Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH 2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH 2 ) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.
Proteinase‐activated receptor‐2 (PAR‐2) may participate in epithelial ion transport regulation. Here we examined the effect of mouse activating peptide (mAP), a specific activator of PAR‐2, on electrogenic transport of mouse distal colon using short‐circuit current (ISC) measurements. Under steady‐state conditions, apical application of amiloride (100 μm) revealed a positive ISC component of 74.3 ± 6.8 μA cm−2 indicating the presence of Na+ absorption, while apical Ba2+ (10 mm) identified a negative ISC component of 26.2 ± 1.8 μA cm−2 consistent with K+ secretion. Baseline Cl− secretion was minimal. Basolateral addition of 20 μm mAP produced a biphasic ISC response with an initial transient peak increase of 11.2 ± 0.9 μA cm−2, followed by a sustained fall to a level 31.2 ± 2.6 μA cm−2 (n= 43) below resting ISC. The peak response was due to Cl− secretion as it was preserved in the presence of amiloride but was largely reduced in the presence of basolateral bumetanide (20 μm) or in the absence of extracellular Cl−. The secondary decline of ISC was also attenuated by bumetanide and by Ba2+, indicating that it is partly due to a stimulation of K+ secretion. In addition, the amiloride‐sensitive ISC was slightly reduced by mAP, suggesting that inhibition of Na+ absorption also contributes to the ISC decline. Expression of PAR‐2 in mouse distal colon was confirmed using RT‐PCR and immunocytochemistry. We conclude that functional basolateral PAR‐2 is present in mouse distal colon and that its activation stimulates Cl− and K+ secretion while inhibiting baseline Na+ absorption.
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