Abstract
Although multiple adenosine receptors have been identified, the subtype and underlying mechanisms involved in the relaxation response to adenosine in the urinary bladder remain unclear. The present study investigates changes in the membrane potential, as assessed by fluorescence-based techniques, of bladder smooth muscle cells by adenosine receptor agonists acting via ATP-sensitive potassium (KATP) channels. Membrane hyperpolarization evoked by adenosine and various adenosine receptor subtype-selective agonists was attenuated or reversed by the KATP channel blocker glyburide. Comparison of adenosine receptor agonist potencies eliciting membrane potential effects showed a rank order of potency 5′-N-ethyl-carboxamido adenosine (NECA; −log EC50 = 7.97) ∼ 2-p-(2-carboxethyl)phenethyl-amino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680; 7.65) > 2-chloro adenosine (5.90) ∼ 2-chloro-N6-cyclopentyladenosine (CCPA; 5.51) ∼N6-cyclopentyladenosine ∼N6-(R)-phenylisopropyladenosine > 2-chloro- N6-(3-iodobenzyl)-adenosine-5′-N-methyl-carboxamide (2Cl-IBMECA; 4.78). Membrane potential responses were mimicked by forskolin, a known activator of adenylate cyclase, and papaverine, a phosphodiesterase inhibitor. The A2A-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), and the adenylate cyclase inhibitorN-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A) inhibited the observed change in membrane potential evoked by adenosine and adenosine-receptor agonists. The rank order potency for relaxation of K+-stimulated guinea pig bladder strips, NECA (−log EC50 = 6.41) ∼ CGS-21680 (6.38) > 2-chloro adenosine (5.90) ≫ CCPA ∼ 2Cl-IBMECA (>4.0) was comparable to that obtained from membrane potential measurements. Collectively, these studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A2A receptor-mediated activation of KATP channels via adenylate cyclase and elevation of cAMP.
Adenosine is an endogenous nucleoside that regulates many physiological functions in cardiovascular, respiratory, renal, immune, and central and peripheral nervous systems via G protein-coupled adenosine receptors (P1 receptors). Four receptor subtypes, A1, A2A, A2B, and A3, have been identified based on molecular structure, pharmacology, and mechanisms of G protein-mediated signaling mechanisms (for reviews, see Ralevic and Burnstock, 1998; Fredholm et al., 2000). The A1 receptors are coupled to the inhibition of adenylate cyclase and can act through effector pathways such as stimulation of phospholipase C, activation of potassium channels, and inhibition of N-type calcium channels. The A2 receptor subtypes, A2Aand A2B, are coupled to activation of adenylate cyclase, whereas A3 receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase (Abbracchio et al., 1995; Baraldi et al., 2000).
In the past decade, considerable effort has been invested in elucidating the physiological roles of the various adenosine receptors in a variety of tissues, including cardiac, vascular, and nonvascular tissues. Adenosine and adenosine receptors are thought to play a critical role in regulating urinary bladder function, especially in conditions of bladder ischemia or during obstruction-induced changes in function that underlie lower urinary tract symptoms (Levin et al., 2000). Although evidence for inhibitory P1-type purinergic receptor-mediated effects by adenosine has been reported in the bladder (Nicholls et al., 1992; King et al., 1997), the receptor subtype(s) and their coupling to smooth muscle relaxation events remain poorly understood. mRNA analysis by Dixon et al. (1996) has revealed the presence of subunits corresponding to all adenosine receptor subtypes in the rat bladder, whereas Northern analysis showed relatively higher expression of the A2B receptor subtype (Stehle et al., 1992). Based on the order of potency of adenosine agonists for inhibition of carbachol-evoked contractions in rat bladder, it was noted that the pharmacological profile was more consistent with the participation of adenosine A2 receptor subtypes (Nicholls et al., 1992, 1996). On the other hand, pharmacological analysis of smooth muscle cells isolated from the circular muscle layer of the feline bladder showed that the adenosine receptor subtype mediating contraction resembled that of the A1subtype in this species (Yang et al., 2000). In vascular smooth muscle, adenosine interacts with both A1 and A2 receptor subtypes to activate KATP channels leading to relaxation. In the rabbit mesenteric artery, A2 receptor subtypes are predominantly involved, whereas in pig coronary arteries, adenosine-evoked activation occurs via A1receptors (Quayle et al., 1997). However, it remains unclear which adenosine receptor subtype mediates functional responses in the bladder and whether functional coupling of these receptors to KATP channels might provide a mechanism underlying bladder smooth muscle relaxation.
In the present study, we have investigated the nature of the adenosine receptor subtype(s) involved in coupling to KATPchannels in the guinea pig urinary bladder, a widely used model for studying the physiology of bladder function (Fujii et al., 1990;Herrera et al., 2000). Our studies provide evidence that hyperpolarization of membrane potential via KATPchannel activation is a necessary component of adenosine receptor activation. Based on pharmacological analysis of membrane potential changes and tissue relaxation effects, it was found that adenosine-mediated relaxation of the guinea pig bladder involves the A2A receptor subtype, which appears to be linked to KATP channel activation by activation of adenylate cyclase.
Experimental Procedures
Materials.
Cell culture reagents were purchased from Invitrogen (Carlsbad, CA). The fluorescent imaging plate reader (FLIPR) membrane potential kit was purchased from Molecular Devices (product number R-8034; Sunnyvale, CA). Glyburide (glibenclamide), adenosine, adenosine deaminase, and other adenosine agonists and antagonists, forskolin, and papaverine were purchased from Sigma Chemical (St. Louis, MO). ZM-241385 and 2Cl-IBMECA were purchased from Tocris Cookson Inc. (Ballwin, MO).N-Cyano-N′-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) was synthesized in house. Compounds were prepared as 10 mM stocks in dimethyl sulfoxide and diluted in assay buffer just before use.
Cell Culture.
Urinary bladder smooth muscle cells were prepared as previously described (Gopalakrishnan et al., 1999;Whiteaker et al., 2001). Bladders were removed from anesthetized male guinea pigs (Hartley; Charles River Laboratories, Inc., Wilmington, MA) and cells were isolated by enzymatic digestion with collagenase (1 mg/ml) and pronase (0.2 mg/ml) at 37°C. The suspension was centrifuged at 1300g for 5 min and the cell pellet was resuspended in 5 ml of growth media (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 mg/ml amphotericin B). The suspension was then passed through a fine polypropylene mesh filter, seeded into 175-cm2 cell culture flasks, and maintained in a tissue culture incubator at 37°C under an atmosphere of 10% CO2. When confluent, cells were passed at least four times without loss of activity. For membrane potential studies, the cells were seeded into black 96-well clear-bottomed plates (VWR, West Chester, PA) at a density of 3 × 104cells/well.
Fluorescence Assays.
Assays were carried out using the FLIPR membrane potential dye in the FLIPR (Molecular Devices). A stock solution of the dye was prepared by the addition of 10 ml of phosphate-buffered saline (catalog number 14287-072; Invitrogen; composition 138 mM NaCl, 2.68 mM KCl, 0.9 mM CaCl2, 0.49 mM MgCl2, 8.9 mM Na2HPO4, 1.47 mM KH2PO4, 0.33 mM sodium pyruvate, and 5.56 mM d-glucose) to a vial of the lyophilized dye stock. The stock solution was further diluted 1:20 in phosphate-buffered saline before use. After media were removed from the cell plate, 180 μl of the diluted FLIPR membrane potential dye was added to the wells and incubated at room temperature for 60 min. Assays were carried out in the FLIPR in a total assay volume of 200 μl at an excitation wavelength of 480 nm by using a 550-nm longpass emission filter (catalog number 0310-4027; Molecular Devices). After fluorescence baseline readout for 1 min, 20 μl of test compounds, at a stock concentration 10-fold higher than the final desired concentration in the assay, was added. Changes in fluorescence were measured for 9 min. In cases where glyburide sensitivities were assessed, 20 μl of 50 μM glyburide was added at the end of the 9-min time point to yield a final concentration of 5 μM. Data were collected for an additional 5-min period.
Isolated Tissue Relaxation Studies.
Urinary bladders were removed from anesthetized male guinea pigs (Hartley; Charles River Laboratories, Inc.) weighing 250 to 300 g and immediately placed in Krebs-Ringer bicarbonate solution (composition 120 mM NaCl, 20 mM NaHCO3, 11 mM dextrose, 4.7 mM KCl, 2.5 mM CaCl2, mM 1.5 MgSO4, 1.2 mM KH2PO4, 0.01 mM K2EDTA, equilibrated with 5% CO2, 95% O2; pH 7.4 at 37°C). The trigonal and dome portions were discarded and strips 3 to 5 mm in width and 10 mm in length were prepared from the remaining tissue by cutting in a circular manner. One end of the strip was fixed to a stationary glass rod and the other to a Grass FT03 transducer at a basal preload of 1.0 g. This preload proved to be the best condition for a steady-state baseline and reproducible responses to K+ stimulation. As previously reported (Herrera et al., 2000; Buckner et al., 2002), the contractions evoked by mild depolarization (i.e., 20 mM K+) are largely myogenic in origin as revealed by the lack of sensitivity to the sodium channel blocker tetrodotoxin (0.1 μM). Tissues were allowed to equilibrate for at least 60 min before the assay. Concentration-response curves were generated in a noncumulative manner with a rinse between successive additions.
Data Analysis.
In FLIPR studies, the fluorescence responses were corrected for any background changes in the negative control wells, and data were normalized to those observed with 10 μM adenosine (100% assigned to maximum decrease in fluorescence units). In tissue bath assays, responses were measured as decreases in tension and expressed as percentage of tension evoked by 20 mM K+. Data are expressed as mean ± S.E.M. The EC50 values were calculated from the concentration-response curve generated by nonlinear regression analysis (GraphPad Prism; GraphPad Software, San Diego, CA). Significant differences between group means were assessed by analysis of variance followed by Student-Newman-Keuls test. Significance was accepted at theP < 0.05 level.
Results
Effect on Membrane Potential Responses in Bladder Smooth Muscle Cells
Adenosine evoked concentration-dependent decreases in fluorescence responses in guinea pig urinary bladder smooth muscle cells (Fig.1). Significant changes in membrane potential responses were noted at concentrations as low as 10 nM and maximal responses were obtained with 10 μM adenosine. The EC50 value was calculated to be 467 nM (−log EC50 = 6.33 ± 0.11; n = 9). Adenosine responses were reversed by the addition of 10 μM glyburide or attenuated by glyburide pretreatment (see below), suggesting that these effects could be mediated by activation of KATP channels. Glyburide alone did not alter the basal fluorescence response (data not shown). The maximal efficacy of adenosine (94.9 ± 2.5%) was comparable to that of a prototypical KATP channel opener such as the cyanoguanidine, P1075 under similar conditions.
Effect of Glyburide and Adenosine Deaminase Treatments
To further investigate the involvement of KATP channels, smooth muscle cells were pretreated with glyburide for 30 min before addition of adenosine. As shown in Fig. 2, glyburide pretreatment significantly attenuated the response of adenosine in a concentration-dependent manner. To examine whether the effects of adenosine are directly mediated, experiments were performed in the presence of adenosine deaminase, which catalyzes deamination of adenosine to inosine. Addition of varying concentrations of adenosine (0.03–10 μM) along with adenosine deaminase (0.03 units/ml) completely abolished the responses of adenosine, which demonstrates that adenosine activates KATP channels by itself, and not via its catabolic products.
Effect of Adenosine Receptor Agonists
To address the nature of the adenosine receptor(s) involved in this phenomenon, the effects of various adenosine receptor agonists (Jacobsen et al., 1992; Jacobsen and Knutsen, 2001) were evaluated. In each case, agonist-evoked reductions in membrane potential effects were attenuated by glyburide.
Adenosine A1 Agonists.
TheN6-substituted adenosine derivatives, including N6-cyclopentyladenosine (CPA, A1Ki = 2.3 nM) and its analog 2-chloro-N6cyclopentyladenosine (CCPA) andN6-(R)-phenylisopropyladenosine (R-PIA; A1Ki = 2 nM) are selective agonists at A1 receptors (Jacobson and Knutsen, 2001). The kinetic data of CCPA are shown in Fig. 3A. The concentration-response curves for changes in fluorescence responses by CCPA and other A1 receptor agonists are provided in Fig. 3, B and C. Although the efficacies of these compounds were comparable to that of adenosine, the EC50 values were generally in the micromolar range (Table 1). 2-Chloro-adenosine, a stable analog of adenosine that is only about 7-fold more selective for the A1 receptor (Ki = 9 nM) compared with A2A/A2B receptor subtypes (Fredholm et al., 1994), also elicited concentration-dependent changes in membrane potential responses.
Adenosine A2A/A2B Agonists.
Next, the effects of CGS-21680, a compound that is approximately 170-fold selective for the A2A versus A1 and (A2AKi = 15 nM; A1Ki = 2600 nM) with weak affinity at A2B receptors was examined together with effects of the nonselective agonist NECA (Hutchinson et al., 1990; Jacobson and Knutsen, 2001). Both CGS-21680 and NECA reduced fluorescence responses (Fig. 4, A and B) with comparable EC50 values of 22 nM (−log EC50, 7.65 ± 0.17) and 11 nM (−log EC50, 7.97 ± 0.23), respectively. Other relatively selective agonists, including 2-phenyl amino adenosine, 5′(N-cyclopropyl)-carboxamidoadenosine (CPCA), and methyl carboxamido adenosine were also active, with EC50values in the nanomolar range (Table 1).
Adenosine A3 Agonists.
The effect of A3 receptor-selective agonist 2Cl-IBMECA, which is about 2500- and 1400-fold selective for rat A2A and A1 receptors, respectively (A3Ki = 0.3 nM; A2AKi = 470 nM; A1Ki = 820 nM; Jacobson and Knutsen, 2001), was evaluated. As shown in Table 1 and depicted in Fig.5A, the EC50 value of 2Cl-IBMECA was 16.6 μM (log EC50 = 4.78 ± 0.23), although the affinity of this compound at the A3 receptor itself is in the low nanomolar range. Other moderately selective A3 agonists, including AB-MECA (Ki = 1.39 nM; Jacobson et al., 1993) and N6-benzyl NECA (Ki = 6.8 nM; van Galen et al., 1994) were effective, but considerably less potent compared with their affinities reported at the A3 receptor subtype (Fig. 5B; Table 1).
Effect of Adenosine Receptor Antagonists
To further investigate the adenosine receptor subtype involved in KATP channel activation, the effects of selective receptor antagonists were examined.
Inhibition of Adenosine-Mediated Responses by Selective Antagonists.
DPCPX, an adenosine antagonist with about 500-fold greater selectivity for the rodent A1(Ki = 0.46 nM) than A2 receptors (Halleen et al., 1987), was used to assess the involvement of the A1 receptor. As shown in Fig. 6A, 100 nM DPCPX did not alter adenosine-evoked changes in membrane potential. CGS-15943, another compound that is about 100-fold more selective in antagonizing A2A receptor compared with A2B, but only about 10-fold selective versus the A1 subtype (Ongini et al., 1999) also inhibited responses of adenosine (0.3 and 1 μM). The effects of ZM-241385, an A2A receptor-selective compound with 30- to 80-fold greater selectivity for the A2A receptor subtype compared with the A2B receptor and 1000-fold selectivity compared with the A1receptor (Poucher et al., 1995), was also examined. As shown in Fig.7A, ZM-241385 (0.1, 1, and 10 μM) significantly inhibited adenosine-evoked responses.
The rank order profile of agonists together with the sensitivity to ZM-241385 indicates that the receptor involved in mediating the effect of adenosine in guinea pig bladder belongs to the A2A subtype. However, a role for the A2B subtype cannot be ruled out, especially considering the lower affinity of A2B receptors for agonists and in the absence of highly selective and potent A2B antagonists. To further investigate a potential role for the A2B receptors, the effects of alloxazine, a nonxanthine A2B-selective inhibitor (Brackett and Daly, 1994; Feoktistov and Biaggioni, 1997), were examined. At a concentration that selectively inhibits the A2B receptor (1 μM), alloxazine did not inhibit responses of adenosine. However, significant inhibition was observed with alloxazine at 10 μM, which could possibly be attributable to an effect on the A2A receptor.
Inhibition of NECA-Mediated Responses by Selective Antagonists.
The nonselective adenosine receptor agonist NECA was also used in conjunction with the A2A-selective antagonist ZM-241385 to further clarify the receptor subtype(s) involved (Fig. 7B). ZM-241385 inhibited responses evoked by NECA in a concentration-dependent manner, suggesting involvement of the A2A subtype. Again, similar to the effects observed with adenosine, alloxazine at 10 μM, but not at 1 μM, significantly inhibited NECA-evoked responses. Collectively, these observations confirm the involvement of A2Areceptors in activating KATP channels in the bladder smooth muscle.
Coupling of Adenosine A2A Receptors to KATPChannels
The observation that adenosine activates glyburide-sensitive changes in membrane potential by activation of A2A receptors suggests a role for the involvement of intracellular cAMP and subsequent activation of protein kinase A. To assess the nature of coupling of A2A receptors to KATP channels, agents that modulate adenylate cyclase and/or cAMP levels were examined. First, the effect of MDL-12330A, a known inhibitor of adenylate cyclase, was evaluated. Addition of 10 μM MDL-12330A (Fukushima et al., 2001) shifted the concentration-response curve of adenosine to the right, whereas at 100 μM, the effects were completely abolished. These findings provide evidence that adenosine activates KATP channels in bladder smooth muscle cells by elevation of intracellular cAMP and stimulation of protein kinase A. Consistent with these observations, direct activation of cAMP levels by forskolin also resulted in concentration-dependent decreases in fluorescence responses that were reversed by glyburide (Fig. 8B). In addition to forskolin, papaverine, a phosphodiesterase inhibitor that elevates cellular cAMP levels by preventing its breakdown, was also found to be effective in activating KATPchannels.
Adenosine Agonist-Evoked Relaxation of Bladder Strips
The inhibitory effects of adenosine receptor ligands were evaluated in bladder smooth muscle strips precontracted with 20 mM K+. It is known that spontaneous phasic contractility of the bladder smooth muscle are largely myogenic in nature, insensitive to suppression by 100 nM tetrodotoxin, but inhibited by L-type calcium channel blockers and KATP channel openers (Fujii et al., 1990; Herrera et al., 2000; Buckner et al., 2002). As shown in Fig.9, the nonselective agonist NECA and other A2A receptor-selective agonists, CGS-21680, CPCA, and 2-phenylaminoadenosine, inhibited bladder contractions with −log IC50 values of 6.41 ± 0.05, 6.38 ± 0.22, 6.91 ± 0.15, and 5.53 ± 0.21, respectively (Table2). In contrast, the A1-selective agonist CCPA and the A3-selective agonist 2Cl-IBMECA did not inhibit contractions, even at the highest concentrations (100 μM) tested. 2-Chloroadenosine also suppressed phasic contractility (−log IC50 = 5.92 ± 0.32), which could be attributed to an effect at the A2 receptor subtypes. The rank order potencies of adenosine agonists in suppressing phasic contractility of the bladder strips and for evoking membrane potential effects were comparable.
Discussion
The present study provides evidence that adenosine evokes membrane hyperpolarization in guinea pig bladder smooth muscle cells by activation of KATP channels. Pharmacological characterization with a variety of subtype-selective agonists demonstrates that these effects are mediated predominantly by activation of the adenosine A2A receptor. This is further supported by inhibition of adenosine and NECA-evoked hyperpolarization by the A2A-selective antagonist ZM-241385. Relaxation of myogenic contractions of bladder smooth muscle strips also show a rank order potency for adenosine receptor agonists that further supports the participation of the A2A receptor subtype. Our studies also demonstrate that, like those previously reported in the vasculature (Kleppisch and Nelson, 1995), adenosine receptor-mediated activation of KATP channels in bladder smooth muscle involves activation of adenylate cyclase, leading to an elevation of intracellular cAMP and stimulation of protein kinase A pathways.
Adenosine Receptor-Mediated Activation of KATPChannels.
By using fluorescence-based readout of membrane potential changes, the present study shows that adenosine can hyperpolarize guinea pig bladder smooth muscle cells in a concentration-dependent manner. Fluorescence-based membrane potential assays of KATP channel function serves as a sensitive method to evaluate changes in membrane potential with a response time comparable to ligand-gated channel activation measured by patch-clamp studies. Previous studies have shown that KATP channels in bladder myocytes can be activated by P1075 and other structurally divergent KATP channel openers with a rank order of potency that correlates well with relaxation of bladder smooth muscle strips (Gopalakrishnan et al., 1999; Whiteaker et al., 2001). The reversal of membrane potential effects by adenosine and subtype-selective agonists by glyburide demonstrates their activation of KATP channels in the bladder smooth muscle.
Pharmacological Characterization of Adenosine Receptors.
Functional analysis with subtype-selective agonists of KATP channel-mediated membrane potential changes and relaxation responses support the participation of adenosine A2 receptor subtypes (Table 1). In particular, CGS-21680 that is selective for the A2A versus A2B receptor (Hutchinson et al., 1989; Jacobson and Knutsen, 2001) was found to be as potent as NECA in evoking membrane potential effects (Table 1). The rank order of potencies, NECA (−log EC50 = 7.97) ∼ CGS-21680 (7.65) > 2-chloro adenosine (5.90) ∼ CCPA ∼ CPA ∼ R-PIA > 2Cl-IBMECA (4.78) is consistent with that defining the A2A receptor subtype (Feoktistov and Biaggioni, 1997). As shown in Figs. 3 and 5, agonists with reportedly nanomolar affinity at the A1 and A3 receptor subtype were found to affect membrane potential responses only at relatively higher concentrations (EC50 values in the micromolar range). Similarly, in tissue relaxation studies, NECA and CGS-21680 were equipotent in suppressing myogenic contractions of the bladder strips, whereas the A1 receptor-selective agonist (CCPA) and A3-selective agonist (2Cl-IBMECA) were inactive. Taken together, the data indicate that the A2Areceptor subtype mediates adenosine-evoked relaxation of urinary bladder smooth muscle.
Further support for participation of the A2Areceptor was derived from studies with selective antagonists. The concentration response of membrane potential effects evoked by adenosine was not shifted by the presence of DPCPX, suggesting the lack of participation of adenosine A1 receptors. On the other hand, ZM-241385, a selective A2Areceptor antagonist, suppressed adenosine- and NECA-evoked hyperpolarization responses. In the absence of selective agonists or antagonists, A2B receptors have been typically characterized by the method of exclusion or lack of agonists that are specific to other subtypes. The lack of effect of low concentrations of alloxazine, together with the relative potency of selective agonists, suggests that A2B receptor may not likely be involved. Detailed analysis of mRNA expression by reverse transcription-polymerase chain reaction in rat urinary bladder have shown the presence of mRNA corresponding to A1, A2A, A2B, and A3 subtypes (Dixon et al., 1996). Although comparable studies in guinea pig bladder are currently unavailable, the rank order potency of agonists together with the suppression of adenosine or NECA-evoked membrane potential by ZM-241385 indicates that A2A receptor could be functionally involved in adenosine receptor signaling.
Mechanism of Adenosine A2A Receptor Signaling.
The inhibition of adenosine effects by the adenylate cyclase inhibitor MDL-123390A together with the observation that the effects can be mimicked by direct activation of adenylate cyclase by using forskolin or after inhibition of cAMP-dependent phosphodiesterase, in a glyburide-reversible manner, implicates that the coupling to KATP channels involves elevation of cAMP. Although both A2A and A2Breceptors are coupled through Gsα protein to the activation of adenylate cyclase and elevation of intracellular cAMP (Fredholm et al., 1994), the present study provide evidence that it is the A2A receptor that is linked to the activation of KATP channels in guinea pig bladder smooth muscle. This situation is comparable to that reported in the vasculature, as for example, in arterial myocytes where adenosine acts via the A2A receptor subtype and protein kinase A to stimulate KATP currents (Kleppisch and Nelson, 1995). Adenosine-induced vasodilation in other vascular beds, including the microvasculature of rat diaphragm and afferent arterioles in rat kidney, have also been shown to involve the A2Areceptor, which are coupled to adenylate cyclase activation and opening of the KATP channel (Tang et al., 1999; Chen et al., 2000).
A2A Receptor-KATP Channel Interactions.
KATP channels have been shown to be hetero-octomeric complexes composed of four inward rectifying K+ channels belonging to the Kir 6.0 subfamily that forms the K+-selective pore and four regulatory proteins, the sulfonylurea receptors. The latter serves to catalyze nucleotide binding and hydrolysis and hosts binding sites for KATP channel openers and sulfonylurea blockers (Bryan and Aguilar-Bryan, 1999; Seino, 1999). On the basis of reverse transcription-polymerase chain reaction and pharmacological studies, it has been suggested that KATP channels in guinea pig bladder smooth muscle may be derived from combinations of sulfonylurea receptor 2B and Kir6.2. Although our studies show that adenosine A2A receptor elevates cAMP and possibly activates protein kinase A-mediated phosphorylation processes, the mechanism by which this leads to activation of KATP channels remains to be elucidated by future coexpression studies of the A2A receptor with KATP channel subunits.
A great deal of evidence shows that adenosine receptors can activate KATP channels in vasculature, especially those for the coronary and mesenteric beds (Dart and Standen, 1993; Quayle et al., 1997). More recently, it has been shown that adenosine receptor activation can enhance the potency of cromakalim via A2A receptors, but not that of pinacidil (Davie et al., 1999). Although no comparable studies have been carried out in bladder smooth muscle, it raises the possibility that the sensitivity of KATP channel openers could be enhanced under metabolically compromised conditions, where adenosine release is higher, as for example, in bladder instability subsequent to partial outlet obstruction. It has been reported that hypertrophied bladder secondary to partial outlet obstruction is more resistant to hypoxic ischemic and reperfusion damage than normal tissue (Levin et al., 2000). It is also possible that such local modulation of adenosine levels might contribute to the in vivo bladder selectivity of some of the KATP channel openers reported in models of chronic outlet obstruction where secondary changes, including alterations in adenosine receptor-mediated pathways, may likely occur (Wojdan et al., 1999).
Although adenosine receptor subtypes have been widely exploited as therapeutic targets (Williams and Jarvis, 2000), the difficulty in separating therapeutic effects from potential side effects has somewhat dampened the development of drugs acting at adenosine receptors. Enhanced understanding of the roles and mechanisms underlying adenosine receptor activation and signaling in various tissues could facilitate future exploitation of the potential of targeting adenosine receptor subtypes. In summary, these present studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of urinary bladder smooth muscle are mediated by adenosine A2A receptor-mediated activation of KATP channels via adenylate cyclase and elevation of cAMP.
Footnotes
- Abbreviations:
- KATP channel
- ATP-sensitive K+ channel
- FLIPR
- fluorescent imaging plate reader
- ZM-241385
- 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol
- MECA
- 5′-N-methylcaboxamido adenosine
- P1075
- N-cyano-N′-(1,1-dimethylpropyl)-N"-3-pyridylguanidine
- CPA
- N6-cyclopentyl adenosine
- CCPA
- 2-chloro-N6-cyclopentyladenosine
- R-PIA
- (R)-N6-phenylisopropyladenosine
- CGS-21680
- 2-p-(2-carboxethyl)phenethyl-amino-5′-N-ethylcarboxamidoadenosine hydrochloride
- NECA
- 5′-N-ethyl-carboxamido adenosine
- CPCA
- 5′(N-cyclopropyl)-carboxamido adenosine
- DPCPX
- 8-cyclopentyl-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione
- CGS-15943
- 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine
- MDL-12330A
- N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride
- 2Cl-IBMECA
- 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyl-carboxamide
- Received October 4, 2001.
- Accepted November 19, 2001.
- The American Society for Pharmacology and Experimental Therapeutics