from TDP was evident either before or after trypsin treatment. The coincident tryptic generation of a substance that quenches the levels of AI detected by RIA, combined with significant changes in the levels of endogenous and trypsin generated AI-like substances, may have significant bearing on the measured levels of inactive renin.

Production of angiotensinogen and renin-like activity by rabbit proximal tubular cells in culture.

Recent studies suggest the presence of local angiotensin generating system in the kidney. By using in situ hybridization technique, mRNA for angiotensinogen has been shown to be present in the proximal tubule. In the present study, we have attempted to examine the production of angiotensinogen and renin-like activity by the proximal convoluted (PCT) and straight (PST) tubular cells. PCT and PST cells were obtained from microdissected rabbit proximal tubules and cultured in vitro. Angiotensinogen and renin-like activity were quantitated in culture media and cell lysates. It was found that PCT culture medium contained both angiotensinogen and renin-like activity, whereas only angiotensinogen was detected in PST culture medium. Support for de novo synthesis is provided by the observation that both angiotensinogen and renin-like activity in PCT culture medium increased in a time-dependent and hormone-sensitive manner in defined serum-free medium. These results thus demonstrate the actual production of angiotensinogen and renin-like activity by proximal tubular cells, and indicate that these locally synthesized components may contribute to the regulation of angiotensin generation in renal proximal tubule.

Hormonal correlates of weight loss associated with blood pressure reduction.

This study investigated changes in plasma norepinephrine and the renin-angiotensin-aldosterone system during weight loss. Subjects were maintained on a hypocaloric and low sodium diet for 12 weeks. During weight loss statistically significant decreases in blood pressure, aldosterone, plasma renin activity, and norepinephrine were evident. Plasma renin substrate was suppressed from week one to eight and returned to control levels by week twelve. The data indicate that a reduction in the activity of the renin-angiotensin-aldosterone system, modulated by circulating norepinephrine and plasma renin substrate, may significantly contribute to the fall in blood pressure associated with weight loss.

Production of angiotensinogen by cultured rat aortic smooth muscle cells.

This study was conducted to further investigate angiotensinogen synthesis in rat aortic smooth muscle cells (SMC) grown in culture. tissue cultures maintained in defined medium neither grew nor synthesized angiotensinogen. However, in the presence of 5% homologous serum both cell proliferation and angiotensinogen synthesis became apparent. Substitution of normal control serum with that of bilaterally nephrectomized rats or animals given dexamethasone (10mg/kg, ip) led to a further significant increase in angiotensinogen production. In contrast, serum from adrenalectomized rats suppressed angiotensinogen synthesis below the rate observed with normal serum. A positive linear correlation (r = 0.96, p less than 0.01) was evident between the serum angiotensinogen level and the rate of de novo synthesis of this protein. No correlations were found between cell proliferation and either angiotensinogen synthesis or serum angiotensinogen levels. Dexamethasone added to serum did not stimulate the rate of angiotensinogen synthesis and appeared to inhibit cell proliferation. Stimulation or suppression of angiotensinogen synthesis was not accompanied by a statistically significant change in angiotensinogen specific mRNA. The data indicate a complex regulation of angiotensinogen in vascular smooth muscle cells in culture.

Renin substrate release in response to perturbations of renin-angiotensin system.

The role of the kidney in the regulation of plasma renin substrate and the release of this protein from rat liver slices were investigated. Physiological and pharmacological maneuvers were performed to perturb the renin-angiotensin system, including 24-h bilateral nephrectomy and furosemide administration to change renin and adrenalectomy and administration of dexamethasone to alter renin substrate. In the normal physiological range of plasma renin activity (PRA), no statistically significant linear relationship was evident between PRA and either plasma renin substrate or renin substrate released from liver slices. Stimulation of renin substrate by nephrectomy and dexamethasone administration were additive. Nephrectomy was also shown not to induce the release of a renin substrate-stimulating factor.

Independent change of plasma and tissue renin in response to anesthetics.

It was the purpose of this study to determine whether acute increases of endogenous plasma renin induced by administration of anesthetics cause simultaneous increases of the enzyme in aortic tissue of the rat. Administration of CO2 did not alter plasma or tissue renin. Ether, Innovar and Nembutal increased active plasma renin and had variable effects on the inactive form of the enzyme. Only Innovar, a combination of droperidol and fentanyl, increased aortic renin. The active component of Innovar was shown to be droperidol, which also increased aortic renin in 24 hour nephrectomized animals. The increase of aortic renin was, therefore, independent of changes of circulating active or inactive renin. The increase of tissue renin following droperidol was rapid, suggesting activation of an inactive tissue renin.

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.

The influence of aging on angiotensinogen production by rat vascular smooth muscle cells in vitro.

Aortic smooth muscle cells from 3, 12 and 24 mo old rats were grown in cell culture. All of the cultures produced angiotensinogen, but cultures from 24 mo old rats produced significantly more (p less than 0.01). These results suggest that angiotensinogen is synthesized in arterial smooth muscle. Furthermore, when the activity of the plasma renin system decreases with advancing age, increased activity of a local angiotensin system may contribute to the regulation of vascular tone.

Stimulation of brain and aortic renin substrate by central administration of steroids in the rat.

The influence of central and peripheral injections of dexamethasone and 17 beta-oestradiol on plasma, brain and aortic renin substrate was studied in the rat. Whereas intraperitoneal or intravenous injection of these steroids raised renin substrate in plasma, brain and the aorta, intraventricular injection raised it only in the brain and aorta. Bilateral nephrectomy produced results equivalent to those observed following intraperitoneal administration of steroids. Dissociation of the plasma and tissue renin-substrate levels in response to central administration of steroids indicates local synthesis of this protein, possibly under central control.