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Journal of Physiology (2002), 544.1, pp. 113-125
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.022574

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

A. P. Albert and W. A. Large

	ABSTRACT

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In the present study we have investigated the role of diacylglycerol (DAG) and protein kinase C (PKC) in mediating activation of Ca²⁺-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 Ca²⁺ 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 RÖ-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 Ca²⁺ stores by CPA and BAPTA-AM.

(Received 15 April 2002; accepted after revision15 July 2002; first published online 2 August 2002)
Corresponding author A. P. Albert: Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK. Email: aalbert{at}sghms.ac.uk

	INTRODUCTION

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Store-operated channels (SOCs) are plasmalemmal Ca²⁺-permeable cation channels that are activated by depletion of internal Ca²⁺ stores, usually endoplasmic reticulum or sarcoplasmic reticulum (SR). This Ca²⁺-influx pathway, which has been described in many non-electrically excitable cells, is important for refilling internal Ca²⁺ stores and may be involved in diverse cellular functions including secretion and cell proliferation (see Parekh & Penner, 1997).

In smooth muscle there have been several reports of store-operated non-selective cation currents (mouse anococcygeus, Wayman et al. 1996; mouse aorta, Trepakova et al. 2000, 2001; rabbit choroidal arteriolar myocytes, Curtis & Scholfield, 2001; human and rat pulmonary artery, Golovina et al. 2001; Ng & Gurney, 2001; rabbit portal vein, Albert & Large, 2002). Moreover it was proposed in several of these reports that SOCs are involved in producing smooth muscle contraction (see review by McFadzean & Gibson, 2002). Single channel studies reveal that SOCs in different vascular smooth muscle preparations have markedly different biophysical properties. Thus, the estimated relative permeability of Ca²⁺ to Na⁺ ions (P_Ca/P_Na) was approximately 50 in rabbit portal vein myocytes (Albert & Large, 2002) whereas in mouse aortic myocytes P_Ca/P_Na was ~1 (Trepakova et al. 2001). In addition, the unitary conductance of the SOC in rabbit portal vein is altered in different external Ca²⁺ concentrations (1.5 mM Ca, 2-3 pS; 110 mM Ca, ~1 pS; 0 mM Ca, ~7 pS; Albert & Large, 2002). In contrast, in mouse aortic smooth muscle cells the single channel conductance is not altered by changing [Ca²⁺]_o (~3 pS with all the above Ca²⁺ concentrations; Trepakova et al. 2001). These data show that SOCs in vascular smooth muscle have different properties and may represent separate molecular entities.

Much attention has focused on the mechanism(s) underlying the activation of SOCs. The two main hypotheses are the conformational coupling model in which 1,4,5-inositol trisphosphate (IP₃) receptors located on the Ca²⁺ stores directly couple to the SOCs. Secondly it has been proposed that a diffusible factor (calcium influx factor) is released from the internal Ca²⁺ stores in response to Ca²⁺ depletion (see Parekh & Penner, 1997). In many of these studies inhibitors of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA), such as thapsigargin and cyclopiazonic acid (CPA), are used to deplete internal stores. However physiologically, in vascular smooth muscle, internal Ca²⁺ stores are depleted by agonists, which activate G-protein-coupled receptors to increase the activity of phospholipase C (PLC) and the subsequent production of IP₃ acts on IP₃ receptors on the SR to release Ca²⁺ ions. The other product of phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis is diacylglycerol (DAG) which produces diverse cellular effects, in some cases mediated by protein kinase C (PKC). Previously we have demonstrated that in freshly dispersed rabbit portal vein smooth muscle cells noradrenaline acts on -adrenoceptors to evoke a Ca²⁺-permeable non-selective cation current (I_cat) that is not activated by store depletion (Byrne & Large, 1988; Wang & Large, 1991). The activation of I_cat involves G-protein, PLC and DAG via a PKC-independent mechanism (Helliwell & Large, 1997).

In the present study we have explored whether DAG and PKC are involved in the activation of SOCs by noradrenaline. We have used cell-attached and outside-out patch recording of single channel currents in freshly dispersed rabbit portal vein myocytes to show that noradrenaline stimulates -adrenoceptors to activate SOCs via DAG and PKC, possibly in a store-independent manner. Moreover, activation of SOCs by CPA and BAPTA-AM is also reduced by PKC inhibitors and SOCs can be activated by serine/threonine phosphatase inhibitors suggesting a pivotal role for PKC and phosphorylation in the opening of SOCs in vascular smooth muscle.

	METHODS

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Cell isolation

New Zealand White rabbits (2-3 kg) were killed by an I.V. injection of sodium pentobarbitone (120 mg kg^-1) according to the Animals (Scientific Procedures) Act 1986 and the portal vein was removed and placed into normal physiological salt solution (PSS). The tissue was dissected free of connective tissue and fat before being cut into strips and placed in Ca²⁺-free (0 Ca) PSS. The tissue was enzymatically dispersed in two sequential enzyme steps. First, the strips of tissue were incubated in 0 Ca PSS with 0.1-0.3 mg ml^-1 protease type XIV (Sigma Chemical Co., Poole, UK) for 5 min and then the strips were washed in 0 Ca PSS. In the second step the strips were incubated with 0.5-1 mg ml^-1 collagenase type IA (Sigma) in PSS containing 100 µM Ca (100 µM Ca²⁺ PSS) for 10 min and were then washed in 100 µM Ca PSS. All enzyme and wash procedures were carried out at 37 °C. After the enzyme treatments the strips were incubated in 100 µM Ca PSS at room temperature (20-25 °C) for 10 min before the cells were released into the solution by gentle mechanical agitation of the strips of tissue using a wide-bore Pasteur pipette. The suspension of cells was then centrifuged (100 g) to form a loose pellet which was resuspended in 0.75 mM Ca PSS. The cells were then plated onto glass coverslips and stored at 4 °C before use (1-6 h).

The normal PSS contained (mM): NaCl 126, KCl 6, CaCl₂ 1.5, MgCl₂ 1.2, glucose 10 and Hepes 11; pH was adjusted to 7.2 with 10 M NaOH. 0 Ca²⁺ PSS, 100 µM Ca PSS and 0.75 mM Ca PSS had the same composition except that Ca²⁺ was omitted, or 1.5 mM CaCl₂ was replaced by 100 µM CaCl₂ or 0.75 mM CaCl₂, respectively.

Electrophysiology

Single cation channel currents were recorded with a L/M-PC (List Electronics, Darmstadt, Germany) patch clamp amplifier at room temperature using cell-attached and outside-out patch configurations of the patch clamp technique (Hamill et al. 1981). Patch pipettes were manufactured from borosilicate glass and were routinely coated in Sylgard (Dow Corning, Germany) to reduce stray capacitance and fire polished to increase seal resistance giving pipette resistances of approximately 10 M when filled with the patch pipette solution. To reduce 'line' noise the recording chamber (volume ca 150-200 µl) was perfused using two 10 ml syringes, one filled with external solution and the other used to drain the chamber, in a 'push and pull' technique. The time taken for the holding current to maximally alter on exchanging an external NaCl solution for an external KCl solution was approximately 20 s, indicating that exchange of solutions within the recording chamber occurred within this time period. Once the solution had been exchanged within the recording chamber, the flow was stationary. To evaluate the characteristics of unitary single channel current-voltage (I-V) characteristics the membrane potential was manually stepped between -130 mV and +100 mV.

Single channel currents were initially recorded onto digital audiotape (DAT) using a CDATA digital tape recorder (Cygnus Technology Inc., Delaware, PA, USA) at a bandwidth of 1 kHz (-3 dB, low pass 4-pole Bessel filter, List L/M-PC patch clamp amplifier) and a sample rate of 48 kHz. For off-line analysis, single channel records were filtered at 100 Hz (-3 dB, low pass 8-pole Bessel filter, Frequency Devices, model LP02, Scensys Ltd, Aylesbury, UK) and acquired using a CED 1401plus interface and CED Patch and Voltage Clamp Software (Version 6.0, Cambridge Electronic Design Ltd, Cambridge, UK) at a sampling rate of 1 kHz. Data were captured with a Pentium (P5-100) personal computer (Gateway, Ireland).

Channel current amplitudes were calculated from idealised traces of at least 10 s in duration using the half-amplitude crossing method and from fitting Gaussian curves to amplitude histograms, defined by the area under the curve and reflecting the peak of the majority of events. I-V relationships, from channel current peak amplitudes of an individual patch, were plotted and the unitary conductance and reversal potential (E_r) were calculated from the slope of the relationship using linear regression (Origin software; OriginLab Corp., Northampton, MA, USA). Values from the individual patches were then used to calculate mean slope conductance and mean E_r values. These values were similar to those obtained when data were pooled from individual patches. Therefore the data shown in the results are the mean data from individual patches. Single cation channel currents were not resolved at membrane potentials positive to-20 mV with most solutions and therefore the E_r was estimated using extrapolation. Open time distributions were plotted using a 2-5 ms bin width and where appropriate, were fitted with one or more exponential functions using the maximum likelihood method. Events which lasted for < 6.64 ms (2 rise time (T_r) for a low pass filter of 100 Hz (-3dB)) were excluded from analysis. Figure preparation was carried out using Origin software. Inward channel currents are shown as downward deflections and outward currents as upward deflections. Most patches contained more than one open level suggesting the presence of more than one channel in a patch and therefore the apparent open probability (NP_o) was calculated using the equation:

NP_o = total open time/sample duration/number of

open levels in a patch.

To measure the reduction of noradrenaline-, OAG-, PDBu-and CPA-activated channel activity by PKC inhibitors, the apparent NP_o was calculated from idealised traces of at least 30 s in duration prior to application of the inhibitor and then recalculated 5 min after adminstration of the inhibitor in the presence of the agonist. The reduction in mean apparent NP_o and the mean percentage reduction in apparent NP_o were then calculated. To assess whether outside-out patches contained functional intracellular Ca²⁺ stores, CPA was bath applied to patches for at least 5 min before application of noradrenaline.

Solutions and drugs

In the cell-attached patch experiments, the cells were perfused in a KCl external solution containing (mM): KCl 126, CaCl₂ 1.5, Hepes 10 and glucose 11; pH adjusted to 7.2 with 10 M NaOH to set the membrane potential to approximately 0 mV. Nicardipine (3-5 µM) was also included to prevent smooth muscle cell contraction by blocking Ca²⁺ entry through voltage-dependent Ca²⁺ channels. The standard 126 mM NaCl cell-attached patch pipette solution was K⁺-free and contained (mM): NaCl 126, CaCl₂ 1.5, Hepes 10, glucose 11, TEA 10, 4-aminopyridine (4-AP) 5, and 100 µM DIDS, 100 µM niflumic acid and 3-5 µM nicardipine; pH was adjusted to 7.2 with NaOH which resulted in an increase in external Na⁺ concentration by approximately 5 mM (310 ± 5 mosmol l^-1). In the 0 Ca²⁺ pipette solution, 1.5 mM CaCl₂ was omitted and 1 mM BAPTA-AM was added to chelate the Ca²⁺ to a concentration of < 10 nM (calculated using EqCal software; BIOSOFT, Cambridge, UK). Under these conditions voltage-gated Ca²⁺, K⁺, swell-activated Cl^- and Ca²⁺-activated Cl^- currents are abolished allowing the recording of non-selective single cation currents in isolation. The external 0 K⁺ solution used in the outside-out patch experiments contained (mM): NaCl 126, CaCl₂ 1.5, Hepes 10 and glucose 11; pH was adjusted to 7.2 with 10 M NaOH. With outside-out patches the pipette solution contained (mM): CsCl 18, caesium aspartate 108, MgCl₂ 1.2, Hepes 10, glucose 11, BAPTA-AM 10, CaCl₂ 4.5 (free internal calcium concentration was approximately 100 nM as calculated using EQCAL software), Na₂ATP 1, and NaGTP 0.2; pH was adjusted to 7.2 with Tris. Under these conditions the equilibrium potential of Cl^- ions (E_Cl) was approximately -46 mV and the junction potential was < 3 mV and was not compensated. Propranolol (1 µM) was added to all noradrenaline-containing solutions to prevent stimulation of -adrenoceptors. Stock solutions (10 mM) of CPA, OAG, phorbol esters, chelerethyrine and RÖ-31-8220 were made up in DMSO and were then diluted in external solution to the final concentration at volumes of 0.1 %. In control experiments using cell-attached and outside-out patches (n = 3 for each) application of an external solution containing 0.1 % DMSO had no effect on single channel activity.

3-1-3-(Amidinthio)propyl-1H-indol-3yl)-3 -(1-methyl-1H-indol-3-yl)maleimide, bisindolylmaleimide IX, methanesulfonate (RÖ-31-8220) and microcystin-LF were purchased from Calbiochem (CN Biosciences (UK) Ltd, Nottingham, UK) and all other drugs were from Sigma. The values are the mean of n cells ± S.E.M. Statistical analysis was carried out using Student's t test (paired and unpaired) with the level of significance set at P < 0.05.

	RESULTS

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Noradrenaline activates single cation channel currents with similar characteristics to SOCs

In the first series of experiments we compared the properties of single channel currents evoked by CPA and noradrenaline, recorded with cell-attached patches using a pipette solution containing 1.5 mM Ca.

Figure 1Ai shows that, at a holding potential of -70 mV, bath application of 10 µM CPA activated inward channel currents in a cell-attached patch which previously did not contain any channel activity. In eight cell-attached patches, 10 µM CPA evoked channel activity after a mean delay of 134 ± 22 s and had an apparent NP_O of 0.15 ± 0.04. Figure 1Aii shows the event amplitude histogram of the CPA-evoked channel currents shown in Fig. 1Ai. The amplitudes of the channel currents could be fitted by a single Gaussian curve with a peak amplitude of -0.21 pA. Figure 1B shows that bath application of 10 µM noradrenaline also activated inward channel currents in a different cell-attached patch (Fig. 1Bi), which had a similar peak amplitude of -0.19 pA at -70 mV (Fig. 1Bii). In five patches, 10 µM noradrenaline evoked channel activity with an apparent NP_O of 0.07 ± 0.02 after a mean delay of 53 ± 21 s.

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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.

To measure the unitary conductance of noradrenaline-evoked channel currents in cell-attached patches we plotted the peak amplitude of the channel currents at different membrane potentials, and the original records and amplitude histograms are shown in Fig. 2A and B. Figure 2C shows the current-voltage (I-V) relationship of the noradrenaline-evoked channel currents shown in Fig. 2A which had a slope conductance of 2 pS between -30 mV and -130 mV. In five different patches, noradrenaline-activated channel currents had a mean single channel conductance of 2.0 ± 0.3 pS (Table 1). A feature of CPA-evoked channel activity is that it is not resolved at positive membrane potentials (Albert & Large, 2002). In this study it was not possible, either, to resolve noradrenaline-evoked channel currents at positive membrane potentials and therefore, as with the CPA-evoked channel currents (Albert & Large, 2002), the E_r of the channel currents could not be interpolated but was instead estimated by extrapolation. Figure 2C shows that the extrapolated E_r of the noradrenaline-evoked channel currents was +26 mV and Table 1 shows that the mean E_r was +36 ± 15 mV (n = 5). Figure 2C also shows the I-V relationship of CPA-activated channel currents shown in Fig. 1A, and Table 1 shows that the mean unitary conductance and E_r of noradrenaline-evoked channel currents are similar to those evoked by CPA.

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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 Ca. 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 Er 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 Er of +34 mV.

Single channel conductance of noradrenaline-evoked channel currents is increased using a cell-attached patch pipette solution containing 0 Ca

In our previous study (Albert & Large, 2002) we showed that a notable characterisitic of SOCs in rabbit portal vein myocytes is that using a cell-attached patch pipette solution containing 0 Ca the unitary conductance was increased 2- to 3-fold compared to SOCs recorded in 1.5 mM Ca. In addition, in 0 Ca PSS SOCs are resolved at positive membrane potentials and have a E_r of about 0 mV. To investigate further whether noradrenaline activates the same SOCs as those evoked by depletion of internal Ca²⁺ stores with CPA we measured the single channel conductance and E_r of noradrenaline-activated channel currents with a cell-attached patch pipette solution containing 0 Ca.

Figure 3A shows noradrenaline-evoked channel activity in a cell-attached patch recorded with a pipette solution containing 0 Ca. Figure 3B shows that the peak amplitude of noradrenaline-evoked channel currents is greater in compared to those recorded in 1.5 mM Ca PSS (cf. Fig. 1B and Fig. 2B). In 1.5 mM Ca PSS the mean peak amplitude of noradrenaline-evoked channel currents was 0.21 ± 0.01 pA (n = 5) at -70 mV whereas in 0 Ca PSS the mean peak amplitude was increased significantly (unpaired t test, P < 0.001) to 0.42 ± 0.03 pA (n = 5). Moreover it can be seen that outward single channel currents can be resolved at positive potentials (Fig. 3A) and therefore E_r can be measured by interpolation. Figure 3C shows that the I-V relationship of noradrenaline-evoked channel currents shown in Fig. 3A had a slope conductance of 7.3 pS and E_r was -6 mV. In five patches with 0 Ca PSS, noradrenaline-activated channel currents had a mean single channel conductance of 6.5 ± 0.3 pS and a mean E_r of -6 ± 4 mV. Therefore the properties of the channel currents in 0 Ca PSS are similar in the presence of CPA or noradrenaline indicating that both agents activate the same SOC.

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Figure 3. Single channel conductance of noradrenaline-evoked channel currents in cell-attached patches recorded with a pipette solution containing 0 Ca A, noradrenaline-evoked channel currents recorded at different membrane potentials from the same cell-attached patch with a pipette solution containing 0 Ca. 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 Er of -6 mV.

The diaceylglycerol analogue OAG and the phorbol ester PDBu activate single channel currents with properties similar to SOCs

It is possible that activation of SOCs by noradrenaline was mediated by G-protein activation with subsequent production of IP₃ and release of Ca²⁺ ions from the SR. However it is also possible that the other product of PIP₂ hydrolysis, DAG, may activate SOCs. Therefore we investigated whether the DAG analogue OAG could open SOCs in cell-attached patches.

Figure 4A shows the effect of 20 µM OAG on a cell-attached patch recorded with a pipette solution containing 1.5 mM Ca. Bath application of 20 µM OAG activated inward channel currents at -70 mV (Fig. 4A) with a mean delay of 48 ± 11 s (n = 8). Figure 4C shows the I-V relationship of the OAG-evoked channel currents shown in Fig. 4A, which had a slope conductance of 2.3 pS and a E_r of +32 mV in 1.5 mM Ca, and Table 1 shows the mean data from five patches.

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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 Ca. 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 Ca. In 1.5 mM Ca () the I-V relationship had a slope conductance of 2.3 pS and an extrapolated Er of +32 mV and in 0 Ca () the slope conductance was 5.8 pS and the Er 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 Ca. In 1.5 mM Ca2+ () the slope conductance was 2.2 pS and the extrapolated Er was +34 mV and in 0 Ca () the slope conductance was 6.1 pS and the Er was -3 mV.

Many of the cellular effects of DAG are mediated by PKC and therefore we tested whether phorbol esters, which directly stimulate PKC, evoked SOCs by bath application of the phorbol ester PDBu to cell-attached patches.

Bath application of 1 µM PDBu activated inward channel currents at -70 mV (Fig. 4B) with a mean delay of 59 ± 18 s (n = 10). Figure 4D shows that the PDBu-evoked channel currents shown in Fig. 4B had a unitary conductance of 2.2 pS and a E_r of +34 mV in 1.5 mM Ca²⁺_o, and Table 1 shows the mean data from five patches.

To ensure that the channel currents activated by OAG and PDBu are the same SOCs as those evoked by depletion of internal Ca²⁺ stores with CPA and noradrenaline we measured the single channel conductance in 0 Ca PSS. Figure 4C and D shows I-V relationships of OAG- and PDBu-evoked channel currents recorded with pipette solutions containing 0 Ca. In Fig. 4C the OAG-activated channel currents had a slope conductance of 5.8 pS and a E_r of -2 mV and in five patches the mean slope conductance was 5.7 ± 0.3 pS and the E_r was -2 ± 2 mV. In 0 Ca PSS, the PDBu-evoked channel currents had a slope conductance of 6.1 pS and a E_r of -3 mV (Fig. 4D) and in four patches the mean slope conductance was 5.5 ± 0.5 pS and the mean E_r was -4 ± 2 mV.

CPA-, noradrenaline-, OAG- and PDBu-evoked single channel currents have similar open time distributions

An additional property of SOCs activated by CPA is that the distribution of channel open times can be fitted by two exponentials with time constants of about 5 ms and 30 ms (Albert & Large, 2002). Therefore we compared the open times of channels opened by CPA, noradrenaline, OAG and PDBu. All four open time distributions could be fitted by the sum of two exponentials using the maximum likelihood method with time constants of approximately 6 ms (O₁) and 30 ms (O₂). The mean values are shown in Table 1 and the data provide further evidence that noradrenaline, OAG and PDBu activate the same SOCs as those evoked by CPA, and moreover that stimulation of DAG and PKC may be involved in the transduction pathway linking -adrenoceptors to SOCs.

SOC activity is markedly reduced by PKC inhibitors

The data from experiments with PDBu suggested that PKC played an important role in activating SOCs and therefore we tested this further by investigating the effect of the PKC inhibitors chelerythrine and RÖ-31-8220 on channel activity evoked by noradrenaline, OAG, PDBu and CPA in cell-attached patches with a pipette solution containing 1.5 mM Ca. Figure 5 shows the effect of bath application of 3 µM chelerythrine on channel activity evoked by previously applied noradrenaline, OAG, PDBu and CPA. Figure 5A shows that noradrenaline-evoked channel activity was markedly reduced after 5 min application of 3 µM chelerythrine in the presence of 10 µM noradrenaline. The apparent NP_O of noradrenaline-evoked channel activity was reduced by 93 % from 0.07 to 0.005, but recovered on wash out of chelerythrine. In four patches, application of 3 µM chelerythrine reduced the mean NP₀ of single channel activity evoked by10 µM noradrenaline from 0.05 ± 0.01 to 0.008 ± 0.004, a reduction of 86 ± 6 %. Bath application of 3 µM chelerythrine also markedly reduced the mean NP_O of OAG-evoked (Fig. 5B) and PDBu-evoked (Fig. 5C) channel activity from 0.17 ± 0.07 to 0.004 ± 0.003 (n = 4) and from 0.08 ± 0.03 to 0.012 ± 0.003 (n = 4), respectively, representing reductions of 97 ± 2 and 86 ± 7 %. It is interesting that bath application of 3 µM chelerythrine for 5 min reduced the mean NP₀ of CPA-evoked channel activity (Fig. 5D) from 0.09 ± 0.01 to 0.009 ± 0.002 (n = 4), an inhibition of 92 ± 3 %. In all cases the effect of chelerythrine was statistically significant (paired t test, P < 0.005) and reversible (right-hand traces in Fig. 5A-D).

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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.

The inhibition of CPA-evoked SOC activity by chelerythrine was unexpected and therefore we tested the effect of chelerythrine on SOC activity induced by the cell-permeant Ca²⁺-chelating agent BAPTA-AM. In five cell-attached patches in which SOC activity was induced by 50 µM BAPTA-AM, NP_O was reduced from a mean value of 0.174 ± 0.02 in the presence of BAPTA-AM alone to 0.009 ± 0.001 in the presence of BAPTA-AM and 3 µM chelerythrine (a reduction of 94 ± 2 %).

To provide further evidence for the involvement of PKC in activating SOCs we assessed the effect of another PKC inhibitor, RÖ-31-8220, on channel activity evoked by PDBu and CPA in cell-attached patches. Bath application of 3 µM RÖ-31-8220 for 5 min markedly reduced the mean apparent NP_O of PDBu-evoked channel activity from 0.15 ± 0.04 to 0.004 ± 0.003 (n = 4, a decrease of 96 ± 2 %) and reduced the mean apparent NP_O of CPA-evoked channel activity from 0.21 ± 0.08 to 0.02 ± 0.01 (n = 4, a decrease of 92 ± 3 %).

Noradrenaline activates SOCs in isolated outside-out patches via PKC

The present study suggests that noradrenaline activates SOCs via a transduction pathway involving DAG and PKC. The activation of PKC by DAG is often thought to occur at, or near, the inner surface of the plasma membrane, which prompted the idea that activation of SOCs by noradrenaline via DAG and PKC may occur independently of depletion of internal Ca²⁺ stores. To investigate this possibility we activated SOCs with noradrenaline in isolated outside-out patches. This configuration also has the advantage of controlling the Ca²⁺ concentration at the internal surface of the membrane and in these experiments Ca was clamped at 100 nM (see Methods).

First we tested the effect of bath application of CPA to outside-out patches to see if functional Ca²⁺ stores were attached to the isolated patch. Figure 6A shows that bath application of 10 µM CPA for 5 min did not evoke any channel activity but subsequent application of 10 µM noradrenaline activated single inward channel currents with properties similar to SOCs. In nine outside-out patches, application of 10 µM CPA did not produce SOC activity but subsequent bath application of 10 µM noradrenaline activated inward channel currents with a mean delay of 82 ± 16 s, a mean peak amplitude of -0.35 ± 0.01 pA and a mean apparent NP_O of 0.06 ± 0.01 at -70 mV. Figure 6B shows that the I-V relationship of the noradrenaline-evoked channel currents had a slope conductance of 3.6 pS and the extrapolated E_r was +25 mV. As with cell-attached recordings, the channel currents were not resolved at positive membrane potentials. In four outside-out patches the mean slope conductance was 3.2 ± 0.3 pS and the mean E_r was +24 ± 11 mV. Figure 6B also shows that the I-V relationship of the noradrenaline-evoked channel currents shown in Fig. 6A recorded in 0 Ca PSS had a slope conductance of 9.2 pS and a E_r of +1 mV. In four patches recorded in 0 Ca PSS the mean unitary conductance was 9.3 ± 0.7 pS and the mean E_r was -3 ± 2 mV.

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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 Ca. In 1.5 mM Ca () the I-V relationship had a slope conductance between -40 mV and -130 mV of 3.6 pS and an extrapolated Er of +25 mV. In 0 Ca () the slope conductance was 9.2 pS and the Er 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 6C shows the open time distribution of the noradrenaline-evoked channel currents shown in Fig. 6A. The open time distribution could be described by the sum of two exponentials with time contants of about 6 ms (O₁) and 30 ms (O₂). In five patches the mean faster time constant (O₁) was 5.5 ± 0.3 ms and the mean slower time constant (O₂) was 25 ± 2 ms. These results indicated that noradrenaline stimulates SOCs in isolated outside-out patches where CPA does not evoke channel activity suggesting that noradrenaline evokes SOCs by activating a store-independent pathway.

To investigate whether PKC was involved in the pathway linking the -adrenoceptor to the SOC in isolated patches we studied the effect of chelerythrine on noradrenaline-evoked channel currents activated in outside-out patches and Fig. 7A illustrates a typical experiment. Figure 7Ai shows that in an isolated outside-out patch under control conditions there is no spontaneous SOC activity. However, there are spontaneous larger amplitude channel currents present, which in this patch had a slope conductance of 22 pS, a E_r of +13 mV and an apparent NP_O of 0.03. This spontaneous larger conductance channel is the non-selective cation channel, I_cat, previously described in outside-out patches (Albert & Large, 2001a, b). Figure 7Aii shows that bath application of 10 µM noradrenaline activates SOCs so that the single channel currents underlying both I_cat and the SOC were observed. Figure 7Aiii shows that after subsequent application of 3 µM chelerythrine in the presence of 10 µM noradrenaline the activity of SOCs was markedly reduced. The apparent NP_O of the noradrenaline-evoked SOC activity was reduced from 0.06 to 0.004, representing a 93 % decrease in activity. In four patches, bath application of 3 µM chelerythrine reduced the apparent NP_O of noradrenaline-evoked SOC activity from 0.07 ± 0.01 to 0.004 ± 0.002, a mean decrease of 95 ± 2 %. However, it should be noted that application of 3 µM chelerythrine had no effect on the spontaneous activity of I_cat (Fig. 7Aiii) since I_cat is activated via a PKC-independent mechanism (Helliwell & Large, 1997). This latter result shows that chelerythrine was not acting as a non-selective blocker of Ca²⁺-permeable cation channels. Figure 7Aiv shows that noradrenaline-evoked SOC activity recovered after 3 µM chelerythrine was washed out.

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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 Icat 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 Icat. 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 (Isoc) represents the open level of SOCs and the lower dashed line represents the open level of the channels underlying Icat. B, bath application of 1 µM calyculin-A activated single channel currents recorded in an outside-out patch at -70 mV.

Application of the serine/threonine phosphatase inhibitors calyculin A and microcystin-LF activate channel currents with similar properties to SOCs

The present work suggests that PKC plays a pivotal role in the activation of SOCs by the stimulation of -adrenoceptors and also by depletion of internal Ca²⁺ stores with CPA. Therefore it seems that phosphorylation is important in the opening of SOCs. To investigate whether phosphorylation is involved in activating SOCs, we applied the cell-permeant serine/threonine phosphatase inhibitors calyculin A and microcystin-LF to outside-out patches.

Figure 7B illustrates the representative data from one outside-out patch recorded with a pipette solution containing 100 nM Ca at -70 mV. Figure 7B shows that bath application of 1 µM calyculin A activated inward channel currents with similar properties to SOCs. In four outside-out patches, 1 µM calyculin A evoked channel activity with a mean delay of 165 ± 76 s. In four patches, the mean single channel conductance of calyculin A-evoked channel currents was 2.9 ± 0.6 pS and the mean E_r was +35 ± 10 mV. The open time distributions at -70 mV could be fitted with two exponentials with a mean faster time constant (O₁) of 6.3 ± 1 ms and a mean slower time constant (O₂) of 32 ± 4 ms (n = 4). Bath application of another cell-permeant serine/threonine phosphatase inhibitor, microcystin-LF, activated single channel currents in isolated outside-out patches with similar properties to those of the calyculin-A-evoked channel currents (n = 5, data not shown). In addition, bath application of calyculin-A (n = 5) and microcystin-LF (n = 5) to outside-out patches recorded with a pipette solution containing Ca²⁺ clamped at a concentration of 14 nM also activated single channel currents with similar properties to those recorded with a pipette solution containing Ca²⁺ clamped at a concentration of 100 nM (data not shown).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The present results indicate that in freshly dispersed rabbit portal vein smooth muscle cells noradrenaline activates SOCs via a pathway that appears to involve DAG and stimulation of PKC.

Pathway linking -adrenoceptors to SOCs

The experiments with noradrenaline were carried out in the presence of a -adrenoceptor antagonist and therefore noradrenaline-evoked SOCs are mediated by -adrenoceptors. With cell-attached recording it was demonstrated that noradrenaline, OAG and PDBu activated single channel currents with the same biophysical properties as channel currents evoked by CPA and BAPTA-AM in terms of: (i) unitary conductance in 0 Ca PSS and 1.5 mM Ca; (ii) E_r values and hence corresponding P_Ca/P_Na estimates; and (iii) distribution of open times (cf. present data with Albert & Large, 2002). Therefore it can be concluded that noradrenaline, OAG and PDBu stimulate SOC activity. Moreover since PKC inhibitors markedly reduced the effect of all three agents it seems that PKC plays a pivotal role in these effects.

Also in isolated patches with the Ca²⁺_i concentration clamped at 100 nM, noradrenaline activated SOCs, which had similar biophysical characteristics to SOCs activated in the cell-attached configuration, and were also inhibited by PKC inhibitors. Therefore, taking the cell-attached and isolated patch data together, the simplest explanation for our results is that the classical DAG-PKC branch of the phosphoinositide pathway links the -adrenoceptor to the SOC. In this scheme, G-protein activation increases PLC activity to produce DAG to stimulate PKC which leads to channel opening. A role for a phosphorylation step being involved in the activation of SOCs is supported by the observation that the serine/threonine phosphatase inhibitors, calyculin-A and microcystin-LF, activated SOCs in unstimulated cells.

Can SOCs be opened by a store-independent mechanism?

The accepted mechanism for agonist-evoked SOC activity is that noradrenaline stimulates the production of IP_3, causing the subsequent release of Ca²⁺ ions from the SR, which leads to SOC opening by an unknown mechanism (see Introduction). However, the present results show that noradrenaline stimulates SOC activity via DAG and PKC, which often produce their biological effects within the membrane domain. This prompts the question: can SOCs be activated by a store-independent mechanism? In our experiments CPA did not stimulate SOC opening in isolated outside-out patches, which suggests that functional Ca²⁺ stores are not normally attached to the membrane in outside-out patches from our cells. It is possible that fragments of the SR were attached to the membrane but that the conditions used (e.g. 10 mM BAPTA) prevented signalling between the SR and SOCs. Nevertheless, under the same conditions, subsequent addition of noradrenaline to those patches evoked SOC activity, which was blocked by PKC inhibitors. These data suggest that noradrenaline can open SOCs independently of Ca²⁺ store depletion via a PKC pathway. This is not to imply that the mechanism using PKC is the sole pathway responsible for opening of SOCs by noradrenaline and there may be several pathways involved in SOC activation, including diffusible factors and/or conformational coupling interactions. Nevertheless in our experiments SOC activity induced by Ca²⁺ store depletion by CPA and BAPTA-AM was markedly reduced by PKC inhibitors. Therefore it appears that PKC has an obligatory role in SOC activation by both G-protein-coupled receptors and store depletion in rabbit portal vein myocytes. The present experiments do not elucidate how PKC is involved in SOC activity induced by store depletion, although one explanation is that constitutive PKC activity may be necessary for the store depletion mechanism to open SOCs.

The above comments indicate that noradrenaline may activate SOCs in a membrane-delimited manner. However, it should be noted that it is uncertain what cellular constituents are present in outside-out patches. Moreover the lag between the application of noradrenaline and the onset of channel activity (about 50 s) might argue against a membrane-delimited mechanism. However, the kinetics of the mechanisms underlying SOC activation are unknown. In our proposed model involving PKC there is presumably a balance between kinase and phosphatase activity and it might take some time for avid phosphatase activity to be overcome before channel opening occurs.

Comparison with other tissues

In human carcinoma A431 cells it has been shown that store depletion and PLC-dependent mechanisms converge on the same Ca²⁺-permeable channel (Kaznacheyeva et al. 2001). It is interesting that the unitary conductance in different external cation solutions and the gating properties of SOCs in A431 cells and rabbit portal vein myocytes are similar (cf. Kaznacheyeva et al. 2001 and present data). However, no evidence for the involvement of PKC inactivation of SOCs in A431 cells was provided. Rosado & Sage (2000) demonstrated a non-capacitative Ca²⁺ entry mechanism in human platelets in addition to store-operated influx. This pathway was stimulated by G-protein-coupled receptors, OAG and PKC. However, in these experiments using platelets loaded with the fluorescent Ca²⁺ indicator fura-2 it was not possible to conclude whether store-operated and non-capacitative mechanisms used the same channel. An important discrepancy from the present work was that PKC inhibitors did not reduce store depletion-evoked Ca²⁺ entry in human platelets (Rosado & Sage, 2000) whereas in our work chelerythrine and RÖ-31-8220 markedly reduced the activity of CPA-evoked channel currents. This latter result suggests that in rabbit portal vein smooth muscle cells PKC may play a central role in the opening of SOCs. In relation to this question, in several cell types Broad et al. (2001) found that the PLC inhibitor U73122 blocked capacitative Ca²⁺ entry and concluded that this mechanism required basal PLC activity, although no experiments on PKC were carried out. Compatible with the latter conclusion, as suggested earlier, it is possible that constitutive PKC activity is necessary for CPA-induced SOC activation in rabbit portal vein myocytes. CPA did not induce SOC opening in isolated-patches where noradrenaline was effective and therefore CPA does not directly stimulate PKC.

A further observation that requires comment is that calyculin-A induced SOC activity, since this agent has been commonly used to inhibit capacitative Ca²⁺ influx in several studies (e.g. Ma et al. 2000, 2002; Xie et al. 2002). This discrepancy might be due to the differences in cell types and/or experimental protocols used. However, it should be pointed out that fluorescent dyes were used to monitor SOC activity in those experiments in which calyculin-A inhibited capacitative divalent cation entry. In the present work SOC activity was recorded directly with patch pipettes and, in addition, another phosphatase inhibitor, microcystin-LF, also evoked SOC activity.

-Adrenoceptors are linked to two Ca²⁺-permeable non-selective cation channels in rabbit portal vein myocytes

Noradrenaline activates two types of Ca²⁺-permeable non-selective cation channels in freshly dispersed rabbit portal vein smooth muscle cells. These native conductances have some similarities but some notable differences. The first current to be described was activated by noradrenaline acting on -adrenoceptors but was not stimulated by depletion of internal Ca²⁺ stores and was termed I_cat (Byrne & Large, 1988; Wang & Large, 1991). The unitary conductance of I_cat in 1.5 mM Ca is approximately 23 pS (Inoue & Kuriyama, 1993; Albert & Large, 2001a,b) with an estimated P_Ca/P_Na of about 4.5 (Wang & Large, 1991; Inoue et al. 2001). Noradrenaline also stimulates SOCs with a unitary conductance of 2-3 pS and a P_Ca/P_Na of about 50 in 1.5 mM Ca (Albert & Large, 2002 and present work), i.e. about one tenth of the conductance of I_cat but approximately ten times higher permeability to Ca²⁺ ions than I_cat. It is interesting that in 0 Ca PSS the unitary conductance of I_cat is reduced to about 13 pS (Albert & Large, 2001b) whereas the single channel conductance underlying SOC increases to about 6-7 pS (Albert & Large, 2002 and present work). The transduction mechanisms linking the -adrenoceptor and I_cat utilised G-proteins, increased PLC activity and production of DAG (Helliwell & Large, 1997). However DAG activates I_cat by a PKC-independent mechanism (Helliwell & Large, 1997) that remains to be elucidated, although we have suggested that a kinase with pharmacological properties similar to myosin light chain kinase may be involved (Aromolaran et al. 2000). A similar pathway links the -adrenoceptor to SOC with one notable exception: DAG stimulated SOCs via PKC.

With regard to molecular identity, Inoue et al. (2001) have provided compelling evidence that a member of the transient receptor potential family of proteins (TRPC6) is an essential component of I_cat. The molecular identity of SOCs in portal vein is unknown; however, it has been suggested that TRPC channels are candidates for store-operated conductances (see Harteneck et al. 2000). In rabbit portal vein myocytes, mRNA for TRPC4, TRPC6 and TRPC7 have been detected (Inoue et al. 2001; I. A. Greenwood, personal communication) and therefore homo- or heteromultimers of these TRPC proteins may be responsible for SOCs in this tissue.

Conclusions

These results show that noradrenaline activates SOCs via DAG and PKC, possibly by a store-independent mechanism. This implies that SOCs may be activated when the internal Ca²⁺ stores are 'full' which reinforces the proposal that SOCs in vascular smooth muscle have important physiological roles in addition to refilling internal Ca²⁺ stores.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

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Acknowledgements

This work was supported by The Wellcome Trust.

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