Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures.

Sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 10(-6) M Na2SeO3 resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na2SeO3 concentrations (7.90 X 10(-6) and 1.19 X 10(-5) M) resulted in a three-fold increase in the SCE frequency above background level (6--7 SCEs/cell). Exposure of lymphocytes to 1 X 10(-4) M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 +/- 0.75 while a similar exposure to 2.7 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25--30% and 11--17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Comutagenic and coclastogenic effects of selenium in vitro and in vivo

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.     

Ameliorating effect of selenium on chromium (VI)-induced oxidative damage in the brain of adult rats

Chromium is known for its wide toxic manifestations. This experiment aims to evaluate the effect of selenium against oxidative stress induced by chromium in the cerebrum and cerebellum. Female Wistar rats were randomly divided into four groups of six each: group I served as controls which received the standard diet; group II received drinking water K(2)Cr(2)O(7) alone (700 ppm); group III received both K(2)Cr(2)O(7) and Se (0.5 mg Na(2)SeO(3)/kg of diet); and group IV received Se (0.5 mg/kg of diet) for 3 weeks. The exposure of rats to K(2)Cr(2)O(7) promoted oxidative stress in the cerebrum and cerebellum with an increase in malondialdehyde and a decrease of nonenzymatic antioxidant levels such as glutathione, nonprotein thiol, and vitamin C. An increase of enzyme activities like catalase, glutathione peroxidase, and superoxide dismutase activities was also observed. Acetylcholinesterase activity was inhibited after treatment with K(2)Cr(2)O(7). Co-administration of Se restored the parameters cited above. The histopathological findings confirmed the biochemical results.     

The effectiveness of S9 and microsomal mix on activation of cyclophosphamide to induce genotoxicity in human lymphocytes

Comparative results are presented on the effectiveness of rat-liver S9 or microsomal mix (M mix) in activating cyclophosphamide (CP) and its ability to induce a clastogenic effect in human lymphocytes in vitro. Structural chromosome changes were analysed exclusively in 1st division (M1) metaphases post-exposure. A high genotoxic response was observed for both metabolizing systems used. With an exposure of 2 h to different concentrations of S9 or M mix, the highest aberration yields were always found for the highest protein content. For CP treatment times of 1, 2 or 4 h together with S9 mix (protein content 10 mg/ml) or M mix (4 mg/ml), the latter was more efficient. With both systems, a lower clastogenic effect of CP was found at 4 h exposure than at 1 h or 2 h. Only a weak cytotoxic effect, reflected mainly by the reduction in the percentage of 3rd cycle cells (M3), and measured in terms of the proportion of M1, M2 and M3 cells, was induced by both systems.     

Clastogenic effect of the plant alkaloid ellipticine on bone marrow cells of Wistar rats and on human peripheral blood lymphocytes

Ellipticine (EPC), a natural alkaloid extracted from Aspidosperma williansii (Apocynaceae), is known to have antitumor and cytotoxic activities on various types of tumors. This drug showed a strong clastogenic effect on bone marrow cells of Wistar rats treated in vivo (7.75-31.00 mg/kg body weight). EPC was also tested in vitro using the human peripheral blood lymphocyte system, at concentrations 100 times lower than those used in the in vivo test on rats, since the cytotoxic effect on lymphocytes was very strong. At the 2 highest concentrations used (7.75 X 10(-1) and 1.55 X 10(-1) micrograms/ml culture medium), EPC induced a statistically significant increase in the frequency of chromosome aberrations and sister-chromatid exchanges in lymphocytes. Based on data reported in the literature, we have tried to establish relationships between the clastogenic effect observed and the process of EPC intercalation into DNA and the formation of protein-associated DNA-strand breaks probably promoted by topoisomerase enzymes.     

Chromium is the proximate clastogenic species for lead chromate-induced clastogenicity in human bronchial cells

Hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen with potentially widespread exposure. Solubility is a key factor in the carcinogenicity of Cr(VI), with the water-insoluble or 'particulate' compounds being the more potent carcinogens. Studies have indicated that the component ions are responsible for their clastogenicity, but it is uncertain whether chromium (Cr), lead (Pb) or some combination of the two is responsible for the clastogenic effects. Accordingly, we compared the clastogenicity of lead chromate (LC) with soluble sodium chromate (SC) and lead glutamate (LG) in WTHBF-6 human lung cells. We found that 1436microM was the maximal intracellular level of Pb after exposure to clastogenic concentrations of LC. However, clastogenesis was not observed after exposure to LG, even when intracellular Pb concentrations reached 13,347microM, indicating that intracellular Pb levels did not reach clastogenic levels in WTHBF-6 cells after LC treatment. By contrast, SC was clastogenic damaging 16 and 44% of metaphase cells at intracellular Cr doses of 312 and 1262microM respectively, which was comparable to the clastogenesis observed after LC treatment. LC damaged 10, 27 and 37% of metaphases at intracellular Cr doses of 288, 926 and 1644microM, respectively. These data indicate that with respect to LC-induced clastogenicity, Cr and not Pb is the proximate clastogenic species in human lung cells.     

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds.

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.     

The induction of chromosome aberrations in human purified peripheral blood lymphocytes following in vitro exposure to selenium

Human lymphocyte cultures were treated with increasing concentrations (8.0 X 10(-8) M to 8.0 X 10(-5) M) of sodium selenite and selenomethionine 24 h after stimulation with phytohemagglutinin and were scored for chromosomal aberrations at 48 h. The yield of abnormal metaphases was dependent on the dose and the form of selenium used. At 8.0 X 10(-5) M the proportion of aberrant cells reached 53.5% and 43.0% for selenite and selenomethionine, respectively. The selenium-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. Chromosomal exchanges were less frequent and included triradials and quadriradials. These results confirm that selenium is clastogenic for cultured human lymphocytes.     

Sister-chromatid exchange induction by sodium selenite: reduced glutathione converts Na2SeO3 to its SCE-inducing form

Sodium selenite (Na2SeO3) is an anticarcinogenic/antimutagenic agent that exhibits carcinogenic/mutagenic properties in some short-term test systems used for the detection of DNA-damaging agents. One such test system is sister-chromatid exchange (SCE) induction. Na2SeO3 induces SCEs only if red blood cells (RBCs) are present to 'activate' it to its SCE-inducing form. Here, the ability of reduced glutathione, a major component of RBCs, to serve as an RBC substitute in the activation of Na2SeO3 was determined. Reduced glutathione (10(-4) and 10(-3) M) was shown to be as capable as RBCs in activating Na2SeO3 (7.95 X 10(-6) M) to its SCE-inducing form. These data suggest strongly that the pathway normally utilized by RBCs in the metabolism of Na2SeO3 is the same as that in which Na2SeO3 is converted to its SCE-inducing form.     

Induction of chromosomal aberrations, sister-chromatid exchanges and cell-cycle delay in cow peripheral blood lymphocytes treatment with two N-nitroso compounds in vitro

We investigated the effect of NDMA and DNSGU on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs), as well as the influence of the former compound on cell-cycle kinetics in cultured cow peripheral lymphocytes. A clastogenic effect was observed in treated cell cultures at 6 or 12 × 10⁻⁵ M concentrations of NDMA and DNSGU, respectively, but no increase of chromosomal breaks was seen at the lowest dose. NDMA at 6 × 10⁻⁴ M was toxic to cow lymphocytes. NDMA and DNSGU induced statistical increases of SCEs at the test doses (6 or 12 × 10⁻⁶ and 6 or 12 × 10⁻⁵ M, respectively). In addition, treatment with NDMA at a dose of 6 × 10⁻⁵ M revealed significant heterogeneity of the first, second and third metaphases between treated and untreated groups. A reduction of the proliferation index and proliferation delay per cycle was shown too.     

Persistence of drug-induced chromosome aberrations in peripheral blood lymphocytes of the rat

We have studied the persistence of pre-clastogenic lesions, detected as induced chromosomal aberrations, in rat peripheral lymphocytes at various time intervals after acute treatment with 3 different antineoplastic drugs: cyclophosphamide (CPA), 5-fluorouracil (5-FU) and adriamycin (AM). Single i.p. doses were administered to groups of rats and heart blood samples from each group were taken after 3, 12, 24 or 48 h or weekly up to 20 weeks later. The cytogenetic analysis was performed on lymphocytes cultured for 33 h after sampling. The results for CPA exposure (10 mg/kg) show that the yield of chromosome aberrations is maximal 3 h after the treatment (20 times the control level). For up to 8 weeks the values remain about 6 times the baseline; afterwards a decrease is observed and the control level is reached after 20 weeks. For 5-FU (50 mg/kg) a remarkable increase (13-fold) in chromosomal damage is observed at the first sampling time. Within 48 h the effect is drastically reduced but persistent (3 times the control level), and the level returns to spontaneous values 1 week later. AM treatment (2 mg/kg) induced an increase of about 8 times the control level at 3 h post exposure. The clastogenic effects remained at a detectable level for 1 week (about 6 times the control level at all sampling times); 2 weeks after the treatment the control level was found. A parallel analysis was performed on bone marrow cells. In this tissue the clastogenic effects of the treatments were maximal, as in lymphocytes, at the first sampling time (20-25 times the control level) and were no longer detectable within 72 h after exposure, irrespective of the administered drug.     

硒镧复合作用对巴西蘑菇子实体形态和数量特征的影响

采用混合培养料试验和电子显微技术,观察了不同硒镧配施水平对巴西蘑菇子实体盖皮和菌褶表面形态及孢子和囊状体数量特征的影响.结果表明,低浓度硒镧配施(Na2SeO3 21.905mg/kg LaCl317.667mg/kg)处理时,孢子数极显著增加,随着硒镧配施浓度升高,孢子数极显著增加,达到峰值后极显著减少;低浓度(Na2SeO 21.905mg/kg LaCl17.667mg/kg)和高浓度(Na2SeO 109.524mg/kg LaCl88.340mg/kg)处理后囊状体数极显著减少,中浓度(Na2SeO365.714mg/kg LaCl353.002mg/kg)处理囊状体数显著增加.研究还发现硒镧配施对巴西蘑菇孢子长、宽、囊状体最大直径和最小直径以及菌盖皮菌丝最大宽度具有促进或抑制作用.扫描电镜观察结果表明,与对照相比,中浓度硒镧配施(Na2SeO365.714mg/kg LaCl3 53.002mg/kg)时,孢子由正常椭圆形变成全部萎缩,菌丝粗壮;囊状体在较低硒镧配施浓度(Na2SeO343.810mg/kg LaCl3 35.335mg/kg)处理时表面纹饰模糊、消失,个别囊状体破裂,表面带有藤蔓状的组织.     

Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells

The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator "Quin 2." [Ca++]i rose from approximately 150 nM to greater than 2 microM over a time course that was strongly dependent on the concentration of H2O2 used (5 X 10(-5) to 5 X 10(-3) M). After exposure of P388D1 cells to 5 X 10(-3) M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points greater than 45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 X 10(-4) M H2O2 to 14.0 min at 5.0 X 10(-3) M. When the total [Ca++]i-buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68-fold (1.1 X 10(2) - 7.6 X 10(3) amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6-fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that "surface blebs" appeared during this phase of injury. Both the rise in [Ca++]i and "blebbing" were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; the rise in [Ca++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; the rate of rise of [Ca++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; during the early phase (less than 30 min) of rise of [Ca++]i, only intracellular calcium is involved in the response; these events occur concomitantly with gross morphological changes to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sister chromatid exchanges induced in cultured mammalian cells by chromate

Chromate compounds induced sister chromatoid exchanges (SCEs) and chromosome aberrations in cultured mammalian cells. Similar increases in SCE frequency were observed in human fibroblasts exposed to the compounds K2Cr2O7 and K2CrO4. Marked increases in SCE frequency in cells exposed to chromate for a 48-h period were detected at concentrations between 10(-7) and 10(-6) M. Chromosome aberrations (primarily chromatid breaks) were also produced in human cells exposed to K2CrO4 at concentrations between 8 . 10(-7) and 3 . 10(-6) M. K2CrO4, but not the trivalent compound CrCl3, induced SCEs in Chinese hamster ovary (CHO) cells at low concentrations.     

Chemical dosimetry of ethyl nitrosourea in the mouse testis

[3H-Et]Nitrosourea was administered to male (101 X C3H) mice by i.p. injection at exposure levels of 10 mg/kg or 100 mg/kg. At intervals from 1 h to 6 days following treatment, the ratio of O6-ethylguanine to N7-ethylguanine in testis DNA averaged 1.13 following the 100 mg/kg exposure and 0.72 following the 10 mg/kg exposure. The amount of O6-ethylguanine recovered after the 100 mg/kg exposure was 40% greater than predicted from a linear extrapolation of the amount of O6-ethylguanine recovered after the 10 mg/kg exposure. We suggest that the high (100 mg/kg) exposure to ethyl nitrosourea results in depletion of the O6-alkylguanine acceptor protein within the testis and permits O6-ethylguanine to persist at higher levels than would be predicted from lower exposure data. W.L. Russell et al. (1982), W.L. Russell (1984) have found that specific-locus mutation frequencies induced in mouse spermatogonial stem cells are 5.8-fold greater after a single 100 mg/kg exposure to ethyl nitrosourea than after 10 weekly exposures to 10 mg/kg. The finding that the corresponding ratio for O6-ethylguanine formed in the testis is only 1.4 may be interpreted in a number of possible ways. If O6-ethylguanine is an important lesion for producing specific-locus mutations, then its formation in the stem cells must be at least 4-fold greater than that for the whole testis as the ENU exposure goes from 10 to 100 mg/kg: alternatively, the rate of repair of this lesion by the stem cells must decrease at least 4-fold relative to the average testicular cell. Other explanations for the difference in mutation response of the stem cells to acute vs. chronic ethyl nitrosourea-exposures include the possibility that other DNA lesions may be responsible for many of the mutations or that two hits on the DNA may be required to produce an effect.     

Interactions of nitrilotriacetic acid (NTA) with Cr(VI) compounds in the induction of gene mutations in cultured mammalian cells

We used the V79 Chinese hamster cell line to detect the induction by NTA of 6-thioguanine resistance, due to mutation at the HGPRT locus, with direct and indirect mutagens as positive controls. NTA was tested within the 10(-4)-1.5 X 10(-2) M concentration range: although it was cytotoxic above the 10(-2) M dose, it did not increase the frequency of mutations at any of the tested concentrations, independently of metabolic activation (rat-liver S9 fraction). NTA is known to dissolve heavy metals and therefore to increase their genotoxicity. We found that an insoluble Cr(VI) compound, lead chromate (PbCrO4), was not cytotoxic nor mutagenic on V79 cells, probably because it is taken up by the cells very slowly, whereas the presence of NTA (2.5 X 10(-3) M in water) elicited a direct cytotoxicity and mutagenicity, which was dose-dependent from 5 X 10(-5) M to 10(-4) M PbCrO4. This effect was due to solubilization of the chromate anion by NTA, as determined by comparing spectrophotometric determinations of Cr(VI) in PbCrO4 treatment solutions with a mutagenicity titration curve obtained with a completely soluble Cr(VI) salt (potassium dichromate, K2Cr2O7).     

Apoptosis of lymphocytes induced by chromium(VI/V) is through ROS-mediated activation of Src-family kinases and caspase-3

Mechanistic insights into Cr(VI)-induced carcinogenicity and possible implication of Cr(V) species formed by the redox reactions of chromium-bearing species have attracted interest. We have previously demonstrated that when human peripheral blood lymphocytes are exposed to the Cr(V) complexes, viz., sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr(V)O(ehba)(2)] and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr(V)O(hmba)(2)], apoptosis and formation of reactive oxygen species (ROS) are observed. The molecular mechanisms involving cellular signaling pathways leading to apoptosis are addressed in the present study. Treatment of lymphocytes with Na[Cr(V)O(ehba)(2)] and K(2)Cr(2)O(7) leads to the activation of the Src-family protein tyrosine kinases namely, p56(lck), p59(fyn), and p56/53(lyn), which then activates caspase-3, both of which are under the partial influence of ROS. Inhibition of the Src-family tyrosine kinases activity by PP2 and of caspase-3 by Z-DEVD-FMK reverses apoptosis, thereby suggesting their importance. Antioxidants only partially reverse the apoptosis induced by Cr(VI/V), suggesting that pathways other than those induced by ROS cannot be ruled out. Although the complex, Na[Cr(V)O(ehba)(2)] is known to be relatively stable in aqueous solutions, previous studies have shown that the Cr(V) complex, Na[Cr(V)O(ehba)(2)] disproportionates to Cr(VI) and Cr(III) forms at pH 7.4 through complex mechanistic processes. Dynamics studies employing EPR data show that the Cr(V) state in Na[Cr(V)O(ehba)(2)] is relatively more stable in RPMI-1640 medium containing plasma. Formation of ROS during the reaction of redox partners with Na[Cr(V)O(ehba)(2)] is an early event and compares favorably in kinetic terms with the reported rate processes for disproportionation. This investigation presents evidence for the direct implication of Cr(V) in Cr(VI)-induced apoptosis of lymphocytes.     

Cerebrovascular Permeability Coefficients to Sodium, Potassium, and Chloride

CSF and regional brain concentrations of 42K, 22Na, 36Cl, and [14C]mannitol were determined 3-45 min after intravenous injection of the tracers in pentobarbital-anesthetized rats. Rapid influx of 36Cl and 22Na into ventricular CSF immediately established concentration gradients from CSF to brain extracellular fluid. The CSF contribution to brain uptake of tracers was greatest in periventricular brain regions, where brain 36Cl concentrations were up to ninefold higher than concentrations in regions distant from ventricular CSF. Acetazolamide (20 mg kg-1 i.p.), an inhibitor of CSF formation, decreased 36Cl uptake into CSF and into periventricular brain regions but not into frontal cortex. 36Cl uptake into brain was unidirectional for 10 min after intravenous injection, and, during that period, diffusion from ventricular CSF did not contribute to uptake in the frontal cortex. Therefore, cerebrovascular permeability coefficients could be calculated from tracer concentrations in frontal cortex at 10 min and equaled, in cm s-1, 13.5 X 10(-7) for 42K, 1.4 X 10(-7) for 22Na, 0.9 X 10(-7) for 36Cl, and 1.5 X 10(-7) for [14C]mannitol. The low cerebrovascular permeabilities to K, Na, and Cl, comparable to those of some cell membranes, and the permselectivity (K much greater than Na greater than Cl) suggest that a significant fraction of ion transport across cerebral capillaries is transcellular, i.e., across the endothelial cell membrane.     

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM-500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase activity was studied. The response of enzymatic activity in the presence of various K+ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na+ / K+-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K+ ions below 20 mM in the medium assay. The IC50 values for high/low isoforms 2.77 x 10(-6) M / 8.56 x 10(-5) M and 7.06 x 10(-7) M / 1.87 x 10(-5) M were obtained in the presence of optimal (20 mM) and 2 mM K+, respectively. However, preincubation in the presence of elevated K+ concentration (50-500 mM) in the medium assay prior to Na+ / K+-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC50 values for both isoforms was the same as in the presence of the optimal K+ concentration. On the contrary, addition of 200 mM K+ into the medium assay after 10 minutes exposure of Na+ / K+-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na+ / K+-ATPase by reducing maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction.