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