Cells in freeze-tolerant tissues must survive substantial shrinkage during exposure to the hyperosmolarity that results as solutes are excluded from extracellular ice. We investigated the possibility that this hyperosmotic shock elicits an acute regulatory volume increase (RVI) by monitoring the response of epithelial cell volume in the Malpighian tubules of the New Zealand alpine weta (Hemideina maori) during exposure to low temperature/hyperosmolarity (mimicking freezing conditions) or during an actual freeze/thaw cycle. The cross-sectional area of cells in isolated Malpighian tubules was measured using differential interference contrast microscopy. At 20 degrees C, cells held in saline containing 400 mmol1-1 glucose exhibit an RVI in response to hyperosmotic shock. Cross-sectional area decreased by 30% immediately after a change from iso-osmotic (0.7 osmol1-1) to hyper-osmotic saline (2.1 osmol1-1, equal to the osmotic shock encountered during freezing to -4 degrees C) and then returned to 21% below the control value 30 min after the exposure. Although substantial cellular function of Malpighian tubules was retained at low temperature (the rate of fluid secretion by isolated tubules at 4 degrees C was 72% of that measured at 20 degrees C), no RVI was observed at 0% degrees C; cross-sectional area was 39% below the control value immediately after the hyperosmotic exposure and 36% below the control value 30 min after hyperosmotic exposure. Dibutyryl cyclic AMP potentiated the RVI observed at 20 degrees C, but failed to elicit an RVI at 0 degrees C. A substantial RVI was also absent when the saline contained trehalose rather than glucose, regardless of whether the tubules were held at 20 degrees C or 0 degrees C. The cross-sectional area of cells in saline containing glucose remained at approximately 30% below the control value during an entire 30 min period of actual freezing to -4 degrees C, suggesting that an acute volume regulatory response was in fact inhibited during mild freezing. The inhibition of an acute RVI during mild freezing may serve to avoid the energetic expenditure associated with volume regulation at a time when the normal defence of cell volume appears to be unnecessary.

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