Cyclic AMP regulates the calcium transients released from IP(3)-sensitive stores by activation of rat kappa-opioid receptors expressed in CHO cells.
We analyzed intracellular Ca(2+)and cAMP levels in Chinese hamster ovary cells expressing a cloned rat kappa opioid receptor (CHO-kappa cells). Although expression of kappa(kappa)-opioid receptors was confirmed with a fluorescent dynorphin analog in almost all CHO-kappa cells, the kappa-specific agonists, U50488H or U69593, induced a Ca(2+) transient only in 35% of the cells. The Ca(2+) response occurred in all-or-none fashion and the half-maximal dosage of U50488H (812.1nM) was higher than that (3.2nM) to inhibit forskolin-stimulated cAMP. The kappa-receptors coupled to G(i/o)proteins since pertussis toxin significantly reduced the U50488H actions on intracellular Ca(2+) and cAMP. The Ca(2+) transient originates from IP(3)-sensitive internal stores since the Ca(2+) response was blocked by a PLC inhibitor (U73122) or by thapsigargin depletion of internal stores while removal of extracellular Ca(2+) had no effect. Interestingly, application of dibutyryl cAMP (+ 56.2%) or 8-bromo-cAMP (+ 174.7%) significantly increased the occurrence of U50488H-induced Ca(2+) mobilization while protein kinase A (PKA) inhibitors, Rp-cAMP (-32.3%) or myr-psi PKA (-73.9%) significantly reduced the response. Therefore, it was concluded that cAMP and PKA activity can regulate the Ca(2+) mobilization. These results suggest that the kappa receptor-linked cAMP cascade regulates the occurrence of kappa-opioid-mediated Ca(2+) mobilization.
Ikeda
M
M
Advanced Research Institute for Science and Engineering, Waseda University, Okubo, Shinjuku-ku, Tokyo, Japan. msikeda@aol.com
Nelson
C S
CS
Shinagawa
H
H
Shinoe
T
T
Sugiyama
T
T
Allen
C N
CN
Grandy
D K
DK
Yoshioka
T
T
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000700:Analgesics / Q000494:pharmacology; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D044482:Benzeneacetamides; D003994:Bucladesine / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D010455:Peptides; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D000700:Analgesics; D018712:Analgesics, Non-Narcotic; D000701:Analgesics, Opioid; D044482:Benzeneacetamides; D004791:Enzyme Inhibitors; D010455:Peptides; D011759:Pyrrolidines; D017473:Receptors, Opioid, kappa; D013873:Thionucleotides; C057337:dynorphin A-analogue kappa ligand; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016957:adenosine-3',5'-cyclic phosphorothioate; D003994:Bucladesine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D015544:Inositol 1,4,5-Trisphosphate; D020881:Enkephalin, D-Penicillamine (2,5)-; D000242:Cyclic AMP; C045444:U 69593; D002118:Calcium