Vol. 302, Issue 3, 972-982, September 2002

Pituitary Adenylate Cyclase-Activating Polypeptide Induces a Sustained Increase in Intracellular Free Ca²⁺ Concentration and Catecholamine Release by Activating Ca²⁺ Influx via Receptor-Stimulated Ca²⁺ Entry, Independent of Store-Operated Ca²⁺ Channels, and Voltage-Dependent Ca²⁺ Channels in Bovine Adrenal Medullary Chromaffin Cells

Katsuya Morita, Akira Sakakibara, Shigeo Kitayama, Kei Kumagai, Kazuo Tanne and Toshihiro Dohi

Department of Dental Pharmacology, Division of Integrated Medical Science (K.M., S.K., K.K., T.D.), and Department of Orthodontics and Craniofacial Developmental Biology, Division of Cervico-Gnathostomatology (A.S., K.T.), Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

Characteristics of pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increase of Ca²⁺ entry and catecholamine (CA) release were studied in bovine adrenal medullary chromaffin cells. PACAP induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i), showing an initial transient [Ca²⁺]_i rise followed by a sustained rise and CA release, which were not blocked by the blocking agents for nicotinic acetylcholine receptor (nAChR) channel, the voltage-dependent Ca²⁺ channel (VOC), or the Na⁺ channel. The sarcoendoplasmic Ca²⁺-ATPase inhibitors thapsigargin and cyclopiazonic acid did not affect the PACAP-induced sustained rise of [Ca²⁺]_i, but did inhibit the initial [Ca²⁺]_i rise. In cells pretreated with cyclopiazonic acid or membrane-permeable, low-affinity Ca²⁺ chelator N',N',N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, PACAP further stimulated the entry of Ca²⁺ or Mn²⁺, whereas these treatments masked [Ca²⁺]_i dynamics induced by bradykinin. PACAP-induced sustained [Ca²⁺]_i rise and Mn²⁺ entry were enhanced by acidic extracellular solution and reduced by alkalinization, whereas thapsigargin-induced Mn²⁺ entry was regulated by the opposite. PACAP-induced [Ca²⁺]_i rise and Mn²⁺ entry were not affected by blockers of cAMP-dependent protein kinase, phospholipase C, or protein kinase C. All store-operated Ca²⁺ channel (SOC) blocking agents tested inhibited thapsigargin-induced Mn²⁺ entry. 1{-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenylethyl}-1H-imidazole hydrochloride (SK&F 96365), (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide, and econazole inhibited PACAP-induced Ca²⁺ or Mn²⁺ entry, whereas GdCl₃, 7,8-benzoflavone, nor-dihydroguaiaretic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, fulfenamic acid, and niflumic acid did not. SK&F 96365 and econazole but not GdCl₃ inhibited PACAP-induced CA release. These results suggest that PACAP activates a novel Ca²⁺ entry pathway associated with sustained CA release independent of the nAChR channel, VOC and SOC, activated by acid pH, with different sensitivity to blockers of SOC. This pathway may provide a useful model for the study of receptor-operated Ca²⁺ entry.