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| 015P London, UK Pharmacology 2016 |
The importance of Rho-associated kinase-induced Calcium sensitization as a component of Bradykinin -induced pharmacomechanical coupling in rat ureteric smooth muscle
Introduction: Bradykinin (BK) was described to be found in urine and cause ureteric stimulant action more than 30 years ago (Marin-Grez et al., 1980). However, the mechanisms of its action on ureteric smooth muscle have not been explored. Thus, the aim of the present study was to investigate the possible role of Rho-associated kinase-induced Ca2+ sensitization as a component of Bradykinin (BK) -induced pharmacomechanical coupling in rat ureteric smooth muscle.
Method: The experiments were performed on Wistar rats which were humanly killed using CO2 anaesthesia followed by cervical dislocation. Force was measured using 12 wells plate mounted on a moveable spring system to enable easy transfer of the tissue between wells and filled with different solutions. Ureter strips were clipped with aluminium foil clips and mounted on two stainless steel hooks. One of the hooks was attached to a stainless steel rod, while the other to the calibrated force transducer. The force was recorded using Digidata 1320x data translation board using Axoscope software (Axon Instruments). The force-Ca2+ relationship was studied using combined Nipkow disc based confocal imaging system with force measurements of Fluo-4 loaded ureteric segments . All the experiments were performed in the presence of L-type Ca2+ channel blocker, Nifedipine, to exclude steps of electro-mechanical coupling.
Result: Inhibition of voltage gated L-type Ca2+ channels by Nifedipine (10µM) selectively blocked intercellular Ca2+ oscillations and phasic contraction, but had no effect on initial phasic and sustained components of Ca2+ transient and force, respectively. Inhibition of Rho-kinase by Y-27632 (10µM) had no effect on BK-induced Ca2+ transient, but strongly reduced contractile response. The amplitude of both phasic and tonic component of BK-induced force in the presence of Y-27632 were reduced from 63.3±9.8 to 29.5±5.7% and 22.3±6.7 to 7.1±3.5% of peak high-K force (n=16), respectively.
Conclusion: The data obtained suggest that both phasic and tonic contraction induced by BK is highly dependent on activity of Rho-kinase, leading to inhibition of MLCP and increased LC20 phosphorylation and force.
Reference: Ureteral contractions induced by rat urine in vitro: probable involvement of renal kallikrein Marin-Grez M., Bonner G and F.Gross, et al., Experientia, 1980, 36, 865-866