Osmotic and thermal effects on in situ ATPase activity in permeabilized gill epithelial cells of the fish Gillichthys mirabilis
Long-jawed mudsuckers (Gillichthys mirabilis) were acclimated to sea water (SW) at 7 &deg;C, SW at 26 &deg;C or dilute sea water (DSW) at 26 &deg;C for 5 months. Gill cells were isolated and the proportion of mitochondria-rich (MR) cells was determined. The number of cells harvested amounted to 4.7x10(7)&plusmn;0.6x10(7) to 10.6x10(7)&plusmn;1.1x10(7) and the yield was between 7.1x10(8)&plusmn;0.6x10(8) and 10.7x10(8)&plusmn;1.4x10(8) cells g-1 gill epithelial mass. Cell viability was 96.8&plusmn;0.4 to 97.8&plusmn;0.6 %. The number, size and volume of MR cells decreased significantly during DSW acclimation, but did not change during thermal acclimation. The protein content was not influenced by osmotic or thermal acclimation and ranged between 20.0&plusmn;1.6 and 22.1&plusmn;1.5 pg cell-1. Using a new method, which is based on the formation of plasma membrane channels by alamethicin, we were able to permeabilize gill cells. For the first time, the Na+/K+-ATPase and H+-ATPase activities of fish gills were determined in intact cells in situ. The activity of both ATPases was dependent on alamethicin concentration (optimum 100 &micro;g mg-1 protein) and on preincubation time (optimum 10 min). The in situ activity of both ATPases was influenced by osmotic, but not thermal, acclimation. A positive linear correlation was found between in situ Na+/K+-ATPase activity and total MR cell volume. However, we show, for the first time, that a negative linear correlation exists between H+-ATPase activity and total MR cell volume, suggesting a localization of H+-ATPase in pavement cells. In permeabilized cells, the activity of both ATPases was 2.6&shy;3.9 times higher than that of crude homogenates and 1.6&shy;2.1 times higher than that of permeabilized homogenate vesicles. We hypothesize that in crude homogenates three-quarters of Na+/K+-ATPase and two-thirds of H+-ATPase activity are not detectable both because of a mixture of inside-out and right-side-out vesicles and because of the disruption of membrane and enzyme integrity.
K&Uuml;Ltz
D
Somero
G