At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries.
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.



Confocal Ca2+imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+oscillations were inhibited by blocking IP3receptors 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.



These results indicate that Cx43 hemichannels contribute to SMC Ca2+dynamics and contractility, by facilitating Ca2+entry, ATP release, and purinergic signalling.
Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium.
Mélissa
M
Bol
Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium.
Nan
N
Wang
Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium.
Marijke
M
De Bock
Unité de Chronobiologie Théorique, Faculté des Sciences, Université Libre de Bruxelles (ULB), 1000 Brussels, Belgium.
Benjamin
B
Wacquier
Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium.
Elke
E
Decrock
Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium.
Ashish
A
Gadicherla
Department of Pharmacology, Vascular Research Unit, Faculty of Medicine & Health Sciences, Ghent University, 9000 Ghent, Belgium.
Kelly
K
Decaluwé
Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine & Health Sciences, Ghent University, De Pintelaan 185 (Block B, Room 031), 9000 Ghent, Belgium.
Bert
B
Vanheel
Department of Medical Physiology, Division of Heart and Lungs, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands.
Harold Victor Maria
HV
van Rijen
Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, 9000 Ghent, Belgium.|Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium.
Dmitri Vadim
DV
Krysko
Laboratory of Molecular and Cellular Signalling, Department of Cellular and Molecular Medicine, KULeuven, 3000 Leuven, Belgium.
Geert
G
Bultynck
Unité de Chronobiologie Théorique, Faculté des Sciences, Université Libre de Bruxelles (ULB), 1000 Brussels, Belgium.
Geneviève
G
Dupont
Department of Pharmacology, Vascular Research Unit, Faculty of Medicine & Health Sciences, Ghent University, 9000 Ghent, Belgium.
Johan
J
Van de Voorde
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.
Luc
L
Leybaert
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002450:Cell Communication / Q000187:drug effects*; D003198:Computer Simulation; D018031:Connexin 43 / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D005838:Genotype; D066298:In Vitro Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000819:agonists / Q000378:metabolism; D008638:Mesenteric Arteries / Q000187:drug effects / Q000378:metabolism; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008955:Models, Cardiovascular; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D010641:Phenotype; D058914:Purinergic Antagonists / Q000494:pharmacology; D017208:Rats, Wistar; D013997:Time Factors; D014661:Vasoconstriction / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology*; D000097006:Gap Junction alpha-4 Protein
D000255:Adenosine Triphosphate; D002118:Calcium; D018031:Connexin 43; D017630:Connexins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009638:Norepinephrine; D010455:Peptides; D058914:Purinergic Antagonists; D014662:Vasoconstrictor Agents; D014665:Vasodilator Agents; D000097006:Gap Junction alpha-4 Protein; C543076:GJA1 protein, mouse; C580592:Gja1 protein, rat
Connexin channels • Hemichannel block • Cytoplasmic calcium • Contractility • Blood flow