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EFFECT OF INTRAVESICAL NITRIC OXIDE THERAPY ON CYCLOPHOSPHAMIDE-INDUCED CYSTITIS
0022-5347/99/1626-2211/0 THE JOURNAL OF UROLOGY® Copyright © 1999 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 162, 2211–2216, December 1999 Printed in U.S.A.
EFFECT OF INTRAVESICAL NITRIC OXIDE THERAPY ON CYCLOPHOSPHAMIDE-INDUCED CYSTITIS HIDEO OZAWA,* MICHAEL B. CHANCELLOR, SUK-YOUNG JUNG, TERUHIKO YOKOYAMA, MATTHEW O. FRASER, YONGBEI YU, WILLIAM C. DE GROAT AND NAOKI YOSHIMURA From the Division of Urologic Surgery and Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
ABSTRACT
Purpose: This study was conducted to examine effects of nitric oxide (NO) donors on bladder hyperactivity induced by cyclophosphamide (CYP)-induced cystitis. Materials and Methods: Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (100 mg./kg.), and then their micturition pattern including mean micturition volume and the number of micturitions during 24 hours was recorded in a metabolic cage before and after CYP treatment. Forty-eight hours after CYP injection, bladder function under urethane anesthesia was evaluated by cystometry with continuous saline infusion (0.04 ml. per minute) or under isovolumetric conditions (0.8 ml. bladder volume). NO donors, S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or sodium nitroprusside (SNP, 1 mM), and an NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 20 mM) were administered intravesically. Direct action of SNAP on bladder afferent neurons was also tested in a patch-clamp recording study. Results: The number of micturitions significantly increased during the first 24 hours after CYP injection (19.0 6 0.88 versus 92.1 6 16.3 micturitions/24 hours, mean 6 SE, n 5 25) (p ,0.001). There was no significant difference in total micturition volume before (12.3 6 1.0 ml./24 hours) and after CYP treatment (15.6 6 1.5 ml./24 hours). During continuous infusion cystometry, intercontraction interval (ICI) was smaller in CYP-injected rats than in control rats. In CYPinjected animals, NO donors increased the ICI, but did not change the amplitude of bladder contractions. Continuous intravesical infusion of the NOS inhibitor did not alter the cystometric parameters. During cystometry under isovolumetric conditions, contraction frequency was decreased after NO donor administration. NO donors did not influence bladder activity in control rats. In patch clamp recordings, when SNAP (500 mM) was directly applied to dissociated afferent neurons innervating the urinary bladder, high-voltage-activated Ca21 channel currents were suppressed by approximately 30%. Conclusions: Intravesical NO donors can suppress CYP-induced bladder hyperactivity. We hypothesize that the effect of NO donors is not due to smooth muscle relaxation, but rather due to an inhibitory effect on bladder afferent pathways that was manifested by an increase in intercontraction interval without changes in contraction amplitude. NO donors may be considered as a possible treatment of CYP-induced and other types of bladder inflammation. KEY WORDS: nitric oxide, cyclophosphamide, urinary bladder, inflammation, detrusor hyperactivity, bladder afferent neurons
Cyclophosphamide (CYP) is a nitrogen mustard-type chemotherapeutic agent, which is used for the treatment of neoplastic diseases such as Hodgkin’s disease, Burkitt’s lymphoma, leukemias and cancer of the breast, lung, cervix, ovary and brain. However, there are major side effects of CYP therapy, including hemorrhagic cystitis which is difficult to treat. According to Stillwell et al,1 major symptoms of hemorrhagic cystitis are gross hematuria (78%) and irritative voiding symptoms (45%). A significant number of patients (9%) with CYP-induced cystitis are refractory to conventional therapies and undergo emergency life-saving cystectomy. Thus, better therapy for CYP-induced cystitis is a high medical priority. Nitric oxide (NO), a toxic gas with free-radical properties,
has been implicated as a neurotransmitter or neuromodulator at various sites in the mammalian nervous system.2 Pharmacological studies have provided evidence that NO is a transmitter mediating penile erection and relaxation of the urethral smooth muscle, but the effect of NO on the urinary bladder is uncertain.3 It was initially thought that NO directly mediates detrusor relaxation, but this effect was found to be small.3–7 However, recent studies in rats indicate that bladder inflammation upregulates the expression of NO synthase (NOS) in bladder afferent neurons, and that NO produced in bladder afferent pathways might be involved in the regulation of micturition reflex following cystitis,6, 8, 9 although there have been no reports on the topical effect of intravesically administrated NO donors on the bladder function. The present study was therefore undertaken to examine the direct effect of NO donors on bladder function under inflammatory conditions. The effects of NO donors, S-nitrosoN-acetyl-penicillamine (SNAP), and sodium-nitroprusside (SNP), as well as the NOS inhibitor, N-nitro-L-arginine
Accepted for publication July 12, 1999. * Requests for reprints: Urologic Surgery, University of Pittsburgh School of Medicine, Suite 700, Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213. Supported by a Grant from the National Institutes of Health (DK-49430). 2211
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methyl ester (L-NAME) were evaluated in rats with CYPinduced cystitis. We also performed an electrophysiological study using patch-clamp recording techniques to examine the direct effect of SNAP on bladder afferent neurons. MATERIALS AND METHODS
Animals. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 250 to 300 gm. were used, at a minimum of 2 days after their arrival. A 12:12 hour lightdark cycle was used. Animals received a single intraperitoneal injection of CYP (100 mg./kg.) dissolved in distilled water in a concentration of 40 mg./ml., and then the micturition pattern was monitored in a metabolic cage. Since reduced voided volume per micturition was detected on day 1 (0 to 24 hours) and day 2 (24 to 48 hours) after CYP injection, bladder function was monitored under urethane anesthesia during continuous infusion cystometrograms (CMG) or under isovolumetric conditions 48 hours after CYP injection. The research protocol was approved by our Institutional Animal Care and Use Committee, and adhered to the guidelines set forth in the U.S. Department of Health and Human Services’ “Guide for the Care and Use of Laboratory Animals.” Micturition pattern. Rats were placed in a metabolic cage (Nalgene metabolic cage, Nalge Co., Rochester, NY) that measures voided urine for a 24-hour period.10 Urine was collected by a specially equipped cup on a transducer (Force displacement transducer FT03, Grass Instruments Co., Quincy, MA) connected to a microcomputer for the recording of micturition frequency, duration and volume. Data were recorded and stored using data acquisition software (Windaq, DATAQ Instruments Inc., Akron, OH). Micturition parameters evaluated were: 1) total urine output per 24 hours; 2) number of micturitions per 24 hours; 3) voided volume/micturition. In 25 animals, micturition patterns were recorded before and during 48 hours after CYP injection; and in 7 of these animals the micturition patterns were recorded for 7 consecutive days after CYP injection. Continuous infusion CMG. Animals were anesthetized with subcutaneous injection of urethane (1.2 gm./kg.; Sigma Chemical Co., St. Louis, MI). Animals with no treatment were used as controls. A cannula (PE-50, Clay-Adams, Parsippany, NJ) was placed in the external jugular vein as an venous line. In some animals, a cannula was also inserted in the carotid artery for monitoring blood pressure. A transure-
thral bladder catheter (PE-50) connected to a pressure transducer was used to record bladder pressure. The bladder was filled with a constant infusion of saline or drug solutions and allowed to empty around the catheter. A continuous CMG was performed by constant infusion (0.04 ml. per minute) of fluid into the bladder to elicit repetitive micturitions, which allowed rapid collection of data for a large number of voiding cycles. The parameters evaluated were amplitude and duration of reflex bladder contractions, and intercontraction interval (ICI), which is defined as the time between two voiding cycles. SNAP, SNP and L-NAME were administered into the bladder for 30 minutes. CMG parameters were monitored for 25 minutes beginning at 5 minutes after switching of the infusion fluid, and compared with the recordings before drug application. Isovolumetric CMG recording. Anesthesia and surgical procedures were the same as those described above. After inserting a PE-50 catheter into the urethra, the urethra was ligated to prevent the leakage around the catheter. The ureters were tied and cut, and then the proximal ends were cannulated (PE-10) and drained externally to prevent the accumulation of urine in the bladder. After the bladder was filled by 0.8 ml. saline, repetitive isovolumetric contractions were observed. To administer drugs, the bladder was punctured with a 30G needle connected to PE-10 cannulae (20 ml. volume dead space). Before drug administration, 20 ml. of bladder contents was removed, and 40 ml. of drug solution (20 ml. for bladder injection and 20 ml. for filling the dead space of connected cannulae) was then injected through the 30G needle using a Hamilton syringe. In these conditions, the final drug concentrations approximated those used during continuous infusion CMGs, after diluted by saline in the bladder. The following parameters were evaluated: amplitude and duration of isovolumetric bladder contractions and frequency of contractions. SNAP, SNP and L-NAME were applied for 30 minutes. CMG parameters were monitored for 25 minutes beginning at 5 minutes after an injection of drug, and compared with the recordings before drug application. After completion of recordings with drug-containing solutions, the bladder was emptied by aspiration and refilled with 0.8 ml. saline. Administration of drugs. Drugs used in this study included S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME)
FIG. 1. Twenty four-hour micturition pattern. A, before injection. B, one day after CYP (100 mg./kg.) injection. Stepwise increments in trace indicate voidings. Note that urinary frequency increased at night.
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(Sigma Chemical Co., St. Louis, MI). SNAP was dissolved in saline with 0.5% DMSO, and SNP and L-NAME were dissolved in saline. All drugs were freshly prepared before each experiment. SNAP and SNP solutions were protected from light during bladder infusion. During continuous infusion CMG, SNAP (2 mM), and SNP (1 mM), as well as L-NAME (20 mM) were administered into the bladder through transurethral catheter. During isovolumetric CMG recording, 20 ml. of SNAP (80 mM) or L-NAME (800 mM) was injected into the bladder, and then diluted by the bladder contents (0.8 ml. of saline) to the same concentration as in a continuous infusion CMG. DMSO (0.5%) alone had no effects on CMG parameters such as bladder contraction pressure and intercontraction interval. Patch clamp recording. Dorsal root ganglion (DRG) neurons which innervate the urinary bladder were labeled by retrograde axonal transport of a fluorescent dye, Fast Blue (4% W/V) injected into the wall of the bladder.11 The dye was injected with a 28 G needle at three to six sites on the dorsal surface of the bladder (5 to 6 ml. per site, total volume of 20 to 30 ml.). DRG at the level of L6 and S1 were then removed under halothane anesthesia, and dissociated in a shaking bath for 25 minutes at 35C with 5 ml. of Dulbecco’ s modified essential medium containing trypsin (0.3 mg./ml.), collagenase (1 mg./ml.) and deoxyribonuclease (0.1 mg./ml.).11 Trypsin inhibitor was then added to neutralize the activity of trypsin. Thereafter, dye-labeled bladder afferent neurons were identified using an inverted phase contrast microscope
FIG. 3. Changes in CMG parameters after CYP injection. A, intercontraction interval in continuous CMG (n 5 12). B, contraction frequency in CMG under isovolumetric conditions (n 5 8).
with fluorescent attachments. Whole-cell recordings were performed with patch electrodes which usually have resistance of 1 to 4 Mohm when filled with the internal solution. The internal solution contained (mM) KCl 140, CaCl2 1, MgCl2 2, EGTA 11, HEPES 10 and Mg-ATP 2 adjusted to pH 7.4 with KOH (310 mosml/l). To isolate Ca21 channel currents, neurons were superfused at a flow rate of 1.5 ml. per minute with an external solution containing BaCl2 5, tetraethylammonium-Cl 155, 4-aminopyridine 5, HEPES 10 adjusted to pH 7.4 with TEA-OH (340 mOsm). An NO donor (SNAP, 500 mM) was administered to neurons using pressure injection methods through a glass pipette positioned close to the cells. Statistical analysis. All data are expressed as mean 6 SE. Student’s t test was used for comparison of 24 hour voiding pattern and CMG parameters. A p-value less than 0.05 was considered significant. RESULTS
FIG. 2. Changes in voiding patterns after CYP injection. A, changes in voiding frequency. B, voided volume per micturition. Data before and on day 1 and day 2 after CYP injection were obtained from 25 animals, and data on days 3, 5 and 7 were from 7 animals.
Micturition pattern. Fig. 1 shows representative 24-hour micturition patterns before (fig. 1, A) and after CYP injection (fig. 1, B). Increased frequency of voiding and decreased volume of voided urine were observed after CYP injection. The number of micturitions per 24 hours was significantly greater for 24 hours after CYP injection (92.1 6 16.3 micturi-
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INTRAVESICAL NITRIC OXIDE THERAPY TABLE 2. Effects of an NO donor (SNAP: 2 mM) and a NOS inhibitor (L-NAME: 20 mM) on reflex bladder contractions under isovolumetric conditions in control and CYP-treated rats
Control
CYP-treated
Saline SNAP (n 5 4) Saline L-NAME (n 5 4) Saline SNAP (n 5 4) Saline L-NAME (n 5 4)
Frequency (Contractions/min.)
Amplitude (cm. H2O)
0.37 6 0.10 0.47 6 0.08 0.32 6 0.09 0.26 6 0.08 0.93 6 0.33 0.14 6 0.09* 0.71 6 0.24 0.51 6 0.33
45 6 10 44 6 9 48 6 10 49 6 10 32 6 5 30 6 4 32 6 5 32 6 6
mean 6 S.E. * p , 0.05; infusion of SNAP versus saline in CYP injected rats.
FIG. 4. Effects of intravesical administration of NO donor (SNAP) on continuous infusion CMG. Upper trace: control period. Regular bladder contractions were observed. Lower trace: 10 minutes after SNAP injection. SNAP increased intercontraction interval without changing detrusor contraction amplitude. Scale bars: 2 minutes (horizontal) and 20 cm. water (vertical).
tions, n 5 25) than before injection (19.0 6 0.88 micturitions, n 5 25) (p ,0.001) (fig. 2). There was no difference between total micturition volumes before (12.3 6 1.0 ml./24 hours) and after CYP injection (15.6 6 1.5 ml./24 hours). The voided volume per micturition was significantly lower after CYP injection (day 1: 0.29 6 0.04 ml., day 2: 0.34 6 0.08 ml.) when compared with that obtained before CYP injection (0.65 6 0.04 ml.). Micturition frequency and volume per micturition returned to normal 3 days after CYP injection (fig. 2). Continuous infusion CMG. ICI was significantly smaller in CYP-treated rats (5.4 6 0.8 minutes, n 5 12) than in control rats (7.4 6 0.7 minutes, n 5 12) (fig. 3, A). In CYP-treated animals, NO donors (SNAP: 2 mM, n 5 5 and SNP: 1 mM, n 5 2) significantly increased the ICI from 5.2 6 0.9 minutes to 9.0 6 2.2 minutes, but did not change the amplitude of bladder contractions (fig. 4 and table 1). This effect was usually observed within 5 minutes after switching to a solution containing NO donors and disappeared 15 to 30 minutes following washout. Intravesical instillation of SNAP and SNP had no effects on blood pressure (n 5 3). Continuous infusion of a NOS inhibitor (L-NAME: 20 mM, n 5 5) did not alter the ICI. The effects of the NO donor (SNAP: 2 mM, n 5 5 and SNP: 1 mM, n 5 2) were not observed in control rats (table 1). Isovolumetric conditions. Under isovolumetric conditions, bladder contraction frequency was significantly greater in CYP-treated rats (0.83 6 0.30 contractions/min, n 5 8) than in control rats (0.37 6 0.10 contractions/min, n 5 8) (fig. 3, B). In CYP-treated animals, bladder contraction frequency significantly (p ,0.05) decreased from 0.93 6 0.33 to 0.14 6 0.09 contractions/min following administration of an NO donor (SNAP: 2 mM., n 5 4) (table 2). This effect was observed within 5 to 10 minutes after NO donor application. After removal of the drug solution from the bladder and replacement with saline (0.8 ml.), the decreased contraction fre-
FIG. 5. Effects of intravesical administration of NO donor (SNAP) on isovolumetric condition CMG. Upper trace: control period. Regular bladder contractions were observed. Puncture of bladder wall with 30G needle did not alter contraction interval. Middle trace; 10 minutes after SNAP administration. Contraction interval was increased and subsequently contractions vanished. Lower trace: 10 minutes after switching bladder contents to 0.8 ml. saline. Bladder contractions were partially restored. These treatments did not change amplitude of bladder contractions. Scale bars: 2 minutes (horizontal) and 20 cm. water (vertical).
quency usually returned to the control value in 10 to 15 minutes (fig. 5). However, these treatments did not change the amplitude of bladder contractions. The effects of the NO donor (SNAP: 2 mM, n 5 4) were not observed in control rats. Intravesical L-NAME (20 mM) did not alter any CMG parameters in CYP-treated rats (table 2). Patch clamp recording. Fig. 6 shows the effect of SNAP (500 mM) on Ca21 channel currents in a bladder afferent neuron. The currents were activated by depolarizing pulses to 0 mV from the holding potentials of – 60 mV. In this
TABLE 1. Effects of NO donors (SNAP: 2 mM and SNP: 1 mM) and a NOS inhibitor (L-NAME: 20 mM) on reflex bladder contractions during continuous infusion CMG in control and CYP-treated rats Control
CYP-treated
Saline NO-donor (n 5 7) Saline L-NAME (n 5 5) Saline NO-donor (n 5 7) Saline L-NAME (n 5 5)
ICI (min.)
Amplitude (cm. H2O)
Duration (min.)
7.2 6 0.8 5.4 6 0.9 7.8 6 1.3 5.7 6 1.3 5.2 6 0.9 9.0 6 2.2* 6.8 6 0.9 5.9 6 1.3
34 6 4 35 6 3 38 6 2 33 6 4 23 6 3 21 6 3 28 6 3 29 6 3
0.78 6 0.11 0.73 6 0.10 0.75 6 0.12 0.75 6 0.08 0.99 6 0.09 1.12 6 0.11 0.90 6 0.10 0.95 6 0.11
mean 6 S.E. ICI: Intercontraction interval. * p , 0.05; infusion of NO donors (SNP: n 5 2 and SNAP: n 5 5) versus saline in CYP injected rats.
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FIG. 6. Effects of NO donor (SNAP) on HVA Ca21 channel currents activated from holding potential of 260 mV in patch clamp recordings. A, superimposed currents before (control) and 3 minutes after SNAP application (SNAP, 500 mM), and after washout (recovery). B, time course of changes in peak current amplitude of Ca21 channel currents following SNAP application.
bladder afferent neuron, 3 minutes after an application of SNAP peak Ca21 channel currents were suppressed by 38%, and then recovered following washout. The mean reduction of Ca21 channel currents by SNAP was 30.5 6 4.1% in 8 bladder afferent neurons. DISCUSSION
Rats with CYP-induced cystitis have frequently been used as an animal model of visceral pain.8, 12–14 Cystitis is always painful, even in the absence of bladder filling or distention. After CYP injection, animals display the characteristic immobile rounded back posture, with the head aligned with the body axis. Histological evaluation of the bladder following intraperitoneal injection of CYP revealed extensive, severe mucosal degeneration accompanied by submucosal edema.15 Other studies as well as the present study also indicated that CYP treatment induces increased voiding frequency in awake rats and bladder hyperactivity in anesthetized rats.12–14 The NO-synthesizing enzyme, nitric oxide synthase (NOS), and NADPH diaphorase, a chemical marker for NOS are localized in afferent and efferent neurons innervating the urinary tract and nerve fibers in the detrusor, trigone, and urethra.16, 17 NOS seems to be colocalized with acetylcholine esterase, vasoactive intestinal polypeptide, and neuropeptide Y, suggesting that NO may have a role both as a directly acting transmitter and as a modulator of efferent neurotransmission.18 It was initially speculated that NO released from nerves in the detrusor could be one factor relaxing bladder smooth muscle during filling; however, it was later shown that the normal detrusor muscle has a low sensitivity to NO as well as other agents acting via the cyclic GMP system, suggesting that NO is unlikely to have a role as an inhibitory neurotransmitter in this tissue.3, 5, 7 At least three types of NOS have been so far identified. Two types (neuronal (nNOS) and endothelial NOS (eNOS)), which are constitutive (cNOS) and Ca21/calmodulindependent, release NO for short periods in response to stimulation. The other enzyme is inducible (iNOS), Ca21independent and, once expressed, generates NO for long periods.19 The cNOS has been identified in vascular endothelium and brain, while iNOS is expressed after stimulation with bacterial lipopolysaccharide (LPS) or some cytokines in endothelial cells, polymorphonuclear leukocytes, macrophages and smooth muscle cells. NOS activity in the normal bladder is mainly due to the cNOS (calcium dependent) isoforms (.95%). nNOS has been detected in urothelial cells
and NO can be released from these cells by various chemical stimuli (for example, norepinephrine, acetylcholine and capsaicin).20, 21 However, CYP administration significantly increases the activity of iNOS (calcium independent), which is evident within 6 hours after injection and remains elevated for up to 48 hours.22 The iNOS in the bladder is mainly expressed in the urothelium and is unregulated with inflammation.23 High basal release of NO mediated by iNOS has been detected in bladder strips from rats treated with CYP.24 Thus, it has been postulated that NO, which is produced by upregulated iNOS, participates in the induction of cystitis following CYP administration.22 However, L-NAME, a NOS inhibitor, administered intravesically did not change any parameter in continuous infusion or isovolumetric CMGs in the present study, suggesting that endogenous NO may not be responsible for the emergence of CYP-induced bladder hyperactivity, although it is possible that intravesically applied L-NAME does not penetrate the urothelium and enter the bladder mucosal layer. On the other hand, the present results indicate that exogenously applied NO can produce detrusor stabilization in rats with CYP-induced cystitis. During continuous infusion CMGs, NO donors reversibly increased the intercontraction interval. Thus it is plausible that topical NO donor application might be beneficial for the treatment of bladder hyperactivity associated with bladder inflammation. Similar therapeutic effects on cystitis-induced bladder hyperactivity were also reported in recent clinical studies in which oral L-arginine that is converted to NO by NOS improved irritative bladder symptoms in patients with interstitial cystitis.25 As for the mechanism of NO-mediated suppression of bladder hyperactivity, one might argue that the effect of NO donors on bladder activity is due to an effect of NO on a urethro-bladder reflex after the drugs enter the urethra. Therefore, we conducted studies under isovolumetric conditions where NO was generated only in the bladder. Since similar results were obtained in the continuous infusion CMG and isovolumetric CMG studies, it is assumed that NO-mediated suppression of bladder hyperactivity in CYPtreated rats was mediated by a direct effect on the bladder. It is possible that NO-induced suppression of bladder hyperactivity is due to a direct effect on bladder smooth muscle. However, this seems unlikely because the amplitude of bladder contractions in control and CYP-treated rats was not changed by NO donors, whereas the ICI was increased in CYP-treated rats. This effect is similar to the effect of capsaicin and ZD6169, a KATP channel agonist, which have been
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INTRAVESICAL NITRIC OXIDE THERAPY 12, 26
shown to act on bladder afferents. Thus it seems reasonable to propose that NO donors may alter CYP-induced bladder hyperactivity by altering the excitability of bladder afferent nerves. Because these drugs did not alter the ICI in control rats, it is also tempting to speculate that the drugs do not influence the mechanoceptive afferents that trigger normal micturition, but rather depress the excitability of capsaicin-sensitive C-fiber afferents which have been activated by CYP and which are responsible for the hyperactive voiding.27 Previous studies shown that capsaicin treatment also reduces CYP-induced bladder hyperactivity.12 The assumption that NO donors can influence afferent excitability is supported by a direct action of an NO donor on bladder afferent neurons in the patch-clamp recording study. SNAP applied to dissociated bladder afferent neurons suppressed high-voltage-activated Ca21 channel currents by approximately 30%, suggesting that NO could have a modulatory effect on the bladder afferent pathway. Calcium ion influx into the afferent neuron terminals can trigger the release of peptides such as substance P or calcitonin gene related polypeptides which induce a local inflammatory response and bladder hyperreflexia.18, 27 Thus it is tempting to speculate that the inhibitory effect of NO donors on CYPinduced bladder hyperactivity is at least in part mediated by a direct modulation of Ca21 channel activity in bladder afferent nerves located adjacent to the urothelium.9, 28 The nitric oxide system is ubiquitous in the body and its regulation is complex. In addition to inhibitory effect of NO donors on bladder hyperactivity as seen in our studies, NO is reportedly involved in the emergence of cystitis-induced bladder hyperactivity by enhancing central neurotransmission in the spinal cord.6 In addition it has been documented that NOS immunoreactivity in bladder afferent neurons increased in the rat with CYP-induced chronic cystitis.8 Thus the long-term effect of CYP on the NO system in the lower urinary tract might be different from the acute effect of CYP shown in this study. We are pursuing further experiments using animals with chronic 2 weeks CYP treatment to determine whether NO therapy also has a beneficial effect on bladder dysfunction following long-term bladder inflammation. CONCLUSIONS
Intravesical application of NO donors can acutely and effectively suppress the CYP-induced bladder hyperactivity. It is likely that the effect of a topical NO donor is due to suppression of bladder afferent nerve function, but not due to a smooth muscle relaxation. NO donors may be considered as a possible treatment of CYP-induced and other types of cystitis. REFERENCES
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8. Vizzard, M. A., Erdman, S. L. and de Groat, W. C.: Increased expression of neuronal nitric oxide synthesis in bladder afferent pathway following chronic bladder irritation. J. Comp. Neurol., 370: 191, 1996. 9. Yoshimura, N. and de Groat, W. C.: Modulation of Ca21 currents by nitric oxide (NO) donors in rat dorsal root ganglion neurons. Soc. Neurosci. Abstr., 23: 1185, 1997. 10. Chancellor, M. B., Rivas, D. A., Huang, B., Kelly, G. and Salzman, S. K.: Micturition patterns after spinal trauma as a measure of autonomic functional recovery. J. Urol., 151: 250, 1994. 11. Yoshimura, N., White, G., Weight, F. F. and de Groat, W. C.: Different types of Na1 and K1 currents in rat dorsal root ganglion neurones innervating the urinary bladder. J. Physiol. (London), 494: 1, 1996. 12. Maggi, C. A., Lecci, A., Santiciolo, P., Bianco, E. D. and Giuliani, S.: Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive afferents. J. Auton. Nerv. Sys., 38: 201, 1992. 13. Lecci, A., Giuliani, S., Santiciolo, P. and Maggi C. A.: Involvement of spinal tachykinin NK1 and NK2 receptors in detrusor hyperreflexia during chemical cystitis in anesthetized rats. Eur. J. Pharmacol., 259: 129, 1994. 14. Lanteri-Minet, M., Bon, K., de Pommery, J., Michael, J. F. and Menetrey, D.: Cyclophosphamide cystitis as a model of visceral pain in rats: Model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins. Exp. Brain Res., 105: 220, 1995. 15. Safron, J., Rice, D., Gordon, D., Leaf, C. and White, R.: Protective effect of L-2-oxothiazolidine-4-carboxylate treatment on cyclophosphamide-induced cystitis in rats. J. Urol., 157: 1946, 1997. 16. Vizzard, M. A., Erdman, S. L. and de Groat, W. C.: Localization of NADPH-diaphorase in bladder afferent and postganglionic efferent neurons of the rat. J. Auton. Nerv. Sys., 44: 85, 1993. 17. Vizzard, M. A., Erdman, S. L., Forstermann, U. and de Groat, W. C.: Differential distribution of nitric oxide synthase in neural pathways to the urogenital organs (urethra, penis, urinary bladder) of rats. Brain Res., 646: 279, 1994. 18. Lundberg, J. M.: Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acid and nitric oxide. Pharmacol. Rev., 48: 113, 1996. 19. Sautebin, L., Ialenti, A., Ianaro, A. and Di Rosa, M.: Modulation by nitric oxide of prostaglandin biosynthesis in the rat. Br. J. Pharmacol., 114: 323, 1995. 20. Birder, L. A., Kanai, A. J. and de Groat, W. C.: DMSO: effect on bladder afferent neurons and nitric oxide release. J. Urol., 158: 1989, 1997. 21. Birder, L. A., Apodaca, G., de Groat, W. C. and Kanai, A. J.: Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am. J. Physiol., 275: F226, 1998. 22. Souza-Filho, M. V. P., Lima, M. V. A., Pompeu, M. M. L., Ballejo, G., Chunha, F. Q. and Ribeiro, R. A.: Involvement of nitric oxide in pathogenesis of cyclophosphamide-induced hemorrhagic cystitis. Am. J. Pathol., 150: 247, 1997. 23. Lundberg, J. O. N., Ehren, I., Jansson, O., Adolfsson, J., Lundberg, J. M., Weitzberg, E., Alving, K. and Wiklund, N. P.: Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis. Urology, 48: 700, 1996. 24. Birder, L. A., Kanai, A. J. and de Groat, W. C.: Porphyrinic microsensor measurements of Ca21 independent nitric oxide release from inflamed urinary bladder. Soc. Neurosci. Abstr., 23: 1523, 1997. 25. Smith, S. D., Wheeler, M. A., Foster, H. E. and Weiss, R. M.: Improvement in interstitial cystitis symptom scores during treatment with oral L-arginine. J. Urol., 158: 703, 1997. 26. Yu, Y. and de Groat, W. C.: Effects of ZD6169, a KATP channel opener, on bladder hyperactivity and spinal c-fos expression evoked by bladder irritation in rats. Brain Res., 807: 11, 1998. 27. Yoshimura, N. and de Groat, W. C.: Neural control of the lower urinary tract. Int. J. Urol., 4: 111, 1997. 28. Lincoln, J. and Burnstock, G.: Autonomic innervation of the urinary bladder and urethra. In: The Autonomic Nervous System, vol. 3, Nervous Control of the Urogenital System, chapt. 2. Edited by C. A. Maggi. London: Harwood Academic Publishers, pp. 33– 68, 1993.
Vol. 162, 2211–2216, December 1999 Printed in U.S.A.
EFFECT OF INTRAVESICAL NITRIC OXIDE THERAPY ON CYCLOPHOSPHAMIDE-INDUCED CYSTITIS HIDEO OZAWA,* MICHAEL B. CHANCELLOR, SUK-YOUNG JUNG, TERUHIKO YOKOYAMA, MATTHEW O. FRASER, YONGBEI YU, WILLIAM C. DE GROAT AND NAOKI YOSHIMURA From the Division of Urologic Surgery and Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
ABSTRACT
Purpose: This study was conducted to examine effects of nitric oxide (NO) donors on bladder hyperactivity induced by cyclophosphamide (CYP)-induced cystitis. Materials and Methods: Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (100 mg./kg.), and then their micturition pattern including mean micturition volume and the number of micturitions during 24 hours was recorded in a metabolic cage before and after CYP treatment. Forty-eight hours after CYP injection, bladder function under urethane anesthesia was evaluated by cystometry with continuous saline infusion (0.04 ml. per minute) or under isovolumetric conditions (0.8 ml. bladder volume). NO donors, S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or sodium nitroprusside (SNP, 1 mM), and an NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 20 mM) were administered intravesically. Direct action of SNAP on bladder afferent neurons was also tested in a patch-clamp recording study. Results: The number of micturitions significantly increased during the first 24 hours after CYP injection (19.0 6 0.88 versus 92.1 6 16.3 micturitions/24 hours, mean 6 SE, n 5 25) (p ,0.001). There was no significant difference in total micturition volume before (12.3 6 1.0 ml./24 hours) and after CYP treatment (15.6 6 1.5 ml./24 hours). During continuous infusion cystometry, intercontraction interval (ICI) was smaller in CYP-injected rats than in control rats. In CYPinjected animals, NO donors increased the ICI, but did not change the amplitude of bladder contractions. Continuous intravesical infusion of the NOS inhibitor did not alter the cystometric parameters. During cystometry under isovolumetric conditions, contraction frequency was decreased after NO donor administration. NO donors did not influence bladder activity in control rats. In patch clamp recordings, when SNAP (500 mM) was directly applied to dissociated afferent neurons innervating the urinary bladder, high-voltage-activated Ca21 channel currents were suppressed by approximately 30%. Conclusions: Intravesical NO donors can suppress CYP-induced bladder hyperactivity. We hypothesize that the effect of NO donors is not due to smooth muscle relaxation, but rather due to an inhibitory effect on bladder afferent pathways that was manifested by an increase in intercontraction interval without changes in contraction amplitude. NO donors may be considered as a possible treatment of CYP-induced and other types of bladder inflammation. KEY WORDS: nitric oxide, cyclophosphamide, urinary bladder, inflammation, detrusor hyperactivity, bladder afferent neurons
Cyclophosphamide (CYP) is a nitrogen mustard-type chemotherapeutic agent, which is used for the treatment of neoplastic diseases such as Hodgkin’s disease, Burkitt’s lymphoma, leukemias and cancer of the breast, lung, cervix, ovary and brain. However, there are major side effects of CYP therapy, including hemorrhagic cystitis which is difficult to treat. According to Stillwell et al,1 major symptoms of hemorrhagic cystitis are gross hematuria (78%) and irritative voiding symptoms (45%). A significant number of patients (9%) with CYP-induced cystitis are refractory to conventional therapies and undergo emergency life-saving cystectomy. Thus, better therapy for CYP-induced cystitis is a high medical priority. Nitric oxide (NO), a toxic gas with free-radical properties,
has been implicated as a neurotransmitter or neuromodulator at various sites in the mammalian nervous system.2 Pharmacological studies have provided evidence that NO is a transmitter mediating penile erection and relaxation of the urethral smooth muscle, but the effect of NO on the urinary bladder is uncertain.3 It was initially thought that NO directly mediates detrusor relaxation, but this effect was found to be small.3–7 However, recent studies in rats indicate that bladder inflammation upregulates the expression of NO synthase (NOS) in bladder afferent neurons, and that NO produced in bladder afferent pathways might be involved in the regulation of micturition reflex following cystitis,6, 8, 9 although there have been no reports on the topical effect of intravesically administrated NO donors on the bladder function. The present study was therefore undertaken to examine the direct effect of NO donors on bladder function under inflammatory conditions. The effects of NO donors, S-nitrosoN-acetyl-penicillamine (SNAP), and sodium-nitroprusside (SNP), as well as the NOS inhibitor, N-nitro-L-arginine
Accepted for publication July 12, 1999. * Requests for reprints: Urologic Surgery, University of Pittsburgh School of Medicine, Suite 700, Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213. Supported by a Grant from the National Institutes of Health (DK-49430). 2211
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methyl ester (L-NAME) were evaluated in rats with CYPinduced cystitis. We also performed an electrophysiological study using patch-clamp recording techniques to examine the direct effect of SNAP on bladder afferent neurons. MATERIALS AND METHODS
Animals. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 250 to 300 gm. were used, at a minimum of 2 days after their arrival. A 12:12 hour lightdark cycle was used. Animals received a single intraperitoneal injection of CYP (100 mg./kg.) dissolved in distilled water in a concentration of 40 mg./ml., and then the micturition pattern was monitored in a metabolic cage. Since reduced voided volume per micturition was detected on day 1 (0 to 24 hours) and day 2 (24 to 48 hours) after CYP injection, bladder function was monitored under urethane anesthesia during continuous infusion cystometrograms (CMG) or under isovolumetric conditions 48 hours after CYP injection. The research protocol was approved by our Institutional Animal Care and Use Committee, and adhered to the guidelines set forth in the U.S. Department of Health and Human Services’ “Guide for the Care and Use of Laboratory Animals.” Micturition pattern. Rats were placed in a metabolic cage (Nalgene metabolic cage, Nalge Co., Rochester, NY) that measures voided urine for a 24-hour period.10 Urine was collected by a specially equipped cup on a transducer (Force displacement transducer FT03, Grass Instruments Co., Quincy, MA) connected to a microcomputer for the recording of micturition frequency, duration and volume. Data were recorded and stored using data acquisition software (Windaq, DATAQ Instruments Inc., Akron, OH). Micturition parameters evaluated were: 1) total urine output per 24 hours; 2) number of micturitions per 24 hours; 3) voided volume/micturition. In 25 animals, micturition patterns were recorded before and during 48 hours after CYP injection; and in 7 of these animals the micturition patterns were recorded for 7 consecutive days after CYP injection. Continuous infusion CMG. Animals were anesthetized with subcutaneous injection of urethane (1.2 gm./kg.; Sigma Chemical Co., St. Louis, MI). Animals with no treatment were used as controls. A cannula (PE-50, Clay-Adams, Parsippany, NJ) was placed in the external jugular vein as an venous line. In some animals, a cannula was also inserted in the carotid artery for monitoring blood pressure. A transure-
thral bladder catheter (PE-50) connected to a pressure transducer was used to record bladder pressure. The bladder was filled with a constant infusion of saline or drug solutions and allowed to empty around the catheter. A continuous CMG was performed by constant infusion (0.04 ml. per minute) of fluid into the bladder to elicit repetitive micturitions, which allowed rapid collection of data for a large number of voiding cycles. The parameters evaluated were amplitude and duration of reflex bladder contractions, and intercontraction interval (ICI), which is defined as the time between two voiding cycles. SNAP, SNP and L-NAME were administered into the bladder for 30 minutes. CMG parameters were monitored for 25 minutes beginning at 5 minutes after switching of the infusion fluid, and compared with the recordings before drug application. Isovolumetric CMG recording. Anesthesia and surgical procedures were the same as those described above. After inserting a PE-50 catheter into the urethra, the urethra was ligated to prevent the leakage around the catheter. The ureters were tied and cut, and then the proximal ends were cannulated (PE-10) and drained externally to prevent the accumulation of urine in the bladder. After the bladder was filled by 0.8 ml. saline, repetitive isovolumetric contractions were observed. To administer drugs, the bladder was punctured with a 30G needle connected to PE-10 cannulae (20 ml. volume dead space). Before drug administration, 20 ml. of bladder contents was removed, and 40 ml. of drug solution (20 ml. for bladder injection and 20 ml. for filling the dead space of connected cannulae) was then injected through the 30G needle using a Hamilton syringe. In these conditions, the final drug concentrations approximated those used during continuous infusion CMGs, after diluted by saline in the bladder. The following parameters were evaluated: amplitude and duration of isovolumetric bladder contractions and frequency of contractions. SNAP, SNP and L-NAME were applied for 30 minutes. CMG parameters were monitored for 25 minutes beginning at 5 minutes after an injection of drug, and compared with the recordings before drug application. After completion of recordings with drug-containing solutions, the bladder was emptied by aspiration and refilled with 0.8 ml. saline. Administration of drugs. Drugs used in this study included S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME)
FIG. 1. Twenty four-hour micturition pattern. A, before injection. B, one day after CYP (100 mg./kg.) injection. Stepwise increments in trace indicate voidings. Note that urinary frequency increased at night.
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(Sigma Chemical Co., St. Louis, MI). SNAP was dissolved in saline with 0.5% DMSO, and SNP and L-NAME were dissolved in saline. All drugs were freshly prepared before each experiment. SNAP and SNP solutions were protected from light during bladder infusion. During continuous infusion CMG, SNAP (2 mM), and SNP (1 mM), as well as L-NAME (20 mM) were administered into the bladder through transurethral catheter. During isovolumetric CMG recording, 20 ml. of SNAP (80 mM) or L-NAME (800 mM) was injected into the bladder, and then diluted by the bladder contents (0.8 ml. of saline) to the same concentration as in a continuous infusion CMG. DMSO (0.5%) alone had no effects on CMG parameters such as bladder contraction pressure and intercontraction interval. Patch clamp recording. Dorsal root ganglion (DRG) neurons which innervate the urinary bladder were labeled by retrograde axonal transport of a fluorescent dye, Fast Blue (4% W/V) injected into the wall of the bladder.11 The dye was injected with a 28 G needle at three to six sites on the dorsal surface of the bladder (5 to 6 ml. per site, total volume of 20 to 30 ml.). DRG at the level of L6 and S1 were then removed under halothane anesthesia, and dissociated in a shaking bath for 25 minutes at 35C with 5 ml. of Dulbecco’ s modified essential medium containing trypsin (0.3 mg./ml.), collagenase (1 mg./ml.) and deoxyribonuclease (0.1 mg./ml.).11 Trypsin inhibitor was then added to neutralize the activity of trypsin. Thereafter, dye-labeled bladder afferent neurons were identified using an inverted phase contrast microscope
FIG. 3. Changes in CMG parameters after CYP injection. A, intercontraction interval in continuous CMG (n 5 12). B, contraction frequency in CMG under isovolumetric conditions (n 5 8).
with fluorescent attachments. Whole-cell recordings were performed with patch electrodes which usually have resistance of 1 to 4 Mohm when filled with the internal solution. The internal solution contained (mM) KCl 140, CaCl2 1, MgCl2 2, EGTA 11, HEPES 10 and Mg-ATP 2 adjusted to pH 7.4 with KOH (310 mosml/l). To isolate Ca21 channel currents, neurons were superfused at a flow rate of 1.5 ml. per minute with an external solution containing BaCl2 5, tetraethylammonium-Cl 155, 4-aminopyridine 5, HEPES 10 adjusted to pH 7.4 with TEA-OH (340 mOsm). An NO donor (SNAP, 500 mM) was administered to neurons using pressure injection methods through a glass pipette positioned close to the cells. Statistical analysis. All data are expressed as mean 6 SE. Student’s t test was used for comparison of 24 hour voiding pattern and CMG parameters. A p-value less than 0.05 was considered significant. RESULTS
FIG. 2. Changes in voiding patterns after CYP injection. A, changes in voiding frequency. B, voided volume per micturition. Data before and on day 1 and day 2 after CYP injection were obtained from 25 animals, and data on days 3, 5 and 7 were from 7 animals.
Micturition pattern. Fig. 1 shows representative 24-hour micturition patterns before (fig. 1, A) and after CYP injection (fig. 1, B). Increased frequency of voiding and decreased volume of voided urine were observed after CYP injection. The number of micturitions per 24 hours was significantly greater for 24 hours after CYP injection (92.1 6 16.3 micturi-
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INTRAVESICAL NITRIC OXIDE THERAPY TABLE 2. Effects of an NO donor (SNAP: 2 mM) and a NOS inhibitor (L-NAME: 20 mM) on reflex bladder contractions under isovolumetric conditions in control and CYP-treated rats
Control
CYP-treated
Saline SNAP (n 5 4) Saline L-NAME (n 5 4) Saline SNAP (n 5 4) Saline L-NAME (n 5 4)
Frequency (Contractions/min.)
Amplitude (cm. H2O)
0.37 6 0.10 0.47 6 0.08 0.32 6 0.09 0.26 6 0.08 0.93 6 0.33 0.14 6 0.09* 0.71 6 0.24 0.51 6 0.33
45 6 10 44 6 9 48 6 10 49 6 10 32 6 5 30 6 4 32 6 5 32 6 6
mean 6 S.E. * p , 0.05; infusion of SNAP versus saline in CYP injected rats.
FIG. 4. Effects of intravesical administration of NO donor (SNAP) on continuous infusion CMG. Upper trace: control period. Regular bladder contractions were observed. Lower trace: 10 minutes after SNAP injection. SNAP increased intercontraction interval without changing detrusor contraction amplitude. Scale bars: 2 minutes (horizontal) and 20 cm. water (vertical).
tions, n 5 25) than before injection (19.0 6 0.88 micturitions, n 5 25) (p ,0.001) (fig. 2). There was no difference between total micturition volumes before (12.3 6 1.0 ml./24 hours) and after CYP injection (15.6 6 1.5 ml./24 hours). The voided volume per micturition was significantly lower after CYP injection (day 1: 0.29 6 0.04 ml., day 2: 0.34 6 0.08 ml.) when compared with that obtained before CYP injection (0.65 6 0.04 ml.). Micturition frequency and volume per micturition returned to normal 3 days after CYP injection (fig. 2). Continuous infusion CMG. ICI was significantly smaller in CYP-treated rats (5.4 6 0.8 minutes, n 5 12) than in control rats (7.4 6 0.7 minutes, n 5 12) (fig. 3, A). In CYP-treated animals, NO donors (SNAP: 2 mM, n 5 5 and SNP: 1 mM, n 5 2) significantly increased the ICI from 5.2 6 0.9 minutes to 9.0 6 2.2 minutes, but did not change the amplitude of bladder contractions (fig. 4 and table 1). This effect was usually observed within 5 minutes after switching to a solution containing NO donors and disappeared 15 to 30 minutes following washout. Intravesical instillation of SNAP and SNP had no effects on blood pressure (n 5 3). Continuous infusion of a NOS inhibitor (L-NAME: 20 mM, n 5 5) did not alter the ICI. The effects of the NO donor (SNAP: 2 mM, n 5 5 and SNP: 1 mM, n 5 2) were not observed in control rats (table 1). Isovolumetric conditions. Under isovolumetric conditions, bladder contraction frequency was significantly greater in CYP-treated rats (0.83 6 0.30 contractions/min, n 5 8) than in control rats (0.37 6 0.10 contractions/min, n 5 8) (fig. 3, B). In CYP-treated animals, bladder contraction frequency significantly (p ,0.05) decreased from 0.93 6 0.33 to 0.14 6 0.09 contractions/min following administration of an NO donor (SNAP: 2 mM., n 5 4) (table 2). This effect was observed within 5 to 10 minutes after NO donor application. After removal of the drug solution from the bladder and replacement with saline (0.8 ml.), the decreased contraction fre-
FIG. 5. Effects of intravesical administration of NO donor (SNAP) on isovolumetric condition CMG. Upper trace: control period. Regular bladder contractions were observed. Puncture of bladder wall with 30G needle did not alter contraction interval. Middle trace; 10 minutes after SNAP administration. Contraction interval was increased and subsequently contractions vanished. Lower trace: 10 minutes after switching bladder contents to 0.8 ml. saline. Bladder contractions were partially restored. These treatments did not change amplitude of bladder contractions. Scale bars: 2 minutes (horizontal) and 20 cm. water (vertical).
quency usually returned to the control value in 10 to 15 minutes (fig. 5). However, these treatments did not change the amplitude of bladder contractions. The effects of the NO donor (SNAP: 2 mM, n 5 4) were not observed in control rats. Intravesical L-NAME (20 mM) did not alter any CMG parameters in CYP-treated rats (table 2). Patch clamp recording. Fig. 6 shows the effect of SNAP (500 mM) on Ca21 channel currents in a bladder afferent neuron. The currents were activated by depolarizing pulses to 0 mV from the holding potentials of – 60 mV. In this
TABLE 1. Effects of NO donors (SNAP: 2 mM and SNP: 1 mM) and a NOS inhibitor (L-NAME: 20 mM) on reflex bladder contractions during continuous infusion CMG in control and CYP-treated rats Control
CYP-treated
Saline NO-donor (n 5 7) Saline L-NAME (n 5 5) Saline NO-donor (n 5 7) Saline L-NAME (n 5 5)
ICI (min.)
Amplitude (cm. H2O)
Duration (min.)
7.2 6 0.8 5.4 6 0.9 7.8 6 1.3 5.7 6 1.3 5.2 6 0.9 9.0 6 2.2* 6.8 6 0.9 5.9 6 1.3
34 6 4 35 6 3 38 6 2 33 6 4 23 6 3 21 6 3 28 6 3 29 6 3
0.78 6 0.11 0.73 6 0.10 0.75 6 0.12 0.75 6 0.08 0.99 6 0.09 1.12 6 0.11 0.90 6 0.10 0.95 6 0.11
mean 6 S.E. ICI: Intercontraction interval. * p , 0.05; infusion of NO donors (SNP: n 5 2 and SNAP: n 5 5) versus saline in CYP injected rats.
INTRAVESICAL NITRIC OXIDE THERAPY
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FIG. 6. Effects of NO donor (SNAP) on HVA Ca21 channel currents activated from holding potential of 260 mV in patch clamp recordings. A, superimposed currents before (control) and 3 minutes after SNAP application (SNAP, 500 mM), and after washout (recovery). B, time course of changes in peak current amplitude of Ca21 channel currents following SNAP application.
bladder afferent neuron, 3 minutes after an application of SNAP peak Ca21 channel currents were suppressed by 38%, and then recovered following washout. The mean reduction of Ca21 channel currents by SNAP was 30.5 6 4.1% in 8 bladder afferent neurons. DISCUSSION
Rats with CYP-induced cystitis have frequently been used as an animal model of visceral pain.8, 12–14 Cystitis is always painful, even in the absence of bladder filling or distention. After CYP injection, animals display the characteristic immobile rounded back posture, with the head aligned with the body axis. Histological evaluation of the bladder following intraperitoneal injection of CYP revealed extensive, severe mucosal degeneration accompanied by submucosal edema.15 Other studies as well as the present study also indicated that CYP treatment induces increased voiding frequency in awake rats and bladder hyperactivity in anesthetized rats.12–14 The NO-synthesizing enzyme, nitric oxide synthase (NOS), and NADPH diaphorase, a chemical marker for NOS are localized in afferent and efferent neurons innervating the urinary tract and nerve fibers in the detrusor, trigone, and urethra.16, 17 NOS seems to be colocalized with acetylcholine esterase, vasoactive intestinal polypeptide, and neuropeptide Y, suggesting that NO may have a role both as a directly acting transmitter and as a modulator of efferent neurotransmission.18 It was initially speculated that NO released from nerves in the detrusor could be one factor relaxing bladder smooth muscle during filling; however, it was later shown that the normal detrusor muscle has a low sensitivity to NO as well as other agents acting via the cyclic GMP system, suggesting that NO is unlikely to have a role as an inhibitory neurotransmitter in this tissue.3, 5, 7 At least three types of NOS have been so far identified. Two types (neuronal (nNOS) and endothelial NOS (eNOS)), which are constitutive (cNOS) and Ca21/calmodulindependent, release NO for short periods in response to stimulation. The other enzyme is inducible (iNOS), Ca21independent and, once expressed, generates NO for long periods.19 The cNOS has been identified in vascular endothelium and brain, while iNOS is expressed after stimulation with bacterial lipopolysaccharide (LPS) or some cytokines in endothelial cells, polymorphonuclear leukocytes, macrophages and smooth muscle cells. NOS activity in the normal bladder is mainly due to the cNOS (calcium dependent) isoforms (.95%). nNOS has been detected in urothelial cells
and NO can be released from these cells by various chemical stimuli (for example, norepinephrine, acetylcholine and capsaicin).20, 21 However, CYP administration significantly increases the activity of iNOS (calcium independent), which is evident within 6 hours after injection and remains elevated for up to 48 hours.22 The iNOS in the bladder is mainly expressed in the urothelium and is unregulated with inflammation.23 High basal release of NO mediated by iNOS has been detected in bladder strips from rats treated with CYP.24 Thus, it has been postulated that NO, which is produced by upregulated iNOS, participates in the induction of cystitis following CYP administration.22 However, L-NAME, a NOS inhibitor, administered intravesically did not change any parameter in continuous infusion or isovolumetric CMGs in the present study, suggesting that endogenous NO may not be responsible for the emergence of CYP-induced bladder hyperactivity, although it is possible that intravesically applied L-NAME does not penetrate the urothelium and enter the bladder mucosal layer. On the other hand, the present results indicate that exogenously applied NO can produce detrusor stabilization in rats with CYP-induced cystitis. During continuous infusion CMGs, NO donors reversibly increased the intercontraction interval. Thus it is plausible that topical NO donor application might be beneficial for the treatment of bladder hyperactivity associated with bladder inflammation. Similar therapeutic effects on cystitis-induced bladder hyperactivity were also reported in recent clinical studies in which oral L-arginine that is converted to NO by NOS improved irritative bladder symptoms in patients with interstitial cystitis.25 As for the mechanism of NO-mediated suppression of bladder hyperactivity, one might argue that the effect of NO donors on bladder activity is due to an effect of NO on a urethro-bladder reflex after the drugs enter the urethra. Therefore, we conducted studies under isovolumetric conditions where NO was generated only in the bladder. Since similar results were obtained in the continuous infusion CMG and isovolumetric CMG studies, it is assumed that NO-mediated suppression of bladder hyperactivity in CYPtreated rats was mediated by a direct effect on the bladder. It is possible that NO-induced suppression of bladder hyperactivity is due to a direct effect on bladder smooth muscle. However, this seems unlikely because the amplitude of bladder contractions in control and CYP-treated rats was not changed by NO donors, whereas the ICI was increased in CYP-treated rats. This effect is similar to the effect of capsaicin and ZD6169, a KATP channel agonist, which have been
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INTRAVESICAL NITRIC OXIDE THERAPY 12, 26
shown to act on bladder afferents. Thus it seems reasonable to propose that NO donors may alter CYP-induced bladder hyperactivity by altering the excitability of bladder afferent nerves. Because these drugs did not alter the ICI in control rats, it is also tempting to speculate that the drugs do not influence the mechanoceptive afferents that trigger normal micturition, but rather depress the excitability of capsaicin-sensitive C-fiber afferents which have been activated by CYP and which are responsible for the hyperactive voiding.27 Previous studies shown that capsaicin treatment also reduces CYP-induced bladder hyperactivity.12 The assumption that NO donors can influence afferent excitability is supported by a direct action of an NO donor on bladder afferent neurons in the patch-clamp recording study. SNAP applied to dissociated bladder afferent neurons suppressed high-voltage-activated Ca21 channel currents by approximately 30%, suggesting that NO could have a modulatory effect on the bladder afferent pathway. Calcium ion influx into the afferent neuron terminals can trigger the release of peptides such as substance P or calcitonin gene related polypeptides which induce a local inflammatory response and bladder hyperreflexia.18, 27 Thus it is tempting to speculate that the inhibitory effect of NO donors on CYPinduced bladder hyperactivity is at least in part mediated by a direct modulation of Ca21 channel activity in bladder afferent nerves located adjacent to the urothelium.9, 28 The nitric oxide system is ubiquitous in the body and its regulation is complex. In addition to inhibitory effect of NO donors on bladder hyperactivity as seen in our studies, NO is reportedly involved in the emergence of cystitis-induced bladder hyperactivity by enhancing central neurotransmission in the spinal cord.6 In addition it has been documented that NOS immunoreactivity in bladder afferent neurons increased in the rat with CYP-induced chronic cystitis.8 Thus the long-term effect of CYP on the NO system in the lower urinary tract might be different from the acute effect of CYP shown in this study. We are pursuing further experiments using animals with chronic 2 weeks CYP treatment to determine whether NO therapy also has a beneficial effect on bladder dysfunction following long-term bladder inflammation. CONCLUSIONS
Intravesical application of NO donors can acutely and effectively suppress the CYP-induced bladder hyperactivity. It is likely that the effect of a topical NO donor is due to suppression of bladder afferent nerve function, but not due to a smooth muscle relaxation. NO donors may be considered as a possible treatment of CYP-induced and other types of cystitis. REFERENCES
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