Characterization of pulmonary sigma receptors by radioligand binding

Abstract

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.

Cocaine occupancy of sigma1receptors and dopamine transporters in mice

“…In agreement with earlier results (Lever et al, ), [ 125 I] E ‐IA‐DM‐PE‐PIPZE displayed good levels of control specific binding (Fig. ) to σ 1 receptors in brain (83%), heart (53%), lung (66%), and spleen (71%).…”

“…Biodistribution studies of [ 125 I] E ‐IA‐DM‐PE‐PIPZE binding to σ 1 receptors (Lever et al, ) and [ 125 I]RTI‐121 binding to the DAT (Desai et al, ; Lever et al, ) were conducted as previously reported. In general, awake animals received tail vein injections of radioligands (2.5 μCi) formulated in saline (0.1 mL) containing 2% ethanol.…”

“…Further, σ 1 receptors are required for acquisition and reinstatement of (−)‐cocaine‐induced conditioned place preference (Maurice and Romieu, ; Romieu et al, ), and σ 1 receptor agonists potentiate (−)‐cocaine's reinforcing effects (Hiranita et al, ; Katz et al, ). (−)‐Cocaine binds with higher affinity to the σ 1 than σ 2 receptor subtype in vitro (Garcés‐Ramírez et al, ; Lever et al, ; Matsumoto et al, ). Nonetheless, (−)‐cocaine functions as a σ 2 receptor agonist (Nuwayhid and Werling, ), and certain selective σ 2 receptor ligands profile as antagonists of (−)‐cocaine‐induced behaviors (Lever et al, ; Matsumoto et al, ).…”

Tactics for preclinical validation of receptor-binding radiotracers

“…[ 125 I]- E -IA-BF-PE-PIPZE uptake was reduced significantly by E -IA-BF-PE-PIPZE (53% – 94%), haloperidol (47% – 90%) and BD1063 (53% – 90%) in brain and peripheral organs, with the exception of the liver. Expression of σ 1 receptors by all of these organs has been confirmed in prior in vitro or in vivo studies [42,55,140,145]. The liver showed increased uptake upon inhibition of σ 1 receptor binding in the other organs.…”

“…However, a 5.7-fold higher affinity ( K d 0.042 nM) was calculated for [ 125 I]- E -IA-BF-PE-PIPZE using rate constants compared to saturation binding ( K d 0.24 nM). Such discrepancies are frequently observed [93,137–140]. Agreement can be improved in some cases by correcting for radioligand depletion, either mathematically or by using low receptor concentrations [137,138].…”