cis-Fatty acids, which activate protein kinase C, attenuate Na+ and Ca2+ currents in mouse neuroblastoma cells

Abstract

1. Activation of protein kinase C (PKC) by phorbol esters or diacylglycerols has been shown to modulate a number of ionic currents carried by Ca2+, K+ and Cl-. Recently, it has been demonstrated that PKC may be activated by cis-fatty acids in the absence of either phospholipid or Ca2+. We wished to determine if this new class of PKC-activating compound would also modulate ionic currents. To this end we applied the whole-cell voltage-clamp technique to N1E-115 neuroblastoma cells. 2. Analysis of families of currents evoked under voltage clamp by depolarizing steps from a holding potential of -85 mV during external application of 5 microM-oleate (a cis-fatty acid) showed a 36% reduction of the peak inward current with no shift in either the peak or the reversal potential of the current-voltage relation and no alteration of outward current. 3. External application of the cis-fatty acids oleate, linoleate and linolenate reversibly attenuated voltage-dependent Na+ current with approximate half-maximal dose values of 2, 3, and 10 microM respectively. Oleate was approximately 2 times more potent when applied internally (ED50 = 1 microM). Externally applied elaidate (a trans-isomer of oleate) and stearate (a saturated fatty acid) which do not activate PKC, had no effect. Since cis-fatty acids are known to fluidize membranes, as well as to activate PKC, we sought to dissociate these functions by applying compounds that fluidize membranes but do not activate PKC: methyloleate and lysophosphatidylcholine. Neither compound affected Na+ current when applied externally at concentrations of 1-50 microM. 4. In contrast to cis-fatty acids, three classical PKC activators, phorbol-12.13-dibutyrate (PDB), phorbol-12.13-diacetate (PDA), and 1.2-oleoylacetylglycerol (OAG) were found to have no effect on the voltage-dependent Na+ current when applied externally at 10 nM-1 microM (phorbol esters) or 1-150 microM (OAG) for incubation periods up to 1 h. 5. External application of the PKC inhibitors polymyxin B, H-7, sphingosine and staurosporine blocked the attenuation of the Na+ current by cis-fatty acid in a dose-dependent manner, with maximal inhibition occurring at doses of 50, 10, 200 and 0.1 microM, respectively. The cyclic nucleotide-dependent protein kinase inhibitor H-8 was much less effective in blocking the cis-fatty acid effect. Polymyxin B and staurosporine were more potent when applied internally. 6. Chronic (24 h) exposure to 1 microM phorbol-12-myristate-13-acetate (TPA) was employed to down-regulate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)