Differential effects of chronic ethanol exposure on ATP- and bradykinin-induced increases in intracellular calcium levels in PC-12 cells

The present study investigated the regulation of intracellular calcium levels ([Ca²⁺]_i) by ethanol, ATP, and bradykinin (BK) in PC-12 cells. Acute addition of 150 mM ethanol increased [Ca²⁺]_i but did not alter ATP- and BK-induced increases in [Ca²⁺]_i. After a 4-day exposure to 150 mM ethanol, the maximal response to ATP was decreased 39.7 ± 8.12% (p < 0.01), whereas that to BK was increased 43.8 ± 6.81% (p < 0.01). There was no change in the EC₅₀ values for either ATP or BK after chronic ethanol exposure. Addition of excess ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to remove extracellular free calcium prevented the ATP-induced increase in [Ca²⁺]_i. In contrast, BK produced an increase in [Ca²⁺]_i in the presence of excess EGTA, suggesting that BK releases calcium from internal stores. Consistent with this suggestion, chronic ethanol exposure enhanced BK-induced phosphoinositide hydrolysis. ATP, however, did not increase phosphoinositide hydrolysis. Pretreatment with 10 μM nifedipine, which blocked depolarization-evoked increases in [Ca²⁺]_i, reduced ATP- and BK-induced increases in [Ca²⁺]_i but did not alter the response to chronic ethanol exposure. Although acute addition of ethanol reduced KCl-stimulated increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ uptake, chronic ethanol exposure did not alter the depolarization-induced increase in [Ca²⁺]_i or ⁴⁵Ca²⁺ uptake. The present study demonstrates that chronic ethanol exposure inhibits calcium influx through voltage-independent cationic channels associated with purinergic receptors and enhances BK-stimulated phosphoinositide hydrolysis, with a subsequent release of calcium from internal stores.