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Inhibition of store-operated Ca²⁺ entry by extracellular ATP in rat brown adipocytes

Mariko Omatsu-Kanbe and Hiroshi Matsuura

1. Modulation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by extracellular ATP was investigated in cultured adult rat brown adipocytes using the fluorescent Ca²⁺ indicator fura-2.

2. Bath application of ATP in micromolar concentrations caused a large increase in [Ca²⁺]_i in cells previously stimulated with noradrenaline. This ATP-induced [Ca²⁺]_i increase exhibited a monotonic decline to near the resting levels within approximately 2 min, even in the continued presence of the agonist.

3. The magnitude and time course of the [Ca²⁺]_i increase in response to ATP were not significantly affected by removal of extracellular Ca²⁺, suggesting that a mobilization of intracellular Ca²⁺ primarily contributes to the increase.

4. The [Ca²⁺]_i increase in response to ATP was sensitive to inhibition by suramin, suggesting the involvement of P2 purinoceptors in the response.

5. Thapsigargin (100 nM) evoked a gradual and irreversible increase in [Ca²⁺]_i which was entirely dependent upon extracellular Ca²⁺, providing functional evidence for the expression of store-operated Ca²⁺ entry in these brown adipocytes.

6. Extracellular ATP at a concentration of 10 µM depressed this thapsigargin (100 nM)-induced [Ca²⁺]_i increase by 92 ± 3 % (n = 8 cells), strongly suggesting that ATP inhibits an influx of Ca²⁺ across the plasma membrane through the store-operated pathway. Bath application of phorbol 12-myristate 13-acetate (PMA, 5 µM) did not affect the thapsigargin-induced [Ca²⁺]_i increase, indicating that the inhibitory action of ATP is not mediated by activation of protein kinase C (PKC).

7. These results indicate that extracellular ATP not only mobilizes Ca²⁺ from the intracellular stores but also exerts a potent inhibitory effect on the store-operated Ca²⁺ entry process in adult rat brown adipocytes.

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