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. 2005 Jan 15;562(Pt 2):395-406.
doi: 10.1113/jphysiol.2004.077602. Epub 2004 Nov 4.

Stimulation of beta-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

M Liu  1 , W A Large, A P Albert
Affiliations

Stimulation of beta-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

M Liu et al. J Physiol. .

Abstract

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca(2+) store depletion these SOCs could also be activated by alpha-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of beta-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective beta-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl-sn-glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of beta-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.

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Figures

Figure 1
Figure 1. Bath application of isoprenaline inhibits CPA-evoked whole-cell currents in rabbit portal vein myocytes
A, bath application of 10 μm CPA induced a whole-cell current that was inhibited by co-application of 1 μm isoprenaline. Horizontal arrow represents zero holding current. Vertical deflections represent ramps from −150 mV to +100 mV from a holding potential of 0 mV. B, individual ramp traces taken before (control) and after application of CPA and co-application with isoprenaline (ISO). C, mean time course of CPA-evoked whole-cell currents at −80 mV before and in the presence of isoprenaline. It should be noted that in this experiment and later ones with isolated patches the intracellular Ca2+ concentration is buffered at 100 nm.
Figure 2
Figure 2. Stimulation of β-adrenoceptors inhibits SOC activity in cell-attached patches
A, bath application of 10 μm CPA evoked SOC activity that was markedly inhibited by co-application of 1 μm isoprenaline. Note that in this and all subsequent figures the holding potential was −80 mV and the downward deflections represent inward current currents. B, mean data of NPo showing that 1 μm isoprenaline significantly reduced SOC activity induced by 10 μm CPA and 20 μm BAPTA-AM. n = 5–8 patches. *P < 0.05.
Figure 3
Figure 3. Forskolin and 8-Br-cAMP reduce SOC activity in cell-attached patches
A and B show, respectively, that bath application of 10 μm forskolin and 100 μm 8-Br-cAMP significantly inhibited SOC activity evoked by 10 μm CPA. C, mean data showing inhibition of CPA-evoked SOC activity by 10 μm forskolin and 100 μm 8-Br-cAMP. n = 6 patches. *P < 0.05.
Figure 4
Figure 4. 8-Br-cAMP and forskolin reduce SOC activity activated by OAG and PDBu in cell-attached patches
A and B show that bath application of 100 μm 8-Br-cAMP significantly reduced SOC activity induced by 10 μm OAG and 1 μm PDBu, respectively. C, mean data showing inhibitory effect of 100 μm 8-Br-cAMP and 10 μm forskolin on SOC activity evoked by OAG and PDBu. n = 6 patches. *P < 0.05, **P < 0.01.
Figure 5
Figure 5. PKA inhibitors stimulate SOC activity
A, bath application of 1 μm H-89 activated channel activity in a cell-attached patch. B, channel current amplitude histogram from the patch shown in A which had a unitary open level of −0.24 pA. The closed level is 0 pA and the peak at −0.46 pA represents two channels in the patch. C, current–voltage relationship of the channel currents shown in A which had a slope conductance of 1.8 pS and an extrapolated reversal potential of +25 mV. Each point is the mean of 4–11 patches.
Figure 6
Figure 6. PBDu-evoked SOC activity in inside-out patches
A shows that bath application of 1 μm PDBu evoked channel currents in an inside-out patch. B, amplitude histogram of channel currents shown in A. The peak at −0.44 pA represents at least two channels in the patch. C, PDBu-evoked channel currents from the patch shown in A recorded at different membrane potentials. Note the absence of resolvable channel currents at +20 mV. D, current–voltage relationships of PDBu-evoked SOC activity from inside-out patches recorded with patch pipette solutions containing either 1.5 mm or 0 [Ca2+]o. Each point is the mean of 5–15 patches.
Figure 7
Figure 7. PKA catalytic subunit and 8-Br-cAMP inhibit PDBu-evoked SOC activity in inside-out patches
A and C show, respectively, that bath application of 100 U ml−1 of a PKA catalytic subunit and 100 μm 8-Br-cAMP had a pronounced inhibitory effect on PDBu-evoked SOC activity. B shows that heated PKA catalytic subunit had no effect and D shows the mean data from these experiments. n = 6 patches. *P < 0.05.
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