Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel.

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.

An ELISA for screening hybridoma cultures for monoclonal antibodies against a detergent solubilized integral membrane protein.

A method is described for the binding of a detergent solubilized integral membrane protein to polystyrene immunoassay plates. Addition of Bouin's fluid, a histochemical fixative, to wells of plates containing the detergent solubilized antigen, followed by low speed centrifugation, is sufficient to promote binding of antigen in the presence of Triton X-100 concentrations as high as 1.75%. The binding of antigen is rapid and the entire binding procedure, including removal of fixative and washing of the plates, can be accomplished in less than 15 min. Immunological specificity of the bound antigen is retained. This method has been used to effectively screen hybridoma cultures for specific antibodies.

Variations in the levels of estrogen receptors in prolactin producing pituitary tumor cells.

The binding of [3H]-17 beta-estradiol in cells and cytoplasmic fractions of three different prolactin producing pituitary tumor lines was compared and found to vary widely. The concentration of estrogen receptors of the MtTW10 rat tumor line was high in early passages, but receptor levels decreased with subsequent passages in animals. Over a period of 12 months, the estrogen binding capacity in low speed supernatant fractions of cell homogenates decreased from 61.5 fmol [3H]-17 beta-estradiol per mg protein to less than 10 fmol [3H]-17 beta-estradiol per mg protein. A similar decrease in receptor concentration was found in MtTW10 cells which were adapted to in vitro culture. The concentration of receptor in low speed supernatant fractions of cultured GH3 rat tumor cells remained between 60 and 80 fmol [3H]-17 beta-estradiol per mg protein throughout the entire period of experimentation. In contrast, no high affinity receptors could be detected in similar fractions of the human pituitary cell line, 18-54, when cultured either in the presence or absence of serum. The Kd for the estradiol:receptor complex was determined to be 1.0 x 10(-10) M for receptors from MtTW10 cells and remained constant as the concentration of receptors declined. The receptors in both MtTW10 and GH3 cells were found to exist as 8s molecular species which are converted to 4s species by a temperature dependent process. The binding of estrogen to 8s and 4s receptors at 4 degrees C was shown to occur in the presence of 3 mg/ml of digitonin.

Methadone induced lysis of mammalian cells.

Methadone induced lysis of human erythrocytes and mouse leukemic cells was studied. The cells lyse without prior swelling that is a necessary step of colloid osmotic lysis. Methadone is accumulated by both cell types, and is widely distributed intracellurly in mouse leukemic cells. The maximum lytic rate is roughly proportional to the amount of methadone uptake and the Q10 for lysis is equal to the Q10 for methadone partitioning between octanol and water. It is concluded that the cells lyse as a result of a non-specific disruption of the plasma membrane.

The metabolism of methadone by cultured mammalian cells.

Rat hepatoma tissue culture cells and mouse leukemic cells were found to metabolize [1-3H] methadone to at least 2 unidentified radioactive compounds. These results suggest that cultured cells may be useful models for studying methadone metabolism by specific cell types.