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(Circulation Research. 1996;78:161-165.)
© 1996 American Heart Association, Inc.

Articles

Adenosine A₁ Stimulation Activates -Protein Kinase C in Rat Ventricular Myocytes

Patrick Henry, Sophie Demolombe, Michel Pucéat, Denis Escande

From the Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Hôpital G & R Laënnec, Nantes, France; the Département de Cardiologie, Hôpital Broussais, Paris, France; and INSERM U-390, Hôpital Arnaud de Villeneuve, Montpellier, France.

Correspondence to Dr Denis Escande, Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Hôpital G & R Laënnec, BP 1005, 44035 Nantes, France.

	Abstract

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Abstract By making use of immunoblotting and immunocytochemical analysis, we explored whether stimulation of adenosine A₁ receptors would promote the activation of -protein kinase C (-PKC) immunolabeled with a polyclonal antibody. Immunoblot analysis of Triton X-100–soluble cell membrane and cytosolic fractions revealed the presence of a specific 75-kD band reactive to the -PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive -PKC was associated with the membrane fraction, whereas 72% was associated with the soluble fraction. Under stimulation with the tumor-promoting phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) used as a positive control, -PKC translocated to the cell membrane, with the membrane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive -PKC. Transverse optical sections performed with confocal laser microscopy showed that immunostaining with anti–-PKC antibody was distributed in the cytosol of unstimulated cells but accumulated in the cell membrane under PMA stimulation. In the membrane fraction of cells pretreated with adenosine (100 µmol/L) or with the adenosine A₁ agonist (-)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA, 1 µmol/L), the 75-kD band corresponding to -PKC increased by 57% and 66%, respectively, when compared with nonstimulated cells processed under the same experimental conditions. In cells exposed to either of the purine agonists, specific fluorescence staining decorated the cell membrane, a pattern that was not observed in control cells. Activation of membrane -PKC produced either by adenosine itself or by its analogue R-PIA was fully antagonized by the specific A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 µmol/L). From these data, we conclude that adenosine A₁ stimulation activates -PKC in freshly isolated rat ventricular myocytes.

Key Words: adenosine • protein kinase C • phorbol ester • preconditioning • rat ventricular myocytes

	Introduction

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Adenosine is a naturally occurring nucleoside that has been implicated in the pathophysiology of ischemic preconditioning, ie, the property of the cardiac myocyte to dramatically tolerate an ordinarily lethal ischemic insult when it is preceded by an initial brief exposure to ischemia.¹ The cellular cascade leading to preconditioning is still a matter of debate, although it is now widely accepted that the adenosine released from the ischemic heart and bound to myocardial adenosine A₁ receptor subtype plays a determinant role.² Because adenosine is extremely labile in the blood with a half-life of seconds,³ this mediator should be washed out during the reflow period separating the preconditioning brief ischemic episode and the prolonged ischemic insult. Thus, adenosine released during preconditioning ischemia should have triggered some secondary mechanism of protection not directly dependent on the presence of the purine. Most recently, the activation of Ser-Thr protein kinase, PKC, has been implicated as follows in the myocardial cellular cascade leading to ischemic preconditioning⁴ : (1) Membrane PKC activity is remarkably enhanced during ischemia.^{5 6} (2) Protection conferred by preconditioning is abolished by PKC inhibitors, eg, calphostin C,⁷ staurosporine,⁸ or chelerythrine,⁹ and is mimicked by PKC activators.^{8 9} PKC exists as a family of at least nine isozymes. Only five different isoforms have been detected at the mRNA level in the myocardium,¹⁰ namely, -PKC (Ca²⁺ dependent) and -, -, -, and -PKC (Ca²⁺ independent). Western blot analysis and immunocytochemical methods have revealed that the predominant Ca²⁺-independent isoforms in cardiomyocytes are - and -PKC.¹¹ Cytosolic PKC is inactive and can phosphorylate proteins only after it has been translocated to the cell membrane, where it activates.

In an attempt to identify a possible link between adenosine receptor activation and the PKC pathway, we explored whether adenosine agonists could translocate one of the major Ca²⁺-independent PKC cardiac isoforms, -PKC, to the cell membrane. -PKC was chosen because it was recently shown to be the only isoform activated by neurohormonal stimulation in rat cardiac cells.¹¹ We used immunoblotting and immunocytochemical analysis with confocal laser microscopy in adult rat ventricular cells to determine the immunoreactive membrane and cytosolic -PKC. Our results demonstrate that adenosine A₁ stimulation increases immunoreactive membrane -PKC.

	Materials and Methods

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Cell Isolation and Drugs
Adult rat ventricular myocytes were enzymatically dissociated with collagenase according to the method described by Irisawa and Kokubun¹² and then stored for 2 hours at 37°C in a Joklik-modified Eagle's minimal essential medium (Sigma Chemical Co) in a cell incubator. Cells were then placed in Eppendorf tubes (300 000 cells per tube) and exposed at 37°C to various adenosine receptor agonists and antagonists for a controlled period of time. PMA (Sigma) was used at a final concentration of 500 nmol/L. Adenosine purchased from Boehringer was used at a final concentration of 100 µmol/L. R-PIA (Sigma) and DPCPX (RBI) were used at a final concentration of 1 µmol/L. All drugs were dissolved in dimethyl sulfoxide, which never exceeded a final concentration of 10 µmol/L.

Immunoblotting
After drug incubation for 1, 5, or 10 minutes, tubes were centrifuged for 5 seconds, the supernatant was removed, and the cell pellet was immediately frozen in liquid nitrogen and stored at -80°C until further use. Membrane and cytosolic fractions were prepared by using digitonin disruption according to Pelech et al.¹³ Briefly, cells were suspended in buffer A and then kept for 10 minutes at 4°C. Buffer A contained (mmol/L) glycerophosphate 50, EDTA 1, EGTA 20, PMSF 1, leupeptin 0.1, E-64 0.01, CaCl₂ 0.34, and sucrose 250, along with 0.05% (wt/vol) digitonin. Cells were centrifuged at 10 000g for 2 minutes. The supernatant containing the cytosolic proteins was saved, and the pellet was resuspended by vortexing in 200 µL buffer B maintained at 4°C and made of (mmol/L) glycerophosphate 50, EGTA 1, PMSF 1, leupeptin 0.1, and E-64 0.01, along with 1% (vol/vol) Triton X-100. After centrifugation at 10 000g for 15 minutes, the supernatant saved as the crude membrane fraction was collected, and a sample (10 µL) was taken for estimation of the total protein content according to the method described by Bradford.¹⁴ Sample electrophoresis buffer made of 100 mmol/L dithiothreitol, 2% (wt/vol) SDS, 60 mmol/L Tris-Cl, and 0.01% (wt/vol) bromophenol blue, pH 6.8, was added to the tubes containing the resulting fractions, which were boiled for 2 minutes and then frozen and stored at -20°C for SDS-PAGE.

Proteins (usually 50 µg per well) were run on 7.5% (wt/vol) SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. Blots were blocked with 5% (wt/vol) nonfat dry milk for 1 hour at 37°C before they were incubated overnight with the primary antibody (anti–-PKC; dilution, 1/1000; GIBCO BRL) in 0.25% (wt/vol) albumin and 0.5% (vol/vol) Tween 20 dissolved in PBS containing (mmol/L) NaCl 137, KCl 2.7, Na₂HPO₄ 8, and KH₂PO₄ 1.5, pH 7.4. The anti–-PKC antibody that we used was generated in rabbits against a peptide from the C terminus of -PKC corresponding to amino acids 662 to 673 (Ser-Phe-Val-Asn-Pro-Lys-Tyr-Glu-Gln-Phe-Leu-Glu). After incubation with primary antibody, blots were then exposed for 1 hour at 20°C to a peroxidase-conjugated secondary antibody (dilution, 1:2000; anti-rabbit IgG, A6154, Sigma). Membranes were rinsed three times with PBS–Tween 20 between each step and finally developed by the enhanced chemiluminescence Western blotting system by use of the ECL kit (Amersham). Hyperfilm (Amersham) was used to reveal -PKC on the blot. Bands were thereafter quantified by using an optical densitometer (Sebia).

Immunostaining and Confocal Laser Microscopy
Ventricular cells were sedimented on 12-mm glass coverslips coated with laminin. The stimulation protocol with PMA, adenosine, and R-PIA, including concentrations and duration of exposure, was similar to that used for immunoblot analysis. Immediately after incubation with drugs, the extracellular medium was removed and replaced by a solution containing 4% formaldehyde (BDH Laboratories Supplies) dissolved in PBS and maintained for 15 minutes to fix the cells. Cells were subsequently washed three times with PBS. The residual aldehydes were inactivated with 50 mmol/L NH₄Cl diluted in PBS. Cells were permeabilized for 10 minutes at room temperature with 0.2% (vol/vol) Triton X-100 (Sigma) diluted in PBS and then rinsed three times with PBS. Nonspecific sites were saturated with a 1-hour incubation at 37°C in PBS containing 5% (wt/vol) albumin (fraction V, Sigma). Coverslips were then incubated with the anti–-PKC primary antibody (1:100 dissolved in PBS-albumin) either for 1 hour at room temperature or overnight at 4°C. Cells were washed three times with PBS and then incubated with fluorescein isothiocyanate–conjugated goat anti-rabbit IgG (1:200 dissolved in PBS-albumin, Sanofi-Pasteur) for 1 hour at room temperature. A mounting medium (Citifluor) was placed on the cells to prevent photobleaching. Cells were examined by confocal microscopy (Bio-Rad MRC-600) on a Nikon x60 PlanApo oil objective for high resolution. Fluorescein was excited through an argon ion laser light at 488 nm. Sequential serial sections were collected as xy and xz sections. Aperture, gain, and black level for imaging acquisition were maintained constant. Images were processed for both intensity and contrast, which were defined at the beginning of the acquisition procedure and kept constant thereafter.

	Results

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Translocation of -PKC in Rat Myocytes Stimulated With PMA
Immunoblot analysis of membrane and cytosolic fractions revealed the presence of a specific 75-kD band reactive to polyclonal antibody (Fig 1). As shown in Fig 1, immunoreactive -PKC was predominantly localized in the cytosol in unstimulated cells. In cardiac myocytes, 28±6% (mean±SEM, n=6) of the total immunoreactive -PKC was associated with the Triton X-100–soluble cell membrane fraction. The remainder (72%) was associated with the soluble fraction. This distribution agrees well with previous findings obtained with the same anti–-PKC antibody.¹¹ The tumor-promoting phorbol ester PMA was used as a positive control to activate -PKC. On exposure to PMA (500 nmol/L), -PKC underwent rapid and almost complete association with the Triton X-100–soluble membrane protein. In cardiac cells exposed to PMA for 5 minutes, the blot showed a large band at 75 kD in the membrane fraction, representing 88±9% (five observations) of total -PKC, whereas the blot of the cytosolic fraction showed a weaker 75-kD band, representing only 12% of total immunoreactive -PKC. Thus, PMA treatment increased by 214% the membrane-associated immunoreactivity (P<.01 versus control) and decreased by 83% the cytosol-associated fraction (P<.01 versus control). This suggests that under PMA stimulation -PKC translocated to the plasma membrane. Increased membrane immunoreactivity was observed after exposure to PMA as brief as 1 minute and persisted for at least 1 hour (not illustrated). Additional experiments were performed to ensure that the dimethyl sulfoxide used to dissolve PMA did not affect the subcellular distribution of -PKC. Immunocytochemical imaging using conventional fluorescence microscopy showed no significant changes in the cellular fluorescence pattern under PMA stimulation because of membrane folding related to T tubules. This difficulty was overpassed by performing transverse optical sections with confocal laser miscroscopy (Fig 2), which confirmed that immunoreactive -PKC was localized in the cytosol of unstimulated cells (n=6), extending from the plasma membrane to the nuclear membrane with no significant intranuclear staining (data not shown). No specific staining was observed in preparations incubated with the secondary antibody alone. In PMA-stimulated cells, transverse optical sections showed that the surface-to-cytosol fluorescence ratio dramatically increased (n=6, Fig 2), with immunostaining predominantly decorating the surface membrane.

View larger version (14K): [in this window] [in a new window]   Figure 1. Expression of -PKC isoform in control unstimulated cells and in cells stimulated for 5 minutes with PMA (500 nmol/L). Immunoblots show Triton X-100–soluble cell membrane and cytosolic fractions prepared as described in "Materials and Methods." Immunoblots were performed by using a polyclonal antibody against -PKC. Each lane was loaded with 50 µg protein. Bound antibody was detected by the enhanced chemiluminescence method, and immunoblots were exposed to film for 5 minutes. The numbers on the left indicate the positions of the prestained molecular mass standards.

View larger version (53K): [in this window] [in a new window]   Figure 2. Confocal laser microscopic imaging of adult cardiac rat myocytes with or without preexposure to PMA (500 nmol/L) for 5 minutes. Each panel is composed of a longitudinal optical section and a magnified transverse optical section. The upper and lower horizontal bars represent 10 µm. Aperture, gain, and black level for imaging acquisition were maintained constant throughout.

Effects of Adenosine and R-PIA on Immunoreactive -PKC
In the membrane fraction of myocytes preexposed to adenosine (100 µmol/L) for 1 minute, the 75-kD band corresponding to -PKC increased by 57±54% (n=8, P<.05 versus control, Fig 3) compared with that in nonstimulated cells processed under the same experimental conditions. R-PIA (1 µmol/L), an adenosine analogue that exhibits 100-fold selectivity for the A₁ over the A₂ receptors,¹⁵ increased membrane-associated immunoreactivity by 66±53% (n=7, P<.05 versus control). The increase in membrane -PKC immunoreactivity as induced by adenosine peaked at 1 minute (+57%), decreased after a 5-minute exposure (+18%), and was not detected after a 10-minute exposure of the cells with the agonist. Thus, adenosine agonists induced consistent changes in immunoreactive -PKC, which were less marked, however, when compared with those induced by the phorbol ester PMA. In particular, neither adenosine nor R-PIA agonists produced a significant decreased signal in the blot obtained from the cytosolic fraction (on average, adenosine decreased the cytosolic fraction by -12±28%; P=NS versus control; Fig 3). To further elucidate the nature of the receptor involved in the effects of adenosine on immunoreactive -PKC, myocytes were pretreated with DPCPX, an adenosine antagonist showing 740-fold selectivity for the A₁ over the A₂ receptors.¹⁵ In the presence of DPCPX (1 µmol/L), exposure to neither adenosine nor R-PIA for 1 minute significantly increased immunoreactive -PKC associated with the Triton X-100–soluble cell membrane fraction (Fig 3). In five additional experiments, pretreatment with DPCPX alone decreased by 6% the intensity of the 75-kD band in the membrane fraction, although this effect did not reach significance. The effects of adenosine receptor stimulation were also clearly visible on immunocytochemistry imaging (Fig 4). When cells were pretreated with adenosine (100 µmol/L) or R-PIA (1 µmol/L) for 1 minute, the fluorescent pattern of -PKC observed in transverse optical sections revealed a consistent peripheral staining not observed in control cells (see Fig 2). However, neither adenosine nor R-PIA appreciably diminished cytosolic immunoreactive -PKC, a finding that is consistent with immunoblots.

View larger version (23K): [in this window] [in a new window]   Figure 3. Expression of -PKC isoform in unstimulated cells and in cells stimulated for 1 minute with adenosine (Ado, 100 µmol/L), R-PIA (1 µmol/L), Ado (100 µmol/L) plus DPCPX (1 µmol/L), or R-PIA (1 µmol/L) plus DPCPX (1 µmol/L). Immunoblots show Triton X-100–soluble cell membrane and cytosolic fractions. Bound antibody was detected by the enhanced chemiluminescence method, and immunoblots were exposed to film for 5 minutes. Each lane was loaded with 50 µg protein. The numbers on the left indicate the positions of the prestained molecular mass standards.

View larger version (49K): [in this window] [in a new window]   Figure 4. Confocal laser microscopic imaging of adult cardiac rat myocytes preexposed to adenosine (ADO, 100 µmol/L) or R-PIA (1 µmol/L) for 1 minute. Each panel is composed of a longitudinal optical section and a magnified transverse optical section performed at the level indicated by the arrow. The upper and lower horizontal bars represent 10 µm. Aperture, gain, and black level for imaging acquisition were maintained constant throughout.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, using both Western blotting and cell immunostaining combined with confocal microscopy, we show that membrane-associated -PKC increases under adenosine stimulation in adult ventricular rat myocytes. In contrast to the sustained activation obtained after phorbol ester nonphysiological stimulation, the increase of -PKC immunoreactivity that we observed with adenosine receptor stimulation was rapid and nonsustained, as previously reported with other physiological agonists.^{11 16 17 18 19} Our data do not firmly prove translocation of -PKC, since the cytosolic fraction of immunoreactive -PKC was not significantly affected by adenosine stimulation. However, translocation remains the most likely mechanism to account for our results; after adenosine stimulation, we measured and determined that -PKC membrane immunoreactivity increased by 57%. If one hypothesized that increased -PKC membrane immunoreactivity was caused by -PKC translocation from the cytosol, the ratio would become 44% in the membrane fraction and 56% in the cytosolic fraction in the presence of adenosine. The method that we used was adequate to detect a 1.57-fold increase (28% to 44%) in membrane immunoreactivity. By contrast, detecting a 0.22-fold decrease (72% to 56%) in cytosolic immunoreactivity may be more difficult, since most of the immunoreactivity still remained in this compartment. An 2-fold increase in membrane PKC isoform immunoreactivity under the effect of various agonists has previously been observed in cardiac cells without an apparent concomitant decrease of the cytosolic fraction.^{11 20} A further limitation of our data is that they do not imply an increase in the biochemical protein kinase activity. However, in previous works in which other agonists were used to activate PKC isoforms, the EC₅₀ for various agonists to increase immunoreactive membrane PKC agreed well with the EC₅₀ for PKC activation assessed by biochemical activity measurement.^{16 19 21}

The antagonistic effects of DPCPX demonstrate the involvement of an adenosine A₁ membrane receptor in the adenosine-induced activation of -PKC. However, the cascade of events that leads to the activation of PKC in response to adenosine binding to A₁ receptors is still unclear. It is well established that cardiac adenosine A₁ receptors are coupled via a pertussis toxin–sensitive G_i protein to adenylate cyclase, resulting in inhibition of this enzyme, reduced cAMP formation, and reduced phosphorylation through protein kinase A.²² The adenosine A₁–activated G_i protein is also negatively linked to the Ca²⁺ channel membrane protein and positively linked to atrial and atrioventricular K⁺ channels. PKC can be activated by two different pathways: (1) phorbol ester or analogues of diacylglycerol, which are able to cross the membrane and directly induce a pronounced and prolonged activation of PKC, and (2) the receptor-mediated pathway, where neurohormones stimulate phospholipase C, the phospholipid turnover, and the generation of endogenous diacylglycerol, which in turn induce a rapid and short-lasting effect.^{16 17 22} Such a pathway has been demonstrated for the ₁-adrenergic receptor and the muscarinic M₂ receptor.²³ Accordingly, it was shown that ₁-adrenergic stimulation increased membrane-associated PKC.^{11 21} By contrast, a similar link between adenosine A₁ receptors and phospholipase C has not yet been firmly proved. Data generated concerning the effects of A₁ stimulation on inositol lipid metabolism are even conflicting. Kohl et al²⁴ reported that in guinea pig cardiac papillary muscle, R-PIA concentration-dependently increased IP₃ and accordingly reduced phosphatidylinositol diphosphate, the precursor of IP₃. These effects, which are in line with our own findings, were antagonized by DPCPX. An increased IP₃ production in response to adenosine was also reported in the rat myocardium.²⁵ By contrast, Leung et al²⁶ found no effects of R-PIA on inositol lipid metabolism in both atrial and ventricular myocytes. Even more conflicting are results obtained in noncardiac tissues, where adenosine A₁ stimulation has been reported to inhibit phosphoinositide breakdown (reviewed in Reference 26). These latter findings suggest major tissue differences.

In perfused, isolated hearts, it has recently been shown that acute ischemia induced the rapid activation of membrane PKC through an ₁-adrenergic receptor–independent mechanism.⁵ Adenosine released during acute ischemia³ may be a good candidate to account for PKC activation, although other mediators such as bradykinin or extracellular ATP may also contribute. This does not rule out the possibility that increased intracellular Ca²⁺ induced by ischemia triggered the activation of Ca²⁺-activated cardiac PKC isozymes such as -PKC, although intracellular Ca²⁺ is usually little affected during the first 15 minutes of acute myocardial ischemia.²⁷ In the context of ischemic preconditioning, our work provides a possible link between the adenosine and the PKC hypothesis.^{2 4} Future works should investigate (1) the exact pathways between adenosine A₁ stimulation and PKC activation, including the possible involvement of a G protein, and (2) the role of the various cardiac PKC isoforms (including -PKC), which are still poorly understood (reviewed in Reference 28).

	Selected Abbreviations and Acronyms

 

DPCPX = 8-cyclopentyl-1,3-dipropylxanthine E-64 = trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane IP3 = inositol tris-phosphate PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate PMSF = phenylmethylsulfonyl fluoride R-PIA = (-)-N6-(2-phenylisopropyl)-adenosine

	Acknowledgments

 
This study was supported by grants-in-aid from the Fondation de France, the Centre National de la Recherche Scientifique, the Assistance Publique/Hôpitaux de Paris, and the Fédération Française de Cardiologie. We gratefully thank Ms Richer for her expert technical assistance and Ms Martens for her kind help in improving the English of the manuscript.

Received March 28, 1995; accepted October 2, 1995.

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