Endogenous cannabinoid anandamide directly inhibits voltage-dependent Ca2+ fluxes in rabbit T-tubule membranes

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Abstract

The effect of the endogenous cannabinoid, anandamide on Ca2+ flux responses mediated by voltage-dependent Ca2+ channels was studied in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca2+ and membrane potentials were generated by establishing K+ gradients across the vesicle using the ionophore, valinomycin. Anandamide, in the range of 1–100 μM, inhibited depolarization-induced efflux responses. Anandamide also functionally modulated the effects of nifedipine (1–10 μM) and Bay K 8644 (1 μM) on Ca2+ flux responses. Pretreatment with the specific cannabinoid receptor antagonist, SR141716A (1 μM), pertussis toxin (5 μg/ml), the amidohydrolase inhibitor, phenylmethylsulfonyl fluoride (0.2 mM) or the cyclooxygenase inhibitor, indomethacin (5 μM) did not alter the inhibition of efflux responses by anandamide. Arachidonic acid (10–100 μM) also effectively inhibited 45Ca2+ efflux from membrane vesicles. In radioligand binding studies, it was found that both anandamide and arachidonic acid inhibited the specific binding of [3H]PN 200-110 to transverse tubule membranes with IC50 values of 4.4±0.7 and 13.4±3.5 μM, respectively. These results indicate that anandamide, independent of cannabinoid receptor activation, directly inhibits the function of voltage-dependent calcium channels and modulates the specific binding of calcium channel ligands of the dihydropyridine class.

Introduction

In the search for endogenous ligands that modulate the activity of voltage-dependent Ca2+ channels (VDCCs), a number of active substances have been identified whose chemical structures vary from novel small peptides to relatively simple chemicals such as ascorbic acid Janis et al., 1988, Callewaert et. al., 1989, Ebersole and Molinoff, 1992. An endogenous lipid soluble substance that modulates the binding of the dihydropyridine class of Ca2+ channel antagonists to rat cortex and brain membranes has been isolated and identified as arachidonylethanolamide (Johnson et al., 1993). Interestingly, the same compound was also identified as anandamide, an endogenous ligand for cannabinoid receptors (Devane et al., 1992).

Inhibition of neuronal Ca2+ currents by anandamide and cannabinoids has been demonstrated in earlier studies Caulfield and Brown, 1992, Mackie et al., 1994, Mackie et al., 1995, Twitchel et al., 1997. However, there has been no report on the effect of anandamide on the function of VDCCs of skeletal muscle which, in terms of its molecular structure, is the best characterized Ca2+ channel subtype (Dunn et al., 1994). Furthermore, since VDCCs are known to be inactivated by rises in intracellular Ca2+, possible secondary effects of anandamide on intracellular Ca2+ homeostasis Martin et al., 1994, Di Marzo and Fontana, 1995 also need to be investigated.

We have previously developed a technique to measure Ca2+ fluxes mediated by VDCCs in purified transverse (T)-tubule membranes Dunn, 1989, Oz et al., 1993. These membranes form sealed, inside-out vesicles that are devoid of intracellular organelles Rosemblatt and Scales, 1989, Dunn, 1989. Thus, studies of Ca2+ fluxes in these vesicles can be used to probe the activity of VDCCs in the absence of intracellular events. In this study, the effect of anandamide on the function of VDCCs in rabbit T-tubule membranes has been investigated.

Section snippets

Preparation of transverse tubule membranes

Microsomal membranes were prepared from the back and hind muscles of small (1–1.5 kg) New Zealand White Rabbits, and T-tubules were isolated by sucrose gradient centrifugation as previously described (Dunn, 1989). The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care (see Guide to the Care and Use of Experimental Animals, Vol. 1 (2nd ed., 1993) and Vol. 2 (1984)). T-tubule membranes were finally resuspended and equilibrated in low K+

Effect of anandamide on voltage dependent Ca2+ fluxes

Fig. 1A shows the results of experiments carried out using the two-step protocol described in the method section. Under control conditions (5–5–5 mM K+ i.e. in the absence of changes in membrane potential) there was no efflux of 45Ca2+ from the membrane vesicles. Upon exposure of vesicles to depolarizing conditions for 10 s (5–120–5 mM K+), the amount of 45Ca2+ remaining in the vesicles was reduced to approximately 30–35% of control values. Both nifedipine (10 μM) and La3+ (1 mM) significantly

Discussion

The main finding of this study is that anandamide, at relatively high concentrations, suppresses the function of VDCCs in T-tubule membranes, and this inhibition is independent of cannabinoid-receptor activation. As is shown in Fig. 1A, these membrane preparations have functional VDCCs i.e., these channels respond to depolarizing voltage changes by opening and are blocked by established inorganic and organic VDCC blockers Dunn, 1989, Oz et al., 1993. Although the inhibitory effect of anandamide

Acknowledgements

This study was in part supported by the Alberta Heritage Foundation for Medical Research and by the Medical Research Council of Canada.

References (41)

  • G. Callewaert et al.

    Modulation of calcium channels in neuronal and cardiac cells by an endogenous peptide

    Science

    (1989)
  • M.P. Caulfield et al.

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

    Br. J. Pharmacol.

    (1992)
  • R. Coronado

    Planer bilayer reconstitution of calcium channels: lipid effects on single-channel kinetics

    Circ. Res.

    (1987)
  • W.A. Devane et al.

    Isolation and structure of a brain constituent that binds to the cannabinoid receptors

    Science

    (1992)
  • R.T. Dirksen et al.

    Single calcium channel behavior in native skeletal muscle

    J. Gen. Physiol.

    (1995)
  • S.M.J. Dunn et al.

    The molecular structure and gating of calcium channels

  • S.M.J. Dunn et al.

    Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: evidence for low-affinity sites for the involvement of G-proteins

    Biochemistry

    (1991)
  • S.M.J. Dunn et al.

    Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in flourescence of felodopine

    Biochemistry

    (1992)
  • B.J. Ebersole et al.

    Identification of ascorbate as an endogenous substance that irreversibly inhibits binding of dhyrdropyridine calcium channel blockers

    J. Neurochem.

    (1992)
  • C. Felder et al.

    Cannabinoid receptors and their endogenous agonists

    Annu. Rev. Pharmacol. Toxicol.

    (1998)
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