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Differential effects of mu-opioid receptor ligands on Ca(2+) signaling
J Mark Quillan1, Kurt W Carlson, Chunyan Song
1Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA, USA.
Abstract:
Activation of mu-opioid receptors (MORs) transfected into human embryonic kidney 293 cells, caused a multiphasic increase in cytosolic free Ca(2+) levels (Ca(2+)i). The first Ca(2+)i maximum (peak 1) between 5 and 7 s depended on the presence of extracellular Ca(2+) (Ca(2+)e). The second phase peaking at approximately 15 s (peak 2) was independent of Ca(2+)e and thus represents Ca(2+) release from intracellular stores. A decrease in temperature from 37 to 25 degrees C also caused reduction of peak 1 but not peak 2, suggesting that the two responses arise from mechanistically distinct pathways. A delayed Ca(2+)e-dependent third response phase is thought to represent capacitative Ca(2+)e influx evoked after release of Ca(2+) 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 Ca(2+) 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 Ca(2+) response to each agonist with greatly varying potencies. Specifically, concomitant injection of diprenorphine failed to affect peak 1 (thought to represent rapid Ca(2+)e influx) stimulated by morphine while fully blocking peak 2 (intracellular Ca(2+) 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 Ca(2+) response patterns resulting from MOR activation.
Insights
Opioid ligands differentially activate mu-opioid receptors (MORs), affecting calcium signaling pathways. Different ligands and antagonists show varying potencies, suggesting distinct binding site conformations for these calcium responses.
Area of Science:
- Pharmacology
- Cellular Biology
- Neuroscience
Background:
- Mu-opioid receptors (MORs) are crucial targets for pain management.
- Opioid receptor activation triggers complex intracellular signaling cascades, including calcium ion (Ca2+) fluxes.
- Understanding these signaling pathways is key to developing safer and more effective analgesics.
Purpose of the Study:
- To investigate the multiphasic calcium (Ca2+) responses elicited by mu-opioid receptor (MOR) activation in human embryonic kidney 293 cells.
- To determine the differential effects of various opioid ligands (agonists and antagonists) on distinct phases of Ca2+ signaling.
- To explore the mechanistic basis of these differential responses, including the role of extracellular Ca2+ and intracellular stores.
Main Methods:
- Transfection of human embryonic kidney 293 cells with mu-opioid receptors (MORs).
- Measurement of cytosolic free Ca2+ levels (Ca2+i) using fluorescence-based assays following agonist stimulation.
- Assessment of the dependence of Ca2+ responses on extracellular Ca2+ (Ca2+e) and intracellular Ca2+ stores.
- Evaluation of the effects of temperature changes on Ca2+ signaling phases.
- Pharmacological characterization using opioid agonists (morphine, etorphine) and antagonists (naloxone, diprenorphine) with varying potencies.
Main Results:
- MOR activation induced multiphasic Ca2+ increases: peak 1 (extracellular Ca2+-dependent), peak 2 (intracellular Ca2+ release-dependent), and a delayed Ca2+ influx.
- Temperature reduction differentially affected peak 1 and peak 2, indicating distinct pathway mechanisms.
- Opioid ligands exhibited differential potencies in activating or blocking Ca2+ signaling phases.
- Antagonist diprenorphine showed distinct effects on peak 1 and peak 2, suggesting ligand-specific interactions with MOR binding site conformations.
Conclusions:
- Opioid ligand binding to MORs can elicit distinct patterns of Ca2+ signaling.
- The differential effects of ligands and antagonists suggest the existence of multiple, functionally distinct MOR binding site conformations.
- These findings provide insights into the complex pharmacology of MORs and their downstream signaling, with implications for opioid drug development.