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The Journal of Neuroscience, May 1, 1999, 19(9):3423-3429
Regulation of Calcitonin Gene-Related Peptide Secretion by a
Serotonergic Antimigraine Drug
Paul L.
Durham and
Andrew F.
Russo
Department of Physiology and Biophysics, University of Iowa, Iowa
City, Iowa 52242
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ABSTRACT |
We have investigated the regulation of calcitonin gene-related
peptide (CGRP) release from trigeminal neurons by the serotonergic antimigraine drug sumatriptan. Serum levels of the neuropeptide CGRP
are elevated during migraine. Treatment with the drug sumatriptan returns CGRP levels to normal coincident with the alleviation of
headache. However, despite this clinical efficacy, the cellular target
and mechanism of sumatriptan action are not well understood beyond the
pharmacology of its recognition of the 5-HT1 class of
serotonin receptors. We have used cultured trigeminal neurons to
demonstrate that sumatriptan can directly repress CGRP secretion from
sensory neurons. The stimulated secretion in response to depolarization
or inflammatory agents was inhibited, but not the basal secretion rate.
Unexpectedly, sumatriptan did not lower cAMP levels, in contrast to the
classical role ascribed to the 5-HT1 receptors. Instead,
activation of 5-HT1 receptors caused a slow and remarkably
prolonged increase in intracellular calcium. The inhibition of CGRP
secretion is attenuated by the phosphatase inhibitor okadaic acid,
suggesting that sumatriptan action is mediated by calcium-recruited
phosphatases. These results suggest that 5-HT1 agonists may
block a deleterious feedback loop in migraine at the trigeminal neurons
and provide a general mechanism by which this class of drugs can
attenuate stimulated neuropeptide release.
Key words:
CGRP; serotonin receptors; trigeminal neurons; calcium; phosphatase; migraine; neuropeptide
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INTRODUCTION |
Calcitonin gene-related peptide
(CGRP) is a 37 amino acid regulatory neuropeptide derived from
alternative splicing of the calcitonin/CGRP gene (Rosenfeld et al.,
1983 ). During migraine, a painful neurological disorder afflicting 16%
of the general population (Stewart et al., 1994), activation of
trigeminal neurons leads to increased secretion of CGRP (Moskowitz,
1993). Together with substance P and neurokinin A, CGRP helps mediate
neurogenic inflammation, a condition characterized by vasodilation,
plasma protein extravasation, and mast cell degranulation (Buzzi et
al., 1995). CGRP is the most potent vasodilatory neuropeptide known (McCulloch et al., 1986) and recently has been shown to cause dural
mast cell degranulation (Ottosson and Edvinsson, 1997). CGRP is also
believed to convey nociceptive information from the vasculature to the
CNS (Van Rossum et al., 1997). On the basis of these data, CGRP is
believed to play a key role in the painful phase of migraine.
This belief has been strongly supported by the clinical efficacy of the
selective 5-HT1 receptor drug sumatriptan (Ferrari, 1998).
Sumatriptan has been shown to decrease the elevated CGRP levels in
migraine patients, coincident with relief of headache pain (Goadsby and
Edvinsson, 1993). Trigeminal nerves play an important role in the
regulation of cerebral blood flow during normal and disease states and
are the major source of sensory and CGRP innervation to the cerebral
vasculature (McCulloch et al., 1986; O'Conner and Van Der Kooy, 1988).
However, because all of the previous studies have used in
vivo model systems and 5-HT1 receptors are expressed
by both cerebral blood vessels and trigeminal nerves (Bouchelet et al.,
1996), the site of sumatriptan's action, let alone the cellular
mechanism, has remained unclear.
In this study, we have demonstrated that sumatriptan and other
5-HT1 receptor agonists can directly repress the
stimulated, but not basal, release of CGRP from cultured trigeminal
neurons. Somewhat surprisingly, we found that sumatriptan did not
mediate a decrease in intracellular cAMP levels, a function typically associated with activation of the 5-HT1 receptors (Boess
and Martin, 1994). Rather, sumatriptan treatment resulted in a slow and
remarkably prolonged increase in intracellular calcium in trigeminal
neurons. We also demonstrated that a phosphatase inhibitor effectively blocked the inhibitory effect of sumatriptan on stimulated CGRP release. These data are suggestive that sumatriptan mediates an increase in phosphatase activity via a calcium-dependent pathway. On
the basis of our results, we have elucidated a novel mechanism by which
the antimigraine drug sumatriptan may block a deleterious feedback loop
in migraine and restore CGRP to normal levels.
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MATERIALS AND METHODS |
Cell culture. Trigeminal ganglia primary cultures
were established as described previously (Durham et al., 1997).
Briefly, ganglia from <1-week-old Sprague Dawley rats were dissociated with Dispase II. The cells from three to four ganglia were plated on
glass coverslips coated with mouse Engelbreth-Holm-Swarm laminin or plastic tissue culture dishes coated with poly-D-lysine
and laminin. Cells were incubated in L15 medium, 10% fetal bovine serum (FBS), and 10 ng/ml mouse 2.5 S nerve growth factor at
37°C in 5% CO2. Penicillin and streptomycin were added
to all media. Cultures of trigeminal ganglia used for CGRP secretion
and calcium studies were subcultured for 24 hr in serum-free medium
(Durham et al., 1997). HeLa cells stably expressing the
5-HT1B receptor (HeLa1B) were kindly provided by Dr. Mark
Hamblin (Seattle Veterans Affairs Medical Center, Seattle, WA)
(Hamblin et al., 1992) and were maintained in F-12 medium supplemented
with 10% FBS. CGS 12066A monomaleate (CGS) and L-694,294 were
purchased from RBI (Natick, MA). Sumatriptan succinate was obtained
from the University of Iowa Pharmacy, methiothepin was from RBI, and
okadaic acid was from Sigma (St. Louis, MO).
Immunohistochemistry. Trigeminal ganglion cells at various
times in culture were fixed and stained as described previously for
neurofilament protein using anti-rat NF-M monoclonal antibodies (Boehringer Mannheim Biochemicals, Indianapolis, IN) and
FITC-conjugated secondary antibodies (Sigma). Expression of CGRP in
trigeminal cultures was detected using CGRP-specific polyclonal
antibodies (RBI) and Cy-3-conjugated secondary antibodies (Sigma).
Calcium measurements. Intracellular calcium levels in
cultured trigeminal neurons were measured essentially as described
previously (Durham et al., 1997). Briefly, dissociated trigeminal
ganglia grown on laminin-coated 25 mm glass coverslips were maintained in phenol- and serum-free medium 24 hr before the start of the calcium
imaging procedure. Cells were incubated in DMEM (high glucose)
containing 0.2% BSA and 1 µM fura-2 AM for 25-30 min at
37°C in 5% CO2. After the cells were washed twice with
DMEM/BSA, they were incubated in the same buffer for 30 min before
analysis. Basal calcium levels were measured for a minimum of 180 sec
before addition of 5-HT1 receptor agonists or other agents.
Statistical analyses were performed using the Student's t
test (two-tailed, unpaired samples).
CGRP and cAMP assays. For the CGRP secretion studies, cells
were incubated in HBS (22.5 mM HEPES, 135 mM
NaCl, 3.5 mM KCl, 1 mM MgCl, 2.5 mM
CaCl, 3.3 mM glucose, and 0.1% BSA, pH 7.4) (Vasko et al.,
1994), and the amount of CGRP released from trigeminal neurons into the
culture media was determined using a specific CGRP radioimmunoassay
(Peninsula Labs, Belmont, CA). As a control, the basal (unstimulated)
rate of CGRP secreted into the media in 1 hr was determined, and these
values were used to normalize for differences between dishes. Cells
were pretreated with the indicated concentrations of 5-HT1
receptor agonists, with or without the 5-HT1 antagonist
methiothepin, or appropriate vehicle for 30 min before addition of
either HBS (control), KCl, or inflammatory cocktail. The inflammatory
cocktail (Strassman et al., 1996) contained 10 µM each of
bradykinin, prostaglandin, and serotonin, and 50 µM
histamine in HBS at pH 5.5. This combination and concentration of
agents was based on previous studies that elicited physiological responses (Steen et al., 1992; Strassman et al., 1996). Although it is
difficult to know what the local concentrations of these agents would
be during neurogenic inflammation, high hydrogen ion concentrations
have been found in inflammation, pH 5.4, and the relatively high,
perhaps unphysiological, concentrations of the chemical agents have
been reported to be necessary for in vitro responses (Steen
et al., 1992).
For the cAMP measurements, trigeminal cultures were incubated with
either 2 µM forskolin (Sigma) in the presence or absence of sumatriptan (20 µM) or CGS (10 µM) for
30 min at 37°C. The HeLa1B cell line was incubated with 100 µM forskolin and 0.1 µM sumatriptan or CGS
under the same conditions. These concentrations were chosen on the
basis of previous studies with this cell line (Hamblin et al., 1992).
Intracellular cAMP was determined using a cAMP-specific
radioimmunoassay (Peninsula Labs). Each experimental condition was
repeated in at least three independent experiments, and statistical
analyses were performed using the Student's t test
(two-tailed, unpaired samples).
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RESULTS |
Regulated release of CGRP from cultured trigeminal
ganglia neurons
To determine whether sumatriptan could directly repress CGRP
secretion from trigeminal neurons, we took a reductionist approach by
establishing primary cultures of rat trigeminal ganglia enriched for
neuronal cells (estimated to be >80%). Expression of a
neuron-specific protein, 160 kDa neurofilament subunit was detected in
all cells exhibiting a neuronal morphology. A unique feature of our
culture conditions is that almost all of the neuronal cells are CGRP
positive (Fig. 1A),
although it is estimated that only 23% of the neurons in the
trigeminal ganglia express CGRP in vivo (O'Conner and Van Der Kooy, 1988). A likely reason for this bias is that only nerve growth factor, and not BDNF or NT-3, which are required for survival of
other neurons, was included in the culture media (Buchman and Davies,
1993). CGRP secretion occurred at a steady-state rate of 148 pg/hr per dish of cells (approximately two ganglia). Treatment with potassium chloride (KCl) to mimic neuronal depolarization caused
approximately a sevenfold increase in the rate of CGRP release (Fig.
1B). Treatment with capsaicin, which selectively activates sensory C-fibers via a vanilloid receptor (Caterina et al.,
1997), resulted in a similar sevenfold increase (Fig. 1B). The rate of CGRP release was also markedly
stimulated by a mixture of agents known to mediate physiological
responses of neurogenic inflammation and sensitization of nociceptive
afferents (Strassman et al., 1996) (Fig. 1B). Because
the release of CGRP during migraine is thought to result in the
production and/or release of agents that escalate and sustain the
inflammatory response, our results indicate that these agents can also
act to maintain CGRP secretion after the initial nerve activation.
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Figure 1.
Expression and regulated release of CGRP from
cultured trigeminal ganglia neurons. A, Fluorescent
micrograph of CGRP-immunoreactive trigeminal neurons 7 d after
plating on poly-D-lysine and laminin. B, The
relative amount of CGRP secreted in 1 hr from untreated control cells
(CON) or cells treated with 60 mM
potassium chloride (KCl), a cocktail of
inflammatory agents (IFC), or 10 µM
capsaicin (CAP) is shown. The mean basal rate of CGRP
release was 148 ± 5 pg/hr per dish (SE, n = 36). The secretion rate for each condition was normalized to the basal
rate for each dish. The means and the SE from at least four independent
experiments are shown. *p < 0.001 when compared
with control levels.
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Sumatriptan represses stimulated CGRP release
Having established that cultured trigeminal neurons exhibit
regulated CGRP secretion, we then asked whether sumatriptan could directly inhibit this release. We found that sumatriptan inhibited potassium-stimulated CGRP secretion in a dose-dependent manner (Fig.
2A). The secretion rate
remained relatively stable throughout 4 hr of potassium stimulation,
and a single dose of sumatriptan was able to maintain a steady
inhibition throughout this period (Fig. 2A). In
contrast, sumatriptan had no significant effect on the basal secretion
of CGRP from unstimulated trigeminal neurons (Fig.
2A). This finding is consistent with clinical reports
that sumatriptan does not lower serum CGRP levels in normal,
nonmigranuer individuals (Goadsby and Edvinsson, 1993). The
specificity of sumatriptan action via the 5-HT1 receptors
was confirmed by addition of a 5-HT1 receptor antagonist,
methiothepin. Methiothepin completely blocked the action of sumatriptan
on CGRP secretion from the cultured neurons (Fig.
2B). Methiothepin treatment alone had little or no
effect on secretion. We have shown previously that this antagonist is
able to block the elevation of intracellular calcium by the 5-HT1 agonist CGS (Durham et al., 1997), and it can also
block the increase in intracellular calcium after the sumatriptan
treatment described below (data not shown). These results demonstrate
that sumatriptan activation of trigeminal 5-HT1 receptors
is sufficient to directly inhibit CGRP release.
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Figure 2.
Effect of 5-HT1 receptor agonists on
CGRP release. A, CGRP secretion as a function of
sumatriptan concentration and treatment time. The effect of sumatriptan
was determined on unstimulated and KCl-stimulated trigeminal neurons
(cultured for 4-7 d). The amount secreted per hour was normalized to
the basal rate determined before addition of buffer, 60 mM
KCl, or sumatriptan (Suma) for 1 hr. Where indicated, 10 µM sumatriptan was added for 2 or 4 hr, and the amount
per hour was normalized to basal. The mean basal CGRP secretion rate
was 131 ± 4 pg/hr per dish. The means and the SE from at least
three independent experiments are shown. *p < 0.001 when compared with control values.
# p < 0.05 when compared with
KCl-only values. B, The 5-HT1 receptor
antagonist methiothepin blocks inhibitory effect of sumatriptan on
KCl-stimulated CGRP release. The relative rates after addition of
buffer (CON), KCl, KCl plus 10 µM
sumatriptan (SUMA) and/or 20 µM
methiothepin (SUMA+MET) are shown. The mean basal
rate was 122 ± 5 pg/hr per dish. The means and the SE from the
two independent experiments are shown for each study.
*p < 0.01 when compared with control values or
sumatriptan values. # p < 0.05 when
compared with KCl values.
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We also investigated whether sumatriptan could inhibit the release of
the vasoactive neuropeptide substance P, because it has been reported
to colocalize with CGRP in sensory neurons (Edvinsson and Goadsby,
1994) and is involved in mediating neurogenic inflammation (Buzzi et
al., 1995). In preliminary studies, we determined that sumatriptan
could also inhibit the KCl-stimulated release of substance P (data not
shown). Thus, the effectiveness of sumatriptan in reducing or
abolishing the pain associated with migraine is likely caused by its
ability to coordinately inhibit the release of vasoactive neuropeptides
from trigeminal ganglion nerves.
Because we had found that a mixture of inflammatory agents caused a
marked increase in the rate of CGRP secretion (Fig. 1), we wanted to
determine whether sumatriptan could also block this type of stimulated
CGRP release. The increase in CGRP release caused by the inflammatory
cocktail was inhibited more than twofold by pretreatment with
sumatriptan (Fig. 3). In addition, we
showed that pretreatment with two other 5-HT1 receptor
agonists, CGS and L-694,294, caused a similar inhibition of CGRP
secretion (Fig. 3). These results demonstrate that multiple
5-HT1 agonists can repress CGRP secretion by at least two
different stimuli, suggesting that these agents target a common
downstream step.
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Figure 3.
Effect of 5-HT1 agonists on stimulated
CGRP release. 5-HT1 repression of CGRP release after
stimulation by inflammatory agents. The amount of CGRP secreted per
hour was normalized to the basal rate determined for 1 hr before
addition of 60 mM KCl or 5-HT1 agonists. The
relative rates after addition of buffer (CON),
inflammatory cocktail (IFC), and IFC plus 10 µM sumatriptan (Suma), 10 µM
CGS, or 10 µM L-694,247 (L694) are
shown. The mean basal rate was 137 ± 2 pg/hr per dish. The means
and the SE from at least three independent experiments are shown.
*p < 0.001 when compared with control values.
# p < 0.05 when compared with IFC
values.
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Sumatriptan does not cause a decrease in intracellular
cAMP levels
We then characterized the signaling pathway(s) used by sumatriptan
in primary trigeminal ganglion cultures. Pharmacological studies have
demonstrated that sumatriptan has high selectivity and potency at the
5-HT1B, 1D, and 1F
receptors, all of which are expressed by trigeminal neurons (Martin,
1997). The classical 5-HT1 signaling pathway based on
studies using brain slices and non-neuronal cell lines overexpressing
5-HT1 receptors has been that these receptors inhibit
adenylate cyclase and decrease cAMP levels via pertussis
toxin-sensitive Gi/o proteins (Boess and Martin, 1994).
However, in contrast to these reports, neither sumatriptan nor CGS
inhibited forskolin-stimulated cAMP accumulation (Table
1). In addition, treatment with the
mixture of inflammatory agents that stimulated CGRP release did not
elevate cAMP levels, nor did the cAMP levels change after cotreatment
with sumatriptan (Table 1). As a positive control, we confirmed that we
would be able to detect inhibition of cAMP production in cells known to
couple 5-HT1B receptors to
Gi/Go. Sumatriptan or CGS treatment essentially blocked forskolin-induced elevation of cAMP levels in HeLa
cells stably expressing the 5-HT1B receptor (Table
2). The degree of inhibition is similar
to previously published results with this cell line (Hamblin et al.,
1992). Thus, sumatriptan could lower cAMP levels in HeLa1B cells but
did not cause a decrease in either the stimulated or unstimulated cAMP
levels in trigeminal neurons.
Sumatriptan mediates an increase in intracellular calcium
We had shown previously that activation of 5-HT1
receptors by sumatriptan and other 5-HT1 receptor agonists
caused a sustained increase in calcium in the neuronal-like CA77
thyroid C-cell line (Durham et al., 1997). With this in mind, we then
tested whether a similar pathway was activated by sumatriptan in
cultured trigeminal neurons. We found that sumatriptan caused a slow,
but markedly prolonged increase in intracellular calcium in the
neurons. There was approximately a fivefold increase in intracellular
calcium when compared with basal calcium levels (Fig.
4, Table
3). The increased calcium levels did not
reach the maximal levels until ~8 min after treatment but were
maintained for at least 30 min (longest time sampled). The calcium
reached a maximum concentration of ~600 nM on average and
as high as 1 µM in some cells. We estimate that ~40%
of the neuronal cells did not respond to sumatriptan treatment. The
viability of these neuronal cells was confirmed after the sumatriptan
treatment by the elevation of calcium levels in response to high
concentrations of KCl. The reason for this heterogeneity is not known
but may indicate that not all of the neurons are expressing sufficient
levels of 5-HT1 receptors. In contrast to the delayed
increase in calcium after sumatriptan treatment, addition of
depolarizing levels of KCl caused a very rapid and transient increase
in calcium (Fig. 4). These data demonstrate that activation of
endogenous trigeminal neuron 5-HT1 receptors is coupled to
a calcium-signaling pathway and not to a Gi/o-coupled decrease in cAMP.
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Figure 4.
Sumatriptan increases the concentration
of intracellular calcium in trigeminal neurons. A,
Intracellular calcium concentrations,
[Ca2+]i, from day 4 cultures
were measured using fura-2 and a microscopic digital imaging system.
The pseudo-color scale indicates the
[Ca2+]i. Basal levels are in a single
neuron with a neurite. B, The same cell 6 min after
addition of 10 µM sumatriptan. C, The same
cell after 12 min. D, A graphic representation of the
change in [Ca2+]i as a function of
time after sumatriptan treatment of a representative cell (same cell as
above). For comparison, a trace of a different cell treated with only
KCl (60 mM) is superimposed.
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Okadaic acid blocks inhibitory effect of sumatriptan
How does a prolonged calcium elevation inhibit secretion? One
possibility is that protein phosphorylation states are changed. It is
generally accepted that changes in calcium can alter protein phosphorylation and that phosphorylation plays an important role in
regulating neuropeptide release from sensory neurons (Greengard et al.,
1993). We have used okadaic acid, a potent inhibitor of serine
threonine protein phosphatases, especially PP1 and PP2A (Denhardt,
1996), to test the possibility that 5-HT1 agonists may be
activating a phosphatase to attenuate stimulated secretion. Okadaic
acid treatment blocked the inhibitory effect of sumatriptan on
stimulated CGRP release (Fig. 5). Okadaic
acid treatment alone increased CGRP release that was similar in
magnitude to that caused by depolarization (Fig. 5), which is in
agreement with previous studies by Vasko and colleagues (Hingtgen and
Vasko, 1994) using cultured sensory neurons from dorsal root ganglia.
Cotreatment with okadaic acid and KCl did not result in a greater
increase in CGRP release than observed with each agent alone. These
results indicate that sumatriptan acts by stimulating a serine
threonine phosphatase. Although the identity of this phosphatase is not known, it is intriguing that okadaic acid has recently been reported to
inhibit a MAP kinase phosphatase activity (Runden et al., 1998), and we
have shown previously that 5-HT1 agonists cause a long-term increase in MAP kinase phosphatase activity in the CA77 cells (Durham
and Russo, 1998).
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Figure 5.
Okadaic acid treatment blocks sumatriptan-mediated
inhibition of potassium-stimulated CGRP release. The relative amount of
CGRP secreted from trigeminal neurons stimulated with 60 mM
KCl, 600 nM (unless indicated as 300 nM)
okadaic acid (OA), or the combination of KCl and OA,
with or without cotreatment with 10 µM sumatriptan
(Suma) is shown. The mean basal rate of CGRP secretion
was 99 ± 4 pg/hr per dish. The means and SE from at least three
independent experiments are shown. *p < 0.001 when
compared with control values. # p < 0.05 when compared with KCl values.
+ p < 0.05 when compared with KCl
plus sumatriptan values.
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DISCUSSION |
Our results support a model in which the trigeminal ganglion
nerves are activated during migraine and release CGRP to cause vasodilation and mast cell degranulation leading to the release of
inflammatory agents (Fig. 6). On the
basis of our data using CA77 cells (Durham and Russo, 1998), these
agents may stimulate MAP kinase pathways leading to an increase in CGRP
synthesis and secretion that could potentially maintain elevated CGRP
levels for the long duration (up to 72 hr) of a migraine. Activation of
this pathway ultimately leads to sensitization of the trigeminal neurons and nociceptive transmission to the CNS contributing to the
pain, nausea, and photophobia associated with migraine (Buzzi et al.,
1995). It is likely that sumatriptan is able to block this pathway via
activation of the 5-HT1 receptors leading to a prolonged
elevation in calcium that mediates the recruitment of phosphatases. The
concentration of sumatriptan required for inhibition in
vitro is higher than the estimated plasma concentration in
patients (~0.2 µM) (Fowler et al., 1991). Possible
explanations are that the effective receptor number may be low because
of the culture conditions and/or lack of colocalization of receptors and secretory machinery at nerve terminals. Alternatively, higher concentrations may be required to counteract chronic stimulation of the
cultures. In either case, the ability to block stimulated CGRP
secretion in the absence of vascular contributions strongly supports
the neurogenic model of migraine.
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Figure 6.
Model of 5-HT1 receptor-mediated
inhibition of CGRP release from trigeminal neurons. A depolarizing
stimulus causes the initial release of CGRP from trigeminal nerves,
leading to neurogenic inflammation, which then further stimulates the
release of CGRP. Activation of 5-HT1 receptors blocks this
cycle by inhibiting CGRP release via an increase in phosphatase
activity that is likely mediated by a sustained elevated level of
intracellular calcium.
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In the process of demonstrating this point, we have uncovered several
unexpected findings. First, activation of endogenous trigeminal
ganglion neuron 5-HT1 receptors did not decrease cAMP levels, which contradicts the commonly held belief (Boess and Martin,
1994). These observations are consistent with our findings that the
5-HT1 receptor agonist CGS also did not decrease cAMP levels in a model neuronal cell line and that CGS actions were pertussis toxin independent (Durham et al., 1997). The simplest explanation is that the cellular context is critical for assigning second messenger pathways to receptors. In support of this conclusion, others have reported that terminal 5-HT1 autoreceptors in
hippocampal neurons may not be coupled to Gi/o proteins
(Blier, 1991).
The second and perhaps most intriguing finding is that the inhibition
of neuropeptide secretion by 5-HT1 receptor activation is
paradoxically coupled to an unusually prolonged intracellular calcium
signal. At face value, this observation is paradoxical because
increased calcium is well known to be a signal to increase secretion
(Matthews, 1996). Indeed, this dogma held true for the potassium
treatment, which caused a more typical transient increase in calcium,
with increased CGRP release from cultured trigeminal ganglia neurons.
There is precedence in parathyroid endocrine cells for coupling of
elevated intracellular calcium with inhibition of peptide secretion
(Shoback et al., 1984). Our data demonstrate that activation of
endogenous trigeminal neuron 5-HT1 receptors is coupled to
a calcium-dependent signaling pathway that differs from
depolarization-induced changes in calcium. This raises the possibility
that the amplitude and duration of increased calcium can differentially
regulate neuropeptide secretion from sensory neurons, analogous to
recent evidence that the amplitude, duration, and localization of
increased calcium can differentially activate transcription factors
(Dolmetsch et al., 1997).
Our working model is that there is a balance between kinase and
phosphatase activity that controls CGRP secretion (Fig. 6). Support for
this type of mechanism is provided by our data showing that the protein
phosphatase inhibitor okadaic acid blocks the inhibitory effect of
sumatriptan on stimulated CGRP release. Under basal conditions the
phosphorylation state is at an intermediate level. Depolarization,
inflammatory agents, and okadaic acid change the balance, leading to
increased secretion. In agreement with this model, okadaic acid
treatment alone stimulated neuropeptide release from cultured
trigeminal (this study) and dorsal root ganglia neurons (Vasko et al.,
1994). Sumatriptan is able to blunt the increased secretion in response
to depolarization and inflammatory agents, but not okadaic acid,
suggesting that specific phosphatases are recruited by
5-HT1 receptor activation. The possibility of coordinated
regulation by phosphatases is suggested by our previous studies showing
that CGRP promoter activity was repressed in CA77 cells by a
calcium-dependent increase in MAP kinase phosphatase-1 activity via
5-HT1 receptor activation (Durham and Russo, 1998). A
remaining question is how sumatriptan selectively inhibits stimulated but not basal release of CGRP. To our knowledge, this is the first report of a drug that selectively targets only one of these events. One
possible explanation would be if sumatriptan causes dephosphorylation of proteins responsible for the assembly, fusion, and/or recycling of
vesicles in response to depolarization or inflammatory agents.
In conclusion, our results have demonstrated that activation of the
5-HT1 receptor class of antimigraine drugs is able to directly block CGRP release from trigeminal nerves. The inhibitory effect of sumatriptan occurs via a paradoxical elevation in calcium and
activation of an okadaic acid-sensitive phosphatase. During migraine,
CGRP helps mediate neurogenic inflammation that may result in the
release of inflammatory agents. These agents could in turn feed back to
sensitize the trigeminal ganglia neurons to sustain an elevated rate of
CGRP release (Fig. 6). On the basis of our data, the effectiveness of
sumatriptan is attributable in part to its ability to break this
deleterious feedback loop at trigeminal ganglia nerve terminals by
inhibiting CGRP secretion.
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FOOTNOTES |
Received Nov. 23, 1998; revised Feb. 1, 1999; accepted Feb. 12, 1999.
This work was supported by grants from National Institutes of Health
(HD25969, NS37386, HL14388) and the American Heart Association (96013860) to A.R., with tissue culture support provided by the Diabetes and Endocrinology Center (DK25295) and an Iowa Cardiovascular Interdisciplinary Research Fellowship (HL07121) to P.D. We thank members of the lab and K. Campbell for comments on this manuscript and
discussions and M. Hamblin for generously providing reagents.
Correspondence should be addressed to Dr. Andrew F. Russo, Department
of Physiology and Biophysics, University of Iowa College of Medicine,
Iowa City, IA 52242.
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TOP
ABSTRACT
INTRODUCTION
