Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca2+ handling of human small arteries

1 Laboratoire de Pharmacologie et Physico-Chimie des Intéractions Cellulaires et Moléculaires, Unité Mixte de Recherche, Centre National pour les Recherches Scientifiques 7034, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, 67401 Illkirch-Cedex; and 2 Centre Hospitalier Universitaire, Hôpital de Hautepierre, Service de Réanimation Médicale, 67098 Strasbourg, France The mechanisms of Ca 2+ handling and sensitization were investigated in human small omental arteries exposed to norepinephrine (NE) and to the thromboxane A 2 analog U-46619. Contractions elicited by NE and U-46619 were associated with an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ), an increase in Ca 2+ -independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca 2+ . The two latter pathways were abolished by protein kinase C (PKC), tyrosine kinase (TK), and Rho-associated protein kinase (ROK) inhibitors. In Ca 2+ -free medium, both NE and U-46619 elicited an increase in tension that was greatly reduced by PKC inhibitors and abolished by caffeine or ryanodine. After depletion of Ca 2+ stores with NE and U-46619 in Ca 2+ -free medium, addition of CaCl 2 in the continuous presence of the agonists produced increases in [Ca 2+ ] i and contractions that were inhibited by nitrendipine and TK inhibitors but not affected by PKC inhibitors. NE and U-46619 induced tyrosine phosphorylation of a 42- or a 58-kDa protein, respectively. These results indicate that the mechanisms leading to contraction elicited by NE and U-46619 in human small omental arteries are composed of Ca 2+ release from ryanodine-sensitive stores, Ca 2+ influx through nitrendipine-sensitive channels, and Ca 2+ sensitization and/or Ca 2+ -independent pathways. They also show that the TK pathway is involved in the tonic contraction associated with Ca 2+ entry, whereas TK, PKC, and ROK mechanisms regulate Ca 2+ -independent signaling pathways or Ca 2+ sensitization. Rho-associated protein kinases; calcium ion * M. C. Martínez and V. Randriamboavonjy contributed equally in this work.