Immunology Letters, 46 (1995) 81-83
01652478/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved
Voltage-dependent ion channels on human basophils: do they exist? ’
Francis Beauvais * , Claude Burtin and Jacques Benveniste
INSERM UZOO, Uniuersit6 Paris&d, Clamart, France
(Received 27 December 1994; accepted 1 February 1995)
Key words: Human basophil; Immediate hypersensitivity; Histamine release; Ca’+ influx; Membrane depolarization
The presence of voltage-dependent ion channels
(particularly Ca* + channels) on the surface of ‘non
excitable’ cells such as human basophils is a matter of
debate. Indeed, in basophils, Ca2+ entry or mobilization
is not sufficient by itself to trigger secretion, although
enhanced cytosolic Ca*+ concentration increases it. In
order to address this question, we used a two-signal
model and we report here experiments which suggest
the presence of voltage-dependent structures directly or
indirectly linked to membrane Ca*+ pathways. Indeed,
it is known that, in the presence of PMA at threshold
concentration (1st signal), elevation of cytosolic Ca*+
(2nd signal) induces histamine release. We observed
that a depolarizing external solution (high K+) induced
a Ca*+-dependent release of histamine from PMA-
treated human basophils. High K+ alone did not induce
Although the voltage-sensitive component and the
physiological relevance of this mechanism remain to be
defined, these results suggest that this voltage-depend-
ent Ca*+ influx in the human basophil could contribute
to the up-regulation of histamine release.
Secretion of granule content in excitable cells, such
as neurons or chromaffin cells, is mainly due to Ca2+
entry through voltage-dependent Ca2+ channels [l]. In
non-excitable cells such as neutrophils and platelets,
increase of cytosolic Ca*+ is due to Ca*+ mobilization
’ This article is the 6th in a series entitled “Regulation of human
* Corresponding author: F. Beauvais, INSERM U312, Salle de
Malte HGpitat, Saint-Louis 1, Avenue Claude Vellefaux, 75010 Paris,
France. Tel.: 33 (1) 4200-0022; Fax: 33 (1) 4200-0029.
from internal stores and Ca*+ influx from external
medium through receptor-operated Ca2’ channels. In
basophils and mast cells, both cells involved in immedi-
ate hypersensitivity, cytosolic Ca*+ plays a major role
in the secretion of histamine [2,3]. However, in these
cells, Ca*+ appears to play an up-regulating rather than
a triggering role [1,4-61.
In this paper we describe a two-signal model where
the depolarization of the human basophil membrane
induced an external Ca’+-dependent release of his-
3. Materials and Methods
Goat Fc-specific anti-human IgE (Nordic Immuno-
logical Laboratories, Tilburg, The Netherlands), phorbol
1Zmyristate 13-acetate (PMA), HEPES (Sigma, St.
Louis, MO) were obtained as mentioned.
3.2. Leukocyte histamine release
Histamine release was measured as previously de-
scribed . Leukocytes in HEPES-buffered saline solu-
tion were suspended at 30-40 basophils/$ and
aliquoted (400 ~1) in plastic tubes containing 50 ~1 of
stimulus (anti-IgE antiserum or PMA) at defined con-
centrations (or buffer alone as control) and 50 ~1 of
CaCl, (2 mM final concentration), or buffer alone
without CaCl,. High Kf solutions were achieved by
isosmotically replacing NaCl with increasing concentra-
tions of KCl. After incubation in a water bath (37°C; 30
min), the tubes were centrifuged (700 X g, 20 mini and
300 ~1 of each supematant was added to 300 ~1 of 0.8
N HClO,. Total histamine content was assessed by
adding 0.8 N HClO, to an equal volume of cell suspen-
sion. After centrifugation, histamine determination was
2 .!!? 10
2.6 30 60 100 100
Fig. 1. High K+-induced histamine release from PMA-treated leuko-
cytes. Leukocyte suspension was added to saline solutions containing
defined K+ concentrations and PMA at threshold concentration (9.7
rt 3.3 ng/ml) in the presence of Ca2+ 2 mM or not. Osmolarity was
maintained with Na+. Spontaneous histamine release in the presence
of K+ alone was less than 5% and was subtracted to give net percent
of histamine release. Results are given as mean f SEM (n = 6 with
CaZf and n = 3 without Ca2+ ).
performed using an automated spectrofluorometric as-
say . At the concentrations used, none of the reagents
interfered with the histamine assay.
It is well documented that high K+ neither induces
histamine release nor modifies IgE-mediated histamine
release in usual saline solutions [2,10]. Since a strong
Fig. 2. Anti-IgE-induced histamine release in the presence of high
K+. Human leukocytes were incubated (30 min, 37T) in the presence
of anti-IgE at defined concentrations in saline solutions containing
K+ at defined concentrations and 2 mM Cazf. Osmolarity was
maintained with Na+. Results are given as mean f SEM (n = 5) of
percent of total histamine release without correction of spontaneous
synergy has been reported between the Ca2+ ionophore
A23187 and PMA on basophil histamine release [ll],
we atterrpted to demonstrate the high K+-induced Ca2+
entry (which here acts like a Ca2+ ionophore) by using
high K+ in synergy with PMA. Indeed, in the presence
of PMA at threshold concentration (9.7 f 3.2 ng/ml;
n = 61, histamine release was observed at Kf concen-
trations above 60 mM (24.9 k 6.8% at 100 mM K+ vs.
3.2 + 2.6% at 2.6 mM K+) (Fig. 1). If no Ca2’ was
added to the suspending medium, the release of his-
tamine was abolished (Fig. 1).
As shown in Fig. 2, external K+ concentrations up
to 140 mM did not affect IgE-mediated histamine re-
lease nor spontaneous basal release of histamine. This
indicates that the release due to high Kf in the presence
of PMA was not due to a lytic effect of high K+.
It seems to be a general rule that in secretory ex-
citable cells an increase of cytosolic Ca*+ is the trigger
event for secretion. In contrast, no influx of Ca*+ in
high K+ solutions has been reported in non excitable
secretory cells such as the rat basophilic leukemia mast
cell line. On the contrary, in these cells, high K+ has
been shown to inhibit Ca2+ uptake and consequently to
inhibit exocytosis .
Previous experiments strongly indicated that it was
reasonable to assume the presence of K+ channels in
human basophil membrane . We thus used high K+
external solutions as a useful way to efficiently depolar-
ize the cell membrane of human basophil. As a result,
basophils pretreated by PMA, behaved as secretory
excitable cells since they released histamine in depolar-
izing medium in a Ca*+-dependent manner (Fig. 1). As
previously reported , high K+ did not increase anti-
IgE-induced histamine release, even at threshold anti-
body concentrations (Fig. 2). Taken together, with the
external Ca*+-dependence, this indicates that high Kf
had not a trivial effect such as lysis of basophil mem-
brane with a non-specific release of histamine. How-
ever, protein kinase C (the target of PMA) has been
shown to be increased in human basophils after IgE-
mediated stimulation . Thus, one could expect a
synergy between anti-IgE and high K+. This is clearly
not the case (Fig. 2). As an hypothesis, it could be
suggested that numerous pathways for Ca2’ were most
probably already open after anti-IgE challenge, thus
high K+ could not further increase this effect.
The present results could appear contradictory with
our previous ones . Indeed, in one case, high K+ is
reported to favor histamine release and, in the other
case, to counteract histamine release. However, in the
former case , high K+-mediated depolarization de-
creases the driving force of external Ca2+ and thus
lowers the IgE-mediated secretion. This Ca2+ entry
occurs through channels opened by an IgE-mediated
mechanism. Moreover, the inhibitory effect of high K+
is evidenced only in low Na+ medium, most probably
because in normal Na+ medium the membrane be-
comes depolarized after anti-IgE challenge and high K+
cannot further depolarize it. We can suppose that, in the
latter case (the present results), high K+-mediated depo-
larization also decreases the Ca2+ driving force but,
above all, allows Ca2+ to enter the cell.
Basophils from allergic subjects have been shown to
release greater amounts of histamine and with weaker
challenges than basophils from normal subjects .
Thus, this possible depolarization-mediated Ca2+ entry
that we describe here could be an element in the
understanding of differences in signal transduction in
basophils from low and high releasers [15,16].
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