Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Currie, Kevin P. M.,
Zhong Zhou, and
Aaron P. Fox.
Evidence for Paracrine Signaling Between Macrophages and Bovine
Adrenal Chromaffin Cell Ca2+ Channels.
J. Neurophysiol. 83: 280-287, 2000.
The adrenal gland
contains resident macrophages, some of which lie adjacent to the
catecholamine producing chromaffin cells. Because macrophages release a
variety of secretory products, it is possible that paracrine signaling
between these two cell types exists. Of particular interest is the
potential paracrine modulation of voltage-gated calcium channels
(ICa), which are the main calcium influx
pathway triggering catecholamine release from chromaffin cells. We
report that prostaglandin E2 (PGE2), one of the
main signals produced by macrophages, inhibited
ICa in cultured bovine adrenal chromaffin
cells. The inhibition is rapid, robust, and voltage dependent; the
activation kinetics are slowed and inhibition is largely reversed by a
large depolarizing prepulse, suggesting that the inhibition is mediated
by a direct G-protein subunit interaction with the calcium
channels. About half of the response to PGE2 was sensitive
to pertussis toxin (PTX) incubation, suggesting both PTX-sensitive and
-insensitive G proteins were involved. We show that activation of
macrophages by endotoxin rapidly (within minutes) releases a signal
that inhibits ICa in chromaffin cells. The
inhibition is voltage dependent and partially PTX sensitive. PGE2 is not responsible for this inhibition as blocking
cyclooxygenase with ibuprofen did not prevent the production of the
inhibitory signal by the macrophages. Nor did blocking the lipoxygenase
pathway with nordihydroguaiaretic acid alter production of the
inhibitory signal. Our results suggest that macrophages may modulate
ICa and catecholamine secretion by releasing
PGE2 and other chemical signal(s).
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INTRODUCTION |
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Interactions between the immune system and the
adrenal gland are well documented. For instance, there is elevated
catecholamine release by the adrenal medulla during periods of stress
and infection due both to increased sympathetic stimulation and
peripheral mechanisms (Zhou and Jones 1993). In
addition, cytokines stimulate the hypothalamic-pituitary-adrenal axis
leading to increased glucocorticoid production by the adrenal cortex;
this results in a negative feedback immunosuppressive effect
(Buckingham et al. 1996
; Turnbull and Rivier
1995
). As opposed to these indirect neurogenic effects of the
immune system on adrenal function, it is becoming increasingly clear
that there are direct paracrine interactions within the adrenal gland
itself (Marx et al. 1998
; Nussdorfer and
Mazzocchi 1998
). Interest in this field has focused largely on
the actions of intra adrenal cytokines, especially on cortical cells.
These cytokines are produced both by adrenal cells themselves and by
macrophages. Both rat and human adrenal glands have a population of
resident macrophages distributed throughout the cortex and medulla
(Gonzalez-Hernandez et al. 1994
; Schober et al.
1998
), some of which lie adjacent to the catecholamine
producing chromaffin cells. This makes them ideally situated to
participate in paracrine signaling to the chromaffin cells and
potentially modulate catecholamine release. Such signaling has been
suggested previously as a subpopulation of the resident macrophages
contain neurotrophin-4 (NT4) and chromaffin cells express the Trk A
receptor that binds NT4 (Schober et al. 1998
). Other
studies have demonstrated that a peptide released by human monocytes
stimulated catecholamine release from cultured adrenal chromaffin cells
(Jones et al. 1993
; Roberts et al. 1996
).
Of particular interest is the question of whether macrophages release
substances that alter calcium signals in chromaffin cells, especially
by modulating voltage-gated calcium channels (ICa) that are the main calcium influx
pathway triggering secretion (Boarder et al. 1987).
Immune-system signals modulate ICa in
other types of cells; ICa is enhanced
by interleukin-1
in vascular smooth muscle cells (Wilkinson
et al. 1996
). In contrast, interleukin 1
(Plata-Salaman and ffrench-Mullen 1992
) and thromboxane
A2 agonists (Hsu et al. 1996
) both
inhibit ICa in hippocampal neurons, whereas prostaglandin E2
(PGE2) inhibits
ICa in sympathetic ganglion neurons
(Ikeda 1992
).
PGE2, one of the main metabolites released by
activated macrophages, has been shown to specifically bind to adrenal
chromaffin cells, release intracellular calcium stores, and stimulate
calcium influx through voltage-independent channels and modulate
catecholamine release (Ito et al. 1991; Karaplis
et al. 1989
; Marley et al. 1988
;
Mochizuki-Oda et al. 1991
; Yokohama et al.
1988
). Prostaglandins are produced by cyclooxygenase (COX),
which catalyzes the first two steps in their synthesis from arachidonic
acid (Vane et al. 1998
). Two different isoforms of the
enzyme have been identified: COX-1, which is constitutively active, and
COX-2, which is inducible. Typically, substantially elevated
PGE2 levels are only observed several hours after
activation of macrophages due to induction of COX-2 activity
(Lee et al. 1992
; O'Sullivan et al.
1992
; Pueringer and Hunninghake 1992
). However,
rapid production of PGE2 through the
constitutively active COX-1 pathway is possible as demonstrated by
application of exogenous arachidonic acid to cultured macrophage cell
lines (Stenson et al. 1981
).
These observations raised the possibility that macrophages could inhibit ICa in adrenal chromaffin cells and thereby modulate catecholamine release. Moreover, this signaling could occur on both a slow and more rapid time scale: the slow pathway mediated by the well-documented induction of synthetic enzymes (such as COX-2) in the macrophages during periods of immune-system activation and the rapid pathway by production of PGE2 or other arachidonic acid metabolites by the constitutively active COX-1, which may play a role under both pathophysiological conditions and normal physiological functioning of the gland. To address the latter possibility, we chose to investigate potential signaling between macrophages and chromaffin cells in two ways. First, as PGE2 is an attractive candidate for this kind of signaling, we investigated the effects of exogenously applied PGE2 on ICa in chromaffin cells. The second approach involved activating a macrophage cell line with lipopolysaccharide (endotoxin) to determine whether these cells rapidly secrete signaling molecules that could alter ICa activity. Our results demonstrate inhibition of ICa in adrenal chromaffin cells by PGE2 and an unidentified signaling molecule(s) that is released rapidly from macrophages.
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METHODS |
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Cell culture
Chromaffin cells were prepared by digestion of bovine adrenal
glands with collagenase and purified by density gradient centrifugation as previously described (Artalejo et al. 1992). The
cells were plated on collagen-coated glass coverslips in 35-mm tissue
culture dishes (2 ml of cell suspension; 0.15-0.2 × 106 cells/ml) and maintained in an incubator at
37°C in an atmosphere of 93% air and 7% CO2
with a relative humidity of 90%. Fibroblasts were suppressed
effectively 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. 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 and 100 µg/ml), cytosine arabinoside (10 µM), and 5-fluorodeoxyuridine (10 µM).
J774.1 cells, a murine macrophage cell line (Snyderman at al.
1977), were grown in tissue culture flasks or dishes. Cells were maintained at 37°C in humidified air containing 5%
CO2 in RPMI culture medium (Gibco) supplemented
with 5% fetal bovine serum and penicillin/streptomycin (100 unit/ ml
and 100 µg/ml). Cells were passaged approximately once a week by
mechanical trituration.
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. 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 compensated partially (
80%) using the series
resistance compensation circuit of the Axopatch-1C amplifier.
Electrodes were pulled from microhematocrit capillary tubes (Drummond)
and coated with silicone elastomer (Sylgard; Dow Corning). After fire
polishing, final electrode resistances when filled with the CsCl-based
patch pipette solution (see following section) were ~1.5-3.0 M
.
Voltage protocols and data analysis were carried out in AxoBasic. Data
are reported as means ± 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 (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) and osmolarity was
~310 mOsm. The NaCl-based extracellular recording medium contained
(in mM) 140 NaCl, 2 KCl, 10 glucose, 10 HEPES, and 10 CaCl2as well as 0.3-1.0 µM tetrodotoxin (TTX),
pH = 7.3 (adjusted with NaOH), and the osmolarity was ~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. Interleukin-1 and interleukin-6 (Sigma) were prepared as
stocks of 10 µg/ml and aliquots frozen until use. Ibuprofen and
nordihydroguaiaretic acid (NDGA; Sigma) were both prepared fresh by
dilution in H2O with the addition of NaOH. Final
dilution yielded no alteration on the pH of the recording or incubation
medium. Lipopolysaccharide (LPS) from Escherichia coli
(serotype 055:B5; Sigma) was dissolved in sterile
H2O (25 mg/ml) and aliquots stored at 4°C for
2-4 wk.
Application of solutions and preparation of conditioned medium
The recording bath was <1 cm in diameter with a volume
of around 250-350 µl. The bath solution was gravity fed from
reservoirs at a flow rate of 3-4 ml per minute, that 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 due to
"dead space" in the tubing leading to the bath. This accounted for
most of the delay seen between agonist application and inhibition of ICa. -conotoxin GVIA (35-50 µl)
was added directly to the bath, with the flow of extracellular solution
stopped, at 10 times the desired final concentration. Thus
-conotoxin GVIA was added at 10 µM to give a final concentration
of ~1 µM.
For experiments in which J774 cells were present in the recording chamber along with the chromaffin cells, the J774 cells were grown on tissue culture dishes until almost confluent. Cells then were removed from the dish by gentle trituration and resuspended at a density of roughly 0.2-1 × 106 in NaCl-based recording medium. Once a chromaffin cell had been voltage clamped, the flow of solution through the recording chamber was stopped, and 50-75 µl of the cell suspension was added directly to the bath. The J774 cells quickly settled and adhered to the coverslip. After several minutes the flow of solution through the bath was resumed to wash away any cells that had not stuck to the coverslip.
For experiments with conditioned media the J774 cells were grown on 10 cm diameter tissue culture dishes until confluent. Cells then were washed several times with NaCl-based recording medium and then incubated for 5-10 min with 7-8 ml of either NaCl-based recording medium (control conditioned media) or NaCl-based recording medium containing 100-250 µg/ml LPS (LPS-conditioned media). The conditioned media was collected and applied directly to chromaffin cells by washing through the bath using the gravity fed perfusion system. TTX and nisoldipine were omitted from the NaCl recording medium before conditioning and were added after the solution was removed from the J774 cells.
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RESULTS |
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PGE2 inhibits ICa in chromaffin cells in a voltage-dependent manner
PGE2, an important metabolite produced by
activated macrophages, was applied to the chromaffin cells by
continuous perfusion through the recording chamber. In virtually every
cell tested, PGE2 produced a rapid, reversible
inhibition of ICa similar to the
response illustrated in Fig. 1. Figure
1A plots peak-current amplitude as a function of time. This
cell was depolarized every 10 s to +20 mV from HP = 80 mV.
Approximately 70% of the current was inhibited when
PGE2 (300 nM) was applied. Figure 1B
shows representative currents obtained during this experiment; the
currents were inhibited and activation was slowed. In other
experiments, PGE2 was applied at concentrations
of 1 nM to 1 µM, and the resulting dose-response curve (not shown)
yielded an EC50 for the inhibition of
ICa of ~10 nM. Application of a
supramaximal dose of PGE2 (300 nM), similar to
that shown in Fig. 1, produced a mean inhibition of 53 ± 4.5%
(n = 20). Multiple applications of
PGE2 to the same cell produced repeated
inhibition of ICa, suggesting there
was little desensitization of the response; however, the washout was often slow and incomplete.
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Activation of G-protein-coupled receptors by various
transmitters/hormones can inhibit ICa
by multiple pathways (Hille 1994) one of which is
thought to involve direct binding of the G-protein
subunits to
the channel (Herlitze et al. 1996
; Ikeda
1996
; for review, see Dolphin 1998
). This type
of inhibition is voltage dependent and characterized by slowed
activation kinetics of ICa, similar to
that shown in Fig. 1B, and relief of the inhibition by a
conditioning prepulse (Bean 1989
; Elmslie et al.
1990
; Penington et al. 1991
). Figure
2A shows an experiment where a
conditioning prepulse (50-ms duration to +100 mV, applied 10 ms before
the activation of ICa) relieved
~65% of the inhibition produced by PGE2. On
average these prepulses relieved 59 ± 3% (n = 13) of the inhibition and reversed the kinetic slowing (Fig.
2B). Please note that 1 µM nisoldipine was present
throughout these experiments, so the prepulse increases in
ICa were due to relief of inhibition not recruitment of L-type channels.
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PGE2 inhibition is mediated by both PTX-sensitive and -insensitive G proteins
Most examples of voltage-dependent inhibition of
ICa are mediated by the Gi/Go family
of G proteins and can be blocked by pertussis toxin (PTX), which
disrupts the coupling of these G proteins with their receptors. We
therefore tested the PTX sensitivity of the PGE2
inhibition. Cells were preincubated for 18-24 h with 300 ng/ml PTX and
then PGE2 applied as before. In control cells (cells from the same cultures and recorded from on the same days as the
treated cells), 300 nM PGE2 inhibited
ICa by 47 ± 3.3% (n = 11), whereas in PTX-treated cells the inhibition
was 21 ± 1.5% (n = 13; P < 1 × 106; Fig.
3). The PTX-insensitive inhibition still
exhibited the voltage-dependent characteristics of slowed activation
kinetics and prepulse reversal. Our results suggest that both
PTX-sensitive and -insensitive G proteins are involved in the
PGE2 response.
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Both N- and P/Q-type ICa were inhibited by PGE2
Under our recording conditions (with L-type channels blocked by
nisoldipine), we have shown previously that
ICa consists of ~50% N-type and
~50% P/Q-type channels (Currie and Fox 1996, 1997
). Furthermore both these current components are inhibited by activation of P2Y purinergic receptors. Consistent with
these data, application of 1-2 µM
-conotoxin GVIA to selectively
block the N-type ICa reduced the
current amplitude by 49 ± 3.1% (n = 4).
PGE2 (300 nM) inhibited the remaining P/Q-type
current by 33 ± 3.7% (n = 4). Our results
suggest that both N- and P/Q-type current components were inhibited by
PGE2 and that the N-type current was inhibited to
a greater extent than the P/Q type current.
J774 macrophages rapidly release an inhibitor of ICa when stimulated with lipopolysaccharide (endotoxin)
From the preceding results it was clear that PGE2 inhibited ICa in adrenal chromaffin cells. Typically there is a delay of several hours after macrophage activation (due to induction of COX-2 activity) before there is a substantial elevation of PGE2 production and release. Macrophages also express the constitutively active form of the enzyme (COX-1) and so have the ability to rapidly produce PGE2 from arachidonic acid. To determine whether macrophages could rapidly produce PGE2 or other paracrine modulators of ICa on exposure to endotoxin (a lipopolysaccharide component of bacterial cell walls commonly used to activate macrophages), a mouse macrophage cell line (J774.1) was exposed to short applications (1-10 min) of high concentrations (100-250 µg/ml) of lipopolysaccharide (LPS) from Escherichia coli; chemical signals secreted by the macrophages were tested on chromaffin cell ICa.
Two approaches were used: addition of J774 cells directly to the recording chamber containing chromaffin cells followed by exposure to LPS and generation of conditioned medium from culture dishes of J774 cells and application of this directly to the chromaffin cells. The first approach is illustrated in Fig. 4. After establishing whole cell recording from a chromaffin cell, the flow of solution through the recording chamber was stopped and a suspension of J774 cells added to the chamber. The cells settled with minutes and flow of the bath was resumed. LPS then was applied to the cells, and the flow of solution through the bath was again stopped to facilitate the accumulation of any substances released from the macrophages. This produced a very rapid inhibition of ICa amplitude and a slowing of the activation kinetics (Fig. 4B). The inhibition was washed out rapidly once flow of solution through the bath was resumed. In 31 cells, the mean inhibition produced in the manner just described was 37 ± 1.2%. Application of LPS to the chromaffin cells in the absence of J774 cells had no effect on ICa (Fig. 5). Similarly when J774 cells were applied to the bath as described in the preceding text and the flow of solution stopped, there was no inhibition of ICa unless LPS was also present (Fig. 5). Prior incubation of the chromaffin cells with PTX reduced the inhibition produced by the same protocol to 12 ± 5% (n = 5).
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Application of conditioned medium collected from J774 cells to chromaffin cells produced similar results (Fig. 6). J774 cells were incubated for 5-10 min with either NaCl-based recording medium to produce control conditioned medium or NaCl-based recording medium containing LPS (100-250 µg/ml) to produce LPS-conditioned medium. This conditioned medium then was collected and applied directly to the chromaffin cells by continuous perfusion through the recording chamber. Control conditioned medium had little or no effect on ICa, but LPS-conditioned medium reversibly inhibited ICa (Fig. 6, A and B). The inhibition slowed the activation kinetics of ICa and was relieved by a depolarizing prepulse (Fig. 6B). Normalizing the data to the control conditioned media showed that LPS-conditioned media inhibited ICa by 18 ± 2.3% (n = 12; P < 0.001) and a prepulse to +100 mV reduced this inhibition to 3 ± 2% (n = 8; Fig. 7). These data are consistent with the idea that a chemical signal was rapidly (within seconds to minutes) released from the J774 cells and acted on a G-protein-coupled receptor to inhibit ICa in the chromaffin cells.
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Please note that the percentage of cells responding was variable from week to week for both the conditioned media experiments and the experiments in which the J774 cells were present in the recording chamber. The reasons for this are not clear but are considered in the discussion.
Rapidly released inhibitor is not PGE2
To determine if the transmitter released by the J774 cells was PGE2, the experiments shown in Figs. 6 and 7 were repeated with conditioned medium from J774 cells that had been pretreated for 1-2 h with 30-100 µM ibuprofen, which blocks activity of both COX-1 and COX-2. Control dishes of J774 cells were treated in the same manner except with the omission of ibuprofen. Ibuprofen also was present during exposure to LPS and so was present in all conditioned medium applied to the chromaffin cells. Ibuprofen itself had no direct action on ICa. The amplitude of ICa was 1,403 ± 268 pA before application and 1,359 ± 264 pA (n = 5) during application of control conditioned medium (no LPS) containing 30 µM ibuprofen.
Chromaffin cells first were exposed to LPS-conditioned medium from untreated J774 cells. After obtaining a response the cell then was washed and LPS-conditioned medium from ibuprofen-treated J774 cells was applied (Fig. 8A). There was no difference in the inhibition of ICa produced by conditioned media from control cells (17 ± 4%, n = 4) or ibuprofen-treated cells (18 ± 3%, n = 4; Fig. 8B).
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Arachidonic acid also can be metabolized by lipoxygenase enzymes to produce signaling molecules such as leukotrienes or other active metabolites. The activity of both 12-lipoxygenase and 5-lipoxygenase can be blocked by nordihydroguaiaretic acid (NDGA). To determine if a product of this pathway was responsible for the rapidly produced inhibition, experiments were performed as described in the preceding text except J774 cells were incubated for ~2 h with 30 µM NDGA. There was no difference in the mean inhibition of ICa produced by conditioned medium from control (17.5 ± 2.1%; n = 4) or NDGA-treated (19 ± 1.9%; n = 4) J774 cells (Fig. 8, C and D), suggesting that these pathways are not involved in the response.
It seemed unlikely that cytokines were responsible for the rapid
signaling observed because it takes hours rather than minutes after
activation of macrophages before there is an up-regulation of cytokine
synthesis (Lin et al. 1994; Yoo et al.
1995
). However, interleukins (including IL-1 and IL-6) have
been detected in the adrenal gland (Nussdorfer and Mazzocchi
1998
), and IL-1
is known to inhibit
ICa in hippocampal neurons
(Plata-Salaman and ffrench-Mullen 1992
). Therefore
IL-1
and IL-6 (30-100 ng/ml) were tested to determine whether they
produced an inhibition of ICa in the
chromaffin cells. Chromaffin cells were exposed to the interleukins by
continuous perfusion through the recording chamber. Neither interleukin
had an effect on ICa. In four cells
tested, the amplitude of ICa was 1,126 ± 119 pA under control conditions and 1,101 ± 101 pA
in the presence of IL-1
. Similarly, in five different cells, the control ICa amplitude was 995 ± 32 pA and in the presence of IL-6 was 984 ± 33 pA.
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DISCUSSION |
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It is known that resident macrophages within the adrenal gland lie
next to chromaffin cells. Macrophages are secretory cells that
synthesize over 100 distinct products (Nathan 1987). The aim of this study was to investigate possible paracrine signaling between macrophages and adrenal chromaffin cells. In particular, these
studies were focused on modulation of
ICa as these channels are important
targets for regulation of catecholamine release. PGE2, synthesized from arachidonic acid by the
cyclooxygenase pathway, is one of the primary products secreted by
macrophages. PGE2 is known to have actions on
adrenal chromaffin cells including elevation of intracellular calcium
and modulation of catecholamine release (Ito et al.
1991
; Karaplis et al. 1989
; Mochizuki-Oda et al. 1991
; Yokohama et al. 1988
) but its
actions on ICa were unknown. However,
PGE2 does inhibit
ICa in sympathetic ganglion neurons
(Ikeda 1992
), which are ontogenetically similar to
chromaffin cells. It therefore seemed to be an attractive candidate to
participate in paracrine inhibition of
ICa in chromaffin cells.
Our results demonstrate that PGE2 produced a
robust inhibition of both N- and P/Q-type
ICa in virtually every cell tested. In
addition to the reduction in current amplitude, the activation kinetics
of ICa were slowed. Conditioning
prepulses reversed the slowing of activation and partially relieved the
reduction in current amplitude. These features are characteristic of
G-protein-mediated inhibition of N- and P/Q-type
ICa in chromaffin cells, neurons, and
other cell types (Bean 1989; Currie and Fox
1996
; Dolphin 1995
; Elmslie et al.
1990
; Hille 1994
; Penington et al.
1991
). Most examples of this type of inhibition are mediated by
PTX-sensitive G proteins. In the chromaffin cells, it appears that
PGE2 couples to both PTX-sensitive and
-insensitive G proteins to inhibit ICa similar to results obtained in sympathetic neurons (Ikeda
1992
). It is not clear whether the same
PGE2 receptor couples to multiple G proteins or
whether there are multiple receptors activated concomitantly. There are
very few readily available, selective pharmacological agents for
prostanoid receptors so the subtype(s) involved in this response were
not characterized.
Macrophages express the constitutive (COX-1) as well as the inducible
(COX-2) form of cyclooxygenase. Substantially elevated production of
PGE2 is usually not seen until several hours
after activation of macrophages in part due to induction of COX-2
(Lee et al. 1992; O'Sullivan et al.
1992
; Pueringer and Hunninghake 1992
). However,
it is possible for PGE2 to be produced rapidly by
COX-1 as demonstrated by addition of exogenous arachidonic acid to
macrophage cell lines (Stenson et al. 1981
). This raised the possibility that macrophages could signal rapidly to chromaffin cells through the production of PGE2 (or other
arachidonic acid metabolite) in addition to slower signaling through
induction of synthetic enzymes such as COX-2 or synthesis of cytokines
and related products.
This rapid signaling pathway was investigated using a mouse macrophage cell line (J774.1) stimulated with high concentrations of lipopolysaccharide (LPS), commonly used to activate macrophages. Addition of J774 cells to a recording chamber containing chromaffin cells had no effect on ICa unless LPS also was added to the bath. With LPS and J774 cells present, and the flow of solution through the bath stopped to allow accumulation of any released chemical signals, ICa amplitude was inhibited rapidly and activation kinetics were slowed; prepulses relieved a portion of the inhibition. PTX pretreatment of chromaffin cells significantly reduced the inhibition. Thus these results suggest the existence of rapid signaling between the immune system and chromaffin cells.
To confirm these observations, conditioned medium was collected from J774 cells and applied directly to chromaffin cells. This produced a comparable inhibition of ICa. Interestingly, responses both to LPS-activated macrophages present alongside chromaffin cells and to conditioned media were much less consistent than direct application of PGE2. The proportion of cells responding to conditioned medium was low and variable from week to week. The reasons for this variability are uncertain but may include: only a subpopulation of the chromaffin cells respond to the macrophage-derived chemical mediator; variability in J774 cell density and/or properties; variability between batches of LPS; and variable amounts of the macrophage-derived mediator produced and/or potential degradation or decay of this chemical once released. Nonetheless despite the problems arising from this variability, responses from many cells were obtained allowing the inhibition to be characterized.
The rapid inhibition produced by the LPS-conditioned medium was not
mediated by PGE2 as blocking COX activity in the
J774 cells using ibuprofen did not suppress the inhibition. The
lipoxygenase pathway, which metabolizes arachidonic acid into
leukotrienes, did not appear to be involved in the rapid signaling, as
blockade of this pathway did not prevent inhibition of
ICa by the LPS-conditioned medium. It
is possible that there are multiple signaling molecules involved in the
response and at present the identity of the inhibitor(s) remains
unknown. The observation that the macrophage-derived inhibitor was
stable in LPS-conditioned medium suggests it is not nitric oxide, which
has a short half life once released from macrophages. The same is true
for the epoxyeicosatrienoic acids, which are short-lived arachidonic
acid metabolites produced by the cytochrome P450 pathway (Imig
1999). Activation of macrophages stimulates the synthesis of
cytokines so it appears unlikely that large amounts could be released
rapidly enough to account for the inhibition reported in this paper.
However, IL-1 and IL-6 have been reported in the adrenal gland
(Nussdorfer and Mazzocchi 1998
) and IL-1
inhibits
ICa in hippocampal neurons
(Plata-Salaman and ffrench-Mullen 1992
). Both IL-1
and IL-6 were applied directly to chromaffin cells but neither was
found to have an effect on ICa.
Paracrine signaling within the adrenal gland is likely to be a complex
web of interactions. Chromaffin cells are known to influence the
functioning of adrenocortical cells; these cells send signals back to
the chromaffin cells as well (Nussdorfer 1996;
Pignatelli et al. 1998
). It is also becoming apparent
that resident macrophages can participate in paracrine signaling within the adrenal gland; locally produced cytokines act on the adrenal cortex
to stimulate steroid production (Marx et al. 1998
;
Nussdorfer and Mazzocchi 1998
). Conversely,
glucocorticoids are known to suppress cytokine production and induction
of COX-2 (Buckingham et al. 1996
; Turnbull and
Rivier 1995
), which contributes to the negative feedback
immunosuppressive actions of the hypothalamic-pituitary-adrenal axis.
There are fewer reports of paracrine interactions between macrophages
and chromaffin cells, but these also may operate bidirectionally; catecholamines are known regulators of immune-system function (Coffey and Hadden 1985
; Johnson et al.
1981
) and histogranin, a recently described peptide released
from chromaffin cells, has been shown to stimulate immune cells
including macrophages (Lemaire et al. 1995
). Conversely,
stimulation of catecholamine secretion by a peptide released from
monocytes (Jones et al. 1993
; Roberts et al.
1996
) and the induction of c-fos immunoreactivity in chromaffin cells by NT4, which is contained in a subpopulation of the resident macrophage cells (Schober et al. 1998
) have been
reported. PGE2 also has been reported to augment
or directly stimulate catecholamine release possibly by stimulating
extracellular calcium influx through voltage-independent channels
(Karaplis et al. 1989
; Marley et al.
1988
; Yokohama et al. 1988
). However,
concentrations of PGE2 in the nanomolar range
suppress catecholamine release stimulated by nicotine (Karaplis
et al. 1989
). The results reported in this paper suggest that
PGE2 also may suppress catecholamine release by
reducing calcium influx through ICa.
Further study will be required to elucidate the precise balance between
these two seemingly opposing mechanism on catecholamine release.
In summary, this paper identifies a novel paracrine signaling pathway between macrophages and adrenal chromaffin cells that may regulate catecholamine release through modulation of ICa. It is possible that during periods of immune-system activation (infection), induction of COX-2 may elevate locally produced PGE2, suppress calcium influx into the chromaffin cells, and oppose the increased sympathetic stimulation to help prevent excessive elevations in circulating catecholamines. The rapid production of a paracrine inhibitor(s) of ICa also lends a further dimension to macrophage-chromaffin cell interactions. It may facilitate a more dynamic signaling pathway that potentially could play a role in the normal physiological functioning of the adrenal medulla as well as during periods of immune-system activation.
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FOOTNOTES |
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Address for reprint requests: K. Currie, The University of Chicago, Dept. of Pharmacological and Physiological Sciences, 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 13 July 1999; accepted in final form 15 September 1999.
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