AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 279: H2685-H2693, 2000;
0363-6135/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lester, J. W.
Right arrow Articles by Hofmann, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lester, J. W.
Right arrow Articles by Hofmann, P. A.
Vol. 279, Issue 6, H2685-H2693, December 2000

Role for PKC in the adenosine-induced decrease in shortening velocity of rat ventricular myocytes

J. William Lester and Polly A. Hofmann

Department of Physiology, University of Tennessee, Memphis, Tennessee 38163


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that both adenosine receptor activation and direct activation of protein kinase C (PKC) decrease unloaded shortening velocity (Vmax) of rat ventricular myocytes. The goal of this study was to further investigate a possible link among adenosine receptors, phosphoinositide-PKC signaling, and Vmax in rat ventricular myocytes. We determined that the adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, 100 µM) and the alpha -adrenergic receptor agonist phenylephrine (Phe, 10 µM) increased turnover of inositol phosphates. PKC translocation from the cytosol to the sarcolemma was used as an indicator of PKC activation. Western blot analysis demonstrated an increased PKC-varepsilon translocation after exposure to R-PIA, Phe, and the PKC activators dioctanoylglycerol (50 µM) and phorbol myristate acetate (1 µM). PKC-alpha , PKC-delta , and PKC-zeta did not translocate to the membrane after R-PIA exposure. Finally, PKC inhibitors blocked R-PIA-induced decreases in Vmax as well as Ca2+-dependent actomyosin ATPase in rat ventricular myocytes. These results support the conclusions that adenosine receptors activate phosphoinositide-PKC signaling and that adenosine receptor-induced PKC activation mediates a decrease in Vmax in ventricular myocytes.

R-phenylisopropyladenosine; protein kinase C-varepsilon ; inositol (1,4,5)trisphosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PHOSPHATIDYLINOSITOL-PHOSPHOINOSITIDE signaling pathway in the myocardium can be activated by alpha -adrenergic (42), ANG II (1), and kappa -opioid (40) receptor agonists. In this signaling pathway, receptor-coupled G proteins activate phospholipase C, which in turn catalyzes hydrolysis of phosphatidylinositol bisphosphate [PtdIns(4,5)P2] into diacylglycerol and inositol (1,4,5) trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 acts to liberate Ca2+ from intracellular storage sites, and diacylglycerol activates protein kinase C (PKC). Activation of PKC involves translocation of inactive PKC from the cytosol to the cell membrane where diacylglycerol activates it. In addition, some PKC isoforms require the presence of Ca2+ (19). The PKC-varepsilon isoform appears to be the predominant isoform of PKC in adult rat ventricular myocytes; PKC-alpha , PKC-delta , and PKC-zeta are also observed (3, 33). Upon activation, PKC-varepsilon has been shown to translocate to cardiac myofilaments (9, 20). PKC activation in the myocardium has been associated with phosphorylation of the myofilament proteins C protein, troponin I, and troponin T in vitro (22) and increased phosphorylation of a 15-kDa sarcolemma protein, a 28-kDa cytosolic protein (37), and myosin light chain 2 (7) in vivo. Previous work has suggested phosphorylation of troponin I by PKC-varepsilon decreases Ca2+-dependent actomyosin ATPase (21).

Activation of adenosine receptors mediates decreases in heart rate (30) and developed pressure (5) and protects the heart from ischemic damage (38). In myocardial preparations, adenosine has been shown to decrease the velocity of unloaded shortening (Vmax ) (26, 36) and activate ATP-sensitive K+ channels (43). Studies suggest that adenosine receptors may be coupled to the activation of PKC in smooth muscle (13). However, adenosine has also been shown to decrease adenylate cyclase activity in the myocardium (34). In a previous study, we found the adenosine agonist R-phenylisopropyladenosine (R-PIA) and the diacylglycerol analog dioctanoylglycerol (diCg) decrease Vmax to the same extent in isolated rat ventricular myocytes (26). This suggests the functional effects of activation of the adenosine receptor may be mediated by activation of the phospholipase-PKC signaling pathway. Therefore, the purpose of the present study was to determine whether adenosine receptors are coupled to the phosphoinositide pathway and whether activation of PKC is the basis for a decrease in Vmax in isolated ventricular myocytes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biochemical Assays

Ins(1,4,5)P3 assay. Cells were isolated as described by Lester and co-workers (26). Treatment groups were the following: control, 10 µM phenylephrine (Phe, alpha -agonist), and 100 µM R-PIA. R-PIA is an A1- and A3-receptor agonist, and treatment with 100 µM R-PIA ensures activation of both A1 and A3 receptors, which have affinities of 1.17 and 1,210 nM, respectively (27). Only isolations showing a positive Phe response >= 1.15 times control were included in the analysis because alpha -adrenergic stimulation is known to liberate Ins(1,4,5)P3 in ventricular myocytes (4).

To prepare samples for Ins(1,4,5)P3 determination, cells were exposed to drugs or vehicle for 1 min, and the reactions were stopped by adding a 5:1 dilution of 1 M trichloroacetic acid or a 0.2:1 dilution of 100% trichloroacetic acid to cells. In early experiments the suspension was homogenized, but this was deemed unnecessary in later experiments. Samples were incubated on ice for 15 min. After 1 min of centrifugation (10,000 g), the supernatant was decanted and incubated for 15 min at room temperature. Trichloroacetic acid was extracted from samples by adding a 3:1 dilution of 1,1,2-trichloro-1,2,2-trifluoroethane and trioctylamine in a 2:1 solvent-to-sample ratio. After 15 s of mixing, samples were incubated for 3 min. The aqueous layer was removed and frozen at -80°C.

The Ins(1,4,5)P3 3H-Radioreceptor Assay Kit from NEN-DuPont was used to determine Ins(1,4,5)P3 levels. The kit is supplied with a preparation of calf cerebellum membrane containing Ins(1,4,5)P3 receptor, [3H]Ins(1,4,5)P3, and appropriate buffers. The kit has a sensitivity range of 0.12-12.0 pmol/100 µl of sample. The assay is based on Ins(1,4,5)P3 from experimental samples displacing known concentrations of labeled Ins(1,4,5)P3 bound to the membrane-receptor preparation. Bound radioactivity in the experimental samples is then compared with a standard curve to determine Ins(1,4,5)P3 levels.

PKC isoform translocation. Cells were prepared as described by Lester and co-workers (26) and incubated for 5 min with 10 µM Phe, 100 µM R-PIA, 50 µM diCg (a PKC activator), or 1 µM phorbol-12-myristate-13-acetate (PMA). PMA at a 1 µM concentration is known to activate PKC in addition to other second messengers. After drug treatment, samples were centrifuged, and pellets were frozen at -80°C. Membrane fractions of cells were isolated using the protocol described by Puceat and colleagues (33).

Samples were run on SDS polyacrylamide gels using 5% acrylamide stacking gels and 12% acrylamide resolving gels. Gel proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed as described in a protocol included with chemiluminescent reagents (NEL-102, NEN-DuPont). The primary antibodies anti-PKC-varepsilon (1:100 dilution; GIBCO-BRL, Grand Island, NY), anti-PKC-delta (1:500 dilution; Transduction Labs, Lexington, KY), anti-PKC-alpha (1:1,000 dilution; Transduction Labs), anti-PKC-zeta (1:1,000 dilution; Transduction Labs), and peroxidase-conjugated secondary antibody (1:4,000 dilution; Sigma, St. Louis, MO) were utilized. Membranes were incubated in chemiluminescent reagents and exposed to X-ray film. PVDF blots were also stained with india ink to establish protein load and relative amount of protein transferred for each sample. Staining of the blot and normalizing to relative protein load were necessary to control for variations in gel loading and transfer. Such variations can ultimately lead to apparent changes in PKC content (31).

Densities of PKC isoform bands from X-ray film and densities of india ink stain of an ~46 kDa protein from corresponding PVDF blots were determined using National Institutes of Health Image software. PKC density was weighted to the corresponding protein transferred on each lane (31). To do this, the average density of the india ink stain for the 46-kDa protein was obtained, and the ratio of density of the 46-kDa protein on a given lane to the average density of the 46-kDa protein was calculated. This value indicates the relative protein transferred onto a given lane of a blot. The density of the PKC band on each lane of the X-ray was also normalized to the average density of PKC of control-treated cells and then multiplied by the relative protein transferred onto the blot. When the PKC contents from multiple measurements were obtained, the normalized PKC band densities were averaged to give a single value for that myocyte isolation and treatment group. Only isolations with a positive Phe response were used in analysis because alpha -adrenergic stimulation is known to activate PKC translocation (33).

Actomyosin ATPase. Cells were prepared as described by Lester and co-workers (26) and treated with 100 µM R-PIA, 500 µM 8-sulfophenyltheophylline (8-SPT), R-PIA plus 8-SPT, 10 µM chelerythrine (Chel), 100 µM R-PIA plus 10 µM Chel, 100 nM bisindolylmaleimide I (Bis), or 100 nM Bis plus 100 µM R-PIA. 8-SPT is an adenosine receptor antagonist; Chel and Bis are PKC inhibitors. Chel was used at 10 µM because the concentration required for 50% inhibition (IC50) of PKC is 0.7 µM, and IC50 values are 0.17 mM for PKA and 0.1 mM for tyrosine kinase and the Ca2+-calmodulin-dependent kinase (16). Chel inhibits PKC by binding to the substrate binding site of PKC (16), whereas Bis inhibits PKC by binding to the ATP binding site on PKC (39). Chel (1 µM) has been shown to inhibit PKC-varepsilon translocation in the rat myocardium (24). Bis (50 nM) has been shown to inhibit the PKC-alpha -, PKC-delta -, and PKC-varepsilon -dependent phosphorylation of proteins in the particulate fraction of the ischemic rat myocardium (2).

Myocytes isolated from a given heart were used in one of the following studies, each of which was divided into three study groups: 1): control, R-PIA, 8-SPT, 8-SPT plus R-PIA; 2): control, R-PIA, Chel, Chel plus R-PIA; and 3): control, R-PIA, Bis, Bis plus R-PIA. PKC inhibitors and 8-SPT were added 3 min before the addition of R-PIA. R-PIA treatment was for 5 min. All groups had an equal amount of DMSO.

After drug treatment, myofibrils were isolated from cells based on the procedures of Murphy and Solaro (28). During myofibrillar isolation, all solutions contained 100 nM calyculin A to inhibit phosphatase activity and help maintain phosphorylations induced by the original drug treatment of cells. Once myofibrils were isolated, 30-240 µg of protein was added to ATPase assay buffers, either pCa 9.0 or pCa 4.0, containing the following (in mM): 2 EGTA, 20 imidazole, 3 NaATP, and 5 MgCl2 and KCl to give a constant ionic strength of 60 mM (pH 7.0). The ATPase assay was carried out for 12 min at 32°C before the reaction was stopped with 20% trichloroacetic acid. Pi was measured using the method of Fiske and Subarrow (12). Pi release was found to be linear with time under the conditions stated (data not shown).

Vmax of Ventricular Myocytes

Cell preparation. Cells were isolated as described by Lester and co-workers (26) and divided into control, 100 µM R-PIA, 10 µM Chel, and 100 µM R-PIA plus 10 µM Chel treatment groups. The 10 µM Chel and 100 µM R-PIA plus 10 µM Chel groups were pretreated with Chel for 3 min. After Chel pretreatment, R-PIA was added to the R-PIA and R-PIA plus Chel groups, and control cells were treated with vehicle. All groups were then incubated for 5 min at room temperature. After drug treatment, cells were exposed to 0.3% ultrapure Triton X-100 in relaxing solution (see Solutions) for 5 min to disrupt all lipid membranes. Cells were then washed in relaxing solution and stored on ice for immediate use. Cells were used up to 48 h postisolation.

For this portion of the study, initial isolations were performed using type 4 collagenase (Worthington lot S2P350N), and later isolations were performed using type 2 collagenase (Worthington lot 45P862). Because we had no experience with collagenase lot 45P862, a positive control for intact receptor-signal transduction pathways was included in isolations performed with this collagenase. Cells were treated with the beta -adrenergic receptor agonist isoproterenol (100 nM), because beta -adrenergic receptor activation is well known to decrease the Ca2+ sensitivity of tension of ventricular myocytes. Only isolations showing a beta -adrenergic-dependent decrease in the Ca2+ sensitivity of tension were included in the final analysis of Vmax.

Myocyte attachment and experimental apparatus. Myocytes were attached via glass pipettes to a force transducer (model 403, Cambridge Technology, Watertown, MA) and a piezoelectric translator (model 173, Physik Institute, Waldbronn, Germany) using Great Stuff foam insulation (Insta-Foam, Marietta, GA) as the adhesive (17, 26). Sarcomere lengths were adjusted to ~2.1-2.3 µm as judged by Filar micrometer. Myocytes were maximally activated in a pCa 4.5 (pCa = -log[Ca2+]) solution and relaxed in a pCa 9.0 solution. Photographs were taken of a given myocyte in both pCa 4.5 and 9.0 solutions to assess change in sarcomere length and to determine the exact length between pipettes (Lo) and cell width. Cell width was averaged from three measurements taken from each photomicrograph at positions near the center of the length of the cell and 5-10 µm from the tips of the two attachment micropipettes.

Measurement of unloaded shortening velocity. The slack-test method was used to determine Vmax of cardiac myocytes (10, 18). In brief, cardiac myocytes were activated in pCa 4.5 solution, and, after developing a steady tension, a shortening length step was introduced. Shortening steps fully unloaded the cells such that the tension initially fell to zero. Once the introduced slack was taken up, tension redevelopment began. The cell was then returned to a solution containing a nonactivating level of Ca2+, pCa 9.0. Each cell underwent four to eight shortening steps of different lengths in a pCa 4.5 solution. To more clearly define the exact time point of tension redevelopment, tension records from a shortening step in pCa 9.0 and pCa 4.5 solutions were computer overlaid. The point at which these tension tracings diverged was taken as the onset of tension redevelopment.

For a given cell, each length of shortening step (µm) was plotted versus the corresponding time to onset of tension redevelopment or duration of unloaded shortening (ms). The least-squares method was used to fit a straight line to the data. The slope was then divided by Lo to calculate the velocity of shortening in muscle lengths per second (ML/s). Cells meeting the following criteria were included in cumulative velocity analysis: final value of pCa 4.5 tension/initial pCa 4.5 tension >0.70, goodness of fit to a straight line >= 0.80, change in sarcomere length <0.15 µm, and y-intercept percentage >5% of Lo.

Solutions

Ringer solution contained (in mM) 1.2 MgCl2, 4.8 KCl, 118 NaCl, 2 KH2PO4, 5 pyruvate, 25 HEPES, 11 glucose, and 1 insulin (pH 7.4). The relaxing solution used to wash cells after isolation had a pCa of 9.0 and contained (in mM) 100 KCl, 10 imidazole, 1 MgCl2, 2 EGTA, and 4 ATP (pH 7.0). Solutions containing varied concentrations of free Ca2+ were used to activate skinned cells. Free-Ca2+ concentrations ranged from pCa 4.5 to pCa 9.0 and were calculated using the computer program of Fabiato (11). Ca2+-activating solutions contained (in mM) 7 EGTA, 1 free Mg2+, 20 imidazole, 4.42 ATP, and 14.5 creatine phosphate along with various free-Ca2+ concentrations and KCl to adjust ionic strength to 180 mM (pH 7.00).

Statistical Analysis

ANOVA and t-test were performed (Systat, Evanston, IL) to determine whether average, normalized PKC isoform band densities or Ins(1,4,5)P3 values of treatment groups were significantly different from 1, i.e., control. For Vmax studies, groups were subjected to an ANOVA and a t-test to determine differences. The level of significance was considered to be P < 0.05 in each analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To establish whether adenosine receptor stimulation activates the phosphatidylinositol signaling pathway in ventricular myocytes, we examined the effects of 10 µM Phe (alpha -adrenergic receptor activation) and 100 µM R-PIA (adenosine-A1 and -A3 receptor activation) treatment to increase Ins(1,4,5)P3 levels normalized to untreated cells. The average ± SE control concentration of Ins(1,4,5)P3 was 9.04 ± 2.67 pmol/mg protein. Treatment with Phe or R-PIA resulted in a significant increase in Ins(1,4,5)P3 production relative to this control (Fig. 1).


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 1.   Inositol (1,4,5) trisphosphate [Ins(1,4,5)P3] mass in isolated rat ventricular myocytes. Treatment groups are 10 µM phenylephrine (Phe; alpha -adrenergic receptor agonist) and 100 µM R-phenylisopropyladenosine (R-PIA; A1- and A3-adenosine receptor agonist). Values from three hearts were normalized to control and expressed as means ± SE; *P < 0.05 vs. control.

Inactive PKC-varepsilon is found in the cytosol, and membrane association is necessary for PKC-varepsilon activation (19). Differential centrifugation and immunoblotting techniques were used to detect translocation of PKC-varepsilon from the cytosol to the membrane and indirectly demonstrate PKC activation. Positive control experiments demonstrated that treatment with the phorbol ester PMA results in decreased cytosolic PKC-varepsilon and increased membrane-associated PKC-varepsilon (Fig. 2). Translocation of PKC-varepsilon to the membrane fraction was also observed with 10 µM Phe, 50 µM diCg, and 100 µM R-PIA treatment of isolated ventricular myocytes. A typical immunoblot of membrane-bound PKC-varepsilon is shown in Fig. 3. Blots were also stained with india ink to establish the relative protein level transferred. Although the initial protein concentration loaded onto a gel and the transfer conditions were identical, some variability in the amount of protein that was ultimately transferred to the blots existed. As such, PKC-varepsilon density for a given lane on the immunoblots was weighted to reflect protein transferred onto that lane (31). For example, in Fig. 3, for Phe treatment, the final membrane-bound PKC-varepsilon would be recorded as 145% [(158 ×0.99) + (140 ×0.95)/2] relative to control for this isolation. Figure 4 presents the final average membrane-bound PKC-varepsilon relative to control using the various treatments. Treatment with the PKC activators PMA and diCg, the alpha -adrenergic agonist Phe, or the adenosine A1 and A3 agonist R-PIA resulted in a significant increase in membrane-associated PKC-varepsilon .


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 2.   Western blot of protein kinase C-varepsilon (PKC-varepsilon ) associated with cytosolic (Cyto) and membrane (Mem) fractions of isolated ventricular myocytes. Treatment groups were control (CON) or 1 µM phorbol-12-myristate-13-acetate (PMA, a PKC activator). The mobility of immunoreactive bands of cell fractions were compared with a purified PKC standard (PKC Std).



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 3.   Typical Western blot of membrane-bound PKC-varepsilon (top) and india ink staining of corresponding immunoblot (bottom) from isolated rat ventricular myocytes. Densities relative to the average control density for the PKC-varepsilon immunoreactive band (top) and relative to the average density of a stained protein slightly >46 kDa on the blot (bottom) are shown. Treatment groups are control (CON), 10 µM Phe, 1 µM PMA, 50 µM dioctanoylglycerol (DOG), and 100 µM R-PIA. PKC represents a commercially available PKC standard. Marker proteins of known molecular weight (MW) were utilized to help identify proteins.



View larger version (42K):
[in this window]
[in a new window]
  		Fig. 4.   Relative membrane-bound PKC-varepsilon in isolated rat ventricular myocytes. PKC-varepsilon membrane content is an indicator of PKC translocation and activation. Treatment groups are control, 10 µM Phe (n = 8), 1 µM PMA (n = 8), 100 µM R-PIA (n = 6), and 50 µM DOG (n = 3). Values are weighted for protein transferred onto the blot and normalized to control (see MATERIALS AND METHODS). Values expressed as means ± SE; *P < 0.05 vs. control.

A 5-min exposure to receptor agonists did not alter total immunoreactive PKC-varepsilon content in the intact myocyte measured immediately after drug treatment. Relative total cell PKC-varepsilon under control conditions was 1.00 ±0.08 (n = 12), after R-PIA exposure was 1.00 ± 0.08 (n = 6), after Phe exposure was 1.01 ± 0.08 (n = 6), after diCg exposure was 0.81 ± 0.11 (n = 6, P < 0.05 compared with control), and after PMA exposure was 1.05 ± 0.16 (n = 6). The small decrease in total immunoreactive PKC-varepsilon after diCg treatment would lead to an underestimate of the diCg-induced increase in the membrane-associated PKC-varepsilon (Fig. 4).

Western blot analysis of the membrane fractions after R-PIA exposure was also completed using antibodies to PKC-alpha , PKC-delta , and PKC-zeta . No significant differences were observed in these isoforms between membranes of untreated and R-PIA-exposed cells. Membranes of R-PIA-treated ventricular myocytes had a PKC-alpha value of 0.93 ± 0.11, a PKC-delta value of 0.91 ± 0.22, and a PKC-zeta value of 0.83 ± 0.14 relative to membranes from untreated myocytes (n = 6 for all groups).

Figure 5 presents photomicrographs of single myocytes in low Ca2+ (pCa 9.0) solution and during maximum activation (pCa 4.5). Among all the treatment groups of control, 100 µM R-PIA, 10 µM Chel, and 100 µM R-PIA plus 10 µM Chel, average sarcomere lengths were not significantly different between pCa 4.5 and pCa 9.0 solutions, suggesting a low compliant attachment. Table 1 summarizes the characteristics of cells used to determine Vmax, including sarcomere length and maximum isometric tension. Maximum isometric tension was not statistically different between any of the drug treatment groups and control (Table 1). However, the SE of the maximum isometric tension determination accounted for ~15% of the total tension; thus small changes in maximum isometric tension may be obscured.


View larger version (85K):
[in this window]
[in a new window]
  		Fig. 5.   Photomicrographs of enzymatically isolated, agonist-treated, detergent-skinned rat ventricular myocytes. Cells are pictured during relaxation, pCa 9.0 (left), and during maximum activation, pCa 4.5 (right). Sarcomere length was not significantly different between groups or upon exposure to maximum activating levels of Ca2+.



                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Characteristics of isolated rat ventricular myocytes used to determine effects of R-PIA, Chel, and R-PIA plus Chel on unloaded shortening velocity

Representative tension traces recorded immediately after various shortening steps during maximum activation are shown in Fig. 6 (see MATERIALS AND METHODS for protocol). As illustrated in Fig. 6, the duration of unloaded shortening is longer for R-PIA treatment compared with the control and for R-PIA plus Chel-treated cells for nearly identical lengths of cell shortening. The means ± SE Vmax values for control, 100 µM R-PIA-, 10 µM Chel-, and 100 µM R-PIA plus 10 µM Chel-treated cells are shown in Fig. 7. Treatment with R-PIA significantly decreased Vmax, and that decrease was blocked by the PKC inhibitor Chel. Chel treatment alone had no effect on Vmax.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6.   Representative tension records of cells obtained immediately after decreases in length during maximum activation (pCa 4.5) or at rest (pCa 9.0). Treatment groups are untreated (A), 100 µM R-PIA (C), or 100 µM R-PIA + 10 µM chelerythrine (Chel) (B). Arrows denote point of tension redevelopment after shortening step. Peak steady-state tension and point of initial release are not shown. Shortening step lengths (Delta L) are expressed as percentage of cell length (Lo). R-PIA increased the time required to take up a slackening step of a given length. Chel attenuated this effect. All recordings obtained from these cells are not shown.



View larger version (55K):
[in this window]
[in a new window]
  		Fig. 7.   Unloaded shortening velocity in isolated ventricular myocytes that were untreated (control), treated with 100 µM R-PIA, 10 µM Chel, or 100 µM R-PIA + 10 µM Chel and were subsequently skinned. Values are expressed as means ± SE; *P < 0.05 vs. control, vs. Chel, and vs. R-PIA/Chel.

To establish whether the decrease in cross-bridge cycling rate was via the activation of adenosine receptors, isolated ventricular myocytes were treated with 100 µM R-PIA, 500 µM 8-SPT (an adenosine receptor antagonist nonselective for adenosine receptor subtype), or R-PIA plus 8-SPT. After drug treatment, myofibrils were isolated from skinned cells. Figure 8A presents the mean Ca2+-dependent actomyosin ATPase from these various preparations. R-PIA treatment led to a significant decrease in the Ca2+-dependent myofibrillar ATPase, and this effect was blocked with the addition of 8-SPT.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 8.   Ca2+-dependent actomyosin ATPase in cardiac myofibrils. Myofibrils were isolated after treatment of myocytes with the after treatments. A: 100 µM R-PIA (an adenosine A1- and A3-receptor agonist), 500 µM 8-sulfophenyltheophylline (SPT, a nonselective adenosine receptor antagonist), R-PIA + 8-SPT, or vehicle (control). B: 100 µM R-PIA, 10 µM Chel (a PKC inhibitor), R-PIA + Chel, or vehicle (control). C: 100 µM R-PIA, 100 nM bisindolylmaleimide I (Bis, a PKC inhibitor), R-PIA + Bis, or vehicle (control). Values are expressed as means ± SE; *P < 0.05 vs. all other groups in that study.

To further establish whether the R-PIA-induced decrease in cross-bridge cycling rate was through activation of PKC, isolated ventricular myocytes were treated with R-PIA (100 µM) and the PKC inhibitors Chel (10 µm) or Bis (100 nM). After drug treatment, myofibrils were isolated from skinned cells. Figure 8, B and C, presents the mean Ca2+-dependent actomyosin ATPase from these various preparations. R-PIA treatment led to a significant decrease in the Ca2+-dependent myofibrillar ATPase, and this effect was blocked with the addition of either Chel or Bis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Evidence suggests that adenosine-mediated cardioprotection requires activation of PKC (35). This implies a link between adenosine receptors, phospholipase C or D activation, and activation of PKC. Therefore, the goal of this study was to establish a link between adenosine receptors and phosphoinositide signaling and functional changes in ventricular myocytes. We found that treatment of isolated ventricular myocytes with the adenosine agonist R-PIA significantly increased turnover of inositol phosphates and led to translocation of PKC-varepsilon . We also demonstrated that the R-PIA-induced decrease in ventricular myocyte Vmax and myofibrillar actomyosin ATPase could be blocked by PKC inhibitors. Finally, we show that the R-PIA-induced decrease in myofibrillar actomyosin ATPase could be blocked by the adenosine receptor antagonist 8-SPT.

Because myocyte membranes can be damaged during isolation, positive control treatment groups were included in these experiments to demonstrate that known receptor-coupled signaling pathways were intact. alpha -Adrenergic receptor activation increases inositol phosphate turnover (4) and stimulates PKC translocation (33) in the rat myocardium; thus the alpha -adrenergic receptor agonist Phe was used as a positive control in the Ins(1,4,5)P3 and PKC translocation assays. beta -Adrenergic receptor PKA activation decreases Ca2+ sensitivity of tension in isolated ventricular myocytes (17). Thus the beta -adrenergic-induced decrease in calcium sensitivity of tension was used as a positive control in studies measuring Vmax.

Activation of the phosphoinositol signaling pathway results in phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to Ins(1,4,5)P3 and diacylglycerol. Diacylglycerol activates conventional and novel isoforms of PKC. We found basal levels of Ins(1,4,5)P3 at ~9 pmol/mg protein. Others have found unstimulated Ins(1,4,5)P3 concentrations from 4 to 40 pmol Ins(1,4,5)P3/mg protein in rat ventricular myocytes (41, 23). We demonstrated that Ins(1,4,5)P3 concentrations increased by 25-30% after Phe or R-PIA treatment of ventricular myocytes. The ability of R-PIA to stimulate Ins(1,4,5)P3 in rat ventricular myocytes has not been previously demonstrated. However, others have shown alpha -adrenergic receptor stimulation increases Ins(1,4,5)P3 levels by 60% in adult rat ventricular myocytes, and R-PIA increased Ins(1,4,5)P3 concentration by 40% relative to control in guinea pig papillary muscle (25, 41). We also found that membrane-bound PKC-varepsilon increased by ~100% with PMA, 30% with Phe, and 20% with R-PIA treatment relative to control. The ability of R-PIA to stimulate PKC-varepsilon translocation in rat ventricular myocytes has not been previously demonstrated. However, others (3, 33) have reported that PMA treatment increased membrane-bound PKC-varepsilon by three- to fivefold, and Phe treatment increased membrane-bound PKC-varepsilon almost twofold. Thus our observations of an R-PIA-induced increase in Ins(1,4,5)P3 and PKC-varepsilon translocation in rat ventricular myocytes are qualitatively consistent with work by others. Our data supports a link between adenosine receptors and phosphoinositide-PKC signaling. It should be noted there is evidence for phospholipase D being coupled to adenosine receptors and PKC activation in smooth muscle cells (13) and whole hearts (8). The present studies do not eliminate the possibility that adenosine receptor-dependent activation occurs through the phospholipase D pathway.

Previous Western blot analysis of rat adult ventricular myocytes has demonstrated the presence of PKC-alpha , PKC-delta , PKC-varepsilon , and PKC-zeta (33) but not PKC-beta 1, PKC-beta 2, PKC-gamma (33), or PKC-eta (3). Thus we looked to see whether there was an increase in membrane-bound PKC-alpha , PKC-delta , or PKC-zeta with 5 min of R-PIA exposure. No statistical differences were found compared with membranes from untreated cells. Henry and colleagues (15) demonstrated an R-PIA-dependent increase in membrane PKC-delta by 66 ± 58% (mean ± SE, n = 7, P < 0.05 vs. controls) at 1 min of R-PIA stimulation of rat ventricular myocytes. However, with 5 min of R-PIA exposure, only an 18% increase of PKC-delta was observed (15). This raises the possibility that in the present study a change in membrane-bound PKC-alpha , PKC-delta , or PKC-zeta with R-PIA stimulation may have been detected with shorter time exposures to R-PIA.

In a previous study, we found that treatment of isolated ventricular myocytes with R-PIA and the PKC activator diCg resulted in decreased Vmax (26). We postulated that the decrease in Vmax was due to adenosine-mediated activation of PKC. In the present study, we again demonstrated that R-PIA decreases Vmax. Novel findings of the present study include the observation that the R-PIA-induced decrease in Vmax is blocked by the PKC inhibitor Chel. In addition, we demonstrated that Ca2+-dependent actomyosin Mg2+-ATPase is decreased through activation of adenosine receptors and that this adenosine-induced reduction in cross-bridge cycling can be blocked by PKC inhibitors. This finding is consistent with that of Noland and Kuo (29), who demonstrated that exposure of cardiac myofibrils to exogenously applied PKC phosphorylates troponin I and results in decreased Ca2+-dependent myofibrillar ATPase. Together, these findings support the hypothesis that PKC activation is involved in the adenosine receptor-dependent slowing of cross-bridge cycling. Because R-PIA-dependent PKC-varepsilon translocation data shows only a coincidence with the observed decrease in cross-bridge cycling, the exact mechanism responsible for the decrease in cross-bridge cycling has yet to be definitively established. It is possible that adenosine receptor activation stimulates PKC-varepsilon or another isoform of PKC (with a rapid transient), and this initiates a cascade of kinases or phosphatases ultimately leading to modification of a myofilament protein and subsequent decrease in Vmax.

Although the present experiments do not specifically address the physiological significance of the adenosine receptor-PKC-induced decrease in Vmax, we hypothesize that the decrease in Vmax is related to the ability of adenosine to protect the heart from ischemic damage (38). We speculate that the 30% decrease in Vmax observed in the present study would significantly reduce ATP consumption because actin-myosin cycling during contraction accounts for 85% of ATP utilized in the rat myocardium (6). This decrease in actomyosin ATPase and conservation of ATP would ensure adequate ATP levels for continued function of cellular ion pumps. Maintenance of ion homeostasis would attenuate intracellular Ca2+ overload and subsequent damage due to activation of Ca2+-dependent proteases.


    ACKNOWLEDGEMENTS

This study was supported by National Institute Heart, Lung, and Blood Grant HL-48839 and an American Heart Association Established Investigatorship (to P. A. Hofmann).


    FOOTNOTES

Address for reprint requests and other correspondence: P. A. Hofmann, Dept. of Physiology, Univ. of Tennessee, 894 Union Ave., Memphis, TN 38163 (E-mail: phofmann{at}physio1.utmem.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 September 1999; accepted in final form 18 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abdellatif, M, Neubauer D, Lederer W, and Roger T. Angiotensin-induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level of the receptor. Circ Res 69: 800-809, 1991[Abstract/Free Full Text].

2.   Albert, CJ, and Ford DA. Protein kinase C translocation and PKC-dependent protein phosphorylation during myocardial ischemia. Am J Physiol Heart Circ Physiol 276: H642-H650, 1999[Abstract/Free Full Text].

3.   Bogoyevitch, MA, Parker PJ, and Sugden PH. Characterization of protein kinase C isotype expression in adult rat heart: protein kinase C-varepsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin. Circ Res 72: 757-767, 1993[Abstract/Free Full Text].

4.   Brown, JH, Buxton IL, and Brunton LL. Alpha1-adrenergic and muscarinic cholinergic stimulation of phosphoinositide hydrolysis in adult rat cardiomyocytes. Circ Res 57: 532-537, 1985[Abstract/Free Full Text].

5.   Cano, C, Slavik KJ, and Malik KU. Loss of adenosine-induced negative inotropic effect in hyperexcited rabbit hearts: relationship to PKC. Am J Physiol Heart Circ Physiol 268: H1037-H1044, 1995[Abstract/Free Full Text].

6.   Challoner, DR. Respiration in myocardium. Nature 217: 78-79, 1968[Medline].

7.   Clement, O, Puceat M, Walsh MP, and Vassort G. PKC enhances MLCK effects on force development and ATPase activity in rat single skinned cardiac cells. Biochem J 285: 311-317, 1992.

8.   Cohen, MV, Liu Y, Liu GS, Wang P, Weinbrenner C, Cordis GA, Das DK, and Downey JM. Phospholipase D plays a role in ischemic preconditioning in rabbit heart. Circulation 94: 1713-1718, 1996[Abstract/Free Full Text].

9.   Disatnik, M-H, Buraggi G, and Mochly-Rosen D. Localization of protein kinase C isozymes in cardiac myocytes. Exp Cell Res 210: 287-297, 1994[ISI][Medline].

10.   Edman, KAP The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J Physiol (Lond) 291: 143-159, 1979[Abstract/Free Full Text].

11.   Fabiato, A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378-417, 1988[ISI][Medline].

12.   Fiske, CH, and Subarrow Y. The colorimetric determination of phosphorus. J Biol Chem 66: 375-400, 1925[Free Full Text].

13.   Gerwins, P, and Fredholm BB. Activation of adenosine A1 and bradykinin receptors increases protein kinase C and phopsholipase D activity in smooth muscle cells. Naunyn Schmiedebergs Arch Pharmacol 351: 186-189, 1995[ISI][Medline].

14.   Haworth, RA, Goknur AB, Warner TF, and Berkoff HA. Some determinants of quality and yield in the isolation of adult heart cells from rat. Cell Calcium 10: 57-62, 1989[ISI][Medline].

15.   Henry, P, Demolombe S, Puceat M, and Escande D. Adenosine A1 stimulation activates delta-protein kinase C in rat ventricular myocytes. Circ Res 78: 161-165, 1996[Abstract/Free Full Text].

16.   Herbert, JM, Augereau JM, Gleye J, and Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 72: 993-999, 1990.

17.   Hofmann, PA, and Lange JH. Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats. Circ Res 74: 718-726, 1994[Abstract/Free Full Text].

18.   Hofmann, PA, and Moss RL. Effects of calcium on shortening velocity in frog chemically skinned atrial myocytes and in mechanically disrupted ventricular myocardium from rat. Circ Res 70: 885-892, 1992[Abstract/Free Full Text].

19.   Hug, H, and Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J 291: 329-343, 1993.

20.   Huang, XP, Pi Y, Lokuta AJ, Greaser ML, and Walker JW. Arachidonic acid stimulates protein kinase C-epsilon redistribution in heart cells. J Cell Sci 110: 1625-1634, 1997[Abstract].

21.   Jideama, NM, Noland TA, Raylor RL, Blobe GC, Fabbro D, Kazanietz MG, Blumberg PM, Hannun YA, and Kuo JF. Phosphorylation specificities of protein kinase C isozymes for bovine troponin I and troponin T sites within these proteins and regulation of myofilament properties. J Biol Chem 271: 23277-23283, 1996[Abstract/Free Full Text].

22.   Katoh, N, Wise BC, and Kuo JF. Phosphorylation of cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T) by cardiac phospholipid-sensitive Ca2+-dependent protein kinase. Biochem J 209: 189-195, 1983[ISI][Medline].

23.   Kawaguchi, H, Shoki M, Sano H, Kudo T, Sawa H, Mochizuki N, Okamoto H, Endo Y, and Kitabatake A. Polyphosphoinositide metabolism in hypertrophic rat heart. J Mol Cell Cardiol 24: 1003-1010, 1992[ISI][Medline].

24.   Kawamura, S, Yoshida K, Miura T, Mizukami Y, and Matsuzaki M. Ischemic preconditioning translocates PKC-delta and -epsilon , which mediate functional protection in isolated rat heart. Am J Physiol Heart Circ Physiol 275: H2266-H2271, 1998[Abstract/Free Full Text].

25.   Kohl, C, Linck B, Schmitz W., Scholz H, Scholz J, and Toth M. Effects of carbachol and (-)-N6-phenylisopropyladenosine on myocardial inositol phosphate content and force of contraction. Br J Pharmacol 101: 829-834, 1990[ISI][Medline].

26.   Lester, JW, Gannaway KF, Reardon RA, Koon LD, and Hofmann PA. Effects of adenosine and protein kinase C stimulation on mechanical properties of rat cardiac myocytes. Am J Physiol Heart Circ Physiol 271: H1778-H1785, 1996[Abstract/Free Full Text].

27.   Linden, J, Hollen CE, and Patel A. The mechanism by which adenosine and cholinergic agents reduce contractility in rat myocardium: correlation with cyclic adenosine monophosphate and receptor densities. Circ Res 56: 728-735, 1985[Abstract/Free Full Text].

28.   Murphy, AM, and Solaro RJ. Developmental differences in the stimulation of cardiac myofibrillar Mg-ATPase activity by calmidazolium. Pediatr Res 28: 46-49, 1990[ISI][Medline].

29.   Noland, TA, and Kuo JF. PKC phosphorylation of cardiac troponin I and troponin T inhibits Ca2+-stimulated Mg-ATPase activity in reconstituted actomyosin and isolated myofibrils and decreases actin-myosin interactions. J Mol Cell Cardiol 25: 53-65, 1993[ISI][Medline].

30.   Olsson, RA, and Pearson JD. Cardiovascular purinoceptors. Physiol Rev 70: 761-845, 1990[Free Full Text].

31.   Ping, P, Zhang J, Qiu Y, Tang X-L, Manchikalapudi S, Cao X, and Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms varepsilon  and eta  in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res 81: 404-414, 1997[Abstract/Free Full Text].

32.   Puceat, M, Clement O, Lechene P, Pelosin JM, Ventura-Clapier R, and Vassort G. Neurohormonal control of calcium sensitivity of myofilaments in rat single heart cells. Circ Res 67: 517-524, 1990[Abstract/Free Full Text].

33.   Puceat, M, Hilal-Dandan R, Strulovici B, Brunton LL, and Brown JH. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. J Biol Chem 269: 16938-16944, 1994[Abstract/Free Full Text].

34.   Romano, FD, MacDonald SG, and Dobson JG. Adenosine receptor coupling to adenylate cyclase of rat ventricular myocyte membranes. Am J Physiol Heart Circ Physiol 257: H1088-H1095, 1989[Abstract/Free Full Text].

35.   Sakamoto, J, Miura T, Goto M, and Iimura O. Limitation of myocardial infarct size by adenosine A1 receptor activation is abolished by protein kinase C inhibitors in the rabbit. Cardiovasc Res 29: 682-688, 1995[ISI][Medline].

36.   Strang, KT, Mentzer RM, and Moss RL. A1-purinergic receptor stimulation decreases maximum shortening velocity (Vmax) of rat skinned single ventricular myocytes independent of beta -adrenergic stimulation. Circulation 88: 241-246, 1993.

37.   Talosi, L, and Kranias EG. Effect of alpha-adrenergic stimulation on activation of protein kinase C and phosphorylation of proteins in intact rabbit hearts. Circ Res 70: 670-678, 1992[Abstract/Free Full Text].

38.   Thornton, JD, Liu GS, Olsson RA, and Downey JM. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation 85: 659-665, 1992[Abstract/Free Full Text].

39.   Toullec, D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, and Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 15771-15781, 1991[Abstract/Free Full Text].

40.   Ventura, C, Guarnieri C, Stefanelli C, Cirielli C, Lakatta DG, and Capogrossi MC. Comparison between alpha -adrenergic-and kappa -opioidergic-mediated inositol(1,4,5)P4 formation in adult cultured rat ventricular cardiomyocytes. Biochem Biophys Res Commun 179: 972-978, 1991[ISI][Medline].

41.   Viko, H, Sandness D, Skomedal T, and Osnes J-B. Effect of concomitant beta -adrenoceptor stimulation of alpha 1-adreneoceptor mediated increases in inositol 1,4,5 triphosphatate mass in adult rat cadiomyocytes. Pharmacol Toxicol 83: 23-28, 1998[ISI][Medline].

42.   Woodcock, EA, Tanner J, Fullerton M, and Kuraja I. Differential pathways of inositol phosphate metabolism in intact neonatal rat hearts and isolated cardiomyocytes. Biochem J 281: 683-688, 1992.

43.   Yao, Z, and Gross GJ. A comparison of adenosine-induced cardioprotection and ischemic preconditioning in dogs: efficacy, time course, and role of KATP channels. Circulation 89: 1229-1236, 1994[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 279(6):H2685-H2693
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
B. C. Blunt, Y. Chen, J. D. Potter, and P. A. Hofmann
Modest actomyosin energy conservation increases myocardial postischemic function
Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1088 - H1096.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Miyazaki, S. Komatsu, M. Ikebe, R. A. Fenton, and J. G. Dobson Jr.
Protein kinase C{epsilon} and the antiadrenergic action of adenosine in rat ventricular myocytes
Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1721 - H1729.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
N. Chaudary, Z. Naydenova, I. Shuralyova, and I. R. Coe
The Adenosine Transporter, mENT1, Is a Target for Adenosine Receptor Signaling and Protein Kinase C{epsilon} in Hypoxic and Pharmacological Preconditioning in the Mouse Cardiomyocyte Cell Line, HL-1
J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 1190 - 1198.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Chen, R. Rajashree, Q. Liu, and P. Hofmann
Acute p38 MAPK activation decreases force development in ventricular myocytes
Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2578 - H2586.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W. G. Pyle, Y. Chen, and P. A. Hofmann
Cardioprotection through a PKC-dependent decrease in myofilament ATPase
Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H1220 - H1228.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. M. Burkart, M. P. Sumandea, T. Kobayashi, M. Nili, A. F. Martin, E. Homsher, and R. J. Solaro
Phosphorylation or Glutamic Acid Substitution at Protein Kinase C Sites on Cardiac Troponin I Differentially Depress Myofilament Tension and Shortening Velocity
J. Biol. Chem., March 21, 2003; 278(13): 11265 - 11272.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
Y. Pi, K. R. Kemnitz, D. Zhang, E. G. Kranias, and J. W. Walker
Phosphorylation of Troponin I Controls Cardiac Twitch Dynamics: Evidence From Phosphorylation Site Mutants Expressed on a Troponin I-Null Background in Mice
Circ. Res., April 5, 2002; 90(6): 649 - 656.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (11)
Right arrow