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 (Denver, Colorado). The cells were grown in minimum essential medium (MEM) (Sigma, St-Louis, Missouri) containing 25 mM sodium bicarbonate, 2 mM glutamine, 5% fetal bovine serum and 1% penicillin-streptomycin equilibrated at 37°C with 5% carbon dioxide/95% oxygen. Cells were used between in-house passages 4 and 10 and were grown for 24 h on round glass coverslips. Materials: Insulin was prepared as a stock solution of 50 pg/mL in 0.01 N hydrochloric acid and diluted daily from frozen aliquots into standard bath solution. A final concentration of 10 nM was used because this is the lowest dose found to yield near-maximal responses, whether at the level of receptor binding, signal transduction or metabolism in cultured rat hepatocytes, as shown by the authors’ laboratory and others. Nystatin was prepared as a stock solution of 100 mg/mL in dimethyl sulphoxide and used at a final concentration of 200 pg/mL in the pipette solution. Fura-2 acetoxymethylester (FURA-2 AM) was purchased from Molecular Probes (Eugene, Oregon). Insulin, nystatin and ionomycin were obtained from Sigma. All other chemicals were of reagent grade.
Patch clamp recording: Membrane currents were measured at room temperature using standard patch clamp recording techniques. Coverslips with adhered HTC cells were placed in a plastic perfusion chamber (100 pL trough) set on the stage of an inverted microscope (Olympus IMT-2, Carsen Medical, Markham, Ontario) and single cells were selected for study. Cells were continuously superfused (approximately 2 mL/min) at room temperature with a standard bath solution (Bath standard) containing (in mM): sodium chloride 138, potassium chloride 3.8, magnesium sulphate 1.2, mono potassium phosphate 1.2, calcium chloride 1.8, 4-(2-hydroxyethyl)-1- -piperazineethanesulfonic acid (HEPES) 10 (pH 7.4 with sodium hydroxide). An eight-way solenoid valve allowed rapid administration and washout of pharmacological agents and various buffers. Patch pipettes typically exhibited resistances of 4 to 6 MO when filled with a standard pipette solution (pipette standard) containing (in mM): potassium chloride 140, magnesium chloride 1.2, ethylene glycol-bis (beta-aminoethylether)-N,N,N’,N’-tetra- acetic acid (EGTA) 1, HEPES-KOH 10 (pH 7.35). For ion substitution experiments, the following bathing solutions were used: a potassium-rich solution (potassium-gluconate bath solution) containing (in mM) potassium-gluconate 113.4, potassium chloride 25.4, magnesium sulphate 1.2, mono potassium phosphate 1.2, calcium chloride 5 (to keep ionized calcium near normal in view of the chelating effect of gluco- nate), HEPES-KOH 10 (pH 7.4); and a nonpermeant cation-rich solution containing (in mM) N-methyl-D-gluca- mine-hydrochloric acid (NMDG-hydrochloric acid) 141.8, magnesium sulphate 1.2, calcium chloride 1.8, HEPES- NMDG 10 (pH 7.4). All bath solutions contained 5 mM glucose and 1 mM pyruvate as energy sources. In these same ion substitution studies, patch pipettes were filled with the following solutions as required: a potassium-rich solution (potassium-gluconate pipette solution) containing (in mM) potassium-gluconate 132.9, potassium chloride 7.1, magnesium chloride 1.2, EGTA 1, HEPES-KOH 10 (pH 7.35); a nonpermeant cation-rich solution containing (in mM) NMDG-hydrochloric acid 140, magnesium chloride 1.2, EGTA 1, HEPES-NMDG 10 (pH 7.35). All solutions were filtered through 0.22 pm cellulose membranes before use.
Whole cell recordings were carried out using the perforated patch method described by Horn and Marty. Briefly, the tip of the pipette was capillary filled with the appropriate pipette solution (approximately 3 s immersion), and the rest of the pipette was then backfilled with the same solution containing nystatin (200 pg/mL). After formation of a seal between the pipette and cell membrane, the membrane potential was held at -40 mV until continuity with the cell interior was fully established (20 to 40 mins), as shown from capacitative transients induced by short 20 mV voltage steps given every 5 mins. Membrane potential was measured in the current clamp (I=0) mode.
After Giga-seal formation, electrical signals were amplified and filtered (3 kHz) with a L/M-EPC-7 patch clamp amplifier (Medical Systems Corp, Greenvale, New York), continuously monitored on an oscilloscope and displayed on a chart recorder. Simultaneously, currents were digitized using a two-channel A/D converter (Instrutech, Elmont, New York) and recorded on standard VHS video magnetic tape. A TL-1 DMA Interface and the PClamp software (v 5.5.1) were used for data acquisition, and PClamp v 6.0 was used for data analysis (Axon Instruments, Foster City, California).
To construct current-voltage (I/V) curves, voltage clamp protocols were used where cells were held at -40 mV (close to the resting potential of HTC cells – see ‘Results’ section) and stepped from -80 to +60 mV in consecutive 20 mV voltage steps lasting 250 ms each. In all cases presented, I/V relationships were found to be linear with a correlation coefficient greater than or equal to 0.9.
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