Bradykinin (Bk) is an important inflammatory mediator in the gut, it is responsible for pain and induces epithelial electrogenic ion transport that leads to secretory diarrhoea. Kinins also have a potential relevance in inflammatory bowel diseases. In this study we characterised the mechanisms underlying the Bk action on submucosal neurons of rat colon descendens. We examined which receptors, subsequent signalling proteins and calcium channels are involved and which events initiate their opening. To obtain a submucosal tissue preparation, the serosa and muscularis propria were stripped away and the mucosa was removed. A primary cell culture was achieved by mechanical dissociation and digestion with collagenase II and tryspin.

Immunocytochemical staining revealed that submucosal neurons as well as dissociated cultured neurons express the Bk-2 receptor. The results of calcium imaging experiments showed that Bk increases the cytosolic calcium concentration in submucosal neurons exclusively via the Bk-2 receptor in a concentration-dependent manner, the Bk-1 receptor is not involved. In contrast to many studies, the Bk-response in these cells is indomethacin resistant and so not prostaglandin-mediated. The Bk-2 receptor is coupled to a Gq/11 protein which is unexpectedly not linked to the classical signal transduction pathway involving the phospholipase C and the associated inositoltrisphophate-induced calcium release from the ER. Experiments in calcium free solution demonstrated the strong dependency on the presence of extracellular calcium. Additionally, divalent cations such as lanthanum and nickel, which are unselective blockers of voltage-operated calcium channels (voccs), reduced the effect of Bk. So the further interest was to identify the different types of voccs through which calcium enters the cell. Inhibition of each subtype (L;N;P;Q;R;T) with more selective drugs revealed that the L-, N- and Q-subtypes take part in the Bk-induced calcium increase, whereas the P-, R- and T-subtypes are not involved. In order to investigate the mechanisms that underlie the opening of the voccs, we established a primary cell culture of dissociated submucosal neurons. Whole-cell patch-clamp measurements showed that Bk leads to a depolarisation of the membrane potential. Experiments concerning the question which ions generate the depolarisation will follow.

In summary we conclude that Bk binds to the Gq/11 protein-coupled Bk-2 receptor. This binding leads to a depolarisation of the cell by a still unknown mechanism. The depolarisation activates the L-, N- and Q-voccs which induce an increase in cytosolic calcium concentration.