The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

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.Chromaffin cells are derived from the neural crest and constitute the adrenal medulla. In the adrenal medullary chromaffin cell, secretory granules contain a complex mixture of proteins and peptides that are co-released with catecholamines into the circulation in response to splanchnic nerve stimulation (1) . 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 (1-3). Recently, using highly sensitive proteomic techniques we have characterized the maturation products of proenkephalin-A (4) and established the presence of other unexpected proteins (5).Phosphatidylethanolamine-binding protein (PEBP) 1 (6, 7)/ Raf-1 kinase inhibitor protein (8) has previously been described in brain and adrenal gland (9, 10). PEBP is a 21-kDa protein initially described as a cytoplasmic protein and later found to be associated with plasma or reticulum membranes (11). This protein has been found in numerous tissues of many species, including rat testis, liver, kidney, and human platelets (6, 9, 10), indicating its ubi...

Raf kinase inhibitory protein knockout mice: Expression in the brain and olfaction deficit

“…It has been suggested that RKIP-1 protein is a precursor of HCNP [29,30,37]. The reporter expression pattern in the mouse brain is consistent with potential endocrine relevance of the gene [1,15]. However, the mutation used herein does not disrupt exon I, preserving the N-terminal HCNP sequence for processing and function.…”

The Emerging Cardioinhibitory Role of the Hippocampal Cholinergic Neurostimulating Peptide

“…In addition, PEPB expression has been detected on Western blots of heart and liver extracts (Frayne et al, 1999). Recently, an estimation of PEBP concentration in bovine serum, obtained after protein sequencing, indicated a value of 35 nM (Goumon et al, 2004). More recently, using chromaffin cells from bovine adrenal medulla as a well characterized secretion model, we demonstrated that PEBP and HCNP are present in chromaffin cells (intragranular matrix), from which they are secreted into the circulation with catecholamines (Goumon et al, 2004).…”

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

“…56 In the brain, an N-terminal fragment of PEBP known as the "hippocampal cholinergic neurostimulating peptide" (HCNP) seems to serve as a transmitter and an endocrine factor. 57 PEBP is primarily a cytosolic protein, but is able to bind to cellular and model membranes through its N-and C-terminal ends. 58 In addition, a cavity at the protein surface binds phosphatidylethanolamine (PE) with high affinity; 58 this site can also bind other phospholipids (albeit with lower affinity) or opioids.…”