It is still unknown how extracellular hyperosmolarity suppresses exocytosis. To evaluate the possibility that extracellular hyperosmolarity affects one of the most important second messenger system, Ca
2+ signal, we evaluated the effect of hyperosmolarity on the thyrotropin releasing hormone (TRH) -induced changes in both intracellular Ca
2+concentration ([Ca
2+]
i) and prolactin (PRL) secretion in GH
4C
1 cells. TRH caused two phases of [Ca
2+]
i: an initial high-amplitude phase (first phase), which was not inhibited by Ca2+ free medium, and a sustained low - amplitude phase (second phase), which was abolished by Ca
2+ free medium. Medium hyperosmolarity (isotonic=300mOsm, hypertonic=338, 375, 450, and 600mOsm) suppressed both TRH-induced phases of [Ca
2+]
i in a dose dependent manner, however, the suppressive effect was clearly stronger in the second phase of [Ca
2+]
i than in the first phase of [Ca
2+]
i. Low doses of medium hyperosmolarity (338 and 375mOsm) suppressed PRL secretion, which was dependent on Ca
2+ influx. However, high doses of medium hyperosmolarity (450 and 600mOsm) also blocked PRL secretion, which was dependent on Ca
2+ mobilized from cytosolic Ca
2+ pools. These data indicate that in GH
4C
1 cells medium hyperosmolarity may inhibit PRL secretion by both blocking Ca
2+ influx and a mechanism unrelated to Ca
2+.
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