Pump and Channel K^+ (Rb^+) Fluxes in Apoptosis of Human Lymphoid Cell Line U937

Abstract

Ouabain-sensitive (OS) and -resistant (OR) Rb⁺ influx was examined in three sublines of U937 cells to compare alterations of K⁺ channel permeability and the Na⁺,K⁺-ATPase pump leading to the shift in ion and water balance during apoptosis induced by 0.2 and 1µM staurosporine (STS) for 4-5 h. Cell K⁺, Rb⁺, Na⁺ and Cl^- content was determined by flame photometry and ³⁶Cl distribution. Changes in cell water content were monitored by measurement of buoyant cell density and distribution of [³H]-glycerol or 3-O-methyl-D-[³H]glucose. Apoptosis was detected by DNA flow cytometry and light microscopy of the native cells stained with acridine orange. Treatment with 0.2 µM STS for 5 hours led to mild apoptosis with 10-13 % cell dehydration and either moderate increase of channel mediated Rb⁺ influx without significant changes in the pump activity or moderate decrease of pump Rb⁺ influx without significant change of channel influx, depending on the cell line used. Treatment with 1 µM STS was followed by 18-23 % cell dehydration, a decrease of the pump activity and a small or insignificant increase in the OR Rb⁺ influx in all studied sublines. It is concluded that moderate apoptotic cell shrinkage may be associated with both an increase in K⁺ channel permeability and inhibition of the pump whereas more remarkable shrinkage occurs presumably due to inhibition of the pump.

Computation of Pump-Leak Flux Balance in Animal Cells

“…This inverse problem is much harder than finding concentrations from known parameters, and we approach it by trial and error. In the case of U937 cells, [Na] i , [K] i , [Cl] i , cell water content per unit of A , the pump coefficient β and OSOR were measured as described previously [11,12,13,14,15]. These data proved sufficient to find a unique set of parameters corresponding to the balanced state for a model that includes the pump, Na + , K + , Cl - channels and a single type of cotransport.…”

“…(C) Time course of changes in cell buoyant density (g ml -1 ) and cell water content (relative units, means ± SE, n=8, 2, 3, 3 for t=0, 240, 360, 720 min, respectively). Cell water was calculated by density as described [13,16]. (D) Changes in [Na] i , [K] i , and [Cl] i in the vicinity of the balanced state starting from na0 140, k0 5.8, cl0 116, B0 48.2, kv 1, na 58, k 136, cl 63 and calculated for the parameters: beta 0.039, pna 0.00317, pk 0.023, pcl 0.00354, inc 3E-5, ikc = inkcc = 0. …”

“…Salts were of analytical grade and were from Reachem (Russia). The experimental methods used in this work have been described in detail earlier [11,12,13,14,15]. Briefly, intracellular K + , Na + and Rb + content was analyzed by flame emission spectrometry, Cl - content was measured using 36 Cl - isotope.…”

“…For our study we chose the proliferating human lymphoid U937 cell line, which better represents the typical animal cell owing to its faster metabolism and intermediate (-40÷-60 mV) MP. The monovalent ion flux balance in U937 cells has been comprehensively characterized in our laboratory [11,12,13,14]. In the present paper we have developed an improved computational model which, compared to the previous models, uses phenomenological parameters derived from the balanced state.…”

Correlational Patch-Clamp Spectrometry of the Ion Channels

“…На данный момент имеются десятки статей по: замещающему действию ионов Rb + на калиевые каналы (см., напр. : [88][89][90][91][92][93][94][95][96][97][98][99][100][101][102] [154,[156][157][158][159][160][161][162][163][164][165] и др. металлов.…”