Abstract
Activation of μ-opioid receptors (MORs) transfected into human embryonic kidney 293 cells, caused a multiphasic increase in cytosolic free Ca2+ levels (Ca2+i). The first Ca2+i maximum (peak 1) between 5 and 7 s depended on the presence of extracellular Ca2+ (Ca2+e). The second phase peaking at ∼15 s (peak 2) was independent of Ca2+e and thus represents Ca2+ release from intracellular stores. A decrease in temperature from 37 to 25°C also caused reduction of peak 1 but not peak 2, suggesting that the two responses arise from mechanistically distinct pathways. A delayed Ca2+e-dependent third response phase is thought to represent capacitative Ca2+e influx evoked after release of Ca2+ from internal stores. Agonists and antagonists of two major classes of opioid ligands, oxymorphinans (morphine and naloxone) and oripavines (etorphine and diprenorphine), had differential effects on Ca2+ currents. Although morphine activated both phases with equal potency, etorphine was 20-fold less potent at stimulating peak 1 over peak 2. Similarly, the antagonists, naloxone and diprenorphine, blocked the Ca2+ response to each agonist with greatly varying potencies. Specifically, concomitant injection of diprenorphine failed to affect peak 1 (thought to represent rapid Ca2+e influx) stimulated by morphine while fully blocking peak 2 (intracellular Ca2+ release). However, diprenorphine potently inhibited peak 1 as well when added to the cells before morphine, indicating limited or slow access of diprenorphine to these morphine binding sites. The existence of multiple, functionally distinct binding site conformations could account for these findings. In conclusion, different opioid ligands can differentially affect Ca2+ response patterns resulting from MOR activation.
Footnotes
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This study was supported by Grant DA 04166 from the National Institute on Drug Abuse and Grant GM 43102 from the National Institutes of Health.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- Ca2+i
- intracellular Ca2+
- SOC
- store-operated channel
- Ca2+e
- extracellular Ca2+
- MOR
- μ-opioid receptor
- HEK
- human embryonic kidney
- PTX
- pertussis toxin
- DAMGO
- [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
- HEK-μ
- human embryonic kidney cells stably transfected with MOR
- BAPTA
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- Received November 30, 2001.
- Accepted April 12, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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