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ANESTHETIC PHARMACOLOGY

Modulation of Xenopus laevis Ca-Activated Cl Currents by Protein Kinase C and Protein Phosphatases: Implications for Studies of Anesthetic Mechanisms

Klaus Hahnenkamp, MD^*, Marcel E. Durieux, MD PhD^*, Hugo van Aken, MD PhD^*, Sascha Berning^*, Thomas J. Heyse^*, Christian W. Hönemann, MD^*, and Bettina Linck, MD PhD

*Department of Anesthesiology and Intensive Care, and Institute of Pharmacology and Toxicology, University Hospital, Muenster, Germany

Address correspondence to Marcel E. Durieux, MD, PhD, Department of Anesthesiology, University of Virginia, PO Box 800710, Charlottesville, VA 22908–0710. Address email to durieux{at}virginia.edu

	Abstract

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Ca-activated Cl currents (I_Cl(Ca)) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I_Cl(Ca) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1 and PP2A on I_Cl(Ca) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1 and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP₃), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl₂ were directly injected into the oocyte. Responses to agonists (LPA 10^–6 M, IP₃ 10^–4 M, CaCl₂ 0.5 M) were measured at a holding potential of –70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 and PP2A inhibited I_Cl(Ca) from 7.6 ± 0.9 µC to 2.5 ± 0.9 µC and 3.2 ± 1.4 µC, respectively. PP inhibition enhanced I_Cl(Ca) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl₂-induced I_Cl(Ca). Our data indicate that the Xenopus I_Cl(Ca) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.

IMPLICATIONS: The Xenopus I_Cl(Ca), commonly used as a reporter current in studies of anesthetic effects on G protein-coupled signaling, is modulated by phosphorylation. Anesthetic effects on channel phosphorylation state can therefore be misinterpreted as effects on receptor signaling.

	Introduction

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Oocytes of the frog Xenopus laevis have become a commonly used model for the study of anesthetic effects on G protein-coupled receptors (GPCR). Particularly receptors that couple to G_q/11/14 proteins, which induce intracellular Ca release, are easily investigated in this model (1,2), as an endogenous Ca-activated Cl current (I_Cl(Ca)) provides a convenient end-point assay of changes in intracellular Ca concentration.

Anesthetics could potentially affect I_Cl(Ca) directly, and such interactions of the anesthetic with the reporter channel could be misinterpreted as interactions with the receptor signaling pathway. To exclude such an action, it has generally been considered sufficient to demonstrate that anesthetics do not affect I_Cl(Ca) induced by a receptor-independent increase in intracellular Ca concentrations (e.g., by microinjection of CaCl₂). For many anesthetics (general as well as local) a lack of effect on receptor-independent I_Cl(Ca) has been demonstrated. However, we suggest that this may not be sufficient evidence to exclude anesthetic modulation of I_Cl(Ca). Activation of G_q/11/14-coupled receptors will, in addition to increasing intracellular Ca levels, result in generation of diacylglycerol (DAG), which will activate protein kinase C (PKC). If PKC were to regulate I_Cl(Ca) by channel phosphorylation, an effect of anesthetics on PKC activity could be misinterpreted as an effect on receptor signaling. Importantly, receptor-independent I_Cl(Ca) would not be affected by anesthetic in this scenario. In addition to affecting PKC, anesthetics might interact with protein phosphatases (PP), which dephosphorylate targets of PKC. This might also result in effects on I_Cl(Ca) that could be misinterpreted as actions on receptor signaling.

We hypothesized that the X. laevis I_Cl(Ca) is modulated by PKC and/or PP. To test this hypothesis, we investigated the effects of activation and inhibition of PKC, as well as overexpression and inhibition of PP, on receptor-, inositoltrisphosphate (IP₃)-, or Ca-induced I_Cl(Ca) in Xenopus oocytes.

	Methods

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Isolation of X. laevis Oocytes
The study protocol was approved by the local Animal Care and Use Committee. Xenopus oocytes were removed as described previously (3). In brief, frogs were anesthetized by immersion in cold 0.2% 3-amino-benzoic-methyl-ester until fully immobile. After abdominal incision an ovarian lobule, containing approximately 200 cells, was removed under sterile conditions. The frogs were allowed to recover from anesthesia and operation in a separate tank. Oocytes were maintained at 20°C in modified Barth’s solution (containing 88 mM NaCl, 2.4 mM NaHCO₃, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 0.3 mM Ca₂NO₃, 0.1 mM gentamycin, and 15 mM HEPES, pH adjusted to 7.4). Oocytes were defolliculated by gentle shaking in 1 mg/mL solution of collagenase type A in calcium-free OR2 solution (containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH adjusted to 7.4). After 2 h the cells were returned to modified Barth’s solution. Microscopic observation confirmed that the follicle cells had been removed.

PP Overexpression
The plasmids with cDNA inserts for the catalytic subunit of rat PP1 and PP2A were obtained by large-scale preparations. Inserts were isolated by digestion with XbaI and BamHI for PP1 and with PstI and EcoRI for PP2A. The cDNA inserts were purified from 1% agarose gels. Sizes were approximately 1000 bp for PP1 and 1300 bp for PP2A. Oocytes were injected with 10 ng cDNA in 50.6 nL sterile water using an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA). The area of the germinal vesicle was targeted, and sufficient injection was confirmed by noting a slight increase in cell size. The cells were then cultured in modified Barth’s solution for 72 h to allow time for expression of the corresponding PP.

Electrophysiology
A single defolliculated cell was positioned in a continuous-flow chamber with 0.5 mL volume, perfused (5 mL/min) by Tyrode’s solution (containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 10 mM dextrose, and 10 mM HEPES, pH adjusted to 7.4). Microelectrodes were pulled in one stage from capillary glass on a vertical computer-controlled electrode puller (Sutter Instrument Company, Novato, CA). Electrode tips were broken to a diameter of approximately 10 µm, providing a resistance of 1–3 M, and filled with 3 M KCl. The oocytes were voltage clamped using a two-electrode voltage clamp amplifier (OC725C; Warner Instruments Corp., New Haven, CT) connected to an IBM-compatible personal computer for data acquisition and analysis (software by J. Kardeous, PhD, University of Muenster, Germany). All measurements were performed at a holding potential of –70 mV. We sampled membrane current at 125 Hz and recorded during 5 s before and 85 s after drug administration. This allowed sufficient time for currents to return to baseline levels. Responses were quantified by integrating the current trace and reported in µC. All experiments were performed at room temperature.

Receptor agonist (lysophosphatidate, LPA) was delivered as a 30 µL aliquot (Tyrode’s solution containing 0.1% fatty acid free bovine serum albumin [BSA]) over a period of 2 s using a hand-held micropipette positioned approximately 2 mm in front of the oocyte.

Intracellular Microinjection
PP, IP₃, CaCl₂, or the PP inhibitor okadaic acid (OA) were diluted in 150 mM of KCl and applied by direct injection into the oocyte using a third electrode inserted into the oocyte. This electrode was connected to an automated microinjector (Nanoject, Drummond Scientific). Under voltage clamp condition, 50.6 nL of 10^–4 M IP₃, 0.5 M CaCl₂, or OA (10^–9 M or 10^–5 M) were injected. The volume of a Xenopus oocyte is approximately 500 nL; therefore we injected an additional volume of approximately 10%. The final concentration of the administered drug was estimated from these numbers, but it should be taken into account that distribution inside the cell will not be homogeneous. Injection of 50.6 nL KCl 150 mM was used as control. To inhibit PKC, 50 nL of 5 x 10^–4 M chelerythrine diluted in 150 mM KCl (or 150 mM KCl as control) was microinjected into oocytes 2 h before experiments; estimated final concentration was 50 µM.

PP Assay
Assays for protein phosphatase activity were performed as described previously (4), using [³²P]-phosphorylase a as substrate. The incubation mixture contained 5 mM caffeine, 0.1% (v/v) ß-mercaptoethanol, 0.1 mM EDTA, and 20 mM Tris HCl (pH 7.0). The reaction was started by adding aliquots of homogenates and terminated after 10 min by the addition of 50% trichloroacetic acid. Precipitated protein was sedimented by centrifugation and the supernatant counted in a liquid scintillation counter.

Data Analysis
Unless stated otherwise, all results are reported as mean ± SEM. Differences among treatment groups were analyzed using Student’s t-test or Mann-Whitney U-test. If multiple comparisons were made, analysis of variance was conducted, followed by Student’s t-test corrected for multiple comparisons (Bonferroni). P < 0.05 was considered significant. Concentration-response curves were fit to the following logistic function, derived from the Hill equation:

where y_max and y_min are the maximum and minimum response obtained, n is the Hill coefficient, and x₅₀ is the half-maximal effect concentration/half-maximal inhibitory effect (EC₅₀ for agonist/IC₅₀ for antagonist).

Materials
LPA was obtained from Avanti Polar Lipids (Alabaster, AL) and was diluted in 0.1% fatty acid-free BSA (ICN Pharmaceuticals, Costa Mesa, CA) in Tyrode’s solution to the appropriate concentration. Cantharidin, OA, and IP₃ were obtained from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Collagenase A was acquired from Boehringer Mannheim GmbH (Mannheim, Germany).

	Results

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PKC Modulates ICl(Ca)
The phospholipid LPA induces I_Cl(Ca) by activating an endogenous receptor in the oocyte and was used in this investigation as a method for inducing receptor-mediated I_Cl(Ca). We demonstrated previously that LPA signaling in oocytes is modulated by PKC (2). We now compared the effect of altering PKC activity on LPA-induced I_Cl(Ca) with its effect on receptor-independent activation of I_Cl(Ca) by intracellular injection of CA.

LPA induced transient I_Cl(Ca), as described previously. EC₅₀ was calculated from the concentration-response relationship and was 5.4 ± 0.2 x 10^–7 M (Fig. 1A). A concentration of 10^–6 M was used for all subsequent experiments.

View larger version (15K): [in this window] [in a new window]   Figure 1. A, lysophosphatidic acid (LPA) signaling in Xenopus laevis oocytes. LPA activates the ICl(Ca) in a concentration-dependent manner. n 7 for each data point. B, effects of 2 h intracellular protein kinase C (PKC) inhibition with 5 x 10–4 M chelerythrine on lysophosphatidic acid (LPA) signaling. Inhibition of PKC-enhanced (LPA 10–6 M) responses by 65%. ICl(Ca) induced by intracellular injection of CaCl2 (CaCl2 ic)) as similarly enhanced in the presence of chelerythrine (91%), indicating that ICl(Ca) is modulated by phosphorylation by PKC. *P < 0.05. Values are mean ± SEM. n 7.

PP Overexpression Modulates LPA-Induced I_Cl(Ca)
To study the effects of PP on I_Cl(Ca), we overexpressed PP1 or PP2A in oocytes. To assure functional expression, we determined phosphatase activity in cells overexpressing PP. Injection of either PP1 or PP2A resulted in an approximate twofold increase in PP activity compared with KCl-injected control oocytes (Fig. 2A), confirming that the overexpressed proteins were functional. In cells overexpressing PP, LPA-induced I_Cl(Ca) was diminished as compared with control oocytes (Fig. 2B). The currents declined by 67% in PP1-injected oocytes and by 58% in PP2A injected cells. To confirm that this inhibitory effect was specific, we also determined the effect of injection of purified PP1 protein (Fig. 2C). The injection of purified PP1 protein also reduced the LPA-induced I_Cl(Ca) as compared with KCl-injected control cells, suggesting that the inhibitory effect is most likely a direct result of PP activity. In PP1-injected oocytes, the relatively nonselective PP inhibitor cantharidine (10^–5 M) abolished the inhibitory effect of PP overexpression on LPA-induced I_Cl(Ca) (Fig. 3A). OA 10^–5 M also abolished the inhibitory effect of PP1 overexpression on LPA-induced I_Cl(Ca) (Fig. 3A). Similar results were obtained in PP2A-injected oocytes (Fig. 3B). Therefore, receptor-mediated I_Cl(Ca) can be modulated by PP.

View larger version (19K): [in this window] [in a new window]   Figure 2. A, protein phosphatase activity in preparations from oocytes overexpressing protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) in comparison with KCl-injected oocytes (control). Phosphatase activity was measured as described in Methods. *P < 0.05, n = 3. B, effects of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) on lysophosphatidic acid (LPA)-induced (10–6 M) ICl(Ca). ICl(Ca) were inhibited in oocytes expressing PP1 or PP2A compared with control. There was no difference between control and KCl-injected cells. *P < 0.05. Values are mean ± SEM. n 7. C, effect of protein injection compared with cDNA injection of protein phosphatase 1 (PP1) in Xenopus oocytes. The injection of purified PP1 protein also reduced the LPA-induced ICl(Ca) as compared with KCl-injected control cells. *P < 0.05. Values are mean ± SEM. n > 7.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effects of the protein phosphatase (PP) inhibitors cantharidine (Cant, 10–5 M) and okadaic acid (OA, 10–5 M) on native oocytes and protein phosphatase (PP) injected oocytes. A, PP1 overexpression inhibited lysophosphatidic acid (LPA)-induced ICl(Ca) responses compared with KCl injected oocytes (KClic). The PP inhibitors Cant and OA abolished the inhibitory effect of PP1 overexpression (values are mean ± SEM, n 7, n 3 for experiments with OA, *P < 0.05 versus PP1 , #P < 0.05 PP1 versus KClic). B, in PP2A overexpressing oocytes Cant and OA abolished the inhibitory effect of PP overexpression (values are mean ± SEM, n 7, n 3 for experiments with okadaic acid, *P < 0.05 versus PP2A, #P < 0.05 PP2A versus KClic). C, the PP inhibitors Cant and OA increased LPA-induced ICl(Ca) in KCl-injected oocytes (control(KClic)), suggesting the presence of endogenous PP in Xenopus laevis oocytes. Values are mean ± SEM, n 7, n 3 for experiments with okadaic acid, *P < 0.05 versus control(KClic).

Endogenous PP Modulate IP₃- or Ca-Induced I_Cl(Ca)
To delineate the site of action of endogenous PP on I_Cl(Ca), we next determined if I_Cl(Ca) induced by either IP₃ or CaCl₂ would be affected by PP also. Both IP₃ and CaCl₂ elicited I_Cl(Ca) when injected directly into oocytes (Fig. 4A and 4B). In cells overexpressing PP1 or PP2A, I_Cl(Ca) induced by IP₃ (10^–4 M) was reduced by 46% or 71%, respectively. Responses to CaCl₂ (0.5 M) were reduced by 63% or 75% in cells expressing PP1) or PP2A, respectively. These results demonstrate that the effects of PP do not take place in the LPA signaling pathway but are most likely mediated by alteration of the phosphorylation state of the Ca-activated Cl channel.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effects of inositoltrisphosphate (IP3) and CaCl2 on ICl(Ca) in protein phosphatase 1 (PP1A) or protein phosphatase 2A (PP2A) injected oocytes compared with control (KCl-injected oocytes). A, ICl(Ca) generated by the injection of 100 µM IP3 were inhibited in PP1 or PP2A overexpressing oocytes compared with control (KCl-injected oocytes) (values are mean ± SEM, n 7, *P < 0.05 versus control). B, ICl(Ca) induced by the injection of 500 mM CaCl2 were inhibited in PP1 or PP2A overexpressing oocytes compared with control (KCl-injected oocytes) (values are mean ± SEM, n 7,*P < 0.05 versus control).

	Discussion

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These findings are of relevance for studies of anesthetics on signaling systems using the Xenopus oocyte I_Cl(Ca) as reporter. In such studies, the target of interest is usually a G protein-coupled receptor expressed in oocytes by injection of complementary RNA or DNA. Such receptors couple effectively to endogenous Xenopus G proteins, and those receptors that activate G_q, G₁₁ and/or G₁₄ proteins will induce an IP₃-mediated increase in intracellular Ca levels. This, in turn, will activate I_Cl(Ca). Whereas the expressed receptor is often of mammalian origin, and the G protein—phospholipase C—IP₃ receptor pathway in oocytes has been shown to be functionally similar to its mammalian counterpart, the frog Ca-activated Cl channel is not a part of this signaling system in mammals. Thus, an anesthetic effect on I_Cl(Ca) would be of limited relevance, and it is therefore appropriate to assure that any anesthetic effects observed take place upstream of the Cl channel. To determine the anesthetic site of action, control experiments are often performed in which I_Cl(Ca) is activated by direct injection into the oocyte of either IP₃ or Ca. If I_Cl(Ca) induced by this means is not affected by the anesthetic, this is taken to imply that the anesthetic is not affecting the Ca-activating Cl channel.

Our data suggest that this control experiment may not be sufficient. Activation of Ca-signaling G proteins also induces the release of DAG, which in turn will activate PKC. Our findings indicate that PKC activation by this means is likely to increase phosphorylation of the Cl channel, resulting in decreased currents. Many anesthetics, volatile as well as local, affect PKC (5,6). Interactions are complex and depend both on the specific anesthetic studied and the PKC subtypes present in the model under study. In Xenopus oocytes, it appears that at least some volatile anesthetics increase PKC activity. For example, halothane inhibits muscarinic signaling in this model (7), but this effect is abolished completely by pretreatment of the cells with PKC antagonists (8). In contrast, local anesthetics appear to inhibit PKC (9). If receptors are activated in the presence of anesthetic, PKC activity induced by receptor signaling will be modulated by the anesthetic, and I_Cl(Ca) will be altered. If, however, the channel is activated directly by injection of Ca, no PKC activation will occur, and I_Cl(Ca) will therefore not be modulated by the presence of anesthetics. Therefore, differences in I_Cl(Ca) measured in the presence and absence of anesthetic could be misinterpreted as an anesthetic effect on the proximal receptor signaling pathway.

A similar argument could be made for the role of PP. However, essentially nothing is known of the interactions between anesthetics and PP. We suggest that the appropriate control experiments would be to determine the effect of anesthetics on I_Cl(Ca) induced by receptor signaling or by Ca injection in the presence of PKC and PP inhibitors. Alternatively, a more proximal (and admittedly less convenient) end-point (e.g., IP₃ production) could be used.

Our findings also have some implications for studies of receptor physiology in oocytes, as it is possible to misconstrue an effect of PKC on I_Cl(Ca) as an effect on the receptor. For example, we have shown previously that LPA signaling in oocytes is regulated by PKC: LPA signaling was affected by either PKC inhibition or activation (2). In view of the current findings, it can not be excluded that such effects on signaling are an action on the Cl channel, rather than on the LPA receptor. In this particular instance, however, this seems not to be the case. Kim et al. (1) also investigated PKC regulation of LPA signaling and observed that PKC activation not only abolished LPA-induced I_Cl(Ca) but also the increase in IP₃ levels induced by LPA signaling. Conversely, inhibiting PKC enhanced IP₃ production. This indicates that the role of PKC is indeed proximal in the signaling pathway.

The modulation of the Xenopus I_Cl(Ca) by channel phosphorylation has not been studied in detail. The effect appears to be somewhat specific to PKC, as Chen et al. (10) reported that protein kinase A is without effect on the Ca-activated Cl channel. They did observe an effect of increased cyclic adenosine monophosphate (cAMP) concentrations on I_Cl(Ca) but demonstrated that this action was indirect and occurred by enhancement of endogenous Ca currents. Of regulation by PP virtually nothing is known. Chen et al. (10) demonstrated previously that injection of PP 1 and 2A attenuated a cAMP-induced increase of I_Cl(Ca) (10), and our observations that inhibition of PP increases I_Cl(Ca) above baseline indicates that regulation of channel activity by PP occurs under basal conditions. However, we have not conclusively shown that it is the channel itself that is being directly phosphorylated and dephosphorylated. It is conceivable that the activity of another regulatory molecule would be modulated by phosphorylation, and in turn would regulate the channel.

In summary, we have demonstrated that the X. laevis oocyte I_Cl(Ca) is modulated by both PKC and PP. These findings should be taken into account in the interpretation of studies using I_Cl(Ca) as a reporter for determining effects of anesthetics on GPCR, as well as in the design of such studies.

	Acknowledgments

 
Supported, in part, by the Department of Anesthesiology and Critical Care, University Hospital, Muenster, Germany.

	References

Top Abstract Introduction Methods Results Discussion References

 

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