Inhibitory regulation of constitutive transient receptor potential‐like cation channels in rabbit ear artery myocytes
Copyright policy )Inhibitory regulation of constitutive transient receptor potential‐like cation channels in rabbit ear artery myocytes
In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca2+‐permeable non‐selective cation conductance (Icat) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of Icat was recorded in cell‐attached and inside‐out patches with similar unitary conductance values. In inside‐out patches with relatively high constitutive activity the G‐protein activator GTPγS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G‐protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G‐protein inhibitor GDPβS suggesting a G‐protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to Gαq/Gα11 enhanced channel activity whereas anti‐Gα1−3/Gαo antibodies decreased basal channel activity which suggests that Gαq/Gα11 and Gαι/Gαo proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1‐oeoyl‐2‐acetyl‐sn‐glycerol (OAG) decreased the probability of channel opening (NPo) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13‐dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NPo. These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving Gαq/Gα11‐mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NPo and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPγS and OAG caused marked increases in NPo. Together with inhibitory effects of GDPβS and anti‐Gα1−3/Gαo antibodies the results suggest that there is constitutive Gαi/Gαo protein activity leading to channel opening via a DAG‐mediated but PKC‐independent mechanism. Finally, with whole‐cell recording it is shown that noradrenaline increases Icat and the noradrenaline‐evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist‐evoked Icat in these arterial myocytes.
- University of East Anglia United Kingdom
- St George's Hospital United Kingdom
- St George’s University Hospitals NHS Foundation Trust United Kingdom
Phosphodiesterase Inhibitors, Myocytes, Smooth Muscle, 610, Ear, Arteries, Ion Channels, Muscle, Smooth, Vascular, Pyrrolidinones, Membrane Potentials, Diglycerides, Norepinephrine, Cations, Type C Phospholipases, Carcinogens, GTP-Binding Protein alpha Subunits, Gs, Animals, GTP-Binding Protein alpha Subunits, Gq-G11, Rabbits, Estrenes, Phorbol 12,13-Dibutyrate, Protein Kinase C
Phosphodiesterase Inhibitors, Myocytes, Smooth Muscle, 610, Ear, Arteries, Ion Channels, Muscle, Smooth, Vascular, Pyrrolidinones, Membrane Potentials, Diglycerides, Norepinephrine, Cations, Type C Phospholipases, Carcinogens, GTP-Binding Protein alpha Subunits, Gs, Animals, GTP-Binding Protein alpha Subunits, Gq-G11, Rabbits, Estrenes, Phorbol 12,13-Dibutyrate, Protein Kinase C
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