Bradykinin-induced depolarisation and Ca 2+ influx through voltage-gated Ca 2+ channels in rat submucosal neurons

Abstract

The aim of the present study was the investigation of the mechanism, by which bradykinin B 2 receptor stimulation evokes an increase of the cytosolic Ca 2+ concentration in rat submucosal plexus. In ganglionic cells within the intact submucosal plexus, the Ca 2+-response evoked by bradykinin was suppressed by Ni 2+, suggesting that Ca 2+ enters the cell through voltage-gated Ca 2+ channels (Ca v channels). Inhibition of Ca v channel subtypes P, T and R with ω-agatoxin IVA, flunarizine, and SNX-482 did not affect the response to bradykinin. In contrast, verapamil, ω-conotoxin GVIA, and ω-conotoxin MVIIC attenuated the actions of bradykinin, indicating the involvement of the L-, N- and Q-subtypes of Ca v channels. The combination of these three blockers had a strong inhibitory action on the bradykinin response. In order to study the mechanism of activation of Ca v channels by bradykinin, isolated submucosal neurons in culture were used. Immunocytochemical stainings revealed that these neurons expressed the bradykinin B 2 receptor, while the B 1 receptor was absent. Isolated submucosal glial cells did not express the bradykinin B 2 receptor. Whole-cell patch-clamp measurements of submucosal neurons showed that bradykinin induced a depolarisation of the membrane in average of 14 mV. The ionic mechanism underlying the depolarisation was identified with current measurements at two different membrane potentials (− 81 and 0 mV). The current associated to Na + influx was not changed by bradykinin, whereas the current representing K + outflux was reduced by 26%. The present results suggest that at submucosal neurons from the rat colon bradykinin induces a depolarisation by decreasing the K + conductance, followed by activation of the Ca v channels, which mediates the increase of the cytosolic Ca 2+ concentration.