The physiological significance of the endogenous cannabinoid anandamide as a vanilloid (TRPV1) receptor agonist is controversial due to its low intrinsic efficacy. The rationale for this study is that the binding site for anandamide on the TRPV1 receptor is at an intracellular domain and that access to this site limits drug potency. The aim of this study was to use the whole-cell patch clamp recording technique and fura-2 Ca²⁺ imaging to compare the potencies of anandamide as a TRPV1 receptor agonist when applied intracellularly via the patch pipette solution and extracellularly by low pressure ejection. The investigations were carried out in neonatal rat cultured dorsal root ganglion (DRG) neurones.

Using fura-2 Ca²⁺ imaging we characterised and compared the actions of anandamide and capsaicin in DRG neurones. Anandamide (1 µM) evoked responses in only 20 out of 159 neurones (n= 18 preparations) and application of capsaicin to the same neurones produced a rise in intracellular Ca²⁺ in 114 out of 159 neurones. The amplitudes of the Ca²⁺ transients evoked by capsaicin (100nM) were significantly larger (P<0.001, Students, unpaired t-test) than those produced by 1 µM anandamide, the mean peak amplitudes being 1.89 ± 0.17 and 0.27 ± 0.08 respectively. Interestingly, we found that 5 neurones that did not respond to capsaicin, did respond to anandamide (peak amplitude 0.26 ± 0.07).

In whole-cell patch clamp electrophysiology experiments, extracellular application of 100 nM anandamide inhibited high voltage-activated Ca²⁺ currents (VACCs) in 5 out of 8 neurones, the mean inhibition being 33 ± 9%. This is consistent with the cannabinoid receptor-mediated inhibition of VACC also observed with cannabinoid receptor agonist WIN 55212 (Ross et al., 2001). In contrast, extracellular application of 100 nM anandamide failed to evoke inward currents in 7 out of 8 neurones that responded to capsaicin (1µM) with a mean inward current of -0.94 ± 0.21 nA. A further 20 neurones failed to respond to anandamide or capsaicin. At the higher concentration of 10µM, anandamide evoked inward currents in ~27% of neurones (n=15, 4 responding neurones), the amplitudes of the responses ranged from -0.2 nA to -1.9 nA.

Intracellular application of 100 nM anandamide elicited robust inward currents in ~62% of neurones, the mean population response was -0.85 ± 0.21 nA (n=21; 13 responding neurones). These currents appear to be mediated by TRPV1 receptor because when anandamide was applied to the intracellular environment with capsazepine (1 µM) the mean population inward current was -0.01 ± 0.01 nA (n=14; 1 responding neurone). Under control conditions, in the absence of anandamide, small mean population current fluctuations of -0.09 ± 0.05 nA were observed (n=13; 3 spontaneously active neurones).

The results suggest that TRPV1 receptors in DRG neurones are more effectively activated when anandamide is applied to the intracellular environment compared with the extracellular environment.

Ross et al., 2001, Neuropharmacology 40, 221-232.

Acknowledgements: The Wellcome Trust.