Abstract
Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.
Highlights
Chromaffin cells are derived from the neural crest and constitute the adrenal medulla
Because Phosphatidylethanolamine-binding protein (PEBP) has been previously immunodetected in adrenal gland, in this report we looked for the localization of PEBP and hippocampal cholinergic neurostimulating peptide (HCNP) in chromaffin cells using confocal laser and electronic microscopy, subcellular fractionation, Western blot analysis, purification by reverse-phase HPLC, sequencing, and mass spectrometry analysis techniques
Immunoelectron microscopy experiments performed on adrenal gland sections have shown that several subcellular compartments of adrenal medullary chromaffin cells were labeled for PEBP (Fig. 1B, middle panel)
Summary
Chromaffin cells are derived from the neural crest and constitute the adrenal medulla. Among the high molecular mass water-soluble proteins, proenkephalin-A and the chromogranins family constitute the major constituents of chromaffin granules with other neuropeptides (neuropeptide Y, vasointestinal peptide, and others). These proteins are actively processed in the intragranular matrix to peptides with various molecular weight [1,2,3]. Because upon a stimulation catecholamines display a positive inotropic effect on heart physiology (for review, see Ref. 20) and on the basis of our recent data showing the negative inotropic effect on frog heart [21] of a natural intragranular N-terminal chromogranin A (CGA)-secreted peptide, vasostatin-I, we have examined the effect of HCNP, the natural N-terminal PEBP-derived fragment on cardiac mechanical performance. We have shown that HCNP acts on the cardiac physiology, indicating that it may be considered, with its precursor, as new endocrine factors
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