Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism.

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.

Chromogranin A in familial pheochromocytoma: diagnostic screening value, prediction of tumor mass, and post-resection kinetics indicating two-compartment distribution.

PURPOSE: Chromogranin A, co-released with catecholamines from the adrenal medullary and sympathetic neuronal vesicles, is elevated in plasma from patients with pheochromocytoma. We assessed its diagnostic screening value, its plasma level in correlation with tumor mass, and its disposition kinetics in familial pheochromocytoma and sporadic pheochromocytoma. PATIENTS AND METHODS: The sensitivity and specificity of chromogranin A's diagnostic value for pheochromocytoma were established through one kindred with familial pheochromocytoma associated with von Hippel-Lindau syndrome (13 available members) and in seven subjects with sporadic pheochromocytoma. Serial postoperative plasma samples were also obtained (5 minutes to 4 days) from eight subjects with pheochromocytoma in order to study chromogranin A post-resection kinetics. Chromogranin A was measured by radioimmunoassay based on purified pheochromocytoma chromogranin A. RESULTS: In this kindred, elevations of chromogranin A (greater than 52 ng/mL) were sensitive (83%, five of six) and specific (100%, 10 of 10) in detecting familial pheochromocytoma; these diagnostic values comparable to those achieved by conventional evaluations for pheochromocytoma, such as urinary catecholamines, urinary catecholamine metabolites or imaging methods. Elevated levels of plasma chromogranin A specifically indicated pheochromocytoma, rather than von Hippel-Lindau syndrome gene carrier status. In 13 preoperative subjects with either familial or sporadic pheochromocytoma, plasma chromogranin A concentration predicted tumor size (r = 0.81, p less than 0.01). The change in chromogranin A plasma concentration after pheochromocytoma resection best fit a two-compartment model, with an initial rapid half-life time of 16 minutes, followed by a longer half-life time of 520 minutes. The model also predicted a 23.8:1 compartmental ratio of extravascular/intravascular chromogranin A, suggesting substantial tissue sequestration or binding of chromogranin A. CONCLUSIONS: (1) Plasma chromogranin A is a valuable (sensitive and specific) diagnostic tool in detecting both familial and sporadic pheochromocytoma. (2) The concentration of plasma chromogranin A predicts the size of the pheochromocytoma. (3) Chromogranin A post-resection kinetics suggest extravascular sequestration of chromogranin A.