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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 We have investigated the regulation of calcitonin gene-related tion rate. Unexpectedly, sumatriptan did not lower cAMP levels, peptide (CGRP) release from trigeminal neurons by the seroto- in contrast to the classical role ascribed to the 5-HT receptors.
nergic antimigraine drug sumatriptan. Serum levels of the neu- Instead, activation of 5-HT receptors caused a slow and re- ropeptide CGRP are elevated during migraine. Treatment with markably prolonged increase in intracellular calcium. The inhi- the drug sumatriptan returns CGRP levels to normal coincident bition of CGRP secretion is attenuated by the phosphatase with the alleviation of headache. However, despite this clinical inhibitor okadaic acid, suggesting that sumatriptan action is efficacy, the cellular target and mechanism of sumatriptan ac- mediated by calcium-recruited phosphatases. These results tion are not well understood beyond the pharmacology of its suggest that 5-HT agonists may block a deleterious feedback recognition of the 5-HT class of serotonin receptors. We have loop in migraine at the trigeminal neurons and provide a general mechanism by which this class of drugs can attenuate stimu- sumatriptan can directly repress CGRP secretion from sensory lated neuropeptide release.
neurons. The stimulated secretion in response to depolarization Key words: CGRP; serotonin receptors; trigeminal neurons; or inflammatory agents was inhibited, but not the basal secre- calcium; phosphatase; migraine; neuropeptide Calcitonin gene-related peptide (CGRP) is a 37 amino acid of the previous studies have used in vivo model systems and 5-HT1 regulatory neuropeptide derived from alternative splicing of the receptors are expressed by both cerebral blood vessels and tri- calcitonin/CGRP gene (Rosenfeld et al., 1983). During migraine, geminal nerves (Bouchelet et al., 1996), the site of sumatriptan's a painful neurological disorder afflicting 16% of the general action, let alone the cellular mechanism, has remained unclear.
population (Stewart et al., 1994), activation of trigeminal neurons In this study, we have demonstrated that sumatriptan and other leads to increased secretion of CGRP (Moskowitz, 1993). To- 5-HT1 receptor agonists can directly repress the stimulated, but gether with substance P and neurokinin A, CGRP helps mediate not basal, release of CGRP from cultured trigeminal neurons.
neurogenic inflammation, a condition characterized by vasodila- Somewhat surprisingly, we found that sumatriptan did not medi- tion, plasma protein extravasation, and mast cell degranulation ate a decrease in intracellular cAMP levels, a function typically (Buzzi et al., 1995). CGRP is the most potent vasodilatory neu- associated with activation of the 5-HT1 receptors (Boess and ropeptide known (McCulloch et al., 1986) and recently has been Martin, 1994). Rather, sumatriptan treatment resulted in a slow shown to cause dural mast cell degranulation (Ottosson and and remarkably prolonged increase in intracellular calcium in Edvinsson, 1997). CGRP is also believed to convey nociceptive trigeminal neurons. We also demonstrated that a phosphatase information from the vasculature to the CNS (Van Rossum et al., inhibitor effectively blocked the inhibitory effect of sumatriptan 1997). On the basis of these data, CGRP is believed to play a key on stimulated CGRP release. These data are suggestive that role in the painful phase of migraine.
sumatriptan mediates an increase in phosphatase activity via a This belief has been strongly supported by the clinical efficacy calcium-dependent pathway. On the basis of our results, we have of the selective 5-HT1 receptor drug sumatriptan (Ferrari, 1998).
elucidated a novel mechanism by which the antimigraine drug Sumatriptan has been shown to decrease the elevated CGRP sumatriptan may block a deleterious feedback loop in migraine levels in migraine patients, coincident with relief of headache and restore CGRP to normal levels.
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 MATERIALS AND METHODS
CGRP innervation to the cerebral vasculature (McCulloch et al., Cell culture. Trigeminal ganglia primary cultures were established as 1986; O'Conner and Van Der Kooy, 1988). However, because all 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 Received Nov. 23, 1998; revised Feb. 1, 1999; accepted Feb. 12, 1999.
mouse Engelbreth-Holm-Swarm laminin or plastic tissue culture dishes This work was supported by grants from National Institutes of Health (HD25969, coated with poly-D-lysine and laminin. Cells were incubated in L15 NS37386, HL14388) and the American Heart Association (96013860) to A.R., with medium, 10% fetal bovine serum (FBS), and 10 ng/ml mouse 2.5 S nerve tissue culture support provided by the Diabetes and Endocrinology Center growth factor at 37°C in 5% CO2. Penicillin and streptomycin were (DK25295) and an Iowa Cardiovascular Interdisciplinary Research Fellowship added to all media. Cultures of trigeminal ganglia used for CGRP (HL07121) to P.D. We thank members of the lab and K. Campbell for comments on secretion and calcium studies were subcultured for 24 hr in serum-free this manuscript and discussions and M. Hamblin for generously providing reagents.
Correspondence should be addressed to Dr. Andrew F. Russo, Department of medium (Durham et al., 1997). HeLa cells stably expressing the 5-HT1B Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA receptor (HeLa1B) were kindly provided by Dr. Mark Hamblin (Seattle Veterans Affairs Medical Center, Seattle, WA) (Hamblin et al., 1992) Copyright 1999 Society for Neuroscience 0270-6474/99/193423-07$05.00/0 and were maintained in F-12 medium supplemented with 10% FBS. CGS

3424 J. Neurosci., May 1, 1999, 19(9):3423–3429
Durham and Russo • Serotonergic Repression of CGRP Secretion 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 Mann- heim 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 trigem- inal 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 mM 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 radioim- munoassay (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 me- thiothepin, 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 mM each of bradykinin, prostaglan- din, and serotonin, and 50 mM 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., Figure 1. Expression and regulated release of CGRP from cultured 1996). Although it is difficult to know what the local concentrations of trigeminal ganglia neurons. A, Fluorescent micrograph of CGRP- these agents would be during neurogenic inflammation, high hydrogen immunoreactive trigeminal neurons 7 d after plating on poly- ion concentrations have been found in inflammation, pH 5.4, and the laminin. B, The relative amount of CGRP secreted in 1 hr from untreated relatively high, perhaps unphysiological, concentrations of the chemical control cells (CON ) or cells treated with 60 m agents have been reported to be necessary for in vitro responses (Steen et M potassium chloride (KCl ), a cocktail of inflammatory agents (IFC), or 10 m M capsaicin (CA P) is shown. The mean basal rate of CGRP release was 148 6 5 pg/hr per dish For the cAMP measurements, trigeminal cultures were incubated with (SE, n 5 36). The secretion rate for each condition was normalized to the either 2 mM forskolin (Sigma) in the presence or absence of sumatriptan basal rate for each dish. The means and the SE from at least four (20 mM) or CGS (10 mM) for 30 min at 37°C. The HeLa1B cell line was independent experiments are shown. *p , 0.001 when compared with incubated with 100 mM forskolin and 0.1 mM sumatriptan or CGS under control levels.
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 (Pen- insula Labs). Each experimental condition was repeated in at least three occurred at a steady-state rate of 148 pg/hr per dish of cells independent experiments, and statistical analyses were performed using (approximately two ganglia). Treatment with potassium chloride the Student's t test (two-tailed, unpaired samples).
(KCl) to mimic neuronal depolarization caused approximately a sevenfold increase in the rate of CGRP release (Fig. 1B). Treat- ment with capsaicin, which selectively activates sensory C-fibers Regulated release of CGRP from cultured trigeminal
via a vanilloid receptor (Caterina et al., 1997), resulted in a similar sevenfold increase (Fig. 1B). The rate of CGRP release To determine whether sumatriptan could directly repress CGRP was also markedly stimulated by a mixture of agents known to secretion from trigeminal neurons, we took a reductionist ap- mediate physiological responses of neurogenic inflammation and proach by establishing primary cultures of rat trigeminal ganglia sensitization of nociceptive afferents (Strassman et al., 1996) (Fig.
enriched for neuronal cells (estimated to be .80%). Expression 1B). Because the release of CGRP during migraine is thought to of a neuron-specific protein, 160 kDa neurofilament subunit was result in the production and/or release of agents that escalate and detected in all cells exhibiting a neuronal morphology. A unique sustain the inflammatory response, our results indicate that these feature of our culture conditions is that almost all of the neuronal agents can also act to maintain CGRP secretion after the initial cells are CGRP positive (Fig. 1A), although it is estimated that nerve activation.
only 23% of the neurons in the trigeminal ganglia express CGRP in vivo (O'Conner and Van Der Kooy, 1988). A likely reason for Sumatriptan represses stimulated CGRP release
this bias is that only nerve growth factor, and not BDNF or NT-3, Having established that cultured trigeminal neurons exhibit reg- which are required for survival of other neurons, was included in ulated CGRP secretion, we then asked whether sumatriptan could the culture media (Buchman and Davies, 1993). CGRP secretion directly inhibit this release. We found that sumatriptan inhibited Durham and Russo • Serotonergic Repression of CGRP Secretion J. Neurosci., May 1, 1999, 19(9):3423–3429 3425
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 mM sumatriptan (Suma), 10 mM CGS, or 10 mM L-694,247 (L694 ) are shown. The mean basal rate was 137 6 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.
normal, nonmigranuer individuals (Goadsby and Edvinsson, 1993). The specificity of sumatriptan action via the 5-HT1 recep- tors 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 Figure 2. Effect of 5-HT1 receptor agonists on CGRP release. A, CGRP to directly inhibit CGRP release.
secretion as a function of sumatriptan concentration and treatment time.
We also investigated whether sumatriptan could inhibit the The effect of sumatriptan was determined on unstimulated and KCl- release of the vasoactive neuropeptide substance P, because it has stimulated trigeminal neurons (cultured for 4–7 d). The amount secreted been reported to colocalize with CGRP in sensory neurons (Ed- 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 vinsson and Goadsby, 1994) and is involved in mediating neuro- mM sumatriptan was added for 2 or 4 hr, and the amount per hour was genic inflammation (Buzzi et al., 1995). In preliminary studies, we normalized to basal. The mean basal CGRP secretion rate was 131 6 4 determined that sumatriptan could also inhibit the KCl- pg/hr per dish. The means and the SE from at least three independent stimulated release of substance P (data not shown). Thus, the experiments are shown. *p , 0.001 when compared with control values.
effectiveness of sumatriptan in reducing or abolishing the pain , 0.05 when compared with KCl-only values. B, The 5-HT1 receptor antagonist methiothepin blocks inhibitory effect of sumatriptan on KCl- associated with migraine is likely caused by its ability to coordi- stimulated CGRP release. The relative rates after addition of buffer nately inhibit the release of vasoactive neuropeptides from tri- (CON ), KCl, KCl plus 10 mM sumatriptan (SUMA) and/or 20 mM me- geminal ganglion nerves.
thiothepin (SUMA1MET) are shown. The mean basal rate was 122 6 5 Because we had found that a mixture of inflammatory agents pg/hr per dish. The means and the SE from the two independent exper- iments are shown for each study. *p , 0.01 when compared with control caused a marked increase in the rate of CGRP secretion (Fig. 1), values or sumatriptan values. # p , 0.05 when compared with KCl values.
we wanted to determine whether sumatriptan could also block this type of stimulated CGRP release. The increase in CGRP potassium-stimulated CGRP secretion in a dose-dependent man- release caused by the inflammatory cocktail was inhibited more ner (Fig. 2A). The secretion rate remained relatively stable than twofold by pretreatment with sumatriptan (Fig. 3). In addi- throughout 4 hr of potassium stimulation, and a single dose of tion, we showed that pretreatment with two other 5-HT1 receptor sumatriptan was able to maintain a steady inhibition throughout agonists, CGS and L-694,294, caused a similar inhibition of this period (Fig. 2A). In contrast, sumatriptan had no significant CGRP secretion (Fig. 3). These results demonstrate that multiple effect on the basal secretion of CGRP from unstimulated trigem- 5-HT1 agonists can repress CGRP secretion by at least two inal neurons (Fig. 2A). This finding is consistent with clinical different stimuli, suggesting that these agents target a common reports that sumatriptan does not lower serum CGRP levels in downstream step.
3426 J. Neurosci., May 1, 1999, 19(9):3423–3429
Durham and Russo • Serotonergic Repression of CGRP Secretion Table 1. cAMP levels in cultured trigeminal neurons
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 FSK 1 sumatriptan 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 IFC 1 sumatriptan 1 mM in some cells. We estimate that ;40% of the neuronal cells cAMP levels were measured from untreated control cultures or cells treated for 30 did not respond to sumatriptan treatment. The viability of these min with forskolin (FSK) or with a cocktail of inflammatory agents (IFC). The neuronal cells was confirmed after the sumatriptan treatment by cultures were cotreated with vehicle, sumatriptan, or CGS. The means and SE from five independent experiments with duplicate samples, and the fold increases in the elevation of calcium levels in response to high concentrations cAMP levels relative to control cells are shown.
of KCl. The reason for this heterogeneity is not known but may *p , 0.001 when compared with control levels.
indicate that not all of the neurons are expressing sufficient levels of 5-HT1 receptors. In contrast to the delayed increase in calcium Table 2. cAMP levels in 5-HT1B-expressing HeLa cells
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 path- way and not to a Gi/o-coupled decrease in cAMP.
FSK 1 sumatriptan Okadaic acid blocks inhibitory effect of sumatriptan
How does a prolonged calcium elevation inhibit secretion? One cAMP levels were measured from untreated control cultures or cells treated for 15 possibility is that protein phosphorylation states are changed. It is min with forskolin (FSK). The cultures were cotreated with vehicle, sumatriptan, or CGS. The means and SE from two independent experiments with duplicate samples, generally accepted that changes in calcium can alter protein and the fold increases in cAMP levels relative to control cells are shown.
phosphorylation and that phosphorylation plays an important role *p , 0.02 when compared with control levels.
in regulating neuropeptide release from sensory neurons (Green- gard et al., 1993). We have used okadaic acid, a potent inhibitor of serine threonine protein phosphatases, especially PP1 and Sumatriptan does not cause a decrease in intracellular
PP2A (Denhardt, 1996), to test the possibility that 5-HT1 agonists cAMP levels
may be activating a phosphatase to attenuate stimulated secre- We then characterized the signaling pathway(s) used by tion. Okadaic acid treatment blocked the inhibitory effect of sumatriptan in primary trigeminal ganglion cultures. Pharmaco- sumatriptan on stimulated CGRP release (Fig. 5). Okadaic acid logical studies have demonstrated that sumatriptan has high se- treatment alone increased CGRP release that was similar in lectivity and potency at the 5-HT1B, 1D, and 1F receptors, all of magnitude to that caused by depolarization (Fig. 5), which is in which are expressed by trigeminal neurons (Martin, 1997). The agreement with previous studies by Vasko and colleagues (Hingt- classical 5-HT1 signaling pathway based on studies using brain gen and Vasko, 1994) using cultured sensory neurons from dorsal slices and non-neuronal cell lines overexpressing 5-HT1 receptors root ganglia. Cotreatment with okadaic acid and KCl did not has been that these receptors inhibit adenylate cyclase and de- result in a greater increase in CGRP release than observed with crease cAMP levels via pertussis toxin-sensitive Gi/o proteins each agent alone. These results indicate that sumatriptan acts by (Boess and Martin, 1994). However, in contrast to these reports, stimulating a serine threonine phosphatase. Although the identity neither sumatriptan nor CGS inhibited forskolin-stimulated of this phosphatase is not known, it is intriguing that okadaic acid cAMP accumulation (Table 1). In addition, treatment with the has recently been reported to inhibit a MAP kinase phosphatase mixture of inflammatory agents that stimulated CGRP release activity (Runden et al., 1998), and we have shown previously that did not elevate cAMP levels, nor did the cAMP levels change 5-HT1 agonists cause a long-term increase in MAP kinase phos- after cotreatment with sumatriptan (Table 1). As a positive con- phatase activity in the CA77 cells (Durham and Russo, 1998).
trol, 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 Our results support a model in which the trigeminal ganglion expressing the 5-HT nerves are activated during migraine and release CGRP to cause 1B receptor (Table 2). The degree of inhibi- tion is similar to previously published results with this cell line vasodilation and mast cell degranulation leading to the release of (Hamblin et al., 1992). Thus, sumatriptan could lower cAMP inflammatory agents (Fig. 6). On the basis of our data using CA77 levels in HeLa1B cells but did not cause a decrease in either the cells (Durham and Russo, 1998), these agents may stimulate stimulated or unstimulated cAMP levels in trigeminal neurons.
MAP kinase pathways leading to an increase in CGRP synthesis and secretion that could potentially maintain elevated CGRP Sumatriptan mediates an increase in
levels for the long duration (up to 72 hr) of a migraine. Activation of this pathway ultimately leads to sensitization of the trigeminal We had shown previously that activation of 5-HT1 receptors by neurons and nociceptive transmission to the CNS contributing to sumatriptan and other 5-HT1 receptor agonists caused a sus- the pain, nausea, and photophobia associated with migraine tained increase in calcium in the neuronal-like CA77 thyroid (Buzzi et al., 1995). It is likely that sumatriptan is able to block C-cell line (Durham et al., 1997). With this in mind, we then this pathway via activation of the 5-HT1 receptors leading to a

Durham and Russo • Serotonergic Repression of CGRP Secretion J. Neurosci., May 1, 1999, 19(9):3423–3429 3427
Figure 4. Sumatriptan increases the concentration of intracellular calcium in trigeminal neurons. A, Intracellular cal- cium concentrations, [Ca 21]i, from day 4 cultures were measured using fura-2 and a microscopic digital imaging system.
The pseudo-color scale indicates the [Ca 21]i. Basal levels are in a single neu- ron with a neurite. B, The same cell 6 min after addition of 10 mM sumatriptan.
C, The same cell after 12 min. D, A graphic representation of the change in [Ca 21]i as a function of time after sumatriptan treatment of a representa- tive cell (same cell as above). For com- parison, a trace of a different cell treated with only KCl (60 mM) is superimposed.
prolonged elevation in calcium that mediates the recruitment of In the process of demonstrating this point, we have uncovered phosphatases. The concentration of sumatriptan required for several unexpected findings. First, activation of endogenous tri- inhibition in vitro is higher than the estimated plasma concentra- geminal ganglion neuron 5-HT1 receptors did not decrease tion in patients (;0.2 mM) (Fowler et al., 1991). Possible expla- cAMP levels, which contradicts the commonly held belief (Boess nations are that the effective receptor number may be low because and Martin, 1994). These observations are consistent with our of the culture conditions and/or lack of colocalization of recep- findings that the 5-HT1 receptor agonist CGS also did not de- tors and secretory machinery at nerve terminals. Alternatively, crease cAMP levels in a model neuronal cell line and that CGS higher concentrations may be required to counteract chronic actions were pertussis toxin independent (Durham et al., 1997).
stimulation of the cultures. In either case, the ability to block The simplest explanation is that the cellular context is critical for stimulated CGRP secretion in the absence of vascular contribu- assigning second messenger pathways to receptors. In support of tions strongly supports the neurogenic model of migraine.
this conclusion, others have reported that terminal 5-HT1 auto- 3428 J. Neurosci., May 1, 1999, 19(9):3423–3429
Durham and Russo • Serotonergic Repression of CGRP Secretion Table 3. Effect of sumatriptan on calcium levels in cultured
1.1 6 0.8a Intracellular calcium levels were measured from untreated control neurons or neurons treated with 10 mM sumatriptan. The means and SE are given.
*p , 0.001 when compared with control levels.
aThere was no significant change in calcium in control cells, so for comparison with the sumatriptan-treated cells, calcium levels at 488 sec were used to calculate peak and fold increase values for the control cells.
Figure 6. Model of 5-HT1 receptor-mediated inhibition of CGRP release from trigeminal neurons. A depolarizing stimulus causes the initial re- lease of CGRP from trigeminal nerves, leading to neurogenic inflamma- tion, 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.
the amplitude, duration, and localization of increased calcium can differentially activate transcription factors (Dolmetsch et al., 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.
Figure 5. Okadaic acid treatment blocks sumatriptan-mediated inhibi- tion of potassium-stimulated CGRP release. The relative amount of In agreement with this model, okadaic acid treatment alone CGRP secreted from trigeminal neurons stimulated with 60 mM KCl, 600 stimulated neuropeptide release from cultured trigeminal (this nM (unless indicated as 300 nM) okadaic acid (OA), or the combination of study) and dorsal root ganglia neurons (Vasko et al., 1994).
KCl and OA, with or without cotreatment with 10 mM sumatriptan Sumatriptan is able to blunt the increased secretion in response to (Suma) is shown. The mean basal rate of CGRP secretion was 99 6 4 pg/hr per dish. The means and SE from at least three independent depolarization and inflammatory agents, but not okadaic acid, experiments are shown. *p , 0.001 when compared with control values.
suggesting that specific phosphatases are recruited by 5-HT1 # p , 0.05 when compared with KCl values. 1 p , 0.05 when compared receptor activation. The possibility of coordinated regulation by with KCl plus sumatriptan values.
phosphatases is suggested by our previous studies showing that CGRP promoter activity was repressed in CA77 cells by a receptors in hippocampal neurons may not be coupled to Gi/o calcium-dependent increase in MAP kinase phosphatase-1 activ- proteins (Blier, 1991).
ity via 5-HT1 receptor activation (Durham and Russo, 1998). A The second and perhaps most intriguing finding is that the remaining question is how sumatriptan selectively inhibits stim- inhibition of neuropeptide secretion by 5-HT1 receptor activation ulated but not basal release of CGRP. To our knowledge, this is is paradoxically coupled to an unusually prolonged intracellular the first report of a drug that selectively targets only one of these calcium signal. At face value, this observation is paradoxical events. One possible explanation would be if sumatriptan causes because increased calcium is well known to be a signal to increase dephosphorylation of proteins responsible for the assembly, fu- secretion (Matthews, 1996). Indeed, this dogma held true for the sion, and/or recycling of vesicles in response to depolarization or potassium treatment, which caused a more typical transient in- crease in calcium, with increased CGRP release from cultured In conclusion, our results have demonstrated that activation of trigeminal ganglia neurons. There is precedence in parathyroid the 5-HT1 receptor class of antimigraine drugs is able to directly endocrine cells for coupling of elevated intracellular calcium with block CGRP release from trigeminal nerves. The inhibitory effect inhibition of peptide secretion (Shoback et al., 1984). Our data of sumatriptan occurs via a paradoxical elevation in calcium and demonstrate that activation of endogenous trigeminal neuron activation of an okadaic acid-sensitive phosphatase. During mi- 5-HT1 receptors is coupled to a calcium-dependent signaling graine, CGRP helps mediate neurogenic inflammation that may pathway that differs from depolarization-induced changes in cal- result in the release of inflammatory agents. These agents could cium. This raises the possibility that the amplitude and duration in turn feed back to sensitize the trigeminal ganglia neurons to of increased calcium can differentially regulate neuropeptide se- sustain an elevated rate of CGRP release (Fig. 6). On the basis of cretion from sensory neurons, analogous to recent evidence that our data, the effectiveness of sumatriptan is attributable in part to Durham and Russo • Serotonergic Repression of CGRP Secretion J. Neurosci., May 1, 1999, 19(9):3423–3429 3429
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