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Biomedical subjects

J Christophe

Publications and source records attributed to J Christophe.

At least 19 recordsLinked to original sources
PubMed1997

[Molecular endocrinology of hereditary obesity].

The hypothalamic disorders of obesity include hyperphagia, a low central orthosympathetic tone (with reduced thermogenesis), vagal hyperinsulinism, low serotonin efficacy, a hyperactive hypothalamo-hypophyseal-adrenal axis, a hypoactive GHRH-GH-IGF axis and hypogonadism of central origin. Hyperlipogenesis, glucose intolerance and excessive gluconeogenesis are secondary features. Most frequently the hypothalamic ARC reacts poorly to the leptin hypersecreted by adipose tissue, so that the local synthesis of NPY is unchecked. Fortunately, two prostaglandins derived from dietary arachidonic acid bind fat cell PPAR gamma and hepatic PPAR alpha. Both nuclear proteins are phosphorylated through an insulin pathway, thereby inhibiting the expression of genes favoring obesity and stimulating that of genes accelerating fatty acid oxidation. The array of dietetic and pharmacologic tools considered today is analyzed.

Adipose Tissue↗
PubMed1994

Characterization of ocular receptors for pituitary adenylate cyclase activating polypeptide (PACAP) and their coupling to adenylate cyclase.

Pituitary adenylate cyclase activating polypeptide (PACAP), a recently discovered neuropeptide, has large structural homology with vasoactive intestinal polypeptide (VIP). Two molecular forms exist, one with 27 (PACAP-27) and one with 38 (PACAP-38) amino acids. PACAP-27 is identical to the N-terminal of PACAP-38. Two major types of PACAP receptors have been identified; selective PACAP receptors, which bind PACAP with a much higher affinity than VIP, and non-selective VIP/PACAP receptors, which bind PACAP and VIP with equally high affinity. In the present investigation, PACAP receptors in different parts of the albino rabbit eye, and their coupling to adenylate cyclase were characterized. Crude tissue homogenates from iris, ciliary body, retina and choroid were used. Competition binding curves were established for VIP, PACAP-27 and PACAP-38, with [125I]VIP or [125I-Acetyl-His1]PACAP-27 as tracer. The effects on adenylate cyclase activity were determined by plotting dose-response curves (10(-10)-10(-6) M) for VIP, PACAP-27 and PACAP-38. The anterior uvea had mainly (approximately 80%) non-selective VIP/PACAP receptors, but a small amount of selective PACAP receptors was detected. In the retina, the selective PACAP receptor predominated (approximately 85%), while the choroid (including the retinal pigment epithelium) had approximately 60% selective PACAP receptors. PACAP-27 and PACAP-38 stimulated the formation of cAMP with the same efficacy: 6.9-fold in the ciliary body, 3.6-fold in the iris, 5.1-fold in the retina and 2.3-fold in the choroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1994

Pancreatic tumoral cell line AR42J: an amphicrine model.

AR42J cells derive from azaserine-induced malignant nodules from the rat pancreas. They differ from normal acinar cells for at least three reasons: 1) they proliferate rapidly; 2) they synthesize, store, and secrete digestive enzymes but the regulation of their exocrine function is abnormal, from the emergence of atypical receptors (e.g., cholecystokinin octapeptide type B and pituitary adenylate cyclase-activating polypeptide type I receptors) to unusual inositol phosphate metabolism and cytoskeleton disorganization; and 3) they possess an added neuroendocrine-regulated pathway characterized by voltage-sensitive ionic currents, post-translational processing of peptidic prohormones (and possibly autocriny), and the release of small neurotransmitters (gamma-aminobutyric acid, glycine, and glutamic acid). These amphicrine cells represent, therefore, a cancerous version of the primordial pancreatic ductular epithelium. Dexamethasone favors their differentiation toward the exocrine phenotype. The mitogenic pathway is favored by the occupancy of receptor tyrosine kinases, adenosine 3',5'-cyclic monophosphate, ornithine decarboxylase expression, and Na(+)-H+ exchange. Somatostatin opposes proliferation through protein phosphatases.

Animals↗
PubMed1994

[Molecular cloning and functional expression of glucagon receptors in the liver, the heart and various other tissues].

The mRNA for a glucagon receptor is present in five glucagon-responsive tissues of rat, including the liver, heart, pancreatic islets, kidneys and adipose tissue. The mature mRNA of this receptor is likely to derive from one gene only in all tissues investigated. The maturation of the pre-mRNA requires the splicing of 11 introns. This proceeds stepwise at the 5' end but a single intronless ORF is finally operative. The receptor is a glycoprotein endowed with 485 amino acids (including the signal peptide).

Adipose Tissue↗
PubMed1993

New evidence for a membrane-bound pathway in hormone receptor binding.

The fully active cholecystokinin analog (Thr,Nle)-CCK-9 was lipo-derivatized by N-terminal grafting of a dimyristoylglycerol moiety to induce tight interdigitation with cell membrane bilayers. While the parent CCK peptide was shown to interact only transiently with small unilamellar phospholipid vesicles, the lipo-CCK peptide, although self-aggregating into vesicles, inserts rapidly and quantitatively into phospholipid bilayers. Fluorescence and, even more so, NMR data are supportive for a chain reversal of the CCK moiety of the lipo derivative with embedment of the C-terminus into hydrophobic compartments of the bilayer. MD simulations allowed for a proposal of the folded form of CCK in bilayers with a helical array parallel to the interface and an amphipathic display of the side chains. In this model, the phenylalanine aromatic ring is heading the peptide molecule and may thus play a decisive role in the lateral penetration of the receptor at the water/lipid interface. In fact, despite the membrane-bound state, its binding affinity for rat pancreatic acini is comparable to that of the CCK peptide when tested after a 3-h equilibration period but 5-6-fold lower at 45 min, suggesting that the association rate is significantly lower than that of the unmodified CCK peptide. This can rationally be attributed to the tight interdigitation of the double-tailed lipo moiety with the membrane bilayer. Moreover, an escape of the lipopeptide into the extracellular aqueous phase is energetically highly unfavored; therefore, the receptor can only be reached by a membrane-bound two-dimensional migration. The observed difference in amplification between binding and amylase secretion may result from inadequate occupation of low-affinity CCK receptors, which leads then to poor couplings to G-proteins. Nevertheless the data confirm that lateral penetration of receptor structures is possible, and thus, preadsorption of peptide (neuro)hormones at the cell membrane bilayer may indeed represent the first step in the receptor recognition process.

Amino Acid Sequence↗
PubMed1993

Purification and amino acid sequence of motilin from cat small intestine.

Motilin was isolated from cat small intestine by a series of chromatographic steps. Using a radioreceptor assay, based upon binding of iodinated porcine motilin to rabbit antral smooth muscle membranes, it was shown that cat duodenal mucosa contains about 495 ng/g tissue, the jejunal mucosa 161 ng/g tissue and the ileal mucosa 95 ng/g tissue motilin. The duodenal mucosa was extracted with 6% acetic acid and concentrated on a cation exchange Whatman CM-52 gel. After lyophilization the material was further purified by gel filtration (Sephadex G-50), followed by reverse phase (C18), cation exchange HPLC (Mono S) and three runs on a reverse phase HPLC (Nucleosil 300-5C18) column. The UV absorbance and the radioreceptor assay were used to monitor the purification. After Mono S chromatography two forms of motilin were detected. The major peak corresponded to a 22 amino acid peptide, which differed only from canine motilin at position 12, where Lys is replaced by Arg. The smaller peak probably corresponds to a deamidated form of this peptide. The sequence homology between cat and porcine/human motilin or cat and rabbit motilin is 81.8% and 72.7%, respectively. The conservation of the first six amino acids in all five species studied is striking, confirming that the biological activity of the peptide resides in the N-terminal part.

Amino Acid Sequence↗
PubMed1993

Type I receptors for PACAP (a neuropeptide even more important than VIP?).

Among vertebrates, there is an extreme conservation in amino acid sequence for the neuropeptide PACAP-38 and its C-terminal shortened derivative PACAP-27. The PACAP gene is assigned to chromosome 18 in man and its organization has been characterized. PACAP-38 and its minor derivative PACAP-27 are widely distributed in the central nervous system. PACAP-38 is particularly abundant in hypothalamus. The mapping of the afferentation and efferentation of PACAP systems are progressively delineated, including a search for the colocalization with other neurotransmitters. In several peripheral organs positive neuronal perikarya and fibers are also seen. PACAP acts through two types of receptors: (1) the highly selective type I that displays a 500 to 2000 selectivity for PACAP-38 and PACAP-27 as compared to VIP; (2) type II is the so-called VIP receptor showing similar high affinity for PACAP-38, PACAP-27 and VIP. It is less selective, therefore, than previously thought. This is why this second receptor, qualifying as an unspecific VIP-PACAP receptor, is hardly considered here. Type I receptors can stimulate two enzymes: the adenylate cyclase and phospholipase C (whose activation leads to the inositol phosphate-cytosolic Ca2+ cascade). This dual coupling may have several distal consequences including on gene expression, cell growth and differentiation. Although a relatively comprehensive spectrum of pharmacological activities has already been established we still need to limit the physiological roles of PACAP as neurotransmitter and/or neuromodulator. Concerning the hypothalamo-pituitary axis, PACAP reduces food intake in mice and raises plasma arginine vasopressin in rat, probably through PACAP-ir neurons in paraventricular and supraoptic nuclei projecting to the neurohypophysis. PACAP originating in the hypothalamus may also be transported to the anterior pituitary through portal vessels. Data on the antehypophysis suggest a role on i.a. reproduction and growth. PACAP stimulates adenylate cyclase and increases [Ca2+] in gonadotropes, somatotropes, and folliculo-stellate cells. It elevates the secretion of alpha-MSH from melanotropes, and that of interleukin-6 from pituitary folliculo-stellate cells. PACAP potentiates the effects of LHRH on LH and FSH secretion. More clearly perhaps, PACAP increases the synthesis of LH, GH, PRL and ACTH after 1-2 days. In human pathology, PACAP-27 and PACAP-38 stimulate adenylate cyclase activity in membranes from 'null'-, gonadotropin-, GH-, and ACTH-producing pituitary adenomas but are inactive in prolactinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Cloning of a splice variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor.

The rat pancreatic acinar cell line AR 4-2J is endowed with numerous PACAP type I receptors (PACAPR1). The cDNA of this receptor was PCR amplified at low stringency using two degenerate primers based on conserved sequences in the TM2 and TM6 segments of secretin, parathormone and calcitonin receptors. One additional amplified band of 574 bp possessed an original 84 bp insert. This fragment, when used as probe for Northern blot analysis, revealed a high M(r) (about 7.5 kb) transcript in AR 4-2J cells and also in rat brain. Screening a lambda Uni-Zap bacteriophage library of AR 4-2J cell cDNA yielded one hybridizing clone with an ORF of 1254 bp. The translated 418 amino acid peptide showed 206 identities with rat VIP receptors and 176 identities with secretin receptors. This sequence homology, together with the mRNA distribution, and the expression study of a similar cDNA published very recently (8) indicated that we had cloned PACAPR1 except for its 77 N-terminal amino acids. Its putative I3 loop contained a unique additional 28 amino acid sequence (with four hemi-cystines and several serine, threonine and basic residues). Using RT-PCR we then demonstrated the coexistence of a second form of mRNA, without this added insert, in DNAse-pretreated RNAs from both AR 4-2J cells and normal rat brain. This indicated that common alternative splicing provokes the diversification of PACAP type I receptors into PACAPR1A (unspliced) and PACAPR1B (spliced) variants.

Alternative Splicing↗
PubMed1993

A cDNA construct allowing the expression of rat hepatic glucagon receptors.

We constructed a full open reading frame (ORF) for the rat hepatic glucagon receptor using two non functional pCDM8 clones of a putative glucagon receptor. Clone I was complete but contained, in addition, two small introns interrupting the frame, while clone II lacked 104 bp at the 5' end. We isolated Hind III/Dra III and BamH I/Not I fragments from clone I and a Dra III/BamH I fragment from clone II then ligated the three fragments with pBluescript SK(+) digested with Hind III/Not I. Following this 4 fragment ligation procedure, we obtained one correct insert in a clone we subcloned in the Hind III/Not I sites of pCDM8. This plasmid was used to transiently transfect COSGs1 cells with the lipofectin method. In COSGs1 membranes transfected with this correct plasmid (but not with a truncated plasmid) [125I]iodoglucagon binding was selectively displaced with glucagon (IC50 = 4 nM) and adenylate cyclase was stimulated with glucagon (K(act) = 10 nM) while related peptides were inefficient at 1 microM. This demonstrated that the receptor previously described is indeed the rat hepatic glucagon receptor.

Animals↗
PubMed1993

Small introns in a hepatic cDNA encoding a new glucagon-like peptide 1-type receptor.

A cDNA fragment was PCR amplified at low-stringency from rat DNA, using two degenerated primers corresponding to consensus sequences in the second and third transmembrane segments of members of the secretin receptor family. A synthetic oligodeoxynucleotide based on the 3 end of this fragment, when used in Northern-blot analysis, hybridized to a broad 1.5-3.0 kb band in rat liver. It was then used as a probe for screening a cDNA library generated from size-fractionated rat liver poly(A)+ RNA. Overlapping alignment of sequences from 3 clones identified a 1455 residue open reading frame encoding a 485 amino acid peptide showing 7 putative transmembrane segments and 42% identity with the glucagon-like peptide 1 (GLP-1) receptor. In addition, four small (85-300 bp) introns were variously represented in the isolated clones.

Amino Acid Sequence↗
PubMed1993

New functionally selective muscarinic agonists.

The muscarinic pharmacology of two novel agonists related to McN-A-343, 4-F-PyMcN and 4-F-PyMcN+, has been studied by the use of pharmacological and radioligand binding techniques. Both compounds were potent agonists at M1 receptors in rabbit vas deferens (pEC50 = 6.24 and 6.96) and rat duodenum (pEC50 = 5.47 and 6.38), but very weak partial agonists or competitive antagonists at guinea-pig cardiac M2 and ileal M3 receptors. There was no receptor reserve for 4-F-PyMcN in rabbit vas deferens, for which the potency (pEC50 = 6.24) and apparent affinity (pKA = 5.99 and 6.21) were similar. 4-F-PyMcN+ showed only limited binding selectivity between four muscarinic receptor binding assays with apparent affinity constants (pKi) of 5.8, 5.2, 5.6 and 5.7 for M1, M2, M3 and M4 muscarinic receptor subtypes. The two novel functionally M1-selective agonists may provide useful tools with which to study muscarinic receptor mechanisms. The non-quaternary compound, 4-F-PyMcN, might become a starting point for the development of drugs that selectively affect M1 receptors involved in central cholinergic function.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1993

Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila-pridinol, diphenidol and sila-diphenidol type.

1. We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using [3H]-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide. 2. The affinity of the antagonists either increased or decreased with temperature. van't Hoff plots were linear in the 278-310 degrees K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to -29 kJ mol-1) to large positive values (up to +30 kJ mol-1). 3. (R)-configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed. 4. When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes. 5. Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change of drug binding. 6. Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects. 7. Taken together, these results suggest that hydrogen bond-forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and M2 muscarinic receptors.

Animals↗
PubMed1993

Regulation of Na-K-Cl cotransport, Na,K-adenosine triphosphatase, and Na/H exchanger in human neuroblastoma NB-OK-1 cells by atrial natriuretic peptide.

The occupancy of atrial natriuretic peptide (ANP) receptors of the ANPA type in human neuroblastoma NB-OK-1 cells elevates cGMP. In this study, ANP concentrations of 10 nM or more increased total K+ uptake. Data obtained in the presence of bumetanide and/or ouabain demonstrated that 1 microM ANP induced a primary stimulation (by 82%) of Na-K-Cl cotransport and a subsequent indirect stimulation (by 15%) of Na,K-ATPase. ANP also inhibited Na/H exchange through an amiloride-sensitive mechanism, as shown by intracellular pH measurement in cells challenged or not by an acid or alkaline load. (Bu)2cGMP mimicked all ANP effects, suggesting that ANP acted through a cGMP-dependent mechanism.

Amiloride↗
PubMed1993

Receptors for pituitary adenylate cyclase activating peptides in human pituitary adenomas.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors coupled to adenylate cyclase was investigated in four types of human pituitary adenomas: three null adenomas and five gonadotropin-, three ACTH-, four GH-, and four PRL-producing adenomas. In all samples, except in prolactinomas, PACAP(1-27) and PACAP(1-38) stimulated adenylate cyclase activity equally well and potently (K(act) around 3 nmol). Vasoactive intestinal polypeptide (VIP) was systematically 100- to 300-fold less potent than both PACAPs. In prolactinomas, PACAP(1-27), PACAP(1-38), and VIP were inactive despite a response of the enzyme to guanosine 5'-triphosphate, Gpp(NH)p, forskolin, and fluoride. [125I-AcHis1]PACAP(1-27) binding was detected in all samples except in prolactinomas. In addition, a detailed analysis of receptors was feasible in all five gonadotropin- and in two ACTH-producing adenomas, confirming the existence of selective PACAP receptors that recognized PACAP(1-27) and PACAP(1-38) with similar high affinity (IC50 0.8-1.5 nmol) and VIP with a low affinity (IC50 100 nmol/L).

Adenoma↗
PubMed1993

[The neuropeptide PACAP: its presence, its mode of action and its receptors].

Specific PACAP type I receptors show 200- 1000-fold higher affinity for both PACAPS as compared to VIP. Their occupancy stimulates adenylate cyclase and frequently also the inositol phosphate-calcium cascade. Considering a few PACAP-38 and PACAP-27 effects exerted on the hypothalamo-hypophyseal axis and elsewhere, there appears already that these neuropeptides may play a physiological role even more important than that of VIP.

Animals↗
PubMed1992

Discovery of a potent atrial natriuretic peptide antagonist for ANPA receptors in the human neuroblastoma NB-OK-1 cell line.

The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).

Atrial Natriuretic Factor↗