The mechanisms of exocytosis induced by neurokinin peptides in mast cells

Abstract

Neurokinin peptides consisting of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) are tachykinin family peptides isolated from mammals that have a wide variety of physiological functions such as the depolarization of primary sensory neurons, the contraction of smooth muscle, the exocrine secretion from pancreas and hypotension [1]. Most of biological functions of neurokinin peptides are mediated by three pharmacologically distinct cell membrane receptors classified as NK1 , NK 2 and NK3 neurokinin receptors [1]. It has been already cloned functional cDNAs for three neurokinin receptors. Neurokinin peptides have a common Phe–X–Gly–Leu–Met–NH 2 structure (X is Phe or Val) at the C-terminus, and this structure is required for the activation of known neurokinin receptors. SP is also known to stimulate the secretion of histamine from peritoneal mast cells. As regard the mechanisms of exocytosis in mast cells induced by SP, it is already demonstrated that exocytosis stimulated by SP is not due to the activation of three known neurokinin receptors by the structure-activity studies using SP and its derivative peptides on the stimulation of exocytosis, that positively charged amino acid residues are important for the stimulation of exocytosis and a common Phe–X–Gly–Leu– Met–NH 2 structure at C-terminus is not required. In addition, it was suggested the direct activation of G i type of G protein in mast cells by SP as well as mastoparan (MP), that also stimulates exocytosis in mast cells [2,3]. It is already demonstrated that SP and MP induce the production of IP3 and the activation of voltage-independent Ca2+ channels indicating the importance of cytosolic Ca 2 + on the exocytotic pathway. However, for the sake of the difficulty in measuring the concentration of intracellular free Ca2 + ([Ca2+] i ) n mast cells, it has not been clear the involvement of cytosolic Ca2+ on the exocytotic pathway. In this study, we found the conditions that we could measure [Ca 2+ ] i change in rat peritoneal mast cells, and measured [Ca 2 + ] i increase induced by SP and MP to investigate the relationship between [Ca 2 + ] i increase and exocytosis in rat peritoneal mast cells. The secretion of β -hexosaminidase from mast cells was measured as the exocytotic secretion, because this enzyme was found in the same secretory vesicles that histamine was contained. Increase of [Ca2+ ]i was measured using fura-2, a sensitive luminescent Ca2+ chelator.