Cholera toxin-A subunit blocks opioid excitatory effects of sensory neuron action potentials indicating mediation by Gs-linked opioid receptors

Our previous studies indicated that opioid-induced prolongation of the Ca 2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances. In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system. In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses. After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A ( 1pg/ml-1ng/ml; > 15min) or whole toxin (1 pg/ml-1 μg/ml) the APD prolongation elicited in DRG neurons by 1–10 nM δ/μ (DADLE) or κ (U-50, 488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by μM opioid concentrations was unaffected. Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD. The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium. The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses. Nevertheless, the results of the present study provide strong evidence that opioid-induced APD prolongation in DRG neurons is mediated by excitatory opioid receptors that are coupled to a CTX-sensitive, Gs-like regulatory protein. Together with our previous studies, these results suggest that excitatory opioid receptors on DRG neurons are positively coupled via Gs to the adenylate cyclase/cyclic AMP second messenger system (resembling, for example, ß-noradrenergic receptor functions), in contrast to inhibitory opioid receptor linkage to Gi/Go (resembling α 2-noradrenergic receptors).