Modulation of calcium channels by norepinephrine in internally dialyzed avian sensory neurons.

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Abstract
Modulation of voltage-dependent Ca channels by norepinephrine (NE) was studied in chick dorsal root ganglion cells using the whole-cell configuration of the patch-clamp technique. Cells dialyzed with K+ and 2-10 mM EGTA [ethyleneglycol-bis-(.beta.-aminoethylether-N,N,N'',N''-tetraacetic acid] exhibited Ca action potentials that were reversibly decreased in duration and amplitude by NE. Ca channel currents were isolated from other channel contributions by using: tetrodotoxin (TTX) to block gNa, internal K channel impermeant ions (Cs or Na/N-methylglucamine mixtures) as K substitutes, external tetraethylammonium (TEA) to block K channels, and internal EGTA to reduce current contribution from Ca-activated channels. A marked decline (rundown) of Ca conductance was observed during continual dialysis, which obscured reversible NE effects. The addition of 2-5 mM MgATP to the intracellular solutions greatly retarded Ca channel rundown and permitted a clear assessment of modulatory drug effects. The inclusion of an intracellular creatine phosphate/creatine phosphokinase nucleotide regeneration system further stabilized Ca channels, which permitted recording of Ca currents for up to 3 h. NE reversibly decreased both steady state Ca currents and Ca tail currents in Cs/EGTA/MgATP-dialyzed cells. A possible role of several putative intracellular 2nd messengers in NE receptor-Ca channel coupling was investigated. cAMP or cGMP, added to the intracellular solutions at concentrations several orders of magnitude higher than the Kd for activation of cyclic nucleotide-dependent protein kinases, did not block or mask the expression of the NE-mediated decrease in gCa. Addition of internal EGTA to a final concentration of 10 mM also did not affect the expression of the NE response. Neither cyclic AMP nor cGMP, nor Ca were acting as a second messenger coupling the NE receptor to the down-modulated Ca channel population.