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Biomedical subjects

J Christophe

Publications and source records attributed to J Christophe.

At least 91 records · Page 5Linked to original sources
PubMed1989

Stereoselectivity of the interaction of muscarinic antagonists with their receptors.

The stereoselectivity of the interaction with muscarinic receptors of enantiomers of a series of chiral antagonists is receptor subtype dependent. There is no overall relationship between stereoselectivity and receptor affinity. Depending on the antagonist studied, receptor stereoselectivity may indeed reflect: (1) the weakening or loss of a single interaction involving one of the four groups bound to the asymmetric carbon; (2) steric hindrance preventing optimum interaction of the low affinity steroisomer with the receptor; and/or (3) the inversion of the relative positions of two moieties of the ligand with similar structural and electronic properties i.e. comparable affinities for the two corresponding subsites in the receptor.

Animals↗
PubMed1989

Determination of the association and dissociation rate constants of muscarinic antagonists on rat pancreas: rank order of potency varies with time.

Antagonist binding to rat pancreatic muscarinic receptors was relatively slow at 25 degrees (tracer dissociation half-life, 50 to 60 min). We, therefore, chose this system to investigate the errors induced by nonequilibrium incubations on the estimates of receptor capacity and selectivity, in binding studies. We took advantage of the fact that muscarinic antagonists recognize only one receptor subtype in rat pancreatic homogenates and that association and dissociation kinetics conform to the law of mass action to analyze quantitatively the binding kinetics of [3H]N-methylscopolamine and of several unlabeled progressive to these receptors. We observed no correlation between the affinities of drugs for muscarinic receptors and their dissociation rate constants. As a result, the apparent receptor specificity (based on relative affinities for different antagonists) varied markedly with the incubation period. We, therefore, strongly recommend that in general competition curves established for receptor classification should be compared at different incubation periods to ensure that equilibrium is attained. The association rate constants of muscarinic antagonists for rat pancreas receptors were remarkably low, when compared with other ligand-receptor systems. This suggests that the antagonist-pancreatic muscarinic receptor association reaction included a rate-limiting conformational change of the drug-receptor complex. This isomerization step was not directly detectable in our kinetic studies, due to the very low affinity and rapid dissociation rate of the initial nonisomerized complex.

Animals↗
PubMed1988

CCK and gastrin inhibit adenylate cyclase activity through a pertussis toxin-sensitive mechanism in the tumoral rat pancreatic acinar cell line AR 4-2J.

(Thr28,Nle31)CCK(23-33) (CCK-9) and gastrin(1-17)I (gastrin) inhibited adenylate cyclase activity in membranes from the tumoral rat pancreatic acinar cell line AR 4-2J through a Bordetella pertussis toxin-sensitive mechanism. This contrasted with the stimulatory effect exerted by CCK-9 on adenylate cyclase activity in membranes from normal rat pancreas. The relative potency of CCK-9, gastrin, and related peptides in inhibiting adenylate cyclase, when confronted with previous evidence, suggests that 'non-selective CCK-gastrin CCK-B receptors' predominating over 'selective CCK-A receptors' in the AR 4-2J cell line, favored the coupling of the first receptors to adenylate cyclase through Gi, while CCK-A receptors capable of stimulating the enzyme through Gs were detected only after Bordetella pertussis toxin pretreatment.

Adenylate Cyclase Toxin↗
PubMed1988

Stereoselectivity of the enantiomers of trihexyphenidyl and its methiodide at muscarinic receptor subtypes.

High stereoselectivity was observed for the enantiomers of trihexyphenidyl and trihexyphenidyl methiodide at muscarinic M1-receptors in field-stimulated rabbit vas deferens and at M2 alpha- and M2 beta-receptors in guinea-pig atrium and ileum, respectively. Considerably higher affinities (up to 1700-fold) were found for the (R)-(-)-enantiomers. The stereochemical demands made by the muscarinic receptor subtypes were most stringent at the M1-receptors. The (R)-(-)-enantiomers were found to be potent M1-selective antagonists (pA2 = 10.1/10.6). They showed a 91- and 45-fold selectivity for M1- over M2 alpha-receptors, respectively.

Animals↗
PubMed1988

Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J.

1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]iodo-VIP-receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4 degrees C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half-life of 2 days for VIP receptors and the necessity of N-glycosylation for proper translocation. 2. For chemical [125I]iodo-VIP cross-linking bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross-linked peptide had Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo-VIP-cross-linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross-linked radioactivity migrated as a smear of Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.

Animals↗
PubMed1988

Functional characterization of muscarinic receptors in rat parotid acini.

The muscarinic agonist, carbamylcholine, stimulated amylase secretion in rat parotid acini 6-fold, the 86Rb efflux 5-fold, the 45Ca efflux 5-fold and the accumulation of inositol monophosphate, bisphosphate, trisphosphate and tetrakisphosphate 4-, 4-, 3- and 3-fold, respectively. The EC50 of carbamylcholine on these parameters were 0.4, 0.5, 1.3, 12, 12, 6 and 9 microM, suggesting spareness between phospholipase C activation and amylase secretion. These muscarinic responses were inhibited by four muscarinic antagonists with an order of potency on all parameters and on receptor occupancy (using N-[methyl-3H]scopolamine as a tracer): atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than AF-DX 116. The pA2 of these antagonists on carbamylcholine-stimulated amylase secretion were 9.72 for atropine, 8.14 for hexahydrosiladifenidol, 7.16 for pirenzepine and 6.22 for AF-DX 116, indicating that the parotid muscarinic receptors were of an M2 subtype 83-fold more sensitive to hexahydrosiladifenidol than to AF-DX 116.

Amylases↗
PubMed1988

Failure of Leiurus quinquestriatus venom to affect potassium movements in pancreatic islets.

The venom from the Israeli scorpion Leiurus quinquestriatus failed to affect 86Rb and 45Ca outflow from rat pancreatic islets perifused in the presence of tetrodotoxin and stimulated by the Ca2+-ionophore A23187 or the hypoglycaemic sulfonylurea tolbutamide. In non-stimulated islets, the venom components whose effects are insensitive to tetrodotoxin did not affect 45Ca and 86Rb outflow. Last, the venom did not alter 86Rb inflow. These findings suggest that 86Rb, 45Ca fluxes and more specifically the Ca2+-activated K+ permeability in the pancreatic B-cell are insensitive to the venom.

Animals↗
PubMed1988

Species differences in the molecular characteristics of vasoactive-intestinal-peptide receptors in the pancreas from rat and guinea-pig.

1. The receptors for vasoactive intestinal peptide (VIP) present in dispersed acini and membranes from rat and guinea-pig pancreas differed in selectivity pattern, i.e. in the displacement of [125I]iodo-VIP by parent peptides, as revealed by a VIP:secretin IC50 ratio at least ten times higher in rat than in guinea-pig preparations. The molecular properties of these VIP receptors were therefore investigated. 2. When comparing six succinimidyl ester cross-linkers, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone proved to be the most universal [125I]iodo-VIP cross-linker for all pancreatic preparations. 3. In intact rat acini the main labeled peptide had an Mr of 80,000, whereas the main labeled peptide in intact guinea-pig acini was a smear of Mr 160,000. In both rat and guinea-pig pancreatic membranes, the main labeled peptide ([125I]iodo-VIP-binding-protein complex) had an Mr of 66,000. In addition, variable proportions of an Mr-80,000 peptide and an Mr-83,000 peptide were visualized in, respectively, rat and guinea-pig membranes. The labeling of all peptides was suppressed by VIP and by GTP. Reducing conditions allowed only a better resolution, making the presence of intermolecular disulfide bridges unlikely. 4. Taking into account the Mr of VIP it is thus plausible that the main native Mr-77,000 VIP-binding site present in rat acini could be easily converted to an Mr-63,000 peptide during membrane preparation, while in guinea-pig acini Mr-80,000 and/or Mr-63,000 VIP-binding sites were often closely associated with another membrane component in the native state. These molecular differences between VIP receptors in intact rat and guinea-pig acini are in keeping with functional differences.

Animals↗
PubMed1988

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts.

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.

Adenylyl Cyclases↗
PubMed1988

80% of muscarinic receptors expressed by the NB-OK 1 human neuroblastoma cell line show high affinity for pirenzepine and are comparable to rat hippocampus M1 receptors.

The NB-OK 1 human neuroblastoma cell line expressed muscarinic cholinergic receptors that could be labeled with N-[3H]methylscopolamine (a nonselective antagonist). 80% of these receptors showed high affinity for pirenzepine, i.e. belonged to the M 1 subtype found in neuronal tissues. Their binding properties were identical to those of rat hippocampus M 1 receptors, and differed from those of rat pancreas and heart muscarinic receptors. The remaining (20%) muscarinic receptors showed low affinity for pirenzepine and AF-DX 116, being therefore of an M2 beta (or B) subtype, and were similar to rat pancreatic receptors.

Animals↗
PubMed1988

Effect of freezing on the coupling of VIP receptors to adenylate cyclase in rat liver membranes.

In fresh rat liver plasma membranes, high affinity VIP receptors were specifically labelled with [125I] helodermin and were well coupled to adenylate cyclase while low affinity VIP receptors were not. After freezing and thawing low affinity VIP receptors were also coupled to adenylate cyclase. This modification of adenylate cyclase activation was specific for the VIP response as freezing and thawing did not modify Gpp (NH)p, NaF and glucagon stimulations.

Adenylyl Cyclases↗
PubMed1988

Is helodermin produced by medullary thyroid carcinoma cells and normal C-cells? Immunocytochemical evidence.

Helodermin is a VIP/secretin-like 35-amino acid peptide originally isolated from the venom of the lizard Gila monster. Recently, helodermin-immunoreactive material was demonstrated in mammalian salivary glands, brain and gut. In the present study 8 human medullary thyroid carcinomas as well as 4 normal thyroid glands were examined immunocytochemically for the presence of helodermin using an antiserum raised against helodermin-(5-35) that does not cross-react with VIP or secretin. Cells displaying helodermin-like immunoreactivity were found in all tumours examined except one. On the whole the helodermin-immunoreactive cells had the same distribution as those storing calcitonin, suggesting coexistence of the two peptides in most of the tumour cells. Also normal human C-cells displayed helodermin immunoreactivity. The results suggest that a peptide chemically related to helodermin is a constituent of human medullary thyroid carcinoma cells as well as of normal C-cells.

Fluorescent Antibody Technique↗
PubMed1988

Receptor occupancy and adenylate cyclase activation in rat liver and heart membranes by 10 glucagon analogs modified in position 2,3, 4, 25, 27 and/or 29.

Rat liver and heart membranes were tested for adenylate cyclase activation by glucagon and 10 glucagon analogs mono- or polysubstituted in positions 2-4, 25, 27 and/or 29. The first membranes were, in addition, examined for the capacity of glucagon analogs to inhibit the binding of [125I]iodoglucagon. The monophasic slope of dose-effect curves suggested interaction with one class of glucagon receptors in both tissues, receptors in liver being more sensitive to the ligands and more efficiently coupled to adenylate cyclase than heart receptors. Structure-activity studies on liver membranes revealed that modifications of the beta-turn potential in the 2-4 region by single residue substitutions could lead to partial agonists (with D-Gln3 or Phe4) or to a superagonist (with D-Phe4). The importance of a proper alpha-helix conformation in the C-terminal part of glucagon for binding affinity was also obvious: replacing Trp25, Met27 and Thr29 in combination by Phe25, Leu27 and Thr29-NH2 increased the affinity while single or combined substitutions with Gly25 and/or Nle27 sharply decreased the affinity. Similar trends were less evident but still obvious on heart membranes.

Adenylyl Cyclases↗
PubMed1988

Secretin receptors in human pancreatic membranes.

Crude membranes (27,000 g pellets) from five normal human pancreases were prepared. In the presence of GTP, the peptides of the secretin family stimulated adenylate cyclase activity, their order of potency being: secretin greater than helodermin greater than peptide histidine isoleucinamide (PHI) greater than or equal to vasoactive intestinal peptide (VIP) greater than growth hormone releasing factor (GRF) (1-29)-NH2. In addition, helodermin and PHI were more efficient than secretin. Secretin (3-27) inhibited fully the secretin stimulation and partially only the helodermin and PHI stimulation of the enzyme. Secretin receptors were investigated by the ability of secretin and related peptides to inhibit tracer binding. [125I]Secretin binding was fully inhibited by secretin (Kd 0.8 nM), helodermin (Kd 200 nM), and PHI (Kd 250 nM). VIP and GRF(1-29)-NH2 induced partial (20%) inhibition at a high 10 microM concentration. The fragments secretin (2-27), (3-27), (4-27), and (7-27) showed the same low potency and efficacy based on their ability to stimulate adenylate cyclase and to occupy secretin receptors. The analogues [Val5]secretin and [Ala2]secretin had a higher potency than secretin. Based on this comparison of adenylate cyclase stimulation and [125I]secretin binding inhibition, it is tempting to conclude that the human pancreas: (a) possesses highly specific secretin receptors and (b) such receptors could not fully account for the whole pattern of adenylate cyclase activation by related peptides, so that the presence of an added type of "helodermin-PHI-preferring" receptors is suggested.

Adenylyl Cyclases↗
PubMed1988

Characterization of muscarinic receptors in human pancreatic membranes.

Crude membranes (27,000 x g pellets) from three normal human pancreata were prepared. Muscarinic receptors were investigated by the ability of three antagonists (atropine, pirenzepine, and AF-DX 116) and three agonists (carbamylcholine, oxotremorine, and pilocarpine) to inhibit [3H]NMS binding. These receptors showed for pirenzepine and AF-DX 116 a M2 beta specificity, typical of secretory glands and smooth muscle, that was comparable to that of rat pancreatic membranes, i.e., a low affinity for the two antagonists (Ki of 0.4 and 0.2 microM, respectively). In addition, these receptors were predominantly in a low affinity state for the agonist carbamylcholine (Ki of 100 microM).

Carbachol↗
PubMed1988

Identification by immunoblotting of somatostatin proforms in a rat pancreatic cell line.

The immunoblotting technique is applied to the analysis of "somatostatin" compounds secreted by R.I.N. T3 cells. We can confirm that two proforms of 15,300 +/- 750 and 29,000 +/- 1,100 Da accumulate in the extracellular medium. The unexpected form of 29 kDa, probably a dimeric form, disappears in reducing conditions. However, the 15 kDa peptide is characterized by several antibodies directed against either the intramolecular cycle of somatostatin-14 or the N-terminal extension of somatostatin-28. The 15 kDa form presents the same electrophoretic mobility in SDS-PAGE than the prosomatostatin isolated from a hypothalamic extract. Furthermore, this compound corresponds to the calculated mass of 10,388 Da deduced from the cDNA sequence. The detection of an immunoreactive 6 kDa peptide in the gel filtration fractions suggests an intermediate step in the prosomatostatin processing in these cells.

Animals↗
PubMed1988

Vasoactive intestinal peptide receptors in pancreas and liver. Structure-function relationship.

In purified rat pancreatic plasma membranes, (D-Phe4)PHI interacts as a selective VIP agonist for rat pancreatic VIP-preferring receptors, based on binding selectivity and adenylate cyclase activation, therefore allowing us to discriminate between the participation of VIP-preferring and secretin-preferring receptors in VIP stimulation. VIP-preferring receptors also bind GRF. They rely on disulfide bridges for their functional integrity. Their coupling with adenylate cyclase, based on the intrinsic activity of VIP analogues, is poor when compared to that of hepatic VIP receptors. In fresh rat liver plasma membranes, high-affinity VIP receptors are specifically labeled with [125I]helodermin and [125I]His1, D-Ala NLeu27)GRF and are well coupled to adenylate cyclase while low-affinity VIP receptors are not. The first subtype of VIP receptors is highly responsive to guanyl nucleotides and is easily altered by dithiothreitol. Only after freezing and thawing are low-affinity hepatic VIP receptors coupled to adenylate cyclase. Concerning the chemical characterization of VIP receptors, 66- and 35-kDa peptides are detected after specific [125I]VIP cross-linking with double agents in rat pancreatic membranes. In contrast, in intact pancreatic acini, the main source of radioactivity has a molecular mass of 130-180 kDa (with no contribution of intramolecular disulfide bridges), and an 80-kDa peptide is also detectable. The 66-kDa species in membranes can conceivably derive from the 80-kDa species observed in intact cells. Its molecular mass is higher than that of the 56-kDa [125I]VIP cross-linked protein previously observed in rat liver membranes. Besides, species differences between rat and guinea pig pancreas are also evident.

Adenylyl Cyclases↗