Angiotensin I to angiotensin II conversion in the human forearm and leg. Effect of the angiotensin converting enzyme gene insertion/deletion polymorphism.

OBJECTIVE: The angiotensin-converting enzyme (ACE) gene I/D polymorphism accounts for part of the variation in ACE concentration; subjects with one or two D alleles have approximately 25 and 50% higher ACE levels, respectively, than subjects with two I alleles. Data from studies on the pressor effects of angiotensin (Ang) I in DD compared with II subjects are inconsistent, because enhanced conversion in DD subjects may have been masked by a decreased responsiveness to Ang II. Here we quantify ACE genotype-related Ang I to Ang II conversion in the human forearm and leg using non-pressor 125I-Ang I infusions. DESIGN AND METHODS: Infusions were given to 12 women and 17 men (age 24-67 years) who were undergoing renal vein sampling followed by renal angiography for diagnostic purposes. 125I-Ang I was infused for 20 min into the right antecubital vein, and blood samples for the measurement of 125I-labelled and endogenous Ang I and Ang II were taken from the aorta, the left antecubital vein and a femoral vein under steady-state conditions. Genotype frequencies were determined by polymerase chain reaction. RESULTS: Fractional conversion (i.e. the percentage of arterially delivered 125I-Ang I that is converted to 125I-Ang II) in the forearm (38+/-4, 30+/-3 and 31+/-6% in 8 II, 16 ID and 5 DD subjects, respectively; mean +/- SEM) and leg (52+/-4, 48+/-3 and 42+/-5%) was similar in all three groups. In addition, no genotype-related differences in plasma Ang II/I ratio (a measure of ACE activity) were observed at the three sampling sites. CONCLUSIONS: Regional Ang I to Ang II conversion does not parallel the previously described D allele-related differences in ACE concentration, suggesting that effects other than enhanced conversion may underlie the reported associations between the D allele and various cardiovascular diseases.

AT1 receptor A/C1166 polymorphism contributes to cardiac hypertrophy in subjects with hypertrophic cardiomyopathy.

The development of left ventricular hypertrophy (LVH) in subjects with hypertrophic cardiomyopathy (HCM) is variable, suggesting a role for modifying factors such as angiotensin II. We investigated whether the angiotensin II type 1 receptor (AT1-R) A/C1166 polymorphism, the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism, and/or plasma renin influence LVH in HCM. Left ventricular mass index (LVMI) and interventricular septal thickness were determined by 2-dimensional echocardiography in 104 genetically independent subjects with HCM. Extent of hypertrophy was quantified by a point score (Wigle score). Plasma prorenin, renin, and ACE were measured by immunoradiometric or fluorometric assays, and ACE and AT1-R genotyping were performed by polymerase chain reactions. The ACE D allele did not affect any of the measured parameters except plasma ACE (P<0.04). LVMI was higher (P<0.05) in patients carrying the AT1-R C allele (190+/-8.3 g/m2) than in AA homozygotes (168+/-7.2 g/m2), and similar patterns were observed for interventricular septal thickness (23.0+/-0.7 versus 21. 6+/-0.7 mm) and Wigle score (7.0+/-0.3 versus 6.3+/-0.3). Plasma renin was higher (P=0.05) in carriers of the C allele than in AA homozygotes. Multivariate regression analysis, however, revealed no independent role for renin in the prediction of LVMI. Plasma prorenin and ACE were not affected by the AT1-R A/C1166 polymorphism, nor did the ACE and AT1-R polymorphisms interact with regard to any of the measured parameters. We conclude that the AT1-R C1166 allele modulates the phenotypic expression of hypertrophy in HCM, independently of plasma renin and the ACE I/D polymorphism.

Activation of overexpressed receptors for insulin and epidermal growth factor interferes in mitogenic signaling without affecting the activation of p21ras.

Activated receptors with a tyrosine kinase activity induce a variety of responses like changes in the differentiation and mitogenic status of cells. These responses are mediated in part by p21ras. Some of these activated receptors induce in certain cell types a pronounced, but transient, increase in Ras-GTP. We have stimulated cells with insulin, epidermal growth factor (EGF), and fetal calf serum (FCS), and the mitogenic response, as reflected by stimulation of [3H]thymidine incorporation, was compared with the magnitude of the transient increase in Ras-GTP levels. Cell lines were used that expressed both physiological and elevated numbers of p21ras and receptors for insulin and EGF, respectively. In all the examined cell lines 9% FCS did not induce a marked increase in Ras-GTP despite its high mitogenic potency. Pronounced increases in Ras-GTP levels were observed in insulin-stimulated CHO cells which overexpress insulin receptors whereas in the parental CHO cells only a small increase is seen. Insulin (1 microM) and FCS (9%) stimulate [3H]thymidine incorporation in parental CHO cells to a similar high level whereas in insulin receptor overexpressing CHO cells the maximum of insulin-stimulated [3H]thymidine incorporation is only 55% of the level reached by 9% FCS. In those cells the maximum is already reached at low (1 nM) insulin concentrations. Remarkably, at higher insulin concentrations stimulation of [3H]thymidine incorporation starts to decrease strongly despite the fact that the magnitude of the transient increase in Ras-GTP and subsequent MAPkinase activation increases. Similarly, when EGF receptors are overexpressed in Rat-1 cells, the mitogenic response is also decreased at higher EGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal-growth-factor receptors generate Ras.GTP more efficiently than insulin receptors.

Activation of the Ras proto-oncogene contributes in general to mitogenic activation of cells. We show here that epidermal growth factor (EGF) stimulates Ras.GTP formation very efficiently in a variety of cell lines expressing endogenous EGF receptors only. Maximal activation of the receptor converts up to 65% of cellular p21ras from the GDP form into the active GTP-bound state. This efficient activation occurs also in cultured primary human fibroblasts. Maximal insulin-induced Ras.GTP formation is less but in cells overexpressing the insulin receptor a similar high response of Ras.GTP formation is observed after insulin stimulation. Not only the efficiency but also the kinetics by which the EGF and insulin receptors stimulate Ras.GTP formation are quite distinct. In the Rat-1-derived cell line, H13IR2000, overexpressing both p21Ha-ras and the insulin receptor, the activated insulin receptor generates approximately 1 mol Ras.GTP/mol activated insulin receptor. The activated EGF receptor amplifies the signal, resulting in the activation of approximately 40 mol p21ras/mol receptor. Moreover, EGF-stimulated generation of Ras.GTP is transient with a maximum after 2 min of hormone stimulation and diminishes to near basal levels within 1 h whereas the insulin-induced Ras.GTP levels are maximal at 5-10 min and decline only slowly to half-maximal in 1 h. Desensitization of the EGF pathway by prolonged EGF stimulation, prevents subsequent stimulation of Ras.GTP formation by newly added EGF but not by insulin. Vice versa, in cells preincubated with insulin for 1 h, EGF stimulates Ras.GTP formation to near maximal values. These observations indicate that desensitization by prolonged hormone incubation does not involve inactivation of common signaling intermediates but rather components, specific for each pathway, like the particular receptors. The rapid down regulation of EGF receptors compared to insulin receptors corroborate this possibility. The observed high potency of EGF receptors to generate Ras.GTP may explain the, in general, stronger mitogenic activity of EGF compared to insulin.

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation.

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell line, most of the p21ras was rapidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109). To determine whether this process also occurs in cells expressing physiologically relevant numbers of insulin receptors, insulin stimulated Ras.GTP formation was quantitated in Chinese hamster ovary (CHO)-derived cell lines expressing varying numbers of insulin receptors. In the parental CHO9 cells, expressing only 5.10(3) insulin receptors, insulin stimulation for 3 min increased Ras.GTP levels with 10%. Upon increasing the number of insulin receptors in these cells, Ras.GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. These data show that receptor overexpression is not a prerequisite for insulin-stimulated Ras.GTP formation. The yield of Ras.GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CHO9- and NIH3T3-derived cell lines, respectively. These values argue against signal-amplifying processes between the insulin receptor and p21ras. To determine whether receptor autophosphorylation is required for Ras.GTP formation, NIH3T3 cells overexpressing insulin receptors were stimulated with a monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser1200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects in CHO cells, were used. In both cases, no Ras.GTP formation was observed. Furthermore, Rat-1-derived cell lines expressing mutant p21ras, which is permanently in the active GTP-bound form, still responded to insulin by increasing the glucose uptake. These results support our hypothesis that Ras.GTP formation is activated by the tyrosyl-phosphorylated insulin receptor and suggest that an active Ras.GTP complex does not mediate metabolic signaling.

The role of ras proteins in insulin signal transduction.

Ras-proteins are guanine nucleotide binding proteins, which, in the GTP bound state emit a strong mitogenic signal. In the GDP bound state, the protein appears inactive. We have found that stimulation by insulin of cells expressing elevated levels of insulin receptors results in a rapid conversion of Ras-GDP into Ras-GTP. This process is part of the signalling pathway leading to immediate-early gene expression and a mitogenic response. There seems to be no involvement of Ras-GTP formation in the process of insulin stimulated glucose transport. Though the precise mechanism by which Ras is converted to the GTP bound state remains to be established, a tight correlation exists between receptor autophosphorylation and Ras-GTP formation.

Analysis of doxorubicin, 4'-epidoxorubicin, and their metabolites by liquid chromatography.

An analytical method based on isocratic HPLC separation and fluorescence detection was developed to allow for sensitive and specific analysis of anthracyclines and their metabolites in plasma and urine. The method is particularly advantageous when comparing the metabolism and/or pharmacokinetics of analogues, such as doxorubicin [(8S, 10S)-10-[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)- oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-meth- oxy-5,12-naphthacenedione] (1) and 4'-epidoxorubicin (2), since both drugs and their metabolites can be analyzed under identical conditions. The analytical properties of 1, 2, and eight metabolites were studied in plasma, serum, buffer solution, and urine. The detection limit in plasma was 4 X 10(-8) M for the glucuronides, 7 X 10(-9) M for the glycosides, and 1 X 10(-9) M for the aglycones. In plasma, 1, 2, doxorubicinol (3), 4'-epidoxorubicinol (4), doxorubicinone (5), and doxorubicinol aglycone (6) showed a linear concentration-response relationship from their detection limit up to 5 X 10(-6) M. A linear calibration graph for plasma samples was also obtained for 7-deoxydoxorubicinone (7) and 7-deoxydoxorubicinol (8); however, these compounds had a significantly lower upper limit (5 X 10(-7) M). Urine samples were acidified to pH 2.5 and analyzed by HPLC without further purification. A linear calibration curve was obtained in the clinically relevant range. The detection limit in urine was approximately 5 X 10(-8) M. Plasma and urine of two patients who had received 4'-epidoxorubicin by iv bolus injection were analyzed.