Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings

0 Present address: Department of Internal Medicine, Massachusetts General Hospital , Fruit Street, Boston, Massachusetts 02114 , U.S.A 1 Departments of Cell Biology and Pathology, Yale University School ofMedicine , New Haven, CT 06510 , U.SA. and Department of Pathology, VA. Hospital, Medical Center , West Haven, CT 06516, U.SA During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1-9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+-ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis. CORRELATION OF Na + ,K + -ATPase CONTENT AND PLASMA MEMBRANE SURFACE AREA IN ADAPTED AND DE-ADAPTED SALT GLANDS OF DUCKLINGS The avian salt gland possesses characteristics that permit its use as a model system for the study of plasma membrane biogenesis (Barrnett, Mazurkiewicz, & Addis, 1983). Significant cellular and biochemical changes involving the basolateral plasma membrane of principal secretory cells constitute a major response during adaptation (salt stress) and de-adaptation (return to fresh water) (Peaker & Linzell, 1975). Adaptive membrane amplification includes development of basal infoldings, increase in lateral plicae (Benson & Phillips, 1964; Ellis, Goertemiller, DeLellis & Kablotsky, 1963; Ernst & Ellis, 1969) and increase in levels of Na+,K+-ATPase activity (Ernst, Goertemiller & Ellis, 1967; Holmes & Stewart, 1968). De-adaptation of salt-stressed animals results in gland involution (Addis, Eager, & Barrnett, unpublished; Fletcher, However, these events have not been correlated quantitatively, especially with regard to the various secretory cell stages. In other systems, ATPase content and biosynthesis are not necessarily synonymous with fluctuations in plasma membrane content (Lo & Edelman, 1976; Pollack, Tate & Cook, 1981). In this study morphometric analysis of the amount of plasma membrane in the principal and peripheral secretory cells from control, adapted and de-adapted glands is related to changes in Na+,K+-ATPase content. The numerical density of mitochondria (suggested to increase during adaptation (Ernst & Ellis, 1969) and decrease during de-adaptation (Addis et al. unpublished; Hossler et al. 1978a)) as well as lipid droplets are also quantified per cell using stereological principles. The plasma membrane results form the basis for further analysis of the regulation of Na+,K+-ATPase content during adaptation and de-adaptation (Merchant & Barrnett, unpublished). Twenty nine white 1-day-old Pekin ducklings (C & R Duck Farm, Long Island, NY) were fed on duck mash and fresh drinking water for an adjustment period of at least 15 days and then divided into three groups. Controls (11) remained on freshwater. Adapted ducklings (10) were given 1 % NaCl in drinking and wading water for 10 days before being killed. Twenty days before death, the de-adapted group (8) was given consecutively 1% NaCl and freshwater (10 days each). All 29 ducklings were killed at 35 days of age. Twenty ducklings (8 control, 6 adapted and 6 de-adapted) were decapitated and their salt glands were rapidly removed for morphometric analysis and enzyme measurements. Oblique slices were cut from the anterior, middle and posterior portions of all glands. Six random slices (derived from a pool of slices from both glands in each animal) were cut into 1 mm3 blocks and fixed in cold 2-5% glutaraldehyde, 2 % paraformaldehyde, 0-05% (w/v) CaCb and 0-lM-sodium cacodylate (pH 7-4) for 2h. The fixed tissue blocks were rinsed (3 times) in 0-15M-cacodylate containing CaCk, refixed for 1 h at 25 C in 1 % osmium tetroxide in 0-lM-cacodylate, dehydrated through an ethanol series and embedded in Spurr's (1969) medium. From seven embedded blocks/animal, three were chosen randomly for sectioning. Thick sections (2/xm) were cut with an LKB Ultratome and stained with Toluidine Blue for light microscopy. Silver sections (from areas selected on the basis of light microscopy) were placed on uncoated 200 mesh grids, stained with uranyl acetate and lead citrate, and examined with an Hitachi electron microscope. Thus, 48 control, 36 adapted, and 36 de-adapted grids were used to generate morphometric studies. The process of sampling was influenced by the morphological organization of the gland of young ducklings as previously described (Ellis, 1965; Ernst & Ellis, 1969). To ensure adequate sampling of both peripheral and principal cells, the percentage contribution of each cell stage in control, adapted and de-adapted glands was first determined by examining randomly chosen light micrographs. These percentages were obtained by point counting positive transparencies projected onto a 240 mm X 180 mm grid at a final counting magnification of X1040. Results obtained (10% peripheral cells in control and adapted gland; 20 % in de-adapted) were then applied to the sampling of electron micrographs of principal and peripheral cells in each of the three groups of animals. For electron microscopy, 17 tissue fields from three grids per animal were photographed (X 3600) using the stratified sampling procedure (Weibel & Bolender, 1973) along with one calibration grid. To maintain the percentages of cells described above, ~ 10 % of the micrographs (2 of 17) were of peripheral cells in the control and adapted groups and ~ 2 0 % (3 of 17) were of peripheral cells in the de-adapted group. The remainder of the micrographs were of principal cells. To calculate membrane surface area and other cytoplasmic profiles on a per cell basis, using formulas described by Weibel & Bolender (1973), peripheral and principal numerical cell densities (Ny, number of cells per tissue volume) were scored from positive transparencies of light micrographs projected (magnification of X2080) onto a 60mm X 60mm test grid. The average numbers of peripheral and principal cells per test volume (MO were counted separately, and their individual cell densities computed on an Apple II plus computer using VisiCalc software and the following formulas: ./VAT (cell number/test area of thickness, T)=NX (Mag)2/dz, wh (...truncated)