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Store-operated influx of Ca²⁺ in pancreatic ß-cells exhibits graded dependence on the filling of the endoplasmic reticulum

¹ Department of Medical Cell Biology, Uppsala University, Biomedicum, Box 571, SE-751 23 Uppsala, Sweden
² Department of Biophysics, National T. Shevchenko University of Kiev, Kiev, Ukraine

View larger version (22K): [in a new window]   Fig. 1. Estimation of store-operated Mn2+ influx in an individual ß-cell. The pancreatic ß-cell was loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiment. The medium was then supplemented with 200 µM Mn2+ (upper bar) and 30 µM carbachol (Carb; lower bars). The cytoplasmic Mn2+ concentration (A) is shown above the calculated Ca2+-independent fluorescence of fura-2 compensated for fading and loss of indicator (B); the calculated Ca2+-independent fluorescence of fura-2 without such compensation (C) and [Ca2+]i (D), which is not reliable after the introduction of Mn2+ (shaded area). The broken lines indicate the rate of change in Mn2+ concentration (A) and Ca2+-independent fluorescence of fura-2 (B,C) before addition of Mn2+ (0), immediately after addition of Mn2+ (1) and after subsequent addition of 30 µM carbachol (2).

View larger version (17K): [in a new window]   Fig. 2. Effect of carbachol concentration on elevation of [Ca2+]i due to store-operated influx of Ca2+ in individual ß-cells. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the representative experiment shown in A. The Ca2+ concentration was then changed between 1.28 (gray), 0 (white; Ca2+-free+2 mM EGTA) and 10 mM (black), as indicated by the upper bars; 0.3-100 µM carbachol was introduced as shown by the lower bars. B shows the dose-response relationship for carbachol-induced elevation of [Ca2+]i in the presence of 10 mM Ca2+. A singe observation is shown at 0.3 µM carbachol and means±s.e.m. for six observations at the other concentrations. The solid line shows a fit of the 25 individual data points in the 0.3-30 µM carbachol range to a logistic function (r=0.977; P<0.0001); the broken line shows that the effect decreases at 100 µM carbachol.

View larger version (23K): [in a new window]   Fig. 3. Store-operated influx of Mn2+ in response to carbachol in an individual ß-cell. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiment. The medium was then supplemented with 200 µM Mn2+ (upper bar) and 3.6 or 30 µM carbachol (Car; lower bars). The cytoplasmic Mn2+ concentration (A) is shown above [Ca2+]i (B), which is not reliable after the introduction of Mn2+ (shaded area). The broken lines indicate the rate of change in Mn2+ concentration immediately after addition of Mn2+ (1) and after subsequent addition of 3.6 (2) and 30 µM carbachol (3). The results are representative of three independent experiments.

View larger version (15K): [in a new window]   Fig. 4. Effect of CPA concentration on elevation of [Ca2+]i due to store-operated influx of Ca2+ in individual ß-cells. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the representative experiment shown in A. The Ca2+ concentration was then changed between 1.28 (gray) and 0 (white; Ca2+-free+2 mM EGTA), as indicated by the upper bars, and 0.3-30 µM CPA was introduced as shown by the lower bars. B shows the dose-response relationship for CPA-induced elevation of [Ca2+]i in the presence of 1.28 mM Ca2+. Means±s.e.m. for 4-5 observations are shown. The solid line shows a fit of the 19 individual data points in the 0.3-10 µM CPA range to a logistic function (r=0.984; P<0.0001) and the broken line that the effect decreases at 30 µM CPA.

View larger version (24K): [in a new window]   Fig. 5. Store-operated influx of Mn2+ in response to CPA in an individual ß-cell. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiment. The medium was then supplemented with 400 µM Mn2+ (upper bar) and 2 or 20 µM CPA (lower bars) as indicated. The cytoplasmic Mn2+ concentration (A) is shown above [Ca2+]i (B), which is not reliable after the introduction of Mn2+ (shaded area). The broken lines indicate the rate of change in Mn2+ concentration immediately after addition of Mn2+ (1) and after subsequent addition of 2 (2) and 20 µM CPA (3). The results are representative of five independent experiments.

View larger version (17K): [in a new window]   Fig. 6. Effect of Gd3+ on elevation of [Ca2+]i due to store-operated and voltage-dependent influx of Ca2+ in individual ß-cells. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+ (A,B), or in medium containing 3 mM glucose and 1.28 mM Ca2+ (C). The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiments shown in A and B, whereas there were no further additive in C. One µM Gd3+ was present as indicated by the upper bars. Ca2+ was omitted during the periods shown by the middle bars. The presence of 100 µM carbachol (Carb), 50 µM CPA and 31 mM K+ are shown by the lower bars. The results are representative of seven (A) or five (B,C) independent experiments.

View larger version (25K): [in a new window]   Fig. 7. Gd3+ inhibits store-operated influx of Mn2+ in response to carbachol in an individual ß-cell. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiment. The medium was then supplemented with 200 µM Mn2+ (upper bar), 100 µM carbachol (Carb; middle bar) and 1 µM Gd3+ (lower bar). The cytoplasmic Mn2+ concentration (A) is shown above [Ca2+]i (B), which is not reliable after the introduction of Mn2+ (shaded area). The broken lines indicate the rate of change in Mn2+ concentration immediately after addition of Mn2+ (1), after subsequent addition of 100 µM carbachol (2) and 1 µM Gd3+ (3). The results are representative of nine independent experiments.

View larger version (21K): [in a new window]   Fig. 8. Effect of 2-APB on elevation of [Ca2+]i due to store-operated and voltage-dependent influx of Ca2+ in individual ß-cells. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+ (A,B) or in medium containing 3 mM glucose and 1.28 mM Ca2+ (C,D). The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiments shown in A and B, whereas there was no further additive in C and D. 100 µM 2-APB was present as indicated by the upper bars. Ca2+ was omitted and 2 mM EGTA added during the periods shown by the middle bars. The presence of 100 µM carbachol (Carb), 50 µM CPA and 31 mM K+ are shown by the lower bars. The results are representative of five (A), six (B), four (C) or three (D) independent experiments.

View larger version (25K): [in a new window]   Fig. 9. 2-APB inhibits store-operated influx of Mn2+ in response to carbachol in an individual ß-cell. Pancreatic ß-cells were loaded with fura-2 in hyperpolarizing medium containing 400 µM diazoxide, 20 mM glucose and 1.28 mM Ca2+. The same medium lacking indicator but containing 50 µM methoxyverapamil was present at the beginning of the experiment. The medium was then supplemented with 200 µM Mn2+ (upper bar), 100 µM carbachol (Carb; middle bar) and 100 µM 2-APB (lower bar). The cytoplasmic Mn2+ concentration (A) is shown above [Ca2+]i (B), which is not reliable after the introduction of Mn2+ (shaded area). The broken lines indicate the rate of change in Mn2+ concentration immediately after addition of Mn2+ (1), after subsequent addition of 100 µM carbachol (2) and 100 µM 2-APB (3). The results are representative of ten independent experiments.

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