Jean-Pierre Galizzi

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor.

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5′-O-(3-[35S]thiotriphosphate ([35S]- GTPγS) binding. The dodecapeptide MCH-(6–17) (MCH ring between Cys7 and Cys16, with a single extra amino acid at the N terminus (Arg6) and at the C terminus (Trp17)) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [35S]- GTPγS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met8, Arg11, and Tyr13, were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys7 and Cys16 were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [35S]- GTPγS binding, these compounds behaved as weak antagonists (K B 1–4 μm). Finally, substitution in MCH-(6–17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K B = 0.1–0.2 μm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.

Genetic Deletion of Trace Amine 1 Receptors Reveals Their Role in Auto-Inhibiting the Actions of Ecstasy (MDMA)

“Ecstasy” [3,4-methylenedioxymetamphetamine (MDMA)] is of considerable interest in light of its prosocial properties and risks associated with widespread recreational use. Recently, it was found to bind trace amine-1 receptors (TA1Rs), which modulate dopaminergic transmission. Accordingly, using mice genetically deprived of TA1R (TA1-KO), we explored their significance to the actions of MDMA, which robustly activated human adenylyl cyclase-coupled TA1R transfected into HeLa cells. In wild-type (WT) mice, MDMA elicited a time-, dose-, and ambient temperature-dependent hypothermia and hyperthermia, whereas TA1-KO mice displayed hyperthermia only. MDMA-induced increases in dialysate levels of dopamine (DA) in dorsal striatum were amplified in TA1-KO mice, despite identical levels of MDMA itself. A similar facilitation of the influence of MDMA upon dopaminergic transmission was acquired in frontal cortex and nucleus accumbens, and induction of locomotion by MDMA was haloperidol-reversibly potentiated in TA1-KO versus WT mice. Conversely, genetic deletion of TA1R did not affect increases in DA levels evoked by para-chloroamphetamine (PCA), which was inactive at hTA1 sites. The TA1R agonist o-phenyl-3-iodotyramine (o-PIT) blunted the DA-releasing actions of PCA both in vivo (dialysis) and in vitro (synaptosomes) in WT but not TA1-KO animals. MDMA-elicited increases in dialysis levels of serotonin (5-HT) were likewise greater in TA1-KO versus WT mice, and 5-HT-releasing actions of PCA were blunted in vivo and in vitro by o-PIT in WT mice only. In conclusion, TA1Rs exert an inhibitory influence on both dopaminergic and serotonergic transmission, and MDMA auto-inhibits its neurochemical and functional actions by recruitment of TA1R. These observations have important implications for the effects of MDMA in humans.

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[3H]verapamil binding to muscle tubule membrane has the following properties. KD = 27 +/- 5 nM and maximum binding capacity Bmax = 50 +/- 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [3H]verapamil versus [3H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k1) and dissociation (k-1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [3H] verapamil binding with KD values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [3H]verapamil binding with half-maximum inhibition values (K0.5) between 1 and 5 nM.

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (±)bepridil and (−)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).

NPY receptor subtype in the rabbit isolated ileum

The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. NPY analogues produced a concentration‐dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP>rPP>PYY[Leu31,Pro34]‐NPY>NPY>>NPY13–36. Pre‐exposure to rPP, PYY, [Leu31,Pro34]‐NPY or NPY (but not NPY13–36) inhibited the effect of subsequent administration of hPP suggesting cross‐desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10u2003μM) which also, by itself, produced intrinsic inhibitory effects. 1229U91, a non‐selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration‐dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose‐dependent inhibition of the response to hPP (apparent pKB: 7.2). These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

NPY receptor subtypes involved in the contraction of the proximal colon of the rat

Experiments were designed to determine the receptor subtype(s) involved in the contraction of the rat proximal colon to NPY. In this tissue, mRNA of Y2 and Y4 NPY receptor subtypes were highly expressed, whereas Y5 mRNA levels were very low and Y1 mRNA levels were intermediate. NPY analogues induced contractions with the following order of potency: rPP > hPP = PYY = NPY = [Leu31,Pro34]NPY > NPY(2-36) = [D-Trp32]NPY > NPY(33-36). Responses to NPY, PYY and NPY(13-36) were not or partially affected by tetrodotoxin, in contrast to the responses to [Leu31,Pro34]NPY, rPP, hPP and [D-Trp32]NPY which were fully blocked. Atropine did not inhibit the contractions to NPY, PYY and [Leu31,Pro34]NPY but significantly affected those to NPY(13-36), [D-Trp32]NPY, rPP and hPP. The specific Y1 receptor antagonist BIBP 3226 was ineffective but JCF 104 and JCF 105 (two compounds with preferential affinity toward the hY5 receptor versus the hY1 or hY2 receptor) abolished the contractions provoked by the NPY analogues. These results suggest that NPY activates three receptor subtypes, a Y2 subtype possibly by a direct action on the smooth muscle cells, as well as a Y4 and a Y5 (or Y5-like) subtype which, respectively, release acetylcholine and an unknown neurotransmitter.

Receptors for diphenylbutylpiperidine neuroleptics in brain, cardiac, and smooth muscle membranes. Relationship with receptors for 1,4-dihydropyridines and phenylalkylamines and with Ca2+ channel blockade.

Neuroleptic molecules of the diphenylbutylpiperidine series (DPBP), such as fluspirilene, penfluridol, pimozide and clopimozide, antagonize binding of (-)[3H]desmethoxyverapamil ((-)[3H]D888) and (+)[3H]PN 200-110 to rabbit brain, heart and smooth muscle membranes. The diphenylbutylpiperidine binding site in all these tissues is distinct but is allosterically related to the 1,4-dihydropyridine binding site and to the phenylalkylamine binding site. High and low affinity binding sites for (-)D888 were identified. (-)[3H]D888 binding at both types of sites was inhibited following the saturation of a single type of diphenylbutylpiperidine binding site. Half-maximal inhibition (K0.5) of brain, heart and smooth muscle membranes binding by different diphenylbutylpiperidines was in the range of 10-100 nM. These K0.5 values were one to two orders of magnitude higher than those found for the high affinity diphenylbutylpiperidine receptor in skeletal muscle membranes. The K0.5 values found in binding experiments in smooth muscle were similar to the (IC50) values for half-maximal inhibition by diphenylbutylpiperidine of voltage-dependent 45Ca2+ influx through the slow Ca2+ channel.

Molecular Properties of Structure and Regulation of the Calcium Channel

Voltage-dependent calcium channels are known to play important roles in excitationcontraction coupling in cardiac and smooth muscles and also in a number of neurosecretory processes. They have recently become accessible to biochemical study as a result of the availability of a number of tritiated calcium channel inhibitors belonging to the dihydropyridine and the phenylalkylamine series.’ Dihydropyridine derivatives such as nitrendipine and other calcium inhibitors such as verapamil, bepridil, and diltiazem are very important therapeutic agents in the treatment of cardiovascular disorders.’ One of the difficulties associated with the biochemical characterization of macromolecules that confer electrical excitability to biological membranes, such as the voltage-sensitive calcium channel, is that they are almost always present in very low amounts in membranes. For most membrane preparations in which the voltagesensitive calcium channel has been characterized, the dihydropyridine receptor is only present at a density in the range of 0.1 to 1 pmol/mg protein.’ Therefore, the transverse tubule (T-tubule) membrane preparation isolated from rabbit skeletal muscle appears to be exceptional in that it contains more than 50 pmol [3H]dihydropyridine-binding sites/mg p r ~ t e i n . ~ Voltage-clamp analyses of skeletal muscle have shown that essentially all specific Ca’+ conductances are localized in the transverse tubular system and that the channel responsible for these conductances is inhibited by low concentrations