ABSTRACT
Angiotensin II (Ang II) is a potent vasoconstrictor with an important role in controlling blood pressure; however, there is little information on cellular mechanisms underlying Ang II-evoked vasoconstrictor responses. The aim of the present study is to investigate the effect of Ang II on cation conductances in freshly dispersed rabbit mesenteric artery myocytes at the single-channel level using patch-clamp techniques. In cell-attached patches, bath application of low concentrations of Ang II (1 nM) activated cation channel currents (Icat1) with conductances states of about 15, 30 and 45 pS. At relatively high concentrations, Ang II (100 nM) inhibited Icat1 but evoked another cation channel (Icat2) with a conductance of approximately 2 pS. Ang II-evoked Icat1 and Icat2 were inhibited by the AT1 receptor antagonist losartan and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol (DAG) lipase inhibitor RHC80267 initially induced Icat1 which was subsequently inhibited to reveal Icat2. The DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (1 microM) activated Icat1 and Icat2 but inositol 1,4,5-trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 microM) potentiated Ang II-evoked Icat1 and inhibited Icat2 whereas the PKC activator phorbol-12,13-dibutyrate (1 microM) reduced Ang II-induced Icat1 but activated Icat2. Moreover in cell-attached patches pretreated with chelerythrine, application of 100 nM Ang II activated Icat1. These data indicate that PKC inhibits Icat1 but stimulates Icat2. Agents that deplete intracellular Ca2+ stores also activated cation channel currents with similar properties to Icat2. Bath application of anti-TRPC6 and anti-TRPC1 antibodies to inside-out patches inhibited Icat1 and Icat2, respectively. Also flufenamic acid and zero external Ca2+ concentration, respectively, potentiated and reduced Ang II-evoked Icat1. Immunocytochemical studies showed TRPC6 and TRPC1 expression with TRPC6 preferentially distributed in the plasma membrane and TRPC1 expression located throughout the myocyte. These results indicate that Ang II activates two distinct cation conductances in mesenteric artery myocytes by stimulation of AT1 receptors linked to PLC. Icat1 is activated by DAG via a PKC-independent mechanism whereas Icat2 involves DAG acting via a PKC-dependent pathway. Higher concentrations of Ang II inhibit Icat1 by activating an inhibitory effect of PKC. It is proposed that TRPC6 and TRPC1 channel proteins are important components of Ang II-induced Icat1 and Icat2, respectively.
Subject(s)
Angiotensin II/metabolism , Calcium/metabolism , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , TRPC Cation Channels/drug effects , Vasoconstrictor Agents/metabolism , Alkaloids/pharmacology , Angiotensin II/pharmacology , Animals , Antibodies/pharmacology , Benzophenanthridines/pharmacology , Diglycerides/metabolism , Dose-Response Relationship, Drug , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Flufenamic Acid/pharmacology , Immunohistochemistry , In Vitro Techniques , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Mesenteric Arteries/drug effects , Muscle, Smooth, Vascular/chemistry , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/chemistry , Myocytes, Smooth Muscle/metabolism , Phorbol 12,13-Dibutyrate/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Pyrrolidinones/pharmacology , Rabbits , Receptor, Angiotensin, Type 1/drug effects , Signal Transduction/drug effects , TRPC Cation Channels/analysis , TRPC Cation Channels/immunology , TRPC Cation Channels/metabolism , Time Factors , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism , Vasoconstrictor Agents/pharmacologyABSTRACT
Calcium (Ca2+) is an almost universal intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in the dimensions of space, time and amplitude. To generate the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling 'toolkit', which comprizes an array of signalling, homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology.
