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¹ Pharmacology and Clinical Pharmacology Department of Basic Medical Sciences St. George's Hospital Medical School Cranmer Terrace London SW17 0RE, UK

	Abstract

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Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca²⁺ store depletion these SOCs could also be activated by -adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of ß-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective ß-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 ß-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.

(Received 19 October 2004; accepted after revision 2 November 2004; first published online 4 November 2004)
Corresponding author AP Albert: Pharmacology and Clinical Pharmacology, Department of Basic Medical Sciences, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. Email: aalbert{at}sghms.ac.uk

	Introduction

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A mechanism by which Ca²⁺ influx across the plasmalemma is triggered by depletion of intracellular Ca²⁺ stores was first proposed for non-excitable cells (Putney, 1986). Subsequently electrophysiological experiments indicated that the influx of Ca²⁺ ions occurred through Ca²⁺-selective ion channels (Hoth & Penner, 1992) and these are commonly described as store-operated channels (SOCs). SOCs have also been described in smooth muscle where it has been proposed that these channels may be involved in functions such as contraction and cell proliferation in addition to replenishing the internal Ca²⁺ stores subsequent to depletion (see review by Albert & Large, 2003). Previously we have described SOCs in freshly dispersed smooth muscle cells from rabbit portal vein (Albert & Large, 2002a), which appear to have different biophysical properties to SOCs recorded in aortic myocytes (cf. Trepakova et al. 2001). It was therefore concluded that there are different classes of SOCs in vascular smooth muscle which probably reflects different molecular identities (see Albert & Large, 2003).

Little is known about G-protein regulation of SOCs in smooth muscle cells. Previously we have provided evidence to demonstrate that noradrenaline, which is released from sympathetic nerves onto vascular smooth muscle, acts on -adrenoceptors to activate SOCs via protein kinase C (PKC) in rabbit portal vein myocytes (Albert & Large, 2002b). Moreover it appeared that this effect was membrane delimited and it was also evident that PKC was pivotal for SOC activation in response to Ca²⁺ store depletion (Albert & Large, 2002b). Importantly the data illustrated that SOCs could be activated by G-protein-coupled receptors independently of store depletion. Therefore in rabbit portal vein myocytes SOCs seem to have multi-modal gating mechanisms, i.e. SOCs are activated by store depletion and also by -adrenoceptor stimulation in the absence of depletion of Ca²⁺ stores. It is well known that vascular smooth muscle cells possess ß-adrenoceptors, which mediate vasodilator responses to catecholamines and these functional responses also occur in rabbit portal vein (Holman et al. 1968). Therefore in light of the role of SOCs in producing smooth muscle contraction we have investigated whether ß-adrenoceptor stimulation modifies SOC activity. It is shown that ß-adrenoceptor stimulation reduces SOC activity and that this effect is mimicked by agents that stimulate cAMP-dependent protein kinase (PKA) and by a catalytic subunit of PKA itself. The study provides further information on SOC regulation by G-protein-coupled receptors in freshly dispersed vascular smooth muscle cells.

	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) and the portal vein was removed 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’ PSS. The tissue was enzymatically dispersed in two sequential enzyme steps. First, the strips of tissue were incubated in ‘Ca²⁺-free’ PSS with 0.3 mg ml^–1 protease type XIV (Sigma) for 5 min and then the strips were washed in ‘Ca²⁺-free’ PSS. In the second step the strips were incubated with 1 mg ml^–1 collagenase type IA (Sigma) in 50 µM Ca²⁺-PSS for 10 min and were then washed in 50 µM Ca²⁺-PSS. All enzyme and wash procedures were carried out at 37°C. After the enzyme treatments the strips were incubated in 50 µM Ca²⁺-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 (1000 r.p.m.) to form a loose pellet which was resuspended in 0.75 mM Ca²⁺-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) and the pH was adjusted to 7.2 with 10 M NaOH. ‘Ca²⁺-free’ PSS, 50 µM Ca²⁺-PSS and 0.75 Ca²⁺-PSS had the same composition except that either Ca²⁺ was omitted or 1.5 mM CaCl₂ was replaced by 50 µM CaCl₂ and 0.75 mM CaCl₂, respectively.

Electrophysiology

Whole-cell and single cation channel currents were recorded with a HEKA EPC-8 patch clamp amplifier at room temperature using whole-cell recording and cell-attached and inside-out patch configurations of the patch clamp technique (Hamill et al. 1981). Patch pipettes were manufactured from borosilicate glass and were fire polished; we used pipettes with resistances of about 6 M for whole-cell and between 10 and 15 M for cell-attached and inside-out patch recording when filled with the standard patch pipette solution. To reduce ‘line’ noise the recording chamber (vol. 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 external solution could be exchanged twice within 30 s. Whole-cell currents were evoked by applying voltage ramps from –150 mV to +100 mV (0.5 V s^–1) every 20 s from a holding potential of 0 mV and filtered at 5 kHz (–3 db, low pass 4-pole Bessel filter, HEKA EPC-8 patch clamp amplifier) and sampled at 1 kHz (Digidata 1322 A and pCLAMP 9.0 Software, Axon instruments, Inc., CA, USA). When recording single channel currents the holding potential was routinely set at –80 mV and to evaluate current–voltage (I–V) characteristics of single channel currents the membrane potential was manually changed between –120 and +100 mV.

Single channel currents were initially recorded onto digital audiotape (DAT) using a biologic DRA-200 digital tape-recorder (BioLogic Science Instruments, France) at a bandwidth of 5 kHz (–3 db, low pass 4-pole Bessel filter, HEKA EPC-8 patch clamp amplifier) and a sample rate of 48 kHz. For off-line analysis single cation 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 Digidata 1322 A and pCLAMP 9.0 Software at a sampling rate of 1 kHz. Data were captured with a Pentium III personal computer (Research Machines).

Single channel current amplitudes were calculated from idealized traces of at least 10 s duration using the 50% threshold method with events lasting for >6.664 ms (2 x rise time for a 100 Hz (–3 db) low pass filter) being excluded from analysis (Colquhoun, 1987). I–V relationships, calculated from pooled single channel current amplitudes, were plotted and slope conductance and reversal potential (E_r) were calculated using linear regression (Origin software, OriginLab Corp., USA). In cell-attached patches single channel currents could not be resolved at positive membrane potentials and therefore the E_r was estimated using extrapolation (see Albert & Large, 2002a). Figure preparation was carried out using Origin software (version 6.0). Inward channel currents were shown as downward deflections and outward currents as upward deflections. The number of channels in a patch was unknown and therefore open probability (NP_o) was calculated using the equation: NP_o= total open time of all channel levels in the patch / sample duration.

Solutions and drugs

In cell-attached patch experiments the membrane potential was set to approximately 0 mV by perfusing cells in a KCl external solution containing (mM): KCl (126), CaCl₂ (1.5), Hepes (10) and glucose (11), pH to 7.2 with 10 M NaOH. Nicardipine (3–5 µM) was also included to prevent smooth muscle cell contraction by blocking Ca²⁺ entry through voltage-dependent Ca²⁺ channels. The bathing solution used in inside-out experiments (intracellular solution) was K⁺ free and contained (mM): CsCl (18), caesium aspartate (108), MgCl (1.2), Hepes (10), glucose (11), BAPTA (1), CaCl₂ (0.48) (free internal Ca²⁺ concentration approximately 100 nM as calculated using EQCAL software), Na₂ATP (1), NaGTP (0.2), pH 7.2 with Tris. The bathing solution used in whole-cell recording experiments contained (mM): NaCl (126), CaCl₂ (1.5), Hepes (10), glucose (11), 100 µM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS), 100 µM niflumic acid and 3–5 µM nicardipine, pH 7.2 with NaOH. The standard pipette solution used for both cell-attached and inside-out recording (extracellular solution) was K⁺ free and contained (mM): NaCl (126), CaCl₂ (1.5), Hepes (10), glucose (11), TEA (10), 4-AP (5), 200 nM iberiotoxin, 100 µM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS), 100 µM niflumic acid and 3–5 µM nicardipine, pH to 7.2 with NaOH (310 ± 5 mosmol l^–1). The standard pipette solution used for whole-cell recording had the same composition as the bathing solution for inside-out patches except that 10 mM BAPTA and 4.8 mM CaCl₂ were included (free internal Ca²⁺ concentration of about 100 nM). Under these conditions voltage-gated Ca²⁺ currents, K⁺ currents, swell-activated Cl^– currents and Ca²⁺-activated conductances are abolished and non-selective cation currents could be recorded in isolation. All drugs including the PKA catalytic subunit and the PKA inhibitory peptide (6–22 amides) were purchased from Sigma (UK). In control experiments the catalytic subunit of PKA was heated to 95°C for 10 min before being applied to excised patches. The values are the mean of n cells ±S.E.M. Statistical analysis was carried out using Student's t test with the level of significance set at P < 0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Stimulation of ß-adrenoceptors inhibits store-operated whole-cell and single cation channel currents (SOCs) in rabbit portal vein myocytes

We have previously shown that experimental procedures that deplete Ca²⁺ stores in the sarcoplasmic reticulum with the Ca²⁺-ATPase inhibitor, cyclopiazonic acid (CPA), activate whole-cell cation currents in rabbit portal vein myocytes (Albert & Large, 2002a). Therefore our initial experiments investigated the effect of the ß-adrenoceptor agonist isoprenaline on whole-cell currents evoked by CPA. Figure 1A shows that bath application of 10 µM CPA evoked whole-cell cation currents within 1–2 min that were markedly inhibited by co-application of 1 µM isoprenaline. Figure 1B illustrates that the CPA-evoked current–voltage (I–V) relationships shown in Fig. 1A had dual rectifying properties and a reversal potential (E_r) of about +50 mV, which are properties similar to those previously described for CPA-induced whole-cell currents in portal vein myocytes (Albert & Large, 2002a). Moreover Fig. 1C shows that in seven cells the mean CPA-induced whole-cell current amplitude was reduced from –23 ± 5 pA to –4 ± 3 pA at –80 mV corresponding to an inhibition of about 85%.

View larger version (20K): [in this window] [in a new window]   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.

View larger version (18K): [in this window] [in a new window]   Figure 2.  Stimulation of ß-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.

Spontaneous SOC activity in cell-attached patches is inhibited by isoprenaline

We have previously shown that some cell-attached and outside-out patches contain spontaneous SOC activity and in the present study about 25% of all cell-attached (19 out of 81 patches) and 25% of all inside-out patches (11 out of 45 patches) contained spontaneous activity with similar properties to those previously described (and see later data on inside-out patches and Albert & Large, 2002a). In six cell-attached patches spontaneous SOC activity had a mean NP_o of 0.366 ± 0.088 which was reduced by bath application of 1 µM isoprenaline by over 95% to 0.013 ± 0.007 (P < 0.01). All other cell-attached and inside-out patches containing spontaneous SOC activity were not included in the following experiments.

Inhibition of SOC activity by agents that activate adenylate cyclase and mimic the action of cAMP

It is well recognized that agonist binding to ß-adrenoceptors stimulates G-proteins which then activate adenylate cyclase leading to production of cAMP that acts as a second messenger to stimulate protein kinase A (PKA). We therefore examined whether this signal transduction pathway was involved in the isoprenaline-induced inhibition of SOC activity by investigating the effect of forskolin, an activator of adenylate cyclase, and 8-Br-cAMP, a cell-permeable non-hydrolysable analogue of cAMP, on CPA-induced SOCs in cell-attached patches. Figure 3A shows that bath application of 10 µM forskolin produced marked inhibition of CPA-evoked SOC activity and Fig. 3C shows that the mean NP_o of CPA-evoked channel activity was reduced by about 95%. Moreover Fig. 3B and C shows that bath application of 100 µM 8-Br-cAMP also produced pronounced inhibition of CPA-induced SOC activity by about 95%.

View larger version (24K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

Constitutive PKA activity inhibits SOC activity in cell-attached patches

It was our intention to study the role of PKA in mediating the inhibitory effect of isoprenaline, adenylate cyclase and 8-Br-cAMP on CPA-induced channel activity by investigating the effect of PKA inhibitors on these inhibitory responses in cell-attached patches. However, preliminary experiments showed that bath application of the PKA inhibitors H-89 and KT5720 on their own stimulated channel activity. Figure 5A shows that bath application of 1 µM H-89 evoked channel currents in a previously quiescent cell-attached patch which was reversed upon wash-out and in 11 patches this activity had a mean peak NP_o value of 0.433 ± 0.148. Figure 5B shows that the amplitude histogram of these H-89-evoked channel currents could be fitted with three Gaussian curves with peaks of 0, –0.24 and –0.46 pA, corresponding to a closed level and two open levels, respectively, with the two open levels indicating at least two channels in the patch. In 11 patches H-89 evoked channel currents with mean amplitude of –0.22 ± 0.01 pA at –80 mV. Figure 5C shows the I–V relationship of H-89-evoked channel currents which had a slope conductance between –120 mV and –20 mV of 1.8 pS and an extrapolated E_r of +25 mV. These characteristics are similar to the channel currents evoked by CPA and BAPTA-AM in cell-attached patches (see data in Albert & Large, 2002a).

View larger version (22K): [in this window] [in a new window]   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.

These data suggest that inhibiting PKA activity activates SOCs which implies that constitutive PKA activity has a pronounced inhibitory influence on SOC activity in portal vein myocytes.

The phorbol ester PDBu activates SOC activity in inside-out patches

In our next series of experiments we intended to further investigate the inhibitory effect of PKA on SOC activity by studying the effect of a PKA catalytic subunit and 8-Br-cAMP on SOCs in inside-out patches. However, before we carried out these experiments we investigated the properties of SOCs in inside-out patches since we have not previously studied these channel currents in this configuration of the patch-clamp technique.

Figure 6A shows that bath application of 1 µM PDBu to an inside-out patch evoked sustained channel activity at –80 mV which in 15 patches had a mean NP_o value of 0.378 ± 0.09. Figure 6B shows that the channel current amplitude histogram of these PDBu-evoked channel currents could be fitted with three Gaussian curves with peaks of 0, –0.21 and –0.44 pA indicating one closed level and two open levels due to at least two channels in the patch. In 15 patches the PDBu-evoked channel currents had a mean amplitude of –0.21 ± 0.01 pA at –80 mV. Figure 6C illustrates PDBu-evoked channel currents recorded at different membrane potentials and Fig. 6D shows I–V relationships of PDBu-evoked channel currents.

View larger version (25K): [in this window] [in a new window]   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.

In 0 [Ca²⁺]_o PDBu-evoked channel currents had an ohmic I–V relationship at all membrane potentials tested (• in Fig. 6D, –120 to +80 mV) that had a slope conductance of about 5 pS and an interpolated E_r of approximately 0 mV.

In both 1.5 mM and 0 [Ca²⁺]_o PDBu-induced channel currents were recorded in inside-out patches with the same internal CsCl/caesium aspartate bathing solution (see Methods). Therefore at positive membrane potentials PDBu-induced channel currents in these two conditions had similar outward slope conductances of about 5 pS, as shown by the superimposed points at +40 mV and +80 mV in Fig. 6D, and a similar E_r of about 0 mV (Fig. 6D). At negative membrane potentials the reduced slope conductance of 1.9 pS and more positive E_r (about 25 mV) in 1.5 mM[Ca²⁺]_o compared with the corresponding values of 5.2 pS and about 0 mV in 0 [Ca²⁺]_o may be explained by a significant permeability of SOCs to Ca²⁺ ions as suggested previously from studies with cell-attached and outside-out patches (Albert & Large, 2002a, 2000b).

Bath application of H-89 (1 µM) and a PKA inhibitory peptide (6–22 amides, 1 µM) also induced channel currents in inside-out patches at –80 mV with mean NP_o values of, respectively, 0.183 ± 0.05 (n= 5) and 0.367 ± 0.153 (n= 5) and mean unitary amplitudes of –0.19 ± 0.01 pA (n= 5) and –0.18 ± 0.01 pA (n= 5), respectively. Similar data were obtained with bath application of 1 µM KT5720 (data not shown).

These data indicate that PDBu can activate SOCs in inside-out patches from portal vein myocytes and PKA inhibitors also stimulate SOC activity in inside-out patches.

PKA catalytic subunit and 8-Br-cAMP inhibit SOC activity in inside-out patches

Figure 7A and D shows that bath application of 100 U ml^–1 of a PKA catalytic subunit reversibly inhibited PDBu-evoked SOC activity in inside-out patches by about 90%. Figure 7B shows that PKA catalytic subunit which was heated to inactivate kinase activity had no effect on PDBu-induced channel activity. Figure 7C and D also shows that bath application of 100 µM 8-Br-cAMP reversibly inhibited PDBu-evoked SOC activity in inside-out patches by about 90%.

View larger version (28K): [in this window] [in a new window]   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.

	Discussion

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The present work demonstrates that ß-adrenoceptor stimulation produces marked inhibition of SOC activity in freshly dispersed rabbit portal vein myocytes and that this effect is mimicked by PKA stimulation. Forskolin, which stimulates adenylate cyclase, and 8-Br-cAMP reduced SOC activity evoked by Ca²⁺-store depletion induced by CPA and BAPTA-AM as well as by OAG and phorbol esters. Moreover a catalytic subunit of PKA also markedly decreased SOC activity evoked by phorbol esters in inside-out patches. These data can be summarized into a scheme where ß-adrenoceptor stimulation activates adenylate cyclase to increase intracellular cAMP concentration which then activates PKA to reduce the probability of opening of SOCs. It was not possible to test whether inhibitors of PKA prevented the effect of isoprenaline since these compounds produced marked activation of SOC activity when applied on their own (see below).

There is very little information of the regulation of SOC activity by G-protein-coupled receptors in vascular smooth muscle. Therefore the present work describing stimulation of a pharmacological receptor linked to activation of PKA and subsequent inhibition of SOC activity represents a novel finding in vascular smooth muscle which may also be important in other tissues. In vascular smooth muscle this mechanism may have a role in reducing Ca²⁺ influx, membrane depolarization and consequent physiological processes such as vasoconstriction, intracellular Ca²⁺ store replenishment and vascular smooth muscle proliferation. With regard to PKC-evoked excitation and PKA-mediated inhibition of SOCs in portal vein myocytes there is some similarity to data obtained in Xenopus oocytes. In a study on the latter preparation it was demonstrated that phorbol esters first increased and then decreased thapsigargin-induced store-operated Ca²⁺ influx (Peterson & Berridge, 1995). Also low concentrations of dibutyryl cAMP (1–10 µM) inhibited Ca²⁺ influx although higher concentrations (1–10 mM) potentiated store-operated Ca²⁺ entry (Peterson & Berridge, 1995). In portal vein myocytes we have only observed phorbol ester-evoked activation of SOCs and PKA-mediated inhibition of SOC activity but the differences may be accounted for by differences in preparations or conditions used. It should be noted that in corneal epithelial cells PKA has also been shown to inhibit an epidermal growth factor-evoked Ca²⁺ influx pathway, attributed to opening of SOCs (Yang et al. 2003).

It is well recognized that PKA can exist in both cytosolic and membrane cellular fractions with levels of membrane-bound PKA thought to be determined by a group of A kinase anchoring proteins (AKAPS) which link PKA to the membrane (Francis & Corbin, 1994). In the present work the observation that 8-Br-cAMP reduced and H-89 or KT5720 evoked SOC activity in inside-out patches indicates that membrane-bound PKA is involved in this inhibitory pathway in portal vein myocytes. However, H-89 and KT5720 also evoked SOCs with a slightly greater effect in cell-attached patches where the intracellular milieu remains intact compared with inside-out patches (NP_o of about 0.2 versus 0.4, see Results) and therefore cytosolic PKA may also be involved in the inhibitory response.

Is there constitutive inhibitory PKA activity?

A surprising result was that PKA inhibitors produced significant activation of SOCs recorded with cell-attached and inside-out patches which indicates that there is constitutive PKA activity keeping the channels closed. Previously we reported that spontaneous SOC activity is recorded in some patches in cell-attached and outside-out patches (Albert & Large, 2002a, 2002b). Also in the present study spontaneous channel currents were observed in cell-attached and inside-out patches, with spontaneous SOC activity in cell-attached patches being markedly inhibited by ß-adrenoceptor stimulation by isoprenaline. Therefore there must be a constitutive driver mechanism to produce SOC opening which is inhibited by tonic PKA activity. We have observed constitutive SOC activity frequently in all configurations of patch recording which suggests that this is a genuine physiological phenomenon that will contribute to basal Ca²⁺ influx and perhaps resting membrane conductance.

Role of phosphorylation in regulating SOC activity

There are several findings which indicate that phosphorylation processes are important in regulating SOC activity. Previously we showed that OAG and phorbol esters which activate PKC evoked SOC opening and that PKC inhibitors reduced SOC activity evoked not only by OAG and PDBu but also by CPA and BAPTA-AM (Albert & Large, 2002b). These data indicate that a phosphorylation process mediated by PKC is important in producing channel opening. Moreover since these effects occurred in isolated patches it is likely that PKC is membrane bound. This idea is supported by the observation that omission of ATP from the intracellular solution prevents channel opening by PDBu in inside-out patches (data not shown). A similar conclusion was reached regarding SOC activation by PKC in human glomerular mesangial cells (Ma et al. 2002). We have also observed that a PKC catalytic subunit activates SOCs in portal vein myocytes (authors' unpublished data). It has been suggested that a member of the transient receptor potential channel family (TRPC1) is a subunit of SOCs in vascular smooth muscle (Xu & Beech, 2001).

Recently strong evidence has been provided to show that store-operated Ca²⁺ influx through expressed TRPC1 in human umbilical vein endothelial cells depends on PKC-mediated phosphorylation of TRPC1 (Ahmmed et al. 2004). Together these data support a role for PKC-mediated phosphorylation in activation of SOCs in vascular smooth muscle. Moreover the present work shows that another phosphorylation pathway involving PKA leads to SOC inhibition. Therefore it is possible that there are two phosphorylation sites on the SOC in rabbit portal vein smooth muscle cells.

Previously we have demonstrated that inhibitors (calyculin A and microcystin-LF) of serine/threonine phosphatases stimulate SOC opening in portal vein myocytes (Albert & Large, 2002b). This was interpreted by the proposal that these agents would potentiate the PKC-mediated opening of SOCs. However, these phosphatase inhibitors would also be expected to enhance the PKA-mediated inhibition of SOC, i.e. the opposing action, and therefore it seems that the net effect is to increase SOC opening.

Comparison of PDBu-evoked SOCs in portal vein myocytes with SOCs from aortic myocytes

The present study shows that PDBu activates SOCs in inside-out patches which further suggests that PKC is a pivotal agent in the transduction mechanism leading to channel opening in portal vein myocytes. In contrast to our studies in rabbit portal vein a mechanism involving Ca²⁺-independent phospholipase A₂ leading to subsequent production of lysophospholipids has been proposed to be important in activating SOC activity in mouse aortic smooth muscle cells (Smani et al. 2004). These data suggest that more than one signal transduction mechanism is involved in activating SOCs in vascular smooth muscle which may also infer that vascular smooth muscle preparations can contain different SOCs with possibly different molecular identities. Moreover the present study provides further evidence that vascular smooth preparations contain different SOCs since the unitary conductance of PDBu-evoked SOCs in inside-out patches increased 2- to 3-fold when Ca²⁺ ions were removed and that this was associated with a shift in E_r from about +25 mV to 0 mV indicating that SOCs in portal vein myocytes are highly permeable to Ca²⁺ ions. In mouse aortic myocytes external Ca²⁺ ions had no effect on the unitary conductance or E_r of SOCs indicating that these channels have a relatively low permeability to Ca²⁺ ions. Therefore SOCs in rabbit portal vein and mouse aortic myocytes appear to have both different biophysical properties and activation mechanisms.

Whole-cell versus single channel recording

Finally it is worth commenting on the method used to record SOCs. Generally in smooth muscle the amplitude of whole-cell currents in response to depletion of intracellular stores by agents such as CPA and BAPTA-AM is rather small (less than –10 pA at –50 to –60 mV, see Albert & Large, 2003). In contrast single channel currents can be recorded with relative ease from patches with any configuration of patch pipette recording. Making the normal assumption about the area of membrane under a pipette of about 5 M resistance it would be expected to observe a much larger whole-cell current representing SOC activity. We have no precise explanation for this discrepancy but it might be due to the patch pipette configuration, e.g. with patch recording the membrane from which the signals are recorded is under a degree of tension (perhaps stimulating a mechanosensitive element to facilitate channel opening) which is not the case with whole-cell recording in which an isolated dispersed cell is relaxed. Of course this is not the case in vivo where the vascular smooth muscle cell in the vessel wall is under significant tension due to the intralumenal pressure.

Conclusions

The results show that ß-adrenoceptor stimulation inhibits SOC activity which is mediated by the classical adenylate cyclase–cAMP–PKA cascade. The data increase our understanding of how G-protein-coupled receptors regulate SOC activity in vascular smooth muscle and show that SOCs may be activated and inhibited by store-independent and membrane-delimited mechanisms.

	References

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	Acknowledgements

 
This work was supported by The British Heart Foundation and The Wellcome Trust.

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