Proton inhibition of [ 3H]resiniferatoxin binding to vanilloid (capsaicin) receptors in rat spinal cord

Abstract

Protons and capsaicin activate overlapping subsets of sensory nerves by opening ion conductances of similar properties. We have used the [ 3H]resiniferatoxin binding assay utilizing rat spinal cord membranes to elucidate the possible interaction of protons at the vanilloid (capsaicin) receptor. Using low pH (pH 6.0 and pH 5.0) buffers, a time-dependent gradual decrease was observed in specific resiniferatoxin binding. Protons inhibited resiniferatoxin binding with an IC 50 of pH 5.3 ± 0.1. In experiments in which the concentration of [ 3H]resiniferatoxin was varied, protons reduced the max value by approximately 40% with a corresponding 2-fold decrease in affinity. No change however, was observed in binding cooperativity (the Hill coefficients were 1.7 ± 0.1 and 1.6 ± 0.2 in the presence of pH 7.4 and pH 5.0 buffers, respectively). These changes in binding parameters are consist with a non-competitive or, alternatively, mixed inhibitory mechanism. The remaining resiniferatoxin binding sites bound capsaicin with an affinity ( K i=5.0 ± 1.0 μM) very similar to that determined in the presence of a pH 7.4 buffer ( K i = 3.0 ± 1.5 μM). A cyclooxygenase inhibitor, indomethacin (up to 10 μM), did not prevent the action of protons on resiniferatoxin binding; neither was it mimicked by prostanoids (prostaglandin I 2 and E 1, both at 100 μM). We conclude that protons interact at vanilloid receptors in the rat spinal cord; this interaction is either non-competitive or mixed in nature, and probably is not related to prostanoid generation. Protons and/or putative proton-generated mediators might represent endogenous modulators of the vanilloid receptor.