Anandamide is a partial agonist at native vanilloid receptors in acutely isolated mouse trigeminal sensory neurons

Abstract

1. The endogenous fatty acid anandamide (AEA) is a partial agonist at cannabinoid CB1 receptors and has been reported to be a full agonist at the recombinant vanilloid receptor, VR1. 2. Whole cell voltage clamp techniques were used to examine the efficacy of AEA and related analogues methanandamide and N-(4-hydroxyphenyl)-arachidonylamide (AM404) at native VR1 receptors in acutely isolated mouse trigeminal neurons. 3. Superfusion of the VR1 agonist capsaicin onto small trigeminal neurons voltage clamped at +40 mV produced outward currents in most cells, with a pEC(50) of 6.3+/-0.1 (maximum currents at 10-30 micro M). 4. AEA produced outward currents with a pEC(50) of 5.6+/-0.1. Maximal AEA currents (30-100 micro M) were 38+/-2% of the capsaicin maximum. AEA currents were blocked by the VR1 antagonist capsazepine (30 micro M), but unaffected by the CB1 antagonist SR141716A (1 micro M). 5. Methanandamide and AM404 were less potent than AEA at activating VR1. Methanandamide (100 micro M) produced currents 37+/-6% of the capsaicin maximum, the highest concentration of AM404 tested (100 micro M) produced currents that were 55+/-9% of the capsaicin maximum. 6. Capsazepine abolished the currents produced by AM404 (100 micro M) and strongly attenuated (>70%) those produced by methanandamide (100 micro M). 7. Co-superfusion of AEA (30 micro M, methanandamide (100 micro M) or AM404 (100 micro M) with capsaicin (3 micro M) resulted in a significant reduction of the capsaicin current. 8. These data indicate that AEA, methanandamide and AM404 activate native VR1 receptors, but that all three compounds are partial agonists when compared with capsaicin.

Figure 1

Anandamide activates outward currents in capsaicin-sensitive mouse trigeminal sensory neurons. Selected trigeminal neurons were voltage clamped at +40 mV, as described in Methods. Superfusion of AEA (100 μM) produced a slowly developing outward current that reversed slowly on wash. Subsequent superfusion of capsaicin produced a large, rapidly developing outward current in the same neuron. This experiment is typical of 56 small trigeminal neurons challenged with both AEA and capsaicin, five additional cells did not respond to either agonist.

Figure 2

The activity of anandamide, but not capsaicin, is antagonized by bovine serum albumin. Trigeminal sensory neurons were voltage clamped at +40 mV and superfused with anandamide and capsaicin in the presence or absence of 0.05% BSA. (a) Concentration-response relationships for AEA in the absence and presence of BSA (0.5 mg ml⁻¹). BSA strongly inhibited the outward currents produced by submaximally effective concentrations of AEA. Each point represents the mean±s.e.mean of six cells, the currents produced by AEA were normalized to that produced by 10 μM capsaicin. (b) Concentration-response relationships for capsaicin in the absence and presence of BSA (0.5 mg ml⁻¹). Capsaicin currents were not affected by the inclusion of BSA. Each point represents the mean±s.e.mean of six cells.

Figure 3

Anandamide acts via vanilloid and not cannabinoid receptors to produce outward currents in mouse trigeminal neurons. Trigeminal sensory neurons were voltage clamped at +40 mV and superfused with AEA and capsaicin alone or in the presence of the VR1 antagonist capsazepine or the CB1 receptor antagonist SR141716. (a) Representative trace showing that capsazepine (30 μM) blocks AEA-induced outward currents in a AEA and capsaicin-sensitive neuron. Typical of six similar experiments. (b) Summary data of the effect of a 2-min preincubation of SR141716 on currents induced by AEA (10 μM). The currents were normalized to those produced by 10 μM capsaicin, each bar represents the mean±s.e.mean of six cells. (c) Summary data of the effect of a 2-min preincubation of SR141716 on currents induced by capsaicin. The same cells were challenged with capsaicin, then superfused with SR141716 and rechallenged with SR141716+capsaicin. The bars represent the mean±s.e.mean of six cells.

Figure 4

Anandamide activation of VR1 is concentration-dependent, but less efficacious than capsaicin. Selected trigeminal neurons were voltage clamped at +40 mV, as described in Methods. Concentration-response curves for capsaicin and AEA to activate VR1 in trigeminal neurons. In each cell, capsaicin or AEA responses were normalized to that produced by 10–30 μM capsaicin in that cell. The EC₅₀ for capsaicin was 300 nM, for AEA 3 μM. AEA produced maximal currents 38±2% of maximal capsaicin currents. Each point represents the mean±s.e.mean of at least six cells, error bars are contained within the symbol for several points.

Figure 5

Anandamide analogs methanandamide and AM404 activates VR1 in sensory neurons. Trigeminal sensory neurons were voltage clamped at +40 mV and superfused with methanandamide or AM404 and the currents produced compared with capsaicin (10 μM). (a) Concentration-response curves for methanandamide and AM404 to activate outward currents in trigeminal neurons, each response was normalized to that produced by 10–30 μM capsaicin in the same cell. Methanandamide produced maximum currents of 37±6% of maximal capsaicin currents, at the highest concentration of AM404 tested the currents were 55±9% of the capsaicin maximum. Each point represents the mean±s.e.mean of at least six cells. (b) Representative trace showing that capsazepine (30 μM) blocks most of the methanandamide-induced outward currents in a capsaicin-sensitive neuron. (c) Summary data of the effect of a 2-min preincubation of capsazepine (30 μM) on currents produced by methanandamide (100 μM) and AM404 (100 μM), determined as illustrated in (b). Capsazepine significantly (P<0.01) blocked the currents produced by both agonists. Outward currents were normalized to those produced by 10 μM capsaicin, each bar represents the mean±s.e.mean of six cells.

Figure 6

Anandamide, methanandamide and AM404 inhibit capsaicin-stimulated VR1 currents. Trigeminal sensory neurons were voltage clamped at +40 mV and superfused with capsaicin (3 μM) alone until the outward current reached a steady state and then AEA (30 μM), methanandamide (100 μM) or AM404 (100 μM) were co-superfused with capsaicin. (a) An example trace illustrating the effects of co-superfusion of AEA (30 μM) with capsaicin (3 μM). Approximately 4 min of wash have been removed for clarity. This is typical of six cells. (b) Bar chart summarizing the effect of co-superfusion AEA, methanandamide and AM404 on the currents induced by capsaicin. Each drug significantly (P<0.01) reduced the capsaicin (3 μM) current. Each bar represents the mean±s.e.mean of 5–6 cells.