Abstract
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+ chelatorN′,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{β-[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.
Footnotes
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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DOI: 10.1124/jpet.102.033456
- Abbreviations:
- PACAP
- pituitary adenylate cyclase-activating polypeptide
- CA
- catecholamine
- [Ca2+]i
- intracellular free Ca2+ concentration
- ACh
- acetylcholine
- nAChR
- nicotinic acetylcholine receptor
- VOC
- voltage-dependent Ca2+ channel
- SOC
- Ca2+ store depletion-operated Ca2+ channel
- IP3
- inositol trisphosphate
- PKC
- protein kinase C
- U-73122
- 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione
- ω-CgTx
- ω-conotoxin
- H-89
- N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfon-amide
- HA1004
- N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride
- SK&F 96365
- 1{β-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenylethyl}-1H-imidazole hydrochloride
- TPEN
- N′,N′,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine
- NDGA
- nor-dihydroguaiaretic acid
- NPPB
- 5-nitro-2-(3-phenylpropylamino)benzoic acid
- LOE 908
- (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl) ethyl]-acetamide
- BSA
- bovine serum albumin
- ω-AgTx IVA
- ω-agatoxin IVA
- TTX
- tetrodotoxin
- PKA
- cAMP-dependent protein kinase
- TG
- thapsigargin
- Received January 22, 2002.
- Accepted April 24, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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