title: Chromostatin receptors control calcium channel activity in adrenal chromaffin cells.
abstract: One of the functions of chromogranin A (CGA), the major soluble component of secretory granules in both adrenal medullary chromaffin cells and many other endocrine cell types appears to be that of a prohormone. CGA is the precursor of several peptides including pancreastatin, a 49-residue peptide, and a 20-residue peptide, chromostatin, which have been identified as biologically active peptides. Chromostatin produces a dose-dependent inhibition (ID50 of 5 nM) of the secretagogue-evoked catecholamine secretion from chromaffin cells. Here we report that chromostatin potently inhibits L-type calcium currents recorded with the nystatin-perforated patch technique in cultured chromaffin cells. This inhibitory effect of chromostatin on calcium currents was not observed in experiments using the classical patch-clamp whole-cell approach which induces the leakage of cytoplasmic components. Using 125I-chromostatin, we show that chromostatin exhibits a fully reversible and saturable binding to the plasma membrane of cultured chromaffin cells. Analysis of binding experiments at equilibrium indicates the existence of one class of binding sites with a Bmax of 2.7 pmol/mg of chromaffin cell proteins and an apparent Kd of 6.5 nM. This high affinity is in good correlation with the half-maximal concentration (ID50 5 nM) of chromostatin inhibiting catecholamine secretion from chromaffin cells. Specificity of the chromostatin binding was further assessed by displacement experiments with unlabeled CGA-related or -unrelated peptides. We found an excellent quantitative correlation between the affinities of the various peptides determined by binding assays and their functional potency tested on catecholamine secretion: bovine chromostatin greater than human chromostatin greater than CGA much greater than rat chromostatin, pancreastatin, CAP-14, substance P, and Leu-enkephalin. Cross-linking experiments reveal that chromostatin associates specifically with an 80-kDa plasma membrane protein. These results together with the patch-clamp experiments support the idea that chromaffin cells possess specific chromostatin receptors and that activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels through an intracellular second messenger pathway.
authors.affiliation: Unité Institut National de la Santé et de la Recherche Médicale, U-338 Biologie de la Communication Cellulaire, Strasbourg, France.
authors.forename: E
authors.initials: E
authors.lastname: Galindo
authors.forename: M
authors.initials: M
authors.lastname: Mendez
authors.forename: S
authors.initials: S
authors.lastname: Calvo
authors.forename: C
authors.initials: C
authors.lastname: Gonzalez-Garcia
authors.forename: V
authors.initials: V
authors.lastname: Ceña
authors.forename: P
authors.initials: P
authors.lastname: Hubert
authors.forename: M F
authors.initials: MF
authors.lastname: Bader
authors.forename: D
authors.initials: D
authors.lastname: Aunis
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000378:metabolism*; D053379:Chromogranin A; D002864:Chromogranins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D053379:Chromogranin A; D002864:Chromogranins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C067412:chromostatin; C072203:chromostatin receptor