Further experiments were thus carried out to characterize better the pathways of calcium entry stimulated by insulin in HTC cells. In rat hepatocytes, calcium influx has commonly been estimated either by the manganese quench method or by a protocol of external calcium withdrawal and readmission. By each of these respective approaches, insulin induced a statistically significant increase of 1.54- and 1.58-fold in the rate of external calcium influx. By comparison, the influx triggered by the purinergic agonist ATP amounted to 2.2- and 2.3-fold over control baseline values, for manganese quench and calcium withdrawal and readmission, respectively. When probed with several cal­cium channel blockers, the insulin-induced calcium influx (acceleration of manganese quench rate) was inhibited by gadolinium and SFK96365, whereas verapamil was without effect. This is similar to the results obtained recently with primary rat hepatocytes using a different approach. In that study, insulin-induced calcium responses were inhibited by nickel and gadolinium, but not by verapamil. As men­tioned earlier, nonselective cation channels in HTC cells were also shown to be sensitive to nickel and SFK96365, but not to verapamil. Such nonselective cation channels could thus be one of the molecular targets of insulin action on hepatocellular membrane conductance.

However, we were unable to observe direct activation of such channel activity in cell-attached recordings, whether insulin was introduced in the bathing or pipette solution. Positive control experiments with the P2U purinergic ago­nist ATP confirmed that nonselective channels were indeed present in our HTC cells and responded as previously reported by Fitz et al. In view of the reported single- channel conductances of 18 and 28 pS for the two types of nonselective cation channels described in HTC cells, and of the small peak amplitude of insulin-induced inward currents (663 pS whole-cell slope conductance), we would expect insulin to activate at most 24 to 37 such channels per cell. Hence, it is not surprising that direct activation of single- channel activity could not be observed with insulin in cell- attached patches. In comparison, ATP activated a whole- cell linear slope conductance of 10.1 nS, which corresponds to the activation of 360 to 561 of the channels mentioned above. This is consistent with the responses involving one to 13 channels (average of 5.3±0.7 channels, n=17) that we could successfully and reproducibly observe in cell-attached patches with ATP. In contrast, the chance of isolating under the patch pipette one or more of the 24 to 37 channels acti­vated per cell with insulin as the agonist was very slim. This holds particularly true if close proximity between the insulin receptor and the channels is required for activation. Further experiments will be required to address this question.

Nevertheless, our results show clearly that HTC cells are an appropriate model in which to study further the molecular targets of insulin action at the level of liver cell membrane conductance as well as the coupling mechanisms with the in­sulin receptor. Indeed, evidence from several laboratories supports this notion. First, as mentioned, these cells possess nonselective cation channels very similar to those present on primary hepatocytes. HTC cells also exhibit volume-sensitive increases in membrane chloride and sodium fluxes, as do normal hepatocytes. Sec­ondly, the HTC liver cell line has successfully been used to characterize several components of insulin receptor signalling cascades. Notably, insulin-induced stimulation of gly­cogen synthesis in HTC cells, as in primary hepato­cytes, was found to implicate phosphatidylinositol-3- kinase. Most interestingly, this signalling pathway, as well as that involving mitogen-activated protein (MAP) ki­nase, were shown clearly by our laboratory to require insulin-induced calcium influx for their full expression in rat hepatocytes. Finally, we found recently that insulin also stimulates p44/42 MAP kinase activity in HTC cells. When external calcium was chelated by 4 mM EGTA, the stimulation of p44/42 MAP kinase activity by 10 nM insulin was reduced by 50%. This strongly indicates that cal­cium influx in HTC cells, like that in primary hepatocytes, plays a physiological role in mediating a significant portion of insulin action.
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Our results demonstrate clearly that HTC cells respond to insulin with inward cationic currents and that nonselective cation currents are the principal source of insulin-induced cation influx. This action causes a depolarization of mem­brane potential and is associated with an increased influx of extracellular calcium through gadolinium- and SKF96365- sensitive but verapamil-insensitive pathways. The present study thus supports the notion that cation influx is an impor­tant component of insulin action in liver cells.