Serge St-Pierre

Neurobehavioral profile of neuropeptide Y

In order to better delineate the profile of central actions of neuropeptide Y (NPY), the effects of intracerebroventricular administration of several doses (2.5-20 micrograms) of the peptide on spontaneous activity, muscular tone, body temperature, food intake, nociception and cataleptic manifestations were examined in rats. Results indicate that, starting at 5 micrograms. NPY significantly decreased motor activity of animals in a dose-related fashion. NPY also significantly lowered body temperature of animals. The hypothermic effect was obtained following injections of 10.0 and 20.0 micrograms of the peptide. Administration of the same two doses of NPY resulted in significant increases in food intake, muscular tone and induced a significant catalepsy in animals. On the other hand, nociceptive response times of animals in the hot plate test were not affected by any of the NPY doses tested. Together, these results indicate that the profile of NPYs neurobehavioral actions is more complex than previously reported and suggest that the peptide might be implicated functionally in a variety of neurophysiological processes.

Two new peptides to improve post-operative gastric ileus in dog.

Peptides can influence gastrointestinal motility, and from data obtained earlier in rats, we hypothesized that MTL-RP/Ghrelin, as well as CGRP receptor antagonist 8-37, could improve gastric post-operative ileus in dog. Dogs submitted to laparotomy were perfused with or saline or CGRP 8-37 or MTL-RP/Ghrelin on days 1-4 post-operatively while gastric emptying was estimated by measuring the postprandial increase in plasma acetaminophen ingested with a meal. As expected, in saline-treated animals the gastric emptying function was impaired post-operatively. The total amount of acetaminophen emptied (AUC over 150 min) on post-operative days 1-4 reached respectively 31+/-5%, 65+/-8%, 60+/-8% and 62+/-8% of the normal emptying capacity. CGRP antagonist increased the total emptying of acetaminophen to 52+/-5% on day 1, 95+/-2% on day 2 and 103+/-3% (P<0.05) on day 3. The delayed emptying of acetaminophen seen post-operatively in saline-treated animals could be completely reversed by MTL-RP/Ghrelin (P<0.01) whether it was given at 100 microg/kg on day 2 (102+/-7% of the normal emptying capacity), 4 microg/kg on day 3 (106+/-7%) or 20 microg/kg on day 4 (132+/-8%). As found earlier in rodents, CGRP receptor antagonist 8-37 as well as MTL-RP/Ghrelin are potent prokinetics to improve post-operative gastric ileus in dog.

Structure-function studies of motilin analogues

Over 100 motilin fragments and analogues, including monosubstituted C-terminal-deleted analogues, conformationally restricted analogues, analogues with peptide bond isoteres (CH2-NH), and N-terminal fragments adjunct to an amphiphilic helix, were synthesized by solid-phase methodology, purified by reverse-phase HPLC, and assayed in vitro in a muscle strip bioassay (rabbit duodenum) and in a radioligand binding assay on crude membrane extracts from smooth muscle (rabbit antrum). The data suggest the existence of three distinct regions involved in the interaction of motilin with its receptor: the N-terminal region (amino acids 1-7) constitutes the minimal basic unit of binding and activity; the transition region (amino acidsa 8-9) links the N-terminal and C-terminal regions; and the C-terminal region (amino acids 10-22) forms an alpha-helix that stabilizes the interaction of the N-terminal residues at the active site.

Neural and muscular receptors for motilin in the rabbit colon.

Motilin receptors were classically recognized in the gastroduodenal area, where they help to regulate interdigestive motility. More recently, motilin receptors were identified in the colon where their biologic significance remains unclear. We aimed here to characterize the motilin receptors of the rabbit colon. Distal colon and duodenum were obtained from sacrificed rabbits. Tissues homogenized by Polytron were submitted to differential centrifugation to obtain neural synaptosomes or smooth muscle plasma membranes enriched solutions. Motilin binding to these membranes was determined by the displacement of (125)I MOT by the native peptide MOT 1-22, or by peptide analogues MOT 1-12 [CH(2)NH](10-11) or GM-109 and by erythromycin derivative GM-611. Motilin binding capacity was maximum in colon nerves (49.5 +/- 6.5 fmol/mg protein vs. 19.9 +/- 2.5 in colon muscles or 9.4 +/- 2.8 and 6.6 +/- 1.2 in duodenal muscles and antral nerves respectively); all tissues expressed similar affinity for MOT 1-22, and the motilin agonist GM-611 bound equally to neural or muscle tissues from the rabbit colon; the synthetic antagonist MOT 1-12 [CH(2)NH](10-11) showed greater affinity for colon nerves than for colon muscles (plC50: 7.23 +/- 0.07 vs. 6.75 +/- 0.03). Similar results were obtained with the peptide antagonist GM-109; receptor affinity toward MOT 1-12 [CH(2)NH(10-11)] was always five times superior in neural tissues, whether they came from the colon or the antrum, than in muscle tissues, whether they were obtained from colon or from duodenum. Motilin receptors are found in very high concentration in nerves and in muscles from rabbit colon; specific motilin receptor subtypes are identified in nerves (N) and muscles (M) of the rabbit colon; N and M receptor subtypes seem independent of the organ location.

Comparative distribution of neuropeptide Y immunoreactivity and receptor autoradiography in rat forebrain

The distributions of neuropeptide Y (NPY) and NPY receptors in rat brain have been compared. High densities of NPY-like fibers and terminals are present in the hypothalamus and the endopiriform nucleus with corresponding low densities of NPY receptor binding sites. Conversely, low densities of fibers and terminals are observed in the thalamus, stria terminalis and hippocampus with corresponding high densities of binding sites. Various hypotheses are discussed to explain those apparent mismatches including the existence of other classes of receptors and possible paracrine actions of NPY-like peptides in the brain.

Human syncytiotrophoblast NPY receptors are located on BBM and activate PLC-to-PKC axis

Neuropeptide Y (NPY) is abundant in plasma and amniotic fluid of women throughout pregnancy, during which its involvement in placental hormonogenesis has been proposed. In accordance with its putative role, the aim of this study was to characterize the human placental syncytiotrophoblast receptivity to NPY. Thus we performed this study on brush-border membranes (BBM) and basal plasma membranes (BPM). Specific125I-labeled NPY (125I-NPY) binding to BBM was rapid (20 min), saturable, with a maximum binding capacity of 604 ± 100 fmol/mg protein, and of high affinity, with a dissociation constant of 11 ± 3 nM. No saturable binding could be shown in BPM. The rank order of affinity of NPY and related peptides to compete for 125I-NPY binding sites was peptide YY (PYY) > NPY = [Leu31,Pro34]NPY > 13-36NPY >> pancreatic polypeptide (PP). It is noteworthy that PYY displaced only 45% of the binding sites. In BBM, both NPY and PYY were potent phospholipase C (PLC) stimulators, leading to a four- to fivefold increase of control phosphodiesterase activity. The latter effect could be prevented by preincubation of membranes with 5 μM U-73122, a known inhibitor of G protein-linked receptor activation of PLC-β. Furthermore, 5 μM BIBP-3226, a Y1-receptor antagonist, shifted both dose-response curves to the right in a similar fashion for both peptides. In accordance with the PLC stimulation, both peptides also induced stimulation of protein kinase C (PKC) activity, which could be partially but additively prevented by U-73122 and LY-294002, a selective inhibitor of phosphatidylinositol-3 kinase (PI3K). Taken together, these data suggest that placental and blood-derived NPY binds to a mixed population of receptors composed of Y1 and Y3 subtypes on the maternal side of the syncytiotrophoblast, where it can mediate its physiological purposes via PLC-β and PI3K activation, both of which lead to PKC activation. However, because BIBP-3226 antagonized both effects, the physiological relevance of the apparent Y3 fraction is still unsolved.

Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit

The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. The rabbit C5a receptor stably expressed in RBL cells binds human 125I‐C5a (2 nM). Unlabelled C5a and the C‐terminal analogue N‐acetyl‐Tyr‐Ser‐Phe‐Lys‐Pro‐Met‐Pro‐Leu‐D‐Ala‐Arg (Ac‐YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. The order of potency human C5a>Ac‐YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).

Micellar electrokinetic chromatography separations of dynorphin peptide analogs

Prodynorphin is a precursor that has multiple cleavage sites to release various dynorphin opioid peptides. The dynorphin analogs used in this study have 18 amino acid residues. A series of dynorphin‐like peptides, differing by a single residue (alanine substitution) were assembled by Fmoc solid‐phase procedures and purified by preparative high performance liquid chromatography (HPLC). Separation of the Ala‐scan dynorphin analogs was investigated by micellar electrokinetic chromatography (MEKC) employing anionic, cationic and zwitterionic surfactants. The role of electrostatic and hydrophobic forces in analyte‐surfactant interactions is discussed with respect to the observed elution patterns. Separation of all dynorphin analogs by MEKC using a zwitterionic surfactant shows this technique to be powerful for separating closely related peptide species. It also demonstrates the potential for using MEKC for the prescreening of peptide libraries to determine their biological activity toward specific receptors. Results from the separation of dynorphin analogs by free solution and ion‐pairing capillary electrophoresis are also presented.

Capsaicin-sensitive structures as potential target sites for neurotensin and bradykinin in guinea pig atria☆

We tested the influence of capsaicin (CAP) desensitization on the positive chronotropic and inotropic effects of neurotensin (NT), bradykinin (BK), calcitonin gene-related peptide (CGRP) and noradrenaline (NA) in guinea pig isolated atria. The positive chronotropic and inotropic effects of NT and BK were completely inhibited, whereas those elicited by CGRP and NA were either slightly reduced (CGRP) or unaffected (NA), in CAP-desensitized compared to control atria. Cross-desensitization studies using CAP, NT and BK showed that the positive chronotropic and inotropic effects of CAP are slightly affected, whereas those evoked by BK are markedly reduced in NT-desensitized atria. On the other hand, the positive chronotropic and inotropic effects of CAP and NT were similar in BK-desensitized and control atria. The results were interpreted as an indication that NT, BK and CAP produce their excitatory effects in guinea pig atria by interacting with a common population of CAP-sensitive sensory nerve fibers (presumably substance P (SP)- and CGRP-containing nerve fibers). The absence of cross-desensitization between NT or BK and CAP, or between NT and BK, suggests that the activation and desensitization of atrial, CAP-sensitive sensory nerve fibers by the latter agents involve different receptors and/or mechanisms.

The coronary vasodilator effect of neurotensin in the guinea pig isolated heart

Neurotensin (NT) infusions into isolated, electrically-driven hearts of guinea pigs, elicited concentration-dependent reductions of myocardial perfusion pressure accompanied by proportional increases of myocardial tension. The decrease of myocardial perfusion pressure caused by NT (attributed to the coronary vasodilator effect of NT) was highly dependent on basal (pre-NT infusions) levels of perfusion pressure, being larger at high perfusion pressure (e.g., 75 mmHg) values than at lower ones (e.g., 50 and 25 mmHg). The perfusion pressure-lowering effect on NT was potentiated and inhibited by neostigmine and atropine, respectively. It was slightly inhibited by methysergide. However, it was not affected by propranolol, indomethacin or a mixture of diphenhydramine and cimetidine. The decreases of myocardial perfusion pressure caused by NT were abolished by NT receptor desensitization, while those evoked by acetylcholine or vasoactive intestinal peptide (VIP) were minimally affected by the desensitization. These results indicate that NT exerts a vasodilator effect in guinea pig coronary vessels. This effect is likely to involve the participation of acetylcholine released from NT-stimulated cardiac cholinergic (vagal) neurons and/or nerve terminals and to be mediated by specific NT receptors. The possible contribution of intracardiac serotonin and/or its receptors to the coronary vasodilator effect of NT is discussed.