J Physiol Volume 562, Number 3, 839-846, February 1, 2005 DOI: 10.1113/jphysiol.2004.073692
Ca2+-independent hypoxic vasorelaxation in porcine coronary artery
Min Gu1,
George D Thorne1,
Robert L Wardle1,
Yukisato Ishida1 and
Richard J Paul1
1 Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
To demonstrate a Ca2+-independent component of hypoxic vasorelaxation and to investigate its mechanism, we utilized permeabilized porcine coronary arteries, in which [Ca2+] could be clamped. Arteries permeabilized with ß-escin developed maximum force in response to free Ca2+ (6.6 µM), concomitant with a parallel increase in myosin regulatory light chain phosphorylation (MRLC-Pi), from 0.183 ± 0.023 to 0.353 ± 0.019 MRLC-Pi (total light chain)–1. Hypoxia resulted in a significant decrease in both force (–31.9 ± 4.1% prior developed force) and MRLC-Pi (from 0.353 to 0.280 ± 0.023), despite constant [Ca2+] buffered by EGTA (4 mM). Forces developed in response to Ca2+ (6.6 µM), Ca2+ (0.2 µM) + GTP
S (1 mM), or in the absence of Ca2+ after treatment with ATPS (1 mM), were of similar magnitude. Hypoxia also relaxed GTPS contractures but importantly, arteries could not be relaxed after treatment with ATPS. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATPS suggests that this Ca2+-independent mechanism(s) may operate through alteration of MRLC-Pi or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 µM) relaxed GTPS and Ca2+ contractures; but the latter required a higher concentration (10 µM) for consistent relaxation. Relaxations to N2 and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca2+-independent O2-sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca2+ is clamped at a constant value.
(Received 11 August 2004;
accepted after revision 28 November 2004;
first published online 25 November 2004)
Corresponding author R. J. Paul: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576, USA. Email: richard.paul{at}uc.edu
M. Gu and G. D. Thorne contributed equally to this work.
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