Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, C60

Oral Communications

Rho-associated kinase-mediated modulation of Ca2+ signaling, myosin and MYPT1 phosphorylation, and force induced by membrane depolarization and carbachol of rat ureteric smooth muscle

L. Borisova1, M. P. Walsh2, T. Burdyga1

1. Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom. 2. Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.


Ureteric peristalsis ensures the unidirectional flow of urine from the kidney to the bladder and is regulated by myogenic mechanisms and neurogenic factors. Unilateral ureteric obstruction of ureter in rabbits exhibited increased expression of both isoforms of ROKβ and ROKα as well as enhanced contractile responses to different stimuli which were attenuated by the ROK inhibitor Y-27632. ROK inhibitors, therefore, represent a promising novel therapy for the prevention of renal colic-associated pain and kidney damage. In the present study, we examined the effects of ROK inhibition on Ca2+ signaling, LC20 phosphorylation, MYPT1 phosphorylation (at T697 and T855) and force activated by high [K+]-induced depolarization (electromechanical coupling) and the carbachol (pharmacomechanical coupling) in rat ureteric smooth muscle. Rats (~200 g) were humanely killed using CO2 anaesthesia followed by cervical dislocation, in accordance with UK legislation. We used fast Nipkow disc-based confocal imaging attached to a high sensitivity (iXon Andor) CCD camera, which allowed acquisition of images at 60 - 200 fps and thereby accurate measurement of temporal and spatial characteristics of Ca2+ signaling in intact ureteric strips. Inhibition of ROK activity decreased the amplitude of the plateau component of the action potential induced by high [K+], which resulted in a decrease in the amplitude of the phasic component of both the Ca2+ transient and force, but did not affect the rapid rise in LC20 phosphorylation. The sustained component of the K+-induced Ca2+ transient was reduced by ROK inhibition by 18.0 ± 0.5%, whereas LC20 was completely dephosphorylated and force returned to baseline. MYPT1 was found to be partially phosphorylated at both ROK sites under resting conditions. Phosphorylation increased only at T855 in response to K+, and ROK inhibition reduced the level of phosphorylation below that at rest. Inhibition of ROK also had no effect on Ca2+ transient, the rapid CCh-induced rise in LC20 phosphorylation or the initial rate of force development ; however, it markedly increased the rate of LC20 dephosphorylation and relaxation. CCh induced MYPT1 phosphorylation at T855, but not T697, under these conditions. ROK inhibition prevented the CCh-induced increase in T855 phosphorylation and reduced the basal level of phosphorylation at both sites. We conclude that ROK, acting via phosphorylation of MYPT1 at T855, inhibition of MLCP and increased LC20 phosphorylation, plays an important role in ureteric smooth muscle contraction.

Where applicable, experiments conform with Society ethical requirements