Activation of store-operated channels by noradrenaline via protein kinase C in rabbit portal vein myocytes

Abstract

In the present study we have investigated the role of diacylglycerol (DAG) and protein kinase C (PKC) in mediating activation of Ca(2+)-permeable store-operated channels (SOCs) by noradrenaline in rabbit portal vein smooth muscle cells. With cell-attached recording, bath application of noradrenaline, 1-oleoyl-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu) evoked single channel currents. The biophysical properties of these channel currents were similar to those of the channel currents activated by depletion of internal Ca(2+) stores with cyclopiazonic acid (CPA). The activation of SOCs in cell-attached recording by noradrenaline, OAG, PDBu, CPA and the acetoxymethyl ester form of BAPTA (BAPTA-AM) was markedly inhibited by the PKC inhibitors chelerythrine and RO-31-8220. In isolated outside-out patches CPA did not evoke SOCs but noradrenaline stimulated SOC activity, which was reduced by about 90 % by PKC inhibitors. The addition of the serine/threonine phosphatase inhibitors calyculin A and microcystin also stimulated SOCs in isolated outside-out patches. It is concluded that in rabbit portal vein myocytes, noradrenaline activates SOCs via DAG and PKC, possibly by a store-independent mechanism. In addition in this cell type it appears that PKC and phosphorylation may play an important role in stimulating SOC activity in response to depletion of internal Ca(2+) stores by CPA and BAPTA-AM.

Figure 1. Noradrenaline activates single inward channel currents with similar amplitudes to CPA-evoked channel currents in cell-attached patches

Ai, application of 10 μM CPA induced single inward channel currents in a cell-attached patch at −70 mV. Here and in subsequent figures, in the expanded records the continuous line represents the closed level and the horizontal dashed lines represent open levels. Aii, amplitude histogram for the channel currents shown in Ai. The histogram could be fitted with one Gaussian curve with a peak amplitude of −0.21 pA. Bi, application of 10 μM noradrenaline activated single inward channel currents in a different cell-attached patch at −70 mV. Bii, the noradrenaline-evoked channel current amplitudes had a peak amplitude of −0.19 pA.

Figure 2. Current-voltage relationship of single channel currents evoked by noradrenaline in cell-attached patches

A, noradrenaline-evoked channel currents recorded at different membrane potentials from the same cell-attached patch with a pipette solution containing 1.5 mm ${\text{ca}}_0^{2+}$. B, amplitude histograms plotted from the corresponding channel currents shown in A. C, I-V relationship of the noradrenaline-evoked channel currents (○) shown in A. The I-V relationship had a slope conductance between −30 mV and −110 mV of 2 pS and an extrapolated E_r of +26 mV. For comparison the I-V relationship of CPA-evoked channel currents (•) shown in Fig. 1A is plotted, and this had a slope conductance of 2.3 pS and an extrapolated E_r of +34 mV.

Figure 3. Single channel conductance of noradrenaline-evoked channel currents in cell-attached patches recorded with a pipette solution containing 0 ca02+

A, noradrenaline-evoked channel currents recorded at different membrane potentials from the same cell-attached patch with a pipette solution containing 0 ${\text{ca}}_0^{2+}$. B, amplitude histograms plotted from the corresponding channel currents shown in A. C, I-V relationship of the noradrenaline-evoked channel currents shown in A. The I-V relationship had a slope conductance of 7.3 pS and a E_r of −6 mV.

Figure 4. Activation of single channel currents in cell-attached patches by the diacylglycerol analogue (OAG) and phorbol ester PDBu

A, application of 20 μM OAG activated single inward channel currents in a cell-attached patch recorded with a pipette solution containing 1.5 mm ${\text{ca}}_0^{2+}$. B, application of 1 μM PDBu activates single inward channel currents in a different cell-attached patch. C, I-V relationships of OAG-evoked channel currents recorded with pipette solutions containing either 0 or 1.5 mm ${\text{ca}}_0^{2+}$. In 1.5 mm ${\text{ca}}_0^{2+}$ (○) the I-V relationship had a slope conductance of 2.3 pS and an extrapolated E_r of +32 mV and in 0 ${\text{ca}}_0^{2+}$ (•) the slope conductance was 5.8 pS and the E_r was −2 mV. D, I-V relationship of PDBu-evoked channel currents recorded with a cell-attached patch pipette solution containing either 0 or 1.5 mm ${\text{ca}}_0^{2+}$. In 1.5 mm Ca²⁺ (○) the slope conductance was 2.2 pS and the extrapolated E_r was +34 mV and in 0 ${\text{ca}}_0^{2+}$ (•) the slope conductance was 6.1 pS and the E_r was −3 mV.

Figure 5. Effect of the PKC inhibitor chelerythrine on noradrenaline-, OAG-, PDBu- and CPA-evoked SOCs in cell-attached patches

Bath application of 3 μM chelerythrine (middle traces) for 5 min markedly reduced SOC activity evoked by 10 μM noradrenaline (A), OAG (B), PDBu (C) and CPA (D) in four different cell-attached patches. The left-hand traces show channel currents activated by 10 μM noradrenaline, 20 μM OAG, 1 μM PDBu and 10 μM CPA before application of chelerythrine and the right-hand traces show recovery of channel currents after wash out of chelerythrine. The holding potential in all cases was −70 mV.

Figure 6. Stimulation of SOCs by noradrenaline in isolated outside-out patches

A, current trace from an isolated outside-out patch recorded at −70 mV. The patch did not contain any spontaneous activity and bath application of 10 μM CPA for 5 min did not induce any SOC activity whereas subsequent bath application of 10 μM noradrenaline did evoke SOC activity. The dashed lines between current traces represent a time period of 1 min. B, I-V relationships of currents evoked by noradrenaline from outside-out patches which were recorded in 0 or 1.5 mm ${\text{ca}}_0^{2+}$. In 1.5 mm ${\text{ca}}_0^{2+}$ (○) the I-V relationship had a slope conductance between −40 mV and −130 mV of 3.6 pS and an extrapolated E_r of +25 mV. In 0 ${\text{ca}}_0^{2+}$ (•) the slope conductance was 9.2 pS and the E_r was +1 mV. C, open time distribution of the noradrenaline-evoked channel currents shown in A. The open time distributions could be described by the sum of two exponentials with time constants of 6.4 ms and 34 ms. Note that the seal resistance was smaller in isolated, compared to cell-attached, patches which made the records appear slightly ‘noisier’.

Figure 7. Noradrenaline-activated SOCs in outside-out patches are inhibited by chelerythrine and SOC activity is evoked by bath application of calyculin-A

Ai, control current trace showing an outside-out patch that did not contain any spontaneous SOC activity but did contain spontaneous I_cat activity. Aii, bath application of 10 μM noradrenaline activated SOC activity in the same patch shown in Ai. Aiii, bath application of 3 μM chelerythrine in the presence of 10 μM noradrenaline for 5 min markedly reduced SOC activity shown in B but did not inhibit the activity of I_cat. Aiv, recovery of SOC activity after wash out of chelerythrine. All current traces are at a holding potential of −70 mV. The continuous horizontal line is the closed level and the upper dashed line (I_soc) represents the open level of SOCs and the lower dashed line represents the open level of the channels underlying I_cat. B, bath application of 1 μM calyculin-A activated single channel currents recorded in an outside-out patch at −70 mV.