Anadamide is a putative endogenous cannabinoid. It has been shown to produce the characteristic tetrad of behaviours associated with cannabinoid receptor ligands in mice. In CB1 receptor knockout mice, anandamide is analgesic whilst the antinociceptive effects of Δ9-THC are lost (Di Marzo et al. 2001). Another endocannabinoid candidate oleamide has been shown to inhibit SRF, secondary to block of the voltage-gated Na+ channel (Verdon et al. 2000). Here we hypothesise that anandamide may elicit the same block.
Cultured rat cortical neurones from humanely killed animals were used after 14Ð21 days in vitro. Cells were current clamped at 24 °C and superfused with cobalt-containing saline solution into which drugs were dissolved using 0.1 % DMSO as a vehicle. Only cells with a membrane potential < -50 mV were examined. Stimuli (750 ms) sufficient to produce SRF (over-shooting action potentials) were applied at 0.1 Hz. Results are expressed as percentage of mean ± S.E.M. Analysis was by Student’s unpaired t test and one-way repeated-measures ANOVA where appropriate. Results are expressed as percentage of residual secondary spikes at t = 15 min compared with 0 time in the presence and absence of drugs.
Time-matched vehicle blanks produced a small reduction in the percentage of secondary spikes (7.87 ± 6.93 %, n = 7). Anandamide (10 mM) produced a significant (P = 0.012, n = 7) block of 31.27 ± 4.72 % when compared with time-matched controls. At 20 mM, block was increased to 85.65 ± 10.38 % (P = 0.0001, n = 4). The block produced by 20 mM anandamide could not be inhibited by the CB1 receptor antagonist AM251 (1 mM). 1,1,1-Trifluoro-10(Z)-nonadecen-2-one (TFNO) and phenylmethylsulphonyl fluoride (PMSF) did not significantly (P > 0.05) block SRF compared with control with 9.03 ± 3.66 % (n = 4) and 16.27 ± 11.58 % (n = 4) block, respectively. The percentage block produced by a suprathreshold concentration of anandamide (10 mM) could be significantly potentiated by 320 nM TFNO (71.06 ± 8.93% P = 0.0009, n = 5) and 100 mM PMSF (73.83 ± 11.76% P = 0.002, n = 6) compared with anandamide alone.
The voltage-gated Na+ channel may underpin some of the non-CB1-dependent effects of anadamide. FAAH limits the potency of exogenous anandamide in vitro and may represent an attractive target for drug design (Walker et al. 1999).
Thanks to The Wellcome Trust, UK and Schwarz Pharma AG, Germany for equipment support.
All procedures accord with current UK legislation.