Localization of alpha 1,2,3-subunit isoforms of Na,K-ATPase in cultured neonatal and adult rat myocardium: the immunofluorescence and immunocytochemical study.

By indirect immunofluorescence and preembedding peroxidase-diaminobenzidine technique the localization of polyclonal and monoclonal antibodies against alpha 1, alpha 2 and alpha 3 isoforms of the Na,K-ATPase were studied in rat myocardium. The alpha 1-subunit was identified predominantly on sarcolemma of cultured myocytes, neonatal, as well as adult cardiocytes. The alpha 2 signal was localized around nuclei of cultured cardiocytes, very weak signals were seen in neonatal and more intense signal, were dispersed throughout the adult myocytes. The alpha 3-subunit immunoreactivity was weak and localized in cell processes connecting individual cultured cells, on sarcolemma and intercalated discs of neonatal cells and very weak in adult working myocytes. Cytochemically demonstrated ouabain resistant Na,K-ATPase localized in junctional sarcoplasmic reticulum may represent alpha 1 isoenzyme which is directly involved in modulation of action potential fluxes.

Overcoming of P-glycoprotein mediated vincristine resistance of L1210/VCR mouse leukemic cells could be induced by pentoxifyline but not by theophylline and caffeine.

Effects of xanthine derivatives (pentoxifylline, caffeine, theophylline, 1-methyl-3-isobutylxanthine) on P-glycoprotein mediated vincristine resistance of L1210/VCR mouse leukemic cell subline were studied. From the applied xanthines only PTX was found to reverse the vincristine resistance of the above cells. Moreover, only PTX, but not other xanthine, increased the accumulation of [3H]vincristine by L1210/VCR cells. Thus it may be concluded that PTX-induced reversal of vincristine resistance could not be explained from the point of known pharmacological effects of PTX that are common for other xanthines such as inhibition of phosphodiesterase activity, calcium mobilizing effect, inhibition of tumor necrosis factor alpha (TNF), etc.

Effect of phorbol myristate acetate (PMA) on P-glycoprotein mediated vincristine resistance of L1210 cells.

Effect of phorbol myristate acetate (PMA) on P-glycoprotein (P-GP)-mediated vincristine resistance of the multidrug resistant mouse leukemic cell line L1210/VCR was studied by one hour lasting incubation of cells in the presence of PMA, and after three days of cultivation in the presence of the same substance. After the incubation with 100 micrograms.1-1 PMA the accumulation of [3H]-vincristine by the above cells was significantly depressed. Moreover, full reverse of verapamil-induced stimulation of [3H]-vincristine accumulation was observed in the presence of PMA. In contrary, when cells were cultivated three days in the presence of PMA, only slight but non-significant increase of [3H]-vincristine accumulation was observed. Slight increase of vincristine accumulation by cells cultivated in the presence of PMA was also supported by higher sensitivity of these cells to vincristine.

Time dependence of [3H]-vincristine accumulation by L1210 mouse leukemic cells. Effect of P-glycoprotein overexpression.

Overexpression of P-glycoprotein (P-GP) accompanied by multidrug resistance (MDR) to diverse groups of cytostatics was developed by long-term adaptation of mouse leukemic cell line L1210 to vincristine. Two resistant sublines of cells characterized by ID50 values for vincristine 1.05 mg/l (L1210/VCR-1) and 2.3 mg/l (L1210/VCR-2), respectively, were used. The sensitive parental cell line L1210 had the ID50 value for vincristine around 0.01 mg/l. Overexpression of P-GP induced by the adaptation procedure was found to be accompanied by an increase in the mean cell diameter from 10.28 +/- 1.60 microns (mean +/- SD, n = 122) for sensitive L1210 cells to 17.82 +/- 2.59 microns (n = 120) and 37.26 +/- 5.72 microns (n = 121) for L1210/VCR-1 and L1210/VCR-2 resistant cell sublines, respectively. Significant decrease in ability to accumulate [3H]-vincristine from cultivation medium was observed for both resistant cell sublines in comparison to sensitive cells. Accumulation of [3H]-vincristine by sensitive cells is secured only by passive diffusion of the drug across the plasma membrane. Contrary to that, active efflux of drug operating against its diffusion across the plasma membrane should be assumed as a factor influencing the [3H]-vincristine accumulation by resistant cells. Indeed, the time dependence of [3H]-vincristine accumulation by sensitive cells could be fitted using simple monoexponential kinetic dependence in contrast to biexponential kinetic dependences that are necessary for fitting [3H]-vincristine accumulation by both resistant cell sublines. Kinetic analysis of the experimental data indicates that accumulation of [3H]-vincristine by sensitive cells grows to a plateau reflecting probably the equilibrium of drug concentration in the intracellular and extracellular space. On the contrary, accumulation of [3H]-vincristine by both resistant cell sublines was stabilized after an initial growth on a considerably lower level than it was observed for the sensitive cells in the equilibrium.

Effect of pentoxifylline on P-glycoprotein mediated vincristine resistance of L1210 mouse leukemic cell line.

Effect of pentoxifylline (PTX) on vincristine (VCR) resistance of multidrug resistant L1210/VCR mouse leukemic cell line was studied. Reversal effect of PTX (in concentration 50-150 mg/l) on vincristine resistance, i.e. potentiation of vincristine cytotoxicity on L1210/VCR cells by PTX was found. PTX alone in the above concentration did not exert any significant cytotoxic effect on sensitive or resistant cell lines in the absence of vincristine. Resistance of L1210/VCR cell line was found previously to be accompanied with overexpression of drug transporting P-glycoprotein. Indeed, lower level of 3H-vincristine accumulation by resistant L1210/VCR cell line in comparison with sensitive L1210 cell line was observed. Accumulation of 3H-vincristine by L1210/VCR cell line was significantly increased in the presence of PTX. PTX in the same condition did not exert any considerable effect on accumulation of 3H-vincristine by nonresistant L1210 cells. Observable morphological damage was observed in L1210/VCR cells cultivated in medium containing vincristine (0.2 mg/l) and pentoxifylline (100 mg/l) in comparison with the non-damaged cells in the presence of vincristine or pentoxifylline alone. The results obtained indicate that pentoxifylline may be considered as a reversal agent in multidrug resistance.