Abstract
This study determined whether phosphodiesterase (PDE) was activated by protein kinase C (PKC) upon κ-receptor stimulation, and if so, to identify the isozyme. We first studied the effects oftrans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-opioid receptor (OR) agonist, and phorbol-12–myristate-13-acetate (PMA), a PKC activator, on cAMP accumulation and PDE activity in rat ventricular myocytes when PKC and PDE were inhibited by respective inhibitors. Like PMA, U50,488H decreased the forskolin-stimulated cAMP accumulation and dose-dependently stimulated the PDE activity, which were antagonized by 10−6 M chelerythrine and bisindolylmaleimide I, selective PKC antagonists. In addition, 3-isobutyl-1-methylxanthine, a PDE inhibitor, dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and abolished the stimulation on PDE activity by U50,488H and PMA. The observations suggest that PKC may enhance cAMP degradation through activating PDE upon κ-OR stimulation. To identify the isozyme(s) mediating the effect of PKC upon κ-OR stimulation, selective inhibitors were used. We found that 10−5 M Ro-20–1724, a selective cAMP-specific PDE (PDE-IV) inhibitor, abolished the inhibitory effects of U50,488H and PMA, whereas 8-methoxymethyl-3-isobutyl-1-methylxanthine,erythro-9-(2-hydroxy-3-nonyl) adenine, cilostamide, and zaprinast, selective inhibitors of Ca2+/calmodulin-dependent PDE (PDE-I), cGMP-stimulated PDE (PDE-II), cGMP-inhibited PDE (PDE-III), and cGMP-specific PDE (PDE-V), respectively, had no effect. Moreover, rolipram, another selective PDE-IV inhibitor, also dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and stimulation on PDE activity by U50,488H and PMA. In conclusion, this study has provided evidence for the first time that PKC and PDE-IV mediate the action of κ-OR.
Previous studies in our (Bian et al., 1998) and other (Ventura et al., 1991) laboratories have shown that effects of κ-opioid receptor (OR) stimulation in the heart are mediated by protein kinase C (PKC). We have found that staurosporine, a nonspecific PKC inhibitor, dose-dependently attenuates the inhibitory effect of bothtrans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-OR agonist, and phorbol-12-myristate-13-acetate (PMA), a selective PKC activator, on forskolin-stimulated accumulation of cAMP, suggesting that PKC mediates the action of κ-OR stimulation. The mechanisms are, however, not fully understood. Previous studies have shown that PKC either stimulates (Bode and Brunton, 1988; Michael and Webley, 1991;Tetsuka et al., 1995) or inhibits (Le Goas et al., 1991; Yingling et al., 1994) phosphodiesterase (PDE) in different tissues. In the heart,Schluter et al. (1995) reported that the PKC, activated by parathyroid hormone, stimulates PDE. It is likely that PDE may mediate the action of PKC on cAMP accumulation upon κ-OR stimulation in the heart.
More than 20 isoenzymes have been identified and are classified into five subfamilies (Beavo and Reifsnyder, 1990). They are differentially distributed. Growing evidence has shown that cAMP-specific PDE (PDE-IV) is one of the most important PDE isoenzymes in regulating cAMP breakdown in the ventricular tissue (Prigent et al., 1984, Dubois et al., 1990). However, PKC also has been shown to enhance the Ca2+/calmodulin-dependent PDE (PDE-I) activity in the hypertrophic cardiomyopathic hamster heart (Yu et al., 1996) and cGMP-stimulated phosphodiesterase (PDE-II) in a liver Golgi-endosomal fraction (Geoffroy et al., 1999).
The purpose of this study was therefore twofold. First, we determined the role of PDE in mediating the action of PKC on cAMP accumulation upon κ-OR stimulation. Second, we screened and identified the isozyme(s) of PDE involved. We determined the forskolin-stimulated cAMP accumulation and the PDE activity in ventricular myocytes upon κ-OR stimulation when PKC or PDE was inhibited with respective inhibitors. For inhibition of PDE, we used a nonselective PDE inhibitor as well as selective PDE-I, II, III, IV, and V inhibitors. The results indicate that PDE-IV mediates the action of PKC on cAMP accumulation upon κ-OR stimulation.
Materials and Methods
Isolation of Ventricular Myocytes.
Ventricular myocytes were isolated from the heart of male Sprague-Dawley rats (190–210 g), with a collagenase perfusion method described previously (Dong et al., 1993). Immediately after decapitation, the hearts were rapidly removed from the rat and mounted to the Langendorff apparatus for perfusion. Perfusion was performed in a retrograde manner at a constant flow rate (10 ml/min) with oxygenated Joklik modified Eagle's medium supplemented with 1.25 mM CaCl2 and 10 mM HEPES, pH 7.2, at 37°C for 5 min. This was followed by 5 min with the same medium free of Ca2+. Collagenase was then added to the medium to a concentration of 125 U/ml with 0.1% (w/v) BSA. After 35 to 45 min of perfusion with a medium containing collagenase, the atria were discarded. The ventricular tissue was shaken in the same oxygenated collagenase solution for 5 min at 37°C and cut into small pieces with a pair of scissors after stirring with a glass rod for 5 min. The procedure separated the ventricular myocytes from each other. The residue was filtered through 250-μm mesh screens, sedimented by centrifugation at 100g for 1 min and resuspended in fresh Joklik solution with 2% BSA. More than 70% of the cells were rod-shaped and impermeable to trypan blue. Ca2+concentration of the Joklik solution was increased gradually to 1.25 mM in 30 min.
Assay of cAMP.
Samples containing 3×106 to 6×106 freshly isolated ventricular myocytes after Ca2+ loading were incubated in an atmosphere of 5% CO2/95% air for 2 h. One hour and 45 min after the addition of specific inhibitors chelerythrine and bisindolylmaleimide I (BSM), respectively, U50,488H was added and incubated for 10 min followed by addition of forskolin for another 5 min. In experiments for determining the role of PDE, the PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX), 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX),erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), cilostamide, Ro-20–1724, rolipram, and zaprinast were added 15 min before U50,488H, respectively. At the end of treatment, the cells were centrifuged for 5 s at 100g. The medium was aspirated, the sediment was resuspended in ice-cold Krebs solution, and centrifuged again for 5 s at 100g. The supernatant was aspirated. Ice-cold ethanol/HCl (1 ml) was added, mixed, and left to stand for 5 min at room temperature. The supernatant was centrifuged at 3000gfor 5 min and collected with a pipette. The precipitate was washed with 1 ml of ethanol/water (2:1), mixed, and centrifuged at 3000gfor 5 min. The supernatant was evaporated to dryness at 55°C under a stream of nitrogen. The sediment was stored at −20°C for assay of cAMP. The pellets were neutralized in 0.1 N NaOH for protein determination by the method of Lowry et al. (1951), with BSA as a standard.
For determination of cAMP, a competitive binding assay with a kit from Amersham Corp. (Amersham, UK) was used. Briefly, 50 μl of 0.5 M Tris (hydroxymethyl) aminomethane (4 mM EDTA) was added to 50 μl of each sample on ice, followed by 50 μl of [3H]cAMP and 100 μl of binding protein. The samples were vortexed for 5 s and placed in an ice bath where they were incubated for 2 h. A charcoal suspension of 100 μl was added. The samples were vortexed again for 10 s and centrifuged at 12,000g for 2 min at 4°C. Samples of 200 μl of supernatant were used for scintillation counting.
Assay of PDE Activity.
Cells received the same pharmacological treatments as described in the measurement of cAMP. U50,488H at 1 × 10−5, 2 × 10−5, 4 × 10−5 M, and PMA at 10−8, 10−7, and 10−6 M were given, respectively. It has recently been shown that rat left ventricle possesses only the soluble form of cAMP-specific PDE (Weishaar et al., 1987), therefore, enzyme activity was determined in the soluble cytosolic fraction of myocytes. The enzyme preparation consisted of the soluble cytosolic components of a suspension of myocytes after lysis of the sarcolemmal membrane with digitonin (62 μg/mg cell protein). It has been previously demonstrated that >95% of cellular lactate dehydrogenase is released from cardiac myocytes after treatment with this detergent (Murphy et al., 1982). The soluble cellular contents containing the crude enzyme were separated from the particulate material by centrifugation at 13,000 rpm. The supernatant was collected and used directly in the PDE assay without further purification of the enzyme.
PDE activity was measured at a substrate concentration of 0.25 μM cAMP according to methods of Boudreau and Drummond (1975) and Thompson and Appleman (1971). The assay consists of a two-step isotopic procedure. [3H]cAMP is hydrolyzed to [3H]5′-AMP by phosphodiesterase and then further hydrolyzed to [3H]adenosine by the nucleotidase. The reaction was initiated by adding 50 μl of the crude enzyme to 200 μl of reaction mixture containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.25 μM cAMP, and [3H]cAMP. The reaction was carried out at 30°C for 10 min and terminated by placing the tubes in a dry ice-ethanol bath. The tubes were then placed in a boiling water bath for 3 min followed by chilling in ice. Following reequilibration to 30°C, 50 μl of a 1.0-mg/ml solution of 5′-nucleotidase in H2O was added. After a 20-min incubation, the reaction was terminated by the addition of 1.0 ml of the resin slurry (3 H2O:1 resin). The resin binds all charged nucleotides and leaves [3H]adenosine as the only labeled compound to be counted. The tubes were allowed to stand at least 10 min, and centrifuged at 100g for 10 min. Then 0.5 ml was removed for scintillation counting.
Drugs and Chemicals.
U50,488H, PMA, type I collagenase, and forskolin were purchased from Sigma Chemical Co.(St. Louis, MO); EHNA from Research Biochemicals International (Natick, MA); and chelerythrine chloride, BSM, IBMX, MIBMX, cilostamide, Ro-20–1724, rolipram, and zaprinast from Calbiochem (La Jolla, CA). [3H]cAMP assay system and [8-3H]cAMP (250 μCi) were from Amersham Corp. PMA, chelerythrine chloride, BSM, IBMX, MIBMX, EHNA, cilostamide, Ro-20–1724, rolipram, zaprinast, and forskolin were dissolved in dimethyl sulfoxide (DMSO) and the rest in distilled water. The final concentration of DMSO was 0.1%, at which DMSO had no effect on cAMP level and PDE activity.
The doses of U50,488H used were 10−5 to 4 × 10−5 M based on previous studies in our (Sheng et al., 1997; Bian et al., 1998; Zhang and Wong, 1998) and other laboratories (Ventura et al., 1991). At this dose range U50,488H increases cytosolic Ca2+ and decreases forskolin-induced cAMP accumulation, and the effects are blocked by nor-binaltorphimine (nor-BNI), indicating the actions are κ-opioid receptor-mediated. The dose of PMA used also was based on previous studies (Ventura et al., 1991; Bian et al., 1998). The concentration of the two PKC antagonists chelerythrine (Kandasamy et al., 1995) and BSM (Chulak et al., 1995; Zhu et al., 1997; Berts et al., 1999) used in this study was 1 μM based on previous studies. The concentrations of PDE antagonists MIBMX (Shimizu et al., 1999), EHNA (Mery et al., 1995), cilostamide (Schwartz et al. 1993), Ro-20–1724 (Hichami et al., 1996), and zaprinast (Liu et al., 1992) used were 10−5, 10−5, 10−6, 10−5, and 10−6 M, respectively, which also were chosen according to previous studies. With the exception of BSM, which has been shown to produce selective actions at the dose range of 10−8 to ∼5 × 10−5 (Toullec et al., 1991; Weissmann et al., 1999), the concentrations of PKC and PDE inhibitors used were slightly higher than the corresponding IC50 values provided by the respective drug firms. For IBMX and rolipram, the dose ranges chosen for dose response studies also were based on previous studies (Shahid and Rodger, 1989; Turner et al., 1993).
Statistical Analysis.
One-way ANOVA was used to determine the difference among groups. Student's t test was used to compare the difference between two groups. Significance level was set at P < .05
Results
Effects of U50,488H and PMA on Forskolin-Induced cAMP Accumulation in Presence and Absence of Specific PKC Inhibitors Chelerythrine and BSM.
In agreement with well established observations (Martinez et al., 1995; Zhang and Wong, 1998), 10−5 M forskolin, an activator of adenylate cyclase (AC), increased cAMP level significantly after 5 min of incubation (Fig.1). Pretreatment with 2 × 10−5 M U50,488H and 10−6M PMA decreased the forskolin-stimulated cAMP accumulation significantly. The effects of U50,488H and PMA were significantly attenuated by 10−6 M chelerythrine and 10−6 M BSM, which themselves had no effect on cAMP (Fig. 1). This is in agreement with our previous study that showed that the inhibitory effect of U50,488H and PMA were dose-dependently attenuated by the nonspecific PKC inhibitor staurosporine (Bian et al., 1998).
Effects of U50,488H and PMA on Forskolin-Induced cAMP Accumulation and PDE Activity in Presence of PDE Inhibitor IBMX.
To determine whether PKC acted at PDE upon κ-OR stimulation, the effects of U50,488H and PMA on the forskolin-induced accumulation of cAMP were studied in the presence of IBMX, a nonspecific PDE inhibitor. IBMX at 10−6 M did not further increase the forskolin-induced increase in cAMP accumulation (Fig.2), indicating that at this concentration it nearly has no effect on PDE. Nor did 10−6 M IBMX alter the effect of either U50,488H or PMA on cAMP (Fig. 2). At 10−5 to ∼10−3 M, IBMX dose-dependently enhanced forskolin-induced cAMP accumulation, indicating that at this dose range IBMX is effective in inhibiting the action of PDE. At this dose range, IBMX abolished completely the inhibitory effect of 2 × 10−5 M U50,488H and 10−6 M PMA (Fig. 2).
In agreement with the inhibitory effects of U50,488H and PMA on forskolin-stimulated cAMP accumulation, U50,488H at 1 × 10−5 to ∼4 × 10−5M (Fig. 3A) and PMA at 10−8 to ∼10−6 M (Fig.3B) dose-dependently stimulated PDE activity, and these effects were abolished by 10−6 M chelerythrine and 10−3 M IBMX (Fig. 3).
Effects of U50,488H and PMA on Forskolin-Induced cAMP Accumulation and PDE Activity in Presence of PDE Isoform Inhibitors.
To screen the isozyme(s) of PDE involved in the κ-OR stimulation, 10−5 M MIBMX, a selective PDE-I inhibitor, 10−5 M EHNA, a selective PDE-II inhibitor, 10−6 M cilostamide, a selective PDE-III inhibitor, 10−5 M Ro-20–1724, a specific PDE-IV inhibitor, and 10−6 M zaprinast, a specific PDE-V inhibitor, were used. We found that 10−5 M Ro-20–1724 abolished, whereas inhibitors of other PDE isoforms did not alter, the inhibitory effect of 2 × 10−5 M U50,488H and 10−6 M PMA on cAMP accumulation (Fig. 4).
To further confirm the role of PDE-IV, rolipram, another selective PDE-IV inhibitor, was used. Rolipram at 10−7 M did not further enhance the forskolin-induced cAMP accumulation nor did it affect the effect of U50,488H or PMA. At 10−6to ∼10−4 M, rolipram dose-dependently attenuated the effects of 2 × 10-5 M U50,488H and 10−6 M PMA on forskolin-induced cAMP accumulation (Fig. 5A) and PDE activity (Fig. 5B).
Discussion
The most important observations in this study are 1) κ-OR stimulation and activation of PKC decrease forskolin-induced cAMP accumulation and stimulate PDE activity, and the effects are antagonized by inhibition of PKC or PDE; and 2) inhibition of PDE-IV with its selective inhibitors Ro-20–1724 and rolipram significantly attenuates the effects of κ-OR stimulation and activation of PKC on cAMP and PDE activities. These observations demonstrate for the first time that PDE-IV mediates the action of PKC on cAMP modulation upon κ-OR stimulation in the rat heart.
Our previous study has shown that a nonspecific PKC inhibitor staurosporine significantly attenuates the suppression of cAMP by κ-OR stimulation (Bian et al., 1998). Because staurosporine has been reported to inhibit not only PKC but also tyrosine kinase, cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Nakano et al., 1987; Yanagihara et al., 1991), the role of PKC in mediating the action of κ-receptor stimulation on cAMP accumulation needs confirmation. In this study, we found that two specific PKC inhibitors, 10−6 M chelerythrine and BSM, significantly attenuated the inhibitory effects of U50,488H and PMA on forskolin-stimulated cAMP accumulation. Chelerythrine (Kandasamy et al., 1995) and BSM (Toullec et al., 1991; Weissmann et al., 1999) at this concentration have been shown to selectively inhibit PKC activity. The result confirms our previous finding that the inhibitory effect of κ-OR stimulation on cAMP accumulation results at least partly from activation of PKC (Bian et al., 1998).
In this study, we found that the effects of U50,488H and PMA on cAMP accumulation and PDE activity were completely abolished by IBMX. These results indicate that PKC activates PDE upon κ-OR stimulation in the heart. Similar observations have been reported when the heart was stimulated by the parathyroid hormone (Schluter et al., 1995) and an α-adrenergic receptor agonist (Buxton and Brunton, 1985). In the neuroblastoma × glioma NG108–15 cell,d-la2,d-Leu5-enkephalin, a δ-OR agonist, also inhibits cAMP accumulation via activation of PDE (Law and Loh, 1993). The isomer of PDE involved is PDE-IV because we found that the effects of U50,488H and PMA were significantly attenuated by Ro-20–1724 and rolipram, two specific PDE-IV inhibitors, but not by MIBMX, EHNA, cilostamide, and zaprinast, selective inhibitors of PDE-I, PDE-II inhibitor, PDE-III, and PDE-V, respectively. The results indicate clearly that PDE-IV is the isoenzyme that mediates the actions of PKC upon κ-OR stimulation in the heart. The finding is in agreement with the previous observation that activation of phospholipase C, known to convert phosphatidylinositol 4,5-biphosphate into inositol trisphosphate and diacylglycerol, which activates PKC, induces a marked stimulation of PDE-IV in the rat heart microsome (Prigent et al., 1984; Dubois et al., 1990). So, in the heart PKC activates PDE-IV. This is similar to the situation in renal collecting tubule (Tetsuka et al., 1995).
In this study, we found that IBMX at 10−5 to ∼10−3 M, but not at 10−6 M, further increased the forskolin-induced cAMP accumulation, indicating that only 10−5 to ∼10−3 M IBMX significantly inhibited the effect of PDE. This is in agreement with the previous observations ofTurner et al. (1993). Shahid and Rodger (1989) also found that IBMX at this concentration inhibits cAMP hydrolyzing in the rabbit heart. Similarly, rolipram at 10−6 to ∼10−4 M, but not at 10−7 M, increased the forskolin-induced cAMP accumulation, indicating that only 10− 6 to ∼10−4 M rolipram inhibited the effect of PDE in agreement with previous findings (Lerner et al., 1986; Turner et al., 1993).
Previous study has found that a positive feedback relationship exists between the synthesis of κ-opioid peptides and κ-OR stimulation in the heart (Ventura and Pintus, 1997). The observations suggest that a large amount of κ-opioid peptides may be produced during myocardial ischemia/reperfusion. So, the high concentrations of κ-OR peptides are not necessarily unreal in our body. In these studies, the effects of the κ-OR agonist at as high as 5 × 10−5 M are blocked by the selective κ-OR antagonist nor-BNI, indicating that the effects are κ-OR-mediated (Ventura et al., 1991; Sheng et al., 1997; Bian et al., 1998; Zhang and Wong, 1998).
Two pathways have been shown to mediate κ-OR stimulation on cAMP accumulation. A previous study in our laboratory has shown that activation of the phosphoinositol/Ca2+ pathway by κ-receptor stimulation with U50,488H leads to inhibition of cAMP in the heart (Zhang and Wong, 1998). This study has clearly provided unequivocal evidence that PKC/PDE pathway also mediates the inhibitory action of κ-OR stimulation on cAMP. In addition to PDE, PKC also has been shown to stimulate AC in the neonatal and adult cardiac myocytes (Strasser et al., 1992; Reupcke et al., 1993). Further study is needed to delineate the role of AC in mediating the action of PKC upon κ-receptor stimulation in the heart. In conclusion, this study has provided evidence for the first time that upon κ-OR stimulation PKC activates PDE-IV, leading eventually to a reduction in cAMP accumulation in the heart.
Acknowledgments
We thank C. P. Mok for technical assistance. The study was performed when J.S.B. and W.M.Z., respectively, were on leave from Nanjing Medical University and Liu Hua Qiao Hospital, Guangzhou, China.
Footnotes
-
Send reprint requests to: T. M. Wong, Ph.D., Department of Physiology, The University of Hong Kong, Li Shu Fan Building, Sassoon Rd., Hong Kong, China. E-mail: wongtakm{at}hkucc.hku.hk
-
↵1 This study was supported by a grant from the Research Grant Council, Hong Kong.
- Abbreviations:
- OR
- opioid receptor
- PKC
- protein kinase C
- U50,488H
- trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate
- PMA
- phorbol-12-myristate-13-acetate
- PDE
- phosphodiesterase
- PDE-IV
- cAMP-specific PDE
- PDE-I
- Ca2+/calmodulin-dependent PDE
- PDE-II
- cGMP-stimulated PDE
- PDE-III
- cGMP-inhibited PDE
- PDE-V
- cGMP-specific PDE
- BSM
- bisindolylmaleimide I
- MIBMX
- 8-methoxymethyl-3-isobutyl-1-methylxanthine
- EHNA
- erythro-9-(2-hydroxy-3-nonyl) adenine
- DMSO
- dimethyl sulfoxide
- nor-BNI
- nor-binaltorphimine
- AC
- adenylate cyclase
- Received June 22, 1999.
- Accepted November 3, 1999.
- The American Society for Pharmacology and Experimental Therapeutics