COLD INHIBITION OF CELL-VOLUME REGULATION DURING THE FREEZING OF INSECT MALPIGHIAN TUBULES

Cells in freeze-tolerant tissues must survive substantial shrinkage du ring exposure to the hyperosmolarity that results as solutes are exclu ded from extracellular ice. We investigated the possibility that this hyperosmotic shock elicits an acute regulatory volume increase (RVI) b y monitoring the response of epithelial cell volume in the Malpighian tubules of the New Zealand alpine weta (Hemideina maori) during exposu re to low temperature/hyperosmolarity (mimicking freezing conditions) or during an actual freeze/thaw cycle, The cross-sectional area of cel ls in isolated Malpighian tubules was measured using differential inte rference contrast microscopy. At 20 degrees C, cells held in saline co ntaining 400 mmol l(-1) glucose exhibit an RVI in response to hyperosm otic shock. Cross-sectional area decreased by 30% immediately after a change from iso-osmotic (0.7 osmol l(-1)) to hyper-osmotic saline (2.1 osmol l(-1), equal to the osmotic shock encountered during freezing t o -4 degrees C) and then returned to 21% below the control value 30 mi n after the exposure. Although substantial cellular function of Malpig hian tubules was retained at low temperature (the rate of fluid secret ion 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 w as 39% below the control value immediately after the hyperosmotic expo sure and 36% below the control value 30 min after hyperosmotic exposur e. 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, regar dless of whether the tubules were held at 20 degrees C or 0 degrees C, The cross-sectional area of cells in saline containing glucose remain ed at approximately 30% below the control value during an entire 30 mi n 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 avoi d the energetic expenditure associated with volume regulation at a tim e when the normal defence of cell volume appears to be unnecessary.