Lymphocyte membrane potential assessed with fluorescent probes

Rink, T J; Montecucco, Cesare; Hesketh, T R; Tsien, Roger Y.

doi:10.1016/0005-2736(80)90243-6

Cited by 329 publications

(189 citation statements)

References 15 publications

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“…7, yet again capped normally. (1) found that RaMIg applied to mouse spleen lymphocytes reduced the cell-associated 4SCa by 20 to 30% within the first 2 rain. This loss could have been due to a primary acceleration of Ca effiux which would lower [Ca2+]i, or to internal release of Ca 2÷ raising [Ca2+]i with subsequent Ca effiux.…”

Section: The Relationship Between [Ca 2 + ]J and Cappingmentioning

confidence: 93%

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

Pozzan¹,

Arslan²,

Tsien³

et al. 1982

The Journal of Cell Biology

208

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This paper examines, in mouse spleen lymphocytes, the effect of anti-immunoglobulin (anti-Is) on the cytoplasmic free calcium concentration, Capping of surface Ig represents a dramatic reorganization of membrane components apparently involving cytoskeletal elements and requiring metabolic energy (2, 12). Formation of caps is followed by contractile activity and cell motility. Rather little is known of how ligand binding stimulates these processes although changes in ion flux and potential difference across the plasma membrane play no essential part (7). Ca 2+ ions are well known to trigger muscle contraction and are implicated in the regulation of various forms of nonmuscle motility, so it has been attractive to suppose that a rise in cytoplasmic free Ca z+ mediates the B cell response to anti-Ig (2). The evidence hitherto available is entirely indirect. It is generally agreed that external Ca z÷ is not needed. The ionophore A23187, which in other cell types raises cytoplasmic free [Ca 2+] and stimulates Ca2+-activated processes, actually inhibits capping in B lymphoeytes. However this inhibitory effect, like those of local anesthetics (5) and cis-unsaturated fatty acids (3), is at least in part explainable (10) by depletion of cellular ATP. Recently anti-Ig has been found to cause a rapid loss of 45Ca from mouse spleen cells (l). This efflux was interpreted as mobilization of Ca 2+ from internal stores causing a rise in the level of free cytoplasmic Ca 2+ and subsequent extrusion of the mobilized ions. There was no evidence that the Ca 2+ movements caused capping but the correlations between the two were felt to suggest "that Ca 2+ may have a physiological role in contractile dependent capping" (2).In the present paper the intracellular Ca 2+ indicator, quin2 (13, 14), has been used to assess more directly the importance of Ca 2+ by monitoring and manipulating the cytoplasmic free Ca 2+ concentration, [Ca2+]i, in mouse spleen lymphocytes. The changes in [Ca2+]i brought about by anti-Ig on cap formation are analyzed and the effects of preventing such changes are examined. MATERIALS AND METHODSBALB/c mouse spleen lymphocytes and thymocytes were prepared as previously described (6-10) in HEPES-buffered culture medium, either H-M EM or RPM 1-1640. Viability, assessed by eosin exclusion, exceeded 95%. B-cell enriched

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Section: The Relationship Between [Ca 2 + ]J and Cappingmentioning

confidence: 93%

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

Pozzan¹,

Arslan²,

Tsien³

et al. 1982

The Journal of Cell Biology

208

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“…The method of Rink et al [20] was used with a slight modification. The reaction mixture (2.5 ml), which contained NaCl buffer, 0.1 pM 3,3'-dipropylthiadicarbocyanine and ionophores, was incubated for 2 min at 25°C in a quartz cuvette in a Hitachi MPF-4 spectrophotometer.…”

Section: Measurement Of the Membrane Potential By 33'-dipropylthiadimentioning

confidence: 99%

“…The effect of ionophores on the membrane potential of bovine lymphocytes was examined also. Generation of membrane potential was monitored by quenching of 3,3'-dipropylthiadicarbocyanine fluorescence [20]. The quenching of fluorescence was blocked by carbonylcyanide m-chlorophenylhydrazone, gramicidin D and A23187, but not by monensin and valinomycin.…”

Section: Requirement Of Membrane Potential For Polyarnine Uptake In Bmentioning

confidence: 99%

Characterization of the inducible polyamine transporter in bovine lymphocytes

Kakinuma

Hoshino

Igarashi

1988

European Journal of Biochemistry

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The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The K, values for uptake activities of putrescine, spermidine and spermine were 3.7 pM, 0.38 pM and 0.23 pM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM IS+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as cr-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 pM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.Polyamines (putrescine, spermidine and spermine) are organic cations widely distributed in nature. The significance of polyamines in cell growth and metabolic processes has been well recognized (for reviews, see [l-31). Increases in polyamine contents are observed in various cells stimulated to proliferate.While cellular polyamines are mainly produced via de nova synthesis, the specific polyamine transport system has been demonstrated in both prokaryotes [4, 51 and eukaryotes [6 -91. Pohjanpelto (61 reported that putrescine uptake is greatly increased in human fibroblasts stimulated to proliferate. In mouse mammary explants [7] it has been shown that polyamine transport can be stimulated by insulin and prolactin, hormones that stimulate growth of explants. In human skin fibroblasts [8], epidermal growth factor stimulated both ornithine decarboxylase and polyamine transport. Thus, the physiological significance of polyamine uptake as well as polyamine biosynthesis is considered to increase intracellular polyamine levels during cellular responses to proliferative stimuli.The polyamine biosynthetic pathway is very highly regulated by the polyamines themselves [3, 101. An increase in the intracellular content of polyamines reduces the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis, and this reduction by polyamines is controlled mainly at the translational level [ll]. However, the molecular basis of regulation of the polyamine transport system is still not known.In the present study we examined the iiiducibility of the polyamine uptake system in bovine lymphocytes, and found that induction of the polyamine transporter is regulated by the intracellular level of spermidine and spermine.Correspondence to K. Igarashi, Faculty of Pharmaceutical Sciences, Inohana Campus, Chiba University, 1-8-1 Inohana, Chiba, Japan 280Abbreviations. ConA, concanavalin A; DFMO, cr-difluoromethylornithine...

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“…The oxonol dyes have been used for indirect measurement of membrane potential in the cells with inside-negative potentials (5,8). In crease in the oxonol fluorescence intensity in dicates a depolarization of the cell, whereas the decrease shows a hyperpolarization.…”

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confidence: 99%