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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Effect of Neocuproine, a Copper(I) Chelator, on Rat Bladder Function

Department of Pharmacology, School of Medicine, University of Çukurova, Adana, Turkey (C.G.); and Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania (B.G., W.C.d.G.)

Received August 19, 2004; accepted October 21, 2004.

	Abstract

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The effects of a specific copper(I)-chelator, neocuproine (NC), and a selective copper(II)-chelator, cuprizone, on nonadrenergic-noncholinergic transmitter mechanisms in the rat urinary bladder were studied by measuring nerve-evoked contractions of bladder strips and voiding function under urethane anesthesia. After blocking cholinergic and adrenergic transmission with atropine and guanethidine, electrical field stimulation induced bimodal contractions of bladder strips. An initial, transient contraction that was blocked by the purinergic antagonist, suramin, was significantly enhanced by NC (20 and 200 µM applied sequentially) but not affected by cuprizone. The facilitating effect, which was blocked by suramin and reversible after washout of the drug, did not occur following administration of neocuproine-copper(I) complex (NC-Cu). NC (20 µM) significantly increased the second, more sustained contraction, whereas 200 µM decreased this response. These effects of NC on the sustained contractions were not elicited by NC-Cu and not blocked by suramin. The nitric oxide synthase inhibitor, L-nitroarginine, did not alter the responses to NC. NC (20 µM) elicited a marked increase in basal tone of the strips. This effect was less prominent after the second application of 200 µMNC or with NC-Cu treatment or in the presence of suramin. In anesthetized rats, during continuous infusion cystometry, intravesical infusion of 50 µM NC but not NC-Cu or cuprizone significantly decreased the intercontraction interval (ICI) without changing contraction amplitude. The ICI returned to normal after washout of NC. Suramin blocked this effect. These results indicate that NC enhances bladder activity by facilitating purinergic excitatory responses and that copper(I)-sensitive mechanisms tonically inhibit purinergic transmission in the bladder.

Nitric oxide (NO) released from afferent nerves or from the urothelium (Birder et al., 1997, 2001) may be involved in inhibitory modulation of sensory mechanisms in the bladder. Intravesical administration of NO donors suppresses detrusor hyperactivity (Ozawa et al., 1999), whereas intravesical administration of oxyhemoglobin, an NO scavenger, stimulates bladder activity (Pandita et al., 2000). Patch-clamp studies revealed that NO donors can suppress voltage-gated Ca²⁺ channels in bladder sensory neurons (Yoshimura et al., 2001).

The present study that focused on the effect of copper chelators on bladder function was prompted by recent reports that copper-dependent mechanisms can modulate and/or mediate the actions of ATP or NO. For example, copper ions can alter the function of P2X₄ purinergic receptors expressed in Xenopus oocytes (Xiong et al., 1999; Acuna-Castillo et al., 2000), raising the possibility that other types of P2X receptors that are expressed in the bladder may also be sensitive to copper ions. Some of the effects of NO in the bladder may also be dependent on copper ions as suggested for nitrergic transmission at other sites in the urogenital system such as the mouse corpus cavernosum (Göçmen et al., 1997, 1998, 2000) and sheep urethra (Garcia-Pascual et al., 2000). In these tissues, it has been speculated that the nitrergic transmitter may not be free NO but rather a superoxide-resistant, nitric oxide-carrying molecule, such as an S-nitrosothiol. A copper(I)-dependent mechanism has been implicated in the effect of S-nitrosothiols (De Man et al., 1999, 2001; Göçmen et al., 2000).

To investigate the contribution of copper-dependent mechanisms in modulation of purinergic and nitrergic mechanisms in the rat bladder, we studied the effects of a selective Cu(I) chelator, neocuproine (2,9-dimethyl-1,10-phenanthroline) (De Man et al., 1999; Göçmen et al., 2000), and a selective Cu(II) chelator, cuprizone (De Man et al., 1999), on neurally evoked contractions of bladder strips and on voiding function in urethane-anesthetized rats. The results indicate that Cu(I) mechanisms tonically suppress purinergic excitatory transmission in the bladder. Preliminary observations have been reported in an abstract (Göçmen et al., 2003).

	Materials and Methods

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Animals. Female Sprague-Dawley rats (200–250 g) were used in this study. The experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

In Vitro Studies. Halothane anesthetized rats were sacrificed by decapitation. The bladders were harvested, and bladder strips (5- x 1-mm longitudinal sections) were prepared from the midportion of bladder body. Strips were subsequently mounted in 5-ml jacketed organ baths containing Krebs' solution (119 mM NaCl, 4.6 mM KCl, 1.25 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24.8 mM NaHCO₃, and 10 mM dextrose) maintained at 37°C and bubbled with a mixture of 95% O₂ and 5% CO₂, pH 7.4. Tissues were allowed to equilibrate for 1 h, during which the preparation was washed with fresh Krebs' solution at 15-min intervals. The responses were recorded with B40 type 373 isotonic transducers (Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten, Germany). Data were recorded and stored using Windaq data acquisition software (DATAQ Instruments Inc., Akron, OH). After the equilibrium period, neurally evoked isotonic contractions were induced using trains of electrical field stimulation (EFS; 10 Hz, 50 V, 0.12-ms duration, 10-s trains) delivered from a Grass S88 stimulator (AstroMed, Inc., West Warwick, RI) at 2-min intervals through platinum electrodes positioned on the top and the bottom of the organ bath. In experiments in which electrical field stimulation was used, atropine (5 µM) and guanethidine (2 µM) were always present in the bathing medium to block adrenergic and cholinergic transmission. EFS induced bimodal contractions of bladder strips. In control experiments, EFS-evoked responses were recorded for a 3-h time period (no drug was added) to examine the stability of the nerve-evoked responses. In other experiments after the control responses to EFS were recorded for approximately 1 h, neocuproine, a selective Cu(I) chelator, was applied at two concentrations (either 5 and 10 or 20 and 200 µM) to the same tissue consecutively at intervals ranging from 20 to 25 min. In some experiments, the effects of various agents including, preprepared neocuproine-copper(I) complex, a nitric oxide synthase inhibitor L-nitroarginine (L-NOARG; 0.5 mM), a selective Cu(II) chelator cuprizone (10 or 20 µM), or a purinergic antagonist suramin (100 µM) were examined on bladder contractions induced by electrical field stimulation in the presence or absence of neocuproine. The neocuproine-copper(I) complex was prepared by reacting glutathione, neocuproine, and CuSO₄ in molar ratios of 1:2:1, respectively (Dicks et al., 1996). In another series of experiments, we studied the effect of exogenous ATP on basal tone of bladder strips. ATP (10, 50, or 100 µM) was applied four times to the same tissue consecutively at intervals ranging from 10 to 15 min. Exposure time of ATP was 60 s for each application. In some experiments, either ATP (10 or 100 µM) or neocuproine (5 or 10 µM) was applied to the bladder strips after prolonged (30-min) ATP treatment (10 or 100 µM) to examine the effects on the EFS-evoked contractions or basal tone in preparations in which the purinergic receptors were desensitized (Driessen et al., 1993; Sahin et al., 2000).

Cystometrogram Recording. Animals were anesthetized with subcutaneous injection of urethane (1.2 g/kg; Sigma-Aldrich, St. Louis, MO). The urinary bladder was exposed via a midline abdominal incision and was catheterized. A PE-50 catheter, the bladder end of which was heated to create a collar, was inserted through a small incision in the bladder dome, and a suture was tightened around the collar. The other end was connected via a T-stopcock to a pump for continuous infusion of physiological saline and to a pressure transducer to record bladder pressure. Physiological saline was infused at room temperature into the bladder at a constant rate of 0.04 ml/min to elicit repeated voiding responses. In all experiments, control cystometrograms (CMGs) were recorded for about 2 h prior to intravesical drug administration. The parameters evaluated were amplitude of bladder contractions and intercontraction interval (ICI), which is defined as the time between two voiding cycles. Neocuproine (25–200 µM), preprepared neocuproine-copper (I) complex, L-NOARG (0.5 mM), cuprizone (20 µM), or suramin (100 µM) were administrated in saline into the bladder for 1 h. CMG parameters were monitored for 1 h and compared with the recordings before drug application.

Drugs. Stock solutions of atropine sulfate, guanethidine, 2,9-dimethyl-1,10-phenanthroline (neocuproine), L-nitroarginine, suramin, ATP, CuSO₄, and glutathione were prepared in distilled water. Cuprizone was dissolved in 50% ethyl alcohol (final concentration in the bath medium was 0.4%). All drugs were administered in saline solutions. Drugs were obtained from Sigma-Aldrich.

Statistical Analysis. All data are expressed as mean ± S.E. Data were analyzed by one-way analysis of variance followed by Student's paired t test. A p value of less than 0.05 is considered significant.

	Results

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Effect of Neocuproine on the EFS-Induced Biphasic Contractions of Rat Urinary Bladder Strips. After blocking cholinergic and adrenergic responses with atropine and guanethidine, EFS (10 Hz, 50 V, 0.12-ms duration) evoked bimodal contractions of bladder smooth muscle strips (Fig. 1) consisting of an initial large amplitude transient response followed by a lower amplitude more sustained response that persisted following the termination of the stimulus. Both responses were blocked by tetrodotoxin (1.5 µM). The amplitude of the EFS-evoked contractions was constant for a 3-h time period (3.21 ± 0.32 mN at the beginning of the experiment and 3.01 ± 0.42 mN at the end of the experiment, n = 4). The initial contraction to EFS was significantly enhanced by two concentrations of neocuproine (3.04 ± 0.33 mN for control and 5.81 ± 0.47 mN at 20 µM; 4.20 ± 0.23 mN for control and 8.85 ± 0.86 mN at 200 µM; Figs. 1 and 2, A and B) but not affected by lower concentrations of neocuproine (5 and 10 µM) or by 10 and 20 µM cuprizone or its solvent (4.8 ± 0.9 mN for control; 5.23 ± 0.7 mN for solvent; 5.65 ± 0.95 mN for 10 µM cuprizone; and 5.97 ± 0.89 mN for 20 µM cuprizone; n = 18–20). Neocuproine (20 µM), applied at the end of experiments after cuprizone or its solvent, significantly enhanced the contractile responses (9.23 ± 1.2 mN for 20 µM neocuproine, n = 12). The repeated application of 20 µM neocuproine produced a reproducible effect on the initial contraction (3.87 ± 0.52 mN for control and 7.56 ± 0.73 mN at the first application; 3.21 ± 0.33 mN for control and 5.97 ± 0.43 mN at the second application, n = 4). This facilitating effect of neocuproine, which was reversible in 15 to 20 min after washout of the drug, did not occur following the application of the preprepared neocuproine-copper (I) complex (Fig. 2, A and B) and was blocked by the purinergic antagonist, suramin (100 µM, Fig. 3). The NOS inhibitor, L-NOARG (0.5 mM), did not affect the control contractions or the responses to neocuproine (initial control contraction, 2.3 ± 0.8 mN; 5.6 ± 0.7 mN for 0.5 mM L-NOARG + 20 µM neocuproine; 6.7 ± 1.1 mN for 0.5 mM L-NOARG + 200 µM neocuproine; n = 4–6).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Representative tracings showing the effects of neocuproine (NC, 20 and 200 µM) and neocuproine-copper(I) complex (NC-Cu) on the nerve-evoked bladder strip contractions induced by electrical field stimulation (10 Hz, 50 V, 0.12 ms).

View larger version (30K): [in this window] [in a new window]   Fig. 2. Effects of 20 µM (A) and 200 µM (B) NC and NC-Cu on the nerve-evoked bladder strip contractions induced by electrical field stimulation (10 Hz, 50 V, 0.12 ms). The bars marked with hatched and cross-hatched bars represent the first and the second phasic responses, respectively. *, p < 0.05; ***, p < 0.001, significantly different from control (n = 6–8).

View larger version (44K): [in this window] [in a new window]   Fig. 3. Effects of 20 or 200 µM NC in presence or absence of suramin (SUR; 100 µM) on the nerve-evoked bladder strip contractions induced by electrical field stimulation (10 Hz, 50 V, 0.12 ms). The bars marked with hatched and cross-hatched bars represent the first and the second phasic responses, respectively. ***, p < 0.001, significantly different from control (n = 6–8).

The sustained contraction was also affected by neocuproine. The high concentration of neocuproine (200 µM) significantly decreased (33.2 ± 6.3%) the sustained component of contraction, whereas the low concentration enhanced (242 ± 21.9%) the sustained component as well as the initial contraction but did not alter the shape of bimodal response (Fig. 1). Also, the repeated application of 20 µM neocuproine did not affect the shape of bimodal response (not shown). These effects of the two concentrations of neocuproine on the sustained contraction did not occur following the administration of neocuproine-copper(I) complex (Fig. 2B). Suramin (100 µM) inhibited the facilitatory effect of 20 µM neocuproine on the sustained component, whereas it failed to alter the depressant effect of the higher concentration of neocuproine (Fig. 3).

The first application of neocuproine (20 µM) caused a marked increase in the basal tone of the strips (Table 1), which persisted for 22.4 ± 4.2 min (n = 24). A second application of the higher concentration of the drug (200 µM) elicited a considerably smaller effect (Table 1). Also, the enhancement of basal tone by 20 µM neocuproine was significantly less after repeated application of this agent at the same concentration (16.23 ± 5.7% of the enhancement due to first application; n = 4). Neocuproine (200 µM) also induced small amplitude (<1 mN) spontaneous contractions in some strips. Neocuproine-copper(I) complex did not elicit these responses. Pretreatment with suramin (100 µM) prevented the effects of neocuproine (20 µM) (Table 1). Application of ATP (10–100 µM) elicited an increase in basal tone in a concentration-dependent manner (2.43 ± 0.41 mN at 10 µM; 4.36 ± 0.53 mN at 50 µM; 5.30 ± 1.01 at 100 µM). Repeated application of the same concentration of ATP at 15-min intervals produced a significant desensitization, e.g., the response to 100 µM ATP declined to 27 ± 5.4% of the control response. Prolonged exposure (30 min) to ATP (10 or 100 µM) also suppressed the EFS-induced contraction in a dose-dependent manner (to 40.2 ± 6.9% of control at 10 µM and to 24.6 ± 4.8% of control at 100 µM). Neocuproine (5 or 10 µM), which alone had no effect, enhanced the suppression of contractile responses after 10 µM ATP (to 30.8 ± 4.3% of control at 5 µM and to 24.2 ± 6.7% of control at 10 µM) or 100 µM ATP (to 19.3 ± 5.6% of control at 5 µM and to 12.1 ± 7.3% of control at 10 µM). Neocuproine-copper(I) complex, CuSO₄, glutathione, or CuSO₄ + glutathione did not elicit significant changes in basal tone or the response to ATP.

Effect of Neocuproine on CMG Parameters in Anesthetized Rats. During continuous infusion cystometry under urethane anesthesia, intravesical administration of neocuproine at a concentration of 50 µM significantly decreased the ICI (Figs. 4 and 5A) but did not change the amplitude of bladder contractions. The decrease in the ICI was maintained for 30 to 40 min during the continuous infusion of neocuproine but returned to normal within 50 to 60 min after washout of the drug. Repeated administration of neocuproine (50 µM) 30 min after the first application produced a similar effect. Preprepared neocuproine-copper(I) complex (Figs. 4 and 5A) did not alter the ICI. Suramin (100 µM) infused with neocuproine application significantly reduced but did not completely block the effect of neocuproine (p < 0.05, Fig. 5B). However, pretreatment with suramin completely blocked the effect of neocuproine on the ICI (Fig. 6). Suramin alone did not alter the ICI. The CMG and/or the effect of neocuproine on the CMG was not affected by infusion of 0.5 mM L-NOARG (ICI, 510 ± 42 s for control; 567 ± 53 s for 0.5 mM L-NOARG; 274 ± 16 s for 0.5 mM L-NOARG + 50 µM neocuproine) or 20 µM cuprizone (ICI, 523 ± 64 s for control; 558 ± 51 s for 20 µM cuprizone). Also, CuSO₄, glutathione, or CuSO₄ + glutathione did not alter the ICI.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Continuous cystometrogram tracing during control (A), neocuproine (50 µM) (B and D), washout after first application of neocuproine (C), and neocuproine-copper (I) complex (E).

View larger version (61K): [in this window] [in a new window]   Fig. 5. Effects on the ICI during continuous infusion cystometrograms of NC (50 µM) or NC-Cu (A) and/or neocuproine (50 µM) plus suramin (100 µM) (NC + SUR) (B) on ICI. *, p < 0.05; **, p < 0.01; ***, p < 0.001, significantly different from control (C); +, p < 0.05, significantly different from the second application of NC (n = 5–7). w, washout.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Effects of suramin (SUR; 100 µM, intravesically) or suramin (100 µM) pretreatment plus neocuproine (50 µM, intravesically) (SUR + NC) on the ICI during continuous infusion cystometrograms (n = 5–7). w, washout.

	Discussion

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The present experiments revealed that a copper(I) chelator, neocuproine, decreased the voiding interval during continuous cystometry in anesthetized rats without altering the amplitude of voiding contractions. This agent also enhanced the electrical field stimulation-induced nonadrenergic-noncholinergic contractions of rat bladder strips. These effects were blocked by a purinergic receptor antagonist, suramin, and did not occur following the administration of a neocuproine-copper(I) complex in which neocuproine was saturated with excess copper ions. These findings suggest that neocuproine can stimulate bladder activity by facilitating purinergic excitatory responses and that copper(I)-sensitive mechanisms play a role in this effect.

Neocuproine could modulate purinergic mechanisms at several sites in the bladder including postjunctional purinergic receptors in the smooth muscle, as well as purinergic receptors in afferent nerves and urothelial cells (Namasivayam et al., 1999; de Groat and Yoshimura, 2001; Vlaskovska et al., 2001; Andersson, 2002; Rong et al., 2002; Birder et al., 2003). The initial component of the neurally evoked contractions of detrusor smooth muscle that persisted in the presence of atropine and guanethidine was suppressed by suramin, a P2X purinergic receptor antagonist as reported by other investigators (Hoyle et al., 1990; Calvert et al., 2001; Burnstock, 2002; Benko et al., 2003). Neocuproine significantly enhanced this initial component, and suramin suppressed this effect of neocuproine. The first application of neocuproine also elicited an increase in basal tone of the smooth muscle strips, an effect that was suppressed by suramin. These data suggest that neocuproine enhances the postjunctional effect of ATP or enhances the release of ATP from nerves. The finding that the second application of neocuproine (20 or 200 µM) did not cause an enhancement of the basal tone supports this speculation because repeated ATP applications elicited a desensitizing effect on the contractile responses (Husted and Nedergaard, 1985) and suppressed EFS-induced contractions (Sahin et al., 2000). Thus, repeated application of neocuproine can induce a desensitizing response similar to that induced by ATP (Namasivayam et al., 1999). Also, neocuproine application after ATP treatment enhanced the suppression of the contractile responses to EFS. These results support the data obtained from suramin experiments that neocuproine enhances purinergic mechanisms, and when purinergic receptors are desensitized, the neocuproine facilitatory effect is reduced. Further studies are necessary to determine whether this effect is due to prejunctional or postjunctional mechanisms.

In addition to purinergic transmission, peptidergic transmission involving substance P or other neurokinins may be involved in nonadrenergic-noncholinergic transmission in the bladder and contribute to the sustained component of the EFS-evoked contraction (Benko et al., 2003). The low concentration of neocuproine enhanced the sustained part of the EFS-evoked bimodal contraction, whereas the high concentration decreased the contraction. This differential effect of neocuproine at two concentrations was not blocked by suramin, raising the possibility that neocuproine influences peptidergic mechanisms in the bladder.

In our study, intravesical application of neocuproine decreased the ICI during continuous cystometry in anesthetized rats, and suramin reversed this effect. ATP can be released by the urothelium, and P2X₃ receptors seem to be critical to the normal physiologic regulation of afferent pathways controlling volume-induced reflexes in the urinary bladder (Namasivayam et al., 1999; Cockayne et al., 2000; Vlaskovska et al., 2001; Andersson, 2002; Pandita and Andersson, 2002). ATP given intravesically induced a pronounced bladder overactivity in unanesthetized rats via action on P2X receptors located on afferent nerve endings in the urothelium, and P2X receptors antagonists counteracted this effect (Hoyle et al., 1990; Chen et al., 1995; Namasivayam et al., 1999; Andersson, 2002). Neocuproine may enhance ATP release from urothelium or the actions of ATP on afferent nerves and thereby induce bladder overactivity and a decrease in the ICI. The finding that suramin prevented this effect of neocuproine supports this possibility.

Preprepared neocuproine-Cu complex did not mimic the facilitating effect of neocuproine on EFS-induced contractions and on basal tone of bladder strips or elicit the decrease in the ICI caused by intravesically infused neocuproine. Cuprisone, a copper(II) chelator, also did not mimic the effects of neocuproine. These results indicate that a copper(I)-sensitive mechanism has a role in the facilitating effect of neocuproine on bladder activity. It has been suggested that copper may modulate the function of another subtype of purinergic receptor, P2X₄,in Xenopus oocytes (Xiong et al., 1999; Acuna-Castillo et al., 2000). The present study indicates that a copper-sensitive mechanism may play a role in the modulation of purinergic efferent and afferent pathways in rat bladder.

The present experiments also examined the possibility that neocuproine might influence NO functions in the bladder. It has been suggested that NO can be released from urothelium, and it can affect the excitability of adjacent afferent nerves (Andersson and Persson, 1995; Ozawa et al., 1999; Yoshimura et al., 2001; Birder et al., 2002). However, the nitric oxide synthase inhibitor L-NOARG (Bennett et al., 1995) did not affect the responses elicited by intravesical application of neocuproine in vivo or the effect of neocuproine on bladder strips in vitro. This finding indicates that an NO pathway does not play a role in the facilitating effect of neocuproine. Previous studies indicated that NO was involved in the actions of other ions (nickel and manganese) on neurally evoked bladder contractions (Liu and Lin-Shiau, 1997, 1998; Sahin et al., 2000). We considered the possibility that NO released in the bladder is not free NO but a sulfhydryl-containing compound, such as an S-nitrosothiol (Thornbury et al., 1992; Garcia-Pascual et al., 2000) and that neocuproine could affect the responses of S-nitrosothiols by its ability to chelate copper (De Man et al., 1999; Göcmen et al., 2000). However, this mechanism seems unlikely because the NOS inhibitor, L-NOARG, did not alter the effect of neocuproine on contractile responses to EFS.

In conclusion, our experiments with selective copper chelating agents suggest that neocuproine enhances bladder activity, presumably by facilitating purinergic excitatory responses, and that copper(I)-sensitive mechanisms can modulate purinergic transmission in the bladder. NO does not mediate the effect of neocuproine on bladder activity. Further studies are needed to determine the mechanisms that underlie the interactions between copper ions and purinergic excitatory responses in the bladder.

	Footnotes

 
This work was supported by National Institutes of Health Grant DK49430.

doi:10.1124/jpet.104.076398.

ABBREVIATIONS: NO, nitric oxide; EFS, electrical field stimulation; L-NOARG, L-nitroarginine; CMG, cystometrogram(s); ICI, intercontraction interval; NC, neocuproine; NC-Cu, neocuproine copper(I) complex.

Address correspondence to: Cemil Göçmen, University of Cukurova, School of Medicine, Department of Pharmacology, Adana, TR-01330, Turkey. E-mail: cgocmen{at}mail.cu.edu.tr

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