Water and electrolyte contents, cell pH and membrane potentials of cultured turtle thyroid cells

Abstract

Water and electrolyte contents, cell pH, membrane potential and 125I- uptake were determined in cultured follicular cells of turtle thyroid. The Na+, K+ and Cl- concentrations in the cultured thyroid cells were 59.2, 119.0 and 50.9 mmol/l cell water respectively. Treatment with TSH (10 mu./ml for 24 h) increased the K+ and Cl- and decreased the Na+ concentrations in cells. The water and protein contents of these cells were 81.6 and 8.7 g/100 g cells respectively. The cell pH was 6.91. With glass micro-electrodes, the resting membrane potential of thyroid cells cultured in Medium 199 averaged 33.9 +/- 0.63 mV which is slightly higher than 29.8 +/- 1.6 mV as calculated from the data on the uptakes of [14C]methyltriphenylphosphonium and 3H2O by the cells. The potential varied linearly with the log of external K+ concentration (between 15 and 120 mmol/l) with a slope of about 24 mV per tenfold change in K+ concentration. Both TSH and cyclic AMP depolarized the cell membrane. Calculations based on the values for the electrolyte concentrations in cells and in culture medium indicated that Na+, K+ and Cl- were not distributed according to their electrochemical gradients across the cell membrane. Na+ was actively transported out of the cells and K+ and Cl- into the cells. Follicular cells of turtle thyroid cultured in the medium without addition of TSH formed a monolayer. Their iodide-concentrating ability was low and they did not respond to TSH with an increase in iodide uptake. In contrast, cells cultured in medium containing TSH tended to aggregate and organize to form follicles. They had higher ability to concentrate iodide and respond to TSH.

Effects of 4,4′-di-isothiocyano-2,2′-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells

“…The methods for preparation of cell cultures have been described previously (Chow et al 1985). The cells were plated on Falcon 3001 (35 15 mm) or 3002 (60 15 mm) culture dishes with 2-5-4 ml Medium 199 supplemented with 10% (v/v) newborn calf serum, 1 mu.…”

“…About 12-16 days after plating, cells were either used directly for 125I~u ptake or for [14C]dimethyloxazolidine-2,4-dione (DMO) and [3H]antipyrine studies or treated with 0-125% (w/v) trypsin in Ca2 + -and Mg2+-free Dulbecco's phosphate-buffered saline solution (DPBS) and replated onto the surface of collagen gel for measurement of intracellular ionic activities and other electrophysiological parameters (Yen-Chow, Chow, Jee & Woodbury, 1984;Chow et al 1985Chow et al , 1986). Determination of ' 2T " uptake by cultured cells Procedures for the determination of 125I~u ptake have been reported previously (Chow et al 1985(Chow et al , 1986. After washing with fresh Hanks' balanced salt solution containing lOmmol HEPES/1 (HBSS, pH adjusted to 7-4), 20 (35 15 mm culture dishes) or 3-0 (60 15 mm dishes) ml HBSS was added to each dish and allowed to equilibrate at 37°C for 20 min.…”

“…The results are expressed as 125It hyroid/medium (T/M) ratios (c.p.m./g protein)/ (c.p.m./ml medium solution). The protein content of each sample was obtained by homogenizing the cells in each counting tube (after determining the 125Iã ctivity) and determined spectrophotometrically by the method of Lowry, Rosebrough, Farr & Randall (1951 Waddell & Butler (1959) and Chow et al (19836) and modified for cell cultures by Chow et al (1985).…”

“…They suggested that the primary effect of DIDS is a stimulatory effect on iodide influx rather than blockade of anion channels, but the transport system utilized was not defined. Recently, we have established an experimental model to study ion transport systems in primary cultures of turtle thyroid follicular cells, and some of their biochemical and physiological properties have been published else¬ where (Chow, Yen-Chow & Woodbury, 1985;Chow, Yen-Chow, White & Woodbury, 1986). The present investigation was undertaken to study further the effects of DIDS on I" uptake in our experimental model (cultured turtle thyroid cells) by manipulating the ion concentration in culture medium and by adding different agents that are known to have significant effects on iodide transport mechanisms in thyroid glands.…”

Studies on pH Regulatory Mechanisms in Cultured Astrocytes of DBA and C57 Mice

“…The pHi of astrocytes in culture was determined with [I4C]DMO and [3H]MDG by the method of Waddell and Butler (1959) as modified for cell cultures (Chow et al, 1983(Chow et al, , 1985(Chow et al, , 1991 Tables 1 and 2. H C 0 3 -/ C 0 2 HBSS contained 25 or 5 mM NaHCO, instead of HEPES and was equilibrated with a mixture of 5% CO, and 95% 0,.…”