| pA2 online © Copyright 2004 The British Pharmacological Society |
140P
GKT, University of London Winter Meeting December 2003 |
|
The effects
of sumatripan on relaease of substance P ans CGRP from guinea-pig
and dorsal root ganglion cells in culture |
|
Print abstractIt is believed that in migraine sumatriptan (Sum) acts in part by inhibition of neuropeptide release from sensory nerve terminals (Goadsby et al, 1990). This study investigated this effect in vitro using primary cultures of cells of the dorsal root (DRG) and trigeminal ganglia (TRG) of the guinea-pig, a species with 5-HT1D receptors similar to those of man.
Male guinea-pigs (Hartley strain, 200-240g) were killed by overdose of halothane and exsanguination. Both TRG and DRG from levels C1 to C3, were collected. Ganglia were dissociated at 37°C in Hank's balanced salt solution (HBSS) using papain (20 U ml-1) followed by a combination of dispase (25 mg ml-1) and collagenase (730 U ml-1). The cells were washed in Dulbecco's modified Eagle's medium (DMEM) containing glutamine, foetal calf serum, uridine, 5-fluoro-2-deoxy-uridine, penicillin, streptomycin and resuspended in DMEM containing nerve growth factor (NGF, 250 ng ml-1) and aliquoted into 16 well collagen-coated plates. Cells were maintained (37o C, 5% CO2) for nine days after which they were washed in HBSS and after 2h depolarized by exposure to HBSS containing KCl (80 mM) for 20 min. The depolarizing HBSS was removed and normal HBSS added. After 40 min Sum (1.0 or 10.0 µM) or vehicle was added to four wells on each plate. After a further 60 min cells were again exposed to HBSS with KCl or, in separate experiments, capsaicin (Caps, 1.0 µM). Samples were collected, stored (-20ºC) and assayed for CGRP and substance P immuno-reactivity (iCGRP, iSP; McGregor & Bloom, 1983; Mulderry et al., 1988).
In the absence of Sum, release of iCGRP from TRG cells was similar when evoked by KCl (61.0±5.3) or subsequently by capsaicin (65.6±13.2 fmole/well, n=8). The values for iCGRP in DRG cells were 51.5±7.4 (KCl) and 53.1±14.4 (Caps) fmoles/well. In contrast, KCl was less effective in releasing iSP than was Caps with release in TRG of 2.6±0.7 (KCl) and 7.2±0.9 (Caps) fmole/well and in DRG 0.8±0.1 (KCl) and 4.6±0.7 (Caps) fmole/well (n=5-6). Sum (1 and 10 µM) significantly reduced KCl-evoked release of iCGRP in DRG (Table 1). A similar trend with Sum in TRG was not significant. Sum did not inhibit Caps-evoked release (Table 1).
Table 1. Release of neuropeptides evoked by capsaicin (1 µM) or KCl (80 mM) in the presence of sumatripan as a ratio to the release evoked by exposure to KCl (80mM) in the same cells before addition of sumatriptan.
|
CGRP
|
Substance
P
|
|||
| Sum |
TRG
|
DRG
|
TRG
|
DRG
|
| (µM) |
ratio
S2 (KCl) : S1 (KCl)
|
|||
| 0 |
0.42
± 0.07
|
0.64
± 0.10
|
1.32
± 0.66
|
0.77
± 0.14
|
| 1 |
0.21
± 0.03
|
0.36*
± 0.05
|
0.58
± 0.10
|
0.79
± 0.25
|
| 10 |
0.27 ± 0.08
|
0.34*
± 0.04
|
0.67
± 0.29
|
1.07
± 0.19
|
|
ratio
S2 (capsaicin) : S1(KCl)
|
||||
| 0 |
0.99 ± 0.14
|
0.95
± 0.16
|
3.22
± 0.48
|
7.27
± 1.48
|
| 1 |
0.93
± 0.13
|
0.95
± 0.18
|
3.02
± 0.47
|
5.26
± 0.92
|
| 10 |
1.08
± 0.12
|
1.05
± 0.11
|
3.16
± 0.71
|
6.78
± 1.44
|
Mean ratios ± s.e. mean are shown * P < 0.05 compared to cells not treated with Sum (ANOVA and t test. n =5-8 wells).
Sum inhibited KCl-evoked release of iCGRP from DRG and possibly TRG cells in culture but did not inhibit Caps-evoked release. Caps was more effective than KCl in releasing iSP but not iCGRP from TRG and DRG cells.
Goadsby, P. J, Edvinsson,
L. & Ekman, R. (1990) Annals Neurol., 28, 183-187.
McGregor, G.P. & Bloom, S.R. (1983) Life Sci., 32, 655-662.
Mulderry, P.K., Ghatei, M.A., Spokes, R.A. et al. (1988) Neurosci.,
25, 195-205.