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Department of Physiology, Faculty of Medicine, Kyoto University, Japan.

1. Hypotonic stimulation (180 +/- 5 mosmol l-1) increased [Ca2+]i in fura-2-loaded Green monkey kidney cells (COS-7 cells) and depolarized the membrane. 2. COS-7 cells were depolarized up to -3.5 +/- 4.4 mV from a resting membrane potential of -35.2 +/- 2.3 mV in response to hypotonic stimulation, when the patch electrode was filled with a 160 mM KCl-0.5 mM EGTA-based intracellular medium. 3. The increase in [Ca2+]i induced by hypotonic stimulation was divided into two phases. One was transient and oscillatory, and observed in Ca(2+)-free medium; the other was persistent, blocked by 100 microM La3+, and observed only in Ca(2+)-containing medium. 4. The increase in [Ca2+]i in Ca(2+)-free medium was blocked by pretreatment with 10 microM thapsigargin. The increase in [Ca2+]i induced by 10 microM thapsigargin was reduced after hypotonic stimulation which induced an increase in [Ca2+]i in Ca(2+)-free medium. 5. The increase in [Ca2+]i in Ca(2+)-free medium was not affected by treatment with 5 mM caffeine or 1-10 microM ryanodine. Neither caffeine nor ryanodine induced an increase in [Ca2+]i. 6. Adenosine 5'-O-2-thiodiphosphate (ADP-beta-S; a P2Y receptor agonist) induced an increase in [Ca2+]i in Ca(2+)-free medium and caused phosphoinositide breakdown in COS-7 cells. Exposure to 10 microM ADP-beta-S blocked the increase in [Ca2+]i induced in the Ca(2+)-free medium by hypotonic stimulation. The results of summary points 4, 5, and 6 suggest that the increase in [Ca2+]i induced by hypotonic stimulation is due to Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive internal stores. 7. The hypotonic stimulation-activated hydrolysis of phosphoinositides was decreased by pertussis toxin (PTX) in a dose-dependent manner. 8. These observations strongly suggest that hypotonic stimulation induced an increase in [Ca2+]i in Ca(2+)-free medium through activation of cascades using PTX-sensitive guanine nucleotide binding protein (G protein) and IP3.

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