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.
Highlights
Major soluble componentof secretory granules in both kDa plasma membrane protein
chromostatin potentlyChromogranin A (CGA) is the precursorof several peptides includ- tors and that activationof such receptors leads to the ing pancreastatin,a 49-residue peptide, anda 20-res- inhibition of L-type voltage-sensitive calcium channels idue peptide, chromostatin, which have been identifiedthrough an intracellularsecond messenger pathway
We report that chromostatin potentlyChromogranin A (CGA)’is an ubiquitous48-kDa secretory inhibitsL-type calcium currentsrecordedwiththe protein first described in the adrenal medulla [1]which has nystatin-perforated patch technique in cultured chro- subsequently been found in both the central and peripheral maffin cells
Summary
Locke's solution. [3H]Noradrenaline secretion was calculated as the Effect of Chromostatin on Voltage-dependent Calcium Curpercentage of total radioactivity present in the cells at thebeginning rent-Previous work fromourlaboratoryhas shown that of the experiment assessed from the amountreleased plus the amount. Inactivation of Ca2+currents is minimized as it occurs using Specificity of the High Affinity Chromostatin-binding the nystatin-perforated patch technique[20] This procedure Sites-The specificity of the high affinity chromostatin-bindof normalization is shown in Fig. 1 E where the timecourse of ing sites was assessed by displacing the binding of lZ5I-chrothe inhibitory actionof the peptidewas studied by repeating mostatin on intact cultured chromaffcienlls with structurally I-V curves every 3 min for both control and peptide-treated and functionally related peptides. Addition of chromos- peptide consisting of chromostatin to which the four amino tatin (1MM) caused amarked decrease in this ratio, indicating acids EVEK were added to the N-terminsailde These results that chromostatinblocks L-type voltage-dependentCa2+cur- are consistent with the fact that neither pancreastatin, nor rents in chromaffin cells (Fig. 1E).
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