Mélissa Bol1, Nan Wang1, Marijke De Bock1, Benjamin Wacquier2, Elke Decrock1, Ashish Gadicherla1, Kelly Decaluwé3, Bert Vanheel1, Harold Victor Maria van Rijen4, Dmitri Vadim Krysko5,6, Geert Bultynck7, Geneviève Dupont2, Johan Van de Voorde3, Luc Leybaert8. 1. Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium. 2. Unité de Chronobiologie Théorique, Faculté des Sciences, Université Libre de Bruxelles (ULB), 1000 Brussels, Belgium. 3. Department of Pharmacology, Vascular Research Unit, Faculty of Medicine & Health Sciences, Ghent University, 9000 Ghent, Belgium. 4. Department of Medical Physiology, Division of Heart and Lungs, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands. 5. Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, 9000 Ghent, Belgium. 6. Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium. 7. Laboratory of Molecular and Cellular Signalling, Department of Cellular and Molecular Medicine, KULeuven, 3000 Leuven, Belgium. 8. Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium; Luc.Leybaert@UGent.be.
Abstract
AIMS: Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca2+/purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca2+ entry and ATP release contributes to smooth muscle cell (SMC) Ca2+ dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca2+ and ATP signalling to investigate their influence on SMC Ca2+ dynamics and vessel contractility. METHODS AND RESULTS: Confocal Ca2+ imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+ oscillations were inhibited by blocking IP3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca2+ concentration, indicating it facilitated Ca2+ entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC. CONCLUSIONS: These results indicate that Cx43 hemichannels contribute to SMC Ca2+ dynamics and contractility, by facilitating Ca2+ entry, ATP release, and purinergic signalling. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca2+/purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca2+ entry and ATP release contributes to smooth muscle cell (SMC) Ca2+ dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca2+ and ATP signalling to investigate their influence on SMC Ca2+ dynamics and vessel contractility. METHODS AND RESULTS: Confocal Ca2+ imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+ oscillations were inhibited by blocking IP3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca2+ concentration, indicating it facilitated Ca2+ entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC. CONCLUSIONS: These results indicate that Cx43hemichannels contribute to SMC Ca2+ dynamics and contractility, by facilitating Ca2+ entry, ATP release, and purinergic signalling. Published on behalf of the European Society of Cardiology. All rights reserved.
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