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 w ater (SW) at 7 degrees C, SW at 26 degrees C or dilute sea water (DSW) at 26 degrees C for 5 months, Gill cells were isolated and the propor tion of mitochondria-rich (MR) cells was determined, The number of cel ls harvested amounted to 4.7 X 10(7) +/- 0.6 X 10(7) to 10.6 X 10(7) /- 1.1 X 10(7) and the yield was between 7.1 X 10(8) +/- 0.6 X 10(8) a nd 10.7 X 10(8) +/- 1.4 X 10(8) cells g(-1) gill epithelial mass. Cell viability was 96.8 +/- 0.4 to 97.8 +/- 0.6%. The number, size and vol ume of MR cells decreased significantly during DSW acclimation, but di d not change during thermal acclimation, The protein content was not i nfluenced by osmotic or thermal acclimation and ranged between 20.0 +/ - 1.6 and 22.1 +/- 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 cell s in situ. The activity of both ATPases was dependent on alamethicin c oncentration (optimum 100 mu g mg(-1) protein) and on preincubation ti me (optimum 10 min). The irt situ activity of both ATPases was influen ced by osmotic, but not thermal, acclimation, A positive linear correl ation was found between in situ Na+/K+-ATPase activity and total MR ce ll volume, However, we show, for the first time, that a negative linea r correlation exists between H+-ATPase activity and total MR cell volu me, suggesting a localization of H+-ATPase in pavement cells. In perme abilized cells, the activity of both ATPases was 2.6-3.9 times higher than that of crude homogenates and 1.6-2.1 times higher than that of p ermeabilized homogenate vesicles, We hypothesize that in crude homogen ates three-quarters of Na+/K+-ATPase and two-thirds of H+-ATPase activ ity are not detectable both because of a mixture of inside-out and rig ht-side-out vesicles and because of the disruption of membrane and enz yme integrity.