The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.     

Somatomedin levels in the diagnosis and therapy of growth hormone deficiency

Serum somatomedin-C (SM-C) and somatomedin (SM) concentrations were measured by, respectively, radioimmuno (SM-C RIA) and radioreceptor assays (SM RRA) in 3 groups of children with short stature. The patient population was different from previously reported series in that it was urban Brazilian, low income, and significantly older. Group A consisted of 6 male and 3 female children, aged 7.7-16.0 years, whose average peak plasma immunoreactive growth hormone (GH) was above 10 ng/ml. Group B contained 8 male and 5 female untreated GH-deficient patients, ranging in age from 9.5 to 21.0 years. In Group C there were 4 male and 1 female GH-deficient subjects treated with I.M. injections of GH (0.1 U/kg) from 1 month to 7 years. The mean +/- SE basal RIA SM-C (ng/ml) concentrations were significantly lower in groups B (34.2 +/- 8.8) and C (43.8 +/- 13.7) than A (214.3 +/- 42.7): A X B, P less than 0.001 and A X C, P less than 0.02. Likewise the mean +/- SE basal RRA SM (ng/ml) concentrations were significantly lower in groups B (78.9 +/- 17.6) and C (90.8 +/- 19.3) than group A (316.3 +/- 43.0): A X B, P less than 0.001 and A X C, P less than 0.002. A significant linear correlation was observed between RIA and RRA in group B (r = 0.84; P less than 0.001) and C (r = 0.96; P less than 0.01), but not for A (r = 0.61; P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential levels of "urotensin-II-like" activity determined by radio-receptor and radioimmuno-assays

Plasma and urinary levels of "urotensin(U)-II-like" substances determined in healthy human volunteers were 12.4 +/- 0.6 ng/ml and 2.2 +/- 0.3 ng/ml by RIA, an order of magnitude lower than that seen by RRA, 167.5 +/- 9.5 ng/ml and 65.2 +/- 4.3 ng/ml. HPLC demonstrated the existence of at least three prominent activity peaks in plasma and urine, the more hydrophobic of which did not co-elute with U-II, degradation products or URP. RRA and RIA recognized these peaks with contrasting efficacy. As such, published levels of "U-II-like" activity should be interpreted with caution until a better understanding is obtained regarding what species specific RIA and RRA assay reagents interact with.     

Angiotensin-(1--7) in the ovine fetus

In the adult animal, ANG-(1-7) may counterbalance some effects of ANG II. Its effects in the fetus are unknown. Basal ANG-(1-7), ANG I, ANG II, and renin concentrations were measured in plasma from ovine fetuses and their mothers (n = 10) at 111 days of gestation. In the fetus, concentrations of ANG I, ANG-(1-7), and ANG II were 86 +/- 21, 13 +/- 2, and 14 +/- 2 fmol/ml, respectively. In the ewe, concentrations of ANG I were significantly lower (20 +/- 4 fmol/ml, P < 0.05) as were concentrations of ANG-(1-7) (2.9 +/- 0.6 fmol/ml), whereas ANG II concentrations were not different (10 +/- 1 fmol/ml). Plasma renin concentrations were higher in the fetus (4.8 +/- 1.1 pmol ANG I x ml(-1) x h(-1)) than in the ewe (0.9 +/- 0.2 pmol x ml(-1) x h(-1), P < 0.05). Infusion of ANG-(1-7) (approximately 9 microg/h) for a 3-day period caused a significant increase in plasma concentrations of ANG-(1-7) reaching a maximum of 448 +/- 146 fmol/ml on day 3 of infusion. Plasma levels of ANG I and II as well as renin were unchanged by the infusion. Urine flow rate, glomerular filtration rate, and fetal arterial blood pressure did not change and were not different than values in fetuses receiving a saline infusion for 3 days (n = 5). However, the osmolality of amniotic and allantoic fluid was significantly higher in fetuses that received ANG-(1-7). Also, compared with the saline-infused animals, mRNA expression levels of renin, the AT(1) receptor, and AT(2) receptor were elevated in kidneys of fetuses that received infusions of ANG-(1-7). Infusion of an ANG-(1-7) antagonist ([D-Ala(7)]-ANG-(1-7), 20 microg/h) for 3 days had no effect on fetal blood pressure or renal function. In conclusion, although infusion of ANG-(1-7) did not affect fetal urine flow rate, glomerular filtration rate, or blood pressure, changes in fetal fluids and gene expression indicate that ANG-(1-7) may play a role in the fetal kidney.     

Measurement of human FSH by radioreceptor assay

We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15 x 10(-10) M. The standard curve was obtained with 0.2-25.6 ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the non-specific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intra-assay and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.     

Radioreceptor assay for atrial natriuretic factor

Interest in accurate measurement of atrial natriuretic factor (ANF) in biological fluids and various tissues has been stimulated by recent data indicating the possible role of ANF in the homeostasis of salt and water. The presence of high-affinity binding sites for ANF in rat glomeruli has allowed us to develop a rapid, sensitive, and simple radioreceptor assay (RRA). A saturable high-affinity binding site on the membranes of rat glomeruli has been characterized by a dissociation constant of 33 pM and binding capacity of 396 fmol/mg protein. Rat plasma extracts or atrial homogenates or standards were incubated with radioiodinated ANF and a preparation of rat glomerular membranes. The receptor-bound and free radioactivity were separated by filtration on Whatman GF/C paper after 1 h incubation at room temperature. The sensitivity of the RRA was 2.08 fmol. The effective concentration of standard ANF that displaced 50% of labeled receptor-bound ANF (EC50) was 43.3 +/- 2.6 fmol/ml (n = 7). Both intra- and interassay coefficients of variation were smaller than 11%. This RRA assay has been compared with radioimmunoassay (RIA). High correlations for 19 plasma extracts and 34 atrial homogenates (r = 0.973 and r = 0.954, respectively) tested by RRA and RIA were obtained. This good correlation between the two methods suggests that the immunoreactive material found in rat plasma and atrial homogenates also displays biological activity.     

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 μl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC₅₀ values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC₅₀ values obtained correlated well with those obtained using EIA methodology (r² = 0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.     

Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1

It is well known that nonselective, nonsteroidal anti-inflammatory drugs inhibit renal renin production. Our previous studies indicated that angiotensin-converting enzyme inhibitor (ACEI)-mediated renin increases were absent in rats treated with a cyclooxygenase (COX)-2-selective inhibitor and in COX-2 -/- mice. The current study examined further whether COX-1 is also involved in mediating ACEI-induced renin production. Because renin increases are mediated by cAMP, we also examined whether increased renin is mediated by the prostaglandin E(2) receptor EP(2) subtype, which is coupled to G(s) and increases cAMP. Therefore, we investigated if genetic deletion of COX-1 or EP(2) prevents increased ACEI-induced renin expression. Age- and gender-matched wild-type (+/+) and homozygous null mice (-/-) were administered captopril for 7 days, and plasma and renal renin levels and renal renin mRNA expression were measured. There were no significant differences in the basal level of renal renin activity from plasma or renal tissue in COX-1 +/+ and -/- mice. Captopril administration increased renin equally [plasma renin activity (PRA): +/+ 9.3 +/- 2.2 vs. 50.1 +/- 10.9; -/- 13.7 +/- 1.5 vs. 43.9 +/- 6.6 ng ANG I x ml(-1) x h(-1); renal renin concentration: +/+ 11.8 +/- 1.7 vs. 35.3 +/- 3.9; -/- 13.0 +/- 3.0 vs. 27.8 +/- 2.7 ng ANG I x mg protein(-1) x h(-1); n = 6; P < 0.05 with or without captopril]. ACEI also increased renin mRNA expression (+/+ 2.4 +/- 0.2; -/- 2.1 +/- 0.2 fold control; n = 6-10; P < 0.05). Captopril led to similar increases in EP(2) -/- compared with +/+. The COX-2 inhibitor SC-58236 blocked ACEI-induced elevation in renal renin concentration in EP(2) null mice (+/+ 24.7 +/- 1.7 vs. 9.8 +/- 0.4; -/- 21.1 +/- 3.2 vs. 9.3 +/- 0.4 ng ANG I x mg protein(-1) x h(-1); n = 5) as well as in COX-1 -/- mice (SC-58236-treated PRA: +/+ 7.3 +/- 0.6; -/- 8.0 +/- 0.9 ng ANG I x ml(-1) x h(-1); renal renin: +/+ 9.1 +/- 0.9; -/- 9.6 +/- 0.5 ng ANG I x mg protein(-1) x h(-1); n = 6-7; P < 0.05 compared with no treatment). Immunohistochemical analysis of renin expression confirmed the above results. This study provides definitive evidence that metabolites of COX-2 rather than COX-1 mediate ACEI-induced renin increases. The persistent response in EP(2) nulls suggests involvement of prostaglandin E(2) receptor subtype 4 and/or prostacyclin receptor (IP).     

Biological activity of the seminal plasma of alpacas: stimulus for the production of LH by pituitary cells

South American camelids are induced ovulators and require a stimulus to trigger the LH surge responsible for the ovulation. Seminal plasma (SP) of fertile alpacas (Lama pacos) was tested using a bioassay of pituitary cells to study the effect of seminal plasma on LH release. Plates containing rat pituitary cells (2 x 10(5) cells/90-95% viability) were cultured adding: (A) whole SP (WSP) treated with charcoal-dextran, or 1:2 or 1:4 proportions diluted in culture medium (DMEM/HEPES + antibiotics), or (B) 1:2 SP + anti-GnRH rabbit serum (inhibitory potency 10(-5) M), or (C) 1:2 SP + anti-GnRH + 100 nM synthetic GnRH (buserelin acetate) or (D) 100 nM, 50 nM, 10 nM, and 1 nM synthetic GnRH. Concentration (ng/ml) of LH secreted (Sec) and contained (Con) was analyzed using RIA 125I and the percentage of Sec and Con in each experiment was determined. The results of LH Sec for the cells treated with 50, 10, and 1 nM GnRH were 39, 13, and 1.5%, respectively (r2 = 98.41%, r = 0.9920) but cells treated with 100 nM GnRH secreted 10% of LH. With WSP, 1:2, or 1:4 SP the LH Sec was of 44.5% (3.25 ng/ml), 27% (1.9 ng/ml), and 18% (1.2 ng/ml), respectively. The exposure of cells to 1:2 SP + anti-GnRH, or to 1:2 SP + anti-GnRH/100 nM GnRH produced 31% (2.20 ng/ml) and 30% (1.8 ng/ml) of LH Sec, respectively. These results suggest that the SP of alpacas could have some factor(s) different from GnRH that would contribute to the mechanisms of LH secretion and to the induced ovulation in the female alpaca.     

Serum prolactin and growth hormone determined by radioimmunoassay and a two-site immunoradiometric assay: comparison with the Nb2 cell bioassay

Serum levels of the two lactogenic hormones prolactin (PRL) and growth hormone (GH) were compared when determined by radioimmunoassay (RIA) and two-site immunoradiometric (IRMA) assays in 83 normal premenopausal women. The mean values for the PRL and GH results determined by RIA were higher than those obtained by IRMA, despite strong correlations between the two (PRL, r = 0.92; GH, r = 0.79). The lactogenic hormones were also determined together by the Nb2 cell bioassay (BA) in 38 of these same women, and the results compared with the sum of the PRL and GH immunoassays. There was a strong correlation between the BA and RIA (r = 0.75), and the BA/PRL+GH RIA ratio averaged 1.6 +/- 0.5. Corresponding values for IRMA were r = 0.66, and BA/PRL + GH IRMA 3.3 +/- 1.1. Thus, the polyclonal RIA antisera appeared to recognize bioactive hormone components not determined by the double monoclonal antibody IRMA. Another 23 women at risk for familial breast cancer, and 14 cystic breast disease patients were also studied. High BA, but normal RIA results, giving mean ratios of 2.4 +/- 1.1 and 3.6 +/- 3.0 respectively, suggest the presence of a further variant with high bioactivity not detected by RIA in these two clinical situations.     

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland

1. To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10-1,000 nM NPY in the presence or absence of 10 microM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA). 2. NPY (10-1,000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1,000 nM), enhanced NE stimulation of melatonin production. 3. NPY (1,000 nM) impaired 3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with 3H-NE. 4. These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.     

A rapid simple method for the assay of renin in rabbit plasma

1. EDTA (10mm), 2,3-dimercaptopropan-1-ol (10mm) and chlorhexidine gluconate (0.005%, w/v) cause complete inactivation of plasma enzymes that degrade angiotensin I, but have no effect on the reaction of renin with its substrate. The reagents were termed the selective inhibitors. 2. Thus it is possible to measure renin in plasma by its ability to catalyse the release of angiotensin I. 3. Sterile plasma, treated with the selective inhibitors, is incubated with renin substrate (500-1000ng. of angiotensin content/ml.) at pH6 at 42 degrees for 6hr. 4. Under these conditions the reaction obeys first-order kinetics. Renin activity is calculated in terms of the percentage release of the angiotensin content/hr. 5. As described, the assay is sufficiently sensitive to measure renin in the plasma of all normal rabbits. By extending the length of the incubation, much lower activities can be measured.     

Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.     

Homologous radioimmunoassay and radioreceptorassay of gonadotropic hormone for an Indian carp Catla catla

A homologous radioimmunoassay (RIA) for Indian carp (C. catla) gonadotropin (GtH) was developed by using purified Catla GtH and its specific antiserum. In Ouchterlony agar diffusion, antisera raised against purified Catla GtH (cGtH), showed clear crossreaction. Catla-anti GtH (anti-cGtH) did not cross-reacted with Catla TSH enriched fraction. Immunocrossreaction was further confirmed with competitive binding inhibition studies where displacement of radiolabelled cGtH was precisely linear against increasing concentrations of cGtH, hence this was later used as standard curve for RIA. Competitive binding inhibition was also observed with purified murrel GtH, silver carp GtH and Cyprinus GtH, using varied doses. Both murrel and silver carp GtH showed clear parallelism with cGtH, while Cyprinus GtH inhibition slope was less steeper. Mammalian GtHs (hCG, oLH, oFSH), bTSH and bPRL had no crossreaction with anti-cGtH. Radioreceptorassay (RRA) for cGtH was developed by preparing plasma membranes from Catla oocytes. Binding of 125I-cGtH to oocyte plasma membranes showed saturability with high affinity (Ka = 0.11 X 10(13)M-1) and low capacity (17 fmol/mg protein). Displacement of 125I-cGtH in receptorassay by cold cGtH was linear and therefore served as standard curve. The interassay and intrassay variability in RIA was 7.9% and 3% while that of RRA was 5% and 3% respectively. Sensitivity of RIA was in the picogram level whereas it was in nanogram level by RRA. Determination of carp pituitary and serum GtH content by RIA and RRA showed the consistency, precision and validity of these assays. Although RRA was comparatively less sensitive than RIA, it was convenient, quick and less expensive.     

A novel and sensitive radioreceptor assay for serum melatonin levels.

A simple and sensitive radioreceptor assay (RRA) has been developed to measure melatonin levels in serum. The assay is based on competition between 2-[125I]iodomelatonin ([125I]MEL) and melatonin for binding to high-affinity binding sites in chick forebrain. To measure the amount of melatonin present in a serum sample, it was extracted with dichloromethane and added to the assay medium. The percentage inhibition of radioligand binding in the presence of the extracted serum was determined and compared to the percent displacement by known amounts of melatonin in a standard curve. There was little or no cross-reactivity with other structurally related compounds. The sensitivity of the assay is approximately 1.5pg/0.15 mL and the intra- and inter-assay variations are approximately 8%. Since the RRA results are comparable to that of an established radioimmunoassay (RIA), it provides a sensitive and rapid alternative to the more time consuming RIA.     

Characterisaton of human plasma thiol proteinase inhibitors.

Earlier, we had reported purification of three thiol proteinase inhibitors (TPI-1 of 70 kDa, TPI-3 of 195 kDa and TPI-4 of 497 kDa) from human plasma. In the present study we report that TPI-1 binds to papain in the stoichiometry ratio (E/I) of 1:1 while TPI-3 and TPI-4 bind in the ratio of 1.5:1 and 3.2:1 respectively. The K(m) for papain with BAPNA as substrate and Kcat/K(m) values for TPI-1, TPI-3 and TPI-4 were 2.7 x 10(-6) M, 0.84 nM/sec; 3.2 x 10(-6) M, 0.75 nM/sec; and 3.6 x 10(-6) M, 0.72 nM/sec respectively. The Ki values were found to be 1.48 nM for TPI-1, 0.133 nM for TPI-3 and 0.117 nM for TPI-4. The UV absorption and fluorescence emission spectra study suggest involvement of aromatic residues in the binding process. This study suggests that TPI-4 is the most potent inhibitor of thiol proteinases.     

Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3alpha-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3alpha-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 microl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 +/- 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 +/- 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3alpha-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.     

Processing of angiotensin peptides by NG108-15 neuroblastoma x glioma hybrid cell line

The metabolism of angiotensin (Ang) peptides was studied in NG108-15 neuroblastoma x glioma hybrid cells which express Ang II receptors, renin, dipeptidyl carboxypeptidase A (converting enzyme), as well as Ang I and Ang II. In these experiments, 0.2 nM of either 125I-Ang I or 125I-Ang II was incubated with intact cell monolayers and the medium was analyzed for 125I-products by high performance liquid chromatography. The major product generated from the metabolism of labeled Ang I or Ang II was identified as the amino-terminal heptapeptide Ang-(1-7). N-benzyloxycarbonyl-prolyl-prolinal (ZPP), a specific inhibitor of prolyl endopeptidase, inhibited the formation of Ang-(1-7) from Ang I by 35%. Complete inhibition of Ang-(1-7) generation was attained with p-chloromercuriphenyl-sulfonate, which suggests that a sulfhydryl-containing peptidase other than prolyl endopeptidase is also involved in Ang-(1-7) formation. Ang II was observed to be a minor product resulting from Ang I metabolism. Although the converting enzyme inhibitor enalaprilat (MK-422) significantly reduced Ang II formation, it had no effect on the levels of Ang-(1-7). These findings demonstrate a preferential processing of Ang I into Ang-(1-7) which is not dependent on the prior formation of Ang II.     

A primate bioassay for the determination of renin inhibitory peptides in serum

The study of renin inhibitory peptides (RIPs) in rodents and primates requires the establishment of a simple, high volume method for determining the concentration of RIPs in serum after intravenous or oral dosing. The human renin inhibition assay useful for rodents is not directly applicable to primates due to inherent production of angiotensin I from the primate serum angiotensinogen and added recombinant human renin. Therefore, a novel approach to analyze the serum concentrations of RIPs in primates is described based on in vitro studies with monkey serum. The procedure involves the inactivation of monkey angiotensinogen and monkey renin by thermal denaturation prior to analysis. Application of this assay was demonstrated by analyzing serum samples from an in vivo study in monkeys using ditekiren (U-71,038), a renin inhibitory peptide, and by validation of the assay and results using a tritium-based radioimmunoassay (RIA) for ditekiren. The minimum detectable limit of ditekiren for both the RIA and the bioassay for primates was 10ng/ml serum. The reported bioassay should be of value for monitoring serum levels of thermostable RIPs from pharmacokinetic, bioavailability, and pharmacodynamic studies in primates as well as in humans.     

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg(-1) and 326 U mg(-1) respectively. The optimum pH range was from 7-8.5 at 37 degrees C and the optimum temperature was 50 degrees C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608 +/- 0.07 mM and 249 sec(-1) respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68 +/- 12.55 nM and 6.763 +/- 0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17beta-estradiol, progesterone, and prostaglandins E2 and F2alpha was determined. The data showed a modulation of 17beta-estradiol, progesterone and prostaglandin E2 production by ovary ACE.