Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Currie, Kevin P. M. and
Aaron P. Fox.
Voltage-Dependent, Pertussis Toxin Insensitive Inhibition of
Calcium Currents by Histamine in Bovine Adrenal Chromaffin Cells.
J. Neurophysiol. 83: 1435-1442, 2000.
Histamine is a known secretagogue in adrenal chromaffin cells.
Activation of G-protein linked H1 receptors stimulates
phospholipase C, which generates inositol trisphosphate leading to
release of intracellular calcium stores and stimulation of calcium
influx through store operated and other channels. This calcium leads to
the release of catecholamines. In chromaffin cells, the main physiological trigger for catecholamine release is calcium influx through voltage-gated calcium channels
(ICa). Therefore, these channels are
important targets for the regulation of secretion. In particular N- and
P/Q-type ICa are subject to inhibition by transmitter/hormone receptor activation of heterotrimeric G-proteins. However, the direct effect of histamine on
ICa in chromaffin cells is unknown. This
paper reports that histamine inhibited ICa
in cultured bovine adrenal chromaffin cells and this response was blocked by the H1 antagonist mepyramine. With high levels
of calcium buffering in the patch pipette solution (10 mM EGTA),
histamine slowed the activation kinetics and inhibited the amplitude of ICa. A conditioning prepulse to +100 mV
reversed the kinetic slowing and partially relieved the inhibition.
These features are characteristic of a membrane delimited,
voltage-dependent pathway which is thought to involve direct binding of
G-protein subunits to the Ca channels. However, unlike virtually
every other example of this type of inhibition, the response to
histamine was not blocked by pretreating the cells with pertussis toxin
(PTX). The voltage-dependent, PTX insensitive inhibition produced by
histamine was modest compared with the PTX sensitive inhibition
produced by ATP (28% vs. 53%). When histamine and ATP were applied
concomitantly there was no additivity of the inhibition beyond that
produced by ATP alone (even though the agonists appear to activate
distinct G-proteins) suggesting that the inhibition produced by ATP is
maximal. When experiments were carried out under conditions of low
levels of calcium buffering in the patch pipette solution (0.1 mM
EGTA), histamine inhibited ICa in some cells
using an entirely voltage insensitive pathway. We demonstrate that
activation of PTX insensitive G-proteins (most likely Gq) by
H1 receptors inhibits ICa. This may represent a mechanism by which histamine exerts inhibitory (in
addition to previously identified stimulatory) effects on catecholamine release.
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INTRODUCTION |
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The role of histamine as a secretagogue in adrenal
chromaffin cells is well documented. Chromaffin cells express high
affinity H1 histaminergic receptors (Chang
et al. 1979) which generally couple to the pertussis toxin
(PTX) insensitive Gq type G-protein (Hill et al. 1997
).
In chromaffin cells H1 receptor activation leads
to the generation of inositol trisphosphate
(IP3), release of intracellular calcium stores,
and calcium influx through store operated and other channels
(Bunn and Boyd 1992
; Cheek et al. 1993
,
1994
; Noble et al. 1988
; Zerbes et al.
1998
). Sustained elevation of intracellular calcium
([Ca2+]i) and
catecholamine release is dependent on this influx of extracellular calcium. It has been suggested that voltage-gated calcium channels (ICa) contribute to the calcium influx
(O'Farrell and Marley 1999
) but this may be a secondary
consequence caused by depolarization of the cells. The direct effects
(if any) of histamine on ICa in
chromaffin cells have not been studied.
Physiologically, calcium influx through
ICa is the main trigger for
catecholamine release from chromaffin cells (Boarder et al.
1987). On stimulation, the preganglionic sympathetic neurons of
the splanchnic nerve release acetylcholine (ACh), which activates nicotinic ACh receptors, depolarizes the cell and thereby activates ICa (Douglas 1968
).
Regulation of ICa is an important
mechanism for the control of catecholamine secretion.
One major example of ICa regulation is
the inhibition of N-type channels via activation of heterotrimeric
G-proteins by seven transmembrane receptors. There are several distinct
pathways by which the channels are inhibited. One of these pathways,
which is only apparent under conditions of low intracellular calcium buffering and often utilizes PTX insensitive G-proteins, is thought to
involve an unidentified diffusible second messenger (for review see
Hille 1994). A different inhibitory pathway is membrane
delimited and thought to involve direct coupling of the G-protein with
the channel (for reviews see Dolphin 1995
; Hille
1994
; Ikeda and Dunlap 1999
; Zamponi and
Snutch 1998
). In addition to suppressing the peak amplitude of
the current, this type of inhibition is characterized by a slowing of
the activation kinetics of ICa. The
kinetic slowing and most of the current suppression are relieved at
very depolarized potentials (Bean 1989
; Elmslie
et al. 1990
; Penington et al. 1991
). These
features have been incorporated into models in which the channels exist
in two functional gating states, one in the absence (willing) and one
in the presence (reluctant) of inhibition (Bean 1989
;
Boland and Bean 1993
; Elmslie et al.
1990
; Golard and Siegelbaum 1993
). P/Q-type
ICa are inhibited by the same
mechanisms but to a lesser extent in chromaffin cells, neurons, and
recombinant systems (Bourinet et al. 1996
; Currie
and Fox 1997
; Mintz and Bean 1993
; Roche
and Treistman 1998
; Zhang et al. 1996
).
At present, the voltage-dependent inhibition of
ICa is thought to result from direct
binding of the G-protein subunit to the channel (De Waard
et al. 1997
; Herlitze et al. 1996
; Ikeda 1996
; Zamponi et al. 1997
). In virtually every
case this inhibition can be prevented or attenuated by pretreatment of
the cells with PTX which ADP ribosylates the
subunit of Gi/Go type
G-proteins and disrupts the coupling to neurotransmitter/hormone
receptors (Gierschik 1992
). This suggests that there is
a preferential coupling between PTX sensitive G-proteins and N-type Ca
channels, although the reasons why this should be the case are not
clear. There are a few exceptions found in the literature where
voltage-dependent inhibition is mediated by PTX insensitive G-proteins
including LHRH and ATP inhibition of
ICa in frog sympathetic ganglion
neurons (Elmslie 1992
) and VIP inhibition of
ICa in rat sympathetic ganglion neurons (Zhu and Ikeda 1994
).
In chromaffin cells ICa is inhibited
in a voltage-dependent manner by several transmitters including ATP,
opioids, and GABA (Albillos et al. 1996; Currie
and Fox 1996
; Doroshenko and Neher 1991
;
Gandia et al. 1993
; Kleppisch et al.
1992
). The inhibition produced by all of these transmitters is
blocked by pretreatment of the cells with PTX. However, we recently
reported that PGE2 also inhibits
ICa in chromaffin cells but that this
inhibition is only partially blocked by PTX (Currie and Fox
1998
). In this paper we report that histamine, which likely
couples to the PTX insensitive G-protein Gq, inhibits
ICa in chromaffin cells in a
PTX-insensitive fashion. This outlines a previously unidentified mechanism by which histamine may participate in the autocrine/paracrine regulation of catecholamine release.
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METHODS |
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Culture of cells
Chromaffin cells were prepared by digestion of bovine adrenal
glands with collagenase and purified by density gradient centrifugation as previously described (Artalejo et al. 1992a). The
cells were plated on collagen-coated glass coverslips in 35 mm tissue
culture dishes (2 ml of cell suspension; 0.15-0.3 × 106 cells/ml) and maintained in an incubator at
37°C in an atmosphere of 92.5% air and 7.5%
CO2 with a relative humidity of 90%. Fibroblasts were effectively suppressed with cytosine-arabinoside (10 µM), leaving relatively pure chromaffin cell cultures. Although mixed, the
cultures were somewhat enriched for epinephrine containing over
norepinephrine containing cells. One-half of the incubation medium was
exchanged every day. This medium consisted of DMEM/F12 (1:1, Gibco)
supplemented with fetal bovine serum (10%), glutamine (2 mM),
penicillin/streptomycin (100 unit ml
1/100 µg
ml
1), cytosine arabinoside (10 µM), and
5-fluorodeoxyuridine (10 µM).
Electrophysiology
Chromaffin cells were voltage clamped in the whole cell
configuration of the patch clamp technique (Hamill et al.
1981) using an Axopatch 1C amplifier (Axon Instruments) at a
holding potential of
80 mV and ICa
were activated by step depolarizations. Current voltage curves were
generated by voltage ramps of 100 ms duration from the holding
potential (
80 mV) to +100 mV. Peak inward calcium channel currents
were activated at approximately +20 mV. Leak currents were generated by
averaging 16 hyperpolarizing sweeps (steps or ramps). All the data
reported in this paper were capacitance and leak subtracted. The data
were filtered at 2 kHz and then digitized at 100 µs/point. Series
resistance was partially compensated (
80%) using the series
resistance compensation circuit of the Axopatch-1C amplifier.
Electrodes were pulled from microhaematocrit capillary tubes (Drummond)
and coated with sylgard (Dow Corning). After fire polishing, final
electrode resistance when filled with the CsCl based patch pipette
solution (see Solutions) was ~1.5-2.5 M
.
Voltage protocols and data analysis were carried out in AxoBasic. Data
are reported as mean ± SE and statistical significance
was determined using paired or independent Student's
t-test. All recording was performed at room temperature
(~23°C)
Solutions
Electrodes were filled with solution composed of (in mM) 110 CsCl, 4 MgCl2, 20 HEPES, 10 EGTA, 0.35 GTP, 4 ATP, and 14 creatine phosphate (pH = 7.3 adjusted by CsOH;
osmolarity ~310 mOsm). Note that in some experiments (Fig. 4) the
patch pipette solution contained 0.1 mM EGTA. The NaCl based
extracellular recording medium contained 140 mM NaCl, 2 mM KCl, 10 mM
glucose, 10 mM HEPES, 10 mM CaCl2, and 0.3-1.0 µM
tetrodotoxin (TTX) (pH = 7.3 adjusted with NaOH, osmolarity ~310
mOsm). In a few experiments the TTX was omitted. Nisoldipine was
prepared as a stock solution (10 mM) in ethanol and stored, protected
from light, at 20°C. It was added to all extracellular solutions (1 µM) to block any facilitation ICa
(L-type) present. PGE2 (Calbiochem) was prepared as a stock solution in DMSO and aliquots frozen. Final dilutions yielded DMSO concentrations of <0.03% which had no effect on the currents by itself.
-Conotoxin GVIA (Alomone Labs) was diluted in sterile
H2O and aliquots (300 µM) frozen until use. ATP
(Boehringer Mannheim), histamine, and reactive blue-2 (RBI) were
prepared as stock solutions in sterile H2O daily
and diluted to final concentrations in recording medium immediately
before use. Pertussis toxin (RBI) was prepared as a stock solution in
sterile H2O and stored at 4°C until dilution in
culture medium before use.
The recording bath was <1 cm in diameter with a volume of ~250-350 µl. The bath solution was gravity fed from reservoirs at a flow rate of 3-4 ml/min which ensured efficient perfusion of the recording chamber. Agonists and antagonists were applied to the cells by including them in the recording solution and washing them into the bath. There was a latency between switching solutions at the reservoirs and the drugs reaching the cell as a result of "dead space" in the tubing leading to the bath. This accounted for most of the delay seen between agonist application and inhibition of ICa. Cgtx (35-50 µL) was added directly to the bath, with the flow of extracellular solution stopped, at 10 times the desired final concentration. Thus Cgtx was added at 10 µM to give a final concentration of ~1 µM.
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RESULTS |
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Histamine inhibited ICa in a voltage-dependent manner
In these experiments, histamine was applied by continuous
perfusion through the recording chamber. In most cells tested (59 of
64) histamine produced an inhibition of
ICa like that shown in Fig.
1. In this experiment, about one quarter
of the whole cell ICa was inhibited by
a supramaximal dose of histamine (300 µM). Histamine (300 µM)
suppressed the peak amplitude of ICa
by 28 ± 1.5% (n = 31, see Fig. 1) and slowed the
activation kinetics. A 50 ms prepulse to +100 mV relieved 71 ± 4% (n = 9) of the inhibition of
ICa amplitude and reversed the kinetic
slowing (Fig. 1B). The slowing of activation kinetics by
histamine and the voltage-dependent reversal of its inhibition are
characteristic of direct G-protein mediated inhibition of
ICa (Bean 1989;
Elmslie et al. 1990
; Penington et al.
1991
). In some cells, prolonged application of histamine (>30
s) resulted in a desensitization of the response which was also seen
with repeated applications in some cells.
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Pharmacology of histamine response
Because prolonged application of histamine produced a desensitization of the histamine response, cells were first exposed to a brief application of histamine (100 nM-100 µM) and then exposed a second time to 300 µM histamine to determine the dose response curve for inhibition of ICa. Exposing cells to two brief histamine applications reduced possible complications from desensitization. The dose response curve (Fig. 2A) yielded an EC50 of 585 nM and a maximal inhibition of 28%.
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Activation of H1 histaminergic receptors is known
to elicit release of intracellular calcium stores and to trigger
catecholamine release in chromaffin cells (Bunn and Boyd
1992; Noble et al. 1988
). To determine if these
receptors were responsible for coupling to
ICa, histamine was applied to cells
either in the absence or presence of mepyramine (1 µM), a selective
H1 receptor antagonist (Fig. 2B). In
the absence of mepyramine 300 µM histamine produced an inhibition of
26 ± 3.5% (n = 4) and in the presence of
mepyramine the response to histamine was abolished (1 ± 0.7%,
n = 4; P < 0.001).
ATP, which is stored at high concentrations in chromaffin cell
secretory granules, is coreleased with catecholamines and causes a
negative feedback inhibition of ICa
through activation of P2Y purinergic receptors
(Currie and Fox 1996). Thus exposing cells to histamine,
a known secretagogue, likely results in the release of ATP from
surrounding cells in the recording chamber which may lead to the
inhibition of ICa. This was unlikely
because the recording chamber was continuously perfused with fresh
medium which should have washed away any ATP released from the
surrounding chromaffin cells before it could concentrate sufficiently
to inhibit ICa (see Currie and
Fox 1996
). However, to rule out involvement of ATP in the
inhibition, histamine was applied to chromaffin cells in the presence
of reactive blue-2 (RB-2), a purinergic receptor antagonist, which
completely inhibits the ATP-mediated inhibition of
ICa. Before application of RB-2,
histamine inhibited ICa by 23 ± 0.9% and in the presence of RB-2 the inhibition was not significantly different at 29 ± 3.3% (n = 5). Hence the
inhibition produced by histamine was not caused by the release of ATP
from surrounding cells in the recording bath.
Inhibition was not blocked by pertussis toxin treatment
Neurotransmitters and hormones inhibit
ICa in neurons (and other cells) using
several pathways (Hille 1994). One of these pathways is
thought to involve direct binding of the G-protein
subunits to
the channel producing a voltage-dependent inhibition and kinetic
slowing. Virtually every example of this type of voltage-dependent inhibition can be blocked by pretreating cells with pertussis toxin
(PTX) which selectively ADP-ribosylates Gi/Go type G-proteins and
prevents their coupling to and activation by neurotransmitter/hormone receptors.
To determine the type of G-proteins involved in the inhibition of
ICa produced by histamine, cells were
incubated for 18-24 h with 300 ngml1 PTX.
Control cells were from the same cultures and were recorded from on the
same days as PTX treated cells. The inhibition produced by 300 µM
histamine was not significantly different in PTX treated cells compared
with control cells (26 ± 2.7%, n = 12 and
20 ± 3%, n = 11, respectively; Fig.
3). As a positive control for the action
of PTX, the cells were also tested with ATP (100 µM) which we have
shown previously to inhibit ICa in a
PTX sensitive fashion (Currie and Fox 1996
). In control
cells ATP inhibited ICa by 50 ± 1.9% (n = 12) and in PTX treated cells by 3 ± 0.7% (n = 14; Fig. 3). Therefore the
H1 receptors use a PTX insensitive G-protein as
part of the signaling pathway that inhibits calcium channels in
chromaffin cells.
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Voltage-independent inhibition of ICa in cells recorded using low intracellular EGTA
Hille and colleagues (1994) have demonstrated the
existence of at least 5 pathways by which neurotransmitters and
hormones can modulate ICa in rat
sympathetic ganglion neurons. In addition to the fast, membrane
delimited, voltage-dependent pathway like the inhibition described so
far in this paper, there is a pathway which involves an unidentified
diffusible messenger (Beech et al. 1991
, 1992
;
Bernheim et al. 1991
); this pathway is
voltage-independent and involves PTX insensitive G-proteins. It is only
seen under conditions of low intracellular calcium buffering. All the
data presented above were recorded with high intracellular calcium buffering (10 mM EGTA) in the patch pipette solution. To determine whether this second pathway could be observed in chromaffin cells some
experiments were performed using a patch pipette solution in which the
concentration of EGTA was reduced to 0.1 mM.
Under these recording conditions histamine (300 µM) inhibited
ICa in all 11 cells tested (average
inhibition was 35 ± 6%, n = 11). Although this
was not significantly greater than the inhibition reported in cells
recorded using 10 mM EGTA, the voltage dependence of the inhibition was
significantly different. Of the 11 cells tested only 7 exhibited
voltage-dependent inhibition as illustrated in the example shown in
Fig. 4A. In the presence of
histamine the current amplitude was suppressed and the activation kinetics were slowed. A 50-ms prepulse to +100 mV relieved ~30% of
the inhibition and reversed the kinetic slowing. In these seven cells
the mean inhibition produced by histamine (300 µM) was 27 ± 5%
and a prepulse relieved 34 ± 5% of this inhibition. In the remaining four cells ICa was inhibited
by histamine in a voltage-independent fashion as illustrated in the
example shown in Fig. 4B. In this cell the current amplitude
was inhibited by ~70% but there was no slowing of activation
kinetics and a prepulse produced no relief of the inhibition. The mean
inhibition in these four cells was 49 ± 13%. The onset of this
voltage independent inhibition and its washout was rather slow (Fig.
4C), which is consistent with the involvement of a
diffusible messenger as suggested previously in sympathetic ganglion
neurons (Beech et al. 1991, 1992
; Bernheim et al.
1991
).
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Both N- and P/Q-type ICa are inhibited
The remaining experiments were all performed using 10 mM EGTA in
the patch pipette solution. As illustrated above (Fig. 3) the maximal
inhibition produced by histamine is about one-half that of ATP. The
calcium current in chromaffin cells is carried by three types of
channels. The L-type channels, also called facilitation Ca channels,
can be recruited by depolarizing prepulses similar to those used to
relieve the G-protein inhibition of the N- and P/Q-type channels
(Artalejo et al. 1992b; Fenwick et al.
1982
; Hoshi et al. 1984
) and so have been
blocked by the inclusion of nisoldipine in all recording solutions. In
the presence of nisoldipine the remaining current is approximately
one-half N-type (blocked by 1 µM
-conotoxin GVIA) and one-half P/Q
type (blocked by 400 nM
-Agatoxin IVA) (Currie and Fox 1996
,
1997
). Both the N- and P/Q type channels are inhibited by ATP
in a PTX sensitive, voltage-dependent manner. One explanation for the
difference in the magnitude of the inhibition produced by histamine
could be that one or other of the channel types was selectively
inhibited by the PTX insensitive G-proteins. Therefore we tested the
ability of histamine to inhibit ICa
before and after block of N-type channels by
-conotoxin GVIA (
-CgTx).
Consistent with our previous studies -CgTx (1-2 µM) blocked about
one-half of the whole cell current (Fig.
5A). On average
-CgTx
blocked 49 ± 3.6% (n = 7) of
ICa. After block with
-CgTx, 300 µM histamine was still able to inhibit part of the remaining P/Q-type
ICa (Fig. 5A). On average
histamine inhibited the remaining (P/Q-type) current by 26 ± 3.3% (n = 7) (Fig. 5). In the presence of histamine
activation kinetics of ICa were
slowed; a conditioning prepulse relieved 44 ± 6%
(n = 5) of the inhibition and reversed the kinetic
slowing (not shown). Thus both channel types are inhibited by
histamine.
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Additivity of the inhibition produced by histamine with that produced by ATP or PGE2
As shown in Fig. 3, ATP inhibits ICa by using a PTX sensitive G-protein whereas histamine uses a PTX insensitive G-protein. Activation of both receptor types at the same time could lead to a higher concentration of activated G-proteins in the vicinity of the channels. To see if the inhibition produced by the two pathways was additive, cells were exposed to a supramaximal dose of histamine (300 µM) followed immediately by histamine plus a supramaximal dose of ATP (100 µM). Histamine caused an inhibition of 21 ± 1.9% whereas histamine + ATP inhibited the current by 50 ± 2.4% (n = 6; P < 0.001; Fig. 6, A and C). The cells were washed and subsequently exposed to ATP alone which inhibited the current by 47 ± 2.6%. In a different group of cells the same experiment was repeated except the order of agonist application was reversed (Fig. 6, B and D). The mean inhibition produced by ATP was 53 ± 3.8% (n = 4) and by ATP + histamine was not significantly different at 54 ± 4.3% (n = 4). These results suggest the channels are maximally inhibited by ATP and are saturated by the PTX sensitive G-proteins activated by the P2Y receptors.
|
Prostaglandin E2 (PGE2)
also inhibits ICa in chromaffin cells
using both PTX sensitive and insensitive G-proteins (Currie and
Fox 1998). Cells treated for 18-24 h with 300 ngml
1 PTX were exposed to histamine (300 µM)
or histamine + PGE2 (300 nM; Fig.
7). The inhibition produced by histamine
alone was 17 ± 4.6% and by histamine + PGE2 was significantly greater at 30 ± 5.1% (n = 5; P < 0.0001). The same
was true if the order of agonist application was reversed so that
PGE2 alone produced an inhibition of 22 ± 1.9% and PGE2 + histamine was 33 ± 2.9%
(n = 6; P < 0.0001). This partial
additivity suggests that the submaximal inhibition produced by PTX
insensitive G-proteins activated either by histamine or by
PGE2 is due, at least in part, to insufficient levels of activated G-protein in the vicinity of the channel.
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DISCUSSION |
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In chromaffin cells, activation of H1
histaminergic receptors leads to the generation of
IP3, release of intracellular calcium stores, and
calcium influx through both store operated and other less well defined
channels which triggers catecholamine secretion (Bunn and Boyd
1992; Cheek et al. 1993
, 1994
; Noble et
al. 1988
; Zerbes et al. 1998
). However, as with
neurotransmission, the main physiological trigger for catecholamine
release is calcium influx through ICa
(Boarder et al. 1987
). Ca2+
channels are important sites for the regulation of catecholamine release. In this paper we demonstrate that histamine inhibits ICa in adrenal chromaffin cells. In
experiments with 10 mM EGTA in the patch pipette solution inhibition of
ICa occurred in >90% of the cells
tested and was blocked by mepyramine, indicating the involvement of
H1 receptors. Histamine not only suppressed ICa amplitude but also slowed its
activation. The kinetic slowing and most of the current suppression
could be reversed by depolarizing prepulses, features which are
characteristic of N- and P/Q-type ICa
inhibition (Dolphin 1995
; Hille 1994
;
Ikeda and Dunlap 1999
; Zamponi and Snutch
1998
). Although multiple pathways are known to cause inhibition
of ICa, the voltage-dependent pathway
is thought to result from a direct interaction between the activated
G-protein
subunit complex and the
1
subunit of the calcium channel (De Waard et al. 1997
;
Herlitze et al. 1996
; Ikeda 1996
;
Zamponi et al. 1997
).
Modulation of ICa by transmitters is a
widespread phenomenon. In adrenal chromaffin cells a variety of
transmitters inhibit ICa including
ATP, dopamine, opioids, and GABA (Bigornia et al. 1990;
Doroshenko and Neher 1991
; Gandia et al.
1993
; Kleppisch et al. 1992
). However, the
inhibition produced by histamine reported in this paper is unique in
that it is not mediated by PTX sensitive G-proteins. In virtually every
case, voltage-dependent inhibition is blocked or substantially
diminished by pretreatment of the cells with PTX. There are few
instances in the literature where the voltage-dependent inhibition has
been reported to be PTX insensitive; these include VIP inhibition in
rat sympathetic ganglion neurons (Zhu and Ikeda 1994
)
and LHRH and ATP inhibition of ICa in
frog sympathetic ganglion neurons (Elmslie 1992
). As a
positive control for the action of PTX we showed the inhibition of
ICa produced by ATP, which is known to
be PTX sensitive in chromaffin cells (Currie and Fox
1996
; Gandia et al. 1993
), was abolished in the same cells that responded to histamine. Typically
H1 receptors couple to the PTX-insensitive Gq
type G-protein (Hill et al. 1997
) and in chromaffin
cells the elevation of intracellular calcium and catecholamine release
are not blocked by PTX (Bunn and Boyd 1992
). It seems
likely that the same PTX insensitive G-protein (most likely Gq) couples
to ICa.
Because histamine can initiate secretion from chromaffin cells we were
concerned that the inhibition of ICa
could be secondary to the release of other transmitters such as ATP or
catecholamines from the chromaffin cells. In a previous study
(Currie and Fox 1996) we showed that when chromaffin
cells in the bath were stimulated (using tetraethylammonium chloride to
block K+ channels and depolarize the cells) they
released sufficient ATP to suppress
ICa. However, the flow of solution
through the recording chamber had to be stopped to allow ATP to
accumulate. If the solution through the bath was kept flowing, as in
the experiments reported in this paper, then the inhibitor never
accumulated sufficiently to suppress
ICa. Furthermore, RB2, a purinergic
receptor antagonist, blocked the "endogenous inhibition" produced
by ATP. In this study RB2 had no significant effect on the inhibition
produced by histamine. The PTX data also rule out an involvement of
locally released ATP in producing the inhibition because this
inhibition is mediated by PTX-sensitive G-proteins.
Hille and colleagues have identified a number of different pathways by
which neurotransmitters inhibit ICa in
rat sympathetic ganglion neurons (reviewed by Hille
1994). In addition to the membrane delimited, voltage-dependent
pathway that is typically sensitive to PTX there is another pathway
that typically uses PTX insensitive G-proteins. This pathway is not
voltage-dependent, is blocked by higher concentrations of intracellular
calcium buffers, and involves an unidentified diffusible second
messenger (Beech et al. 1991
, 1992
; Bernheim et
al. 1991
). Our results indicate the presence of a second
inhibitory pathway in chromaffin cells. With 0.1 mM EGTA in the patch
pipette solution, histamine inhibited ICa in all 11 cells tested but only 7 of these cells showed kinetic slowing and prepulse relief of
inhibition. In addition the proportion of the inhibition that was
relieved by a prepulse was only about one-half of that in cells
recorded with 10 mM EGTA. The other four cells were strongly inhibited
by histamine but there was no kinetic slowing evident and a prepulse
failed to relieve any of the inhibition.
Both N- and P/Q-type channels were inhibited to the same extent by
histamine. The mean inhibition of the total current (both N- and
P/Q-type) by histamine was 28%. After block of N-type
ICa with -CgTx about one-half the
current remained. Histamine inhibited this P/Q-type current by 26%.
Assuming that the voltage-dependent inhibition is produced by direct
binding of the G-protein subunit to the channel, the smaller
inhibition produced by histamine (compared with ATP) could be explained
by there being fewer channels occupied by
subunits after
H1 receptor activation than by
P2Y receptor activation. It has been shown that
some
subunits are less effective at producing inhibition than
others and that this correlated with weaker interactions with a
sequence encoding the I-II linker of the channel where the
subunits bind (Garcia et al. 1998
). Another explanation
could be that the species of
activated by the two agonists bind
to the channels equally well but elicit the inhibition with different
efficacies once bound. To begin to probe these questions, the
additivity of the inhibition produced by ATP and histamine was
investigated. Because ATP and histamine utilize different G-proteins in
their respective signaling pathways (PTX sensitive and insensitive
respectively), when both agonists are applied simultaneously one would
expect there to be a higher concentration of activated G-proteins in
the vicinity of the channels. Our results show that when ATP was
applied in the continued presence of histamine there was an increased
inhibition of ICa. However, the
inhibition produced by both agonists together was not significantly
different from that produced by ATP alone. Similarly if ATP was applied first and then histamine added, there was no further inhibition of
ICa. This suggests that the channels
are maximally inhibited and are saturated by the
subunits
released by P2Y receptor activation. It also
suggests the reason for the smaller inhibition produced by histamine is
partially caused by nonsaturating amounts of
subunits being
activated near the channels. If the
subunits activated by
histamine saturated the channels but were less effective at producing
the inhibition once bound one might expect a competitive block of the
ATP inhibition (because the
binding sites would already be
occupied). Furthermore, we show that in PTX treated cells
PGE2 inhibits
ICa by about 20%. This inhibition and
that produced by histamine were partially additive no matter what the order of agonist application. This again suggests that the amounts of
subunits generated by activation of PTX insensitive G-proteins is nonsaturating and that this accounts at least in part for the sub-maximal inhibition of ICa.
Physiologically it is not clear what role the inhibition of ICa by histamine may have. Histamine is produced by adrenal chromaffin cells and may also be produced locally by mast cells or reach the adrenal gland through the bloodstream. Inhibition of ICa will almost certainly reduce catecholamine secretion. However, it is clear that histamine also has the ability to induce secretion through elevation of [Ca2+]i by release of intracellular calcium stores and calcium influx through channels other than ICa. There may be a balance between these seemingly opposing mechanisms whereby acute histamine exposure could inhibit catecholamine release by suppressing ICa. Although our results suggest that desensitization is possible in some cells with exposures >30 s, it is still possible that prolonged exposure may augment release due to summation of the calcium influx pathways stimulated by histamine with the calcium influx through the noninhibited ICa. These complicated interactions will require further investigation, but this study identifies multiple pathways that result in the inhibition of ICa and which may modulate the functioning of adrenal chromaffin cells.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grants to A. P. Fox.
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FOOTNOTES |
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Address for reprint requests: K. Currie, Dept. of Neurobiology, Pharmacology and Physiology, The University of Chicago, 947 E. 58th St., Chicago, IL 60637.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 September 1999; accepted in final form 30 November 1999.
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REFERENCES |
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