Research and Development, Department of Veterans Affairs Medical Center, West Roxbury and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02132
Submitted 19 February 2003 ; accepted in final form 5 August 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
eicosanoids; calcium; vascular smooth muscle
Among the methods of treatment of coronary artery disease, Ca2+ channel blockers such as verapamil and diltiazem are often recommended to decrease coronary vasospasm and thereby reduce the incidence of angina and myocardial infarction (1, 2, 13, 35). However, certain forms of angina and coronary artery disease do not respond adequately to treatment with Ca2+ channel blockers (26, 38). The causes of the Ca2+ antagonist-insensitive forms of coronary vasospasm are not clearly understood, but they could be caused by decreased sensitivity of the coronary smooth muscle contraction induced by certain endogenous coronary vasoconstrictors to Ca2+ channel blockers. For example, although PGF2 is a potent coronary vasoconstrictor (3, 37), the sensitivity of the PGF2
-stimulated mechanisms of coronary smooth muscle contraction to Ca2+ channel blockers is unclear.
Another potential cause of the Ca2+ antagonist-insensitive forms of coronary vasospasm is possible activation of a Ca2+ sensitization, or perhaps Ca2+-independent, mechanism(s) of smooth muscle contraction. It is widely accepted that vascular smooth muscle contraction is triggered by increases in intracellular free Ca2+ concentration ([Ca2+]i) caused by Ca2+ release from the intracellular stores and Ca2+ entry from the extracellular space (12, 23, 36). Also, the interaction of a vasoconstrictor agonist with its specific receptor is coupled to increased breakdown of plasma membrane phospholipids and increased production of diacylglycerol (DAG) (17, 34). DAG binds to and activates protein kinase C (PKC). PKC is a family of Ca2+-dependent and Ca2+-independent isoforms that have different enzyme properties, substrates, and functions and exhibit different subcellular distributions in the same blood vessel type from different species and in different blood vessels from the same species (17, 20, 27, 34). PKC is mainly cytosolic under resting conditions and undergoes translocation to the particulate fraction when it is activated by endogenous DAG or exogenous phorbol esters (17, 34). In addition, direct activation of PKC by phorbol esters causes sustained contraction of vascular smooth muscle, suggesting a role for PKC in regulating smooth muscle contraction (14, 22). However, the role of PKC as a potential signaling mechanism of the Ca2+ antagonist-insensitive forms of coronary smooth muscle contraction has not been fully investigated.
The purpose of the present study was to test the hypothesis that a significant component of coronary smooth muscle contraction is Ca2+ antagonist insensitive and involves activation of specific PKC isoforms. We used PGF2, a potent coronary vasoconstrictor, to induce coronary smooth muscle contraction. Because the PKC family includes both Ca2+-dependent and Ca2+-independent isoforms, any PGF2
-induced changes in coronary smooth muscle [Ca2+]i may determine which PKC isoform would be activated. Therefore, experiments were designed to investigate the effects of PGF2
on coronary smooth muscle cell contraction, [Ca2+]i, and PKC activity. The effects of PGF2
were compared with those of phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, and with membrane depolarization by high-KCl solution, an activator of Ca2+ entry from the extracellular space. The sensitivity of coronary smooth muscle cell contraction, [Ca2+]i, and PKC activity to two Ca2+ channel antagonists, namely verapamil and diltiazem, and mechanistically distinct PKC inhibitors, namely, GF-109203X, calphostin C, Gö-6976, and selective
-PKC V1-2 inhibitory peptide (5-7, 25, 28, 39, 41), was also investigated.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Single-cell isolation. Coronary artery strips (50 mg) were placed in a tissue digestion mixture containing collagenase type II (236 U/mg protein; Worthington, Freehold, NJ), elastase grade II (3.25 U/mg protein; Boehringer Mannheim, Indianapolis, IN), and trypsin inhibitor type II (10,000 U/ml; Sigma, St. Louis, MO) in 7.5 ml of Ca2+- and Mg2+-free Hanks' solution supplemented with 30% bovine serum albumin (Sigma) as previously described (21, 30, 31). The tissue was incubated three times in the tissue digestion mixture to yield three batches of cells. For batch 1, the tissue was incubated with 5 mg of collagenase, 4 mg of elastase, and 147 ml of trypsin inhibitor for 60 min. For batches 2 and 3, the tissue was incubated with 2.5 mg of collagenase, 4 mg of elastase, and 122 ml of trypsin inhibitor for additional 30 min. The tissue preparation was placed in a shaking water bath at 34°C in an atmosphere of 95% O2-5% CO2. At the end of each incubation period, the preparation was rinsed with 12.5 ml of Hanks' solution with albumin. The first batch containing digested cells, damaged smooth muscle, and other unwanted material was discarded. Cells from both batches 2 and 3 were used and were poured over glass coverslips placed in wells and cooled to 2°C. The cells were allowed to settle by gravity and adhere to the glass coverslips. Ca2+ was gradually added back to the preparation to avoid theö calcium paradox
(32).
Contraction studies. Coverslips with the attached cells were placed on the stage of an inverted Nikon (Diaphot-300) microscope and viewed with a Nikon x100 oil-immersion objective (total magnification x1,000). The cells were bathed in 0.5 ml of Hanks' solution that remained stationary during the data recording. The cell isolation procedure yielded smooth muscle cells of variable lengths. Only viable, healthy, and spindle-shaped cells 60 µm in length were selected. Viable, healthy cells adhered to the glass coverslips and appeared bright, with a halo along the periphery and without a visible nucleus when viewed with phase-contrast optics. The viability of the smooth muscle cells was confirmed by their exclusion of Trypan blue (0.2%; Sigma) and their consistent and significant contraction in response to PGF2
and high-KCl solution. The cells were further characterized and consistently showed significant immunofluorescence signal when fixed and labeled with anti-smooth muscle myosin antibody. Cell images were acquired with a PXL charge-coupled device camera and displayed on a PC with PMIS image analysis software (Photometrics, Tucson, AZ). The number of pixels corresponding to the cell length in the cell image was transformed into micrometers with a calibration bar. The steady-state changes in cell length were consistently measured after 10-min stimulation with PGF2
(10-5 M) or KCl (51 mM) and after 30-min stimulation with the phorbol ester PMA (10-6 M). The magnitude of cell contraction was expressed as the final cell length (L) as a fraction of the initial cell length (Li). All contraction measurements were made at 37°C.
Measurement of [Ca2+]i. Single coronary smooth muscle cells were loaded with the Ca2+ indicator fura 2 for 30 min at 34°C as previously described (19, 30, 31). The fura 2 loading solution was made of normal Hanks' solution, the cell-permeant fura 2 acetoxymethyl ester (fura 2-AM, 10-6 M; Molecular Probes, Eugene, OR), and 0.01% Pluronic F-127 (Sigma). The fura 2-loaded cells were washed twice and further incubated in Hanks' solution for at least 30 min to allow complete deesterification of the fura 2-AM. Precautionary measures were taken throughout the procedure to avoid extensive photobleaching of fura 2.
The fura 2-loaded cells were viewed through a Nikon Fluor x100 oil-immersion objective (numerical aperture 1.3) on an inverted Nikon Diaphot-300 microscope. The Ca2+ indicator was excited alternately at 340 and 380 nm with a filter wheel that alternated at a frequency of 0.5 Hz. The emitted light was collected at 510 nm into a R928 photomultiplier tube (Ludl Electronic Products, Hawthorne, NY) through a pin-hole aperture 1 µm in diameter positioned 1 µm from the plasma membrane and 1 µm from the nucleus. The fluorescent signal was digitized with a module (Mac 2000; Ludl) and analyzed on a PC with data analysis software. The fluorescent signal was background subtracted. Spectral shifts that result from binding of Ca2+ to fura 2 make it possible to use the ratio method, thus rendering the measurements of [Ca2+]i less sensitive to changes in cell thickness or the extent of dye loading and photobleaching. The ratio between the fluorescence intensity at 340 and 380 nm (R) was transformed into the corresponding levels of [Ca2+]i as described by Grynkiewicz and coworkers (10).
![]() |
Tissue fractions. Tissue strips (80 mg) under resting conditions or stimulated with PGF2
, PMA, or KCl for 30 min and in the absence or presence of the Ca2+ channel blockers verapamil or diltiazem or the PKC inhibitors GF-109203X, calphostin C, Gö-6976, or
-PKC V1-2 were used. The tissues were rapidly transferred to ice-cold equilibrating buffer A containing (in mM) 25 Tris·HCl (pH 7.5), 5 EGTA, 0.02 leupeptin, 0.2 phenylmethylsulfonyl fluoride, and 1 dithiothreitol. For measurement of PKC activity, the tissue was transferred to homogenization buffer B, which had the same composition as buffer A plus 250 mM sucrose. For Western blots, the tissue was transferred to a homogenization buffer containing 20 mM 3-(N-morpholino)propanesulfonic acid, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 2.3 mg dithiothreitol, 1.2 mM EDTA, 0.02% bovine serum albumin, 5.5 µM leupeptin, 5.5 µM pepstatin, 2.15 µM aprotinin and 20 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride. The tissue was homogenized with a 2-ml tight-fitting homogenizer (Kontes Glass) at 4°C and centrifuged at 100,000 rpm for 20 min at 4°C (Ultra-Centrifuge TL-100; Beckman), and the supernatant was used as the cytosolic fraction. The pellet was resuspended in homogenization buffer containing 1% Triton X-100 for 20 min, diluted with homogenization buffer to a final concentration of 0.2% Triton, and centrifuged at 100,000 rpm for 20 min at 4°C. The supernatant was used as the particulate fraction. Protein concentrations in tissue fractions were determined with a protein assay kit (Bio-Rad, Hercules, CA).
PKC activity. The cytosolic and particulate fractions were applied to diethylaminoethyl-cellulose columns (0.8 x 4.0 cm; Bio-Rad). The columns were washed with buffer A, and the protein was eluted with 0.1 M NaCl. PKC activity in the aliquots was determined by measuring the incorporation of 32P from [-32P]ATP (ICN) into histone III-S (15, 16). Because histone III-S may not be a good substrate for novel PKC isoforms such as
- and
-PKC, experiments were also performed with myelin basic protein (MBP) as a substrate for PKC. The assay mixture contained 25 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 200 µg/ml histone III-S or MBP, 80 µg/ml phosphatidylserine, 30 µg/ml diolein, [
-32P]ATP (1-3 x 105 cpm/nmol), and 0.5-3 µg of protein. After 5-min incubation at 30°C, the reaction was stopped by spotting 25 µl of the assay mixture onto phosphocellulose disks. The disks were washed 3 x 5 min with 5% trichloroacetic acid and placed in 4 ml of Ecolite scintillation cocktail, and the radioactivity was measured in a liquid scintillation counter.
Immunoblotting. Protein-matched samples of the cytosolic and particulate fractions were subjected to electrophoresis on 8% SDS-polyacrylamide gels and then transferred electro-phoretically to nitrocellulose membranes. The membranes were incubated in 5% dried nonfat milk in phosphate-buffered saline (PBS)-Tween at 22°C for 1 h, washed with PBSTween 3 x 5 min, and then incubated in the primary anti-PKC antibody solution at 4°C overnight. Polyclonal antibodies to -,
-,
-,
-,
-, and
-PKC (GIBCO, Grand Island, NY) were used. These antibodies have been shown to react with the specific PKC isoforms in porcine aortic endothelial cells and airway and coronary smooth muscle (11, 15, 16, 40). The specificity of each antibody was routinely verified by demonstrating that it was blocked by the synthetic peptide to which the antibody was raised and not by other sequences of the PKC molecule, a more stringent control than simple elimination of the primary antibody. To keep the labeling conditions constant, we used the same anti-PKC antibody titer (1:500) and protein concentration (10 µg) in all tissue samples. These antibody titer and protein concentrations gave optimal immunoreactive signals while remaining on the linear portion of the titration curve. To confirm the results with the GIBCO polyclonal antibodies, we also used polyclonal anti-PKC antibodies from Sigma (1:500), polyclonal anti-PKC antibodies from Chemicon International (Temecula, CA; 1:100), and monoclonal anti-PKC antibodies from Seikagaku America (Ijamsville, MD; 1:100) and obtained similar results. The nitrocellulose membranes were washed 5 x 15 min in PBS-Tween and then incubated in horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG for 1.5 h. The blots were washed with PBS-Tween 5 x 15 min and visualized with enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). PBS-Tween contained (in mM) 80 Na2HPO4, 20 NaH2PO4, 100 NaCl, and 0.05% Tween. The reactive bands corresponding to PKC isoforms were analyzed quantitatively by optical densitometry with a GS-700 imaging densitometer (Bio-Rad).
Solutions. Krebs solution contained (in mM) 120 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11.5 dextrose, at pH 7.4. Hanks' solution contained (in mM) 137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 Na2HPO4, 4.17 NaHCO3, 5.55 dextrose, and 10 HEPES, at pH 7.4. For Ca2+-and Mg2+-containing Hanks' solution, 1 mM CaCl2 and 1.2 mM MgCl2 were added. For Ca2+-free solution CaCl2 was omitted and replaced with 2 mM EGTA.
Drugs and chemicals. PGF2 (Sigma), verapamil, and diltiazem (Calbiochem, La Jolla, CA) were dissolved in distilled water. Neomycin sulfate and 1-[6-([17
-3-methoxyestra-1,3,5(10)trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) were purchased from Biomol (Plymouth Meeting, PA). PMA (Alexis, San Diego, CA), GF-109203X (Biomol), Gö-6976 (Biomol), calphostin C (Kamiya, Tukwila, WA), and myristoyl-tagged membrane-permeant
-PKC V1-2 inhibitory peptide (Biomol) were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in solution was
0.1%. All other chemicals were of reagent grade or better.
Statistical analysis. The data were analyzed and presented as means ± SE and compared by Student's t-test for unpaired data, with P < 0.05 considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Pretreatment of the cells with the Ca2+ channel blocker verapamil (10-6 M) for 10 min reduced the transient PGF2-induced [Ca2+]i to 123 ± 14 nM, abolished the maintained PGF2
-induced increase in [Ca2+]i, and partially inhibited the PGF2
-induced cell contraction to 17.2 ± 2.4% (Fig. 1B). Pretreatment of the cells with another Ca2+ channel blocker such as diltiazem (10-6 M) for 10 min also reduced the transient PGF2
-induced [Ca2+]i to 119 ± 12 nM, abolished the maintained PGF2
-induced increase in [Ca2+]i, and partially inhibited the PGF2
-induced cell contraction to 16.8 ± 2.2%. GF-109203X at nanomolar concentrations has been suggested to inhibit mainly Ca2+-dependent PKC isoforms (41). However, at micromolar concentrations, GF-109203X inhibits both Ca2+-dependent and Ca2+-independent PKC isoforms (28). Pretreatment of the cells with GF-109203X (10-6 M) for 10 min partially inhibited the PGF2
-induced cell contraction to 16.5 ± 1.6%, with no change in [Ca2+]i (Fig. 1C). Similar inhibition of PGF2
contraction (16.7 ± 1.4%), with no significant change in [Ca2+]i, was observed in cells pretreated with the PKC inhibitor calphostin C (10-6 M) for 10 min. Pretreatment of the cells with verapamil plus GF-109203X for 10 min abolished both the PGF2
-induced contraction and the maintained increase in [Ca2+]i (Fig. 1D). A small, transient PGF2
-induced increase in [Ca2+]i to 121 ± 13 nM could still be observed in cells pretreated with verapamil plus GF-109203X (Fig. 1D); however, such a small and transient increase in [Ca2+]i did not appear to be sufficient to cause significant cell contraction.
Treatment of the cells with PMA (10-6 M), a direct activator of PKC, caused 20.7 ± 2.1% cell contraction with no significant increase in [Ca2+]i above basal levels (Figs. 2A and 3). Pretreatment of the cells with verapamil or diltiazem (10-6 M) did not affect the PMA-induced contraction. In contrast, pretreatment of the cells with GF-109203X (10-6 M) abolished the PMA contraction, with no significant change in [Ca2+]i (Figs. 2B and 3). Complete inhibition of PMA contraction, with no significant change in [Ca2+]i, was also observed in cells pretreated with the PKC inhibitor calphostin C (10-6 M).
|
|
Membrane depolarization with high-KCl solution (51 mM) caused 35.8 ± 2.9% cell contraction and increased [Ca2+]i to 288 ± 13.7 nM (Figs. 2C and 3). Pretreatment of the cells with verapamil (10-6 M) abolished the KCl-induced cell contraction and [Ca2+]i (Figs. 2D and 3). Similarly, pretreatment of the cells with diltiazem (10-6 M) abolished the KCl-induced cell contraction and [Ca2+]i. On the other hand, treatment of the cells with GF-109203X or calphostin C (10-6 M) did not significantly affect the KCl-induced contraction or [Ca2+]i.
The effect of relatively specific PKC inhibitors on PGF2- and PMA-induced contraction was also tested. Gö-6976 (10-6 M), a relatively specific inhibitor of Ca2+-dependent PKC isoforms (28), did not significantly affect [Ca2+]i, the verapamil-insensitive PGF2
contraction, or PMA contraction (Fig. 3). On the other hand, the membrane-permeant selective
-PKC V1-2 inhibitory peptide (10-4 M) (5, 6, 9) abolished the verapamil-insensitive component of PGF2
contraction and the PMA contraction, with no change in [Ca2+]i (Fig. 3). Neither Gö-6976 nor
-PKC V1-2 caused any significant inhibition of KCl-induced contraction or [Ca2+]i.
In resting tissues PKC activity was greater in the cytosolic than the particulate fraction, and the particulate-to-cytosolic ratio was 0.5 ± 0.1. PGF2 (10-5 M) caused an increase in PKC activity in the particulate fraction and a decrease in the cytosolic fraction (Fig. 4A) and a significant increase in the particulate-tocytosolic ratio to a maximum of 2.1 ± 0.2 (Fig. 4B). In tissues pretreated with verapamil (10-6 M) the PGF2
-induced PKC activity was partially, but significantly, reduced (Fig. 4, B and C). Partial, but significant, reduction of the PGF2
-induced particulate-to-cytosolic PKC ratio to 1.2 ± 0.1 was also observed in tissues pretreated with diltiazem (10-6 M). On the other hand, pretreating the tissues with the PKC inhibitor GF-109203X (10-6 M) abolished the PGF2
-induced increases in PKC activity (Fig. 4, B and C). Complete inhibition of PGF2
-induced PKC activity was also observed in tissues pretreated with the PKC inhibitor calphostin C (10-6 M). Treatment of the tissues for 30 min with Gö-6976 (10-6 M), a relatively specific inhibitor of Ca2+-dependent PKC isoforms (28), did not significantly affect the verapamil-insensitive component of the PGF2
-induced PKC activity ratio (1.3 ± 0.2). On the other hand, the
-PKC V1-2 inhibitory peptide (10-4 M) decreased the verapamil-insensitive PGF2
-induced PKC activity ratio to 0.6 ± 0.1, which was not significantly different from the basal PKC activity ratio.
|
In comparison with PGF2, the phorbol ester PMA (10-6 M) caused significant increases in PKC activity that were inhibited by GF-109203X or calphostin C but not by verapamil (Fig. 4C). In tissues stimulated with KCl depolarizing solution (51 mM) no significant increases in PKC activity could be observed, and pretreating the tissues with verapamil, diltiazem, GF-109203X, or calphostin C did not cause any significant change in PKC activity (Fig. 4C).
Western blot analysis revealed significant amounts of -,
-,
-, and
-PKC. In resting unstimulated tissues,
- and
-PKC appeared to be mainly cytosolic (Fig. 5),
-PKC was slightly more in the particulate fraction, and
-PKC was equally distributed in the cytosolic and particulate fractions as previously described (15, 16). PGF2
caused significant translocation of the Ca2+-dependent
-PKC (Fig. 5A) and the Ca2+-independent
-PKC (Fig. 5B) from the cytosolic to the particulate fraction. Pretreating the tissues with the Ca2+ channel blocker verapamil inhibited the PGF2
-induced translocation of
-PKC (Fig. 5A) but not
-PKC (Fig. 5B). On the other hand, the PKC inhibitor calphostin C, but not GF-109203X, inhibited the PGF2
-induced translocation of both
-PKC (Fig. 5A) and
-PKC (Fig. 5B). The phorbol ester PMA did not cause translocation of
-PKC but induced translocation of
-PKC that was inhibited in tissues pretreated with calphostin C but not GF-109203X.
|
Experiments in Ca2+-free (2 mM EGTA) solution indicated that significant PGF2-induced cell contraction, PKC activity, and
-PKC translocation could still be observed in the absence of extracellular Ca2+ entry (Fig. 6). The PGF2
-induced cell contraction, PKC activity, and
-PKC translocation in Ca2+-free solution were inhibited in tissues pretreated with the selective
-PKC V1-2 inhibitory peptide (Fig. 6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The interaction of an agonist with its receptor is known to activate the enzyme phospholipase C and to increase the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and DAG (16, 17, 34). Inositol 1,4,5-trisphosphate stimulates Ca2+ release from the intracellular stores, and DAG stimulates PKC (17, 34). The inhibition of PGF2-induced cell contraction by the phospholipase C inhibitors neomycin and U-73122 provides evidence that the PGF2
responses involve activation of phospholipase C and the hydrolysis of plasma membrane phospholipids.
The PGF2 (10-5 M)-induced coronary smooth muscle contraction in Ca2+-containing solution was associated with a transient followed by a lower, maintained elevation in [Ca2+]i, suggesting that the increases in [Ca2+]i represent an important mechanism of PGF2
contraction. The agonist-induced transient increase in [Ca2+]i is often thought to be due to Ca2+ release from the intracellular stores (23). The observation that the transient PGF2
-induced increase in [Ca2+]i was abolished in cells pretreated with the phospholipase inhibitors neomycin and U-73122 suggests that it may involve Ca2+ release from the intracellular stores in response to the hydrolysis products of plasma membrane phospholipids. Also, the observation that a small, transient PGF2
-induced increase in [Ca2+]i was still present after Ca2+ entry was blocked with verapamil or diltiazem supports the contention that the [Ca2+]i transient is in part due to Ca2+ release from the intracellular stores. However, the observation that the PGF2
-induced [Ca2+]i transient was significantly smaller in the presence than in the absence of verapamil or diltiazem suggests that a major component of the initial increase in [Ca2+]i is due to Ca2+ influx. In addition, the possibility that prolonged cell incubation with the Ca2+ channel blocker may reduce the available Ca2+ stores by inhibiting their replenishment from the extracellular Ca2+ pool cannot be excluded under the present experimental conditions. On the other hand, the complete inhibition of the maintained PGF2
-induced increase in [Ca2+]i by verapamil or diltiazem suggests that it is mainly due to Ca2+ influx from the extracellular space.
To further investigate the mechanisms involved in the PGF2-induced contraction and [Ca2+]i, we compared the PGF2
response with that of membrane depolarization by high-KCl solution, which mainly stimulates Ca2+ entry from the extracellular space (22, 23). The KCl contraction was associated with increases in [Ca2+]i but not change in PKC activity or distribution, providing support to the notion that KCl contraction is mainly due to increases in [Ca2+]i. Also, verapamil or diltiazem inhibited the KCl-induced contraction and [Ca2+]i but did not affect the PMA contraction or PKC activity, providing evidence that the effects of verapamil and diltiazem are mainly due to inhibition of Ca2+ influx. The observation that verapamil or diltiazem, at a concentration that completely inhibits KCl-induced contraction and [Ca2+]i, abolished the maintained PGF2
-induced [Ca2+]i and caused significant inhibition of PGF2
contraction supports the contention that a component of the PGF2
contraction is dependent on Ca2+ entry and is sensitive to Ca2+ antagonists. On the other hand, a significant component of PGF2
contraction in Ca2+-containing solution was not inhibited by verapamil or diltiazem. Also, a small but significant PGF2
-induced contraction could be observed in Ca2+-free solution. These data suggest that a significant component of PGF2
contraction does not require Ca2+ entry and is thereby Ca2+ antagonist insensitive.
Although [Ca2+]i is a major determinant of smooth muscle contraction (23, 36), other contraction pathways have been suggested, including activation of PKC (12, 17). Several studies have shown that direct activation of PKC by phorbol esters causes significant and sustained contraction of smooth muscle with no significant change in [Ca2+]i (14, 22), suggesting a role for PKC in regulating smooth muscle contraction, at least in part, by increasing the myofilament force sensitivity to [Ca2+]i. To investigate the role of PKC in the Ca2+ antagonist-insensitive component of PGF2 contraction, we compared the PGF2
response with that of a direct PKC activator such as the phorbol ester PMA. PMA caused significant cell contraction that is most likely due to activation of PKC because 1) PMA caused contraction in the absence of detectable changes in [Ca2+]i; 2) PMA caused significant increases in PKC activity; and 3) the PMA-induced contraction and PKC activity were inhibited by the PKC inhibitors GF-109203X and calphostin C but not by verapamil. The observation that GF-109203X or calphostin C, at a concentration that completely inhibits PMA-induced contraction and PKC activity, caused significant inhibition of PGF2
contraction suggests that a significant component of PGF2
contraction involves activation of PKC. Also, the combined pretreatment of the cells with GF-109203X or calphostin C and the Ca2+ antagonist verapamil, which are partial inhibitors of PGF2
contraction, completely inhibited the PGF2
contraction. These data suggest that GF-109203X or calphostin C inhibits the Ca2+ antagonist-insensitive component of PGF2
and provide evidence that the Ca2+ antagonist-insensitive component of PGF2
involves activation of PKC. This is supported by the present observation that PGF2
causes significant increase in PKC activity that is inhibited by the PKC inhibitors GF-109203X and calphostin C.
PKC is a family of Ca2+-dependent and Ca2+-independent isoforms (17, 34). The present immunoblotting data suggest that in Ca2+-containing solution PGF2 causes activation and translocation of both the Ca2+-dependent
-PKC and the Ca2+-independent
-PKC. The PGF2
-induced translocation of
- and
-PKC is inhibited by the PKC inhibitor calphostin C, confirming the specificity of the PKC translocation assay. On the other hand, verapamil inhibited the PGF2
-induced translocation of
-PKC but not that of
-PKC. The inhibition of
-PKC translocation by the Ca2+ antagonist verapamil is consistent with reports that
-PKC is a Ca2+-dependent isoform (17, 34). On the other hand, the lack of inhibition of
-PKC by verapamil is in accordance with the reports that
-PKC is a Ca2+-independent isoform (17, 34) and may therefore explain the Ca2+ antagonist-insensitive component of PGF2
contraction. The role of
-PKC in the Ca2+ antagonist-insensitive PGF2
-induced contraction is supported by the following observations. 1) The Ca2+ antagonist-insensitive PGF2
contraction and PKC activity were not inhibited by Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms, but were inhibited by GF-109203X or calphostin C at concentrations that inhibit both Ca2+-dependent and Ca2+-independent PKC isoforms. 2) The Ca2+ antagonist-insensitive PGF2
contraction and PKC activity were inhibited by the specific
-PKC V1-2 inhibitory peptide. 3) Under conditions in which Ca2+ entry was minimized in Ca2+-free (2 mM EGTA) solution, significant PGF2
contraction, PKC activity, and
-PKC translocation could still be observed. 4) The PGF2
-induced contraction, PKC activity, and
-PKC translocation in Ca2+-free solution were inhibited in tissues pretreated with the selective
-PKC V1-2 inhibitory peptide and thus further support the contention that a portion of PGF2
contraction is Ca2+ insensitive and involves activation of the Ca2+-independent
-PKC.
In the present study, the phorbol ester PMA, a PKC activator, caused contraction with no significant increase in [Ca2+]i and induced significant activation and translocation of -PKC, consistent with a possible role for
-PKC in Ca2+-insensitive smooth muscle contraction. The question arises as to why PMA did not activate
-PKC at basal levels of [Ca2+]i whereas PGF2
caused significant activation of
-PKC when [Ca2+]i was increased above basal levels. This could be related, at least in part, to the level of [Ca2+]i required for activation of Ca2+-dependent PKC isoforms. This is consistent with previous reports that a threshold increase in [Ca2+]i is required for activation of
-PKC in vascular smooth muscle cells of the ferret and the pig (16, 19). We should also note that the PKC inhibitor calphostin C inhibited both the PGF2
- and PMA-induced translocation of
- or
-PKC as well as PKC activity. However, GF-109203X did not inhibit PGF2
-or PMA-induced translocation of
- or
-PKC despite its complete inhibition of PKC activity. This could be explained by the difference in the site of action of calphostin C and GF-109203X on the PKC molecule. GF-109203X and calphostin C are two mechanistically distinct PKC inhibitors. GF-109203X inhibits the PKC enzyme by interfering with ATP binding to the catalytic domain and therefore would inhibit the PKC phosphotransferase activity without inhibiting its translocation (41). On the other hand, calphostin C competes with PMA or DAG for binding to the regulatory domain and therefore inhibits both PKC translocation and phosphotransferase activity (7, 25, 39). This may also explain the observation that
-PKC V1-2, a 14- to 21-peptide sequence derived from the first variable region V1 of the regulatory domain of
-PKC (5, 6), prevented
-PKC translocation and significantly inhibited Ca2+-insensitive PKC activity.
The cellular mechanisms of PGF2 contraction in the absence of significant increases in [Ca2+]i are not entirely clear but may involve G protein-induced activation of phospholipases (4). The observed inhibition of PGF2
-induced cell contraction by the phospholipase C inhibitors neomycin and U-73122 provides evidence that the PGF2
responses involve activation of phospholipase C and the hydrolysis of plasma membrane phospholipids. The increased phospholipid turnover leads to increased DAG production and activation of
-PKC.
-PKC could then cause phosphorylation, either directly or indirectly, of actin-binding proteins such as calponin or caldesmon and thereby lead to increased force sensitivity of the contractile proteins even to basal levels of [Ca2+]i (12).
In conclusion, a significant component of PGF2-induced coronary smooth muscle contraction appears to require both Ca2+ entry and PKC activation. This Ca2+/PKC-dependent component of PGF2
contraction is inhibited by both Ca2+ channel blockers and PKC inhibitors and appears to involve the Ca2+-dependent
-PKC isoform. An additional significant component of PGF2
contraction is Ca2+ antagonist insensitive, appears to involve activation and translocation of the Ca2+-independent
-PKC, and may represent a possible signaling mechanism of the Ca2+ antagonist-resistant forms of coronary vasospasm.
![]() |
DISCLOSURES |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Brogden RN and Benfield P. Verapamil: a review of its pharmacological properties and therapeutic use in coronary artery disease. Drugs 51: 792-819, 1996.[ISI][Medline]
3. Cannon PJ. Eicosanoids and the blood vessel wall. Circulation 70: 523-528, 1984.[ISI][Medline]
4. Cao W, Chen Q, Sohn UD, Kim N, Kirber MT, Harnett KM, Behar J, and Biancani P. Ca2+-induced contraction of cat esophageal circular smooth muscle cells. Am J Physiol Cell Physiol 280: C980-C992, 2001.
5. Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R, Dorn GW 2nd, and Mochly-Rosen D. Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci USA 98: 1114-11119, 2001.
6. Chen L, Wright LR, Chen CH, Oliver SF, Wender PA, and Mochly-Rosen D. Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7). Chem Biol 8: 1123-1129, 2001.[ISI][Medline]
7. Dubyak GR and Kertesy SB. Inhibition of GTPS-dependent phospholipase D and Rho membrane association by calphostin is independent of protein kinase C catalytic activity. Arch Biochem Biophys 341: 129-139, 1997.[ISI][Medline]
8. Fuster V, Badimon L, Badimon JJ, and Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (2). N Engl J Med 326: 310-318, 1992.[ISI][Medline]
9. Gratton JP, Yu J, Griffith JW, Babbitt RW, Scotland RS, Hickey R, Giordano FJ, and Sessa WC. Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo. Nat Med 9: 357-363, 2003.[ISI][Medline]
10. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985.[Abstract]
11. Hempel A, Maasch C, Heintze U, Lindschau C, Dietz R, Luft FC, and Haller H. High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha. Circ Res 81: 363-371, 1997.
12. Horowitz A, Menice CB, Laporte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967-1003, 1996.
13. Jespersen CM. Verapamil in acute myocardial infarction. The rationales of the VAMI and DAVIT III trials. Cardiovasc Drugs Ther 14: 99-105, 2000.[ISI][Medline]
14. Jiang MJ and Morgan KG. Intracellular calcium levels in phorbol ester-induced contractions of vascular muscle. Am J Physiol Heart Circ Physiol 253: H1365-H1371, 1987.
15. Kanashiro CA, Altirkawi KA, and Khalil RA. Preconditioning of coronary artery against vasoconstriction by endothelin-1 and prostaglandin F2 during repeated downregulation of
-protein kinase C. J Cardiovasc Pharmacol 35: 491-501, 2000.[ISI][Medline]
16. Kanashiro CA and Khalil RA. Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids. Can J Physiol Pharmacol 76: 1110-1119, 1998.[ISI][Medline]
17. Kanashiro CA and Khalil RA. Signal transduction by protein kinase C in mammalian cells. Clin Exp Pharmacol Physiol 25: 974-985, 1998.[ISI][Medline]
18. Kawano H, Fujii H, Motoyama T, Kugiyama K, Ogawa H, and Yasue H. Myocardial ischemia due to coronary artery spasm during dobutamine stress echocardiography. Am J Cardiol 85: 26-30, 2000.[ISI][Medline]
19. Khalil RA, Lajoie C, and Morgan KG. In situ determination of [Ca2+]i threshold for translocation of the -protein kinase C isoform. Am J Physiol Cell Physiol 266: C1544-C1551, 1994.
20. Khalil RA, Lajoie C, Resnick MS, and Morgan KG. Ca2+-independent isoforms of protein kinase C differentially translocate in smooth muscle. Am J Physiol Cell Physiol 263: C714-C719, 1992.
21. Khalil RA and Morgan KG. Phenylephrine-induced translocation of protein kinase C and shortening of two types of vascular cells of the ferret. J Physiol 455: 585-599, 1992.[Abstract]
22. Khalil RA and van Breemen C. Sustained contraction of vascular smooth muscle: calcium influx or C-kinase activation? J Pharmacol Exp Ther 244: 537-542, 1988.[Abstract]
23. Khalil RA and van Breemen C. Mechanisms of calcium mobilization and homeostasis in vascular smooth muscle and their relevance to hypertension. In: Hypertension: Pathophysiology, Diagnosis, and Management, edited by Laragh JH and Brenner BM. New York: Raven, 1995, p. 523-540.
24. Kijima Y, Hashimura K, Matsu-ura Y, Kato Y, Yasuda T, Ueda T, Orita Y, and Fukunaga M. Transcardiac 8-iso-prostaglandin F2 generation from acute myocardial infarction heart: insight into abrupt reperfusion and oxidant stress. Prostaglandins Leukot Essent Fatty Acids 64: 161-166, 2001.[ISI][Medline]
25. Kobayashi E, Nakano H, Morimoto M, and Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 159: 548-553, 1989.[ISI][Medline]
26. Kumar A, Chandna H, Santhanam V, and Denes P. Refractory vasospasm with a malignant course. Clin Cardiol 23: 127-130, 2000.[ISI][Medline]
27. Liou YM and Morgan KG. Redistribution of protein kinase C isoforms in association with vascular hypertrophy of rat aorta. Am J Physiol Cell Physiol 267: C980-C989, 1994.
28. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, and Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go6976. J Biol Chem 268: 9194-9197, 1993.
29. Mehrabi MR, Serbecic N, Ekmekcioglu C, Tamaddon F, Wild T, Plesch K, Sinzinger H, and Glogar HD. Enhanced accumulation of the isoprostane, 8-epi-PGF2alpha, in human aortic and pulmonary valves of patients with coronary heart disease. Histol Histopathol 17: 1053-1059, 2002.[ISI][Medline]
30. Murphy JG and Khalil RA. Decreased [Ca2+]i during inhibition of coronary smooth muscle contraction by 17-estradiol, progesterone, and testosterone. J Pharmacol Exp Ther 291: 44-52, 1999.
31. Murphy JG and Khalil RA. Gender-specific reduction in contractility and [Ca2+]i in vascular smooth muscle cells of female rat. Am J Physiol Cell Physiol 278: C834-C844, 2000.
32. Nayler WG, Perry SE, Elz JS, and Daly MJ. Calcium, sodium, and the calcium paradox. Circ Res 55: 227-237, 1984.[Abstract]
33. Nichols WW, Mehta JL, Thompson L, and Donnelly WH. Synergistic effects of LTC4 and TxA2 on coronary flow and myocardial function. Am J Physiol Heart Circ Physiol 255: H153-H159, 1988.
34. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607-614, 1992.[ISI][Medline]
35. Opie LH. Mechanisms whereby calcium channel antagonists may protect patients with coronary artery disease. Eur Heart J 18 Suppl A: A92-A104, 1997.[ISI][Medline]
36. Rembold CM and Murphy RA. Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res 63: 593-603, 1988.[Abstract]
37. Rigel DF and Shetty SS. A novel model of conduit coronary constriction reveals local actions of endothelin-1 and prostaglandin F2. Am J Physiol Heart Circ Physiol 272: H2054-H2064, 1997.
38. Silvestry FE and Kimmel SE. Calcium-channel blockers in ischemic heart disease. Curr Opin Cardiol 11: 434-439, 1996.[ISI][Medline]
39. Spitaler M, Villunger A, Grunicke H, and Uberall F. Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota. J Biol Chem 275: 33289-33296, 2000.
40. Togashi H, Hirshman CA, and Emala CW. Qualitative immunoblot analysis of PKC isoforms expressed in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 272: L603-L607, 1997.
41. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, and Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 15771-15781, 1991.
42. Xuan YT, Guo Y, Zhu Y, Han H, Langenbach R, Dawn B, and Bolli R. Mechanism of cyclooxygenase-2 upregulation in late preconditioning. J Mol Cell Cardiol 35: 525-537, 2003.[ISI][Medline]
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |