Biomedical subjects

H Ong

Publications and source records attributed to H Ong.

At least 37 records · Page 2Linked to original sources

PubMed1998

Identification of a pituitary growth hormone-releasing peptide (GHRP) receptor subtype by photoaffinity labeling.

Hexarelin, an analogue of GHRP-6, in which D-Tryptophan has been replaced by its 2-methyl derivative, is known to release growth hormone (GH) in vivo and in vitro by direct action on receptors present in anterior pituitary cells. Measurement of second messengers (c-AMP, Ca++, IP3) upon somatotrophs stimulation, suggests the existence of more than one GHRP receptor subtype. In order to document such an hypothesis, we have used a new photoactivatable derivative of Hexarelin, Tyr-Bpa-Ala-Hexarelin. This derivative was shown to be fully active in the release of GH in vivo with neonate rats. Using this photoactivatable ligand, we have specifically labeled a protein with an apparent Mr of 57,000 in human, bovine and porcine anterior pituitary membranes. Hexarelin and the spiroindoline sulfonamide MK-0677 displaced the Mr-57,000 photolabeled band with an apparent ED50 of 6x10(-7) M and 2x10(-5) M respectively. Taking into account the high efficiency (>60%) of covalent incorporation of the Bpa residue, this photoactivatable Hexarelin derivative has allowed the identification of a pituitary GHRP receptor subtype, which is apparently distinct from the recently cloned GH secretagogue receptor.

Animals↗

PubMed1998

Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland.

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.

Adult↗

PubMed1997

Radioimmunoassay of meterelin and pharmacokinetics after single injection and implant administration in dogs.

A sensitive and specific radioimmunoassay for a novel luteinizing-hormone-releasing-hormone (LHRH) agonist, [2-Me-D-Trp6, DesGly10]LHRH ethylamide (meterelin), was developed for documenting the pharmacokinetic parameters of this peptide following its intravenous (iv) and subcutaneous (sc) administration in dogs. The assay was also used for monitoring meterelin in plasma following its release from d,l-lactide-glycolide implants in the same species. Rabbit antisera generated against [DespyroGlu1] meterelin and conjugated to bovine serum albumin with glutaraldehyde showed high specificity, whereas crossreactivity to LHRH and its fragments and to analogs with substitutions at residues 6 and 10 was found insignificant. The assay was validated in terms of accuracy (recovery range, 94.0-105.4%), in terms of precision (intra- and interassay variations of 10.0-12.4% and 8.6-11.3%, respectively), and in terms of sensitivity (minimum detectable dose of 2.7 pg/assay). Following iv acute administration, a biexponential decline of plasma meterelin levels was observed, with distribution and elimination half-lives of 5.9 +/- 2.5 and 106 +/- 22 min, respectively. After sc acute administration, the elimination half-life was in the range of 103 to 173 min. The systemic clearance (CLT) ranged from 1.6 to 2.6 mL/min/kg, and the volume of distribution at steady state (Vdss) varied from 285 to 438 mL/kg. The elimination half-life (T1/2 beta), Vdss, and ClT were not significantly different after both routes of administration over the 1-100-microgram/kg dose range of peptide studied. Castrate levels of testosterone were attained 10 days after sc administration of the implant, lasted for up to 247 days, and were well correlated with plasma levels of meterelin.

Amino Acid Sequence↗

PubMed1997

Hepatic extraction of hexarelin, a new peptidic GH secretagogue, in the isolated perfused rat liver.

PURPOSE: To assess the hepatic extraction of hexarelin (HEX), a novel peptidyl GH secretagogue, in the isolated perfused rat model and document the in vitro binding of HEX to plasma proteins using plasma from rats, dogs, pigs, and humans. METHODS: Rat liver was perfused in situ using a recirculating system. The recirculating perfusate consisted of a Krebs Henseleit buffer containing 20% (v/v) prewashed bovine red blood cells, 1% albumin, and lg/L dextrose. Three HEX concentrations of 5, 50, and 500 ng/ml were examined. In vitro plasma binding was determined by the ultrafiltration method. RESULTS: The disappearance rate constant (K), half-life (t1/2), clearance (Cl), and hepatic extraction ratio (E) were: K = 0.013-0.014 min-1, t1/2 = 45-55 min, Cl = 0.345-0.401 ml/min/g liver, and E = 19-21% for the different concentrations of HEX. A linear increase in AUC (270-24334 min pmol/ml) was observed with increasing concentrations. Binding of HEX to plasma proteins of rats, dogs, pigs, and humans was 68.7 +/- 0.8%, 78.7 +/- 0.6%, 67.3 +/- 0.7%, and 65.2 +/- 0.6% respectively. Plasma binding was concentration-independent in the range between 0.003-3 microM for the four species examined. CONCLUSIONS: These results show that 1) the hepatic extraction of HEX is low, 2) the hepatic clearance is concentration independent up to 500 ng HEX/ml of perfusate, and 3) the plasma protein binding of HEX is significant over the dose range studied. HEX exhibits a low hepatic extraction ratio, allowing us to predict that its hepatic clearance may be limited upon HEX protein binding.

Animals↗

PubMed1997

Down-regulation of the hepatic cytochrome P450 by an acute inflammatory reaction: implication of mediators in human and animal serum and in the liver.

1. Infection and inflammation trigger a cascade of mediators that eventually will down-regulate the hepatic cytochrome P450 (P450). The present study aimed to characterize the mediators contained in the serum of rabbits with an acute inflammatory reaction (AIR) induced by the s.c. injection of turpentine (5 ml), and in the serum of humans with an acute upper respiratory tract viral infection. 2. Hepatocytes from control (H(CONT)) rabbits and rabbits with an AIR (H(INFLA)) were isolated and cultured. Compared with H(CONT) in H(INFLA) the production of theophylline metabolites, 3-methylxanthine (3MX), 1-methyluric acid (1MU), and 1,3-dimethyluric acid (1,3DMU) was reduced as was the amount of total P450, while lipid peroxidation was increased. Incubation of H(INFLA) with serum of rabbits with an AIR (RS(INFLA)) for 4 h further reduced the formation of the metabolites of theophylline as well as the amount of P450, and enhanced the lipid peroxidation. RS(INFLA) obtained 6, 12 and 24 h after the injection of turpentine showed the same ability to down-regulate hepatic P450 as the serum obtained at 48 h. 3. The efficacy (Emax) of RS(INFLA) to inhibit the formation of theophylline metabolites differed, i.e. 1,3DMU > 1MU > 3MX, and the potency of serum mediators (IC50) was similar for 3MX and 1MU, but lower for 1,3DMU. 4. Incubation of serum of human volunteers (HS(INFLA)) with a viral infection with H(CONT) or H(INFLA) reduced the production of theophylline metabolites, as well as the amount of P450, and increased the lipid peroxidation. HS(INFLA) depressed 1,3DMU more efficiently than 3MX and 1MU. HS(INFLA) reduced 3MX with greater efficacy than did RS(INFLA). Potency was very variable but not different from rabbits. 5. It is concluded that the serum of rabbits with an AIR or of humans with a viral infection contain several mediators that inhibit noncompetitively various isoenzymes of the hepatic P450. The decrease in P450 induced by HS(INFLA) or RS(INFLA) is closely associated with the increase in lipid peroxidation (r2= 0.8870) suggesting that lipid peroxidation could directly or indirectly be involved in the P450 down-regulation.

Animals↗

PubMed1997

Comparative effect of pituitary adenylate cyclase-activating polypeptide on aldosterone secretion in normal bovine and human tumorous adrenal cells.

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.

Adrenal Cortex Neoplasms↗

PubMed1996

Localization by photoaffinity labeling of natriuretic peptide receptor-A binding domain.

A portion of the ligand binding domain for atrial natriuretic peptide (ANP) was identified as an affinity cross-linked proteolytic fragment of bovine adrenal natriuretic peptide receptor type-A (NPR-A). Affinity purified NPR-A was UV-cross-linked to the amino terminus of 125I-[Tyr2] rat ANP-(2-27). A chymotryptic fragment of the affinity labeled NPR-A was isolated by chromatography and electrophoresis. This fragment yielded a major microsequence corresponding to a region from Met173 to Phe188 of the receptor extracellular domain and containing one N-glycosylation site at Asn180. Bovine NPR-A receptor was then cross-linked to the carboxy terminus of the highly efficient photoaffinity derivative 125I-[Tyr18,Bpa27] rat ANP(1-27). Proteolysis of the affinity labeled NPR-A with cyanogen bromide and trypsin produced radiolabeled and glycosylated fragments of size 15 and 9 kDa, respectively, which contained the epitope Ile181-Phe188 (CS328) and which were detectable by immunoprecipitation with a monospecific polyclonal antibody against CS328. Proteolysis with cyanogen bromide followed by Glu-C produced a shorter photolabeled 6 kDa fragment which was not immunoprecipitable by anti-CS328 antibody and which was not glycosylated. The results lead to the identification of the short segment Asp191-Arg198 as the site of covalent binding of [Tyr18,Bpa27] rat ANP(1-27). This hydrophilic region is adjacent to the epitope Ile181-Phe188 and to the glycosylation site Asn180. It displays the species variability and the high surface probability expected for a portion of the binding domain of NPR-A in contact with ANP.

Affinity Labels↗

PubMed1996

The H295R human adrenocortical cell line contains functional atrial natriuretic peptide receptors that inhibit aldosterone biosynthesis.

The inhibitory effect of atrial natriuretic peptide (ANP) on angiotensin II (AII)-stimulated aldosterone secretion has been previously studied in rat and bovine adrenal zona glomerulosa cells in primary culture. However the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems and by the lack of cell lines from human adrenal cortex. Here we demonstrate the presence of fully functional ANP receptors in the recently characterized AII-responsive adrenocortical carcinoma cell line H295R. Specific saturable binding of 125I-rANP to H295R cell membrane preparations revealed a single class of high affinity binding sites with a density of 20 fmol/mg of protein. The pharmacological profile of this ANP receptor was documented by competitive binding of 125I-rANP with naturally occurring natriuretic peptides. rANP was the most potent with a Kd of 42 pM. pBNP32 was less potent with a Kd of 174 pM. 125I-rANP binding was not competed by pCNP (NPRB-specific ligand) nor by C-ANF (NPRC-specific ligand). Photoaffinity labeling of membrane preparations with 125I-BPA-ANP revealed a single specific protein of molecular weight around 130 kDa. This protein was further identified by immunodetection with a specific antibody directed to the human ANP-specific receptor NPRA. Natriuretic peptides stimulated cGMP production by the receptor-coupled guanylate cyclase with the same specificity. Aldosterone production by AII-stimulated H295R cells was dose-dependently inhibited by rANP with an ED50 of 1.5 nM. In addition, we used this model to test two chimeric analogs of ANP and BNP. pBNP1 and pBNP3 were, respectively, 4- and 2-fold more potent than rANP in competing for 125I-rANP binding with Kd of 10 and 20 pM. pBNP1 was 24-fold more potent in inhibiting AII-stimulated aldosterone production with ED50 of 63 pM. pBNP1 is therefore the most potent natriuretic peptide analog tested. In summary, the human H295R cell line contains NPRA receptors positively coupled to the particulate guanylate cyclase and that antagonize angiotensin II stimulation of aldosterone secretion.

Adrenal Cortex↗

PubMed1996

Arginine vasopressin disposition in the conscious rabbit.

To characterize the urinary kinetics of AVP, and the influence of regional blood flow on the metabolic degradation of AVP, multiple doses of AVP were administered to conscious rabbits. AVP systemic clearance (ClT) was not influenced by changes in dose, in spite of a decrease in AVP urinary clearance following the highest dose. Hepatic blood flow was inversely associated with AVP concentrations, and despite a decrease in hepatic plasma flow of 37% (p < 0.05), following the high dose of AVP, ClT remained unchanged. These results indicate that AVP plasma kinetics are first order and plasma flow independent, and urinary kinetics are zero order.

Animals↗

PubMed1996

Labelling of haptenic drug with digoxigenin for competitive immunoassay: its application to lesopitron, a new anxiolytic agent.

A new labelling approach of haptenic drugs with digoxigenin for the development of competitive enzyme immunoassay (EIA) is reported. It consists of the covalent linking of the hapten to the preactivated digoxigenin derivative and revealing the immune complexes with anti-digoxigenin Fab fragments coupled to alkaline phosphatase. This approach has been applied to the development of an EIA for the pharmacokinetic study of lesopitron (E-4424), a new anxiolytic agent. The assay involves a solid-phase immobilization of IgG purified from polyclonal antiserum developed against the butylamino derivative of lesopitron covalently linked to bovine serum albumin. The tracer consists of the covalent linking of the same butylamino derivative to digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The calibration curve for E-4424 from 12.5 to 6400 pg per well displays an ED50 of 34.5 pg per well, a slope factor of 0.86 and a minimum detectable dose of 4.1 pg per well. The accuracy and the precision of the assay assessed at three different concentrations of E-4424 (500, 1000 and 2000 pg ml-1) give a recovery higher than 95% and intra- and inter-assay RSDs lower than 5 and 10%, respectively. The specificity of the assay was demonstrated by a good correlation of the samples analysed by both HPLC and EIA. A kinetic profile of E-4424 in rats following an oral dose of 50 mg kg-1 has also been established.

Animals↗

PubMed1996

Pituitary adenylate-cyclase activating polypeptide (PACAP) evokes long-lasting secretion and de novo biosynthesis of bovine adrenal medullary neuropeptides.

Recently, the pituitary adenylate-cyclase activating polypeptide (PACAP) has emerged as a potential noncholinergic neuromodulator of adrenal medullary function. In support of this hypothesis, we documented PACAP's effects on the secretion and biosynthesis of neuropeptides by cultured bovine chromaffin cells. Data presented in this study indicate that PACAP is a potent and efficacious secretagogue of leucine-enkephalin which was coreleased with catecholamines with identical profiles. In comparison to nicotinic activation, however, rates of PACAP-induced secretion were substantially slower but persisted for several hours causing a prolonged increase in the tonic release of both transmitters and peptides. Interestingly, renewal of intracellular pools of neuropeptides was also stimulated by PACAP but not the vasoactive intestinal peptide (VIP). Indeed, the higher incorporation of [35S]-labeled amino acids into atrial and brain natriuretic peptides (ANP, BNP) provided strong evidence that PACAP directly activated de novo biosynthesis. Of particular importance was PACAP's net preferential stimulation of the biosynthesis of BNP, similar to the differential regulation by protein kinase A (PK-A) and protein kinase C (PK-C) activators we have previously the differential regulation by protein kinase A (PK-A) and protein kinase C (PK-C) activators we have previously reported. PACAP-induced secretion and biosynthesis appeared to be mediated by the PACAP-specific type I receptors known to activate adenylate cyclase and phospholipase C. We verified that PACAP did indeed stimulate the production of cyclic AMP and inositol phosphates in our cell system. These findings suggest that the dual signaling properties of type I receptors may be important for PACAP's differential effect on the biosynthesis of natriuretic peptides. We conclude that PACAP might assume important noncholinergic trans-synaptic regulation of the adrenal medulla by releasing and modifying intragranular catecholamine and neuropeptide contents.

Adrenal Medulla↗

PubMed1996

Radioimmunoassay of Antarelix, a luteinizing hormone releasing-hormone antagonist, in plasma and its application for pharmacokinetic study in dogs.

A procedure for the radioimmunoassay (RIA) of Antarelix (teverelix) in plasma has been developed for the pharmacokinetic study of this potent LHRH antagonist in dogs. Antiserum was produced by coupling the deamidated Antarelix analog to bovine serum albumin by a carbodiimide reaction and immunizing rabbits with the conjugate. The crossreactivity of the antiserum with LHRH, LHRH agonist Metereline and LHRH antagonists tested was negligible, except for Antide which displayed a crossreactivity of 33%. No crossreactivity with Antarelix metabolites could be detected. The RIA is suitable for the direct determination of Antarelix in plasma, with a minimum detectable level of 1.12 fmol/assay. The accuracy and precision of the method were assessed with plasma samples spiked with Antarelix at concentrations ranging from 0.4 to 6.4 pmol/ml. The recovery was 104.8% with intra- and interassay CV between 1 and 3.7%. Pharmacokinetic profiles of Antarelix in dogs were established following an i.v. or a s.c. dose of 10 micrograms/kg.

Animals↗

PubMed1995

Quantification of des-Arg9-bradykinin using a chemiluminescence enzyme immunoassay: application to its kinetic profile during plasma activation.

There is a renewed interest in the kininase I pathway of kinin metabolism, because des-Arg9-bradykinin (des-Arg9-BK) and des-Arg10-Lys-BK are selective and potent agonists of the B1 receptors, that are apparently upregulated by tissue injury. We have developed a polyclonal rabbit antiserum against des-Arg10-Lys-BK. In a radioimmunoassay for des-Arg10-Lys-BK, this antiserum exhibited high specificity. Notably, native kinins with the C-terminal Arg residue, bradykinin (BK) and Lys-BK, did not cross-react to a significant extent, whereas des-Arg9-BK and digoxigenin (DIG)-des-Arg9-BK exhibited a complete cross-reactivity. The antibodies were used to set up a sensitive chemiluminescence enzyme immunoassay (CLEIA) using the DIG-anti-DIG system as intermediate for the revelation of the immune complexes. The detection limit and the half-maximal saturation concentration for des-Arg9-BK were 27 and 1530 fmol/ml respectively. This assay, as well as another for BK quantification, have been applied in vitro to rabbit plasma activated by kaolin. The conversion of BK into des-Arg9-BK was generally efficient, and the persistence and concentration of both peptides were increased in the presence of enalaprilat an inhibitor of the angiotensin converting enzyme (ACEI). Rabbits treated with bacterial lipopolysaccharide exhibited an increase of plasma immunoreactive des-Arg9-BK that was potentiated in animals also treated with ACEI. This CLEIA for des-Arg9-BK is a new analytical tool applicable to analyze of the kininase I metabolites of kinins in vitro and in vivo. Measurements of des-Arg9-BK may be useful indicators of the kallikrein-kinin system activation.

Amino Acid Sequence↗

PubMed1995

Stoichiometry of the atrial natriuretic factor-R1 receptor complex in the bovine zona glomerulosa.

The atrial natriuretic R1 receptor is a membrane protein that is present as an apparently preassociated noncovalent oligomer in the absence of ligand as suggested by steric exclusion studies and cross-linking experiments in physiological and recombinant receptor expression systems. The association state of this receptor oligomer was studied in the presence of amiloride and ATP, two known modulators of the R1 receptor functions with both the intact receptor and a cytoplasmic domain-deleted form obtained by limited proteolysis with trypsin. It was shown by steric exclusion on Superose 6 column that amiloride increased the affinity of ANF for the native and truncated receptor, in contrast with ATP, whose destabilizing effect on ANF binding was abolished by truncation of the cytoplasmic domain. Neither amiloride nor ATP exerts its effects by altering the aggregation state of the receptor. Comparison of the measured number of ANF binding sites with immunoassayable receptor protein revealed that the stoichiometry of ANF binding to the R1 receptor was 1:2. This was confirmed by using an ANF analog that bears a photoactivatable group at both of its ends, showing that ANF, as for the growth hormone/receptor complex, interacts with both the receptor subunits and specifically cross-links a dimeric form of the receptor. The potential pharmacological consequences of this 1:2 stoichiometric ratio of the ANF-receptor complex are discussed.

Adenosine Triphosphate↗

PubMed1995

Radioimmunoassay for hexarelin, a peptidic growth hormone secretagogue, and its pharmacokinetic studies.

A radioimmunoassay (RIA) method for hexarelin, a peptidic growth hormone secretagogue, has been developed and applied to pharmacokinetic studies in dogs following an IV dose of 1 microgram/kg, and three SC doses of 1, 10, and 100 micrograms/kg. The sensitivity of the assay was determined to be 1.34 fmol/assay. Cross-reactivity of the antiserum with nine hexarelin analogues was less than 1% upon modification of positions 3, 4, or 5 of the peptide. No apparent cross-reaction with endogenous hexarelin metabolites were observed. Intra- and interassay coefficients of variation were less than 3% and 4%, respectively. Intravenous bolus pharmacokinetics of hexarelin displayed a high terminal half-life of 120 min, a fractional plasma clearance of 4.28 ml/min/kg, and a volume of distribution at steady state of 387.7 ml/kg. Following SC administration of hexarelin, despite the increase in dose administered, both clearance (3.93-5.17 ml/min/kg) and volume of distribution (316-544 ml/kg) parameters remained constant over the dose range studied.

Amino Acid Sequence↗

PubMed1995

A chemiluminescence enzyme immunoassay (CLEIA) for the determination of medroxyprogesterone acetate in human serum.

An enzyme immunoassay with chemiluminescence detection (CLEIA) for measuring serum levels of medroxyprogesterone acetate (MPA), a synthetic progestational agent currently used in fertility control and hormonal cancer, is reported. The polyclonal antiserum was obtained by immunizing rabbits with the synthetized 17-hemisuccinate derivative of medroxyprogesterone (MPS) coupled to serum albumin. This antiserum does not display any cross reactivity with extracted metabolites or with corticosteroid analogs with modifications at positions 11 and 16. The same MPS coupled to alkaline phosphatase is used as tracer. For the chemiluminescent detection system, adamantyl-1,2-dioxetane phosphate is selected as substrate. The typical standard curve ranges from 18.5 to 1182 pg per well and displays a slope factor of 0.74, with an ED50 of 143.8 pg of MPA per well and a minimum detectable and maximal level of 0.83 and 12,400 pg per well respectively. The assay has been validated on spiked serum samples in terms of precision (intra- and interassay coefficient variations of less than 8% and 13%, respectively), and of accuracy (mean recovery 105%). The validation on clinical samples demonstrates a good correlation of MPA serum values obtained both by radioimmunoassay and CLEIA. This specific and sensitive CLEIA, which requires less than 100 microliters of serum sample, appears to be an interesting alternative for the monitoring of serum levels of MPA in humans.

Administration, Oral↗

PubMed1995

A role for protein tyrosine kinase in the steroidogenic pathway of angiotensin II in bovine zona glomerulosa cells.

Stimulation of aldosterone synthesis in bovine adrenal zona glomerulosa (ZGB) cells by angiotensin II (AngII) is believed to be mediated by the phospholipase C (PLC) pathway that results in the increase of cytosolic free calcium concentration and in the activation of protein kinase C (PKC). However, the cell proliferation and contraction associated with AngII action are known to be mediated in part by protein tyrosine kinases (PTK). To assess the potential role of PTK in the stimulatory effect of AngII on adrenal steroidogenesis, the actions of a series of PTK inhibitors on this metabolic pathway were examined in isolated ZGB cells. Tyrphostin 23 (TP23) caused a dose-dependent inhibition of AngII-stimulated aldosterone production with an IC50 of 15 microM and reached complete inhibition at 100 microM. Genistein (GS) was more potent with an IC50 of 35 nM and complete inhibition at 10 microM. The stimulation of aldosterone production by the calcium-mobilizing agent thapsigargin (Thaps) was also dose-dependently inhibited by TP and GS with the same potency. A specific PKC inhibitor, calphostin C (0.1 microM) caused only a 51.7% inhibition of AngII-stimulated aldosterone production. In the same way, a specific Ca2+/calmodulin-dependent protein kinase inhibitor, KN-62 (1 microM), reduced aldosterone production stimulated by AngII by 64%. As expected, thapsigargin-stimulated aldosterone biosynthesis was not affected by calphostin C, but was completely inhibited by KN-62. These results demonstrate for the first time that protein tyrosine kinase activity is part of the angiotensin II signalling pathway in bovine zona glomerulosa cells. The activation of this PTK occurs subsequently to the mobilization of intracellular calcium. This calcium-dependent protein tyrosine kinase pathway is essential for the steroidogenic response to AngII in bovine zona glomerulosa cells.

Aldosterone↗

PubMed1995

Natriuretic peptides inhibit nicotine-induced whole-cell currents and catecholamine secretion in bovine chromaffin cells: evidence for the involvement of the atrial natriuretic factor R2 receptors.

There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R1C but not the ANF-R1A receptor subtype. Evidence for the existence of ANF-R2 receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R2-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R2 receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R2 receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that the effect of CNP is predominantly mediated by the ANF-R2 and not the ANF-R1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCl-induced secretions were affected by natriuretic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗