(Received for publication, October 30, 1995; and in revised form, January 3, 1996)
From the
In contrast to excitable tissues where calcium channels are well
characterized, the nature of the B lymphocyte calcium channel is
unresolved. Here, we demonstrate by single cell analysis of freshly
isolated rat B cells that the anti-immunoglobulin (Ig)-induced calcium
influx takes place through a channel which shares pharmacologic and
serologic properties with the L-type calcium channel found in excitable
tissues. It is sensitive to the dihydropyridines nicardipine and Bay K
8644, to calciseptine, and to an anti-peptide antibody raised against
the subunit of the L-type calcium channel, but is
voltage-insensitive. Anti-
and anti-
antibodies stain B but not T lymphocytes. Application of a cGMP
agonist, measurement of cGMP levels in anti-Ig-stimulated B cells, and
examining the effect of a guanylyl cyclase inhibitor on the anti-Ig
response show that cGMP mediates the influx. This possibly involves a
cGMP-dependent protein kinase. The anti-Ig-induced response is not
abolished by prior treatment of B cells with a high dose of
thapsigargin. These findings undermine the widely held belief of a
categorical divide between excitable and non-excitable tissue calcium
channels, demonstrate the limitations of the capacitative calcium
influx theory, and point to a distinction between the calcium response
mechanisms utilized by B and T lymphocytes.
Cross-linking of the B cell antigen receptor can lead to the generation of intracellular signals. The earliest detected biochemical event is the tyrosine phosphorylation of intracellular proteins(1) . This is due to the activation of a number of cytoplasmic tyrosine kinases(2) . The activation of Ras, phosphatidylinositol 3-kinase, and phosphoinositide signaling pathways are among the most significant consequences of tyrosine phosphorylation(3) , with the latter leading to an increase in cytosolic calcium levels(4) . Calcium is the most common signal transduction element in cells. It affects the activity of various enzymes and helps to regulate universal processes such as cell growth and development(5) . In order to sustain a high level of intracellular calcium during activation, calcium conductance through the B cell plasma membrane is transiently increased(6) . However, unlike the case in excitable tissues, where fine structural and functional details of numerous channels have been unraveled(7) , the mechanism of calcium influx in B cells has not been identified. Bearing in mind the conservation of calcium channel structure in excitable tissues, we hypothesized the potential presence of homologous structures in B lymphocytes. Single cell fluorescence imaging techniques, flow-cytometry, and radioimmunoassays were used to explore this possibility. Here, we present our findings about the nature of the B cell calcium channel and its mode of regulation.
Figure 1:
The B
lymphocyte calcium response induced by the addition of (a)
anti-IgD (45) (10 µg/ml) (n = 46 cells) or (b) anti-IgM (10 µg/ml) (ICN) (n = 57
cells); (c) the effect of low calcium (0 Ca,
3 mM EGTA) (n = 51 cells); (d)
dihydropyridine antagonist nicardipine (Sigma) (10 µM) (n = 125 cells); (e) calciseptine (Latoxan)
(10 µM) (n = 154 cells); and (f)
the agonist Bay K 8644 (Calbiochem) (10 µM) (n = 173 cells) on the anti-Ig-induced calcium response. The
antagonists were added at t =
0.
Figure 2:
Two-color
cell surface staining of rat lymphocytes with (a)
anti- or (b) anti-
antibody, and the anti-
light chain monoclonal
antibody(46) , and (c) the effect of the preimmune
serum (n = 125 cells) and anti-
antibody (n = 127 cells) on the anti-Ig-induced calcium
response.
Figure 3: The B lymphocyte calcium response after (a) addition of NO (n = 90 cells) or (b) stimulation with 8-bromo-cGMP (Sigma) (10 µM) and the effect of (c) low calcium and (d) nicardipine (10 µM) on the latter (due to variation in the time of onset of the response induced by 8-bromo-cGMP in different cells, only representative single traces are displayed); (e) the increase in B lymphocyte cGMP levels induced by anti-IgD (10 µg/ml) treatment (n = 4 samples from two separate experiments) (results expressed as mean ± S.E.; background cGMP level = 0.50 pmol/mg protein); (f) the effect of the guanylyl cyclase inhibitor LY83583 (Calbiochem) (10 µM) (n = 84 cells); and (g) the cGMP-dependent protein kinase inhibitor Rp-8-pCPT-cGMPS (100 µM, supplied by Biolog) (n = 125 cells) on the anti-IgD (10 µg/ml) induced response.
Two separate approaches were used to examine the possible mediation of the anti-Ig-induced response by cGMP. First, we investigated the effect of anti-Ig stimulation on the B cell cGMP levels. B lymphocytes were stimulated with anti-IgD (10 µg/ml) for 2 or 5 min. cGMP levels were then estimated by radioimmunoassay and compared to those of unstimulated samples. The stimulation led to an approximate 2.5-fold increase in B lymphocyte cGMP levels (Fig. 3e). In another approach, the effect of manipulating guanylyl cyclase on the anti-Ig-induced response was examined. B lymphocytes were stimulated with anti-IgD (10 µg/ml) in the presence of LY83583 (10 µM)(27) . Treatment of B cells with this guanylyl cyclase inhibitor nearly abolished the anti-Ig-induced calcium influx (Fig. 3f). cGMP is therefore implicated in the anti-Ig-induced calcium response.
To define the target of cGMP in
this pathway, we examined the effect of a nonspecific inhibitor of
cGMP-dependent protein kinase I on the calcium
response(28) . The presence of Rp-8-pCPT-cGMPS (100
µM) led to a delayed and diminished calcium response to
anti-Ig stimulation (Fig. 3g), indicating the possible
involvement of a cGMP-dependent protein kinase in the B cell calcium
influx.
The endogenous production of NO in human B cell lines has
been documented(29) . However, neither the addition of NO
synthase inhibitors N-methyl L-arginine (30) or N
-nitro-L-arginine-methyl
ester (31) to B cells a few minutes prior to anti-Ig
stimulation nor the overnight incubation of the B cells with these
inhibitors (500 µM) had any effect on the anti-Ig-induced
calcium response (data not shown). This would suggest that endogenous
NO is not involved in the coupling of surface immunoglobulin to
guanylyl cyclase.
To investigate the possible role of this mechanism in the B
lymphocyte calcium response, the cells were treated with thapsigargin.
As shown in Fig. 4a, treatment of B lymphocytes with
thapsigargin (1 µM) leads to a substantial increase in
cytosolic calcium levels. The profile of the response is different from
that induced by anti-Ig stimulation, with calcium levels maintained at
a raised plateau throughout the period of experimentation. The elevated
plateau reflects an increased calcium influx through the cell membrane.
We studied the effect of LY83583 on this response. B lymphocytes were
stimulated with thapsigargin (1 µM) in the presence of
LY83583 (10 µM). Under such circumstances, the
thapsigargin-induced response remained unaffected (Fig. 4b). This indicates the lack of cGMP involvement
in the thapsigargin-induced calcium response. Pretreatment of most cell
types with high concentrations of thapsigargin abolishes Ca release by their receptor agonists(33) . B cells were
stimulated with anti-IgD (10 µg/ml) after treatment with 10
µM of thapsigargin. In spite of the high concentration of
thapsigargin used in the experiment, the addition of anti-IgD could
still induce a significant additional increase in the B lymphocyte
calcium levels (Fig. 4c).
Figure 4: B cell calcium response to (a) thapsigargin (1 µM, supplied by Calbiochem) (n = 84 cells), (b) the effect of Ly83583 (10 µM) on the thapsigargin response (n = 105 cells), and (c) anti-IgD (10 µg/ml) induced calcium response after treatment with thapsigargin (10 µM) (n = 64 cells).
The sensitivity of the anti-Ig response to dihydropyridines
and the anti- antibody and the serological
evidence for the existence of
and
-like subunits in B cells indicate the existence of
functional and structural homology between the B lymphocyte channel and
the L-type calcium channel found in excitable tissues. The results
obtained with the calcium channel blockers can be used to argue the
existence of a structure similar to L-type calcium channels as the B
cell calcium channel.
We sought to verify this by an independent
approach. The results obtained with the anti-channel antibodies
adequately serve this purpose. Of these, the data obtained with the
anti- antibody is of paramount
significance. This antibody is targeted against a sequence residing
between the S
and S
segments of the
subunit domain IV, an area thought to form part of the
transmembrane pore and the dihydropyridine binding site. While the
staining of B cells with the antibody signifies the serologic
similarity of the B cell structure with the L-type calcium channel, its
ability to block the anti-Ig-induced calcium response indicates its
functional similarity to that structure.
However, the calcium
response in B cells is not voltage-sensitive. As already mentioned,
treatment of these cells with KCl (50 mM) or gramicidin A (1
µM) gave no calcium response. Moreover, surface staining
of excitable cells with anti-, or
reduction of their calcium conductance by this antibody, is dependent
on prior depolarization with KCl, suggesting that in these cells the
antibody's target epitope is only exposed following a
conformational change in the channel as a result of
depolarization(34, 35) . We have found this to be
unnecessary for B cells. The B cell calcium channel could therefore be
a dihydropyridine-sensitive complex devoid of a membrane voltage
sensor. Two models in the literature serve as precedents to this
hypothesis (36, 37) . (i) The Drosophila
melanogaster trp gene product has a sequence similarity to the
subunit of L-type calcium channels but lacks the
charged residues in the S
segment(36) ; (ii) a
murine erythroleukemia cell line expresses a truncated form of the
subunit, in which the first four transmembrane
segments are absent(37) . This would enable it to interact with
dihydropyridines without having a voltage-dependent gating pattern.
Our results demonstrate a role for cGMP in B cell calcium influx. In theory, this might be by (i) direct cGMP gating of the channel or (ii) its regulation through a cGMP-dependent protein kinase.
Cyclic
nucleotide gated channels are structurally related to voltage-gated
cation channels(38, 39) . The S-S
linkers of the two groups show significant resemblance. Bearing
in mind that the anti-
antibody is raised
against this region, the B cell channel can be potentially seen as a
cyclic nucleotide gated entity. However, direct channel gating by cGMP
in rod photoreceptors is apparently carried out by comparatively low
affinity interactions(40) . If we were to extrapolate this
condition to B lymphocytes, the low cGMP levels found in these cells
(in comparison to rod photoreceptors) would make a direct gating
mechanism implausible.
A better case can be made for the involvement
of a cGMP-dependent protein kinase. The ability of Rp-8-pCPT-cGMPS to
diminish the calcium response favors the role of a kinase as the
immediate target of cGMP. The slow escape from inhibition with this
compound may be due to the dissociation of the inhibitor and recovery
of the cGMP-dependent protein kinase. The 8-substituted analogues of
cGMP (such as 8-bromo-cGMP) are more potent activators of cGMP kinase
I than cGMP itself(41) . This normally accounts for the
biological activity of these compounds in intact cells. In fact, a
reduction in cGMP-dependent protein kinase expression would make cells
less responsive to 8-bromo-cGMP, while the restoration of its
expression would restore the 8-bromo-cGMP response(42) .
Therefore, the B cell calcium response to 8-bromo-cGMP can be seen as
another indicator of cGMP-dependent protein kinase involvement. The
8-bromo-cGMP result also argues against the involvement of cGMP
regulated phosphodiesterases since it cannot interact with the
allosteric binding site of these molecules(41) .
Prior treatment of B cells with a high dose of thapsigargin did not prevent an additional increase in intracellular calcium upon anti-Ig stimulation, nor did the guanylyl cyclase inhibitor LY83583 affect the thapsigargin-induced calcium response. This indicates that the anti-Ig-induced increase in cytosolic calcium cannot be explained by the depletion of calcium from a thapsigargin-sensitive pool. This is in contrast to the reports on T cells where engagement of the T cell receptor after maximal thapsigargin treatment fails to evoke a further calcium response(43, 44) . One can suggest that T and B cell receptor-mediated increases in calcium are mechanistically dissimilar, a notion that concurs with the B cell specificity of anti-channel staining among lymphocytes.
Our findings introduce a novel calcium influx channel in non-excitable cells, provide ample evidence for the role of cGMP as a second messenger in B cell calcium influx, indicate a distinction between B and T cell calcium channels, and demonstrate the limitations of the capacitative calcium theory in explaining calcium influx in non-excitable tissues.