Biomedical subjects

J Christophe

Publications and source records attributed to J Christophe.

At least 73 records · Page 4Linked to original sources

PubMed1990

Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J.

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4-2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38-residue peptide (PACAP-38) and as an N-terminal amidated 27-residue derivative (PACAP-27). The binding sites showed considerable affinity for [125I]PACAP-27 (Kd = 0.4 nM) and PACAP-38, while their affinity for VIP and the parent peptide helodermin was 1000-fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP-38 and PACAP-27 (Kact = 0.2 nM) being much higher than that of VIP (Kact = 100 nM) and helodermin (Kact = 30 nM). Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed a specifically cross-linked peptide with an Mr of 68,000 (including 3000 for one PACAP-27 molecule).

Adenylyl Cyclases↗

PubMed1990

Divergent regulation of beta 2-adrenoceptors and adenylate cyclase in the Cyc- mouse T lymphoma cell line TL2-9.

The radiation leukemia virus-induced murine Cyc- T lymphoma cell line TL2-9 expressed one homogeneous population of beta 2-adrenoceptors based on competition curves of [125I]cyanopindolol with the specific antagonist ICI 118.551 and three beta-adrenergic agonists. These receptors were uncoupled from adenylate cyclase due to the absence of Gs. The catalytical unit was directly stimulated by MnCl2, forskolin, and even more markedly in the simultaneous presence of both reagents. In contrast, the enzyme was inhibited in the presence of Gpp[NH]p, probably through interaction with Gi. Indeed, this inhibitory effect was constrained by preincubating cells in the presence of pertussis toxin and a 41 kDa protein was specifically ADP-ribosylated in the presence of the toxin. This cell line was therefore analogous to the Cyc- cell line derived from the murine S49 lymphoma cell line. When added to the culture medium, butyrate (2 mM) induced beta 2-adrenoceptors, the expression of these uncoupled receptors depending on protein synthesis, as judged by inhibitory effects of cycloheximide. In contrast, dBcAMP (1 mM) and TPA (tumor-promoting agent phorbol ester) increased the rate of disappearance of beta 2-adrenoceptors. Butyrate, dBcAMP and TPA systematically decreased adenylate cyclase activity. Besides, TPA (but neither butyrate nor dBcAMP) reduced the efficacy of Gpp[NH]p in inhibiting adenylate cyclase, suggesting a proportionately higher alteration of Gi. We conclude that beta 2-adrenoceptors, uncoupled from adenylate cyclase, are regulated independently from the catalytical unit and Gi, in this Cyc- T lymphoma cell line.

Adenylyl Cyclases↗

PubMed1990

Stereoselective inhibition of muscarinic receptor subtypes by the enantiomers of hexahydro-difenidol and acetylenic analogues.

1. The affinities of the (R)- and (S)-enantiomers of hexahydro-difenidol (1) and its acetylenic analogues hexbutinol (2), hexbutinol methiodide (3) and p-fluoro-hexbutinol (4) (stereochemical purity greater than 99.8%) for muscarinic receptors in rabbit vas deferens (M1), guinea-pig atria (M2) and guinea-pig ileum (M3) were measured by dose-ratio experiments. 2. The (R)-enantiomers consistently showed higher affinities than the (S)-isomers. The stereoselectivity ratios [(R)/(S)] were greatest with the enantiomers of 1 (vas deferens: 550; ileum: 191; atria: 17) and least with those of the p-Fluoro-analogue 4 (vas deferens: 34; ileum: 8.5; atria: 1.7). 3. The enantiomeric potency ratios for compounds 1-4 were highest in rabbit vas deferens, intermediate in guinea-pig ileum and much less in guinea-pig atria. Thus, these ratios may serve as a predictor of muscarinic receptor subtype identity. 4. (S)-p-Fluoro-hexbutinol [(S)-4] showed a novel receptor selectivity profile with preference for M3 receptors: M3 greater than M2 greater than or equal to M1. 5. These results do not conform to Pfeiffer's rule that activity differences between enantiomers are greater with more potent compounds.

Alkynes↗

PubMed1990

VIP receptors in human SUP-T1 lymphoblasts.

We characterized a new type of vasoactive intestinal peptide (VIP) receptors in the CD4+ Stanford University Pediatric (SUP)-T1 lymphoma cell line, by comparing receptor occupancy [in the presence of (125I)helodermin and (125I)(acetyl-His1)VIP] and adenylate cyclase activation (in the presence of GTP). The order of potency of peptides on both parameters was: helodermin greater than (acetyl-His1)VIP greater than (Phe1)VIP = VIP greater than PHI while secretin was ineffective. In membranes, when Gs was permanently activated by Gpp(NH)p or by ADP-ribosylation (after pretreating intact lymphoblasts for 2 h with cholera toxin), there resulted a variably increased affinity of receptors for VIP-like peptides, suggesting reduced receptor selectivity. Preexposing intact lymphoblasts to the same peptides induced, within 5 min, homologous desensitization (i.e. reduced binding capacity and even more so impaired capability to activate adenylate cyclase), whose extent correlated with the Kd of each peptide at time 0. After prolonged (16 h) exposure to 30 nM VIP that resulted in marked (75%) downregulation, 60% of the adenylate cyclase responsiveness could recover within 30-120 min even in the presence of cycloheximide, but further resensitization was cycloheximide-sensitive. To conclude, VIP receptors coupled to adenylate cyclase showed distinct specificity in human SUP-T1 lymphoblasts. Their specificity decreased when Gs was permanently activated. In intact cells exposed to VIP-like peptides, the receptors were rapidly desensitized, then down-regulated, the resensitization mechanism being not immediately inhibited by cycloheximide.

Adenylyl Cyclases↗

PubMed1990

Binding of selective antagonists to four muscarinic receptors (M1 to M4) in rat forebrain.

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.

Alkaloids↗

PubMed1989

Purification and characterization of five variants of phospholipase A2 and complete primary structure of the main phospholipase A2 variant in Heloderma suspectum (Gila monster) venom.

1. Five increasingly anionic variants (Pa1-Pa5) of Ca2+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspectum (Gila monster). The purification procedure was based on semi-preparative reverse-phase HPLC followed by anion-exchange HPLC and analytical reverse-phase HPLC. 2. Their Mr were 17,000-18,000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 greater than Pa5 greater than Pa4 greater than Pa1 greater than Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase A2 type and were lost upon p-bromophenacyl bromide treatment. 3. All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa2 greater than Pa1 approximately equal to Pa4 greater than Pa3 approximately equal to Pa5. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4. The five variants, Pa1-Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Pa2 and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N-terminal amino acid sequence (31 residues) of Pa1-Pa5 was exactly the same. 5. The full sequence of the major variant, Pa5, showed that this 142-amino-acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class II secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa5 displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half-cystine residues only, an incomplete N-terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C-terminal extension never seen in other phospholipases A2, with the only exception being bee venom.

Amino Acid Sequence↗

PubMed1989

Affinity profiles of hexahydro-sila-difenidol analogues at muscarinic receptor subtypes.

In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a series of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M1-(rabbit vas deferens), M2- (guinea-pig atria) and M3- (guinea-pig ileum) receptors. All compounds were competitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenyl ring with a methoxy group or a chlorine atom as well as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 approximately M3 greater than M2. A different selectivity pattern was observed for p-fluoro-hexahydro-sila-difenidol: M3 greater than M1 greater than M2. This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA2 = 6.68) and lowest affinity for the M2-receptors in guinea-pig atria (pA2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2) and ileum (M3) of the rat. Furthermore, dose ratios obtained with either pirenzepine (M1) or hexahydrosila-difenidol (M2 and M3) and the p-fluoro analogue used in combination suggested that the antagonism was additive, implying mutual competition with a single population of muscarinic receptor subtypes. These results indicate that p-fluoro-hexahydro-sila-difenidol represents a valuable tool for characterization of muscarinic receptor subtypes.

Animals↗

PubMed1989

The phorbol ester TPA stimulates the expression of functional beta-adrenoceptors in human T lymphoblasts Molt 3.

In crude membranes from human T lymphoblasts Molt 3 cultured under standard conditions, the adenylate cyclase system was stimulated by GTP, its beta gamma-imido analogue (p[NH]ppG,) NaF and forskolin, but not by isoprenaline, prostaglandin E1 and vasoactive intestinal peptide. TPA (tumour-promoting agent phorbol ester) added at low concentration (3.2 nM) to the culture medium induced a marked increase in functional beta 2-adrenoceptors. Competition curves of [125I]cyanopindolol with the antagonist ICI 118.551 and four beta-adrenergic agonists indicated that the emergence of functional beta 2-adrenoceptors corresponded to one class of binding sites, shifting from a high-affinity state for agonists to a low-affinity state in the presence of p[NH]ppG. This expression of beta 2-adrenoceptors after a 4 h lag period depended on newly formed mRNA and protein synthesis as judged by the inhibitory effects of actinomycin D and cycloheximide. Further effects of TPA included alterations of the stimulatory G-protein Gs and/or the catalytic unit of adenylate cyclase.

Adenylyl Cyclases↗

PubMed1989

Properties of vasoactive-intestinal-peptide receptors and beta-adrenoceptors in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3.

1. Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with beta 2-adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus. 2. The relative potency of VIP-related peptides to stimulate adenylate cyclase activity was: helodermin greater than VIP greater than peptide histidine isoleucinamide. Five VIP analogs inhibited 125I-iodo-VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-Ala4]VIP = [D-His1]VIP = [D-Phe2]VIP. [D-Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin- and VIP-stimulated adenylate cyclase activity with a similar Ki (0.07-0.10 microM). These data suggest the existence, in this murine T-cell lymphoma, of VIP receptors of the 'helodermin-preferring' subtype that are coupled to adenylate cyclase.

Adenylyl Cyclases↗

PubMed1989

Homologous and heterologous regulation of the helodermin/vasoactive-intestinal-peptide response in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3.

1. Functional vasoactive intestinal peptide (VIP)/helodermin receptors and beta 2-adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal). 2. Short-term (5-30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase. 3. Long-term (16-h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D-Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.

Adenylyl Cyclases↗

PubMed1989

Isolation and primary structure of rat secretin.

A major form of rat secretin was purified to homogeneity from small intestine, being detected with a porcine secretin radioimmunoassay throughout 7 chromatographic steps. The sequence of the heptacosapeptide amide H-S-D-G-T-F-T-S-E-L-S-R-L-Q-D-S-A-R-L-Q-R-L-L-Q-G-L-V-NH2 shows that rat secretin has a glutamine residue in position 14 instead of arginine as in pig secretin.

Amino Acid Sequence↗

PubMed1989

Molecular architecture of secretin receptors: the specific covalent labelling of a 51 kDa peptide after cross-linking of [125I]iodosecretin to intact rat pancreatic acini.

p-Azidophenylglyoxal (APG), a heterobifunctional reagent with one group reacting selectively with arginine residues and another group photoactivable, was used to cross-link [125I]secretin prebound to intact rat pancreatic acini. The best yield was obtained when the [125I]secretin-acini complex was incubated under dim light with 2 mM APG at 37 degrees C and pH 8.0, followed by photolysis at 312 nm. The main secretin binding peptide cross-linked under reducing conditions, when tested by SDS-PAGE and autoradiography: (i) had a molecular mass of 51 kDa and was not a subunit of a larger disulfide-linked structure, and (ii) was distinct from the main VIP binding peptide coexisting in the same preparation.

Animals↗

PubMed1989

Bretylium tosylate binds preferentially to muscarinic receptors labelled with [3H]oxotremorine M (SH or 'high affinity' receptors) in rat heart and brain cortex.

Bretylium tosylate is an antiarrhythmic agent. In guinea pig atria it showed the properties of a competitive muscarinic (cholinergic) antagonist and could distinguish between two muscarinic receptor classes or states in cardiac membranes. We decided to further investigate its binding properties at muscarinic cholinergic receptors of the rat heart and brain (cortex), keeping in mind the recently discovered heterogeneity of muscarinic receptor protein. Bretylium tosylate recognized two receptor classes or states in the heart with Ki values of 0.9 and 11 microM. All cardiac membrane receptors showed a homogeneous (11 microM) Ki value for the drug in the presence of GTP in the incubation medium, or after in vivo pretreatment with islet activating protein (IAP). Bretylium tosylate was able (but only at a high concentration, 1 mM) to slow the dissociation kinetics of the tracer, which suggests that it also bound to an allosteric site on the muscarinic receptor, or that it affected the receptor environment. In the brain cortex, as in the heart, bretylium tosylate displayed a high affinity for receptors labelled with the agonist [3H]oxotremorine M (Ki value: 0.8 microM for the SH-or cardiac-type high-affinity receptors), and a 8- to 10-fold lower affinity for cortex M and L receptors. These data suggest that the antagonist bretylium tosylate had binding properties in rat cardiac membranes analogous to those of the partial agonist pilocarpine and that it interacted with a single type of receptor.

Animals↗

PubMed1989

Variable stimulation of adenylate cyclase activity by vasoactive intestinal-like peptides and beta-adrenergic agonists in murine T cell lymphomas of immature, helper, and cytotoxic types.

We examined several cultured murine T cell lymphomas, induced by a radiation leukemia virus MuRadLV, including cell lines derived from immature T cells (5 clones of the BL/VL3 cell line), antigen-specific T helper cells (5 lines of the TL2 series), and one T cytotoxic cell line (NS8). With one exception (the TL2-9 cell line), these cells showed common characteristics: 1) an efficient adenylate cyclase system; 2) increased cyclic AMP production in response to at least one type of neurotransmitter, i.e., to the catecholamine isoproterenol and/or the neuropeptide VIP; 3) on the basis of adenylate cyclase stimulation, beta-adrenoceptors were of the beta 2 subtype and VIP receptors were of a "helodermin-preferring" subtype previously encountered in a human T lymphoblast cell line. Although we analyzed only a limited number of cell lines, it appeared that the immature T BL/VL3 clones responded to peptides of the VIP family with higher potency and efficacy than T helper and T cytotoxic cells. The membranes from the specific TL2-9 helper cell line were without adenylate cyclase activity in the presence of Gpp[NH]p, NaF, and GTP alone or GTP in the presence of isoproterenol or VIP. They produced cyclic AMP in the presence of Mn2+ and forskolin only, suggesting a defect in Gs as in S49 cyc- mouse lymphosarcoma cells. This was further demonstrated by the absence of cholera toxin-stimulated ADP-ribosylation in TL2-9 membranes.

Adenylyl Cyclases↗

PubMed1989

Binding and functional properties of antimuscarinics of the hexocyclium/sila-hexocyclium and hexahydro-diphenidol/hexahydro-sila-diphenidol type to muscarinic receptor subtypes.

1. In an attempt to assess the structural requirements for the muscarinic receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a series of structurally related C/Si pairs were investigated, along with atropine, pirenzepine and methoctramine, for their binding affinities in NB-OK 1 cells as well as in rat heart and pancreas. 2. The action of these antagonists at muscarinic receptors mediating negative inotropic responses in guinea-pig atria and ileal contractions has also been assessed. 3. Antagonist binding data indicated that NB-OK 1 cells (M1 type) as well as rat heart (cardiac type) and pancreas (glandular/smooth muscle type) possess different muscarinic receptor subtypes. 4. A highly significant correlation was found between the binding affinities of the antagonists to muscarinic receptors in rat heart and pancreas, respectively, and the affinities to muscarinic receptors in guinea-pig atria and ileum. This implies that the muscarinic binding sites in rat heart and the receptors in guinea-pig atria are essentially similar, but different from those in pancreas and ileum. 5. The antimuscarinic potency of hexahydro-diphenidol and hexahydro-sila-diphenidol at the three subtypes was influenced differently by structural modifications (e.g. quaternization). Different selectivity profiles for the antagonists were obtained, which makes these compounds useful tools to investigate further muscarinic receptor heterogeneity. Indeed, the tertiary analogues hexahydro-diphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) had an M1 = glandular/smooth muscle greater than cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M1 preferring (M1 greater than glandular/smooth muscle, cardiac).

Animals↗

PubMed1989

Peptide histidine isoleucinamide (PHI)-(1-27)-Gly as a new major form of PHI in the rat small intestine.

Three immunoreactive peptide histidine isoleucinamide (PHI) forms (I, II, and III) from a rat small intestinal extract were separated on a Fractogel column, using a specific RIA. Peak III was identified as rat PHI-(1-27)-NH2 based on its coelution with a synthetic standard and its amino acid sequence. Peak I was tentatively considered as PHI extended with the connecting peptide preexisting between PHI and vasoactive intestinal peptide in their common precursor, based on its apparent mol wt. Peak II was the most abundant form (based on immunoassay) and has not been described previously. It was purified to homogeneity by using a RIA throughout the first three chromatographic steps, then a fast RRA (on rat liver membranes) during the last three purification steps. This new PHI variant was identified as rat PHI-(1-27)-Gly, as judged by full sequencing amino acid analysis after C-terminal digestion by carboxypeptidase-Y and by coelution with synthetic rat PHI-(1-27)-Gly.

Amino Acid Sequence↗

PubMed1989

Variable distribution of three molecular forms of peptide histidine isoleucinamide in rat tissues: identification of the large molecular form as peptide histidine valine-(1-42).

We previously isolated three forms of peptide histidine isoleucinamide (PHI) from rat small intestine by chromatography on Fractogel. Rat PHI-(1-27)-NH2 and rat PHI-(1-27)-Gly were identified by microsequence analysis. In the present work the third larger form was purified to homogeneity, being detected by RIA in preliminary steps and by RRA in the last steps. This form could be recognized by a PHI antiserum and an antiserum raised against the synthetic rat PHI-(22-27) fragment C-terminally extended by the connecting peptide linking PHI to vasoactive intestinal peptide (VIP) in their common PHI/VIP precursor, as postulated by the reported cDNA analysis of rat brain mRNA. Sequence analysis and C-terminal hydrolysis by carboxypeptidase-Y identified this large form as peptide histidine valine-(1-42), i.e. rat PHI-(1-27) C-terminally extended by the totality of the connecting peptide except for the terminal dibasic residues. We also documented the coexistence of the three PHI forms in rat brain, rachidian bulb, pituitary, adrenal glands, uterine horns, and stomach. Tissue distribution was highly variable. The classical PHI-(1-27)-NH2 form was best represented in rachidian bulb and somewhat less so in brain and uterine horns. PHI-(1-27)-Gly, while being particularly abundant in the small intestine, was very poorly present in rachidian bulb and uterine horns. Peptide histidine valine-(1-42) was the major form in pituitary and adrenal glands and was also well expressed in uterine horns. The three forms coexisted equally in stomach. This uneven distribution suggests a tissue-specific posttranslational processing of rat prepro-PHI/VIP.

Amino Acid Sequence↗

PubMed1989

Synthesis of cholecystokinin-related peptides and their biological properties.

Two tyrosine-O-sulfate containing cholecystokinin-related peptides were synthetized, i.e. the undeca- and the dodecapeptide amide corresponding to the C-terminus of this gut hormone; in the undecapeptide the N-terminal serine was O-substituted with the 2-deoxy-alpha-D-galactosyl group. Within the limits of error of the biological assay systems the two CCK peptides exhibited potencies in stimulating amylase secretion from dispersed rat pancreatic acini comparable to those of the CCK deca-, nona- and octapeptide.

Amino Acid Sequence↗