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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 […]

The present study shows clearly that insulin causes a small inward current in HTC liver cells maintained near the rest­ing membrane potential in physiological cationic condi­tions. This insulin-induced current triggers a gradual depolarization of 5.7 mV. Such a slow depolarizing effect of insulin was observed previously following impalements of cultured rat hepatocytes with glass microelectrodes, […]

Effects of calcium channel inhibitors on insulin-induced increase in calcium influx: To determine the pharmacologi­cal profile of the calcium influx in HTC cells, several inhibi­tors known to interfere with calcium entry pathways were administered using the manganese quench protocol. Inhibi­tors were introduced with a calcium-free standard buffer at the same time as 50 pM manganese […]

Effects of insulin on steady-state intracellular calcium in HTC cell line: When HTC cells were superfused with standard bath solution (see ‘Materials and Methods’), the steady-state intracellular calcium concentration averaged 132±9 nM (n=111). Figure 3 presents representative traces of the cytosolic calcium ([calcium]i) responses to 2 mins administration of insulin (10 nM, panel A) or […]

Effect of insulin on HTC cell membrane potential: Patch clamp experiments were carried out using nystatin- containing pipettes to establish whole-cell recordings by the ‘perforated patch technique’. In the current clamp mode, the baseline membrane potential of HTC cells aver- aged-42.9±1.9 mV (n=59). In physiological cationic condi­tions, insulin (10 nM) induced a slow depolarization that […]

HTC cells were loaded with 1.5 pM Fura-2 AM for 45 mins at room temperature in bicarbonate-free MEM medium. Coverslips with adhered cells were transferred into a 100 pL plastic chamber placed on the stage of an inverted microscope coupled to a spectrofluo- rimeter (Deltascan RF-D4010, Photon Technology Interna­tional Inc, London, Ontario). Cells were superfused […]

Cell culture: Rat hepatoma HTC cells were chosen because they express surface membrane channels very similar to those found in primary cultures of rat hepatocytes while offering a greater stability for measurements of membrane currents by the patch clamp method. HTC cells were provided by Dr J Gregory Fitz of the Colorado Health Science Center […]