Elsevier

Brain Research

Volume 783, Issue 1, 2 February 1998, Pages 77-84
Brain Research

Research report
The cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-mediated and direct pathways in cultured rat hippocampal neurons

https://doi.org/10.1016/S0006-8993(97)01195-5Get rights and content

Abstract

The effects of the cannabinoid receptor agonist Win55,212 on Ca2+ channels were studied in rat hippocampal neurons grown in primary culture. Win55,212-2 inhibited whole-cell Ba2+ currents through Ca2+ channels by both CB1 receptor-mediated and direct mechanisms. The concentration dependent inhibition of the current showed two clear phases, a high-affinity receptor-mediated phase (IC50=14±2 nM) that was stereoselective and sensitive to a CB1 receptor antagonist, 300 nM SR141716, and a non-saturating phase that was neither stereoselective nor inhibited by SR141716. These concentration-dependent effects were paralleled by Win55212-induced inhibition of glutamatergic synaptic transmission. Win55,212-2 (100 nM) inhibited both ω-agatoxin IVA- and ω-conotoxin GVIA-sensitive currents. Thus, activation of cannabinoid receptors inhibits N- and P/Q-type Ca2+ channels. Activation of cannabinoid receptors inhibited only a fraction of the whole-cell Ca2+ channel current (17±2%) even though more than half of the whole-cell Ba2+ current was carried by N- and P/Q-type Ca2+ channels. Concentrations of agonist greater than 1 μM inhibited Ca2+ channels directly.

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).

References (53)

  • W. Regehr et al.

    Synaptically triggered action potentials in dendrites

    Neuron

    (1993)
  • M. Rinaldi-Carmona et al.

    SR141716A, a potent and selective antagonist of the brain cannabinoid receptor

    FEBS Lett.

    (1994)
  • K. Rose et al.

    Magnesium removal induces paroxysmal neuronal firing and NMDA receptor-mediated neuronal degeneration in cortical cultures

    Neurosci. Lett.

    (1990)
  • M. Scanziani et al.

    Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus

    Neuron

    (1992)
  • A.R. Schatz et al.

    Cannabinoid receptors CB1 and CB2 — a characterization of expression and adenylate cyclase modulation within the immune system

    Toxicol. Appl. Pharmacol.

    (1997)
  • S.M. Thompson et al.

    Presynaptic inhibition in the hippocampus

    Trends Neurosci.

    (1993)
  • L.G. Wu et al.

    Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus

    Neuron

    (1994)
  • M. Bidaut-Russell et al.

    Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain

    J. Neurochem.

    (1990)
  • M.P. Caulfield et al.

    Cannabinoid receptor agonists inhibit Ca current in NG108-15 neuroblastoma cells via a pertussis toxin-sensitive mechanism

    Br. J. Pharmacol.

    (1992)
  • S.R. Childers et al.

    Opioid and cannabinoid receptor inhibition of adenylyl cyclase in brain

    Ann. NY Acad. Sci.

    (1992)
  • D.R. Collins et al.

    Prevention by the cannabinoid antagonist, SR141716A, of cannabinoid-mediated blockade of long-term potentiation in the rat hippocampal slice

    Br. J. Pharmacol.

    (1995)
  • D.R. Compton et al.

    Aminoalkylindole analogs: cannabimimetic activity of a class of compounds structurally distinct from (delta)9-tetrahydrocannabinol

    J. Pharmacol. Exp. Ther.

    (1992)
  • D.R. Compton et al.

    Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities

    J. Pharmacol. Exp. Ther.

    (1993)
  • T.E. D'Ambra et al.

    Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor

    J. Med. Chem.

    (1992)
  • A. De Lean et al.

    Simultaneous analysis of families of sigmoidal curves: application to bioasay, radioligand assay, and physiological dose-response curves

    Am. J. Physiol.

    (1978)
  • S.A. Deadwyler et al.

    Cannabinoids modulate potassium current in cultured hippocampal neurons

    Recept. Channels

    (1993)
  • Cited by (131)

    • Endocannabinoid system contributions to sex-specific adolescent neurodevelopment

      2022, Progress in Neuro-Psychopharmacology and Biological Psychiatry
    • Analysis of the pharmacological properties of JWH-122 isomers and THJ-2201, RCS-4 and AB-CHMINACA in HEK293T cells and hippocampal neurons

      2018, European Journal of Pharmacology
      Citation Excerpt :

      Activation of presynaptic CB1 on glutamatergic synapses is associated with reduced pre-synaptic glutamate release, which is observed experimentally as a suppression of Ca2+ spiking. Suppressed Ca2+ spiking is consistently observed in response to cannabinoid exposure (Blair et al., 2006; Costain et al., 2016; Deshpande et al., 2007; Pacico and Mingorance-Le Meur, 2014; Shen et al., 1996; Shen and Thayer, 1999, 1998; Tauskela et al., 2016). Consequently, the ability of emerging synthetic cannabinoids was compared with benchmark synthetic cannabinoids in their ability to suppress Ca2+ spiking in primary hippocampal neuron cultures.

    View all citing articles on Scopus
    View full text