Capsaicin stimulates release of substance P from dorsal root ganglion ...

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177 Capsaicin stimulates dew# of substance P from dorsal root ganglion neurons vla two distinct mechanisms. JOHN R. PURKISS, MARY J. WELCH, SARAH ...
5423 Biochemical Society Transactions (1997) 25

177

Capsaicin stimulates d e w # of substance P from dorsal root ganglion neurons vla two distinct mechanisms.

JOHN R. PURKISS, MARY J. WELCH, SARAH DOWARD and KEITH A. FOSTER CAMR (Centre of Applied Microbiology and Research), Porton Down, Salisbury, Wiltshire, SP4 OJG, U.K. Capsaicin (8-methyl Nvanillyl 6-nonamide), the pungent component of plants.of the capsicum family, binds to, and activates unmyelinated C-fibres and thinly myelinated A6 primary sensory neurons [l]. Functionally these fibres transmit nociceptive information to the central nervous system and in general express neuropeptides, including substance P. Activation of sensitive neurons is due to a depolarisation which occuls when there is a capsaicin-Inducedincrease in membrane permeability to cations, particularly calcium and sodium [2]. The capsaicin-activated ion channel is insensitive to the classical blocking agents such as dihydrcpyridines, w conotoxin.and tetrodotoxin [2]. Capsaicin-induced release of neurotransmitters is also unaffected by these agents and so appears to be directly stimulated by Caz+entry through the capsaicin-activatedchannel [3]. The report of Del Bianco et al 131also describes the complete dependence of capsaicininduced release on the presence of extracellular Ca2+which is in agreement with previous reports on capsaicin-induced substance P releaseJp]. However, in this report we describe both extracellular Ca -dependent and -independent mechanisms of capsaicin-induced release of substance P from cultured embryonic rat dorsal root ganglion neurons. Further, we describe the differingbotulinum toxin A sensitivity of these two mechanisms. Rat dorsal root ganglion neurons (DRGs) were prepared from 14-16 days gestation embryos. Dissected ganglia were treated with trypsinlEDTA and then triturated. Cells were plated onto Matrigel coated 12 well plates at a density of 3-9 x 1O5 cells/well. Cells were maintained In DMEM, 5% inactivated foetal calf serum, 827 supplement, 100ng/ml nerve growth factor, 5mM glutamine and 0.6% dextrose. Cultures were treated with 1OpM cytosine arabinoside between days 2 and 5 and used after 2 weeks in culture. The neuronal identity of the cells was confirmed by immunofluorescencefor neurofilament, syntaxin, SNAP-25 and substance P. Cultures were negative for glial fibrillar acidic protein. Release of substance P was measured by establishing basal release in a balanced salt solution (BSS) without added Ca". Capsaicin or 75mM KCI (with commensurate reduction in NaCI) stimulation was made in the absence of Ca2+(0.5mM EGTA) and then in the presence of Ca" (2mM), each treatment for 4.5 min. Total substance P was measured in 2M acetic acid, 0.1% trifluoroacetic acid cell extracts following dehydration. Substance P immunoreactivity was measured using an enzyme immunoassay kit (Cayman Chemical Company). Botulinum neurotoxin A (BoNTIA) cleavage of SNAP-25 was measured, by Western blotting, in cells following 18-20 hours exposure to toxin. Table 1. YCI- and Caosaicin-inducedSubstance P release Release is expressed as a percentage of total SE (n=4 (KU), n=3 (Capsaicin) from two experiments). Difference from preceding stimulation: 'p>0.05, '*>p0.02. Stimulus

m

&

75mM KCI

0.48k0.13

0.33k0.09

1.63k0.48*

1OpM Capsaicin

0.40.06

1.27+0.38*

3.1 7&0.33**

75mM KCI did not stimulate release above basal until Ca2+was introduced into the medium, when a five fold increase was seen (Table 1.). By contrast 1OpM capsaicin induced a three fold increase in release in the absence of Ca2+ independent) which increased to eight fold over basal when ca& was included. The dose-dependence of capsaicin induced release was found, in the absence of Ca2+,to be a monophasic curve which was not fully described even at 30pM. In the presence of Ca2+a biphasic curve was produced, the second phase corresponded to the curve generated in the absence of Ca". The first phase reached an apparent plateau at 0.3pM capsaicin. To establish the optimal concentration of BoNTIA for cleavage of SNAP-25, cells were exposed to a range of toxin concentrations. A dose-dependent reduction in SNAP-25 was observed, by Westem blotting, between 0.01 nM and 1OnM. The loss of SNAP-25 was accompanied by a dose-dependent reduction in the KCI-stimulated release of substance P (1% 0.037k.016nM). Release was reduced to basal levels by 1OnM BoNT/A. To ensure complete SNAP-25 cleavage cells were treated with 1OOnM BoNT/A then stimulated with 75mM K U or 10pM capsaicin (Table 2.). The KCI-induced release of substance P was completely abolished by neurotoxin,

(a'+-

Table 2. b B A Release is expressed as a percentage of total k SE (n=4, from two experiments). Difference from preceding stimulation: *p>o.oi, *=p>o.o05.

Sh'mulus

&Sa!

g

&

75mM KCI

0.45i0.1

0.3k0.06

0.4d.11

10pM Capsaicin

0.48k0.15

l.l8&0.15'

1.9M.12"'

whereas the Ca2+-independent capsaicin-induced release was unaffected by toxin. However, the Ca'+-dependent component of capsaicin-induced release was markedly reduced by BoNT/A treatment from eight fold over basal to four fold (3.17k0.33 v 1.9+0.12%, p>0.005). From these data we conclude that capsaicin is able to evoke release of substance P from DRGs by two mechanisms. The first mechanism is Ca2+-dep8nden?,maximally stimulated by 0.3pM capsaicin and requires intact SNAP-25 for optimum release. The second mechanism is Ca*+-independent,becomes activated at 3-1OpM capsaicin and is insensitive to BoNT/A so induces release through a mechanismthat does not have SNAP-25 as an essential component. This work was supported by Speywood Pharmaceuticals Ltd. 1. Winter, J., Bevan, S.and Campbell E. A. (1995) Br. J. Anaesth. 75, 157-168. 2. Wood J. N. et al(1988) J Neumsci. 8,3208-3220. 3. Del Bianco, E., et al(1991) Brain Res. 566,46-53. 4. Gamse, R., Molnar, A. and Lembeck, F.(1979) Ufe Sci. 25, 629-636. 5.Theriault, E., Otsuka, M. and Jessell, T. (1979) Brain Re$. 170,209-213.