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

Abstract

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

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

“…Previous studies demonstrated that when the loading is performed in Ca='-containing media, the cell is able to compensate for the generation of the high-affinity Ca 2+ chelator by increasing the total cytoplasmic calcium, so that [Ca 2 +]i remains unchanged (22,30,40,41) . We previously showed that the release response brought about in quint-loaded PC 12 cells by high K+ and a-latrotoxin was not significantly different from that in control cells (22) .…”

“…Quint can be used not only to measure [Ca 2+]i but also to control and modify this parameter . In particular, when the loading is performed in Ca 2 '-free media, homeostasis is not maintained and [Ca 2 +] ; is decreased to very low levels (30) . In 63 0 THE JOURNAL of CELL 131OLOGY -VOLUME 99, 11 184 …”

“…First, cloned neurosecretory cells can be used which, while endowed with features typical of nerve cells, grow rapidly and are homogeneous . The second tool is a fluorimetric technique for measuring [Caz +]; which relies on the use of quin2 (30,40,41). The hydrophobic acetoxymethyl tetraester of this fluorescent Ca 2+ indicator penetrates the plasma membrane by diffusion and is then cleaved within the cytoplasm by unspecific esterases to yield the hydrophilic probe.…”

“…The hydrophobic acetoxymethyl tetraester of this fluorescent Ca 2+ indicator penetrates the plasma membrane by diffusion and is then cleaved within the cytoplasm by unspecific esterases to yield the hydrophilic probe. Thus, quin2 can be loaded into cells of any size, which can then be studied in suspension by conventional fluorimetric analyses (30,40,41).…”

Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

“…One of the earliest responses associated with crosslinking of slg is an increase in the level of cytoplasmic free calcium ([Ca2+]j). This was initially inferred from measurements of 45Ca2" distribution (2) and later confirmed by direct determinations of [Ca2+], using fluorescent probes such as quin2 (3). At least part of the Ca2" that appears in the cytoplasm originates from intracellular stores, inasmuch as anti-slg can elevate [Ca2+], in cells suspended in Ca2+-free media containing EGTA (3,4).…”