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Voltage-dependent ion channels on human basophils: do they exist?

DOI:10.1016/0165-2478(95)00022-W
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Abstract

The presence of voltage-dependent ion channels (particularly Ca2+ 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 Ca2+ 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 Ca2+ pathways. Indeed, it is known that, in the presence of PMA at threshold concentration (1st signal), elevation of cytosolic Ca2+ (2nd signal) induces histamine release. We observed that a depolarizing external solution (high K+) induced a Ca(2+)-dependent release of histamine from PMA-treated human basophils. High K+ alone did not induce histamine release. Although the voltage-sensitive component and the physiological relevance of this mechanism remain to be defined, these results suggest that this voltage-dependent Ca2+ influx in the human basophil could contribute to the up-regulation of histamine release.
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Immunology Letters, 46 (1995) 81-83
01652478/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved
IMLET 2360
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
1. Summary
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
histamine release.
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.
2. Introduction
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
basophil activation”.
* 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.
SSDI 0165-2478(95)00022-4
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-
tamine.
3. Materials and Methods
3.1. Reagents
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 [8]. 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
81
40
E
z 30
ii
T 20
:
._
E
2 .!!? 10
I
0
2.6 30 60 100 100
1
K+ (mM)
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 [9]. At the concentrations used, none of the reagents
interfered with the histamine assay.
4. Results
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
80
$ 80
:
-?
:
40
._
E
0
ti 20
._
I
0
External K+
0 2.6mM
m 60mH
II 140mM
1
Anti-IgE (yg/ml)
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
histamine release.
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+.
5. Discussion
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 [3].
Previous experiments strongly indicated that it was
reasonable to assume the presence of K+ channels in
human basophil membrane [12]. 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 [2], 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 [13]. 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 [12]. 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 [12], high K+-mediated depolarization de-
82
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 [14].
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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83
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Article
  • Apr 1989
We have examined the changes in protein kinase C (PKC) which follow IgE-mediated activation of basophils. Exposure to 0.1 microgram/ml anti-IgE resulted in an increase in total cellular PKC (169 +/- 23% of control, histamine release (HR) = 33 +/- 7%, n = 12) which could be accounted for solely by the increase in membrane-associated PKC. These changes reached a maximum (280 +/- 48%) 1.0 min after challenge and declined to 190 +/- 38% after 5.0 min though histamine release was not complete until 5 to 10 min later. We found a good correlation between the increase in membrane-associated PKC and the eventual release of histamine (rs = 0.902). Donors whose basophils released less than 5% total histamine (n = 3, HR = 3 +/- 1%) showed a partial activation of PKC (173 +/- 18%) though much less than the remaining donors (increase in PKC = 346 +/- 59%, n = 9, HR = 43 +/- 7%). We observed no redistribution of cytosolic PKC at any time following exposure to anti-IgE. In contrast, 0.1 microgram/ml 2-O-tetradecanoyl-phorbol-13-acetate (HR = 36 +/- 3%, n = 3) promoted an increase in total cellular PKC, the loss of 31 +/- 4% of the cytosolic PKC and an 816 +/- 183% increase in membrane-associated PKC. Activation of PKC by anti-IgE was only partially dependent on extracellular calcium. In the absence of calcium, the increase in PKC was approximately 65% (n = 4) of that noted in the presence of 1mM calcium but these levels were sustained over much longer periods, failing to return to base line after 30 min. Higher than normal concentrations of calcium (5 to 10 mM) promoted rapid increases in PKC activity and accelerated the return to base line (back to prechallenge levels by 5 min). Suboptimal concentrations of anti-IgE (0.01 microgram/ml) attenuated the changes in membrane associated PKC and altered the kinetics of the response. The time required to reach maximum activity increased from 1.0 to 5.0 min with a corresponding decrease in the rate at which histamine was released. Higher concentrations of anti-IgE (1.0 microgram/ml) promoted a rapid increase in PKC (maximum increase in PKC = 501 +/- 59%, time = 0.5 min, HR = 28 +/- 2%) followed by an equally rapid return to base line levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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Receptor-Operated, but Not Voltage-Operated, Calcium Channels Are Involved in Basophil Leucocyte Activation and Histamine Release
Article
  • Feb 1989
Transmembrane calcium flux is a critical step in basophil and mast cell activation and subsequent histamine release. This calcium flux is likely to take place through specialized membrane ion channels. Two types of calcium channels have been described so far: the first type is voltage operated and the second type is receptor operated. Depolarization of cell membrane by K+-rich solutions is followed by voltage-operated channel opening in excitable cells, such as smooth muscle cells. We evaluated whether high K+ extracellular concentrations can trigger basophil activation and histamine release. We found that human basophil leucocytes, showing a normal response to activating signals, such as anti-IgE antiserum and formylmethionine peptide, release no histamine when exposed to K+-rich media, alone or in combination with the K+ carrier valinomycin. These results are consistent with there being receptor-operated, but not voltage-operated, calcium channels in the basophil leucocyte plasma membrane.
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Intracellular calcium mobilization triggered by a glutamate receptor in rat cultured hippocampal cells
Article
  • Aug 1989
1. Intracellular free calcium ([Ca2+]i) was monitored by means of Fura-2 fluorescence measurements in hippocampal cells in primary cultures from newborn rats. 2. In external media containing 200 microM-DL-2-amino-5-phosphonovalerate and 1 mM-kynurenate, but no added Ca2+, an increase in [Ca2+]i was observed in 30-40% of cells examined in response to quisqualate or L-glutamate. 3. Under such conditions, [Ca2+]i often increased gradually with a latency of a few seconds after application of the agonists. 4. Pre-treatment of the cultured cells with pertussis toxin reduced the extent of quisqualate-stimulated [Ca2+]i increase in Ca2+-free media, but the percentage of the responsive cells was not affected appreciably. 5. It is concluded that quisqualate and L-glutamate can trigger the release of Ca2+ from intracellular Ca2+ stores, most likely by activating a glutamate receptor coupled to a pertussis toxin-sensitive G-protein.
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The Role of Calcium in Stimulus-Secretion Coupling in Excitable and Non-Excitable Cells
Article
  • Oct 1988
Secretion of vesicular contents by exocytosis is a common feature of excitable (neurones, chromaffin cells, beta cells) and non-excitable cells (platelets, neutrophils, mast cells). The simplistic view that the universal mechanism controlling secretion is elevation of [Ca2+]i -whatever the source of this second messenger may be -is no longer tenable in view of recent reports demonstrating secretion at basal or even reduced [Ca2+]i. It is nevertheless clear that in excitable cells an increase in [Ca2+]i is the triggering event that induces secretion. In non-excitable cells, secretion is presumably triggered by other second messengers, although [Ca2+]i appears to act as an important modulator of the rate of secretion. Conversely, these second messenger systems may serve a regulatory function in excitable cells. Given the relative importance of [Ca2+]i in the regulation of cellular functions in excitable and non-excitable cells, it is not surprising that several mechanisms are expressed in these cells to regulate intracellular calcium concentration. The major pathway for Ca2+ in excitable cells is by voltage-activated Ca2+ channels, but release of Ca2+ from intracellular stores, via second messengers, predominates in non-excitable cells, and may also be important in excitable cells. In addition, receptor-operated channels and second messenger-gated conductances may prove to be important. All of these pathways are subject to regulation by a variety of interactive second messenger systems, which provide necessary tuning for an appropriate control of intracellular calcium level.
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