The overall goal of this research is to characterize the molecular mechanisms that mediate opioid inhibition of voltage-sensitive calcium (Ca2+) channels in mammalian neurons and to determine how they are affected by long-term opiate administration. The work is predicated on recent demonstrations that activation of mu-opioid receptors, in addition to kappa-opioid receptor stimulation, inhibits Ca2+ currents in rat dorsal root ganglion (DRG) sensory neurons via a G protein-dependent pathway. A major objective is to determine whether the suppression in Ca2+ current produced by mu- and kappa-opioid receptors, respectively, demonstrates specificity with respect to the types of channels that are modulated and the particular species of G proteins involved. To accomplish this, we will record Ca2+ currents in acutely isolated DRG neurons from adults rats using the whole-cell variation of the patch- clamp technique and record currents through single Ca2+ channels in cell- attached membrane patches. The Ca2+ channels to which mu-and kappa- opioid receptors are coupled will be identified by comparing the ability of DAMGO and dynorphin or U69593, respectively, to inhibit L-, N- and P- type components of whole-cell current, distinguished by biophysical criteria and their sensitivity to Ca2+ channels antagonists and agonists. We will then look for modulation of the corresponding subtypes of Ca2+ channels isolated in cell-attached patch during bath application of agonists to determine whether mu- or kappa-opioid receptors couple to Ca2+ channels via diffusible second messenger and/or a membrane delimited signaling pathway. The role of conventional second messengers in transduction of the opioid signal will also be assessed by comparing agonist-induced effects on Ca2+ currents evoked at early and late times during intracellular dialysis of the cell interior with specific protein whether distinct G proteins mediate the suppression in Ca2+ current by mu- and kappa-opioid receptors. We will attempt to reconstitute mu- and kappa-opioid responses in PTX-pretreated neurons using purified and recombinant G protein alpha subunits and compare the ability of a panel of specific anti-G/o/alpha and anti-G/i/alpha antibodies to interaction with Go will then be establish by using intranuclear injection of antisense oligonucleotide to selectivity block the expression of specific a subunits. In later studies, the effects of dialysis of the appropriate G protein will be tested in neurons from morphine-tolerate rats for their ability to reinstate the suppression in Ca+ current by full and partial mu-opioid agonists to the levels found in controls. The morphine, might involve modifications in G proteins that couple mu-opioid receptors to ion channels and transduction at the molecular level, should help in the design of therapeutic agents for pain management that possess little of the tolerance and abuse liability characteristic of opiate drugs.
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