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C Levinson

Publications and source records attributed to C Levinson.

At least 19 recordsLinked to original sources
PubMed1996

Chloride accumulation in freshly isolated Ehrlich ascites tumor cells: the role of the Na/K/2Cl cotransporter.

When Ehrlich ascites tumor cells are removed from the peritoneal cavity and incubated in a saline solution, cells lose water, sodium, lactate and hydrogen ions and gain chloride. The gain of intracellular chloride exceeds that predicted from passive distribution. As chloride has been purported to play a role in volume regulation, it was of interest to identify factors responsible for controlling or maintaining intracellular chloride out of electrochemical equilibrium in Ehrlich cells. The results demonstrate that chloride accumulation in freshly isolated Ehrlich cells is sensitive to bumetanide, low extracellular K+ and low extracellular Na+, and is insensitive to DIDS. We conclude that chloride accumulation occurs due to the activity of the Na/K/2Cl cotransporter.

Animals↗
PubMed1993

Regulatory volume decrease in Ehrlich ascites tumor cells is not mediated by a rise in intracellular calcium.

Ehrlich ascites tumor cells suspended in hyposmotic solution initially swell and then shrink back towards normal volume, a process known as regulatory volume decrease (RVD). RVD is characterized by a specific loss of KCl, although the mechanism for this is currently unknown. The hypothesis that a rise in intracellular calcium ([Ca2+]i) activates calcium-sensitive ion conductances to initiate RVD was investigated. The results indicate that in the Ehrlich cell no rise in [Ca2+]i occurs when the extracellular osmolality is reduced from 300 mosM to 180 mosM. These findings were substantiated by the lack of sensitivity of RVD to the Ca(2+)-sensitive K+ channel blockers charybdotoxin (CTX) and nifedipine. In contrast, the ionophore ionomycin induced a cell shrinkage that was sensitive to CTX and nifedipine indicating that a rise in [Ca2+]i could play a role in cell volume reduction but that this occurred by a mechanism different from that observed in RVD. The conclusion from these experiments is that Ca2+ does not act as a second messenger for RVD in the Ehrlich cell.

Animals↗
PubMed1992

Regulatory volume increase in Ehrlich ascites tumor cells is mediated by the 1Na:1K:2Cl cotransport system.

After swelling in hyposmotic solution, Ehrlich ascites tumor cells shrink towards their original volume. Upon restoration of isosmolality (300 mOsM) the cells initially shrink but subsequently recover volume. This regulatory volume increase (RVI) is completely blocked when [Na+]o or [Cl-]o is reduced by 50% in the presence of normal [K+]o. With normal [NaCl]o but less than 2 mM [K+]o, not only is volume recovery blocked but the cells lose KCl and shrink. When [K+]o is increased to 5 mM there is a rapid net uptake of K+ and Cl- which results in volume recovery. This suggests that the reswelling phase requires the simultaneous presence of Na+, K+, and Cl-. Although ouabain has no effect on volume recovery, bumetanide completely blocks RVI by inhibiting a cotransport pathway that mediates the net uptake of Na+, K+ and Cl- in the ratio of 1Na:1K:2Cl. Na+ that accumulates is then replaced by K+ via the Na/K pump.

Animals↗
PubMed1991

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge.

Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOSM initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na+/H+ exchanger, the Na/K pump, the Na+ + K+ + 2Cl cotransporter and by loss of K+ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl-]i (approximately 100 mM) and/or the increase in the outwardly directed gradient of chemical potential for Na+, K+ and Cl-, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl- flux was almost zero suggesting minimal net cotransport activity. We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na+ + K+ + Cl-], as well as the ratio of intra- to extracellular [Cl-]. Both factors appear to influence the activity of the cotransport pathway.

Animals↗
PubMed1990

Regulatory volume increase in Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells, shrunken as a result of KCl-depletion and Na+ loading, re-establish normal ionic concentrations by the combined activity of the Na+/K+ pump and the (2Cl- + K+ + Na+) cotransport system. Restoration of cell volume, however, correlates only with the increase in intracellular Cl-. This along with the finding that the equilibrium volume is linearly related to the steady state [Cl-] suggests that the extent to which cell volume increases is determined by Cl- transport. Net Cl- uptake, which is mediated almost exclusively by the cotransport system, is ultimately responsible for establishing the steady-state intracellular Cl- concentration. Transport mediated by this pathway ceases when the sum of the chemical potentials for Na+, K+ and Cl- approaches zero and corresponds with the establishment of a steady state for Cl-. These findings suggest that Cl- plays a key role in the regulation of net cotransport activity and thereby cell volume.

Animals↗
PubMed1989

Quinine inhibits multiple Na+ and K+ transport mechanisms in Ehrlich ascites tumor cells.

The interaction of quinine with K+ and Na+ transport mechanisms has been investigated in Ehrlich ascites tumor cells. Quinine affects both Ca2+-dependent K+ channel and total K+ influx. Activation of Ca+-dependent K+ channels by propranolol is abolished by quinine (1 mM). In addition, quinine inhibits the ouabain-sensitive component of K+ influx with an apparent Ki of 0.32 +/- 0.02 mM and the furosemide-sensitive component with a Ki of 0.24 +/- 0.01 mM. Furthermore, a significant fraction (52%) of Na+ influx is inhibited by quinine. The same component is sensitive to amiloride, suggesting that it represents Na+/H+ antiport. Concomitant with the inhibition of K+ and Na+ transport, quinine stimulates ATP hydrolysis by 57%. The results suggest that quinine exerts broad, nonspecific effects on cellular mechanisms which serve to regulate cation transport in Ehrlich cells.

Adenosine Triphosphate↗
PubMed1988

Behavior of antithrombin III isoforms on immobilized heparins. Evidence that the isoforms bind to different numbers of low-affinity heparin sites.

Antithrombin III exists in plasma as major and minor isoforms differing in affinity for heparin. The nature of the binding of each purified isoform to immobilized heparins was investigated. Unfractionated, mixed-affinity heparin bound each isoform with both high affinity and concentration-dependent low affinity. The isoforms were resolved when filtered through low-affinity heparin (heparin repeatedly passed over immobilized antithrombin III) columns. Following chemical modification of a specific tryptophan residue required for heparin binding, each isoform failed to bind to either low-affinity or mixed-affinity heparin-agarose, but elution of the modified higher-affinity isoform was retarded on both gels. Because the modified lower-affinity isoform eluted with the similarly sized bovine serum albumin in these experiments, the difference in isoform affinity for heparin appears to be the result of a unique, secondary heparin-binding site in the higher-affinity isoform that can bind a heparin site with low affinity for antithrombin III. This interpretation was supported by the chromatographic behavior of the isoforms on mixed-affinity agarose during reverse gradient elution. Two other populations of each of the tryptophan-modified isoforms were identified. Since these isoforms bound tightly to mixed-affinity heparin-agarose but eluted at lower salt concentrations than the corresponding unmodified isoforms, both isoforms may contain additional secondary sites that interact weakly with heparin. A general model of heparin-antithrombin III interaction is proposed in which a high-affinity heparin site initially interacts with a primary site on antithrombin III. The subsequent conformational change leads to a cooperative, entropy-driven association between secondary sites on the protein and low-affinity sites on heparin, stabilizing antithrombin III in its activated form.

Antithrombin III↗
PubMed1987

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport.

The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na+-enriched cells. Subsequent incubation in K+-containing NaCl medium results in the reaccumulation of K+, Cl-, water and the extrusion of Na+. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (KM for K+o = 3.5 mM; Jmax = 30.1 mEq/kg dry min), which in these experiments was coupled 1K+/1 Na+. The second is the Cl--dependent (Na+ + K+) cotransport system (KM for K+o = 6.8 mM; Jmax = 20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K+:1Na+:2Cl-, the exchange of K+i for K+o. The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.

Animals↗
PubMed1986

Phosphate transport in Ehrlich ascites tumor cells: inhibition by H+.

The effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na+-dependent and Na+-independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHo from 7.30 to 5.50 results first in a transient (approximately 7 min) stimulation of Pi transport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 X 10(-6) M (pHo 6.68). However, Pi transport is progressively inhibited as pHi falls below 6.50. The effect of pHi on Pi transport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+ interaction with an intracellular Na+ site(s) on the Na+-dependent carrier. At pHo 7.4, about 15% of the steady state Pi flux persists in the absence of Na+. However, when pHo is reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+ stimulated Pi transport does not require Na+, raising the possibility that the Na+-independent component is mediated by the anion (Cl-) exchanger.

Animals↗
PubMed1985

Sodium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells.

The Ehrlich tumor cell possesses an anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl- + Na+ and/or K+), is Cl- -selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na+ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na+, ouabain-insensitive K+ and DIDS-insensitive Cl- transport (KCl cotransport) are low and equivalent to that found in 150 mM Na+ medium containing furosemide. Increasing the [Na+] results in parallel increases in K+ and Cl- transport. The maximum rate of each (18 to 20 meq/(kg dry wt) . min) is reached at about 20 mM Na+ and is maintained up to 55 mM. Thus, over the range 1 to 55 mM Na+ the stoichiometry of KCl cotransport is 1:1. In contrast to K+ and Cl-, furosemide-sensitive Na+ transport is undetectable until the [Na+] exceeds 50 mM. From 50 to 150 mM Na+, it progressively rises to 7 meq/(kg dry wt) . min, while K+ and Cl- transport decrease to 9 and 16 meq/(kg dry wt) . min, respectively. Thus, at 150 mM Na+ the stoichiometric relationship between Cl-, Na+ and K+ is 2:1:1. These results are consistent with the proposal that the Cl- -dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na+].

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

1983 AAGL survey. Professional profile of the membership. AAGL Research and Survey Committee.

The average AAGL physician does general obstetrics and gynecology, with 135 deliveries, 41 hysterectomies and 38 IUD insertions a year in addition to about 50 laparoscopies for sterilization and 43 for diagnosis. About 1 in 10 will experience a major complication, and 1 in 25 will undergo medicolegal actions, although their complication rates as a group are no higher than those of the total membership.

Gynecology↗
PubMed1984

Self-inhibition of chloride transport in Ehrlich ascites tumor cells.

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

H+ transport and the regulation of intracellular pH in Ehrlich ascites tumor cells.

The intracellular pH (pHi) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pHi of cells placed in an acidic medium (pHo below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pHo is acutely reduced to 5.5, pHi falls exponentially from 7.20 +/- 0.06 to 6.29 +/- 0.04 with a halftime of 5.92 +/- 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 +/- 0.05 mmol/(kg dry wt X min) and Km is 0.74 +/- 0.09 X 10(-6) M. Steady-state cells with pHi above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pHi 6.3) when returned to pHo 7.3 medium respond by transporting H+, resulting in a rapid rise in pHi. The halftime for this process is 1.09 +/- 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pHi to its steady-state value.

Adenosine Triphosphate↗
PubMed1983

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells.

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.

Animals↗
PubMed1982

Phosphate concentration and transport in Ehrlich ascites tumor cells: effect of sodium.

The effects of extracellular Pi and Na+ on cellular Pi concentration and transport were studied. Steady-state Pi exchange flux was measured by 32P uptake in the presence and absence of Na+. Model experiments were also conducted to assess the possibility that hydrolysis of organic phosphate esters contributes to the chemically measured intracellular Pi concentration of Ehrlich ascites tumor cells. The results of these experiments indicate that hydrolysis of labile organic phosphate esters does not contribute to the measured intracellular pool of Pi. The Pi transport system exhibits an apparent Ks of 0.115 mM Pi and a maximal flux of 1.73 mmole min-1 (kg dry wt)-1. When incubated in a phosphate-buffered choline chloride medium (5 mM Pi) the intracellular Pi and the Pi influx fall by 65 and 88%, respectively. At 5 mM extracellular Pi, the Na+-dependent component of Pi transport fits Michaelis-Menten kinetics with the maximal flux equal to 2.46 mmole min-1 (kg dry wt)-1 and an apparent Ks of 35.4 mM Na+. In addition, a Na+-independent component of Pi transport, comprising about 12% of the total Pi flux, was identified. The data support the hypothesis that a Pi transport system, dependent on Na+, plays a principal role in the maintenance of intracellular Pi concentration.

Animals↗
PubMed1979

Interaction of tritium-labeled H2DIDS (4,4'-diisothiocyano-1,2,diphenyl ethane-2,2'disulfonic acid) with the Ehrlich mouse ascites tumor cell.

The experiments reported in this paper were undertaken to explore the interaction of tritiated H2DIDS (4,4'-diisothiocyano-1,2,diphenyl ethane-2,2'-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (3H)H2DIDS to tumor cell suspension at 21 degrees C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H2DIDS i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H2DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H2DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H2DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H2DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H2DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H2DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H2DIDS into the intracellular phase.

Animals↗