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 ATP (100 pM, panel B), which acts mainly via a G protein-coupled P2U receptor in these cells. As shown, insulin induced a slow monophasic rise in [calcium]i compared with the typical biphasic response to the calcium-mobilizing agonist, ATP. These responses were quantified by the area under the curve of the calcium-versus-time relationship. The total intracellular calcium mobilized by insulin amounted to 3.1±0.3 pM (n=25), whereas that induced by ATP was 24.9±5.1 pM (n=11).

Figure 4) Effect of insulin on intracellular

Figure 4) Effect of insulin on intracellular calcium (Ca2+) in calcium- free bathing conditions. Representative traces of the [calcium ]i response of HTC cells to 2.5 min administrations of 10 nM insulin (—) or 100 цM ATP (■■■■) after 13.5 mins in standard bath solution lacking calcium chloride to which 4 mM ethylene glycol-bis (beta-aminoethylether)- N,N,N’,N’-tetra-acetic acid (EGTA) was added (calcium free, ‘Materials and Methods’) The single sharp response induced by ATP (n=16) corresponds to the mobilization of calcium from internal stores, whereas insulin (n=21) failed to elicit a response in the absence of extracellular calcium

To evaluate the role of extracellular calcium in the insulin-induced rise in [calcium]i, experiments were carried out in standard bath solution lacking calcium chloride to which 4 mM EGTA was added. In such calcium-free condi­tions, the response to 10 nM of insulin (n=21) was not ob­served, whereas the response to 100 pM of ATP (n=16) was reduced to a single sharp peak as expected from the mobiliza­tion of calcium from internal stores (Figure 4). The lack of response to insulin in such calcium-free conditions was not caused by a change in receptor occupancy as confirmed by displacement studies with I-insulin (data not shown). These results suggest that insulin induces an influx of exter­nal calcium into HTC cells. To verify this, the two different approaches described in ‘Materials and Methods’ were used. The first consists of extracellular calcium withdrawal and re- admission as depicted in Figure 4. After a calcium-free period of 13.5 mins, the initial slope of the rise in [calcium]i upon calcium readmission was 4.65± 0.69 nM/s, and this was in­creased 1.58±0.26-fold (n=21, P<0.05 by paired t test) with the prior administration of 10 nM insulin (Figure 5). ATP (100 pM), which had induced depletion of internal stores as depicted in Figure 4, also induced a statistically significant increase in the initial slope of calcium influx that amounted to 2.3±0.4-fold of the respective daily control (n=16, P<0.002 by paired t test, Figure 5A).

Figure 5) The effect of 10 nM insulin

Figure 5) The effect of 10 nM insulin and 100 цM ATP on the initial calcium (Ca2+) influx rate measured by a protocol of external calcium withdrawal and readmission. After a 13.5 min calcium-free bathing period (see Figure 4), extracellular calcium was reintroduced and an influx of calcium was apparent from the sudden rise in intracellular calcium . Initial calcium influx rates (Vi) were assessed by measuring the slope of the calcium -versus-time curve for the initial 15 s where calcium influx was observed after readmission of external calcium. The effects of insulin (10 nM) or ATP (100 цM) were observed when added for 2.5 mins before calcium readmission. Insulin increased the influx Vi by 1.58-fold (n=21) over a baseline value of 4.65±0.69 nM/s, whereas ATP increased this influx Vi by 2.3-fold (n=16). B The effect of 10 nM insulin and 100 цM ATP on the basal quench of FURA-2 fluorescence by manganese ions. Manganese chloride (50 цM) was introduced with a calcium-free standard buffer 2 mins before and throughout the subsequent administration of agonists insulin (10 nM) or ATP (100 цM). Insulin increased the quench rate 1.54-fold (n=56), whereas ATP increased this quench rate by 2.16-fold (n=41)(see Materials and Methods). Note different time scale from panel A

Another approach, which reflects unidirectional calcium in­flux pathways, is to measure the rate of FURA-2 quenching induced by manganese ions, which act as surrogates for calcium ions. During the first minute of administration of 50 pM manganese chloride, the rate of fluorescence quenching was 8.1±0.5 arbitrary units/s (n=103), and this baseline rate was subsequently increased by 1.54±0.08 (n=56) and 2.16±0.33 (n=41)-fold after the addition of 10 nM insulin and 100 pM ATP, respectively (Figure 5B, P<0.0001 by paired t test in both cases). Collectively, these results support the interpreta­tion that insulin induces calcium influx into HTC cells.
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