ABSTRACT
Incubation of L1210 murine leukemia cells in vitro with 10 microM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener 86Rb+ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of 86Rb+ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359-367) as being mediated by a Na+/K+/Cl- cotransporter. Inhibition by 10 microM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of 86Rb+ influx, mediated by Na+/K+-ATPase. After 3 h of incubation with 10 microM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [3H]TPMP+, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K+ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumor activity, did not inhibit 86Rb+ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 +/- 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na+/K+/Cl- cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Chlorides/metabolism , Mechlorethamine/pharmacology , Potassium/metabolism , Sodium/metabolism , Adenosine Triphosphate/metabolism , Alkylating Agents/pharmacology , Animals , Biological Transport/drug effects , Diuretics/pharmacology , Hydrogen-Ion Concentration , In Vitro Techniques , Leukemia L1210 , Membrane Potentials , Mice , Ouabain/pharmacology , Rubidium/metabolism , Sodium-Potassium-Chloride Symporters , Tumor Cells, CulturedABSTRACT
Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.
Subject(s)
Glycoproteins/metabolism , Protein Kinase C/metabolism , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Recombinant Proteins/metabolism , Amino Acid Sequence , Annexins , Cell Line , Fibrinolysin , Humans , Peptide Fragments/analysis , Peptide Mapping , Phosphorylation , Proto-Oncogene Proteins pp60(c-src) , TrypsinABSTRACT
When myo-[3H]inositol-prelabelled primary-cultured murine bone-marrow-derived macrophages were challenged with platelet-activating factor (PAF; 200 ng/ml), there was a rapid (2.5-fold at 10 s) rise in the intracellular concentration of D-myo-[3H]inositol 1,4,5-trisphosphate, followed by a rise in myo-[3H]inositol tetrakisphosphate. myo-[3H]Inositol tetrakisphosphate fractions were isolated by high-performance anion-exchange chromatography from myo-[3H]inositol-prelabelled chick erythrocytes and primary-cultured macrophages. In both cases [3H]iditol and [3H]inositol were the only significant products (greater than 90% of recovered radioactivity) after oxidation to completion with periodic acid, reduction with NaBH4 and dephosphorylation with alkaline phosphatase. The presence of [3H]inositol after this procedure is consistent with the occurrence of [3H]inositol 1,3,4,5-tetrakisphosphate in the cell extracts, whereas [3H]iditol could only be derived from D- or L-inositol 1,4,5,6-tetrakisphosphate. When [3H]inositol tetrakisphosphate fractions obtained from (A) unstimulated macrophages, (B) macrophages that had been stimulated with PAF for 40s or (C) chick erythrocytes were subjected to the above procedure, radioactivity was recovered in these polyols in the following proportions: A, 60-90% in iditol, with 10-40% in inositol; B, total radioactivity increased by a factor of 9.8, 94% being recovered in inositol and 8% in iditol; C, 70-80% in iditol and 20-30% in inositol. [3H]Iditol derived from myo-[3H]inositol tetrakisphosphate fractions from macrophages and chick erythrocytes was oxidized to sorbose by L-iditol dehydrogenase (L-iditol:NAD+2-oxidoreductase, 1.1.1.14) at the same rate as authentic L-iditol. D-[14C]Iditol, derived from D-myo-inositol 1,4,5-trisphosphate, was not oxidized by L-iditol dehydrogenase. This result indicates that the [3H]iditol was derived from L-myo-inositol inositol 1,4,5,6-tetrakisphosphate. The data are consistent with rapid PAF-sensitive synthesis of D-myo-[3H]inositol 1,3,4,5-tetrakisphosphate in macrophages, and demonstrate that L-myo-inositol 1,4,5,6-tetrakisphosphate is synthesized in both mammalian and avian cells. The levels of L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate in primary-cultured macrophages are not acutely sensitive to PAF.
Subject(s)
Erythrocytes/metabolism , Inositol Phosphates/metabolism , Macrophages/metabolism , Sugar Phosphates/metabolism , Animals , Cells, Cultured , Chickens , Chromatography, High Pressure Liquid , Inositol Phosphates/blood , L-Iditol 2-Dehydrogenase/metabolism , Mice , Oxidation-Reduction , Polymers/blood , Polymers/metabolism , Species SpecificityABSTRACT
Incubation of human erythrocytes with Adriamycin prevented their morphological transition from discocytes to echinocytes when they were either depleted of ATP or loaded with calcium. This effect was dependent upon drug concentration and cell density. Adriamycin (10(-5) M) prevented, by greater than 90%, the echinocytosis of 10(7) cells/ml (S. B. Chahwala and J. A. Hickman, Cancer Res., 45: 4986-4989, 1985), and 5 X 10(-4) M prevented that of 10(9) cells/ml. There was a poor correlation between the effects of Adriamycin as a modulator of this morphological transition and its potency as an inhibitor of calmodulin. Using inside-out red blood cell vesicles, Adriamycin inhibited calmodulin dependent Ca2+ uptake with a 50% inhibitory concentration of 5 X 10(-4) M. Adriamycin thus differs from other amphipathic drugs, such as those of the phenothiazine class, where inhibition of calmodulin correlated well with effects on erythrocyte morphology (G. A. Nelson, M. L. Andrews, and M. J. Karnovsky, J. Cell Biol., 96: 730-735, 1983). After 12 h of ATP depletion, levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] extracted from 10(9) erythrocytes/ml fell by 57% and after 48 h they fell by 97%, changes which were coincident with a 100% transition of morphology to echinocytes. Adriamycin, 5 X 10(-4) M-1 X 10(-3) M, maintained 10(9) cells/ml in a discocyte morphology and maintained PtdIns(4,5)P2 levels at 60-70% of the time zero controls, independently of the size of the fall in PtdIns(4,5)P2 levels. The data suggested that Adriamycin inhibited a discrete pool of PtdIns(4,5)P2 which may be responsible for the maintenance of a discocyte morphology. Neomycin, 10(-3) M, had no effect on the ATP depletion-induced discocyte-echinocyte transition of 10(9) erythrocytes/ml or on the fall in PtdIns(4,5)P2 levels. Adriamycin, like neomycin, prevented the calcium-induced breakdown of erythrocyte membrane vesicle PtdIns(4,5)P2 to inositol trisphosphate (50% inhibitory concentration, 7 X 10(-4) M) but, unlike neomycin (50% inhibitory concentration, 4.25 X 10(-4) M) it was able to inhibit breakdown by 100% at higher concentrations.
Subject(s)
Doxorubicin/pharmacology , Erythrocyte Membrane/drug effects , Phosphatidylinositols/blood , Adenosine Triphosphate/metabolism , Calcium/metabolism , Calmodulin/pharmacology , Erythrocyte Membrane/metabolism , Humans , Membrane Lipids/metabolism , Time FactorsABSTRACT
12-O-Tetradecanoylphorbol-13-acetate (TPA) (100 nM) when incubated with human erythrocytes under conditions of ATP depletion, delayed the onset of the morphological transition from discocytes to echinocytes so that at 2 h, when control incubations were estimated to contain 65% echinocytes, those treated with TPA contained 23% echinocytes. TPA did not alter the subsequent rate of the transition which was complete by 3 h in control cells and 5 h in TPA-treated cells. Addition of 100 nM TPA to ATP-depleted erythrocytes at 2.5 h (greater than 80% echinocytes) for 0.5 h at 37 degrees C resulted in 17% reversal to a discocyte morphology, but as the time of incubation under conditions of ATP depletion was extended, the level of the reversal fell. TPA had no significant effect on the fall in ATP concentrations over the time course of the experiments (5 h). Preincubation of discocytes with TPA for 10 min also prevented, by approx. 50%, the echinocytosis induced by the calcium (0.2 mM) loading of discocytes using 5 microM A23187. TPA was unable to reverse the echinocyte morphology of calcium-loaded cells back to discocytes. The less potent tumour promotor 4-phorbol-12,13-didecanoate had no effect on this discocyte-echinocyte transition. Incubation of discocytes with the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) (1-10 microM) had complex effects on morphology, and the ATP-induced morphological transition, ranging from stomatocyte formation to echinocyte formation, depending upon the concentration of the agent and the time of incubation.
Subject(s)
Erythrocytes/ultrastructure , Tetradecanoylphorbol Acetate/pharmacology , Adenosine Triphosphate/blood , Erythrocytes/drug effects , Humans , In Vitro Techniques , Kinetics , Microscopy, Electron, ScanningABSTRACT
Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.
Subject(s)
Guanosine Triphosphate/analogs & derivatives , Phosphatidylinositols/metabolism , Thionucleotides/pharmacology , Adenosine Triphosphate/metabolism , Animals , Cell Line , Guanosine 5'-O-(3-Thiotriphosphate) , Guanosine Triphosphate/pharmacology , Kidney , Kinetics , Mathematics , Models, Biological , Phosphorus Radioisotopes , Phosphorylation , Rats , TritiumABSTRACT
Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2-acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase.
Subject(s)
Blood Proteins/metabolism , Cytoskeletal Proteins/blood , Erythrocyte Membrane/metabolism , Membrane Proteins , Neuropeptides , Phosphoproteins/blood , Protein Kinase C/blood , Cyclic AMP/pharmacology , Diglycerides/pharmacology , Dose-Response Relationship, Drug , Humans , Kinetics , Microfilament Proteins , Protein Kinase C/isolation & purification , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The red cells of a severely anaemic 2-year-old child of a white British family showed high haemolytic fragility with poikilocytosis. The cells showed markedly impaired thermal stability. The mother was phenotypically normal, but the father's red cells showed mild elliptocytosis. The spectrin from the latter, extracted at low temperature, was 30% dimeric (cf. 5-10% in normals). Tryptic digests of the spectrin from both father and daughter showed a reduction in the fragment of 80,000 molecular weight, derived from the terminus of the alpha-chain, and the elevation of a fragment of molecular weight 46,000, as well as one of 53,000. These characteristics and the autosomal recessive inheritance lead to a diagnosis of type II hereditary pyropoikilocytosis, so far reported only in two black American families (Lawler et al, 1983). The spectrin from the father was examined with respect to thermal conformational stability, and was found to be normal. The spectrin from the cells of the daughter gave evidence of the presence of oxidative (disulphide) cross-links, as well as of extensive noncovalent aggregation. Blood was obtained from the umbilical cord vein of the 19-week fetus of the pregnant mother: 250 microliters of blood was used for preparation of red cell membranes for SDS-gel electrophoresis and for extraction of spectrin. Analysis of the spectrin by gel electrophoresis in the native state revealed that the proportion of dimer was within the normal range, and the fetus therefore did not possess the hereditary pyropoikilocytosis phenotype. It is suggested that the procedures described could be generally applied to the prenatal identification of phenotypes associated with severe haemolytic anaemias.
Subject(s)
Anemia, Hemolytic, Congenital/diagnosis , Erythrocyte Membrane/ultrastructure , Erythrocytes, Abnormal/ultrastructure , Prenatal Diagnosis/methods , Anemia, Hemolytic, Congenital/blood , Anemia, Hemolytic, Congenital/pathology , Child, Preschool , Female , Fetal Blood/analysis , Hot Temperature , Humans , Male , Microscopy, Electron, Scanning , Pregnancy , Spectrin/analysisABSTRACT
A suspension of renal tubule fragments from the rat was prepared by a method involving collagenase digestion of the excised renal cortex and dispersion of the digest by passage through a nylon mesh. Through the use of scanning electronmicroscopy it was confirmed that the tubular lumena were patent, thus ensuring access of medium to both the luminal and the contraluminal membranes of the tubular cells. The viability of the tubule fragments was ascertained by measuring the rate of formation of glucose from pyruvate as substrate and the uptake of [14C] L-lysine against a concentration gradient. The uptake of L-lysine was unimpaired in the presence of gentamicin (10(-3) M), suggesting that there is no competition between this basic amino acid and the polycationic aminoglycoside for transport into tubular cells. The uptake of [3H] gentamicin was studied and found to be reduced in the presence of 2,4-dinitrophenol, potassium cyanide, and ouabain. The reduced uptake in the presence of ouabain was interpreted to mean that a component of gentamicin uptake, which occurs at the luminal membrane, may be driven by the Na+ gradient created by Na+-K+ATPase activity at the contraluminal membrane. This renal tubule preparation offers advantages over the kidney-slice technique for studies into the mechanisms of aminoglycoside nephrotoxicity.
Subject(s)
Gentamicins/toxicity , Kidney Diseases/chemically induced , Kidney Tubules/drug effects , Animals , Gentamicins/metabolism , Gluconeogenesis/drug effects , Glucose/metabolism , In Vitro Techniques , Kidney Diseases/metabolism , Kidney Diseases/pathology , Kidney Tubules/ultrastructure , Lysine/metabolism , Male , Rats , Rats, Inbred StrainsABSTRACT
Products of phosphatidylinositol (PI) turnover have recently been implicated as regulators of cell growth and differentiation. Transformation of cells in culture by infection with certain viruses (Rous sarcoma virus, Kirsten sarcoma virus, and polyoma virus) or by transfection with the oncogenes carried by these viruses affect the steady-state level of intermediates in the PI turnover pathway. In addition, immunoprecipitates of the transforming gene products of Rous sarcoma virus and polyoma virus contain activities of certain enzymes in the PI turnover pathway. We have previously reported that polyoma middle T immunoprecipitates can catalyze phosphorylation of PI to phosphatidylinositol-4-phosphate (PIP). This activity is not intrinsic to middle T or pp60c-src but is due to a cellular enzyme that specifically associates with the middle T/pp60c-src complex. The PI kinase is found in immunoprecipitates of the middle t protein from polyoma viruses that are capable of cell transformation but does not associate with mutants of middle t defective in transformation, suggesting that this association may be important for transformation. Two PI kinases from fibroblasts (type I and type II) that are separable by anion exchange chromatography have been partially purified and characterized. These enzymes differ in their Km for ATP as well as their Ki for adenosine and ADP. Only the type I PI kinase specifically associates with the transformation-competent mutants of middle T.
Subject(s)
Cell Transformation, Neoplastic , Phosphotransferases/metabolism , 1-Phosphatidylinositol 4-Kinase , Animals , Oncogenes , Retroviridae/geneticsABSTRACT
Steady-state cellular levels of phosphatidylinositol-4,5-bisphosphate (PIP2), 1,2-diacylglycerol (DAG), and inositol phosphates have been measured in two different fibroblast cell lines (NIH 3T3 and NRK cells) before and after transformation with three different ras genes. At high cell density the ratio of DAG to PIP2 was 2.5- to 3-fold higher in the ras-transformed cells than in their untransformed counterparts. The sum of the water-soluble breakdown products of the polyphosphoinositides, inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate, was also elevated in ras-transformed NRK cells compared with nontransformed NRK cells. These findings suggest that the ras (p21) protein may act by affecting these levels, possibly as a regulatory element in the PIP2 breakdown pathway.
Subject(s)
Cell Transformation, Neoplastic/analysis , Oncogenes , Phosphatidylinositols/analysis , Animals , Cell Line , Diglycerides/analysis , Humans , Inositol 1,4,5-Trisphosphate , Inositol Phosphates/analysis , Phosphatidylinositol 4,5-Diphosphate , RatsABSTRACT
The antitumor drug Adriamycin, when preincubated with human red blood cells (discocytes) for 10 min, prevented the formation of echinocytes induced by the calcium ionophore A23187 in the presence of 0.2 mM calcium. The degree of protection was concentration dependent and was greater than 90% at 10 microM Adriamycin. Adriamycin did not interfere with the accumulation of calcium induced by a 5 microM concentration of the ionophore. Adriamycin reversed echinocyte morphology to the discocyte form in echinocytes which had been formed by adenosine triphosphate depletion but not those formed after treatment with A23187 and Ca2+. Its ability to protect against Ca2+-induced echinocyte formation contrasts with the failure of the local anesthetic procaine to exert such an effect, even at 45 mM (J. Palek et al., Blood, 50: 155-164, 1977), and this difference suggests that Adriamycin may not be acting simply as a chaotropic agent. This hypothesis was supported by the observation that Adriamycin alone did not induce a cup-form morphology in discocytes (stomatocytosis). Wheat germ agglutinin protection of echinocyte formation induced by calcium loading was reversed by 30 mM N-acetylglucosamine, which partially reversed the Adriamycin protection of echinocyte formation. However, desialylation of human red blood cells with Clostridium perfringens type V neuraminidase, while preventing the protection of echinocyte formation by wheat germ agglutinin, had no effect on the protection afforded by Adriamycin. This suggests that Adriamycin does not prevent echinocyte formation via binding to the sialic acid residues of the transmembrane protein glycophorin and that another mechanism or mechanisms are involved in its action to modulate morphological transitions of the red blood cell membrane.
Subject(s)
Doxorubicin/pharmacology , Erythrocytes/drug effects , Acetylglucosamine/pharmacology , Adenosine Triphosphate/metabolism , Calcimycin/pharmacology , Calcium/metabolism , Calcium/pharmacology , Cells, Cultured , Erythrocytes/ultrastructure , Humans , Lectins/pharmacology , Microscopy, Electron, Scanning , Wheat Germ AgglutininsABSTRACT
The membrane potential of L1210 murine leukemia cells was assessed by use of the tritiated lipophilic cation probe triphenylmethylphosphonium bromide. The potassium equilibrium potential of the cells was found to be -71 +/- 7 mV. The resting membrane potential was partly dissipated by the protonophore m-chlorocarbonylcyanidephenylhydrazone (10 microM), but was unaffected by ouabain (1 mM) and apparently by the calcium ionophore A23187 (2.5 microM). Monensin (20 microM) caused a hyperpolarization which, since it was blocked by ouabain, was presumed to be brought about by activation of the Na+K+-ATPase via an elevated cytoplasmic Na+ concentration. Adriamycin at concentrations as high as 5 X 10(-4) M brought about no change in the resting potential of the cells. Also, cytotoxic concentrations of adriamycin, unlike ouabain, had no effect on rubidium-86 transport into L1210 cells, nor upon a monensin-induced increased in rubidium-86 uptake. The results suggest that although adriamycin is capable of interaction with the plasma membrane, and may exert its cytotoxicity at this locus, changes in ion flux mediated by Na+K+-ATPase or those capable of changing the membrane potential do not appear to be implicated in its mechanism of action.
Subject(s)
Doxorubicin/pharmacology , Leukemia L1210/metabolism , Animals , Calcimycin/pharmacology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Line , Cell Membrane/drug effects , Membrane Potentials/drug effects , Mice , Monensin/pharmacology , Onium Compounds/metabolism , Ouabain/pharmacology , Sodium-Potassium-Exchanging ATPase/analysis , Trityl Compounds/metabolismABSTRACT
The antitumor antibiotic adriamycin was found to be a potent modulator of the human erythrocyte discocyte echinocyte transition. Incubation of discocytes for 10 min with 10 microM adriamycin inhibited calcium-induced echinocytosis by 90 per cent. Adriamycin itself had no effect on erythrocyte morphology, a feature which distinguished it from other amphipaths which bring about the formation of a cupped cell morphology. Additionally, adriamycin differed from amphipaths such as the phenothiazines in that concentrations which prevented echinocytosis had no effects on calmodulin, as measured by effects on calmodulin-stimulated 45Ca2+ uptake into inside-out red cell vesicles. Adriamycin, paradoxically, appeared to cause a fall in the levels of erythrocyte polyphosphoinositides, but prevented further breakdown induced by calcium loading. This fall in inositides may be apparent rather than real, as the drug did not cause breakdown of the inositides to either inositol di- or triphosphates in red cell vesicles. Instead, it inhibited breakdown. It is possible that adriamycin may complex out the inositides and thus maintain levels of the inositide polyphosphates, congruent with the maintenance of the discocyte morphology. Interference with inositol lipid metabolism may be an important aspect of the pharmacology of adriamycin.
Subject(s)
Doxorubicin/pharmacology , Erythrocyte Membrane/drug effects , Biological Transport/drug effects , Calcium/blood , Erythrocyte Membrane/metabolism , Humans , Phorbol Esters/pharmacology , Phosphatidylinositols/bloodABSTRACT
N-Methylformamide is an antitumour drug with hepatotoxic properties. Three potential targets for hepatocellular toxic lesions caused by N-methylformamide were investigated: the mitochondrial and microsomal Ca2+ pumps and the functional integrity of the plasma membrane. The administration of N-methylformamide to mice caused a dramatic decrease in the ability of the liver mitochondria to sequester [45Ca2+]. This effect was dose-dependent and was not caused by dimethylformamide, N-hydroxymethylformamide or formamide. The microsomal Ca2+ pump was not affected by N-methylformamide. Incubations of isolated mitochondria with N-methylformamide for 1 hr also led to the inhibition of the Ca2+ sequestration. Incubation of isolated mouse hepatocytes with N-methylformamide did not cause changes in plasma membrane potential as measured using the lipophilic cation triphenylmethylphosphonium. Of the three targets studied, the mitochondrial Ca2+ pump may be the one through which N-methylformamide triggers the events leading ultimately to hepatic necrosis.
Subject(s)
Antineoplastic Agents/toxicity , Calcium/metabolism , Formamides/toxicity , Liver/pathology , Microsomes, Liver/metabolism , Mitochondria, Liver/metabolism , Animals , Cell Membrane/physiology , Kinetics , Liver/drug effects , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred BALB C , Microsomes, Liver/drug effects , Mitochondria, Liver/drug effectsABSTRACT
Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.
Subject(s)
Adenosine Triphosphate/pharmacology , Ions/metabolism , Leukemia, Erythroblastic, Acute/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/pharmacology , Amiloride/pharmacology , Animals , Calcium/metabolism , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Line , Dimethyl Sulfoxide/pharmacology , Furosemide/pharmacology , Mice , Potassium/metabolism , Sodium/metabolismABSTRACT
A study was made of the effects of gentamicin (40 mg/kg/day for 7 days) on the excretion of sodium, potassium, calcium and magnesium in the urine of rats. Volume of urine, protein excretion and the urinary activities of the enzymes alanine aminopeptidase, lactate dehydrogenase and N-acetyl-beta-glucosaminidase were also monitored. There were no significant changes in the excretion of sodium or potassium and the excretion of magnesium was too variable to permit a meaningful interpretation of changes in its excretion. Significant changes occurred in the excretion of calcium and protein and in the 24 hr urine osmolarity. The earliest of these changes, on day 4, was an increase in calcium excretion (P less than 0.01) which progressed until the drug was stopped and persisted throughout the 5 follow-up days. The increase in protein excretion was significant (P less than 0.01) by day 5 and the decrease in osmolarity (P less than 0.01) by day 6. These changes were preceded, on day 1, by a significant (P less than 0.001) rise in the activities in urine of all three enzymes. This early enzymuria suggests that considerable perturbation of cell integrity occurred before the increase in calcium excretion. Further studies are required to elucidate what role, if any, the loss of calcium plays in the genesis of tubular cell injury.
Subject(s)
Calcium/urine , Gentamicins/toxicity , Animals , Drinking , Eating , Electrolytes/urine , Enzymes/urine , Hydrogen-Ion Concentration , Male , Proteinuria/chemically induced , Rats , Rats, Inbred Strains , Time FactorsABSTRACT
Nitrogen mustard, HN2 (10(-5) M), inhibited the transport of the potassium congener 86rubidium into PC6A mouse plasmacytoma cells by 45% after a 4 hr incubation at 37 degree in vitro. HN2 (10(-3) M) had a rapid effect on the profile of 86rubidium transport into PC6A cells when added simultaneously with the 86rubidium whereas a monofunctional analogue of HN2((2-chloroethyl)dimethylamine) had no effect at 10(-3) M. The transport of the amino acid analogues alpha-aminoisobutyric acid and cycloleucine into PC6A cells was inhibited by 19% and 5% respectively after a 4 hr incubation with 10(-5) M HN2. The results suggest that the activity of plasma membrane Na+K+-ATPase may be affected by HN2. This enzyme may play a pivotal role in controlling cell growth and division. Crude cell membrane preparations from PC6A cells had variable Na+K+-ATPase activity which was possibly due to contamination with mitochondrial Mg2+-ATPase. Incubation of a crude cell membrane preparation in the presence of 40 nM dicyclohexylcarbodiimide gave constant Na+K+-ATPase activity which was inhibited by 44% on incubation with HN2 (10(-3) M) for 0.5 hr. The monofunctional analogue of HN2 inhibited this preparation by only 7% under the same conditions. It is suggested that inhibition of Na+K+-ATPase by HN2 may be an important facet of its cytotoxic activity.
Subject(s)
Nitrogen Mustard Compounds/pharmacology , Plasmacytoma/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Aminoisobutyric Acids/metabolism , Animals , Cell Membrane/drug effects , Female , Mice , Mice, Inbred BALB C , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Plasmacytoma/drug therapy , Rubidium/metabolismABSTRACT
An investigation was made of the effect of aminoglycoside antibiotics on Na+-K+ ATPase to determine whether their toxicity might be attributable to an inhibition of this enzyme. Three preparations were used: human erythrocyte ghosts; microsomal fractions of the cortex and outer medulla of the guinea-pig kidney; rat isolated renal tubules. In all three preparations marked inhibition of Na+-K+ ATPase (50-100%) was seen only at very high concentrations of drug (10(-2)M). Although aminoglycoside antibiotics are known to accumulate to high concentrations in vivo, particularly in the renal cortex, it is argued that these findings suggest that an effect on Na+-K+ ATPase is unlikely to be their primary mechanism of toxicity.
Subject(s)
Anti-Bacterial Agents/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Aminoglycosides/pharmacology , Aminoglycosides/toxicity , Animals , Anti-Bacterial Agents/toxicity , Erythrocyte Membrane/enzymology , Female , Guinea Pigs , Humans , In Vitro Techniques , Kidney Cortex/enzymology , Kidney Medulla/enzymology , Kidney Tubules/enzymology , Male , Microsomes/enzymology , Phosphates/blood , Rats , Rats, Inbred StrainsABSTRACT
1. The novel aminoglycoside antibiotic apramycin is shown to be a potent inhibitor of protein synthesis in bacteria both in vivo and in vitro. 2. In cell-free systems from Escherichia coli programmed with poly(U), apramycin induces translation errors, as assayed by incorporation of leucine, isoleucine and serine, although this effect occurs only to a limited extent. 3. Apramycin inhibits the translocation step of protein synthesis both in vivo, in protoplasts of Bacillus megaterium, and in vitro, in cell-free systems from E. coli. It is proposed that this is the primary inhibitory effect of the drug.