Characterization and regulation of basal calcium influx in human peripheral blood lymphocytes.

The basal 45Ca2+ influx into resting human blood lymphocytes was measured. This process showed biphasic kinetics with first rapid phase followed by the second long-lasting and markedly slower phase. Further, it showed signs of saturability and reaches maximal values at 37 degrees C and extracellular pH 7.2. The basal 45Ca2+ influx was stimulated by addition of submicromolar concentrations of 4 beta-phorbol-12-myristate-13-acetate, and this effect was abolished by protein kinase C (PKC) inhibitor Ro-31-8220. In the regulation of basal 45Ca2+ influx is probably only partially involved adenylate cyclase pathway as show results with intracellular c-AMP elevating agents (dB-c-AMP, 3-isobutyl-1-metylxantine and forskolin). Uncoupler 3,3',4',5-tetrachloro-salicylanilide (TCS) in micromolar concentrations stimulated basal 45Ca2+ influx and its effect was more significant in media with high extracellular concentration of K+.

L1210 cells cultivated under the selection pressure of doxorubicin or vincristine express common mechanisms of multidrug resistance based on the overexpression of P-glycoprotein.

Multidrug resistance of neoplastic tissue is often associated with the overexpression and increased drug transport activity of plasma membrane transporters like P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) or breast cancer resistance protein, as well as with the elevation of the glutathione detoxification pathway. We have already described the overexpression of P-gp under the selection pressure of vincristine in L1210 mouse leukemia cells. In the present study, mechanisms of multidrug resistance induced in L1210 cells cultivated in the presence of doxorubicin were analyzed. The selection pressure of both vincristine (yielding a resistant subline of L1210 cells, R(V)) and doxorubicin (yielding a resistant subline of L1210 cells, R(D)) induced a dramatic depression of cell sensitivity to both drugs. Both R(V) and R(D) cells demonstrated a lack of ability to accumulate calcein/AM and fluo-3/AM as fluorescent substrates of P-gp and MRP. The retention of dyes could be reached in both cell sublines by the application of inhibitors of P-gp (like verapamil) but not by probenecid - an inhibitor of anion transporters, including MRPs. Massive protein bands, at a M(r) range of 130-180 kDa that interact with c219 antibody against P-gp, were detected in the crude membrane fraction isolated from both R(V) and R(D) (but not from L1210) cells by Western blot. The cytosolic activity of glutathione S-transferase was found to be similar in R(V) and R(D) cells and did not differ significantly from the activity ascertained in parental L1210 cells. Neither the R(V) nor R(D) cell sublines differed considerably, as measured by cell ultrastructure. In conclusion, based on P-gp overexpression, both doxorubicin and vincristine induce a common multidrug resistance phenotype in L1210 cells.

Expression of P-glycoprotein in L1210 cells is linked with rise in sensitivity to Ca2+.

L1210/VCR cell line (R) was obtained by adaptation of the L1210 mouse leukaemia cells (S) to vincristine and showed P-glycoprotein (P-gp) mediated multidrug resistance (MDR). R cells were observed to be more sensitive to high external calcium as parental S. More pronounced calcium uptake was observed for R cells. Moreover, differences in intracellular calcium cell localization between S and R cells were found ultrastructurally following a calcium precipitating cytochemical method. In S cells, calcium precipitates were found to be localized predominantly along the cell surface coat and within mitochondria delineating the cristae. In R cells, precipitates were also found inside nuclei, at the border of heterochromatin clumps, and scattered within the cytoplasm. High extracellular calcium did not influence the P-gp mediated extrusion of calcein/AM as P-gp substrate. These results indicate that calcium enters and consequently damages the MDR cells to a higher extent than parental cells.

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts.

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.

Properties of the basal calcium influx in human red blood cells.

The basal (45)Ca(2+) influx in human red blood cells (RBC) into intact RBC was measured. (45)Ca(2+) was equilibrated with cells with t(1/2)=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5+/-0.2 micromol/l(packed cells) (n=6) at 37 degrees C. The average value of the Ca(2+) influx rate was 43.2+/-8.9 micromol/l(packed cells) hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 degrees C. The basal Ca(2+) influx was saturable with Ca(2+) up to 5 mmol/l but at higher extracellular Ca(2+) concentrations caused further increase of basal Ca(2+) influx. The (45)Ca(2+) influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3',4',5-tetrachloro-salicylanilide (TCS) 10(-6)-10(-5) mol/l) inhibited in part the Ca(2+) influx. The results show that the basal Ca(2+) influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca(2+) influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca(2+) are coupled via the RBC H(+) homeostasis.

Monitoring of oxidation steps of ascorbic acid redox reaction by kinetics-sensitive voltcoulometry in unsupported and supported aqueous solutions and real samples.

Aqueous solutions of ascorbic acid in unsupported and supported aqueous solutions and real samples were studied by the kinetics-sensitive double-step voltcoulommetric method with the aim to contribute to a better understanding of its behavior in biological systems. The data obtained from measurements made on analytes prepared in the laboratory, as well as those made on real samples (some commercial orange drinks, flash of the fresh fruits) point to the redox reaction of L-ascorbic acid (L-AH2) being very sensitive to both the presence of dissolved gaseous species (O2, CO2) and the ionic strenght in the analyte. Either the dissolved gaseous species, or the higher ionic strength caused by both the presence of supporting electrolyte and increased total concentration of ascorbic acid, respectively, give birth to the degradation of L-AH2. Naturally, the highest percentage of L-AH2 was spotted in fresh fruit.