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M Graber

Publications and source records attributed to M Graber.

At least 37 records · Page 2Linked to original sources
PubMed1992

Electrogenic Na-independent HCO3 transport in OK cells.

We have shown previously that OK cells recover from an acid load in a medium nominally CO2-free by extruding H via a Na/H exchanger and a passive H-conductive pathway. In this work, the regulation of cell pH (pHi) was studied after addition or withdrawal of CO2/HCO3 (5% CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHi after CO2 entry and CO2 exit were found to depend in part on HCO3 entry and exit, respectively. Efflux of H per se also contributed to restoring pHi after CO2 addition, whereas H influx may have played a smaller role to normalize pHi after CO2 removal. DIDS, 0.5 mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHi after CO2 addition and removal. Cl removal also failed to inhibit pHi recovery after CO2 removal. Cell depolarization in the presence of Na moderately stimulated the pHi recovery rate after CO2 addition whereas it markedly inhibited the normalization of pHi after CO2 removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHi recovery after CO2 addition but markedly prevented the pHi recovery after CO2 removal. These results indicate that OK cells lack Na or Cl-dependent HCO3 transport systems. The OK cell possesses a novel stilbene-sensitive electrogenic HCO3 transport system that is involved in the regulation of cell pH.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor.

Several lines of evidence implicate a regulatory tyrosine phosphorylation in the activation of phospholipase C (PLC) by the T cell antigen receptor (TCR). These include studies using inhibitors of protein tyrosine kinases (PTKs). In Jurkat T cells expressing the heterologous human muscarinic receptor (HM1), PLC activity can be induced by either the TCR or HM1. HM1 activates PLC via a guanine nucleotide binding protein. We have studied the selectivity of the effects of the PTK inhibitors, herbimycin A and genistein, in this system. The results indicate that these inhibitors have different mechanisms of action, and suggest that herbimycin A, but not genistein, is a specific inhibitor of PTKs in T cells. Herbimycin A markedly inhibited both the resting and induced levels of phosphotyrosine-containing proteins, including the gamma 1 isozyme of PLC and the zeta chain of the TCR, and prevented activation of PLC by anti-TCR mAb. Herbimycin A did not inhibit activation of PLC by HM1. Genistein had a much less pronounced effect than herbimycin A on the appearance of tyrosine phosphoproteins. Moreover, genistein inhibited activation of PLC by both the TCR and HM1, and inhibition was only partial. Genistein was cytotoxic and markedly inhibited protein synthesis in both Jurkat cells and human peripheral lymphocytes. Herbimycin A was not cytotoxic. These findings confirm the role of a regulatory tyrosine phosphorylation in activation of PLC by the TCR. Herbimycin A was a selective inhibitor of a subclass of PTKs in Jurkat cells. In contrast, inhibition of signal transduction and later events in T cells by genistein may be due to effects other than direct inhibition of PTK activity.

Benzoquinones↗
PubMed1992

Intracellular pH in OK cells. II. Effects of temperature on cell pH.

Intracellular pH (pHi) is known to acidify as temperature rises. It has been proposed that this relationship reflects the temperature dependence of the dissociation constant of intracellular buffers. The purpose of this study was to evaluate whether H+ production and membrane acid/base transport events also contribute to the temperature dependence of pHi. This relationship was studied by means of the 490/450-nm fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, to measure pHi of OK cells grown on cover slips and perfused in a thermostated chamber. As temperature was increased from 12 degrees C, pHi progressively acidified, until a precipitous and large alkalinization appeared at 40-45 degrees C. We specifically examined the range from 25 to 37 degrees C, over which pHi acidified by 0.32 units from 7.50 to 7.18. The three major components that regulate pHi in the OK cell were quantitated as follows: H+ production, H+ extrusion via Na(+)-H+ antiport, and the passive H+ leak from buffer to cell. Relative to the rate at 25 degrees C, acid production at 37 degrees C increased by 129% (3.82 +/- 0.78 vs. 8.76 +/- 1.12 mmol.l-1.min-1). The recovery of pHi after NH4Cl loading was used to measure the rate of Na(+)-H+ antiport, and in Na(+)-free depolarized cells the magnitude of passive H+ conductance through the leak pathway. Compared with the rate at 25 degrees C, at 37 degrees C the Na(+)-H+ antiport increased by 26% (1.25 +/- 0.22 vs. 1.58 +/- 0.25 mmol.l-1.min-1) and the H+ permeability increased by 140% (0.005 +/- 0.001 vs. 0.012 +/- 0.003 cm/s).(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1991

Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number.

Engagement of the TCR initiates at least two transmembrane signaling pathways, the phosphatidylinositol pathway and a tyrosine kinase pathway. The T cell leukemic line Jurkat was used to study the relationship between the number of occupied TCR on the cell surface and the TCR-mediated activation of phosphatidylinositol-specific phospholipase C. We characterized a series of Ti beta-chain transfectants of the Jurkat mutant J.RT3-T3.5, in which surface expression of the TCR is limited by expression of the TCR beta-chain. Calibrated flow cytometry was used to determine the number of binding sites for anti-CD3 mAb on the surface of these cells, which was less than 1.2 x 10(3) to 1.2 x 10(4) sites/cell. In the presence of lithium chloride, the accumulation of inositol phosphates (InsP) in these cell lines in response to saturating concentrations of anti-CD3 mAb was proportional to the calculated surface TCR number. This result was consistent with dose-response studies using anti-CD3 mAb in Jurkat cells, in which ligand concentration, rather than number of binding sites, was limiting. Increase in intracellular free calcium concentration was a sensitive indicator of TCR engagement and correlated with the level of TCR expression, but less closely than did InsP levels. Induction of the early lymphocyte activation marker CD69 by anti-CD3 mAb also correlated with surface expression of TCR. In order to test whether limitation of this signaling pathway by TCR number may be relevant to signal transduction in the wild-type cell, we compared PLC activity in Jurkat cells during soluble anti-CD3 mAb-induced internalization of the TCR and also in response to immobilized mAb. The net accumulation of InsP per min decreased linearly with TCR number during the rapid phase of TCR internalization, confirming the limiting role of TCR number in this system. When internalization was prevented by immobilization of the stimulus, there was no decrease in the net accumulation of InsP per minute over time. In a Jurkat cell line transfected with the heterologous human muscarinic receptor, subtype 1, the InsP response to a muscarinic agonist was unaffected by TCR internalization, indicating that the distal phosphatidylinositol pathway was not affected by prolonged stimulation of the TCR. We conclude that transmembrane signaling through the TCR may be regulated by the number of surface TCR-ligand complexes. This observation has implications for transmembrane signaling in both mature T cells and thymocytes.

Antigens, CD↗
PubMed1991

The electrolytes in hyponatremia.

It is commonly taught that retention of free water is the dominant factor reducing the serum sodium concentration in hyponatremia. To determine whether the concentrations of other electrolytes are similarly diluted, we identified 51 patients with hyponatremia (Na = 121 +/- 1 mmol/L [mEq/L]) and compared electrolyte and laboratory values at the time of hyponatremia with values at a time when serum sodium was in the normal range (138 +/- 1 mmol/L). The medium interval between these measurements was 12 days. At the time of hyponatremia, serum sodium and chloride were substantially and significantly reduced by 12% to 15%. Although many hyponatremic patients had overtly increased or decreased concentrations of the other measured electrolytes, there were no significant changes in the mean concentration for any of these at the time of hyponatremia. Unchanged mean values were found for the plasma concentration of bicarbonate (26.1 +/- 0.6 normal v 25.2 +/- 0.8 mmol/L at the time of hyponatremia), potassium (4.31 +/- 0.10 v 4.33 +/- 0.15 mmol/L), albumin, phosphate, and creatinine. The stability of these laboratory values was observed both in patients with clinically normal extracellular fluid (ECF) volume and in those with true or effective ECF depletion. The urinary sodium (UNa) concentration was found to be a reliable predictor of the ECF volume status, whereas the fractional sodium excretion (FENa) was not. Electrolyte derangements are common in patients with hyponatremia, but are usually confined to patients on diuretics or who have an abnormal ECF volume. In the absence of these complicating situations, the plasma electrolytes are typically normal and are not reduced by dilution to the same extent as Na and CI. Based on a review of both the classic and recent knowledge concerning electrolyte regulation in hyponatremia, we propose that two factors explain these observations. First, the degree of dilution is overestimated because of Na losses in urine and perhaps Na shift into cells. Second, both renal and extrarenal adaptive mechanisms are activated by hyponatremia that stabilizes the concentration of other ions. One of these mechanisms is cell swelling, which triggers a volume-regulatory response leading to the release of ions and water into the ECF. Other adaptive mechanisms are mediated by antidiuretic hormone (ADH) per se, and by atrial natriuretic peptide (ANP).

Adaptation, Physiological↗
PubMed1991

Intracellular pH in the OK cell. I. Identification of H+ conductance and observations on buffering capacity.

The regulation of intracellular pH (pHi) in the opossum kidney (OK) cell line was studied in vitro using the pH-sensitive excitation ratio of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Recovery from an NH4Cl acid load disclosed a Na-dependent component blocked by amiloride and a smaller Na-independent component. The Na-independent recovery rate was proportional to the H+ gradient from cell to buffer and was zero in the absence of an electrochemical gradient. The Na-independent recovery was not affected by N-ethylmaleimide, dicyclohexylcarbodiimide, HCO3, phloretin, or ZnCl2 but was accelerated in depolarized cells and by membrane-fluidizing drugs and was inhibited by glutaraldehyde. The apparent cellular buffering capacity changed in proportion to this H+ conductance. Consistent with an electrogenic H+ leak, steady-state cell pH alkalinized with depolarization and acidified with hyperpolarization. Removal of buffer Na+ produced a profound acidification, as did amiloride. In 0-Na+ buffers, extremely large cell-to-buffer H+ gradients were present and proportional to buffer pH. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on steady-state pHi. Measurements of intracellular buffering capacity were derived from the change of cell pH induced by withdrawing NH4Cl. This buffering capacity was increased threefold in Na-free buffers, whereas the value measured by direct titration of cell lysate was the same or less than that of control cells. The NH4Cl-derived buffering capacity varied in direct proportion to the magnitude of the H+ leak. Drugs that changed H+ permeability produced the apparent changes of the measured buffering capacity within a few minutes. We conclude that, in HCO3-free buffer, the OK cell uses two membrane acid-base transport pathways: a Na-H antiporter active at physiological pH and a substantial passive H+ conductance. The results also reveal that the NH4Cl-derived buffering capacity is subject to artifacts, possibly due to a finite leak of ionic NH4+.

Amiloride↗
PubMed1991

Bubble cells: renal tubular cells in the urinary sediment with characteristics of viability.

The urinary sediment was examined by light microscopy in 65 consecutive inpatients with renal insufficiency (not due to pre- or postrenal factors) referred to a nephrology consult service for evaluation. In the 60 patients in whom a single diagnosis was reached, the sediments of 34 (57%) contained an easily recognized cell, which we have called the "bubble cell". These cells were bizarre, large cells with a single nucleus, which appeared to contain one or more fluid-filled vesicles. Bubble cells were most prevalent in the sediment of patients with acute tubular necrosis but were also seen a variety of other renal diseases. In most patients with acute tubular necrosis, the sediment also contained "normal"-appearing renal tubular cells, muddy brown casts, and oval fat bodies which were indistinguishable from those seen in the nephrotic syndrome. By electron microscopy, the bubble cells appeared to be vacuolated renal tubular epithelial cells, which had characteristics of viable cells. Most bubble cells excluded the vital dye Trypan blue, whereas the normal-appearing renal tubular cells were typically strongly positive. It was concluded that bubble cells, often accompanied by oval fat bodies, are commonly present in the sediment of patients with acute tubular necrosis as well as many other types of renal disease. Most cells which would be classified as "normal" renal tubular cells in these sediments are dead. In contrast, the findings suggest that the bubble cell represents an injured but viable renal tubular cell. The frequent finding of oval fat bodies in the same sediments suggests that the oval fat body is also produced by tubular cell injury.

Acute Kidney Injury↗
PubMed1990

Effect of diffusional resistances on the action pattern of immobilized alpha-amylase.

Alpha-amylase from Aspergillus oryzae has been immobilized onto corn grits and porous silica (specific areas 180 and 440 m2 g-1). Kinetic parameters of immobilized enzyme have been determined. Immobilization of alpha-amylase results in the formation of less polymerized products resulting in an apparent decrease in the number of transglycosylation reactions, for both maltotetraose and starch as substrates, when compared with free enzyme. Diffusional limitations for substrate and products have been quantified in the case of the three supports used. External diffusional resistances were important in all cases for the reaction products, whilst they became negligible for the substrate in the case of silica supports. Moreover, internal transfer limitations were identified with silica 180 m2 g-1 support. It was demonstrated that diffusional resistances were in direct relation to the apparent modification of the enzyme action pattern after immobilization.

Aspergillus oryzae↗
PubMed1990

Why do young infants fail to search for hidden objects?

Recent evidence indicates that infants as young as 3.5 months of age understand that objects continue to exist when hidden (Baillargeon, 1987a; Baillargeon & DeVos, 1990). Why, then, do infants fail to search for hidden objects until 7 to 8 months of age? The present experiments tested whether 5.5-month-old infants could distinguish between correct and incorrect search actions performed by an experimenter. In Experiment 1, a toy was placed in front of (possible event) or under (impossible event) a clear cover. Next, a screen was slid in front of the objects, hiding them from view. A hand then reached behind the screen and reappeared holding the toy. The infants looked reliably longer at the impossible than at the possible event, suggesting that they understood that the hand's direct reaching action was sufficient to retrieve the toy when it stood in front of but not under the clear cover. The same results were obtained in a second condition in which a toy was placed in front of (possible event) or behind (impossible event) a barrier. In Experiment 2, a toy was placed under the right (possible event) or the left (impossible event) of two covers. After a screen hid the objects, a hand reached behind the screen's right edge and reappeared first with the right cover and then with the toy. The infants looked reliably longer at the impossible than at the possible event, suggesting that they realized that the hand's sequence of action was sufficient to retrieve the toy when it stood under the right but not the left cover. A control condition supported this interpretation. Together, the results of Experiments 1 and 2 indicate that by 5.5 months of age, infants not only represent hidden objects, but are able to identify the actions necessary to retrieve these objects. The implications of these findings for a problem solving explanation of young infants' failure to retrieve hidden objects are considered.

Attention↗
PubMed1989

Biochemical and growth inhibitory effects of the erythro and threo isomers of gamma-fluoromethotrexate, a methotrexate analogue defective in polyglutamylation.

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(gamma-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.

Animals↗
PubMed1989

Acetazolamide inhibits acidification by the turtle bladder independent of cell pH.

Acetazolamide (ACZL) inhibits luminal acidification by the turtle urinary bladder, a process thought to be mediated by the drug's ability to inhibit carbonic anhydrase (CA) and thus elevate cell pH. To test the hypothesis that these transport changes are actually mediated by changes of cell pH, we measured this parameter in single, identifiable mucosal cells using 4-methylumbelliferone and fluorescence microscopy. In control bladders 5 X 10(-4) M ACZL inhibited proton transport by 80 +/- 6%, and alkalinized cell pH, especially in a subpopulation of CA cells. A much larger cell alkalinization was induced by serosal HCO3- but proton transport fell only 30 +/- 7%. When cell pH was clamped at approximately 7.0 using 50 mM dimethyloxazolidinedione, or when cell pH was acidified using 7.5 mM propionate, transport rates still declined by 74 +/- 2, and 100 +/- 12%, respectively, in response to ACZL. In propionate-acidified bladders, 1 mM sodium azide blocked the inhibition of transport seen with 5 X 10(-4) M ACZL and reversed the inhibition with 10(-5) M ACZL. The apical endocytosis rate was increased by ACZL in normal and propionate-acidified bladders, but was not stimulated by alkalinizing the cell with NH4Cl. We conclude that ACZL can induce cellular alkalinization in this tissue, but that this pH change is not required for the inhibition of transport, or the ACZL-associated stimulation of endocytosis. The drug's ability to inhibit acidification appears to be the result of an azide-sensitive mechanism that has yet to be defined.

Acetazolamide↗
PubMed1989

Phorbol myristate acetate inhibits acidification by turtle urinary bladder.

The effects of phorbol myristate acetate (PMA) on acid secretion by the turtle urinary bladder were examined to evaluate the importance of protein phosphorylation in modulating the distal acidification system. In HCO3-free PO4 buffer 0.2 mM mucosal PMA inhibited reverse short-circuit current (RSCC) by 42%. The inhibition of RSCC was dose dependent, and RSCC approached zero at high concentrations of PMA. PMA also inhibited acid secretion measured titrimetrically but had no effect on RSCC from bladders in which proton secretion was selectively inhibited by an adverse pH gradient or serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The inhibition by PMA was duplicated by 1-oleoyl-2-acetyl-rac-glycerol, but not by an inactive phorbol ester. The PMA inhibition was much more potent with mucosal compared with serosal application. The PMA inhibition caused a significant 0.15 pH unit reduction of carbonic anhydrase (CA) cell cytoplasmic pH and a change in the CA cell morphology. Unlike the inhibition of proton transport induced by acetazolamide, the PMA inhibition was neither reversed nor prevented by sodium azide. We conclude that PMA decreases basal acid secretion and blocks the stimulatory effects of CO2 by an azide-insensitive mechanism distinct from that of acetazolamide. In view of recent findings that PMA stimulates HCO3 secretion by the turtle bladder, these results suggest that kinase C-mediated protein phosphorylation may be a central event in the transition from the secretion of acid to the secretion of base.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Thrombocytosis elevates serum potassium.

Spurious elevation of the serum potassium can be seen if the platelet count exceeds 1,000 x 10(9)/L in patients with myeloproliferative disease. To see if serum potassium is increased at more modest elevations of the platelet count we studied these parameters in 283 controls and 161 patients with reactive thrombocytosis. The incidence of hyperkalemia was 34% in patients with over 500 x 10(9) platelets/L compared with 9% if the platelet count was below 250 x 10(9)/L. Over this range there was a significant correlation of potassium increasing with the platelet count. The higher potassium values could not be explained on the basis of leukocytosis, renal insufficiency, or acidosis, suggesting that the elevated serum levels were often spurious. In support of this conclusion, the serum potassium exceeded plasma potassium by a larger amount in patients with thrombocytosis. We conclude that serum potassium rises in direct proportion to the platelet count in normal patients and in those with thrombocytosis, and that this increment is an artifact.

Humans↗