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is carried by L-type calcium channels. In B-cells, the predominant calcium channel is Ca V 1.2, whereas Ca V 1.2 and Ca V 1.3 were identified in A-cells. These results were confirmed by using mice carrying A-or B-cell-specific inactivation of the Ca V 1.2 gene. In B-cells, the remaining I Ca flows in equal amounts through Ca V 2.1, Ca V 2.2 and Ca V 2.3. In A-cells, 30 and 15% of I Ca is due to Ca V 2.3 and Ca V 2.1 activity, respectively, whereas Ca V 2.2 current was not found in these cells. Low-voltage-activated T-type calcium channels could not be identified in A-and B-cells. Instead, two TTX-sensitive sodium currents were found: an early inactivating and a residual current. The residual current was only recovered in a subpopulation of B-cells. A putative genetic background for these currents is Na V 1.7.

Direct neuronal activation by IBS mucosal biopsy supernatants is primarily a feature of submucous rather than myenteric neurons. This is associated with a stronger excitation of submucous neurons by serotonin. The plexus-specific effects support the concept that altered mucosa-nerve signaling underlies disturbances in IBS.

Background & aimsThe causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC).MethodEffects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis.ResultsNerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin–the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants.ConclusionProteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS.

New Findings r What is the central question of this study?Supernatants from colonic mucosal biopsies from patients with irritable bowel syndrome (IBS) activate enteric and dorsal root ganglion (DRG) neurons. Based on the discomfort/pain threshold during rectal distension, IBS patients may be subtyped as normo-or hypersensitive. However, the link between neuronal activation and visceral sensitivity remains unknown. r What is the main finding and its importance?We found that supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons than supernatants from normosensitive IBS patients. The level of activation correlated with the individual discomfort/pain threshold pressure values. We therefore conclude that mucosal biopsy supernatants have biomarker potential and may, in the future, help to personalize treatment of IBS patients with different visceral sensitivities.Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo-or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo-and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca 2+ dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both