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Signal Transduction of Arginine Vasopressin-Induced Arachidonic Acid Release in H9c2 Cardiac Myoblasts: Role of Ca²⁺ and the Protein Kinase C-Dependent Activation of p42 Mitogen-Activated Protein Kinase¹

Pharmacological Institute, College of Medicine, National Taiwan University, Taipei 10018, Taiwan

Address all correspondence and requests for reprints to: Ching-Chow Chen, Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan. E-mail: ccchen{at}ha.mc.ntu.edu.tw

	Abstract

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The mechanism of arginine vasopressin (AVP)-induced arachidonic acid (AA) release was examined in the cardiac myoblast cell line, H9c2. Stimulation of cells with AVP induced dose-dependent AA release, and this effect was completely inhibited by the V₁ receptor antagonist, d(CH)₅[Tyr(Me)²]AVP. AVP also produced dose-dependent stimulation of inositol phosphate formation; this was not affected by pertussis toxin, indicating the presence of the V₁ receptor/Gq protein/PLCß pathway in H9c2 cells. The concentration-response curves for these two effects of AVP overlapped. AVP induced a rapid increase in [Ca²⁺]_i, followed by a sustained increase. The Ca²⁺ ionophore, A23187 or ionomycin, mimicked the effect of AVP, whereas the protein kinase C (PKC) activator, TPA, only induced a slight increase in AA release. Both the AVP- or A23187-stimulated AA release and the AVP-induced sustained [Ca²⁺]_i increase were completely blocked in the absence of external Ca²⁺. The receptor-operated Ca²⁺ channel blocker, SKF 96365, and the inorganic Ca²⁺ channel blockers, Co²⁺ and Ni²⁺, also inhibited the AVP-induced AA release. Western blots demonstrated expression of PKC, ßI, , , and in H9c2 cells; PKC inhibitors (staurosporine or Ro 31–8220) or down-regulation of PKC, ßI, , and by long-term (24 h) TPA treatment caused a partial blockade of the AVP-induced response, whereas the A23187-induced AA release was unaffected by down-regulation of these isoforms. AVP-induced, but not A23187-induced, AA release was partially blocked by the p42 MAPK cascade inhibitor, PD 98059. AVP and TPA, but not A23187, induced an increase in activity and tyrosine phosphorylation of p42 MAPK, together with a molecular weight shift, consistent with phosphorylation, of cytosolic PLA₂. AVP- or TPA-induced activation and tyrosine phosphorylation of p42 MAPK were completely blocked by down-regulation of PKC, ßI, , and , but still occurred, together with the cytosolic PLA₂ mobility shift, in the absence of external Ca²⁺. These results show that AVP-induced AA release in H9c2 cells is secondary to activation of the V₁ receptor/Gq protein/PLCß pathway, leading to an influx of extracellular Ca²⁺ and activation of PKC, ßI, , and . The influx of extracellular Ca²⁺ and DAG act, respectively, through PKC-/MAPK-independent or PKC-dependent MAPK pathways to mediate AA release.

	Introduction

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PHOSPHOLIPASES A₂ (PLA₂) are a group of enzymes that catalyze the hydrolysis of the sn-2 ester bond of phospholipids, resulting in the production of free fatty acid and lysophospholipids. These lipid products may serve as intracellular second messengers or can be further metabolized to form potent inflammatory mediators. The PLA₂-induced release of arachidonic acid (AA) from membranes and its subsequent conversion into leukotrienes, PGs, and other eicosanoids play a key role in the process leading to inflammation. PLA₂ enzymes can be divided into two main classes (1), these being the extracellular 14-kDa secretory PLA₂s, which have been extensively characterized and structurally defined (2) and the intracellular class of PLA₂s, composed of a Ca²⁺-independent PLA₂, involved essentially in regulating the incorporation of AA into membrane phospholipids (3, 4), and the group IV cytosolic 85-kDa Ca²⁺-dependent PLA₂ (cPLA₂), responsible for intracellular AA release (4, 5) and activated by both micromolar [Ca²⁺]_i and Ser-505 phosphorylation by MAP kinases (MAPKs) or protein kinase C (PKC) (6).

Arginine vasopressin (AVP) plays important roles in cardiovascular regulation, presumably through its systemic vasoconstrictor effect (7). Numerous studies have demonstrated its importance in the regulation of blood pressure and the pathogenesis of hypertension (8); however, most of these were performed on the vasculature, and little is known concerning the effect of this hormone on the myocardium. Several studies have shown AVP to have a direct effect on the heart (9, 10, 11), and receptors for AVP in the myocardium have been identified as V₁-vasopressinergic receptors (11, 12). H9c2 cells, a permanent cell line derived from rat cardiac tissue, have morphological characteristics similar to those of immature embryonic cardiomyocytes, but they also have several elements of the electrical and hormonal pathway found in adult cardiac cells, such as the L-type Ca²⁺ channel, K channel, and G proteins (13, 14), and these cells are therefore useful as a model for cardiomyocytes in transmembrane signal transduction studies (13).

Based on the fact that Ca²⁺ and PKC are capable of promoting AA release, cPLA₂ activation has been proposed to be a sequela of the PLC-mediated hydrolysis of phosphoinositides (6). Although phosphorylation of Ser-505 is important for cPLA₂ activation in certain cells, it is not sufficient for full activation, leading to AA release (5). In Chinese hamster ovary cells overexpressed cPLA₂, it has been proposed that cPLA₂ phosphorylation by MAPK, together with an increase in intracellular Ca²⁺, is the mechanism whereby the membrane receptor fully activates this enzyme (15). Ca²⁺ may activate cPLA₂ by promoting its association with the nuclear envelope and endoplasmic reticulum (16, 17), and phosphorylation increased the catalytic activity (15). However, in astrocytes, TPA-induced PKC-dependent MAPK activation was able to stimulate AA release without inducing an increase in [Ca²⁺]_i, and the ATP-induced influx of extracellular Ca²⁺ was able to act through a PKC-dependent MAPK pathway to phosphorylate cPLA₂, then induce AA release (18). In the present study, we examined the signal transduction pathway involved in the AVP-induced AA release seen in H9c2 cardiac myoblasts and explored the role of Ca²⁺, PKC isoforms, and MAPK in the regulation of this event. It is apparent that extracellular AVP exerts its effect via the V₁-vasopressinergic receptor/Gq protein/PLCß pathway, leading to capacitative Ca²⁺ influx and PKC activation. Ca²⁺ influx exerts its effect through a PKC-/MAPK-independent mechanism, whereas PKC acts through MAPK-dependent activation of cPLA₂ to induce AA release. However, in H9c2 cardiac myoblasts, the role of Ca²⁺ predominates over that of the PKC-dependent mechanism.

	Materials and Methods

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Materials
Rabbit polyclonal antibodies raised against peptide sequences unique to PKC, or and DMEM, FCS, penicillin and streptomycin were purchased from Gibco BRL (Gaithersburg, MD). Rabbit polyclonal antibodies raised against peptide sequences unique to PKC, ßI, ßII, , , or , external signal-regulated kinase 1 (ERK1) (C-16) or ERK2 (C-14) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibody specific for the phosphorylated form of p44/42 MAPK was from New England Biolabs, Inc. (Beverly, MA). Rabbit polyclonal antibody against cPLA₂ was a gift from the Genetics Institute (Cambridge, MA). TPA was purchased from LC Services Corp. (Woburn, MA). AVP, d(CH)₅[Tyr(Me)²]AVP, pertussis toxin (PTX), staurosporine, protein A Sepharose, and myelin basic protein (MBP) were from Sigma Chemical Co. (St. Louis, MO). SKF 96365 was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Ro 31–8220 and PD 98059 were from Calbiochem (San Diego, CA). Reagents for SDS-PAGE were from Bio-Rad (Hercules, CA). [³H]AA (211.8 Ci/mmol), myo-[³H]inositol (23.5 Ci/mmol), and [-³²P]ATP (30 Ci/mmol) were from DuPont NEN (Boston, MA). The horseradish peroxidase-labeled donkey antirabbit second antibody and the ECL detecting reagent were purchased from Amersham International (Uppsala, Sweden).

Cell cultures
The cardiac myoblast cell line, H9c2, purchased from ATCC (Rockville, MD), was maintained in DMEM, supplemented with 10% FCS, 100 U/ml penicillin, and 100 µl/ml streptomycin, and was grown in an atmosphere of 5% CO₂/95% humidified air at 37 C on 24-well plates (AA release), 145-mm dishes (PKC isoform assay), 24-mm diameter coverslip in 35-mm dishes (intracellular Ca²⁺ measurement), or 6-well plates [phosphatidylinositol (PI) hydrolysis], or in T75 flasks (MAPK activity and cPLA₂ mobility shift assay).

Determination of [³H]AA release
Cells were prelabeled with [³H]AA (0.5 µCi/ml) for 18 h at 37 C, as previously described (18), washed three times with DMEM containing 20 mM HEPES (pH 7.5) and 0.2% BSA to remove unincorporated [³H]AA, and equilibrated by incubation for 10 min at 37 C. AVP at various concentrations, 1 µM TPA, or 5 µM A23187, was then added, and incubation continued for another 10 min; then, the medium was removed and the released radioactivity was determined by scintillation counting. In the Ca²⁺-free experiments, prelabeled cells were washed with Ca²⁺-free physiological salt solution (PSS) containing 1 mM EGTA and 0.2% BSA, and the control experiments were washed with normal PSS (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM KH₂PO₄, 11 mM glucose, 20 mM HEPES, pH 7.4) containing 0.2% BSA.

Measurement of PI hydrolysis
PI hydrolysis was assessed by measuring the accumulation of [³H]inositol phosphate ([³H]IP) in cells labeled during a 24-h incubation period in growth medium containing myo-[³H]inositol (2.5 µCi/ml), as previously described (19).

Measurement of the intracellular Ca²⁺ concentration
Intracellular concentrations of Ca²⁺ were determined using the Ca²⁺-sensitive dye, fura 2-AM, as previously described (18). Cells grown on coverslips were loaded with 5 µM fura 2-AM for 1 h at room temperature, washed twice with PSS. The coverslips were mounted on a chamber (300 µl vol) for fluorescence measurements, and the cells were continuously superfused using a peristaltic pump (Pharmacia, Uppsala, Sweden) (flow rate 1.25 ml/min) during microscopy. Experimental solution was removed from the coverslip chamber using a microaspirator connected to a vacuum pump. Ca²⁺-free experiments were performed using Ca²⁺-free PSS containing 1 mM EGTA. Before starting the experiment, the cells were equilibrated with the buffer for 5 min.

Fura-2 fluorescence was imaged with an inverted Diaphet microscope equipped with a 40x/1.3 NA Fluor DL objective lens (Nikon, Melville, NY). The cells were illuminated using a 100 Watt Xenon lamp with quartz collector lenses. A shutter and a filter wheel containing the two interference filters (340 and 380 nm) were controlled by a computer. The emitted light was passed through a 400-nm dichroic mirror, filtered at 510 nm, and collected using a CCD camera connected to a light intensifier (Photon Technology International, model A1010). Images of as many as 10 cells/field were digitized and averaged in an image-processor (PTI) connected to a computer equipped with software.

Preparation of cell extracts and immunoblot analysis of PKC isoforms
Cells were treated with TPA for periods of 10 min or 24 h before harvesting; the vehicle, dimethyl sulfoxide (0.1%), was added to control cells for 24 h. The cells were then rapidly washed with ice-cold PBS, scraped, and collected by centrifugation at 1000 x g for 10 min, as previously described (20). The preparation of cell extracts and immunoblot analyzes were performed as described previously (19, 20).

Immunoblot analysis of phosphorylated MAPK, ERK1, and ERK2
After cells were treated for 10 min with 30 nM AVP, 1 µM TPA, or 5 µM A23187, they were rapidly washed with ice-cold PBS and lysed with 500 µl ice-cold lysis buffer (50 mM Tris·HCl (pH 7.4), 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethlysulfonylflouride, 5 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM NaF, and 1 mM Na₃VO₄), as previously described (18). After centrifugation at 13,000 g for 10 min, equal amounts of total cell lysate (50 µg protein) were subjected to 10% SDS-PAGE, followed by immunoblotting for phosphorylated MAPK, ERK1 (p44 MAPK), or ERK2 (p42 MAPK) using rabbit polyclonal antibodies specific for phosphospecific p44/42 MAPK (1:1000 dilution), ERK1, or ERK2 (1:500 dilution), as described in the PKC isoform analysis.

Immunoprecipitation of MAPK and MAPK activity assay
Fifty micrograms of total cell lysate was incubated with 0.5 µg anti-ERK2 (p42 MAPK) antibody for 1 h at 4 C and collected using protein A-Sepharose CL-4B beads, as previously described (18). The beads were then washed three times with lysis buffer and incubated in 50 µl kinase reaction mixture containing 20 mM HEPES (pH 7.4), 10 mM MgCl₂, 100 µM Na₃VO₄, 0.3 mg/ml MBP, 50 µM [-³²P]ATP (2000 cpm/pmol) for 30 min at 30 C. MAPK activity was then assayed either by ³²P-incorporation on P81 phosphocellulose paper or by autoradiography. After incubation, half the reaction mixture (25 µl) was stopped by adding 25% trichloroacetic acid and spotted onto P81 phosphocellulose paper. After extensive washes with 75 mM phosphoric acid, the paper was dried, and ³²P-incorporation into MBP was measured with a scintillation counter. The other half of the reaction mixture was stopped by the addition of Laemmli buffer and subjected to 13% SDS-PAGE, and phosphorylated MBP was visualized by autoradiography.

Immunoblot analysis of cPLA₂ and mobility shift of phosphorylation
Fifty micrograms of total cell lysate was treated with 2x Laemmli buffer, boiled for 5 min, and subjected to 7.5% SDS-PAGE, as previously described (18). Electrophoresis (20 mA/gel) was continued for 4 h, after the tracking dye exited the gel, to increase the separation between the native and phosphorylated cPLA₂. Proteins were transferred to nitrocellulose paper, and immunoblot analysis was performed using rabbit polyclonal antibody specific for cPLA₂ (1:1000 dilution), as described in the PKC isoform analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Relationship between the effect of AVP on AA release and on IP formation and cytosolic Ca²⁺ rise in H9c2 cells
AVP induced a dose-dependent increase in both [³H]AA release and [³H]IP formation, with respective EC₅₀ values of 2.19 nM and 0.922 nM (Fig. 1). The AVP-promoted AA release was completely inhibited by the V₁ receptor antagonist, d(CH)₅[Tyr(Me)²] AVP (100 µM), indicating that AVP acts through the V₁ receptor to mediate AA release in these cells (Fig. 2A). Pretreatment of cells with PTX (100 ng/ml) for 24 h had no effect on AVP-stimulated IP formation or AA release (Fig. 2B), indicating the presence of the V₁ receptor/Gq protein/PLCß pathway in these cells. In addition, the concentration-response curve for the effect of AVP on IP formation overlapped with that for AA release (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Figure 1. Concentration-dependent stimulation of [3H]-AA release and [3H]-inositol phosphate formation by AVP in H9c2 cells. A, Prelabeled cells were washed free of unincorporated radioactivity and incubated with the indicated concentrations of AVP at 37 C for 10 min, then the released [3H]AA in the medium was counted; B, prelabeled cells were washed with PSS, and the lithium-dependent accumulation of [3H]IPs was measured in the presence of AVP at the indicated concentrations at 37 C for 15 min. The data are presented as the mean ± SEM of a typical triplicate determination; similar results were obtained in more than three experiments. Basal [3H]AA release and [3H]IP accumulation were 1500 cpm/well and 100 cpm/well, respectively.

View larger version (34K): [in this window] [in a new window]   Figure 2. Effects of the V1 receptor antagonist, d(CH)5[Tyr(Me)2]AVP, on [3H]AA release (A) and PTX pretreatment on AVP-induced [3H]AA release and [3H]IP formation (B) in H9c2 cells. Cells were pretreated with 100 µM d(CH)5[Tyr(Me)2]AVP for 30 min, or 100 ng/ml of PTX for 24 h, before exposure to 10 nM AVP for 10 min. The data are presented as the means ± SEM of a typical triplicate determination; similar results were obtained in three independent experiments.

View larger version (16K): [in this window] [in a new window]   Figure 3. Relationship between AVP-stimulated [3H]AA release and [3H]IP formation in H9c2 cells. Parallel cultures of [3H]AA and myo-[3H]-inositol prelabeled cells were incubated with 10 nM AVP for 10 min or 15 min, respectively. [3H]AA release and [3H]IP formation were measured as indicated in Materials and Methods. The results are expressed as percentage of maximal stimulation.

View larger version (18K): [in this window] [in a new window]   Figure 4. Time course of AVP-induced [3H]AA release (A) and fluorometer tracings of [Ca2+]i, in response to AVP (B) in H9c2 cells. A, Prelabeled cells were incubated with 10 nM AVP at 37 C for 1, 5, 10, or 15 min; and released [3H]AA was counted. The data are presented as the mean ± SEM of a typical triplicate determination; similar results were obtained in three experiments. B, Cells were continuously perfused with AVP in normal Ca2+ PSS or in Ca2+-free PSS containing 1 mM EGTA.

View larger version (37K): [in this window] [in a new window]   Figure 6. Effect of extracellular Ca2+-free or SKF 96365 (A) or Co2+ and Ni2+ (B) on the AVP- or A23187-induced [3H]AA release in H9c2 cells. Prelabeled cells, washed free of unincorporated radioactivity, were incubated with 10 nM AVP or 5 µM A23187 at 37 C for 10 min in PSS, Ca2+-free PSS, or Ca2+-free PSS containing 1 mM EGTA, which have 0.2% BSA, or pretreated with 10 µM SKF 96365 for 20 min (A) or 1 mM Co2+ or Ni2+ for 5 min (B) before challenge with these two agents. The data are presented as the mean ± SEM of three triplicate determinations. *, P < 0.05, as compared with the control.

View larger version (31K): [in this window] [in a new window]   Figure 8. Effect of staurosporine, Ro 31–8220, or long-term TPA pretreatment on AA release (A) and PKC, ßI, , and expression after 10 min or 24 h treatment with TPA (B) in H9c2 cells. A, Cells were pretreated with 100 nM staurosporine or 10 µM Ro31–8220 for 30 min, or with 1 µM TPA for 24 h before exposure to 10 nM AVP, 1 µM TPA, or 5 µM A23187 for 10 min. The data are presented as the mean ± SEM of three determinations. *, P < 0.05, as compared with the control. B, Cells were treated with 1 µM TPA for 10 min or 24 h, then fractionated into cytosolic and membrane fractions, and the proteins were separated by 10% SDS-PAGE, transferred to NC paper, and immunodetected with PKC-, ßI-, -, or -specific antibodies.

View larger version (26K): [in this window] [in a new window]   Figure 5. Fluorometer tracings of [Ca2+]i in response to AVP in H9c2 cells. A, Cells were continuously perfused with 10 nM AVP in normal PSS; B–F, the first arrow indicates the perfusion of 10 nM AVP for 150 sec in PSS and the second arrow indicates a change in perfusion solution to AVP in Ca2+-free PSS containing 1 mM EGTA (B), or to AVP in normal PSS containing 10 µM SKF 96365 (C), 1 mM Co2+ (D), 1 mM Ni2+ (E), or 1 mM Zn2+ (F).

View larger version (30K): [in this window] [in a new window]   Figure 7. Effect of extracellular Ca2+-free or SKF 96365 on AVP-induced [3H]IP formation in H9c2 cells. Prelabeled cells were washed with PSS; and the lithium-dependent accumulation of [3H]IP, in response to 10 nM AVP, was measured either in Ca2+-free PSS or Ca2+-free PSS, containing 1 µM EGTA, or after pretreatment with 10 µM SKF 96365 for 20 min.

To determine which PKC isoform was involved in the regulation of AVP-induced AA release in H9c2 cells, the effect of short-term (10 min) and long-term (24 h) TPA treatment on PKC isoform expression was studied (Fig. 8B). Western blot analysis, using nine PKC isoform-specific antibodies (a, ßI, ßII, , , , , , and ), showed expression of PKC, ßI, , , and in H9c2 cells. Ten-minute exposure to TPA caused marked translocation of PKC, ßI, , and ; whereas complete down-regulation of these isoforms was seen after 24 h of treatment (Fig. 8B).

The AVP-induced AA release is partially mediated by MAPK
To investigate whether the AVP-promoted AA release involved the activation of MAPK, a MAPK kinase inhibitor [PD 98059 (10 µM)] was used, resulting in 54.8% inhibition of the AVP-induced AA release, though not affecting A23187-induced AA release (Fig. 9).

View larger version (23K): [in this window] [in a new window]   Figure 9. Effect of PD 98059 on the AVP- or A23187-induced [3H]AA release in H9c2 cells. [3H]AA release, at basal state or in response to stimulation by 10 nM AVP or 5 µM A23187, was measured as described in Materials and Methods. Before stimulation, cells were pretreated with or without 10 µM PD 98059 for 30 min. The data are presented as the mean ± SEM of three typical triplicate experiments. *, P < 0.05, as compared with the control.

View larger version (21K): [in this window] [in a new window]   Figure 10. Effect of AVP, TPA, or A23187 on MAPK activity, MBP phosphorylation, phospho-MAPK, ERK1, and ERK2 expression and the cPLA2 mobility shift in H9c2 cells. Cells were stimulated with 10 nM AVP, 1 µM TPA, or 5 µM A23187 for 10 min, then lysed with lysis buffer. A and B, Cell lysates were immunoprecipitated with anti-ERK2 (p42 MAPK) antibody, and the MAPK activity was determined (A) or autoradiography of phosphorylated MBP performed (B), as described in Materials and Methods; C–F, cell lysates were subjected to 10% (C, D, and E) or 7.5% (F) SDS-PAGE, and immunoblots were performed using antiphospho-specific p44/42 MAPK (C), anti-ERK1 (D), anti-ERK2 (E), or anti-cPLA2 (F) antibodies, as described in Materials and Methods.

The AVP-induced activation of MAPK is mediated by PKC
Because the AVP-stimulated AA release is correlated with PKC, ßI, and (Fig. 8), and MAPK (Fig. 9) activation, and AVP induced activation of MAPK and cPLA₂ mobility shift (Fig. 10), we examined the effect of PKC down-regulation on the AVP-induced activation of MAPK to determine whether AVP-induced MAPK activation was mediated by PKC. After treatment of cells with 1 µM TPA for 24 h; conditions under which PKC, ßI, , and are completely down-regulated (Fig. 8B); the AVP-promoted activation of p42 MAPK activity and tyrosine phosphorylation were completely blocked, as was the activation effect of TPA (Fig. 11A). Ro 31–8220 and PD 98059 completely blocked AVP-induced p44/42 MAPK activation and cPLA₂ mobility shift as well (Fig. 11B).

View larger version (22K): [in this window] [in a new window]   Figure 11. Effect of PKC, ßI, , and down-regulation on AVP-, TPA-, or A23187-stimulated MAPK activity, MBP phosphorylation, and phospho-MAPK expression (A) and effect of Ro 31–8220 and PD-98059 on AVP-induced MAPK activation and cPLA2 mobility shift (B) in H9c2 cells. A, After overnight (24 h) incubation with 1 µM TPA, cells were stimulated with 10 nM AVP, 1 µM TPA, or 5 µM A23187 for 10 min; B, cells were pretreated with 10 µM Ro 31–8220 or PD 98059 for 30 min before exposure to 10 nM AVP for 10 min. The methods used were as described for Fig. 10.

View larger version (17K): [in this window] [in a new window]   Figure 12. Effect of extracellular Ca2+-free medium on the AVP-, TPA-, or A23187-stimulated MAPK activity (A), MBP phosphorylation (B), phospho-MAPK expression (C), and cPLA2 mobility shift (D) in H9c2 cells. After incubation in Ca2+-free PSS containing 1 mM EGTA for 20 min, cells were stimulated with 10 nM AVP, 1 µM TPA, or 5 µM A23187 for 10 min. The methods used were as described for Fig. 10.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Several investigators have shown that activation of the AA cascade is associated with increased phosphorylation, accompanied by stimulation of cPLA₂ activity in different types of cells and cPLA₂-overexpressed Chinese hamster ovary cells (15, 25, 26). However, others have shown that cPLA₂ phosphorylation does not always correlate with increased AA production in human neutrophils and murine macrophages (27, 28, 29, 30). cPLA₂ possesses a MAPK consensus sequence at Ser-505, and p42 MAPK is able to phosphorylate cPLA₂ both in vitro and in vivo, thereby increasing its activity (15, 21, 25, 31, 32). In this study, AVP stimulated tyrosine phosphorylation and activation of p42 MAPK, induced a cPLA₂ mobility shift, and provoked marked AA release (more than 4-fold of basal). TPA also elicited tyrosine phosphorylation and activation of p42 MAPK and induced a cPLA₂ mobility shift, but it elicited only a slight increase in AA release (1.8-fold of basal). In contrast, A23187 did not stimulate tyrosine phosphorylation and activation of p42 MAPK or induce a cPLA₂ mobility shift, but it did provoke marked AA release (more than 4-fold of basal). PKC has been implicated in the regulation of receptor-mediated AA release and eicosanoid synthesis (18, 33, 34). In this study, the AVP-stimulated AA release was partially mediated by PKC (Fig. 8A). After down-regulation of PKC, ßI, , and by long-term treatment with TPA, the AVP- and TPA-stimulated tyrosine phosphorylation and activation of p42 MAPK were both completely blocked. This result differs from those obtained in astrocytes and MDCK cells expressing PKC; this isoform is not down-regulated by long-term treatment with TPA (35); therefore, the agonist or TPA still induced MAPK activation after treatment in these two types of cells (18). Ro 31–8220 and PD 98059 also completely blocked the AVP-induced p42 MAPK activation and cPLA₂ mobility shift (Fig. 11B). However, the AVP-induced AA release was only partially blocked by down-regulation of PKC, ßI, , and (54.1% inhibition) or by staurosporine (60% inhibitin), or PD 98059 (54.8% inhibition) (Figs. 8A and 9). In the absence of extracellular Ca²⁺, the AVP or TPA-stimulated tyrosine phosphorylation and activation of p42 MAPK was not significantly affected, and the cPLA₂ mobility shift was still apparent. These results indicate that, as seen in astrocytes (18), activation of new PKC isoforms by agonist and TPA was sufficient to activate MAPK and induce cPLA₂ mobility shift, because translocation of the new PKC isoform is Ca²⁺ independent (22). However, AVP-induced AA release was completely blocked, in the absence of extracellular Ca²⁺, despite the cPLA₂ mobility shift. Furthermore, A23187 caused AA release without inducing activation of p42 MAPK and cPLA₂ mobility shift. These results indicate that the PKC-dependent MAPK phosphorylation of cPLA₂ is of minor importance in the AVP-mediatied AA release seen in H9c2 cells, whereas the influx of extracellular Ca²⁺, which is PKC-/MAPK-independent, plays a predominant role.

It has been proposed that phosphorylation of cPLA₂ by MAPK, in coordination with an increase of [Ca²⁺]_i, is the mechanism whereby the membrane receptor fully activates this enzyme (15). In astrocytes (18), however, TPA alone is able to stimulate AA release without inducing an increase in [Ca²⁺]_i, suggesting that PKC-dependent MAPK activation directly stimulates the activity of cPLA₂ (18, 33). Furthermore, this type of cell, A23187, also acts through PKC-dependent MAPK activation to phosphorylate cPLA₂ and induce AA release. However, in macrophages and monocytes, where the A23187-induced AA release is PKC-/MAPK-independent and no mobility shift of cPLA₂ can be detected (27, 32), a rise in [Ca²⁺]_i (but not cPLA₂ phosphorylation) is essential for activation of AA release in macrophages (32). The result of the influx of extracellular Ca²⁺ playing a predominant role in H9c2 cells is similar to that obtained in macrophages and monocytes. However, down-regulation of PKC, ßI, , and , or in the presence of staurosporine, Ro 31–8220 or PD 98059 partially inhibited the AVP-induced AA release, indicating that PKC-dependent MAPK phosphorylation also plays a minor role. Thus, the AVP-stimulated AA release seen in H9c2 cells is secondary to the activation of the V₁ receptor/Gq protein/PLCß pathway. Two signaling components, DAG and the capacitative Ca²⁺ influx, act via different pathways to regulate AA release. DAG could act via activation of PKC, ßI, , and , followed by MAPK activation, leading to cPLA₂ phosphorylation. On the other hand, capacitative Ca²⁺ influx acts via PKC-/MAPK-independent mechanism. A schematic representation of the signaling pathway of AVP-induced AA release in H9c2 cells is shown in Fig. 13. Recently, a report of a pathway involving cPLA₂, which couples AA release to the muscarinic receptor, requires PKC activity and G proteins but may operate in the absence of Ca²⁺ mobilization (36). The differential regulation of cPLA₂ by Ca²⁺ and phosphorylation or G proteins in astrocytes (18), H9c2 cels (present study), macrophages and monocytes (32), and astrocytoma cells (36), suggest that different cPLA₂ isoforms might exist in these types of cells.

View larger version (17K): [in this window] [in a new window]   Figure 13. Schematic representation of the signaling pathway of AVP-induced AA release in H9c2 cells. AVP binds to V1-receptor and activates PI-PLC via Gq protein, resulting in PKC activation, followed by MAPK activation, leading to cPLA2 phosphorylation and AA release. In contrast, capacitative Ca2+ influx acts via a PKC-/MAPK-independent mechanism. PI-PLC, Phosphatidylinositol phospholipase C, DAG, 1,2-diacylglycerol; PIP2, phosphatidylinositol 4,5-biphosphate; G, GTP-binding protein; IP3, inositol-1,4,5-triphos- phate.

2

2

1

2

	Footnotes

 
¹ This work was supported by a research grant from the National Science Council of Taiwan.

Received August 31, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Dennis EA 1997 The growing phospholipase A₂ superfamily of signal transduction enzymes. Trends Biochem Sci 22:1–2[CrossRef][Medline]
2. Davidson FF, Dennis EA 1990 Evolutionary relationships and implications for the regulation of phospholipase A₂ from snake venom to human secreted forms. J Mol Evol 31:228–238[CrossRef][Medline]
3. Balsinde J, Barbour SE, Bianco ID, Dennis EA 1994 Arachidonic acid mobilization in p388D1 macrophages is controlled by two distinct Ca²⁺-dependent phospholipase A₂ enzymes. Proc Natl Acad Sci USA 91:11060–11064[Abstract/Free Full Text]
4. Balsinde J, Dennis EA 1996 Distinct roles in signal transduction for each of the phospholipase A₂ enzymes present in p388D1 macrophages. J Biol Chem 271:6758–6765[Abstract/Free Full Text]
5. Clark JD, Schievella AR, Nalefski EA, Lin LL 1995 Cytosolic phospholipase A₂. J Lipid Med Cell Signal 12:83–117[CrossRef][Medline]
6. Leslie CC 1997 Properties and regulation of cytosolic phospholipase A₂. J Biol Chem 272:16709–16712[Free Full Text]
7. Share L 1998 Role of vasopressin in cardiovascular regulation. Physiol Rev 68:1248–1284[Free Full Text]
8. Jonston CI 1985 Vasopressin in circulatory control and hypertension. J Hypertens 3:557–569[Medline]
9. Schoemaker IE, Meulemans AL, Andries LJ, Brutsaert DL 1990 Role of endocardial endothelium in positive inotropic action of vasopressin. Am J Physiol 259:M1148–M1151
10. Cheng CP, Igarashi Y, Klopfenstein HS, Applegate RJ, Shihabi Z, Little WC 1993 Effect of vasopressin on left ventricular performance. Am J Physiol 264:H53–H60
11. Walker BR, Childs ME, Adams ME 1988 Direct cardiac effects of vasopressin: role of V₁- and V₂-vasopressinergic receptors. Am J Physiol 255:H261–H265
12. Xu YJ, Gopalakrishnan V 1991 Vasopressin increases cytosolic free [Ca²⁺] in the neonatal rat cardiomyocyte-evidence for V₁-subtype receptors. Circ Res 69:239–245[Abstract/Free Full Text]
13. Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, Schultz G 1991 Morphological, biochemical and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res 69:1476–1486[Abstract/Free Full Text]
14. Sipido KR, Marban E 1991 L-type calcium channels, potassium channels and novel nonspecific cation channels in a clonal muscle cell line derived from embryonic rat ventricle. Circ Res 69:1487–1499[Abstract/Free Full Text]
15. Lin LL, Wartmann M, Lin AY, Knopf J, Seth A, Oavis RJ 1993 cPLA₂ is phosphorylated and activated by MAP kinase. Cell 72:269–278[CrossRef][Medline]
16. Schievella AR, Regir MK, Smith WL, Lin LL 1995 Calcium-mediated translocation of cytosolic PLA₂ to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270:30749–30754[Abstract/Free Full Text]
17. Glover S, Bayburt T, Jonas M, Chi E, Gelb MH 1995 Translocation of the 85-kDa phospholipase A₂ from cytosol to the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen. J Biol Chem 270:15339–15367
18. Chen WC, Chen CC 1998 ATP-induced arachidonic acid release in cultured astrocytes is mediated by Gi protein-coupled P2Y₁ and P2Y₂ receptors. Glia 22:360–370[CrossRef][Medline]
19. Chen CC, Chang J, Chen WC 1995 Role of protein kinase C subtypes and in the regulation of bradykinin-stimulated phosphoinositide breakdown in astrocytes. Mol Pharmacol 48:39–47[Abstract]
20. Chen CC 1993 Protein kinase C , , and in C6 glioma cells. TPA induces translocation and down-regulation of conventional and new PKC isoforms but not atypical PKC. FEBS Lett 332:169–173[CrossRef][Medline]
21. Nemenoff RA, Winitz S, Olan NX, Putten VV, Johnson GL, Heasley LE 1993 Phosphorylation and activation of a high molecular weight form of phospholipase A₂ by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem 268:1960–1964[Abstract/Free Full Text]
22. Chen CC 1994 Effect of Ca²⁺ on the activation of conventional and new PKC isozymes and on TPA and endothelin-1 induced translocation of these isozymes in intact cells. FEBS Lett 348:21–26[CrossRef][Medline]
23. Putney JW 1990 Capacitativity calcium entry revisited. Cell Calcium 11:611–624[CrossRef][Medline]
24. Lazarowski ER, Boucher RC, Harden TK 1994 Calcium-dependent release of arachidonic acid in response to purinergic receptor activation in airway epithelium. Am J Physiol 266:C406–C415
25. Sa G, Marugesan G, Jaye M, Ivashchenko Y, Fox PL 1995 Activation of cytosolic phospholipase A₂ by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J Biol Chem 270:2360–2366[Abstract/Free Full Text]
26. Qiu ZH, de Carvalho MS, Leslie CC 1993 Regulation of phospholipase A₂ activation by phosphorylation in mouse peritoneal macrophages. J Biol Chem 268:24506–24513[Abstract/Free Full Text]
27. Xu XX, Rock CO, Qiu ZH, Leslie CC, Jackowski S 1994 Regulation of cytosolic phospholipase A₂ phosphorylation and eicosanoid production by colony-stimulating factor. J Biol Chem 269:31693–31700[Abstract/Free Full Text]
28. Doerfler ME, Weiss J, Clark JD, Elsbach P 1994 Bacterial lipopolysaccharide primes human neutrophils for enhanced released of arachidonic acid and causes phosphorylation of an 85-kDa cytosolic phospholipase A₂. J Clin Invest 93:1583–1591
29. Durstin M, Durstin S, Molski TFP, Becker EL, Sha’afi RI 1994 Cytosolic phospholipase A₂ translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc Natl Acad Sci USA 91:3142–3146[Abstract/Free Full Text]
30. Nahas N, Waterman WH, Sha’afi RI 1996 Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A₂ in human neutrophil. Biochem J 313:503–508
31. Qiu ZH, Leslie CC 1994 Protein kinase C-dependent and -independent pathway of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A₂. J Biol Chem 269:19480–19487[Abstract/Free Full Text]
32. Ambs P, Baccarini M, Fitzke E, Dieter P 1995 Role of cytosolic phospholipase A2 arachidonic acid release of rat liver macrophages: regulation by Ca²⁺ and phosphorylation. Biochem J 311:189–195
33. Chao W, Liu H, Hanahan DJ, Olson MS 1992 Platelet activating factor-stimulated protein tyrosine phosphorylation and eicosanoid synthesis in rat Kupffer cells. J Biol Chem 267:6725–6735[Abstract/Free Full Text]
34. Xing M, Firestein BL, Shen GH, Insel PA 1997 Dual role of protein kinase C in the regulation of cPLA₂-mediated arachidonic acid release by P2u receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and-independent pathways. J Clin Invest 98:805–814
35. Chen CC, Wang JK, Chen WC 1997 TPA induces translocation but not down-regulation of new PKC isoform in macrophages, MDCK cells and astrocytes. FEBS Lett 412:30–34[CrossRef][Medline]
36. Bayon Y, Hernandez M, Alonso A, Nunez L, Garcia-Sancho J, Leslie C, Crespo MS, Nieto ML 1997 Cytosolic phospholipase A₂ is coupled to muscarinic receptors in the human astrocytoma cell line 1321N1: characterization of the transducing mechanism. Biochem J 323:281–287
37. Revtyak GE, Buja LM, Chien KR, Campbell WB 1990 Reduced arachidonate metabolism in ATP-depleted myocaridal cells occurs early in cell injury. Am J Physiol 259:H582–H591
38. Pinson A, Holabi A, Shohami E 1992 Mechanical injury increases eicosanoid production in cultured cardiomyocytes. Prostaglandins Leukot Essent Fatty Acids 16:9–13
39. Lai J, Jin H, Yang R, Winer J, Li W, Yen R, King KL, Zeigler F, Ko A, Cheng J, Bunting S, Paoni NF 1996 Prostaglandin F₂ induces cardiac myocyte hypertrophy in vitro and cardiac growth in vivo. Am J Physiol 271:H2197–H2208
40. Adams JW, Migita DS, Yu MK, Young R, Hellickson MS, Castro-Vargas FE, Domingo JD, Lee PH, Bui JS, Henderson SA 1996 Prostaglandin F₂ stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes. J Biol Chem 271:1179–1186[Abstract/Free Full Text]
41. Moffat MP 1987 Concentration-dependent effects of prostacyclin on the response of the isolated guinea pig heart to ischemia and reperfusion: possible involvement of the slow inward current. J Pharmacol Exp Ther 242:292–299[Abstract/Free Full Text]
42. Oudot F, Grynberg A, Sergiel JP 1995 Eicosanoid synthesis in cardiomyocytes: influence of hypoxia, reoxygenation and polyunsaturated fatty acids. Am J Physiol 268:H308–H315

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