An in vitro model of neurogenic inflammation in rat colon: modulation of CGRP release

R.Kaur, S.McNulty, E.Jarvie & C.O’Shaughnessy, Neurology & GI CEDD, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, CM19 5AW, UK.

In the gastrointestinal tract, CGRP is expressed in extrinsic primary afferents and in enteric neurons of the myenteric and submucosal plexus. CGRP is released in response to neurogenic inflammation in vivo (Jansco et al., 1967). The neurogenic response involves release not only of CGRP but of substance P from primary afferents, as well as a host of inflammatory mediators from mast cell degranulation. The aim of these studies was to try to mimic these conditions in vitro in tissue derived from the rat colon. We additionally explored the possible mechanisms underlying the responses using compounds known to modulate signalling pathways active during neurogenic inflammation.

Male CD rats (200-250g) were killed by cervical dislocation and 2 cm segments of distal colon dissected into synthetic interstitial fluid (SIF) :- (mM) NaCl (108), KCl (3.48), MgSO4 (0.7), NaHCO3 (26), NaH 2PO4 (1.7), CaCl2 (1.5), glucose (5.55), sucrose (7.6) pre-gassed with 95% O2 / 5% CO2 pH7.4. Tissue was equilibrated for 30min at 37 0C and incubated further for 30min ± antagonist prior to release study. Tissue was transferred to the 1 st well containing SIF ±antagonist (basal) and then transferred to 2 nd well containing stimuli for 30mins, 37 0C. CGRP in supernatant were determined using ELISA (SPBio). Statistical analysis was performed using unpaired T-test.

CGRP release was stimulated by either capsaicin, 60mM K+, an inflammatory soup:- (10µM) 5-HT, histamine, bradykinin, PGE 2, (0.1µg/ml) IL-1B, TNFα and IFNγ), acidosis (pH5.2) or mast cell degranulator (compound 48/80), thus demonstrating that conditions of neurogenic activation could indeed be induced in vitro in the rat colon. Of the number of mechanisms evaluated as possible modulators of this response, robust effects were seen with the vanilloid receptor antagonists SB750364 and capsazepine on capsaicin evoked release and the mast cell inhibitor, ketotifen, in the presence of compound 48/80. Tissue acidosis also produced a robust increase in CGRP release and whilst this was insensitive to the ASIC inhibitor, amiloride, inhibition with vanilloid receptor antagonists was seen. Interestingly acidosis was potentiated by the inflammatory soup (pH5.2=546% (n=12) vs inflammatory soup+pH5.2= 899% (n=12)). The summarized data can be seen in Table 1.

In conclusion, we have produced an in vitro model of neurogenic inflammation in the rat colon. This model can be used to determine functional activity of key pharmacological mechanisms in a native tissue setting.

Table 1

Jansco et al., (1967) Br J Pharmacol 31:138-151.