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 (I_cat) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I_cat 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 GTPγ S 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 G_αq/G_α11 enhanced channel activity whereas anti-G_α1-3/G_αo antibodies decreased basal channel activity which suggests that G_αq/G_α11 and G_αi/G_αo 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 (NP_o) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP_o. These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G_αq/G_α11-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP_o 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 NP_o. Together with inhibitory effects of GDPβS and anti-G_α1-3/G_αo antibodies the results suggest that there is constitutive G_αi/G_αo 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 I_cat and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I_cat in these arterial myocytes.