Abstract
The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of κ receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The κ receptor agonist, (±)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective κ receptor antagonist, norbinaltorphimine (10−6M), but the effect of PDBu was not, thus supporting the involvement of κ-opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10−5 M), the inhibitory effect of BRL or PDBu (10−6 M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10−6 M), the inhibitory effect of PDBu or BRL (10−6 M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10−6 M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10−5 M) or chelerythrine (10−6 M) caused significant reversal of both BRL- and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby κ-opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of κ-opioid agonists to suppress aqueous flow rate and to lower intraocular pressure.
Opioid receptors are among the numerous G-protein-coupled receptors which regulate the production of the second messenger, cAMP. It is well documented that alterations in the levels of cAMP can modulate the production of aqueous humor by the ciliary epithelium, thereby affecting intraocular pressure (Korenfeld and Becker, 1989; Wax, 1992). Opioid receptors linked to inhibitory G-proteins reduce levels of cAMP via direct inhibition of the activity of adenylate cyclase, the enzyme responsible for the production of cAMP. Another mode of lowering cAMP levels is by increasing the activity of a family of degradative enzymes, phosphodiesterases (PDEs).
There are at least 11 distinct families of PDE isozymes (Uckert et al., 2001), which can be differentiated by their physical properties, substrate specificity (cAMP versus cGMP) and affinity, regulation by physiological modulators (e.g., calmodulin or cGMP), and sensitivity to selective inhibitors. The presence of distinct profiles of PDE isozymes has been demonstrated in ocular ciliary epithelium (Bode et al., 1993). Because PDE4 hydrolyzes cAMP preferentially, the PDE4-selective antagonist, rolipram, was used in this study as well as the nonspecific PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX).
Recent studies in our laboratory have demonstrated the ability of κ-opioid agonists to lower intraocular pressure in rabbits (Russell et al., 2000) as well as reduce levels of cAMP in isolated iris-ciliary bodies (Moore and Potter, 2001). However, the signal transduction events associated with κ agonist-induced ocular hypotension have not been delineated. Recently, other investigators have provided evidence that PKC and PDE4 mediate the response to κ-opioid receptor activation in isolated ventricular myocytes (Bian et al., 1999). In addition, other studies have demonstrated the involvement of PKC in mediating the actions of κ receptor stimulation on Ca2+ homeostasis in cardiac myocytes (Bian et al., 1998). In this regard, indirect evidence of the involvement of PKC in κ agonist-induced increases in the levels of inositol phosphates in rabbit isolated ICBs has been demonstrated recently in this laboratory (Carnes and Potter, 2000). The goal of the present study was to determine the possible roles of PKC and PDE in κ agonist-induced inhibition of cAMP accumulation in the rabbit isolated ICB. Information from this study should help in delineating the mechanism of κ-opioid agonist-induced ocular hypotension.
Materials and Methods
Isolation of ICBs and Sample Preparation for cAMP Assay.
Iris-ciliary bodies (ICBs) from the eyes of New Zealand White rabbits were removed, and each was cut into six equal pieces. The tissue samples were then placed in Earle's balanced salt solution (EBSS) containing 2% indomethacin (included for the inhibition of prostaglandin synthesis) for 30 min. Control samples were treated with EBSS. Others were treated with either forskolin (Forsk) alone, PDBu and Forsk, or (±)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL) and Forsk. All treatments were done in duplicate. To examine the involvement of κ receptors in the response to BRL, the relatively selective κ-opioid receptor antagonist, nor-BNI, was preincubated for 30 min before addition of BRL and PDBu. In experiments examining the effect of PDE inhibition, IBMX or rolipram was added 15 min before the addition of BRL or PDBu. In the described experiments, the PKC antagonist, chelerythrine (Chel), was allowed to incubate for 1 h before addition of agonists. After completion of the treatment regimens, the tissue samples were rapidly frozen in liquid nitrogen. Frozen tissue samples were homogenized in 10% trichloroacetic acid at 2–8°C, followed by centrifugation at 2000g for 15 min at 4°C. Pellets were neutralized in 1 N NaOH for protein determination by the Bio-Rad method, with bovine serum albumin as standard. The supernatant was recovered and washed four times with 5 volumes of water-saturated diethyl ether. The remaining aqueous extract was dried and frozen at −80°C until assays for cAMP content were performed as described below.
Assay of cAMP.
For determination of cAMP levels in tissue samples, an enzyme immunoassay kit from Amersham Biosciences (Piscataway, NJ) was used. Briefly, antibodies to cAMP were added to each sample and allowed to incubate at 3–5°C for 2 h. Next, samples were treated with cAMP-peroxidase conjugate and allowed to incubate for 60 min, followed by washing. Immediately after the final wash, enzyme substrate was added and samples were incubated at room temperature for 60 min. Reactions were terminated with 100 μl of 1 M H2SO4 and optical densities were determined in a plate reader at 450 nm.
Sample Preparation for Assay of PDE Activity.
Tissue treatment was carried out under the same conditions as described for the measurement of cAMP. Tissue samples were incubated with BRL or PDBu (10−6 M) for 10 min. Control samples received EBSS. To examine the role of PKC and PDE4 in BRL- and PDBu-mediated effects on PDE activity, some tissue samples were treated with Chel (10−6 M) or rolipram (10−5 M) for 1 h or 15 min, respectively, prior to the addition of BRL or PDBu. After treatment, samples were immediately frozen in liquid nitrogen and stored at −80°C until assayed for PDE activity as described below.
Assay of PDE Activity.
Frozen tissue samples were homogenized in 300 μl of cold 20 mM Tris-HCl/10 mM MgCl2 (pH 7.4) protease inhibitor buffer. The homogenate was centrifuged at 13,000 rpm for 1 h at 4°C. The assay used to measure PDE activity is a modification of that described by Thompson and Appleman (1971) and Marchmont and Houslay (1980). The assay consists of a two-step isotopic procedure. In the first step, cyclic [3H]AMP is hydrolyzed to 5′-[3H]AMP by PDE. In the second step, 5′-[3H]AMP is further hydrolyzed to [3H]adenosine by the snake venom nucleotidase. The reaction was initiated by adding 25 μl of the sample containing the crude enzyme to 50 μl of the reaction mixture containing 20 mM Tris-HCl/10 mM MgCl2 buffer (pH 7.4), 0.1 mM cAMP, cyclic [3H]AMP (∼ 1–2 × 105 cpm), and 25 μl of 20 mM Tris-HCl, for a total assay volume of 100 μl. The reaction was carried out at 30°C for 10 min and terminated by immediate boiling for 2 min followed by cooling on ice. Next, 25 μl of 1 mg/ml snake venom (Crotalus atrox) was added to all samples and incubated for 10 min at 30°C. After returning samples to ice, the reaction was terminated by the addition of 400 μl of anion exchange resin slurry (2 H2O/ethanol:1 resin). The resin binds all charged nucleotides and leaves [3H]adenosine as the only labeled compound to be counted. Following addition of resin, samples were vortexed and left on ice for at least 15 min. After the 15 min on ice, the samples were vortexed again, followed by centrifugation at 13,000 rpm for 2 min. A 150-μl aliquot of the supernatant from samples was removed for scintillation counting.
Drugs and Chemicals.
BRL, Forsk, and rolipram were purchased from Tocris Cookson (Ballwin, MO); Chel, IBMX, PDBu, indomethacin,Crotalus atrox (snake venom), Tris-HCl, and the protease inhibitor cocktail were obtained from Sigma Chemical Co (St. Louis, MO); and the resin (AG1-8X) was from Bio-Rad (Hercules, CA). Forsk, Chel, rolipram, and IBMX were dissolved in dimethyl sulfoxide and then diluted appropriately with EBSS. Unless otherwise indicated, all other agents were dissolved in distilled H2O.
Statistical Analysis.
A one-way analysis of variance followed by Tukey's post hoc test was used to determine the difference among groups. P < 0.05 was considered significant.
Results
Concentration Response Effects of BRL and PDBu on cAMP Accumulation in Isolated Iris-Ciliary Bodies.
Basal levels of cAMP in ICBs were 2.59 ± 0.38 pmol/mg protein. In the presence of the adenylate cyclase activator, Forsk (10−5 M), levels of cAMP were increased ∼5-fold (Fig. 1). The κ-opioid receptor agonist BRL and the PKC agonist PDBu (10−7–10−5 M) produced concentration-dependent inhibition of Forsk-stimulated cAMP accumulation in the rabbit ICB (Fig. 1). Exposure of tissue samples to BRL or PDBu alone produced no significant change in the levels of cAMP (Table 1).
Effects of the Selective κ-Opioid Receptor Antagonist, Nor-BNI, on BRL- and PDBu-Induced Inhibition of Forskolin-Stimulated Accumulation of cAMP.
Preincubation of samples with the κ-opioid antagonist, nor-BNI (10−7 and 10−6 M) (Fig. 2) produced a concentration-dependent antagonism of BRL-induced inhibition of Forsk-stimulated cAMP accumulation, indicating that the response was mediated via κ-opioid receptors. On the other hand, the effects of PDBu were not significantly altered by 10−6 M nor-BNI (Fig. 2). Treatment of tissues with nor-BNI alone had no significant effect on cAMP levels (Table2).
Effects of BRL and PDBu on Forskolin-Stimulated cAMP Accumulation in the Presence and Absence of the Specific PKC Inhibitor, Chel.
To determine whether PKC was involved in the action of κ receptor stimulation on cAMP production, the effects of BRL and PDBu in the presence and absence of Chel were examined. The effects of BRL and PDBu (10−6 M) were significantly attenuated by Chel (10−6 M) (Fig. 3). Chelerythrine alone had no significant effect on cAMP levels (Table 2).
Effects of BRL and PDBu on Forskolin-Stimulated cAMP Accumulation in the Presence and Absence of the Nonselective PDE Inhibitor IBMX and the PDE4 Inhibitor Rolipram.
In these experiments, basal levels of cAMP were 2.80 ± 0.46 pmol/mg protein. As in previous experiments, Forsk elevated cAMP levels approximately 5-fold. As shown in Fig. 4, the nonselective PDE inhibitor, IBMX (10−5 M), reversed the inhibitory effect of BRL on Forsk-stimulated cAMP accumulation as well as that of PDBu. Preincubation of samples with the selective PDE4 inhibitor rolipram (10−5 M) also produced significant antagonism of BRL- and PDBu-induced inhibition of Forsk-stimulated cAMP levels (Fig. 5). Exposure of tissue samples to the PDE inhibitors alone had no significant effect on the levels of cAMP compared with basal levels. (Table 2).
Effects of BRL and PDBu on PDE Activity in the Absence and Presence of Rolipram or Chel.
Incubation of tissue samples with BRL or PDBu (10−6 M) caused a significant increase in PDE activity from control values (Fig. 6). Preincubation with rolipram (10−5 M) or Chel (10−6 M) attenuated both BRL- and PDBu-induced increases in PDE activity (Fig. 6).
Discussion
The ciliary epithelium consists of a bilayer containing an outer layer of nonpigmented cells and in inner layer of pigmented cells, and is the site of aqueous humor production (Krupin et al., 1986). It has been postulated that alterations in levels of cAMP can modulate the production of aqueous humor by the ciliary epithelium of the eye, thereby affecting intraocular pressure (Neufeld, 1984; Sears, 1985). Alteration of ocular hydrodynamics by cAMP levels of tissues in the anterior segment suggests the importance of this nucleotide in regulating aqueous humor production (Korenfeld and Becker, 1989; Wax, 1992). Other investigators have found that the ocular hypotensive effects of drugs acting on adrenergic and adenosine receptors in the ciliary process are associated with their inhibitory action on adenylyl cyclase (Cepelik et al., 1997). However, the concentration of cAMP in cells is determined ultimately by the relative rates of activity of the synthetic isozymes of adenylate cyclase and the degradative isozymes of PDE. Because it has been established in our laboratory that κ-opioid agonists suppress aqueous flow rate and intraocular pressure, in part, by decreasing levels of cAMP in the ICB (Russell et al., 2000; Moore and Potter, 2001), the purpose of this study was to determine the roles of PKC and PDE in κ-opioid agonist-induced reductions in cAMP levels.
Other studies in our laboratory have demonstrated the interaction of κ-opioid agonists with the PLC/inositol triphosphate signaling cascade in the ciliary body (Carnes and Potter, 2000). This signal transduction cascade can generate diacylglycerol (DAG), a breakdown product of phosphatidylinositol bisphosphate under the action of PLC. Once DAG is formed, it then activates PKC, which has been postulated to be involved in the inhibitory effect of κ agonists on cAMP production (Ventura et al., 1991; Bian et al., 1998).
The present study has demonstrated significant roles for PKC and PDE4 in κ-opioid receptor-mediated decreases in cAMP accumulation in isolated iris-ciliary bodies. Similar to the effect of the κ receptor agonist, BRL, the PKC activator, PDBu, caused a concentration-dependent inhibition of forskolin-stimulated accumulation of cAMP. Preincubation with the selective PKC antagonist, Chel, caused significant inhibition of the response to both agents, suggesting a role of PKC in mediating their effect on cAMP accumulation. Similarly, these results agree with those of other investigators who reported that two specific PKC inhibitors, chelerythrine and bisindolylmaleimide, significantly attenuated the inhibitory effects of the κ-opioid receptor agonist, U50488H, on forskolin-stimulated cAMP accumulation in rat ventricular myocytes (Bian et al., 1999).
To determine whether PDE activation was involved in κ-opioid receptor-mediated inhibition of cAMP accumulation in the ICB, the nonselective PDE inhibitor, IBMX, and the PDE4-selective inhibitor, rolipram, were utilized. It is important to note that many investigators routinely use IBMX in their studies examining the effects of cAMP in various systems. However, in doing this, the activity of PDE in regulating cAMP levels is masked. Investigators should therefore study cAMP modulation in the absence and presence of PDE inhibition. As shown by the results presented in this study, both PDE inhibitors reversed BRL- and PDBu-induced inhibition of Forsk-stimulated cAMP production, indicating a role of PDE in their responses. Direct evidence of the involvement of PKC and PDE4 in κ agonist-induced reductions in cAMP levels in the ICB was generated in experiments showing the effect of BRL and PDBu on PDE activity in the absence and presence of the PKC inhibitor, Chel, or the PDE4 antagonist, rolipram. Results demonstrate the ability of both Chel and rolipram to significantly reduce BRL- and PDBu-induced augmentation of PDE activity in the rabbit ICB, thus firmly establishing the involvement of PKC and PDE4 in reducing levels of cAMP upon activation of κ-opioid receptors in this preparation. The proposed signaling events leading to reduced levels of cAMP upon κ-opioid receptor activation in the ICB are postulated to involve activation of PKC leading to increased PDE activity, specifically PDE4, and an ultimate reduction in cAMP levels.
The detailed signaling events between PKC activation and increased PDE activity have not been explored in the current study. However, it is known that PDE4 contains phosphorylation sites for various protein kinases in its N-terminal domain (Thompson, 1991). Other investigators have demonstrated the ability of PKC to phosphorylate PDE in vascular smooth muscle cells (Liu et al., 2000), hamster hearts (Yu et al., 1996), and bovine rod outer segments (Udovichenko et al., 1994). Furthermore, other studies have demonstrated the ability of PKC and PDE4 to mediate the effect κ-opioid receptor-stimulated decreases in cAMP levels in cardiomyocytes (Bian et al., 1999). Bian et al. (1999)found that upon stimulation of the κ-opioid receptor, PKC activates PDE4, which leads to a reduction in cAMP accumulation in the heart. The present results have shown that similar κ receptor-mediated events occur in the ICB of the rabbit.
There are two possible explanations for the demonstrated effect of κ-opioids on cAMP accumulation. A previous study in our laboratory has shown that activation of the PLC/inositol triphosphate pathway by κ-opioid receptor stimulation leads to the production of inositol phosphates. This same pathway generates DAG, which is known to activate PKC. An alternative mechanism that may participate involves the release of βγ subunits from the Gi protein linked to κ-opioid receptors. Free βγ subunits could activate PLC, thereby generating the production of DAG, which is followed by activation of PKC. The remaining signaling events following PKC activation are postulated to involve stimulation of PDE, which leads to decreased levels of cAMP.
The most salient observations derived from the present study are: 1) PDBu, a PKC agonist, mimicked κ-opioid receptor stimulation by decreasing Forsk-stimulated accumulation of cAMP in the isolated ICB; 2) the PDE antagonists, IBMX (nonselective) and rolipram (PDE4-selective), as well as the PKC antagonist, Chel, attenuated the effect of PKC activation and κ-opioid receptor stimulation on cAMP accumulation; and 3) BRL and PDBu both induced an increase in the activity of PDE, which was antagonized by rolipram and Chel, clearly indicating a direct association between κ-opioid receptor activation and stimulation of PDE activity in the rabbit ICB. These observations suggest that the effect of κ-opioid receptor stimulation is mediated, at least in part, via the increased activities of PKC and PDE4. In addition, the selective κ-opioid receptor antagonist, nor-BNI, attenuated BRL-stimulated inhibition of cAMP levels but not that mediated by PDBu, indicating the specificity of the κ receptor-mediated response.
In conclusion, this study has demonstrated a role of PKC and PDE in κ-opioid agonist-induced reductions in cAMP levels in the isolated ICB of the rabbit.
The current study provides evidence of an additional tissue in which increased PDE activity plays a significant role in cAMP homeostasis. In view of the fact that most studies examining cAMP regulation include IBMX to inhibit PDE activity, a study such as this one is important because it brings to the forefront an additional, often overshadowed means of regulating levels of this second messenger. Furthermore, this study extends the work of others who demonstrated the presence of a variety of PDEs in an ocular cell type, trabecular meshwork cells (Zhou et al., 2000), and provided evidence for a role of PDE5 in regulating cGMP homeostasis in this cell type. Additional studies are being conducted to elucidate other avenues of cross-talk among signal transduction pathways in ocular tissues that are linked to κ-opioid receptors. One such study involves examining the mechanism whereby PDE is regulated by protein kinases.
Acknowledgments
Mention of brand names does not constitute product endorsement.
Footnotes
-
This work was supported by National Eye Institute Grant EY11977.
- Abbreviations:
- PDE
- phosphodiesterase
- IBMX
- 3-isobutyl-1-methylxanthine
- ICB
- iris-ciliary body
- EBSS
- Earle's balanced salt solution
- PDBu
- phorbol 12,13-dibutyrate
- nor-BNI
- norbinaltorphimine
- Chel
- chelerythrine
- Forsk
- forskolin
- PKC
- protein kinase C
- PLC
- phospholipase C
- DAG
- diacylglycerol
- Received August 29, 2001.
- Accepted January 17, 2002.
- The American Society for Pharmacology and Experimental Therapeutics