Are the antibodies to a peptide complementary to angiotensin II useful to isolate the angiotensin II receptor?
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Angiotensin II↗
Biomedical subjects
M de Gasparo
Publications and source records attributed to M de Gasparo.
Antialdosterones: incidence and prevention of sexual side effects.
The use of spironolactone in the treatment of hypertension has been limited by the occurrence of sexual side effects, mainly menstrual disturbances in women and gynaecomastia in men. In order to minimize this limitation on the use of an effective potassium-sparing antihypertensive agent, two strategies can be proposed: (1) A decrease in the daily dose of spironolactone. In 182 patients with essential hypertension treated with spironolactone alone for a mean follow-up period of 23 months, daily doses of 75-100 mg were as effective on blood pressure as doses of 150-300 mg. In contrast, the development of gynaecomastia--91 cases among 699 men--was dose-related in 6.9% (50 mg/day) to 52.2% (150 mg or more/day) of the cases. (2) An improvement in the receptor-binding specificity of spironolactone. Three 9 alpha, 11 alpha-epoxy derivatives have been characterized in vitro in rats and in rabbits. They exhibited a 3- to 10-fold decrease of the antiandrogenic and progestagenic effect, compared with spironolactone. In humans, one of these derivatives counteracted the fall in urinary Na/K ratio induced by 9 alpha-fluorohydrocortisone at a 25 mg dose.
Blood Pressure↗
Immunoradiometric assays of total renin and gonadotropins in serum during the menstrual cycle.
The contribution of the ovary to the circulating total renin pool is linked to its secretory activity in relation with its reproductive function. In a longitudinal study of 13 normal women, total renin levels measured in serum by an immunoradiometric assay were lower in the midfollicular phase (180 +/- 59 pg/ml) than in the midluteal phase (291 +/- 100 pg/ml). Peak levels were encountered 1 day after the luteinizing hormone (LH) surge (375 +/- 97 pg/ml). Rapid circhoral fluctuations were observed in all periods of the cycle, unrelated to the LH pulses. In case of ovarian function inhibition (induced by gonadotropin-releasing hormone agonists), total renin levels were lower than in the midfollicular phase (126 +/- 56 pg/ml). Low levels also were encountered in the prepubertal period (153 +/- 89 pg/ml). Very high levels (17,600 +/- 11,400 pg/ml) were found in follicular fluids from stimulated cycles. These results show that circulating total renin levels follow a complex pattern in which LH, but possibly also follicle-stimulating hormone and gonadal steroid hormones (estradiol and progesterone), may play a role.
Adult↗
Biochemical effects of prolonged renin inhibition in marmosets.
The renin inhibitor CGP 29,287 was administered continuously for 7 days (30 mg/kg per day, intraperitoneally, via osmotic minipumps) to normotensive marmosets fed a low-salt diet. As a control, another group of marmosets was given vehicle only. After 7 days of treatment, the mean arterial blood pressure of the CGP 29,287-treated marmosets was significantly lowered by about 23 mmHg. Plasma immunoreactive total and active renin were increased 10- and sevenfold, respectively, whereas plasma renin activity (PRA) was reduced by 95%. Plasma angiotensin II (Ang II) and aldosterone were also reduced (by 97 and 85%, respectively). Serum angiotensin converting enzyme activity was unchanged and there were no differences in plasma concentrations of electrolytes, urea or creatinine between CGP 29,287-treated and control marmosets. These results indicate that although renin release is markedly stimulated after continuous administration of a renin inhibitor for 7 days, the formation of Ang II and aldosterone, the active hormones of the renin-angiotensin system, is substantially reduced.
Aldosterone↗
Pharmacological investigations of a new renin inhibitor in normal sodium-unrestricted volunteers.
1. CGP 38 560 A, a low-molecular-weight, non-peptidic renin inhibitor, was well tolerated upon intravenous and oral administration to recumbent healthy volunteers on an unrestricted-sodium diet. 2. After intravenous infusion over 30 min at a rate of 100 ml h-1, doses of 50, 125 and 250 micrograms kg-1 appear to induce a long-lasting inhibition of plasma renin activity. Plasma angiotensin II was decreased in a dose-dependent manner during the infusion and thereafter reverted to the initial level. A concomitant dose-related increase in active plasma renin was observed. Blood pressure was unaffected. The plasma levels of CGP 38 560 reached during infusion were at least 2000-fold higher than the theoretical inhibitory concentration based on in vitro results. 3. After oral administration in doses of 50, 100 and 200 mg CGP 38 560 A, inhibition of plasma renin activity was observed, but plasma active renin was unchanged. Blood pressure also remained unaffected. 4. CGP 38 560 was rapidly cleared from plasma with a half-life of 7.6 min for the first phase and 63 min for the second phase. Plasma levels were 100-fold lower after oral administration than after infusion, indicating a low degree of absorption (less than 1% oral bioavailability).
Administration, Oral↗
Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma.
A radioinhibitor binding assay and an enzyme inhibition assay have been developed to measure plasma levels of CGP 38 560, a potent human renin inhibitor. The detection limit of the assays was between 0.5 and 1 pmol/ml. There was a good correlation (r = 0.989) between the two assays for the measurement of human plasma spiked with CGP 38 560 in concentrations from 1.9 nM to 12 microM. Intra-assay variability was 6.1-17.3% and 4.4-27.2% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Interassay variability was 6.0-28.2% and 3.8-28.4% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Blood samples were collected during a pharmacological study performed in normotensive human volunteers on an unrestricted diet who were infused during a 30-minute period with CGP 38 560 A (50 micrograms/kg). Similar values for the concentrations of renin inhibitor in plasma were obtained with the radioinhibitor binding assay and the enzyme inhibitor assay, and there was a significant correlation between values obtained with the two different methodologies (r = 0.94). The plasma levels of renin inhibitor reached a maximum at the end of infusion and then decreased rapidly, indicating a short plasma half-life. The changes in biochemical parameters, plasma renin activity, and plasma concentration of active renin could be related to the concentrations of CGP 38 560 measured in the plasma.
Angiotensin I↗
CGP 38 560: orally active, low-molecular-weight renin inhibitor with high potency and specificity.
CGP 38 560 is a low-molecular-weight (730) inhibitor of human renin that contains only one natural amino acid. In vitro, it is a potent inhibitor of human renin (Ki with tetradecapeptide, 0.4 X 10(-9) M). It has a high enzyme specificity (Ki values against human pepsin, gastricsin, and cathepsin D are 5 X 10(-6), 3 X 10(-6), and 0.6 X 10(-6) M) and is also species specific (IC50 values against human, marmoset, dog, and rat plasma renins are 7 X 10(-10), 7 X 10(-10), 7 X 10(-9), and 1 X 10(-6) M). In vivo, CGP 38 560 inhibits plasma renin activity (PRA) and lowers blood pressure (BP) after oral administration to conscious, normotensive, furosemide-pretreated marmosets. A dose of 10 mg/kg induces complete inhibition of PRA and a decrease in BP of 23 +/- 3 mm Hg (n = 4) after 30 min. These effects persist for up to 2 h. Blockade of the renin-angiotensin system appears to cause the hypotensive response since it is completely prevented by pretreatment with a converting-enzyme inhibitor. These findings demonstrate that the molecular size of renin inhibitors may be reduced to improve their oral activity without loss of potency or specificity.
Animals↗
Monoclonal antibodies to human renin: properties and applications.
A series of 11 different monoclonal antibodies generated against human kidney renin have been characterised. Their binding affinity, inhibition of renin activity, epitope distribution, crossreactivity with related enzymes and finally in vivo pharmacological effects were analysed. All antibodies were found to be specific for primate renin recognising 6 independent antigenic structures on the renin molecule. They expressed different effects on renin activity namely (1) no inhibition, (2) only partial, or (3) complete inhibition. Partially inhibiting antibodies demonstrated specific degrees of inhibition (30, 60 or 80%). One antibody, R-36-16, demonstrated an IC 50 of 1.3 X 10(-11) M/L and, when injected into marmosets, induced complete inhibition of plasma renin activity and reduction of blood pressure. Using a selected pair of antibodies a radioimmunoassay has been established providing a fast and highly reproducible determination of human and marmoset immunoreactive renin, detecting both active and inactive renin down to concentrations of 10 pg/ml (1.25 X 10(-17) moles of renin per 50 microliter sample).
Animals↗
Modification of the interaction of human renin with different substrates by monoclonal antibodies.
The mechanism by which anti-renin antibody inhibits renin activity was studied by following the kinetics of the reaction with angiotensinogen or a low molecular weight synthetic substrate, tetradecapeptide (TDP). Two monoclonal antibodies (70 pM) inhibited the production of angiotensin I from angiotensinogen but they differed when hog TDP was used as a substrate. R3-47-10 partially and non-competitively inhibited, whereas R3-36-16 stimulated the activity of renin. This is in contrast to the effects of the synthetic renin inhibitor, CGP 29 287, which competitively inhibits the enzyme activity with both substrates. These antibodies probably bind to the renin molecule on the flap which protects the active cleft. Angiotensinogen may be prevented from entering the cleft due to steric hindrance from bound antibody. However TDP, because of its smaller size may still be able to reach the catalytic site. In addition R3-36-16 might freeze the flap in an open position allowing a greater turnover of TDP whereas R3-47-10 may prevent the flap from fully opening and thereby hinder the reaction of TDP with the active site.
Angiotensinogen↗
Rapid measurement of total and active renin: plasma concentrations during acute and sustained converting enzyme inhibition with CGS 14824A.
Total and active renin were measured in plasma of 6 normal volunteers before and after acute and sustained angiotensin converting enzyme (ACE) inhibition with CGS 14824A (2 mg and 10 mg p.o. q.d.) or placebo treatment. The same sandwich technique was used for the measurement of both total and active renin using a polyacrylamide-iron-oxide linked monoclonal antibody to trap renin and 125I-labelled second monoclonal antirenin antibodies without or with specificity for active renin. Normal values for supine subjects ranged for active renin between less than 3 pg/ml and 28 pg/ml and for total renin between 73 and 263 pg/ml. Plasma ACE activity was clearly suppressed during 24 hours following both 2 mg and 10 mg CGS 14824A. Active plasma renin reached 6- and 12-fold normal values on days 1 and 7 of treatment with the 10 mg dose. Total renin rose to 150% and 228% respectively at the same time. Inactive renin continued rising during the first day of 10 mg CGS treatment to a final 141% at 24 hours post-drug and didn't change on day 7. Plasma renin activity correlated well with active renin levels (r = 0.92). We conclude that both total and active plasma renin concentrations can now be directly measured with great accuracy within 6 hours.
Adult↗
Three new epoxy-spirolactone derivatives: characterization in vivo and in vitro.
The use of spironolactone, the most commonly used antimineralocorticoid compound, is limited by the occurrence of sexual endocrine effects. New antagonists are therefore required which lack these unwanted effects. Three 9 alpha,11 alpha-epoxy-derivatives of known aldosterone antagonists (spironolactone, prorenone and mexrenone) have been characterised in vitro and in vivo. In each experiment spironolactone was run as a reference. The introduction of the epoxy-group only marginally affected the binding affinity of these compounds for the mineralocorticoid receptor, whereas it caused a decrease for the androgen and progesterone receptors of between 10- and 500-fold. In vivo, all three epoxy-derivatives (3 mg/kg) were potent aldosterone antagonists, 1 to 2 times the potency of spironolactone in the rat. Parallel to the decreased affinity for the androgen and progesterone receptor in vitro, there was a 3- to 10-fold decrease of the antiandrogenic and progestagenic effect compared to spironolactone in the rat and in the rabbit, respectively. Virtually no disturbance of the vaginal or ovulatory cycle was observed with either epoxymexrenone or epoxyprorenone, although epoxyspironolactone caused a 20% decrease in ovulation. It appears therefore that the 9 alpha, 11 alpha-position of the steroid structure is a site of the molecule which can be modified to improve the specificity of aldosterone-antagonists not only in vitro, but also in vivo.
Androgen Antagonists↗
Chromatographic characterization of adult and foetal rat insulin.
Foetal rat pancreatic rudiments explanted on day 14 of gestation were grown in organ culture in medium enriched with amino acids. The size of the insulin granules was increased, resulting in an insulin granule volume fraction greater than the volume fraction measured in pancreas grown in vivo. The pancreas was extracted and the insulin compared. Serial dilution curves of extracts of adult pancreas and pancreas grown in vitro are parallel in the insulin radioimmunoassay, whereas extracts of pancreas of foetus developing in utero appear immunologically different. Adult and foetal rat insulin (in utero) were purified using chromatography on OPTI UP C12, cellulose thin-layer chromatography plates, cellulose acetate foil electrophoresis and finally high-performance liquid chromatography. The ratio of insulin I to insulin II was found to be 1.5 for the adult and 2.7 for the foetus. These results show that there is an unequal expression of the two non-allelic genes controlling insulin biosynthesis in foetal and adult rat pancreas.
Animals↗
Localization of GABA high-affinity binding sites in the pancreas of neonatal rat.
The localization of gamma-aminobutyric acid (GABA) high-affinity binding sites was investigated in the exocrine and endocrine pancreas of neonatal rats by means of 3H-GABA autoradiography. GABA-binding was identified on Schwann cells and on the cells of the intralobular excretory ducts. In the endocrine part of the pancreas, no labelling was observed except in peripheral islet cells which, on the basis of their scarcity and distribution, could be somatostatin cells. Furthermore, peri-insular innervation showed considerable labelling.
Animals↗
Role of the adrenals in the development of streptozotocin (STR)-induced diabetes in male albino rats.
Diabetes induced by streptozotocin (STR) (55 mg/kg i.v.) in non-fasting, male albino rats is considerably mitigated by adrenalectomy. The timing of the operation is important for the effect of blood glucose. The later the adrenalectomy, the lesser the protection against STR. Plasma insulin in intact, STR-treated rats is significantly lower than in rats that have also been adrenalectomized. Pancreatic insulin in both groups is 10 times lower than in normal controls and adrenalectomized rats. Plasma FFA are distinctly elevated in intact, STR-treated rats. Adrenalectomy in these diabetic animals reduces plasma FFA to levels below those of normal controls. The changes in the glycogen contents of the diaphragm and myocardium of STR-treated rats are reversed by adrenalectomy. The rate of pancreatic insulin degradation might be retarded or the rate of secretion increased. In addition, sensitivity to insulin is exaggerated after adrenalectomy, as is evident from the changes in blood sugar and the glycogen contents of the organs. These changes in the effects of insulin are ascribed to deprivation of adrenal steroids and in particular glucocorticoids. Loss of the adrenal medulla is presumably one of the causes of the changes in plasma FFA.
Adrenal Glands↗
Effect of gliquidone on insulin binding to rabbit erythrocytes.
A study was made of the effects of acute or chronic treatment of rabbits with 10 mg gliquidone (Glurenorm)/kg body weight on erythrocyte insulin receptors. The binding of 125I-labelled insulin to the population of whole red blood cells was not affected by either treatment. Because of the heterogeneity of the erythrocytes, subpopulations were selected according to their specific gravity, which is directly related to cell age. The lowest-density fraction showed a 30% greater capacity of the insulin receptors without any change in affinity after chronic therapy with gliquidone, but no effects were observable after acute treatment. The highest-density fraction was not affected by either acute or chronic administration of gliquidone. Therefore, all other things being equal (blood sugar, plasma insulin levels), young erythrocytes are sensitive to sulphonylurea, whereas old ones are not. Irrespective of the compound tested, this observation suggests that receptor sensitivity might be tissue-specific or age-specific or both, and that the stage of maturation of a cell is a determining factor.
Animals↗
Sulfonyliminoimidazolidines, a new class of oral hypoglycemic agents. 3. Hypoglycemic activity and mode of action of 1-[p-[2-(crotonylamino)-ethyl]-phenylsulfonyl]-3-cyclohexyl-2-imino- imidazolidine (CGP 11 112).
1-[p-[2-(Crotonylamino)-ethyl]-phenylsulfonyl]-3-cyclohexyl-2-imino- imidazolidine (CGP 11 112) is a representative of a new class of oral hypoglycemic agents. It lowers blood glucose in normal animals and in streptozocin (streptozotocin)-diabetic rats. In normal mice, rats and dogs the hypoglycemic effect is more potent (range 3-30 times) and has a shorter duration than that of tolbutamide. CGP 11 112 increases plasma insulin after oral administration in normal animals and stimulates release of insulin in perifused rat islets in vitro (sulfonylurea-like effect). In streptozocin-diabetic rats CGP 11 112 decreases blood glucose with a potency comparable to that of phenformin. In contrast to phenformin, CGP 11 112 does not increase blood lactate in diabetic rats or inhibit intestinal absorption of glucose. An effect of CGP 11 112 on gluconeogenesis and glycogenolysis could not be demonstrated. It is suggested that the hypoglycemic activity in streptozocin-diabetic rats may be due to stimulation of glucose efflux from the circulation. In vitro, CGP 11 112 inhibits glucose oxidation and lipolysis in isolated rat fat cells.
Adipose Tissue↗
Effect of non-essential amino acids on fetal rat pancreatic growth and insulin secretion in vitro.
Fetal rat pancreatic rudiments explanted on day 14 of gestation were grown for 6 days in organ culture in medium containing glucose (5.5 or 16.5 mmol/l) and essential amino acids (3.5 or 13.1 mmol/l). Non-essential amino acids (alanine, aspartic acid, asparagine, glycine, proline and serine) were added to the culture medium in a number of combinations and at a maximum total concentration of 4.0 mmol/l. At the end of the period of culture rudiments were compared for DNA content, insulin concentration and quantitative morphology. The release of insulin from the rudiments was tested during 6-h incubations on day 6 of culture. Enrichment of the culture medium with any combination of non-essential amino acids had a slight or no effect on the growth or cellular composition of the rudiment of insulin release from it. Addition of all the non-essential amino acids to medium containing 16.5 mmol glucose and 13.1 mmol essential amino acids/l caused a dramatic reduction in the net insulin accumulation by the cultured rudiment. Combinations of non-essential amino acids in which one or more were omitted did not have the same effect. These findings suggest that fetal rat pancreas grown in vitro may require both essential and non-essential amino acids for the full expression of insulin biosynthesis and secretion.
Alanine↗
The timing of fetal B cell hyperplasia in diabetic rat pregnancy.
Fetal pancreatic development was measured in terms of total organ DNA and insulin concentration (ng/migrogram DNA) in the offspring of rats made mildly diabetic by intraperitoneal streptozotocin injection (60 mg/kg) on the first and second day of life. On day 20 the mean pancreatic insulin concentration of fetuses of diabetic mothers was significantly higher than that of controls (27.8 versus 20.8 ng/microgram DNA) but no significant difference was observed on day 18 (4.47 versus 4.68 ng/microgram DNA), day 16 (0.64 versus 0.77 ng/microgram DNA) or day 14 (0.04 versus 0.03 ng/microgram DNA). No significant difference in total pancreatic DNA was observed between test and control animals on day 14, 16, 18 or 20 of gestation. It is concluded that maternal streptozotocin diabetes alters the development of the differentiated fetal B cell (20 days) but has no effect on the protodifferentiated B cell (14 days), or during the period of secondary transition (16-18 days).
Animals↗