GABAA receptor subunit mRNA expression in cultured embryonic and adult human dorsal root ganglion neurons.

Previous studies have demonstrated significant pharmacological differences between the GABA(A) receptors expressed by neurons cultured from embryonic and adult human dorsal root ganglia (DRG). GABA(A) receptors of both embryonic and adult neurons are potentiated by diazepam and low concentrations of pentobarbital, and are activated by high concentrations of pentobarbital. However, in contrast to the GABA responses of embryonic neurons, the GABA responses of adult neurons are insensitive to both bicuculline and picrotoxin. We performed RT-PCR using subunit specific primer pairs, followed by Southern blot analysis with a third specific primer, to determine the pattern of subunit mRNA expression in cultures of embryonic and adult human DRG neurons. alpha2 and beta3 mRNA were expressed in all embryonic and adult cultures, while beta2 mRNA was present in all adult cultures but none of the embryonic cultures. Transcripts expressed by at least half of both embryonic and adult cultures were alpha3, alpha5, gamma2S, gamma3, theta, and rho1. Transcripts for gamma1 and delta were expressed in most adult cultures, but only a single embryonic culture. alpha4 mRNA was expressed by a single embryonic culture and pi mRNA was expressed by a single adult culture. We found no evidence for expression of alpha1, alpha6, beta1, gamma2L or rho2 transcripts. Changes in receptor subunit composition may underlie the novel pharmacological properties of GABA(A) receptor responses in adult cells. However, post-translational modification of a known subunit or the expression of a novel subunit may also contribute to the unique pharmacology of these neurons.

Effects of pyridine ring substitutions on affinity, efficacy, and subtype selectivity of neuronal nicotinic receptor agonist epibatidine.

2'-Pyridine ring substituted analogs of epibatidine were assessed for equilibrium binding affinity, functional potency, and efficacy at rat neuronal nicotinic receptors expressed in Xenopus oocytes. Binding affinities were determined in membrane homogenates from oocytes expressing alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, or alpha4beta4. Efficacy (relative to acetylcholine) and potency were measured electrophysiologically with oocytes expressing alpha3beta4, alpha4beta2, and alpha4beta4. Hydroxy, dimethylamino, and trifluoromethanesulfonate analogs had affinities too low for accurate measurement. The bromo analog had affinities 4- to 55-fold greater at beta2 than at beta4-containing receptors, modestly greater efficacy at alpha4beta4 than at alpha4beta2, and 5- to 10-fold greater potency at a4beta4 than at alpha3beta4 or alpha4beta2. The fluoro analog displayed affinities 52- to 875-fold greater at beta2- than at beta4-containing receptors, efficacy at alpha4beta4 receptors 3-fold greater than at alpha4beta2 and alpha3beta4, and was equipotent at all receptors tested. The norchloro analog showed affinities 114- to 3500-fold greater at beta2- than at beta4-containing receptors, 2-fold greater efficacy at alpha4beta2 and alpha4beta4 than at alpha3beta4, and 4- to 5-fold greater potency at alpha4beta4 and alpha3beta4 than at alpha4beta2. The amino analog displayed affinities 10- to 115-fold greater at beta2- than at beta4-containing receptors, 3-fold greater efficacy at alpha3beta4 than at alpha4beta2, and 2- to 4-fold greater potency at alpha3beta4 and alpha4beta4 than at alpha4beta2. Although these compounds displayed a variety of differences in affinity, efficacy, and potency, with one exception (binding affinity and functional potency at alpha4beta4 receptors) there were no significant correlations among these properties.