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

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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.
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-
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
z 30
T 20
2 .!!? 10
2.6 30 60 100 100
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
ti 20
External K+
0 2.6mM
m 60mH
II 140mM
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-
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].
111 Penner, R. and Neher, E. (1988) J. Exp. Biol. 139, 329.
121 Hook, W.A. and Siraganian, R.P. (1981) Immunology 43, 723.
[31 Mohr, F.C. and Fewtrell, C. (19871 J. Cell. Biol. 104, 783.
[4] Warner, J.A. and MacGlashan, D.W., Jr. (19901 J. Immunol.
145, 1897.
151 Mazurek, N., Schindler, H., Schurholz, T.H. and Pecht., I.
(1986) Proc. Natl. Acad. Sci. USA 81, 6841.
[6] Penner, R., Matthews, Cl., and Neher, E. (1988) Nature 334,499.
[71 Furuya, S., Ohmori, H., Shigemoto, T. and Sugiyama, H. (1989)
J. Physiol. (Lond.) 414, 539.
[S] Beauvais, F., HiBblot, C., Burtin, C. and Benveniste, J. (19901 J.
Immunol. 141, 3881.
191 Lebel, B. (1983) Anal. Biochem. 133, 16.
[lo] Tadeschi, A., Miadonna, A., Lorini, M., Arquati, M. and Zanussi,
C. (19891 Int. Archs. Allergy Appl. Immunol. 90, 109.
[ill Schleimer, R.P., Gillespie, E., Daiuta, R. and Lichtenstein, L.M.
(1982) J. Immunol. 128, 136.
[12] Beauvais, F., Shimahara, T., Inoue’, I., Hieblot, C., Burtin, C.
and Benveniste, J. (19921 J. Immunol. 148, 149.
[13] Warner, J.A. and MacGlashan, D.W. (19891 J. Immunol. 142,
1141 Marone, G., Casolaro, V., Ayala, F., Metillo, G. and Condorelli,
M. in: Respiratory Allergy, Cinical Immunology, Vol. 2 (G.
Mellilo, P.S. Norman, G. Marone, eds.) pp. 67-77, Decker,
Philadelphia, PA.
il.51 Nguyen, K.-L., Gillis, S. and MacGlashan, D.W., Jr. (1990) J.
Allergy Clin. Immunol. 85, 1020.
1161 Beauvais, F., Hitblot, C., Burtin, C. and Benveniste, J. (19901 J.
Allergy Clin. Immunol. 90, 52.
... j ISSN: 1552-5783 duced activation of signaling pathways and apoptosis in many cell types including corneal epithelial cells. [23][24][25][26] The Kv channel type activated by UV irradiation and hypoxic stress in corneal epithelial and other cells has been determined to be the Kv3. 4 and Kv2.1 Kv channels by using electrophysiological and biochemical approaches. 27,28 These combined approaches used in corneal epithelial cells include screening K þ channels with a panel of specific anti-K þ channel antibodies, applying specific channel blockers, and analyzing whole-cell and singlechannel characteristics. ...
Full-text available
Purpose The purpose of the study is to understand how extracellular stresses, such as ultraviolet (UV) irradiation, affect corneal epithelial cells. Cell volume changes, damage to corneal epithelial integrity, and cellular responses were assessed after exposure to UVC stresses. Methods Primary human and rabbit corneal epithelial cells were exposed to UVC light in culture conditions. Ultraviolet C irradiation–induced changes in cell size and volume were measured by real-time microscopy and self-quenching of the fluorescent dye calcein, respectively. The effects of UVC irradiation on Src and focal adhesion kinase (FAK) phosphorylation and FAK-dependent integrin signaling were detected by ELISA, immunoblotting, and immunostaining. Results Ultraviolet C irradiation induced both size and volume shifts in human and rabbit corneal epithelial cells. Ultraviolet C irradiation-induced decrease of cell volume elicited activation of Src and FAK, characterized by increased phosphorylations of SrcY416, FAKY397, and FAKY925. In addition, immunostaining studies showed UVC irradiation–induced increases in phosphorylation of FAK and formation of integrin β5 clustering. Application of Kv channel blockers, including 4-aminopyridine (4-AP), α-DTX, and depressing substance-1 (BDS-1), effectively suppressed UVC irradiation–induced cell volume changes, and subsequently inhibited UVC irradiation–induced phosphorylation of Src/FAK, and formation of integrin β5 clustering, suggesting UVC irradiation–induced volume changes and Src/FAK activation. Hyperosmotic pressure–induced volume decreases were measured in comparison with effects of UVC irradiation on volume and Src/FAK activation. However, Kv channel blocker, 4-AP, had no effect on hyperosmotic pressure–induced responses. Conclusions The present study demonstrates that UVC irradiation–induced decreases in cell volume lead to Src/FAK activation due to a rapid loss of K ions through membrane Kv channels.
... Recent evidence implies that the apoptosis-induced K + channel activation plays a critical role in eliciting excessive K + efflux or intracellular K + depletion (Yu et al., 1997). Blocking K + efflux by a K + channel blocker or by increasing extracellular K + inhibited shrinkage and apoptosis in human eosinophil (Beauvais et al., 1995a) human myeloid HL-60 cells (McCarthy et al., 1994), and mouse cortical neurons (Yu et al., 1999a). The K + channel blocker 4-AP can prevent cell shrinkage of human eosinophils undergoing apoptosis induced by cytokine withdrawal, and a combination of two K + channel blockers, TEA and 4-AP, inhibited IL-1b release from lipopolysaccharide (LPS)-stimulated monocytes (Beauvais et al., 1995a,b;Walev et al., 1995). ...
One of the functional roles of the corneal epithelial layer is to protect the cornea, lens and other underlying ocular structures from damages caused by environmental insults. It is important for corneal epithelial cells to maintain this function by undergoing continuous renewal through a dynamic process of wound healing. Previous studies in corneal epithelial cells have provided substantial evidence showing that environmental insults, such as ultraviolet (UV) irradiation and other biohazards, can induce stress-related cellular responses resulting in apoptosis and thus interrupt the dynamic process of wound healing. We found that UV irradiation-induced apoptotic effects in corneal epithelial cells are started by the hyperactivation of K+ channels in the cell membrane resulting in a fast loss of intracellular K+ ions. Recent studies provide further evidence indicating that these complex responses in corneal epithelial cells are resulted from the activation of stress-related signaling pathways mediated by K+ channel activity. The effect of UV irradiation on corneal epithelial cell fate shares common signaling mechanisms involving the activation of intracellular responses that are often activated by the stimulation of various cytokines. One piece of evidence for making this distinction is that at early times UV irradiation activates a Kv3.4 channel in corneal epithelial cells to elicit activation of c-Jun N-terminal kinase cascades and p53 activation leading to cell cycle arrest and apoptosis. The hypothetic model is that UV-induced potassium channel hyperactivity as an early event initiates fast cell shrinkages due to the loss of intracellular potassium, resulting in the activation of scaffolding protein kinases and cytoskeleton reorganizations. This review article presents important control mechanisms that determine Kv channel activity-mediated cellular responses in corneal epithelial cells, involving activation of stress-induced signaling pathways, arrests of cell cycle progression and/or induction of apoptosis.
The purpose of the present study is to determine the role of K+ channel activity as an early event in UV-induced corneal epithelial cell apoptosis. Both cell-attached and nystatin-perforated patch-clamping were performed to record K+ channel activity in rabbit corneal epithelial (RCE) and primary cultured rabbit corneal epithelial (PRCE) cells exposed to UV irradiation. On exposure of corneal epithelial cells or intact corneas to UV-C irradiation or treatment of corneal epithelial cells with etoposide, cell apoptosis was determined by DNA fragmentation, ethidium bromide-acridine orange nuclear stain and TdT-mediated dUTP nick-end labeling (TUNEL). In the present study, UV-irradiation-induced corneal epithelial cell apoptosis through activation of a K+ channel in the cell membrane was an early event in response to UV irradiation. UV-C irradiation (42 microJ/cm(2)) activated robust K+ channel activity in RCE and PRCE cells at both the single-channel and whole-cell levels, when measured with the cell-attached and nystatin-perforated patch clamps, respectively. Suppression of UV-irradiation-induced K+ channel activity with the specific K+ channel blocker 4-aminopurydine (4-AP) prevented UV-irradiation-induced apoptosis in the RCE and PRCE cells, loss of the superficial layer of corneal epithelium, and apoptosis in the basal layer corneal epithelium. However, suppression of K+ channel activity did not protect RCE and PRCE cells from etoposide, a topoisomerase II inhibitor, which induced cell death by bypassing the membrane. Furthermore, application of valinomycin, a K+ ionophore, to mimic the effect of mass activation of the K+ channel in RCE and PRCE cells caused cell apoptosis. The results indicate that UV irradiation induces superactivity of K+ channels in the membrane is an early event mediating signaling transduction and resulting in corneal epithelial cell death in response to UV irradiation.
Full-text available
Na+ and K+ are the major extra- and intracellular cations, respectively. We have thus studied the role of these ions on human basophil histamine release by modifying their transmembrane gradients or by increasing membrane ion fluxes using ionophores. 1) When external Na+ (reduced to 4 mM) was replaced by the nonpermeating Na+ substitute N-methyl-D-glucamine, the release of histamine was enhanced in 2 mM Ca2+ (from 37.5 +/- 8.0% in 140 mM Na+ to 68.5 +/- 9.1% in low Na+) and became possible in the presence of low Ca2+ (at 1 microM Ca2+: from 0.6 +/- 0.7% in 140 mM Na+ to 36.2 +/- 8.0% in low Na+); moreover, in low Na+, the release of histamine became partly independent on Ca2+ influx. 2) Increasing the Na+ influx with the cation channel-forming gramicidin D inhibited the release of histamine by 33.2 +/- 13.6% (n = 6) in an external Na(+)-dependent manner. 3) Decreasing K+ efflux using K+ channel blockers (4-aminopyridine, quinine, sparteine) inhibited histamine release in a dose-response manner. 4) The K+ ionophore valinomycin, which increases K+ efflux, slightly enhanced IgE-mediated histamine release when used alone, whereas it potentiated the release of histamine from leukocytes previously treated with 4-aminopyridine by 57.0 +/- 18.6% (n = 7). 5) Decreasing K+ efflux by increasing external K+ inhibited IgE-mediated release in a similar manner as Na+ did. The inhibitory effects of Na+ and high K+ were not additive, thus suggesting that both cations inhibited the release by a common mechanism. In conclusion 1) our data evidence that histamine release from human basophils is inhibited by Na+ influx and potentiated by K+ efflux; 2) they suggest that K+ channels are present on the basophil membrane and that Na+ and K+ fluxes act on histamine release most probably via modulation of membrane potential.
Full-text available
We recently observed that external Na+ inhibited the IgE-dependent human basophil histamine release (HR) in normal subjects. In this article we report differences in the Na+ effect on basophil HR between normal subjects (n = 16) and age matched patients with allergic rhinitis (AR) (n = 18). As expected, in vitro anti-IgE-stimulated basophils from the group with AR released greater amounts of histamine than basophils from the normal group. However, removal of external Na+ (and replacement by N-methyl-D-glucamine) abolished this difference between the two groups. HR in the normal group increased to the same high level as that of the group with AR. By contrast, the release of histamine in the group with AR was not further increased by Na+ removal. Although high releasers were more frequent in the group with AR, the absence of effect after Na+ removal was not due to the high basal release level (in the presence of Na+) because no effect after Na+ removal was also observed with medium releasers. These results strongly suggest that increased basophil HR in populations with AR, and possibly in other allergic populations, is linked to a defect in the inhibitory effect of Na+.
Full-text available
The role of IgG4 antibodies in allergic disorders is suspected. Yet, their presence on human basophil membrane has not been demonstrated and the mechanism of the degranulation induced by anti-IgG4 antibodies remains unclear. As previously reported, we observed that monoclonal anti-IgG4 (10 to 100 micrograms/ml) induced histamine release in the presence of D2O from leukocytes of normal and atopic subjects. The release was accompanied by a decrease of the number of toluidine blue-positive basophils (TB+). Histamine release and TB+ decrease were also observed with lower concentrations of anti-IgG4 (1 to 100 pg/ml). Since basophil activation assessed by TB+ decrease was more sensitive than histamine release, we thus used the former method to further study the mechanisms of the anti-IgG4- vs anti-IgE-induced basophil activation. Basophil activation by anti-IgG4 at 1 to 100 pg/ml, but not by anti-IgG4 at 10 to 100 micrograms/ml or anti-IgE, required the presence of polymorphonuclear cells. Furthermore, anti-IgG4-stimulated purified eosinophils, but not neutrophils, released basophil-activating factors identified as cationic proteins from eosinophils. Thus, the human basophil can be activated by anti-IgG4 via two different mechanisms according to the antibody concentration. At high concentrations (10 to 100 micrograms/ml) basophil activation does not require the presence of polymorphonuclear cells whereas at lower concentrations (1 to 100 pg/ml) the presence of eosinophils is necessary. We propose that in the latter concentration range, basophil activation is a two-step process: 1) release by anti-IgG4 of eosinophil cationic proteins that 2) will, in turn, activate human basophils. This study lends support to the role of IgG4 and eosinophils in anaphylactic reactions.
Basophils from approximately one fifth of the population were found to be unresponsive (nonreleasers), in terms of both histamine and leukotriene release, to an IgE cross-linking stimulus, such as anti-IgE antibody. Although unresponsive to any IgE-mediated stimulation, these basophils responded to non-IgE-mediated stimuli, such as the phorbol ester, 12-o-tetradecanoyl phorbol-13 acetate, the calcium ionophore, A23187, and to formyl-methionyl-leucyl-phenylalanine peptide. These stimuli produced equal dose-response curves in both releaser (basophils able to respond with greater than 5% histamine release to anti-IgE antibody) and nonreleaser basophils. Nonreleaser basophils possessed statistically similar densities of cell-surface IgE antibody (287,000 versus 400,000 IgE molecules per basophil for releaser and nonreleaser basophils, respectively), and with 12-o-tetradecanoyl phorbol-13 acetate as a probe of anti-IgE-induced cross-linking, the IgE on nonreleaser basophils was found to be cross-linked by the polyclonal anti-IgE antibody used for these studies. Interleukin-3 (IL-3) has previously been demonstrated to enhance markedly both histamine and leukotriene release in human basophils. However, IL-3 was unable to convert nonreleasing basophils into releasing basophils, as measured by anti-IgE antibody. IL-3 equivalently enhanced formyl methionine peptide-induced release in both releaser and nonreleaser basophils, suggesting that the lack of an effect on anti-IgE-induced release was not due to a lack of IL-3 receptors. Although there are several possible interpretations of these data, these results and results of our previous studies of protein kinase C activation and cytosolic Ca++ elevations in human basophils suggest that nonreleasing basophils have a defect in early signal transduction, possibly involving the influx of Ca++.
We have compared the transmembrane signals generated in human basophils by two distinct stimuli, anti-IgE antibody and FMLP (f-met peptide). Although both stimuli resulted in the activation of protein kinase C (PKC) and an increase in intracellular free calcium, there were substantial differences between the two which suggested that distinct signal transduction mechanisms were operating. We have confirmed an earlier observation that the cross-linking of IgE led to an increase in membrane PKC activity with no apparent concomitant loss of cytosolic PKC and established that in contrast, the univalent stimulus, f-met peptide, resulted in the canonical translocation of cytosolic PKC to the membrane. Furthermore, unlike anti-IgE-stimulated basophils, there was no clear relationship between the increase in PKC activity and the subsequent release of histamine. Two PKC inhibitors, staurosporine (0.1 to 1 nM) and sphingosine (25 to 50 microM), inhibited anti-IgE induced release, yet, potentiated the release of mediators after a challenge with 1 microM f-met peptide. Both stimuli led to an increase in the intracellular Ca2+ levels that correlated well with the release of histamine, however, the anti-IgE-induced responses were typically only 50% of those required to give equivalent histamine release when f-met peptide initiated release. Pharmacologic evidence suggested that the up-regulation of PKC was required for a full IgE-mediated Ca2+ response and that PKC contributed to the elevated Ca2+ levels that persist for up to 15 min after the addition of anti-IgE. In contrast, the PKC inhibitor, staurosporine, did not affect the initial increase in Ca2+ after the addition of f-met peptide but reduced the rate at which Ca2+ was removed from the cytosol. Experiments with the phorbol ester, PMA, suggested that substantial degranulation can occur in the absence of any increase in intracellular Ca2+. The addition of 10 ng/ml PMA 10 min before the addition of f-met peptide did not affect the magnitude of the initial Ca2+ transient but increased the rate at which Ca2+ levels returned to a stable baseline. Similar pretreatment with PMA almost completely abolished the anti-IgE antibody-induced Ca2+ response. These experiments, together with other previous data, suggest that the activation of PKC is a prodegranulatory component of the IgE-mediated signal transduction pathway, yet serves principally to modulate the Ca2+ signal when f-met peptide initiates release.
Biphasic increases in the free intracellular calcium concentration, consisting of a large initial transient followed by a sustained elevation, are frequently observed in non-excitable cells following stimulation. In rat peritoneal mast cells a cAMP- and Ca-activated chloride current can interact with IP3-dependent calcium influx to provide the sustained elevation of intracellular Ca concentration following transient IP3-induced release of calcium from intracellular stores. This novel combination of second messenger systems provides a flexible means to modulate calcium-dependent processes such as exocytosis.
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)
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.
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.
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.