Payet Marcel D, Bilodeau Lyne, Breault Lyne, Fournier Alain, Yon Laurent, Vaudry Hubert, Gallo-Payet Nicole
Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
J Biol Chem. 2003 Jan 17;278(3):1663-70. doi: 10.1074/jbc.M206470200. Epub 2002 Nov 11.
Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration (Ca(2+)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in Ca(2+) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.
先前的研究表明，17至20周龄胎儿的人胎儿肾上腺表达垂体腺苷酸环化酶激活多肽（PACAP）受体，其定位于嗜铬细胞上。本研究的目的是鉴定PACAP受体亚型，并确定PACAP是否能影响细胞内钙浓度（[Ca(2+)]i）和儿茶酚胺分泌。通过原代培养和对嗜铬细胞的特异性刺激，我们证明PACAP-38诱导[Ca(2+)]i增加，该增加被PACAP(6-38)阻断，独立于细胞外钙，且起源于对毒胡萝卜素不敏感的内部储存库。PACAP触发的钙增加不受PLCβ抑制（用U-73122预孵育）或用西司他丁C预处理细胞的影响，表明肌醇1,4,5-三磷酸敏感储存库未被动员。然而，升高胞质cAMP的福斯高林（FSK）诱导的钙增加与用PACAP-38记录的相似。H-89或(R(p))-cAMPS对PKA的阻断抑制了PACAP-38和FSK的钙反应。排空咖啡因/ryanodine敏感的钙储存库也消除了PACAP-38的作用。此外，用原钒酸盐（100微摩尔）处理细胞损害了PACAP敏感储存库的钙再装载，表明PACAP-38可从分泌小泡中动员钙。此外，PACAP诱导嗜铬细胞分泌儿茶酚胺。结论是，PACAP-38通过PAC(1)受体，在人胎儿嗜铬细胞中作为神经递质，通过非经典的、最近描述的ryanodine/咖啡因敏感池诱导儿茶酚胺分泌，涉及cAMP和PKA依赖性磷酸化机制。
