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Biomedical subjects

P Robberecht

Publications and source records attributed to P Robberecht.

At least 19 recordsLinked to original sources
PubMed1999

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.

Adenylyl Cyclases↗
PubMed1999

Vasoactive intestinal polypeptide receptor VPAC(1) subtype is predominant in rat prostate membranes.

BACKGROUND: The 28-amino-acid neuropeptide vasoactive intestinal peptide (VIP) might play an important role in the physiology of the prostate, since it stimulates glandular secretion, inhibits muscle contraction, stimulates proliferation of epithelial cells, and increases the secretion of prostate-specific antigen (PSA). This neuropeptide may act through interaction with two types of high-affinity receptors, named VPAC(1) and VPAC(2) receptors. Recently, selective agonists and antagonists for each receptor subtype were synthesized. We used them to identify the VIP receptor subclass expressed in rat prostatic tissue. METHODS: We tested the capacity of selective labeled and unlabeled agonists and antagonists of VPAC(1) and VPAC(2) receptors to bind to rat prostatic membranes and to stimulate or prevent the stimulation of adenylate cyclase activity. RESULTS: The following selective peptides were used: VPAC(1) agonist ([K(15), R(16), L(27)] VIP (1-7)/GRF (8-27)); VPAC(1) antagonist (PG 97-269); and VPAC(2) agonist (RO 25-1553). The IC(50) values of [(125)I]-VIP binding inhibition for the different peptides in rat prostatic membranes were: VIP (1.7 nM) < VPAC(1) agonist (20 nM) < VPAC(1) antagonist (40 nM) < VPAC(2) agonist (329 nM). The EC(50) values of adenylate cyclase stimulation were similar to the IC(50) values for each peptide, and the Ki values for the VPAC(1) antagonist, inhibiting the adenylate cyclase activity stimulated by VIP and the VPAC(1) agonist, were 22 and 35 nM, respectively. Comparison of binding of [(125)I]-VIP and of [(125)I]-RO 25-1553 indicates the presence of 80% of VPAC(1) and 20% VPAC(2) receptors. CONCLUSIONS: In rat prostate membranes, VPAC(1) receptors are largely predominant. Binding studies were compatible with a ratio of 80/20 of VPAC(1)/VPAC(2) receptors, whereas functionally only VPAC(1) receptors were detected.

Adenylyl Cyclases↗
PubMed1999

Human A-type ANP receptor upregulation by PACAP and carbachol in neuroblastoma cells.

Pituitary adenylyl cyclase activating polypeptide (PACAP-27), forskoline and carbachol increased type A atrial natriuretic peptide receptor (NPR-A) density, as well as NPR-A mRNA level, in the human neuroblastoma NB-OK-1 cell line. TPA did not have any effect per se, but blunted the effect of PACAP-27 on both NPR-A density and NPR-A mRNA. The half-life of the NPR-A mRNA was not modified by any of the agents tested. Our data support an original transcriptional upregulation of human NPR-A in response to cAMP-induced agents, and in response to carbachol.

Animals↗
PubMed1999

The role of VIP/PACAP receptor subtypes in spinal somatosensory processing in rats with an experimental peripheral mononeuropathy.

Peripheral nerve damage often results in the development of chronic pain states, resistant to classical analgesics. Since vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are up-regulated in dorsal root ganglion cells following peripheral nerve injury, we investigated the expression and influence of VPAC1, VPAC2 and PAC1 receptors in rat spinal dorsal horn following a chronic constriction injury (CCI). Electrophysiological studies revealed that selective antagonists of VPAC1, VPAC2 and PAC1 receptors inhibit mustard oil-, but not brush-induced activity of dorsal horn neurones in CCI animals, while cold-induced neuronal activity was attenuated by VPAC1 and PAC1, but not VPAC2 receptor antagonists. Ionophoresis of selective agonists for the receptor subtypes revealed that the VPAC2 receptor agonist excited twice as many cells in CCI compared to normal animals, while the number of cells excited by the VPAC1 receptor agonist decreased and responses to PACAP-38 remained unchanged. In situ hybridisation histochemistry (ISHH) confirmed an increase in the expression of VPAC2 receptor mRNA within the ipsilateral dorsal horn following neuropathy, while VPAC1 receptor mRNA was seen to decrease and that for PAC1 receptors remained unchanged. These data indicate that VIP/PACAP receptors may be important regulatory factors in neuropathic pain states.

Animals↗
PubMed1999

Isolation and primary structure of the CCI papain-like cysteine proteinases from the latex of Carica candamarcensis hook.

The dried latex of the mountain papaya, Carica candamarcensis, was chromatographed on CM-Sephadex C50, giving rise to three peaks (CCI, CCII and CCIII) with amidase activity on N-alpha-benzoyl-DL-arginine-4-nitroanilide. The less basic, most active, peak, CCI, was separated into two components, CCIa and CCIb, by reverse-phase HPLC under denaturing conditions. The primary structures of CCIa and CCIb are presented. They were deduced from sequence analysis of the whole proteins and peptides resulting from enzymatic digestions. Both proteinases are made of 213 amino acid residues, CCIb sharing 88-89% similarity with the three subvariants (G90/R212, E90/R212, E90/K212) of CCIa. 139-140 amino acid residues (65.8%) of CCIa and 141 residues (66.5%) of CCIb are common to papain. The seven cysteine residues are aligned with those of papain and the catalytic triad (Cys25, His159, Asn175) of all cysteine peptidases of the papain family is conserved. The similarity with the other cysteine proteases from Carica papaya is discussed.

Amino Acid Sequence↗
PubMed1999

Neuroprotection of the developing brain by systemic administration of vasoactive intestinal peptide derivatives.

Periventricular leukomalacia (PVL), a necrotic and often cystic lesion of the cerebral white matter occurring in very premature babies, is the leading cause of cerebral palsy in this population. Increased glutamate release and the excitotoxic cascade thus triggered may be critical factors in the development of PVL. The glutamatergic analog ibotenate injected intracerebrally into newborn mice produces white matter cysts that mimic human PVL. Concomitant injection of vasoactive intestinal peptide (VIP), a trophic factor, protects the white matter against excitotoxic lesions. The goal of the present study was to assess the protective properties of systemically injected VIP analogs against ibotenate-induced excitotoxic white matter lesions in newborn mice. VIP analogs were selected on the basis of their low susceptibility to endopeptidases and their potential ability to cross biological membranes. RO-25-1553, a long-lasting cyclic VIP analog, and stearyl-norleucine-VIP, a fatty derivative of VIP, reduced ibotenate-induced white matter cysts by up to 87% and 84%, respectively, when injected i.p. immediately after ibotenate. By comparison, i.p. coadministration of VIP and ibotenate was not protective against the excitotoxic insult. Furthermore, RO-25-1553 and stearyl-norleucine-VIP still induced significant neuroprotection of the developing white matter when injected systemically 8 and 12 h, respectively, after ibotenate, establishing these peptides as therapeutic agents in this murine model. VIP analogs may have therapeutic potential in human premature babies at high risk for PVL.

Amino Acid Sequence↗
PubMed1999

Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor.

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.

Adenylyl Cyclases↗
PubMed1999

Properties of a recombinant human secretin receptor: a comparison with the rat and rabbit receptors.

A secretin receptor was cloned from a commercial human pancreatic complementary DNA (cDNA) bank. The amino acid sequence deduced from the nucleotide sequence differed slightly from the three different sequences previously published, suggesting a genetic polymorphism of the human receptor. The binding properties of the receptor were evaluated by testing natural secretin, related peptides, and synthetic analogs or fragments on membranes of Chinese hamster ovary (CHO) cells expressing the receptor after transfection. The second-messenger coupling was evaluated by adenylate cyclase measurement. The human secretin receptor was compared with the rat and the rabbit receptors. In the three animals species, rat and human secretin were equipotent; rabbit secretin was equipotent on human and rabbit secretin receptors and less potent on the rat receptor. Similar data were obtained for the [Arg16]-secretin analog. Deletion of histidine 1 and replacement of aspartate 3 reduced the affinity of the peptides for the three receptors; however, the reduction was more pronounced on rat than on human and rabbit secretin receptors. Finally, the low affinity of the rat and human receptors for vasoactive intestinal peptide (VIP) was identical; the rabbit receptor, however, had a 20-fold higher affinity. Thus the human secretin receptor shows properties of both rat and rabbit receptors. Evaluation of the properties of chimeric receptors will be useful to fit the ligand on the receptors.

Adenylyl Cyclases↗
PubMed1998

A critical view of the methods for characterization of the VIP/PACAP receptor subclasses.

The binding properties of the three cloned VIP/PACAP receptors and their coupling to G proteins and effectors can be studied in cells expressing each recombinant protein. The data obtained in these models must be critically evaluated: the expression of a high receptor density may reveal irrelevant receptors states and coupling to non-cognate G protein, and entail a marked amplification of the response as well as distortions in the selectivity profile of full and partial agonists. These models are, however, of great interest in the design of selective agonists and antagonists for each receptor subtype. The availability of selective ligands will facilitate the identification of the receptor subtype responsible for PACAP and VIP actions in cells and tissues.

Animals↗
PubMed1998

Analogues of VIP, helodermin, and PACAP discriminate between rat and human VIP1 and VIP2 receptors.

Vasoactive intestinal polypeptide (VIP) acts through interaction with two subclasses of seven transmembrane G protein-coupled receptors named VIP1 and VIP2 receptors. These receptors have been cloned in different species, such as rat and human. Considering the different distribution of both receptor subclasses, there is considerable interest in the development of selective agonists and antagonists. The present study compares the binding properties of VIP, PACAP, GRF, secretin, and helodermin analogues on recombinant rat and human VIP1 and VIP2 receptors. On both rat and human receptors, secretin and GRF had a higher affinity for the VIP1 receptor subtypes. The amino-shortened VIP, and the carboxy terminal-shortened VIP and PACAP analogues also presented a higher affinity for the VIP1 receptor. PHI, PHV, helodermin, and helospectin were selective for the human VIP2 receptor subtypes. These results suggest that the helical structure of the carboxy terminal end is necessary for VIP2 recognition. The differences between species were the following: PHI, PHV, helodermin, and helospectin had a higher affinity for the rat VIP1 receptor than for the human VIP1 receptor. On both rat and human receptors, D-Ala4 VIP and D-Phe4 VIP had a high affinity for the VIP1 receptor and a low affinity for the VIP2 receptor. Thus, three domains of the ligand involved in VIP1/VIP2 receptor discrimination were identified: the amino acid residue in position 4 ([D-Ala4], [D-Phe4]VIP), in positions 8 and 9 (the effects of helodermin and helospectin), and the carboxy terminal end (the effects of the shortened VIP and pituitary adenylate cyclase activating polypeptide analogues).

Amino Acid Sequence↗
PubMed1998

Influence of selective VIP receptor agonists in the rat gastric fundus.

The receptor subtypes involved in the relaxant effect of vasoactive intestinal polypeptide (VIP) in the rat gastric fundus were investigated in vitro. The selective VIP2 receptor agonist [Ac-H1,E8,K12,Nle17,A19,D25,L26,K27,28,G29,30,++ +T31]VIP(cyclo21-25) (RO25-1553) induced a concentration-dependent relaxation (EC50 2.8 nM), while the selective VIP1 receptor agonist derived from growth hormone-releasing factor (GRF) [K15,R16,L27]VIP-(1-7)/GRF-(8-27) had no effect up to 1 microM. [R16] chicken secretin, a selective VIP1 receptor agonist, induced relaxation with a potency of 4.8 nM but its maximal effect was clearly lower than that of VIP, pituitary adenylate cyclase-activating peptide [PACAP-(1-27)] and RO25-1553. This effect was reproduced by porcine secretin (EC50 2.1 nM). It is concluded that the rat gastric fundus contains functional VIP2 receptors but not VIP1 receptors, and that specific secretin receptors are also present.

Animals↗
PubMed1998

Interaction of lipophilic VIP derivatives with recombinant VIP1/PACAP and VIP2/PACAP receptors.

Stearyl vasoactive intestinal polypeptide has been reported to be a VIP (vasoactive intestinal polypeptide) receptor agonist of high potency with an original bioavailability and action. We synthesized three fatty acyl derivatives, myristyl-, palmityl- and stearyl-[Nle17]VIP, and tested their capacity to recognize recombinant rat- and human VIP1- and VIP2/PACAP (pituitary adenylate cyclase-activating polypeptide) receptors and to stimulate adenylate cyclase activity. The three lipophilic analogues bound with high affinity (from 0.5 to 20 nM) to both receptor subtypes but did not distinguish between them. In preparations expressing a high density of human VIP1/PACAP receptors, the three lipophilic analogues had the same efficacy as VIP and [Nle17]VIP. In preparations expressing the rat receptors, stearyl-[Nle17]VIP had a lower efficacy than the other peptides tested. In preparations expressing a low level of VIP1/PACAP receptors and in those expressing VIP2/PACAP receptors, all analogues behaved like partial agonists. The lowest efficacy was observed for stearyl-[Nle17]VIP on the VIP2/PACAP receptor subclass. Based on our results, a complex pattern of in vivo biological effects of the lipophilic VIP derivatives should be expected: these compounds might behave as full agonists, partial agonists, or antagonists of the VIP response, depending on the number and the subtype of receptor expressed.

Adenylyl Cyclases↗
PubMed1998

Vasoactive intestinal peptide modification at position 22 allows discrimination between receptor subtypes.

Secretin and growth hormone releasing factor (GRF) have a weak affinity for VIP (vasoactive intestinal peptide)/PACAP (pituitary adenylate cyclase activating polypeptide) receptors, but discriminate between VIP1/PACAP and VIP2/PACAP receptors. This previously allowed us to develop modified secretin and GRF derivatives as high affinity and highly selective VIP1/PACAP receptor ligands. We tested the hypothesis that the presence of a Gln residue at position 24 and a Leu residue at position 22 was responsible for their VIP1/PACAP receptor selectivity. [Gln24]VIP was not different from VIP but [Leu22]VIP had a 100-fold lower affinity for VIP2/PACAP receptors as compared to VIP1/PACAP receptors. The substitution of Tyr22 by Phe22 in VIP had no significant effect on the recognition of both receptors but [Ala22]VIP had a reduced affinity for the VIP2/PACAP receptor. This indicated that an aromatic residue at position 22 of VIP was required for a high affinity for the VIP2/PACAP receptor but not for the VIP1/PACAP receptor.

Amino Acid Sequence↗
PubMed1998

Contribution of the second transmembrane helix of the secretin receptor to the positioning of secretin.

The secretin amino-terminal residues are essential for high affinity binding to its cognate receptor and for its biological activity. Mutation of the [Asp3] residue of secretin to [Asn3] decreased the ligand's affinity for the rat wild-type receptor 100-300-fold. Receptor mutations in the transmembrane 2 domain and the beginning of the first extracellular loop allowed the identification of three residues involved in recognition of the [Asp3] residue: D174, K173 and R166. Mutation of K173 and D174 not only reduced the secretin and [Asn3]secretin affinities, but also changed the receptor's selectivity as judged by a decreased secretin and [Asn3]secretin potency ratio. The most striking effect was observed when R166 was mutated to Q, D or L. This led to receptors with a very low affinity for secretin but an up to 10-fold higher affinity than the wild-type receptor for [Asn3]secretin. This suggested that R166, highly conserved in that subgroup of receptor, is a major determinant for the recognition of the [Asp3] of the ligand.

Adenylyl Cyclases↗
PubMed1998

Expression of transforming growth factor beta receptors in normal human colon and sporadic adenocarcinomas.

BACKGROUND & AIMS: An absence or a presence of mutated transforming growth factor (TGF)-beta receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-beta. Mutations involving microsatellite-like regions of the type II TGF-beta receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-beta receptors in sporadic colorectal cancers and normal tissues. METHODS: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-beta receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. RESULTS: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. CONCLUSIONS: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-beta receptors that could explain a resistance to TGF-beta-mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.

Adenocarcinoma↗
PubMed1998

Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor.

The rabbit secretion receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine-->arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin (2-27), [E3]secretin, [N3]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1-26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.

Adenylyl Cyclases↗