CLINICAL CONCENTRATIONS OF ISOFLURANE SIGNIFICANTLY SUPPRESSES SODIUM CURRENTS IN RAT DORSAL ROOT GANGLIA

Abstract

S519 Introduction: Over the last decade a number of studies have revealed that clinical concentrations of volatile anesthetics alter the discharge pattern and action potential properties of mammalian CNS and peripheral neurons [1]. In thin and unmyelinated neurons, which represent a substantial fraction of intracortical fibers and sensory afferents, anesthetics also have been found to alter signal propagation. [1] These neuronal functions all depend on the proper function of voltage-dependent sodium channels, making these integral membrane proteins potentially important molecular targets of general anesthetics. Although early studies examining anesthetic modification of sodium channel currents in non-mammalian preparations found them to be relatively insensitive targets compared with ligand-gated channels, a recent report has found that rat brain-Ila sodium channels expressed in a mammalian cell line are as sensitive as GABA-A receptors to clinical concentrations of volatile anesthetics [2]. It therefore has become essential to determine whether sodium channels in mammalian neurons have a similar anesthetic sensitivity as the expressed channels. To examine this question, we examined isoflurane modification of sodium currents in cultured rat Dorsal Root Ganglia (DRG). Methods: Freshly dissociated and primary cultured neurons of DRG from 5-30 day rats were obtained after enzymatic digestion with collagenase and/or trypsin. Sodium currents were measured using the whole cell patch clamp configuration; the extracellular Hanks' balanced salt solution additionally contained 5 mM HEPES and 0.1 mM CdCl2, pH 7.2, while the internal solution was 140 mM CsF, 10 mM NaCl, 5 mM HEPES, pH 7.4. Control measurements of the cells elicited normal action potentials and ionic currents. Sodium currents and anesthetic suppression were measured with depolarizing test potentials from a hyperpolarizing holding potential. Aqueous concentrations of isoflurane, which are relatively independent of experimental temperature [3], were measured by gas chromatography. All experiments were conducted at room temperature (22-25[degree sign]C). Results: Similar to results with the rat brain Ila sodium channels, isoflurane suppressed sodium currents through at least two mechanisms: a) a voltage-independent suppression of resting or open channels at hyperpolarized potentials, and b) a hyperpolarizing shift in the voltage-dependence of channel inactivation. Unlike the expressed channels, however, even high concentrations of isoflurane could not completely block sodium currents at potentials more negative than -80 mV. At -80 mV, isoflurane maximally suppressed 54 +/- 7.0% of sodium current (n=11, concentrations up to 5.4 mM isoflurane). The range of maximal suppression was 40-75%. Averaging data from the 11 cells and fitting to a rectangular hyperbola yielded a maximal suppression of 0.49 with an ED50=0.17 mM, slightly lower than aqueous MAC concentrations. The midpoint of sodium channel inactivation was about -50 mV. Similar to rat brain sodium channels, 0.54 mM isoflurane (twice MAC) shifted the inactivation midpoint by about -20 mV, resulting in significantly greater current suppression at these potentials (about 80% block at -60mV, compared with about 20% at -80 mV). Conclusions: Clinical concentrations of isoflurane caused a large depolarizing shift in steady-state channel inactivation, resulting in the block of a significant fraction (>50%) of sodium currents in DRG neurons at resting membrane potentials. These results support the hypothesis that some mammalian neuronal sodium channels are sensitive to clinical concentrations of volatile anesthetics in situ, and that their anesthetic modification may directly alter neuronal function.