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

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 IC50 of pH 5.3 +/- 0.1. In experiments in which the concentration of [3H]resiniferatoxin was varied, protons reduced the Bmax 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 consistent with a non-competitive or, alternatively, mixed inhibitory mechanism. The remaining resiniferatoxin binding sites bound capsaicin with an affinity (Ki = 5.0 +/- 1.0 microM) very similar to that determined in the presence of a pH 7.4 buffer (Ki = 3.0 +/- 1.5 microM). A cyclooxygenase inhibitor, indomethacin (up to 10 microM), did not prevent the action of protons on resiniferatoxin binding; neither was it mimicked by prostanoids (prostaglandin I2 and E1, both at 100 microM). 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.