PubMed Health⌕ Search

Biomedical subjects

S Jard

Publications and source records attributed to S Jard.

At least 37 records · Page 2Linked to original sources
PubMed1992

Pharmacological characterization of inositol 1,4,5-trisphosphate binding sites: relation to Ca2+ release.

Two subcellular fractions, one enriched in plasma membranes and the other in endoplasmic reticulum membranes, were obtained from WRK1 cells using a combination of differential centrifugations and Percoll gradient fractionation. Specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites were detected in these two preparations. Endoplasmic reticulum membranes exhibited a binding capacity which was about 5-fold higher than that of plasma membranes. Dose-dependent Ins(1,4,5)P3 binding was determined. Experimental data obtained with endoplasmic reticulum membranes could be adequately fitted with a two-site model (a high-affinity binding site with Kd and Bmax values of 0.7 +/- 0.15 nM and 12.9 +/- 5 fmol/mg protein and a low-affinity binding site with Kd and Bmax values of 44.2 +/- 14.6 nM and 143 +/- 43 fmol/mg protein). Both the high- and low-affinity binding sites were selective for Ins(1,4,5)P3. Besides Ins(1,4,5)P3, Ins(1,3,4,5)P4 also discriminated between the two populations of sites while heparin interacted with the high- and low-affinity binding sites with the same affinity. Ins(1,4,5)P3-induced calcium release from endoplasmic reticulum vesicles was determined by monitoring the calcium concentration in the extravesicular compartment with fura-2. Under experimental conditions where the degradation of Ins(1,4,5)P3 was reduced (incubation at 0 degrees C), a high-affinity Ins(1,4,5)P3-induced calcium release (apparent Kact around 20 nM) could be demonstrated. These results suggest that in WRK1 cells, the endoplasmic reticulum is a major site for Ins(1,4,5)P3 action and that the high-affinity binding sites located on the endoplasmic reticulum membranes may contribute to the physiological regulation of the cytosolic free calcium concentration.

Binding Sites↗
PubMed1991

Angiotensin receptors from rat liver, brain and pituitary gland. Expression of two subtypes in Xenopus oocytes.

Xenopus laevis oocytes were used to express angiotensin receptors encoded by mRNAs extracted from rat liver, adenohypophysis and brain. Groups of ten mRNA-injected oocytes were loaded with 45Ca2+ and the responsiveness to angiotensin II (A II) and related molecules tested by monitoring 45Ca2+ outflux. A II and angiotensin III (A III) induced a marked and transient increase in 45Ca2+ outflux from mRNA, but not from control, water-injected, oocytes. The increase over basal value of 45Ca2+ outflux during a 5 min application period of A II or A III was used as a response index. Observed responses were of high magnitude, reproducible and dose-dependent. For these reasons, mRNA-injected oocytes constitute a valuable system for investigating the pharmacological properties of angiotensin receptors from tissues of different origin under experimental conditions which eliminate tissue-specific interference which might be encountered in classical binding studies on acellular preparations. We demonstrate a fairly good parallelism between the relative potencies of A I, A II and A III in eliciting an increase in 45Ca2+ outflux from liver and adenohypophyseal mRNA-injected oocytes and the relative affinities of these peptides for binding to liver or adenohypophyseal membranes (A II greater than A III much greater than A I). The predominant receptor subtype expressed by brain mRNA discriminated very poorly between A II and A III, whereas angiotensin receptors expressed by liver or adenohypophyseal mRNA discriminated between AII and AIII very efficiently.

Angiotensins↗
PubMed1991

A radioiodinated linear vasopressin antagonist: a ligand with high affinity and specificity for V1a receptors.

A linear vasopressin antagonist, Phaa-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti-vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (Kd = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (Ki = 2.1 nM), and affinities for the rat kidney- and adenohypophysis-vasopressin receptors were much lower (Ki = 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V1a receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.

Amino Acid Sequence↗
PubMed1990

Labelling of vasopressin and oxytocin receptors from the human uterus.

Four labelled ligands, [3H]arginine vasopressin ([3H]AVP), [3H]oxytocin ([3H]OT), [3H]d(CH2)5[Tyr(Me)2]AVP ([3H]VPA), and [125I]d(CH2)5[Tyr(Me)2-Thr4-Orn8-Tyr(NH2)9]OT([125I]OTA] and nine unlabelled analogues exhibiting enhanced selectivity for rat oxytocin (OT) and vasopressin (VP) receptors were used to characterize OT and VP receptors on myometrial membranes from non-pregnant and pregnant human uteri. On membranes from non-pregnant uteri, [3H]AVP, [3H]VPA, and [125I]OTA labelled with high affinity (Kd values: 3.2, 2 and 0.8 nM, respectively) a major and apparently homogeneous population of sites, the ligand selectivity of which resembled that of rat V1a VP receptors. On membranes from pregnant and non-pregnant uteri, [3H]OT labelled a single population of high-affinity sites that could be distinguished from VP receptors on the basis of ligand selectivity. Several analogues (in particular [125I]OTA) that are highly selective for rat OT receptors exhibited a much less pronounced selectivity for human OT receptors. Experiments with [3H]VPA allowed detection of VP receptors on myometrical membranes from pregnant uteri and confirmed that only OT but not VP receptors increase during pregnancy in humans.

Female↗
PubMed1990

Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes.

We have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH2)2SS(CH2)2CO-[Ala1,Phe(4N3)8]AII, which contains a cleavable disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (Kd approximately 1 nM), proved to be suitable for indirect affinity chromatography of rat liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

Amino Acid Sequence↗
PubMed1990

Oxytocin receptors from LLC-PK1 cells: expression in Xenopus oocytes.

Two selective radioligands for oxytocin receptors, [3H]-[4-threonine,7-glycine]oxytocin [( 3H]-[Thr4,Gly7]OT) and 125I-[1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine, 4-threonine, 8-ornithine, 9-tyrosine amide]-oxytocin (125I-OTA), were used to characterize oxytocin receptors from two pig kidney-derived cell lines, LLC-PK1 and LLC-PK1L. [3H]-[Thr4,Gly7]OT and 125I-OTA bind with high affinity (mean Kd values of 14 and 0.06 nM, respectively) to the same population of sites on LLC-PK1 cell membranes [maximum binding (Bmax) of 100 fmol/mg membrane protein]. These sites had the expected ligand selectivity of oxytocin receptors. [3H]-[Thr4,Gly7]OT and 125I-OTA binding sites could be distinguished from V2 vasopressin receptors present on LLC-PK1 and LLC-PK1L cells on the basis of clearly different maximal capacities and ligand selectivities, different sensitivities to insulin and serum, and absence of heterologous downregulation. Oxytocin receptors from LLC-PK1 cells have no functional relationship with adenylate cyclase. [Thr4,Gly7]OT affected neither the basal adenosine 3',5'-cyclic monophosphate (cAMP) content nor the vasopressin-induced cAMP accumulation by LLC-PK1 cells. Xenopus laevis oocytes injected with LLC-PK1 cell mRNA responded to [Thr4,Gly7]OT by an increase in 45Ca2+ outflux; this effect is antagonized by a highly selective oxytocin antagonist.

Animals↗
PubMed1990

Oxytocin receptors in rat kidney during development.

The development and characteristics of oxytocin (OT) receptors in the rat kidney were studied by light-microscopic autoradiography and on membrane preparations using the iodinated OT antagonist 125I-d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]OT. Specific binding was first detected by autoradiography at embryonic day 17 (E17) in both the cortex and the medulla. Cortical labeling was found thereafter at all ages examined including in the adult (postnatal day 90, PN90). It was localized on the distal tubule at the level of the juxtaglomerular apparatus. Medullary binding was detected only transiently during two stages of development, first, before PN6, and second, at the approximate time of weaning (PN20-PN30). Binding studies on crude membranes prepared from whole kidneys of animals aged between PN1 and PN15 showed a single class of high-affinity binding sites, with a dissociation constant of 0.13 +/- 0.08 nM. Thus transient OT binding sites expressed in the medulla do not differ from cortical OT binding sites; moreover, the ligand selectivity of kidney OT receptors regardless of location and age appears similar to that of previously characterized OT receptors. Our results suggest that OT may play a role in both renal development and renal function.

Animals↗
PubMed1990

Characterization of the angiotensin II receptor.

We present a brief overview of the present knowledge on the structural and molecular properties of angiotensin II receptors and the various attempts to determine their primary structures, with special reference to our strategy for receptor purification. The strategy involves covalent labeling of the receptor with synthetic biotinylated photoactivatable probes, followed by indirect affinity chromatography on immobilized streptavidin. The various applications of these probes to the study of structural and molecular properties and to the cell biology of angiotensin II receptors are discussed.

Amino Acid Sequence↗
PubMed1989

Synthesis of a biotinylated, iodinatable, and photoactivatable probe for angiotensin receptors.

We propose here a biotinyl-aminohexanoyl-[Ala1, Phe(4N3)8]angiotensin II analog as a radioiodinatable and photoactivatable probe for covalent labeling, detection and isolation of angiotensin receptors. A combination of solid phase and minimum-protection segment-coupling strategy using hexafluorophosphate of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium (BOP) as a coupling reagent is proposed for the synthesis of this probe. Optimized yields were obtained by HPLC monitoring of all reactions. A complete n.m.r. study suggests an extended conformation of this molecule, allowing a simultaneous recognition of receptor and avidin. The probe binds with high affinity (Kd = 2 nM) to angiotensin II receptors from rat liver membranes.

Amino Acid Sequence↗
PubMed1989

Angiotensin II stimulates both inositol phosphate production and human placental lactogen release from human trophoblastic cells.

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.

Angiotensin II↗
PubMed1988

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

2,4-Dinitrophenol↗
PubMed1988

125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP: iodination and binding characteristics of a vasopressin receptor ligand.

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non-radiolabelled monoiodinated antagonist was used as a reference for identification. 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28 +/- 0.09 nM (Kd +/- SD) to V1 vasopressin receptors on rat liver membranes.

Animals↗
PubMed1988

125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT: a selective oxytocin receptor ligand.

An oxytocic antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid,2-O-methyltyrosine,4-threonine, 8-ornithine,9-tyrosylamide]vasotocin (d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT [corrected], was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. 125I-labelling was performed with 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl-glycoluril. Iodination resulted in an increased affinity for rat uterine oxytocin receptors. A considerably lower affinity for rat vascular V1- and renal V2-receptors was found, resulting in a highly specific oxytocin receptor ligand. 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT [corrected] was demonstrated to bind selectively to one population of binding sites in rat uterus and ventral hippocampal membrane preparations. Dissociation constants ranged between 0.03 and 0.06 nM. After 3 days of exposure autoradiography revealed binding in regions known to contain oxytocin receptors as well as labelling in some new regions, while no binding was found in the lateral septum, a structure containing mainly [8-arginine]vasopressin receptors. The high specific radioactivity of 125I-labelling allowed important reductions in membrane protein amount, gain in precision of binding analysis as well as considerably lower exposure times for autoradiography.

Animals↗
PubMed1988

Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography.

Sites which bind tritiated vasopressin (AVP) with high affinity were detected in the brain of male, adult rats, by light microscopic autoradiography. Their anatomical localization differed markedly from that of high affinity binding sites for tritiated oxytocin (OT) determined in the same animal. Co-labelling was minimized by using low concentrations of [3H]AVP and [3H]OT. Binding of the former occurred predominantly in several structures of the limbic system (septum, amygdala, bed nucleus of the stria terminalis, accumbens nucleus), in two hypothalamic nuclei (suprachiasmatic and dorsal tuber) and in the area of the nucleus of the solitary tract. Binding of OT was evidenced in the olfactory tubercle, the ventromedial hypothalamic nucleus, the central amygdaloid nucleus and the ventral hippocampus. The ligand specificity of the binding sites was assessed in competition experiments. Synthetic structural analogues were used, allowing to discriminate OT receptors (OH[Thr4,Gly7]OT) from V2 receptors (dDAVP and d[Tyr(Me)2]VDAVP), V1 receptors ([Phe2,Orn8]VT) and V1b receptors (desGly9d(CH2)5AVP). Our main conclusions are, firstly, that AVP and OT binding sites can be readily distinguished, and that there is virtually no overlap in their distribution in the rat brain. Second, we showed that the sites which bind AVP with high affinity in the brain are V1 receptors, different both from the renal V2 receptors and from the anterior pituitary V1b receptors. Our results support the conjecture that AVP and OT play a role in interneuronal communication in the brain.

Animals↗
PubMed1988

Vasopressin potentiates the noradrenaline-induced accumulation of cyclic AMP in the rat superior cervical ganglion.

Previous experiments gave biochemical and electrophysiological evidence for the presence of functional V1-vasopressin receptors coupled to inositol lipid metabolism, but not to cyclic AMP accumulation in the rat superior cervical ganglion. This work was designed to investigate whether there was an action of vasopressin on the noradrenaline-induced cyclic AMP accumulation through the activation of phospholipase C. Our results clearly demonstrate that arginine-vasopressin potentiates cyclic AMP accumulation induced by noradrenaline or isoproterenol in a concentration-dependent manner. The potentiation was unaffected by phentolamine, but was suppressed by the V1-type vasopressin receptor antagonists. Moreover, the phorbol ester 4 beta-phorbol-12-myristate-13-acetate (TPA) did not affect this potentiation which seemed to be Ca2+-dependent. The results suggest that vasopressin may modulate the activity of autonomous functions in the sympathetic ganglia.

Adenylyl Cyclases↗
PubMed1988

Autoradiographic localization of vasopressin and oxytocin binding sites in rat kidney.

The presence of vasopressin receptors of the V1 (vascular) type and of oxytocin receptors in the rat kidney was investigated using an autoradiographical approach. Rat kidney sections were incubated with tritiated vasopressin ([3H]vasopressin, 1.5 nM) or oxytocin ([3H]oxytocin, 3 nM). The ligand selectivity of the [3H]vasopressin binding sites detected was deduced from competition experiments using one selective unlabeled ligand for V2 (antidiuretic) vasopressin receptors (1-deamino-[8-D-arginine]-vasopressin, dDAVP) and one selective unlabeled ligand for V1 receptors (des-glycineamide-[1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid]-arginine vasopressin, des(Gly(NH2)9d(CH2)5-AVP). Specific and dense [3H]vasopressin labeling was observable in the medullopapillary and cortical portions of the kidney. Specific [3H]vasopressin binding in the cortex was insensitive to the V1-selective ligand, des(Gly(NH2)9d(CH2)5-AVP, but was inhibited by dDAVP. Glomerular structures identified as such by microscopical observation of the kidney sections were specifically labeled with [3H]oxytocin and [125I]-SAR1-angiotensin II but not with [3H]vasopressin. It is concluded that V1 receptors which have been evidenced on mesangial cells in culture are not expressed in a detectable quantity on mesangial cells in situ. The specific [3H]oxytocin binding to glomeruli might reflect the presence on glomerular structures of oxytocin receptors involved in the effects of the hormone on renal hemodynamics, and possibly in some of the effects ascribed to vasopressin.

Animals↗
PubMed1988

[3H]-[Thr4,Gly7]OT: a highly selective ligand for central and peripheral OT receptors.

Oxytocin receptors in rat hippocampal synaptic plasma membranes were compared with mammary gland and uterine oxytocin receptors. For this purpose, a highly specific oxytocic agonist [Thr4,Gly7]oxytocin was tritiated. We demonstrated that this ligand labels oxytocin receptors selectively. Scatchard analyses revealed a high affinity for all the oxytocin receptors investigated, with equilibrium dissociation constants between 1.0 and 2.0 nM. Binding appeared to take place at a single population of receptor sites. Competition experiments confirmed the high affinity of arginine vasopressin for hippocampal oxytocin receptors but also revealed that mammary gland and uterine oxytocin receptors do not discriminate more efficiently between oxytocin and arginine vasopressin. This lack in specificity is not affected by applying different concentrations of Mg ions.

Animals↗