Research reportThe cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-mediated and direct pathways in cultured rat hippocampal neurons
Introduction
Cannabinoid receptors are members of the seven helix transmembrane, G-protein-linked receptor superfamily [26]. There are at least two isoforms of the receptor, one predominantly present in neurons (CB1) and the other located in the periphery (CB2) 26, 15, 30, 2, 41. There are several potent drugs that bind to the CB1 receptor that are of particular relevance to this report. SR141716 is a potent antagonist that is selective for the CB1 receptor 36, 37, 8, 21. The aminoalkylindole Win55,212-2 is a cannabinoid receptor agonist that is approximately 1000-fold more potent than its enantiomer, Win55,212-3 9, 11, 20, 23.
Activation of cannabinoid receptors inhibits adenylate cyclase 18, 19, 4, 7, 10, 41, inhibits N- and P/Q-type voltage-gated Ca2+ channels 6, 24, 25, 31and enhances activation of K+ channels 13, 17. Win55,212-2 has been shown to inhibit Ca2+ channel currents in cell lines 6, 24, 25and primary neurons expressing rat brain cannabinoid receptors [31]. In a previous study we reported that cannabinoid receptor agonists acted presynaptically to inhibit glutamatergic synaptic transmission between cultured rat hippocampal neurons [43]. Because Ca2+ channel inhibition may be responsible for the presynaptic inhibition of glutamate release, we tested the effects of cannabimimetics on Ca2+ channel currents in hippocampal neurons.
In this report, we test directly the effects of the cannabinoid receptor agonist Win55,212-2 on Ca2+ channel currents in cultured rat hippocampal neurons. The concentration-dependent inhibition of Ca2+ channels by Win55,212-2 was biphasic. The high affinity component resulted from CB1-mediated inhibition of N- and P/Q-type Ca2+ channels. At micromolar concentrations the drug affected the channel directly. The receptor-mediated inhibition of whole-cell, Ca2+ channel currents was modest relative to the complete block of synaptic transmission produced by Win55,212-2.
Section snippets
Materials
Materials were obtained from the following companies: indo-1 AM and indo-1 pentapotassium salt, Molecular Probes, Eugene, OR; Win 55,212-2 and Win 55,212-3, RBI, Natick, MA; SR141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide], Sanofi, Montpellier, France; ω-agatoxin-IVA, Pfizer, Groton, CT; media and sera, GIBCO, Grand Island, NY; and all other reagents, Sigma, St. Louis, MO.
Cell culture
Rat hippocampal neurons were grown in primary culture as previously
Results
The whole-cell configuration of the patch-clamp technique with Ba2+ as charge carrier [33]was used to study the effects of Win55,212 on voltage-gated Ca2+ channels. Win55,212 inhibited IBa stereoselectively in cultured rat hippocampal neurons. Win55,212-2 (100 nM), the enantiomer active at cannabinoid receptors, inhibited IBa by 17±2% (n=3). Win55,212-3, the inactive stereoisomer, did not affect IBa at this concentration (Fig. 1A). These results indicate that the inhibition of IBa by 100 nM
Discussion
The cannabinoid receptor agonist Win55,212-2 inhibited whole-cell IBa through Ca2+ channels by both CB1 receptor-mediated and direct mechanisms. The concentration dependent inhibition of the current showed two distinct phases, a high-affinity receptor-mediated phase that was stereoselective and sensitive to SR141716 and a non-saturating phase that was neither stereoselective nor inhibited by the CB1 receptor antagonist. Thus, at concentrations greater than 1 μM, Win55,212-2 inhibited Ca2+
Acknowledgements
We gratefully acknowledge Sanofi Recherche for SR141716 and Dr. Nicholas Saccomano at Pfizer Inc. for ω-agatoxin-IVA. This work was supported by grants from the National Institute on Drug Abuse (DA07304, DA09293) and the National Science Foundation (IBN9723796).
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