Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes

Deutsch, Carol; Holian, Andrij; Holian, Sandra K.; Daniele, Ronald P.; Wilson, David F.

doi:10.1002/jcp.1040990110

Cited by 140 publications

(105 citation statements)

References 48 publications

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“…The latter phase has been attributed to SCN oxidation to S0 4 of SCN uptake is extrapolated to zero time to obtain the SCN transmembrane distribution (3,5).…”

Section: Resultsmentioning

confidence: 99%

Membrane potential of Plasmodium-infected erythrocytes.

Mikkelsen¹,

Tanabe²,

Wallach³

1982

The Journal of Cell Biology

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The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP) . The SCN-and TPMPmeasured E m of normal erythrocytes are -6 .5 ± 3 mV and -10 ± 4 mV, respectively . The TPMPmeasured Ern of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Err, = -35 mV and "early" stages (ring and copurifying noninfected) by a low E m (-16 mV) . The SCN-determined Ern of infected cells was -7 mV regardless of parasite stage .Studies with different metabolic inhibitors including antimycin A, a proton ionophore Cells expend energy to maintain specific transmembrane ion gradients and appropriate intracellular ion concentrations necessary for nutrient transport (e.g . Na'-dependent amino acid transport, see reference 1) and cellular enzymatic processes (e.g. protein synthesis, see reference 2). Parasites such as the Plasmodia present an intriguing problem in this regard since they can apparently adjust to the very different ionic environments of the erythrocyte cytoplasm and host plasma . The intraerythrocytic forms develop from ring to schizont stage in the relatively high K+ environment of the erythrocyte whereas the end products of this development, the merozoites and gametocytes, are viable for at least a short time in the high Na' of extracellular plasma . In this publication, the membrane potential (Em) of Plasmodium-infected erythrocytes has been estimated from the transmembrane distributions of radiolabeled lipophilic anion, ["C]thiocyamate (SCN), and lipophilic cation, [3 H]triphenylmethy.phosphonium (TPMP) . This method has been used to measure Em of erythrocytes (3, 4), lymphocytes (3, 5), and MATERIALS AND METHODS Preparation of CellsMaintenance of Sprague-Dawley rats, infection with P. chabaudi, andisolation of erythrocytes are described in the accompanying article (8).

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“…The latter phase has been attributed to SCN oxidation to S0 4 of SCN uptake is extrapolated to zero time to obtain the SCN transmembrane distribution (3,5).…”

Section: Resultsmentioning

confidence: 99%

Membrane potential of Plasmodium-infected erythrocytes.

Mikkelsen¹,

Tanabe²,

Wallach³

1982

The Journal of Cell Biology

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“…Uptake of indo-1 AM and absorption and emission spectra were measured in a LS-5 fluorometer (Perkins-Elmer, Oak Brook, IL). Intracellular concentrations of indo-l were calculated assuming cellular water content to be 1.5 J/107 cells (5,20).…”

Section: Reagents and Cell Loadingmentioning

confidence: 99%

Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti‐IgM and anti‐IgD

et al. 1987

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The accurate measurement of ionized intracellular calcium [Ca++] Full understanding of lymphocyte transmembrane signaling requires detailed analysis of the rapid response to occupancy and/or crosslinking of membrane receptors for antigen, lymphokines and hormones as well as identification of the receptor molecules that mediate these processes. Heterogeneity of cell phenotype and functional responses, as well as clonal distribution of antigen receptors, requires analysis at the single cell level of the intracellular second messengers produced by these interactions. Much recent evidence indicates that the level of cytoplasmic free ionized calcium [Ca++]i is a critical second messenger (1) in the activation of both T (11,23,28) and B (2,3,4,12,16,19) [Ca++]i determination by flow cytometry offers several experimental advantages. It provides fully objective, highly quantitative measurements. The technique is applicable to any type of dissociated cells. Cross-correlation with other physiologic parameters and surface antigens can be obtained. Preparative sorting for functional correlations and selection of mutants to dissect pathways are both feasible.The newly available fluorescent chelator, indo-1 (9), has the properties required for single cell calcium measurement in the flow cytometer. When excited near the optimal wavelength of 356 nm, the fluorescence emission maximum shifts from 485 nm for free dye to 404 nm for the Ca++/indo-l complex with minimal change in fluorescence intensity. The ratio of fluorescence emission at these wavelengths reflects the fraction of Ca++-bound dye molecules and thus provides a quite precise measurement of [Ca++]i that is independent of the individual cell's dye content. Such ratio determinations eliminate variability due to heterogeneity in dye uptake and cell size.

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“…Knowledge of the normal cytoplasmic pH (pHi) and free [Mg2+] ([Mg2+]i) is thus a prerequisite for setting conditions for investigation of intracellular mechanisms, from organelle function to enzyme kinetics. Furthermore, both these ions, though more often H +, have been implicated in various aspects of cell activation, especially cell growth, differentiation, and proliferation (e.g., references 3,6,13,22).…”

mentioning

confidence: 99%

“…Many transductor systems are proposed as mediators of the effects of these surface ligands, including changes in ion flux and cytoplasmic ion composition (e.g. references 2,3,7,25), and there has been considerable biochemical investigation into the processes of stimulation. Yet, reliable measurements of cytoplasmic pH are not available and there seem to be no data relating to free Mg 2÷.…”

mentioning

confidence: 99%

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Cytoplasmic pH and free Mg2+ in lymphocytes.

Rink¹,

Tsien²,

Pozzan³

1982

The Journal of Cell Biology

1,037

449

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Measurements have been made of cytoplasmic pH, (pHi) and free Mg ~÷ concentration, ([Mg2+]~), in pig and mouse lymphocytes, pH~ was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31p nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37°C, pH~ was close to 7.0. Addition of physiological levels of HCO3 and CO2 transiently acidified the cells by ~0.1 U. Mitogenic concentrations of concanavatin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]~ was assessed in three ways: (a) from the external Mg 2÷ null-point at which the ionophore A23187 produced no net movement of Mg 2÷ or H÷; (b) by Mg-sensitive electrode measurements in freezethawed pellets; and (c) from the 31p nuclear magnetic resonance spectrum of the -},-phosphate of intracellular ATP. Total cell Mg 2÷ was ~12 mmol per liter cell water. The NMR data indicated [Mg2÷], >0.5 mM. The null-point method gave [Mg2+]~ -0.9 nM. The electrode measurements gave 1.35 raM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg 2÷.The concentration of H ÷ and free Mg 2÷ can powerfully influence many, probably most, intraceltular processes. Knowledge of the normal cytoplasmic pH (pHi) and free [Mg2+]([Mg2+]i) is thus a prerequisite for setting conditions for investigation of intracellular mechanisms, from organelle function to enzyme kinetics. Furthermore, both these ions, though more often H +, have been implicated in various aspects of cell activation, especially cell growth, differentiation, and proliferation (e.g., references 3, 6, 13, 22).One of the most widely studied model systems is mitogenic stimulation of mammalian lymphocytes by iectins such as concanavalin A (Con A). Many transductor systems are proposed as mediators of the effects of these surface ligands, including changes in ion flux and cytoplasmic ion composition (e.g. references 2, 3, 7, 25), and there has been considerable biochemical investigation into the processes of stimulation. Yet, reliable measurements of cytoplasmic pH are not available and there seem to be no data relating to free Mg 2÷. We therefore attempted to fill this gap, spurred on by a specific need for values for phi and [Mg2÷]i to calibrate our fluorescent dye measurements of cytoplasmic free-[Ca 2÷] ([Ca2+]i) (25).Many of the available techniques seemed inappropriate for lymphocytes, which are too small and elusive for microinjection of indicators or impalement with ion-selective microelectrodes.The equilibrium distribution of weak acids and bases gives not cytoplasmic pH, but some average intracellular pH for all the cell compartments (20). This method has given widely varying values in human peripheral lymphocytes, pH 6.8 to 7.4 with pHo 7.4 according to the probe chosen (3, ...

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Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes

Cited by 140 publications

References 48 publications

Membrane potential of Plasmodium-infected erythrocytes.

Membrane potential of Plasmodium-infected erythrocytes.

Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti‐IgM and anti‐IgD

Cytoplasmic pH and free Mg2+ in lymphocytes.

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