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. 2004 Oct 1;560(Pt 1):169-80.
doi: 10.1113/jphysiol.2004.071738. Epub 2004 Aug 5.

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes

A P Albert  1 , W A Large
Affiliations

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes

A P Albert et al. J Physiol. .

Abstract

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-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 GTPgammaS 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 GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) 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(alphaq)/G(alpha11)-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 GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) 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.

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Figures

Figure 1
Figure 1. Constitutively active cation channel currents in cell-attached patches from rabbit ear artery myocytes
A, a typical cell-attached patch recording showing constitutively active cation channel currents at a holding potential of −50 mV. Openings are represented as downward deflections. On the lower faster time scale channel currents are shown allowing transitions to three different levels to be observed. The continuous line represents the closed state and the dashed lines the three different open levels. B, amplitude histogram from the patch shown in A which could be fitted with Gaussian curves representing three open channel current levels of −0.72, −1.32 and −2.01 pA and the closed state at 0 pA. Note that in all following figures the holding potential was −50 mV. C, current–voltage relationship of the three channel current levels recorded from the patch shown in A showing that the three levels correspond to conductances of 14, 25 and 38 pS.
Figure 2
Figure 2. The role of G-proteins on constitutive cation channel activity in inside-out patches
A, inhibition of constitutively active channel currents by bath application of 0.5 mm GTPγS which is reversed by 3 μm chelerythrine. B, reversible inhibition of constitutive activity by bath application of 0.5 mm GDPβS. C, mean data showing significant reduction of constitutive activity by GTPγS (n = 6) and GDPβS (n = 6) in inside-out patches (*P < 0.05).
Figure 3
Figure 3. The effect of anti-G-protein antibodies on cation channel activity in inside-out patches
A, bath application of an anti-Gαq/Gα11 antibody at 1 : 200 dilution increased channel activity. B, bath application of a combination of anti-Gαι1/Gαι3 and anti-Gαι3q/Gαo antibodies at a dilution of 1 : 200 reversibly inhibited constitutive channel activity. C, mean data of effect of anti-Gαq/Gα11 antibody (n = 10) and anti-Gαι1−3q/Gαo antibodies (n = 10, *P < 0.05).
Figure 4
Figure 4. U73122-sensitive PLC is involved in inhibiting constitutively active cation channel activity in cell-attached patches
A, bath application of 2 μm U73122 increased cation channel activity. B, mean data of the effect of U73122 (n = 6) and U73343 (n = 5) on constitutive activity (*P < 0.05).
Figure 5
Figure 5. The effect of OAG and PKC on cation channel activity
A, bath application of 40 μm OAG reduced constitutive channel activity in a cell-attached patch which was reversed by 3 μm chelerythrine. B, bath application of 1 μm PDBu reversibly inhibited constitutively active channel activity in an inside-out patch. C, mean data of the effects of OAG (n = 6), PDBu (n = 5) and chelerythrine (n = 6) on constitutive activity (*P < 0.05).
Figure 6
Figure 6. Effect of G-protein and PLC activity on inside-out patches containing low channel activity
A, B and C show that bath application of, respectively, 0.5 mm GTPγS, anti-Gαq/Gα11 antibody at 1 : 200 dilution and 2 μm U73122 to three separate patches evoked marked channel activity which had similar amplitude histograms indicating three open channel levels.
Figure 7
Figure 7. Effect of noradrenaline on whole-cell Icat in ear artery myocytes
A, bath application of 100 μm noradrenaline evoked a whole-cell Icat at −50 mV which declined in the continued presence of the agonist. B, bath application of 100 μm noradrenaline to a myocyte pre-incubated for 5 min with 3 μm chelerythrine activated a whole-cell Icat that had a much larger peak amplitude and was sustained in the continued presence of the agonist. In A and B the dashed lines represent zero holding current, and the current responses to voltage ramps have been truncated for the purpose of presentation. C, mean current–voltage relationships of noradrenaline (NA)-evoked whole-cell Icat in the absence and presence of chelerythrine. D, mean data showing peak amplitudes of noradrenaline-evoked Icat in control conditions (n = 7, 1.5 mm Ca2+o), in the presence of chelerythrine (n = 6) and in 0 Ca2+o (n = 6) and 89 mm Ca2+o (n = 5, P < 0.05).
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References

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