Characterization of pulmonary sigma receptors by radioligand binding

This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [3H](+)−pentazocine reached steady state within 6 h at 37 °C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36±0.04 nM; Bmax 967±11 fmol/mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)−N-allylnormetazocine with respect to the (−)−enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [3H]1,3−di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2 min at 25 °C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8±8.3 nM; Bmax 921±228 fmol/mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2 μmol/kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo.