intracellular calcium: RESULTS

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 reached an amplitude of 5.7±0.8 mV above baseline (n=21) within 5 mins and was reversible upon removal of the hor­mone (Figure 1A). In additional experiments, this depolarization remained stable for up to 10 mins of insulin administration (results not shown). This is similar to that observed previously in primary cultures of rat hepatocytes. By comparison, ATP (100 pM) induced a typical tripha­sic response of membrane potential (Vm), as described by Fitz and Sostman (Figure 1B). ATP is a purinergic agonist that acts mainly via G protein-coupled P2U receptors in HTC cells and was used as a positive control for cell respon­siveness.

Effect of insulin on steady-state currents in HTC cells: To study the effect of agonists on membrane currents, HTC cells held in the whole cell configuration were perfused with 10 nM insulin or 100 pM ATP for 5 mins. In physiological cationic conditions, administration of insulin induced a small monophasic inward current at resting potential (n=9, Figure 2A). On average, insulin-induced currents exhibited a whole-cell linear slope conductance of 663±100 pS and a reversal potential (Erev) of-17.9 mV (I/V curve inset in Figure 2A). In the same ionic conditions, ATP induced the expected triphasic response (Figure 2B), whose initial part was clearly shown by Fitz and Sostman to be a nonselec- tive cation current. In the present study, such currents had a mean whole-cell linear slope conductance of 10.1±2.3 nS (n=6) and a reversal potential of-21.6 mV (I/V curve inset in Figure 2B).


Figure 1) The effect of 10 nM insulin

Figure 1) The effect of 10 nM insulin (A) or of 100 цМ ATP (B) on membrane potential in HTC cells. The trace in panel A is a composite graph of the mean ± SEM changes in membrane potential observed in 21 whole-cell experiments for a 5 min administration of insulin and in five such experiments for the return to baseline after removal of the hormone. Panel B presents the mean ± SEM changes in membrane potential observed in 24 whole-cell experiments for a 5 min administration of 100 цМ ATP

Because the Erev of insulin-induced currents indicated a possible mixture of potassium, chloride and nonselective cation components, additional experiments were carried out using ion substitutions. The equilibrium potential for potassium ions (Ek) was first moved to 0 mV, and the equi­librium potential for chloride ions (Eq) to +35 mV (stan­dard pipette solution, potassium-gluconate bath solution, as described in ‘Materials and Methods’). The reversal poten­tial of the insulin-induced currents shifted to -3 mV, and the slope conductance reached 1030±205 pS (n=8, not signifi­cantly different from physiological cationic conditions by unpaired ANOVA). Keeping Ek and the nonselective cat­ion equilibrium potential (Ecation) at 0 mV, Eq was moved to -33 mV (potassium-gluconate bath and pipette solution, as described in ‘Materials and Methods’). This yielded an Erev of +8 mV and a mean slope conductance of 776±284 pS for insulin-induced currents (n=6, not significantly different from physiological cationic conditions by unpaired ANOVA). Finally, experiments were performed where all cations were replaced by nonpermeant species (NMDG, ‘Materials and Methods’). In such conditions, no current was elicited by insulin administration (data not shown). Collec­tively, these data suggest that insulin-induced currents are mainly caused by cationic components.

Figure 2) The effect of 10 nM insulin

Figure 2) The effect of 10 nM insulin (A) and 100 цМ ATP (B) on steady-state currents in HTC cells. Curves are the average change in baseline currents induced by insulin (panel A, n=9) and ATP (panel B, n=6) in physiological cationic conditions at -40 mV during a 5 min administration. Insulin-dependent currents measured over the last 50 s of the 5 min administration of the hormone are depicted by the current- voltage (I/V) curve inset in panel A. Agonist-dependent currents recorded at the peak of the initial response induced by ATP (representing cation influx through nonselective channels) are also depicted by the I/V curve inset in panel B. Error bars are not shown in the current- versus-time traces for the sake of clarity, but coefficients of variation for each point did not exceed 20%. dl pA Change in baseline current in pi- coamperes

Finally, experiments were carried out in the cell-attached mode. Administration of 10 nM insulin in the medium bath­ing the cells (n=14) or inclusion of 10 nM insulin in the pipette solution (n=39) never elicited any channel activity. In contrast, bath addition of 100 pM ATP elicited the acti­vation of one to 13 channels in cell-attached patches (n=17), even in cells that failed to respond to insulin. Chan­nel activity was initiated instantly upon ATP administration and returned toward baseline in a pattern resembling cytosolic calcium responses (Figure 3). This response profile as well as channel conductances were similar to those described previously by Fitz and collaborators. You can afford your pills. Buy cialis professional online

Figure 3) The effect of 10 nM insulin

Figure 3) The effect of 10 nM insulin on intracellular calcium (Ca2+) in physiological cationic conditions. Representative trace of the cytosolic calcium [calcium ]i response to 2-min administrations of insulin (10 nM) and ATP (100 цЫ). Total calcium mobilization (area under the curve over 180 s) was 3.1±0.3 цM (n=25) and24.9±5.1 цM (n=11) for insulin and ATP, respectively