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Urodynamic Effects of Intravesical Resiniferatoxin and Capsaicin in Conscious Rats With and Without Outflow Obstruction
Vol. 154.611-616. August 1995 Printed i n
USA
URODYNAMIC EFFECTS O F INTRAVESICAL RESINIFERATOXIN AND CAPSAICIN IN CONSCIOUS RATS WITH AND WITHOUT OUTFLOW OBSTRUCTION OSAMU ISHIZUKA, ANDERS MATTIASSON
AND
KARL-ERIK ANDERSSON'!'
F r ( m the Depc~rtrnrntsof Uro1og.v arid Clinical Pharrnocolug.~.Lurid Uriicersity Hospital, L u n d , Swederi
ABSTRACT
Purpose: T h e urodynamic effects of intravesical resiniferatoxin a n d capsaicin were investigated in rats. Materials a n d Methods: Continuous cystometry was performed in conscious, female SpragueDawley rats with a n d without outflow obstruction. Results: Intravesical instillation of resiniferatoxin facilitated micturition. The potency of t h e d r u g was approximately 1,000 times higher than that of capsaicin. Repeated instillations of resiniferatoxin for 6 consecutive days caused desensitization to resiniferatoxin. This w a s not found with repeated instillations of capsaicin. Capsaicin w a s also effective in rats with bladder hypertrophy, while resiniferatoxin w a s not. Conclusions: The findings suggest that resiniferatoxin can induce desensitization of vanilloid receptor-mediated release of tachykinins in the rat urinary bladder and that intravesical resiniferatoxin would be a n interesting alternative to intravesical capsaicin in the t r e a t m e n t of selected cases of bladder hypersensitivityhyperactivity. KEY WORDS:bladder, capsaicin, rats, urethral obstruction In the mammalian bladder and urethra, capsaicin-sensi- rats undergoing continuous cystometry and compared them tive primary afferent neurons are involved in the regulation with those of capsaicin; 2) studied whether the effects of of various micturition-related reflexes.' Capsaicin, instilled resiniferatoxin could be inhibited by SR 48,968; 3) tested the intravesically in conscious normal rats, induced a concentra- desensitizing effects of single and repeated treatments with tion-dependent stimulation of micturition, which, in turn, resiniferatoxin in normal, conscious rats; and 4) tested the was initiated by stimulation of sensory afferents.2 These effects of intravesically instilled capsaicin or resiniferatoxin afferents may be of importance in the pathogenesis of detru- in conscious rats with bladder hyperactivity and hypertrophy sor hyperactivity, such as that induced by irritants in rats.3 secondary to outflow obstruction. The effects of intravesical capsaicin were counteracted by the selective NK, receptor antagonist SR 48,968 given intraMATERIALS AND METHODS arterially, whereas the selective NK,-receptor antagonist RP 67,580 was ineffective,, suggesting that the effect was mediAnimals. Female Sprague-Dawley rats, weighing 200 to ated, a t least partly, by release of neurokinin (NK).A. Maggi 285 g., were used in this study. The experimental protocol et al.* provided evidence for the presence of capsaicin-sensi- was approved by the Animal Ethics Committee, University of tive nerves in the human urinary bladder, where these Lund. nerves probably have functions similar to those described in Procedures: Outlet obstruction procedure. The methods rodents. Desensitization of sensory nerves by intravesical used for establishing infravesical outflow obstruction and the capsaicin infusion was shown to have a therapeutic potential technique of cystometry in awake rats have been described in to improve symptoms in otherwise intractable bladder hyper- detail previously.2 Six weeks after partial ligature of the sensitivity.4 Intravesical capsaicin also improved the control urethra, the animals were subjected to cystometrical evaluof detrusor hyperreflexia in patients with multiple s c l e r ~ s i s . ~ation. Partial obstruction of the urethra induces a significant Recent studies have demonstrated that resiniferatoxin, bladder hypertrophy and hyperactivity. One day after rewhich is a compound isolated from some plants of the genus moval of the ligature, when the animals of this study were Euphorbia, has effects similar to those of capsaicin.6,7When investigated, the bladder still exhibits a significant degree of repetitively administered, both capsaicin and resiniferatoxin hypertrophy and hyperactivity.9 For simplicity, the prevican produce desensitization and an inactivation of sensory ously obstructed rats are referred to as rats with bladder neurons, which may explain the beneficial effects of capsaicin hypertrophy. in cases of bladder hyperactivity. Intravesically infused resBladder catheter implantation. Rats were anesthetized iniferatoxin caused behavioral changes in rats similar t o with ketamine (75 mg./kg. intramuscularly) and xylazine (15 those produced by capsaicin,S suggesting that resiniferatoxin mg./kg. intramuscularly). The abdomen was opened through may induce bladder hyperactivity in the same way as capsa- a midline incision, and a polyethylene catheter (Clay-Adams However, the urodynamic effects of intravesical resinif- PE-50, Parsippany, New Jersey) implanted into the bladder eratoxin do not seem to have been investigated. through the dome as described previously., We therefore 1) studied the effects on the cystometrogram Cystometrical investigations. Cystometric investigations of intravesically instilled resiniferatoxin in normal, conscious were performed without any anesthesia 3 days after bladder catheter implantation in normal rats and 1 day afterward in Accepted for publication February 16, 1995. * Requests for reprints: Department of Clinical Pharmacology, obstructed rats. The bladder catheter was connected via a Lund University Hospital, S-22185,Lund, Sweden. T-tube to a pressure transducer (P23 DC, Statham InstruThis project was supported by the Swedish Medical Research Council (no. 6837 and 10399)and by the Medical Faculty of Lund, ment Inc., Oxnard, California) and a microinjection pump (CMA 100, Carnegie Medicine AJ3, Solna, Sweden). The conSweden. 611
612
INTRAVESICAL CAPSAICIN AND RESINIFERATOWN
scious rat was placed, without any restraint, in a metabolic mg., n = 15, p <0.001),compared with normal controls (123 cage, which also permitted measurements of micturition vol- 2 5 mg., range 90 to 170 mg., n = 21). Bladder capacity umes by means of a fluid collector connected to a Grass force increased from 1.06 5 0.05 ml. ( n = 39) to 3.81 5 0.40 ml. (n displacement transducer (FT 03 C, Grass Instrument CO., = 19). Repeated cystometries gave reproducible results in Quincy, Massachusetts). Room-temperature saline (20 % 1c) both control animals and animals with bladder hypertrophy. was infused into the bladder at a rate of 10 ml. per hour in In control animals, the bladder pressure was low (5.8 % 0.9 normal rats. To achieve an approximately equal number of cm. H,O., n = 48) and almost devoid of spontaneous fluctumicturitions in normal rats and rats with bladder hypertro- ations during the period of cystometry. On the other hand, in phy, the rate of infusion was increased to 20 ml. per hour in animals with bladder hypertrophy, cystometry revealed rats with bladder hypertrophy. Intravesical pressure and spontaneous contractile activity during filling. Effects of intravesically infused capsaicin or resiniferatoxin micturition volumes were recorded continuously on a Grass polygraph (Model 7E, Grass Instrument Co.; recording speed: in normal rats. Intravesical capsaicin increased bladder ac10 mm. per minute). Three reproducible micturition cycles, tivity in a concentration-dependent way. At a concentration corresponding to a 20-minute period, were recorded before of 10 pM. (n = 4), no effects on cystometric parameters were drug administration and used as baseline values. The follow- observed. However, in a concentration of 30 pM., capsaicin ing cystometric parameters were investigated? micturition increased micturition pressure (p <0.001), and decreased pressure (the maximum bladder pressure during micturi- bladder capacity (p <0.001) and micturition volume (p tion), bladder capacity (residual volume a t the preceding <0.001; n = 8; table 1). The animals showed no signs of micturition plus volume of infused saline a t the micturi- distress and behaved normally, except for occasional licking capsaicinI., tion), micturition volume (volume of expelled urine) and of the lower abdomen. At a concentration of 100 @ residual volume (bladder capacity minus micturition vol- evoked stimulation of bladder activity was still more proume). The 3 micturition cycles showing the most pronounced nounced (n = 8; table l), and the rats frequently licked their changes (increase or decrease) within 60 minutes after lower abdomen; no other abnormal behavior was observed. switching saline to capsaicin or resiniferatoxin were ana- The effects of capsaicin were reversible, and the cystometric pattern was restored to the preadministration state within lyzed and compared with the baseline values. Groups of normal rats were investigated for 6 consecutive 40 minutes &r the end of the infusion. The effects of capdays. In these animals, capsaicin or resiniferatoxin was in- saicin could be repeated in the same rat at least 3 times and were still reversible. fused intravesically in concentrations of 100 @. and 100 &., Intravesical resiniferatoxin increased bladder activity in a respectively, and the effects on the cystometrogram and beconcentration-dependent way (table 1).At a concentration of havior were recorded. Zntravesical pretreatment with resiniferatoxin in normal 30 nM., micturition pressure increased (p <0.05), and bladrats. Rats were anesthetized as described above. The abdo- der capacity (p C0.05) and micturition volume (p C0.05; n = men was opened through a midline incision, and the urethra 7) decreased. The animals showed no signs of distress and ligated by 3-zero nylon. A 23-gauge needle was put into the behaved normally, except for occasional licking of the lower dome of the bladder, all urine was aspirated, and 0.3 ml. of abdomen. The urodynamic changes were more pronounced at saline or saline containing resiniferatoxin in concentrations a concentration of 100 nM. (fig. 1; table 11, as were the of 100 nM. or 1000 nM. was instilled. After 60 minutes, the changes in behavior (licking of the lower abdomen). SR ligature of the urethra was released, and saline or resinifera- 48,968, in a concentration of 20 nmol./kg. practically aboltoxin was removed. Four days after the pretreatment with ished the actions of resiniferatoxin 30 nM. (n = 5; table 1)on saline or resiniferatoxin, the rat was anesthetized again, and micturition pressure. In a concentration of 100 nmol./kg., SR a polyethylene catheter (Clay-Adams PE-50) was implanted 48,968 significantly attenuated or abolished the changes ininto the bladder. Cystometric investigations were performed duced by resiniferatoxin 100 nM., not only in micturition by infusing capsaicin 1 week after the pretreatment. pressure, but also in bladder capacity and micturition volume Administration of drugs. Stock solutions (100 pM.) of cap- (n = 5; table 1).Only occasionally did the animals lick the saicin (LabKemi, Lund, Sweden) and resiniferatoxin lower abdomen. (LabKemi) were made in absolute ethanol. The drugs were Effects of intravesically infused capsaicin after pretreatment then stored a t -7OC, and subsequently diluted on the day of with resiniferatoxin in normal rats. Under anesthesia, resexperiment to achieve concentrations of 0.03,0.1, 1.0, 10, 30, iniferatoxin, in concentrations of 100 nM. (n = 5 )or 1000 nM. or 100 pM. Capsaicin or resiniferatoxin was infused intra- (n = 5 ) , was instilled and kept intravesically for 1 hour. vesically by changing the syringe of the microinjection pump. Saline was instilled in controls and kept for the same time During the infusion of resiniferatoxin, the syringes were (n = 12). When the rats recovered from the anesthesia, there protected from light. With the tubing used, drugs reached the were no differences in behavior between the resiniferatoxinbladder in 8.0 2 0.7 minutes after switching from saline to and saline-treated rats. the solution containing capsaicin or resiniferatoxin. After 28 One week after treatment with resiniferatoxin or saline, minutes’ administration of capsaicin or resiniferatoxin, sa- there were no differences in the effect of intravesically inline was again infused. SR 48,968 was given immediately fused capsaicin 100 pM. between the two groups (data not after the start of resiniferatoxin infusion. The doses of the shown). drugs used were chosen on the basis of pilot experiments and Effects of repeated instillations of cupsuicin or resirziferafrom previously published data.2 toxin in normal rats. Intravesical infusion of capsaicin in a Statistical analysis. The results are given as mean values z concentration of 100 kM. (infusion time 28 minutes; n = 6) standard error of the mean. For comparisons between values for 6 consecutive days caused bladder hyperactivity from day obtained before and after drug administration, Student’s paired 1 to day 6. There was a consistent, significant increase in t test was used.One way factorial ANOVA was used for com- micturition pressure. From day 3, there was an increase parisons between capsaicin and capsaicin after pretreatment in bladder capacity before capsaicin infusion; capsaicin sigwith resiniferatoxin and was followed by Scheffe’s F-test. A nificantly decreased micturition volume each time i t was probability level of <5% was accepted as significant. administered. The same changes were observed in micturition volume (data not shown). Licking of the lower abdomen was observed after each administration. RESULTS Resiniferatoxin, instilled in a Concentration of 100 nM. Partial obstruction of the urethra led to a significant in- (n = 61, increased micturition pressurc: o n days 1 and 2 crease in bladder weight (759 % 35 mg., range 520 to 1,000 ( p /0.01;fig. 2 ) and decreased bladder cnliacity 011 day 1
INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN
6 13
cystometric parameters in the absence or presence of intmarterial S R 48,968 in normal conscious mts M.P. B.C. M.V. RV.
TAnLE 1. Effects of intravesical infusion of capsaicin or resiniferatoxin on
Capsaicin 30 pM. (n = 8)
Before After
65.0 2 2.7 83.9 .C 3.2."
1.27 f 0.10 0.64 ? 0.14***
0.23 2 0.03 0.12 f 0.06
1.03 f 0.10 0.62 t 0.10***
100 pM. (n = 8 ) 0.22 f 0.03 Before 66.2 z 7.4 1.13 2 0.12 0.91 2 0.10 0.08 2 0.02*** 108.2 f 7.8*** 0.20 t 0.03*** After 0.26 ? 0.03*** Resiniferatoxin 30 nM. (n = 7) 0.15 f 0.03 Before 67.7 f 9.6 1.17 2 0.15 1.04 2 0.13 0.14 2 0.03 86.8 f 12.01 After 0.88 f 0.20' 0.74 f 0.18' 100 nM. (n = 11) 0.09 f 0.03 Before 73.9 f 8.0 0.88 ? 0.09 0.80 ~t. 0.07 0.08 2 0.02 95.2 f 5.6** 0.43 f O.O5** After 0.52 ? 0.06** Resiniferatoxin plus SR 48,968 (20 nmolJkg.) 30 nM. (n = 5) 0.16 2 0.04 Before 1.08 f 0.20 0.92 f 0.17 63.6 f 6.7 0.16 2 0.07 64.3 2 6.4t 1.01 f 0.18 0.86 2 0.13 After Resiniferatoen plus SR 48,968 (100 nmolhg.) 100 nM. (n = 5) Before 0.09 2 0.02 0.92 f 0.20 72.3 2 9.8 1.01 f 0.19 After 70.8 f 11.77 1.02 f 0.22t 0.w f 0.23tt 0.08 2 0.04 M.P.: micturition pressure (em.H,O); B.C.: bladder capacity (ml.k M.V.:micturition volume (ml.); R.V.: reaidual volume (ml.); Before:before administrati00 o f capsaicin or resiniferatoe ARer: after administration of eapsaicin or reainiferntoxin, or reainiferataxin plus SR 48,968. Results are expressed as mean ? standard error of the mean. Comparisons are made before and after drug addrmtmtion: p CO.05, ** p CO.01. *** p CO.001 (paired Student's two tailed t test) and between resiniferatoxin and resiniferatorin in the praaenw of intra-arterial?~ bven SR 48,968:t p C0.06, fi p CO.01 (ANOVA followed by Scheffe's F-test). ~
~~
Resiniferatoxin
200- BP
100
I
,
0=
-5
0
5
10
15
20
25
30
Tme(mio)
FIG. 1. Effects of resiniferatoxin, administered intravesically in concentration of 100 nM., on bladder pressure micturition volume (Mv, ml.). Asterisk (*) indicates adjustment to baseline position.
(p <0.001;fig. 3). The same changes in micturition volume were observed (data not shown). The last 3 days of infusion, the differences in micturition pressure and bladder eapacity were small (figs. 2 and 3), and the animals only occasionally licked the lower abdomen. Effectsof intravesically infrcsed capsaicin or resinifemen in obstructed ruts. Capsaicin infused in a concentration of 30 increased micturition pressure (p <0.05) and decreaeed bladder capacity and micturition volume (p C0.05; n = 7). At a concentration of 100 f l .(n = 8). these changes were still more pronounced (table 2; fig. 4), and the animals frequently licked the lower abdomen. The effects of capsaicin were reversible, and the cystometric pattern was restored to the preadministration state within 40 minutes after the end of the infusion. Resiniferatoxin, infused in a concentration of 100 nM. (n = lo), caused no changes in the cystometric pattern (table 2; fig. 5), and the animals showed no changes in behavior.
a.
DISCUSSION
The present results showed that intravesical resiniferatoxin, like intravesical capsaicin,2 facilitated micturition in normal rats. However, the potency of resiniferatoxin was approximately 1,000 times higher than that of capsaicin. This potency difference is in agreement with that found by de Vries and Blumberg'o concerningthennoregulatory effects in the mouse, and with that found by CraR et al.? who invee-
(BP;cm.H20)and
tigated the effects on behavior of the two drugs given intravesically. CraR et al.,8 instilling single doses of resiniferatoxin of 0.1 to 3.0 nmol. into the bladder (correeponding to concentrations of 03-10 fl.), found a d d e p e n d e n t , increased incidence of abdominal licking and head-turning induced by the drug. With the approach of the present study, using continuous instillation of drug, a distinct increase in the incidence of such behaviors could be induced by resiniferatoxin in a concentration of 100 nM. At this concentration, a distinct bladder hyperactivity was also found in normal animals. A similar degree of hyperactivity and behavioral changes could be induced by capsaicin in a concentration of 100 pM. Capsaicin, in this concentration, also induced bladder hyperactivity and changes in behavior in animals with bladder hypertrophy, but surprisingly, no such effects were observed with 100 nM. resiniferatoxin. The reason for the lack of effect of resiniferataxin(in the concentrationsused) in rats with bladder hypertrophy is not clear, since it is believed that capsaicin and resiniferatoxin have the same mode of action: that is, they release peptides from sensory neurons by interacting with the same type of receptor (the vanilloid receptor). In cultured neurons from the rat dorsal root ganglia, the only difference noted between reainiferataxin and capsaicin, apart from a large Werence in potency, w a a~ prolonged time courae of action for resiniferatoxin, consistent with a much higher affinity for the binding sites for resiniferatoxin.' However, other studies have euggeeted that
INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN
614 140
I
-E,
-
120
-
100
-
TABLE2. Effects of intravesical infusion of capsaicin or resiniferatoxin on cystometric parameters in conscious rats with bladder hypertrophy
*.
T
M.P.
60
R.V.
30 &I.(n = 7) 131.4 2 12.5 2.98 i- 0.55 2.84 2 0.57 0.14 2 0.04 Before 162.7 2 11.2* 2.01 2 0.37' 2.00 2 0.41* 0.06 2 0.03 After 100 &I.(n = 8) Before 107.1 z 7.5 3.88 f 0.58 3.67 2 0.55 0.21 2 0.09 ARer 144.4 -c 7.0** 1.71 2 0.21' 1.63 2 0.20* 0.08 z 0.02 Resiniferatoxin 100 nM. (n = 10) 112.8 2 15.2 4.07 z 0.37 3.87 2 0.38 0.18 2 0.05 Before 109.2 2 15.8 3.91 2 0.65 3.56 2 0.61 0.36 z 0.14 After M.P.: micturition pressure (cm. H,O); B.C.: bladder capacity (ml.); M.V.: micturition volume (ml.);R.V.: residual volume (ml.); Before: before administration of capsaicin or resiniferatoxin;After: after administration of capsaicin or resiniferatouin. Results are expressed as mean 2 standard error of the mean. Comparisons ** p 10.01(paired are made before and aRer drug administration: * p c0.05, Student's two tailed t test).
80 .
3
M.V.
B.C.
~~
ICapsaicin
~
40
-
20
.
0 -t
I
0
1
2
3
4
5
6
7
DAY
FIG. 2. Effects of resiniferatoxin, administered for 6 consecutive days intravesically in concentration of 100 nM., on micturition pressure (MPcm. H 0) = before administration, = after administration. Each vafue'is mean value i. standard error of mean. Comparisons are made before and after drug administration: ** p <0.01 (paired Student's two tailed t test). 1.6
(3 r
200 BP
n
15
10
1.4
f I
20
30
25
~ ~ r (nrnm e )
Saline
1.2 .n
I
A..
I
f
"I A
0
I
t
10
15 T w ( rnin )
20
25
30
FIG.4. Effects of capsaicin, administered intravesically in concentration of 100 pM.,on bladder pressure (BP; cm. H. 0)and micturition volume (Mv,ml.) in rat with bladder hypertropiy. A: control. B: effects of capsaicin. Asterisk ( * ) indicates adjustment to baseline position.
0.4
o.2 n "l
5
*** I
0
I
1
2
3
4
5
6
7
DAY
FIG.3. Effects of resiniferatoxin, administered for 6 consecutive days intravesically in concentration of 100 nM., on bladder capacity (BC;ml.).0 = before administration, = after administration. Each value is mean value C standard error of mean. Comparisons are made before and after drug administration: * p c0.05,*** p <0.001 (paired Student's two tailed t test).
iniferatoxin may be more specific in its action than capsaicin.6." If this is the case, the present results may indicate that the vanilloid receptors in the bladder a r e changed by the process of hypertrophy associated with outlet obstruction and also t h a t capsaicin has additional effects besides its action on the vanilloid receptor. Thus, the present data may reflect a difference in the mode of action between capsaicin and resiniferatoxin. Even if it is generally accepted t h a t capsaicin and resiniferatoxin act on vanilloid receptors on afferent nerves, the location of the nerves t h a t these drugs act upon when given intravesically, has not been established. Szallasi e t al.,'" who used I"H1resiniferatoxin to identifi sDecific bindine sites.,
- .
found t h a t the density of vanilloid receptors was 1.7-fold higher in the neck t h a n in t h e dome of rat urinary bladder. Pelvic nerve resection resulted i n a substantial decrease in specific resiniferatoxin binding, suggesting that the binding sites were localized chiefly to the terminals of this nerve. The extent of penetration of capsaicin and resiniferatoxin through the urothelium is not known, but it is reasonable to assume t h a t nerve terminals within or immediately beneath the urothelium may be affected by the drugs. This implies t h a t released neuropeptides, for example, tachykinins and calcitonin gene-related peptide (CGRP), would have little direct effect on the bladder smooth muscle. I t is known t h a t tachykinins injected intra-arterially near the bladder cause a n increase in basal bladder pressure.13 Such increases were not found after intravesical capsaicin or resiniferatoxin (in the present study), suggesting t h a t the peptides released by the drugs also had a site of action near the urotheliuni and did not have direct effects on t h e detrusor muscle. By autoradiography, it was shown t h a t t h e receptors for substance P, NKA and CGRP in the rat urinary bladder are located on different cell types and may be subject to different neural influences.' Neurokinin-2 receptors have Ileen demonstrated in the suburothelial layer of the rilt hladderl-' and may be the target of t h e released peptides In .,upport of this,
INTRAVESlCAL CAPSAICIN AND RESINIFERATOXIN
@
2oolsp
lo0
.
h
0
M 0
5
10
15
20
25
30
Time ( min )
FIG.5. Effects of resiniferatoxin, administered intravesically in Concentration of 100 nM., on bladder pressure ( B P cm. H20) and micturition volume (MY; ml.) in rat with bladder hypertrophy. A: control. 8:effects of capsaicin. Asterisk (*) indicates adjustment to baseline position.
['2611NKA labelling of the bladder submucosa of the rat was eelectively inhibited by the NK, receptor antagonist MEN 10,207,and the labelling was sensitive to neonatal capsaicin treatment.I6 In a previous study: we found that intravesical capsaicin apparently exerted no direct contractile effect on the bladder but nevertheless induced reflex-mediated hyperactivity, probably via stimulation of NK, receptors, since the effects were significantly reduced by the selective Mc,receptor antagonist SR 48,968(4 nmol. per rat), which is a potent and competitive nonpeptide antagonist of NK, receptors with no activity on NK, or NK, receptors.17 It was therefore speculated that capsaicin released neuropeptides from superficial lrensory nerves in the bladder and/or outflow region but had no effects on the nerves of the bladder smooth muscle. In the present study, it was found that the effects of resiniferatoxin were significantly reduced by SR 48,968.This would be con&tent with the view that intravesically administered resiniferatoxin and capsaicin release primarily neurokinin A &om sensory nerves. Craft et a1.S gave resiniferatoxin and capsaicin intravesically to rats under anesthesia and studied the excitatory and desensitizing effects of the drugs on behavior. They showed that in a dose of 1.0 nmol. (concentration 3 pM.1 resiniferatoxin induced desensitization to a second dose, given 60 minutes later. They also showed that a single exposure to resMeratoxin produced desensitization of bladder d e r e n t a that lasted at least 1 month and that intervening exposures to a lower dose did not significantly enhance desensitization. In the present study, we pretreated rats with intravesical instillation of 0.3 ml. resiniferatoxin in concentrations of 0.1 or 1.0 pM. (doses of 0.03 and 0.3 nmol.) for 1 hour under anesthesia. These concentrations were chosen because the eystometric investigation had shown that, in normal rats, resiniferatoxin in a concentration of 0.1 p M . caused pronounced bladder hyperactivity. One week after treatment, when the animals were challenged with capsaicin, no desensitization to capsaicin was observed. Since resiniferatoxininduced loss of vanilloid receptors is reversible,lS the nerves affected may have recovered from the exposure to the drug. However, when given daily for 6 days, resiniferatoxin produced desensitization effects on both micturition pressure and bladder capacity, as well as on behavior. Daily instillation of capsaicin for 6 days, on the other hand, caused no desensitization effects on cystometric parameters, when b a t e d on the sixth day. This may reflect that capsaicin has
615
effects on the nerves that are not mediated entirely through the vanilloid receptors. Desensitization to capsaicin and resiniferatoxin is a complex phenomenon, dependent on, for example, the concentration of the drug, the time of exposure and the temperature.,O In addition to a tachyphylaxis of the vanilloid receptor,*o,2* other mechanisms may be involved, such as blockade of transmitter release through the inactivation of voltage-sensitive calcium channels,*2 inhibition of axonal conductance23 and intraaxonal transport,*4 ultimately leading to depletion of neurotransmitters from the nerve terminals.26Szallasi et a1.12 pointed out that capsaicin not only was less potent than resiniferatoxin, but showed a different spectrum of action. Thus, capsaicin was much less effective for inducing desensitization relative to its ability to induce acute pain. Therefore the actions of resiniferatoxin may, at least in part, be different from those of capsaicin. The present results thus suggest that there may be differences in sensitivity to intravesical resiniferatoxin between normal and hypertrophic bladders, and differences in mode of action between resiniferatoxin and capsaicin. Intravesical resiniferatoxin would be an interesting alternative to intravesical capsaicin in the treatment of selected cases of bladder hypersensitivityhyperactivity. REFERENCES
1. Maggi, C. A.: The role of peptides in the regulation of the micturition reflex: an update. Gen.Pharmacol., 2 2 1,1991. 2. Ishizuka, O., Igawa, Y., Mattiasson, A. and Andersson, K-E.: Capsaicin-induced bladder hyperactivity in normal conscious rats. J. Urol., 152 525, 1994. 3. Pietra, C., Bettelini, L., Hagan, R. M., Ward, P., McElroy, A. and Trist, D. G.: Effect of selective antagonists at tachykinin NK-1 and NK-2 receptors on xylene-induced cystitis in rats. Neuropeptides, suppl., 2 2 52, 1992. 4. Maggi, C. A., Barbanti, G., Santicioli, P., Beneforti, P., Misuri, D., Meli, A. and Turini, D.: Cystometric evidence that capsaicin-sensitive nerves modulate the afferent branch of micturition reflex in humans. J. Urol., 142: 150, 1989. 5. Fowler, C. J., Jewkes, D., McDonald, W. I., Lynn, B. and de Groat, W. C.: Intravesical capsaicin for neurogenic bladder dysfunction. Lancet, 339: 1239, 1992. 6. Szallasi, A. and Blumberg, P. M.: Resiniferatoxin, a phorbolrelated diterpene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper. Neuroscience, 30:515, 1989. 7. Winter, J., Dray, A, Wood, J. N., Yeats, J. C. and Bevan, S.: Cellular mechanism of action of resiniferatoxin: a potent sensory neuron excitotoxin. Brain Res., 520: 131,1990. 8. CraR, R. M., Carlisi, V. J., Mattia, A, Herman, R. M. and Porreca, F.: Behavioral characterization of the excitatory and desensitizing effecta of intravesical capsaicin and resiniferatoxin in the rat. Pain, 56: 205, 1993. 9. Malmgren, A, Uvelius, B., Anderason, K-E. and Andersson, P. 0.: On the reversibility of functional bladder changes induced by infravesical oufflow obstruction in the rat. J. Urol., 145: 1026,1990. 10. de Vries, D. J. and Blumberg,P. M.: Thermoregulatory effects of resiniferatoxin in the mouse: comparison with capsaicin. Life Sci., 44:711, 1989. 11. Szolcsanyi, J., Szallasi, A., Szallasi. Z., Joo, F. and Blumberg, P. M.:-Resiniferatoxin:an ultrapotent selective modulator of capsaicin-sensitive primary afferent neurons. J. Pharmacol. Exp. "her., 26k 923, 1990. 12. Szallasi, A., Conte, B., Goso, C., Blumberg, P. M. and Manzini, S.: Vanilloid receptors in the urinary bladder: regional distribution, localization on sensory nerves, and species-related differences. Naunyn-SchmiedebergsArch. Pharmacol., 347:624, 1993. Mattiasson, A. and Andersson, K-E.: Neurokinin A 13. Ishizuka, 0.. induced bladder hyperactivity in conscious normal rats. Neurourol. Urcdyn.,13: 404,1994. 14. Banasiak, D. and Burcher, E.: Effect of capsaicin on distribution of binding sites for tachykinins and calcitonin generelated peptide in rat urinary bladder: a quantitative autoradie
616
INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN
graphic study. Peptides, 15: 333, 1994. 15. Carstairs. J. R..Nimmo, A. J. and Morrison, J. F. B.: Autoradiographical characterization of pre-junctional neurokinin receptors in rat bladder. Neuropeptides, suppl., 22: 13, 1992. 16. Carstairs, J. R.,Maggi,C. A, Andersson, P. 0.and Nimmo. A. J.: A novel tachykinin NK, receptor associated with capsaicinsensitive afferent nerves in rat bladder. Neuropeptides, abstract P32,24: 200, 1993. 17. Ma&, C. A,. Patacchini, R., Giuliani, S. and Giachetti, A.: In vivo and in vitro pharmacology of SR 48,968, a non-peptide tachykinin receptor antagonist. Eur.J. Pharmacol., 234: 83.1993. 18. Goeo, C., Piovacari, G. and Szallasi, A.: Resiniferatoxin-induced loss of vanilloid receptors is reversible in the urinary bladder but not in the spinal cord of the rat. Neurosci. Lett., 162 197, 1993. 19. Amann. R.: Desensitization of capsaicin-evoked neuropeptide release-influence of Ca" and temperature. NaunynSchmiedebergs Arch. Pharmacol., 342 671,1990. 20. Dray, A, Bettaney, J. and Forster, P.: Capsaicin desensitization
of peripheral nociceptive fibres does not impair sensitivity to other noxious stimuli. Neurosci. Lett., 99: 50, 1989. 21. Szallasi, A. and Blumberg, P. M.: Vanilloid receptor loss in rat sensory ganglia associated with long term desensitization to resiniferatoxin. Neurosci. Lett., 140: 51, 1992. 22. Bleakman, D., Brorson, J. R. and Miller, R. J.: The effect of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells. Br. J. Pharmacol., 101: 423, 1990. 23. Baranowski, R., Lynn, B. and F'ini, A.: The effects of locally applied capsaicin on conduction in cutaneous nerves in four mammalian species. Br. J. Pharmacol., 8 9 267, 1986. 24. Miller, M. S., Buck, S. H., Sipes, I. G., Yamamura, H. I. and Burks, T. F.: Regulation of substance P by nerve growth factor: disruption by capsaicin. Brain Res., 250 193, 1982. 25. Holzer. P.:Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin generelated peptide and other neuropeptides. Neuroscience, 24: 739,1988.
USA
URODYNAMIC EFFECTS O F INTRAVESICAL RESINIFERATOXIN AND CAPSAICIN IN CONSCIOUS RATS WITH AND WITHOUT OUTFLOW OBSTRUCTION OSAMU ISHIZUKA, ANDERS MATTIASSON
AND
KARL-ERIK ANDERSSON'!'
F r ( m the Depc~rtrnrntsof Uro1og.v arid Clinical Pharrnocolug.~.Lurid Uriicersity Hospital, L u n d , Swederi
ABSTRACT
Purpose: T h e urodynamic effects of intravesical resiniferatoxin a n d capsaicin were investigated in rats. Materials a n d Methods: Continuous cystometry was performed in conscious, female SpragueDawley rats with a n d without outflow obstruction. Results: Intravesical instillation of resiniferatoxin facilitated micturition. The potency of t h e d r u g was approximately 1,000 times higher than that of capsaicin. Repeated instillations of resiniferatoxin for 6 consecutive days caused desensitization to resiniferatoxin. This w a s not found with repeated instillations of capsaicin. Capsaicin w a s also effective in rats with bladder hypertrophy, while resiniferatoxin w a s not. Conclusions: The findings suggest that resiniferatoxin can induce desensitization of vanilloid receptor-mediated release of tachykinins in the rat urinary bladder and that intravesical resiniferatoxin would be a n interesting alternative to intravesical capsaicin in the t r e a t m e n t of selected cases of bladder hypersensitivityhyperactivity. KEY WORDS:bladder, capsaicin, rats, urethral obstruction In the mammalian bladder and urethra, capsaicin-sensi- rats undergoing continuous cystometry and compared them tive primary afferent neurons are involved in the regulation with those of capsaicin; 2) studied whether the effects of of various micturition-related reflexes.' Capsaicin, instilled resiniferatoxin could be inhibited by SR 48,968; 3) tested the intravesically in conscious normal rats, induced a concentra- desensitizing effects of single and repeated treatments with tion-dependent stimulation of micturition, which, in turn, resiniferatoxin in normal, conscious rats; and 4) tested the was initiated by stimulation of sensory afferents.2 These effects of intravesically instilled capsaicin or resiniferatoxin afferents may be of importance in the pathogenesis of detru- in conscious rats with bladder hyperactivity and hypertrophy sor hyperactivity, such as that induced by irritants in rats.3 secondary to outflow obstruction. The effects of intravesical capsaicin were counteracted by the selective NK, receptor antagonist SR 48,968 given intraMATERIALS AND METHODS arterially, whereas the selective NK,-receptor antagonist RP 67,580 was ineffective,, suggesting that the effect was mediAnimals. Female Sprague-Dawley rats, weighing 200 to ated, a t least partly, by release of neurokinin (NK).A. Maggi 285 g., were used in this study. The experimental protocol et al.* provided evidence for the presence of capsaicin-sensi- was approved by the Animal Ethics Committee, University of tive nerves in the human urinary bladder, where these Lund. nerves probably have functions similar to those described in Procedures: Outlet obstruction procedure. The methods rodents. Desensitization of sensory nerves by intravesical used for establishing infravesical outflow obstruction and the capsaicin infusion was shown to have a therapeutic potential technique of cystometry in awake rats have been described in to improve symptoms in otherwise intractable bladder hyper- detail previously.2 Six weeks after partial ligature of the sensitivity.4 Intravesical capsaicin also improved the control urethra, the animals were subjected to cystometrical evaluof detrusor hyperreflexia in patients with multiple s c l e r ~ s i s . ~ation. Partial obstruction of the urethra induces a significant Recent studies have demonstrated that resiniferatoxin, bladder hypertrophy and hyperactivity. One day after rewhich is a compound isolated from some plants of the genus moval of the ligature, when the animals of this study were Euphorbia, has effects similar to those of capsaicin.6,7When investigated, the bladder still exhibits a significant degree of repetitively administered, both capsaicin and resiniferatoxin hypertrophy and hyperactivity.9 For simplicity, the prevican produce desensitization and an inactivation of sensory ously obstructed rats are referred to as rats with bladder neurons, which may explain the beneficial effects of capsaicin hypertrophy. in cases of bladder hyperactivity. Intravesically infused resBladder catheter implantation. Rats were anesthetized iniferatoxin caused behavioral changes in rats similar t o with ketamine (75 mg./kg. intramuscularly) and xylazine (15 those produced by capsaicin,S suggesting that resiniferatoxin mg./kg. intramuscularly). The abdomen was opened through may induce bladder hyperactivity in the same way as capsa- a midline incision, and a polyethylene catheter (Clay-Adams However, the urodynamic effects of intravesical resinif- PE-50, Parsippany, New Jersey) implanted into the bladder eratoxin do not seem to have been investigated. through the dome as described previously., We therefore 1) studied the effects on the cystometrogram Cystometrical investigations. Cystometric investigations of intravesically instilled resiniferatoxin in normal, conscious were performed without any anesthesia 3 days after bladder catheter implantation in normal rats and 1 day afterward in Accepted for publication February 16, 1995. * Requests for reprints: Department of Clinical Pharmacology, obstructed rats. The bladder catheter was connected via a Lund University Hospital, S-22185,Lund, Sweden. T-tube to a pressure transducer (P23 DC, Statham InstruThis project was supported by the Swedish Medical Research Council (no. 6837 and 10399)and by the Medical Faculty of Lund, ment Inc., Oxnard, California) and a microinjection pump (CMA 100, Carnegie Medicine AJ3, Solna, Sweden). The conSweden. 611
612
INTRAVESICAL CAPSAICIN AND RESINIFERATOWN
scious rat was placed, without any restraint, in a metabolic mg., n = 15, p <0.001),compared with normal controls (123 cage, which also permitted measurements of micturition vol- 2 5 mg., range 90 to 170 mg., n = 21). Bladder capacity umes by means of a fluid collector connected to a Grass force increased from 1.06 5 0.05 ml. ( n = 39) to 3.81 5 0.40 ml. (n displacement transducer (FT 03 C, Grass Instrument CO., = 19). Repeated cystometries gave reproducible results in Quincy, Massachusetts). Room-temperature saline (20 % 1c) both control animals and animals with bladder hypertrophy. was infused into the bladder at a rate of 10 ml. per hour in In control animals, the bladder pressure was low (5.8 % 0.9 normal rats. To achieve an approximately equal number of cm. H,O., n = 48) and almost devoid of spontaneous fluctumicturitions in normal rats and rats with bladder hypertro- ations during the period of cystometry. On the other hand, in phy, the rate of infusion was increased to 20 ml. per hour in animals with bladder hypertrophy, cystometry revealed rats with bladder hypertrophy. Intravesical pressure and spontaneous contractile activity during filling. Effects of intravesically infused capsaicin or resiniferatoxin micturition volumes were recorded continuously on a Grass polygraph (Model 7E, Grass Instrument Co.; recording speed: in normal rats. Intravesical capsaicin increased bladder ac10 mm. per minute). Three reproducible micturition cycles, tivity in a concentration-dependent way. At a concentration corresponding to a 20-minute period, were recorded before of 10 pM. (n = 4), no effects on cystometric parameters were drug administration and used as baseline values. The follow- observed. However, in a concentration of 30 pM., capsaicin ing cystometric parameters were investigated? micturition increased micturition pressure (p <0.001), and decreased pressure (the maximum bladder pressure during micturi- bladder capacity (p <0.001) and micturition volume (p tion), bladder capacity (residual volume a t the preceding <0.001; n = 8; table 1). The animals showed no signs of micturition plus volume of infused saline a t the micturi- distress and behaved normally, except for occasional licking capsaicinI., tion), micturition volume (volume of expelled urine) and of the lower abdomen. At a concentration of 100 @ residual volume (bladder capacity minus micturition vol- evoked stimulation of bladder activity was still more proume). The 3 micturition cycles showing the most pronounced nounced (n = 8; table l), and the rats frequently licked their changes (increase or decrease) within 60 minutes after lower abdomen; no other abnormal behavior was observed. switching saline to capsaicin or resiniferatoxin were ana- The effects of capsaicin were reversible, and the cystometric pattern was restored to the preadministration state within lyzed and compared with the baseline values. Groups of normal rats were investigated for 6 consecutive 40 minutes &r the end of the infusion. The effects of capdays. In these animals, capsaicin or resiniferatoxin was in- saicin could be repeated in the same rat at least 3 times and were still reversible. fused intravesically in concentrations of 100 @. and 100 &., Intravesical resiniferatoxin increased bladder activity in a respectively, and the effects on the cystometrogram and beconcentration-dependent way (table 1).At a concentration of havior were recorded. Zntravesical pretreatment with resiniferatoxin in normal 30 nM., micturition pressure increased (p <0.05), and bladrats. Rats were anesthetized as described above. The abdo- der capacity (p C0.05) and micturition volume (p C0.05; n = men was opened through a midline incision, and the urethra 7) decreased. The animals showed no signs of distress and ligated by 3-zero nylon. A 23-gauge needle was put into the behaved normally, except for occasional licking of the lower dome of the bladder, all urine was aspirated, and 0.3 ml. of abdomen. The urodynamic changes were more pronounced at saline or saline containing resiniferatoxin in concentrations a concentration of 100 nM. (fig. 1; table 11, as were the of 100 nM. or 1000 nM. was instilled. After 60 minutes, the changes in behavior (licking of the lower abdomen). SR ligature of the urethra was released, and saline or resinifera- 48,968, in a concentration of 20 nmol./kg. practically aboltoxin was removed. Four days after the pretreatment with ished the actions of resiniferatoxin 30 nM. (n = 5; table 1)on saline or resiniferatoxin, the rat was anesthetized again, and micturition pressure. In a concentration of 100 nmol./kg., SR a polyethylene catheter (Clay-Adams PE-50) was implanted 48,968 significantly attenuated or abolished the changes ininto the bladder. Cystometric investigations were performed duced by resiniferatoxin 100 nM., not only in micturition by infusing capsaicin 1 week after the pretreatment. pressure, but also in bladder capacity and micturition volume Administration of drugs. Stock solutions (100 pM.) of cap- (n = 5; table 1).Only occasionally did the animals lick the saicin (LabKemi, Lund, Sweden) and resiniferatoxin lower abdomen. (LabKemi) were made in absolute ethanol. The drugs were Effects of intravesically infused capsaicin after pretreatment then stored a t -7OC, and subsequently diluted on the day of with resiniferatoxin in normal rats. Under anesthesia, resexperiment to achieve concentrations of 0.03,0.1, 1.0, 10, 30, iniferatoxin, in concentrations of 100 nM. (n = 5 )or 1000 nM. or 100 pM. Capsaicin or resiniferatoxin was infused intra- (n = 5 ) , was instilled and kept intravesically for 1 hour. vesically by changing the syringe of the microinjection pump. Saline was instilled in controls and kept for the same time During the infusion of resiniferatoxin, the syringes were (n = 12). When the rats recovered from the anesthesia, there protected from light. With the tubing used, drugs reached the were no differences in behavior between the resiniferatoxinbladder in 8.0 2 0.7 minutes after switching from saline to and saline-treated rats. the solution containing capsaicin or resiniferatoxin. After 28 One week after treatment with resiniferatoxin or saline, minutes’ administration of capsaicin or resiniferatoxin, sa- there were no differences in the effect of intravesically inline was again infused. SR 48,968 was given immediately fused capsaicin 100 pM. between the two groups (data not after the start of resiniferatoxin infusion. The doses of the shown). drugs used were chosen on the basis of pilot experiments and Effects of repeated instillations of cupsuicin or resirziferafrom previously published data.2 toxin in normal rats. Intravesical infusion of capsaicin in a Statistical analysis. The results are given as mean values z concentration of 100 kM. (infusion time 28 minutes; n = 6) standard error of the mean. For comparisons between values for 6 consecutive days caused bladder hyperactivity from day obtained before and after drug administration, Student’s paired 1 to day 6. There was a consistent, significant increase in t test was used.One way factorial ANOVA was used for com- micturition pressure. From day 3, there was an increase parisons between capsaicin and capsaicin after pretreatment in bladder capacity before capsaicin infusion; capsaicin sigwith resiniferatoxin and was followed by Scheffe’s F-test. A nificantly decreased micturition volume each time i t was probability level of <5% was accepted as significant. administered. The same changes were observed in micturition volume (data not shown). Licking of the lower abdomen was observed after each administration. RESULTS Resiniferatoxin, instilled in a Concentration of 100 nM. Partial obstruction of the urethra led to a significant in- (n = 61, increased micturition pressurc: o n days 1 and 2 crease in bladder weight (759 % 35 mg., range 520 to 1,000 ( p /0.01;fig. 2 ) and decreased bladder cnliacity 011 day 1
INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN
6 13
cystometric parameters in the absence or presence of intmarterial S R 48,968 in normal conscious mts M.P. B.C. M.V. RV.
TAnLE 1. Effects of intravesical infusion of capsaicin or resiniferatoxin on
Capsaicin 30 pM. (n = 8)
Before After
65.0 2 2.7 83.9 .C 3.2."
1.27 f 0.10 0.64 ? 0.14***
0.23 2 0.03 0.12 f 0.06
1.03 f 0.10 0.62 t 0.10***
100 pM. (n = 8 ) 0.22 f 0.03 Before 66.2 z 7.4 1.13 2 0.12 0.91 2 0.10 0.08 2 0.02*** 108.2 f 7.8*** 0.20 t 0.03*** After 0.26 ? 0.03*** Resiniferatoxin 30 nM. (n = 7) 0.15 f 0.03 Before 67.7 f 9.6 1.17 2 0.15 1.04 2 0.13 0.14 2 0.03 86.8 f 12.01 After 0.88 f 0.20' 0.74 f 0.18' 100 nM. (n = 11) 0.09 f 0.03 Before 73.9 f 8.0 0.88 ? 0.09 0.80 ~t. 0.07 0.08 2 0.02 95.2 f 5.6** 0.43 f O.O5** After 0.52 ? 0.06** Resiniferatoxin plus SR 48,968 (20 nmolJkg.) 30 nM. (n = 5) 0.16 2 0.04 Before 1.08 f 0.20 0.92 f 0.17 63.6 f 6.7 0.16 2 0.07 64.3 2 6.4t 1.01 f 0.18 0.86 2 0.13 After Resiniferatoen plus SR 48,968 (100 nmolhg.) 100 nM. (n = 5) Before 0.09 2 0.02 0.92 f 0.20 72.3 2 9.8 1.01 f 0.19 After 70.8 f 11.77 1.02 f 0.22t 0.w f 0.23tt 0.08 2 0.04 M.P.: micturition pressure (em.H,O); B.C.: bladder capacity (ml.k M.V.:micturition volume (ml.); R.V.: reaidual volume (ml.); Before:before administrati00 o f capsaicin or resiniferatoe ARer: after administration of eapsaicin or reainiferntoxin, or reainiferataxin plus SR 48,968. Results are expressed as mean ? standard error of the mean. Comparisons are made before and after drug addrmtmtion: p CO.05, ** p CO.01. *** p CO.001 (paired Student's two tailed t test) and between resiniferatoxin and resiniferatorin in the praaenw of intra-arterial?~ bven SR 48,968:t p C0.06, fi p CO.01 (ANOVA followed by Scheffe's F-test). ~
~~
Resiniferatoxin
200- BP
100
I
,
0=
-5
0
5
10
15
20
25
30
Tme(mio)
FIG. 1. Effects of resiniferatoxin, administered intravesically in concentration of 100 nM., on bladder pressure micturition volume (Mv, ml.). Asterisk (*) indicates adjustment to baseline position.
(p <0.001;fig. 3). The same changes in micturition volume were observed (data not shown). The last 3 days of infusion, the differences in micturition pressure and bladder eapacity were small (figs. 2 and 3), and the animals only occasionally licked the lower abdomen. Effectsof intravesically infrcsed capsaicin or resinifemen in obstructed ruts. Capsaicin infused in a concentration of 30 increased micturition pressure (p <0.05) and decreaeed bladder capacity and micturition volume (p C0.05; n = 7). At a concentration of 100 f l .(n = 8). these changes were still more pronounced (table 2; fig. 4), and the animals frequently licked the lower abdomen. The effects of capsaicin were reversible, and the cystometric pattern was restored to the preadministration state within 40 minutes after the end of the infusion. Resiniferatoxin, infused in a concentration of 100 nM. (n = lo), caused no changes in the cystometric pattern (table 2; fig. 5), and the animals showed no changes in behavior.
a.
DISCUSSION
The present results showed that intravesical resiniferatoxin, like intravesical capsaicin,2 facilitated micturition in normal rats. However, the potency of resiniferatoxin was approximately 1,000 times higher than that of capsaicin. This potency difference is in agreement with that found by de Vries and Blumberg'o concerningthennoregulatory effects in the mouse, and with that found by CraR et al.? who invee-
(BP;cm.H20)and
tigated the effects on behavior of the two drugs given intravesically. CraR et al.,8 instilling single doses of resiniferatoxin of 0.1 to 3.0 nmol. into the bladder (correeponding to concentrations of 03-10 fl.), found a d d e p e n d e n t , increased incidence of abdominal licking and head-turning induced by the drug. With the approach of the present study, using continuous instillation of drug, a distinct increase in the incidence of such behaviors could be induced by resiniferatoxin in a concentration of 100 nM. At this concentration, a distinct bladder hyperactivity was also found in normal animals. A similar degree of hyperactivity and behavioral changes could be induced by capsaicin in a concentration of 100 pM. Capsaicin, in this concentration, also induced bladder hyperactivity and changes in behavior in animals with bladder hypertrophy, but surprisingly, no such effects were observed with 100 nM. resiniferatoxin. The reason for the lack of effect of resiniferataxin(in the concentrationsused) in rats with bladder hypertrophy is not clear, since it is believed that capsaicin and resiniferatoxin have the same mode of action: that is, they release peptides from sensory neurons by interacting with the same type of receptor (the vanilloid receptor). In cultured neurons from the rat dorsal root ganglia, the only difference noted between reainiferataxin and capsaicin, apart from a large Werence in potency, w a a~ prolonged time courae of action for resiniferatoxin, consistent with a much higher affinity for the binding sites for resiniferatoxin.' However, other studies have euggeeted that
INTRAVESICAL CAPSAICIN AND RESINIFERATOXIN
614 140
I
-E,
-
120
-
100
-
TABLE2. Effects of intravesical infusion of capsaicin or resiniferatoxin on cystometric parameters in conscious rats with bladder hypertrophy
*.
T
M.P.
60
R.V.
30 &I.(n = 7) 131.4 2 12.5 2.98 i- 0.55 2.84 2 0.57 0.14 2 0.04 Before 162.7 2 11.2* 2.01 2 0.37' 2.00 2 0.41* 0.06 2 0.03 After 100 &I.(n = 8) Before 107.1 z 7.5 3.88 f 0.58 3.67 2 0.55 0.21 2 0.09 ARer 144.4 -c 7.0** 1.71 2 0.21' 1.63 2 0.20* 0.08 z 0.02 Resiniferatoxin 100 nM. (n = 10) 112.8 2 15.2 4.07 z 0.37 3.87 2 0.38 0.18 2 0.05 Before 109.2 2 15.8 3.91 2 0.65 3.56 2 0.61 0.36 z 0.14 After M.P.: micturition pressure (cm. H,O); B.C.: bladder capacity (ml.); M.V.: micturition volume (ml.);R.V.: residual volume (ml.); Before: before administration of capsaicin or resiniferatoxin;After: after administration of capsaicin or resiniferatouin. Results are expressed as mean 2 standard error of the mean. Comparisons ** p 10.01(paired are made before and aRer drug administration: * p c0.05, Student's two tailed t test).
80 .
3
M.V.
B.C.
~~
ICapsaicin
~
40
-
20
.
0 -t
I
0
1
2
3
4
5
6
7
DAY
FIG. 2. Effects of resiniferatoxin, administered for 6 consecutive days intravesically in concentration of 100 nM., on micturition pressure (MPcm. H 0) = before administration, = after administration. Each vafue'is mean value i. standard error of mean. Comparisons are made before and after drug administration: ** p <0.01 (paired Student's two tailed t test). 1.6
(3 r
200 BP
n
15
10
1.4
f I
20
30
25
~ ~ r (nrnm e )
Saline
1.2 .n
I
A..
I
f
"I A
0
I
t
10
15 T w ( rnin )
20
25
30
FIG.4. Effects of capsaicin, administered intravesically in concentration of 100 pM.,on bladder pressure (BP; cm. H. 0)and micturition volume (Mv,ml.) in rat with bladder hypertropiy. A: control. B: effects of capsaicin. Asterisk ( * ) indicates adjustment to baseline position.
0.4
o.2 n "l
5
*** I
0
I
1
2
3
4
5
6
7
DAY
FIG.3. Effects of resiniferatoxin, administered for 6 consecutive days intravesically in concentration of 100 nM., on bladder capacity (BC;ml.).0 = before administration, = after administration. Each value is mean value C standard error of mean. Comparisons are made before and after drug administration: * p c0.05,*** p <0.001 (paired Student's two tailed t test).
iniferatoxin may be more specific in its action than capsaicin.6." If this is the case, the present results may indicate that the vanilloid receptors in the bladder a r e changed by the process of hypertrophy associated with outlet obstruction and also t h a t capsaicin has additional effects besides its action on the vanilloid receptor. Thus, the present data may reflect a difference in the mode of action between capsaicin and resiniferatoxin. Even if it is generally accepted t h a t capsaicin and resiniferatoxin act on vanilloid receptors on afferent nerves, the location of the nerves t h a t these drugs act upon when given intravesically, has not been established. Szallasi e t al.,'" who used I"H1resiniferatoxin to identifi sDecific bindine sites.,
- .
found t h a t the density of vanilloid receptors was 1.7-fold higher in the neck t h a n in t h e dome of rat urinary bladder. Pelvic nerve resection resulted i n a substantial decrease in specific resiniferatoxin binding, suggesting that the binding sites were localized chiefly to the terminals of this nerve. The extent of penetration of capsaicin and resiniferatoxin through the urothelium is not known, but it is reasonable to assume t h a t nerve terminals within or immediately beneath the urothelium may be affected by the drugs. This implies t h a t released neuropeptides, for example, tachykinins and calcitonin gene-related peptide (CGRP), would have little direct effect on the bladder smooth muscle. I t is known t h a t tachykinins injected intra-arterially near the bladder cause a n increase in basal bladder pressure.13 Such increases were not found after intravesical capsaicin or resiniferatoxin (in the present study), suggesting t h a t the peptides released by the drugs also had a site of action near the urotheliuni and did not have direct effects on t h e detrusor muscle. By autoradiography, it was shown t h a t t h e receptors for substance P, NKA and CGRP in the rat urinary bladder are located on different cell types and may be subject to different neural influences.' Neurokinin-2 receptors have Ileen demonstrated in the suburothelial layer of the rilt hladderl-' and may be the target of t h e released peptides In .,upport of this,
INTRAVESlCAL CAPSAICIN AND RESINIFERATOXIN
@
2oolsp
lo0
.
h
0
M 0
5
10
15
20
25
30
Time ( min )
FIG.5. Effects of resiniferatoxin, administered intravesically in Concentration of 100 nM., on bladder pressure ( B P cm. H20) and micturition volume (MY; ml.) in rat with bladder hypertrophy. A: control. 8:effects of capsaicin. Asterisk (*) indicates adjustment to baseline position.
['2611NKA labelling of the bladder submucosa of the rat was eelectively inhibited by the NK, receptor antagonist MEN 10,207,and the labelling was sensitive to neonatal capsaicin treatment.I6 In a previous study: we found that intravesical capsaicin apparently exerted no direct contractile effect on the bladder but nevertheless induced reflex-mediated hyperactivity, probably via stimulation of NK, receptors, since the effects were significantly reduced by the selective Mc,receptor antagonist SR 48,968(4 nmol. per rat), which is a potent and competitive nonpeptide antagonist of NK, receptors with no activity on NK, or NK, receptors.17 It was therefore speculated that capsaicin released neuropeptides from superficial lrensory nerves in the bladder and/or outflow region but had no effects on the nerves of the bladder smooth muscle. In the present study, it was found that the effects of resiniferatoxin were significantly reduced by SR 48,968.This would be con&tent with the view that intravesically administered resiniferatoxin and capsaicin release primarily neurokinin A &om sensory nerves. Craft et a1.S gave resiniferatoxin and capsaicin intravesically to rats under anesthesia and studied the excitatory and desensitizing effects of the drugs on behavior. They showed that in a dose of 1.0 nmol. (concentration 3 pM.1 resiniferatoxin induced desensitization to a second dose, given 60 minutes later. They also showed that a single exposure to resMeratoxin produced desensitization of bladder d e r e n t a that lasted at least 1 month and that intervening exposures to a lower dose did not significantly enhance desensitization. In the present study, we pretreated rats with intravesical instillation of 0.3 ml. resiniferatoxin in concentrations of 0.1 or 1.0 pM. (doses of 0.03 and 0.3 nmol.) for 1 hour under anesthesia. These concentrations were chosen because the eystometric investigation had shown that, in normal rats, resiniferatoxin in a concentration of 0.1 p M . caused pronounced bladder hyperactivity. One week after treatment, when the animals were challenged with capsaicin, no desensitization to capsaicin was observed. Since resiniferatoxininduced loss of vanilloid receptors is reversible,lS the nerves affected may have recovered from the exposure to the drug. However, when given daily for 6 days, resiniferatoxin produced desensitization effects on both micturition pressure and bladder capacity, as well as on behavior. Daily instillation of capsaicin for 6 days, on the other hand, caused no desensitization effects on cystometric parameters, when b a t e d on the sixth day. This may reflect that capsaicin has
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effects on the nerves that are not mediated entirely through the vanilloid receptors. Desensitization to capsaicin and resiniferatoxin is a complex phenomenon, dependent on, for example, the concentration of the drug, the time of exposure and the temperature.,O In addition to a tachyphylaxis of the vanilloid receptor,*o,2* other mechanisms may be involved, such as blockade of transmitter release through the inactivation of voltage-sensitive calcium channels,*2 inhibition of axonal conductance23 and intraaxonal transport,*4 ultimately leading to depletion of neurotransmitters from the nerve terminals.26Szallasi et a1.12 pointed out that capsaicin not only was less potent than resiniferatoxin, but showed a different spectrum of action. Thus, capsaicin was much less effective for inducing desensitization relative to its ability to induce acute pain. Therefore the actions of resiniferatoxin may, at least in part, be different from those of capsaicin. The present results thus suggest that there may be differences in sensitivity to intravesical resiniferatoxin between normal and hypertrophic bladders, and differences in mode of action between resiniferatoxin and capsaicin. Intravesical resiniferatoxin would be an interesting alternative to intravesical capsaicin in the treatment of selected cases of bladder hypersensitivityhyperactivity. REFERENCES
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