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Protective action of Ruthenium red toward capsaicin desensitization of sensory fibers
Neuroscience Letters, 88 (1988) 201 205
2111
Elsevier Scientific Publishers Ireland Ltd.
NSL05315
Protective action of Ruthenium red toward capsaicin desensitization of sensory fibers Carlo Alberto Maggi l, Riccardo Patacchini l, Paolo Santicioli 1, Sandro Giuliani 1, Pierangelo Geppetti 2 and Alberto M eli l ~Pharmaeology Department, Smooth Muscle Division, Research Laboratories, 'A. Menarini' Pharmaceuticals, Florence (Italy) and "Department of Internal Medicine and Clinical PharmacMogy, University qf Florence, Florence (Italy) (Received 3 November 1987; Revised version received 28 January 1988; Accepted 4 February 1988) Key word~v Capsaicin; Desensitization; R u t h e n i u m red: Rat urinary bladder: Efferent function of sensory nerves In the rat isolated urinary bladder, exposure to capsaicin (1 tiM) produced a contraction thought to involve neuropeptide(s) release from sensory nerves. A second application of the drug had no motor effect indicating desensitization. The establishment of the desensitized state requires the presence of cxtraccllular calcium. In the presence of R u t h e n i u m red (RR, 30 nM-10/.tM) the first response to capsaicin was reduced and a concentration-dependent protection from capsaicin desensitization was observed. R R up to 10(I/tM had no inhibitory effect toward contractions produced by exogenous substance P nor by electrical stimulation of efferent nerves.
Capsaicin exerts a selective excitatory (depolarizing) action on certain sensory nerves [5, 11]. Although the ionic basis of depolarization of sensory terminals is still largely unexplored, some evidence indicates that an increase in conductance to calcium ions (Ca) may be involved [1, 5, 11], This event determines the secretion of stored transmitters from peripheral sensory terminals through a tetrodotoxin-resistant depolarization e.g. the sensory receptor potential-coupled 'effbrent'response [5]. Circumstantial evidence also supports the idea that Ca accumulation into sensory neurons and nerves [3, 10] due to the prolonged depolarization by capsaicin [14], is involved in both the desensitizing and neurotoxic effect ofcapsaicin on primary afterents [6, 11]. At the ultrastructural level, accumulation of radiolabeled Ca within mitochondria of sensory neurons was observed following systemic capsaicin desensitization and this effect was implicated in the neurodegenerative action of capsaicin [3]. Indeed alterations at mitochondrial level were evident also in sensory a x o n s of rats desensitized to capsaicin as adults [12]. We have shown previously that, in the ab-
Correspondence: C.A. Maggi, Pharmacology Department, Smooth Muscle Division, Research Laboratories, "A Menarini' Pharmaceuticals, Via Sette Santi 3, 1-50131 Florence, Italy. 0304-3940/88,,$ 03.50 ~) 1988 Elsevier Scientific Publishers Ireland Ltd.
202 sence of extracellular Ca, there is a protection toward the capsaicin desensitization phenomenon at the level of peripheral terminals of the rat urinary bladder [10]. We now present evidence that Ruthenium red (RR), a compound reported to inhibit Ca mobilization in various tissues [2, 4, 8] and Ca uptake from mitochondria [7], behaves, in a quite large concentration range (30 nM-10/,tM), as a selective antagonist of the 'efferent' response (contraction) mediated by capsaicin-sensitive nerves of the rat urinary bladder and protects sensory fibers from capsaicin desensitization. Male albino rats of the Wistar-Morini strain weighing 340-360 g were killed by cervical dislocation and exsanguinated. The whole urinary bladder was rapidly removed, a strip of detrusor muscle (about I cm long) was dissected out and placed in a 5 ml organ bath (at 37°C) containing a standard Krebs solution (Ca concentration 2.5 raM) gassed with 96% O2~4%CO2, as described previously [9, 10]. Tension was recorded by means of an isometric strain gauge connected to a Basile 7050 Unirecord. Field stimulation was obtained by means of two wire platinum electrodes placed at the top and the bottom of the organ bath. Square wave pulses (60 V, 0.5 ms) were delivered automatically at a frequency of 0.1 Hz by means of a Grass S11 stimulator. In each experiment, capsaicin (1/~M) was added to the organ bath 5 min after the interruption of field stimulation. RR was added to the organ bath 25 min before the first application of capsaicin. On each application, capsaicin was left in contact with the tissue for 5 min and the maximal contractile response observed during this period was expressed as % of the response to field stimulation (FS, 0.5 ms, 60 V, 0.1 Hz) obtained in the same tissue. After each application of capsaicin (at 25 rain intervals) the bathing solution was repeatedly renewed during the first 15 min, e.g. RR was present in the bath only during the first exposure to capsaicin. During the washout period the strips were superfused with capsaicin-free Krebs solution at 20 ml/min for 1 min and then at 2.5 ml/min for 14 min. All data in the text are mean_+S.E.M. Statistical analysis was made by means of Student's t-test or analysis of variance, when applicable. Drugs used were R R (Serva), Ruthenium chloride (Aldrich), substance P (SP, Peninsula), capsaicin (Sigma). RR (30 nM-300/~M) did not inhibit twitches amplitude up to 10/~M. At 30-300 /,M, a 38 + 4 and 9 6 _ 2% inhibition was observed, respectively. At lower concentrations (30 nM-10 ktM) either no effect or a small (10-15%) potentiation or inhibition (Fig. I) of twitches was observed. These latter effects were present in a 40% of cases, were concentration-independent and their nature was not investigated further. In the presence of RR a concentration-dependent inhibition of the response to capsaicin (1 /~M) was observed (Table I, Fig. l) but at 30-300 nM there was no significant inhibitory effect. In control experiments RR up to 10/~M had no inhibitory effect toward contractions produced by SP at a concentration (30 nM, n = 8) which produced a response similar to that induced by capsaicin (1 /~M). At higher concentrations, RR inhibited the SP-induced contraction and this effect was similar to that observed toward twitches (35 + 4 and 86+ 3% inhibition at 30 and 300/~M, respectively, n = 4). In control strips, the first exposure to capsaicin (1 /tM for 5 min) was sufficient to produce desensitization to further applications of the drug (Fig. 1, Table I). On the other hand, exposure to RR before administration of capsaicin resulted in a con-
203 5 rain 10 mN
CONTROL
B
¸¸¸¸¸¸I RUT.EN,UM REO ~3 r "
l
I., RUr.EN,UM REO 3rM
C
1 D
l
• RUTHENIUM
RED
• 10 }JM
1
• •
= CAPSAICIN
• 1 ~M
Fig. 1. Typical tracings showing the effect of increasing concentrations of RR (A, control; B, RR 0.3/~M; C, RR 3 izM; D, RR I0 ,uM) on the contractile response to capsaicin (1/LM at the dots) of the rat isolated urinary bladder and subsequent desensitization. The response to field stimulation is shown before cach application of capsaicin. In each preparation, capsaicin was applied at 25 min intervals with repeated renewals of the bathing solution between each application. After the first application ofcapsaicin the preparations were washed with RR-free Krebs solution.
c e n t r a t i o n - d e p e n d e n t p r o t e c t i o n f r o m c a p s a i c i n d e s e n s i t i z a t i o n ( T a b l e I). T h i s is well e v i d e n t in a t y p i c a l e x p e r i m e n t s h o w n in Fig. 1: at 300 n M o f R R a s e c o n d a p p l i c a t i o n o f c a p s a i c i n p r o d u c e d a large, s l o w l y d e v e l o p i n g c o n t r a c t i o n w h i l e a t h i r d o r f o u r t h a p p l i c a t i o n was ineffective. A t h i g h e r R R c o n c e n t r a t i o n s , the p r o t e c t i o n f r o m
204 TABLE I E F F E C T OF E X P O S U R E TO R U T H E N I U M R E D ON T H E C O N T R A C T I L E RESPONSE OF T H 1 RAT I S O L A T E D B L A D D E R TO R E P E A T E D A D M I N I S T R A T I O N OF C A P S A I C I N Each value is mean -+S.E.M. RR was added to the organ bath 25 rain before the first application ofcapsai cin. After each capsaicin administration there was a 30 min interval between doses during which the Krebs solution was repeatedly renewed. NE, no effect. ~Significantly different from controls, P<0.05. Treatment
Controls R R 0.03 ~M R R 0.3/.tM RR 3/tM R R 10/tM
No. of experiments
Response to FS (raN)
Response to capsaicin ( 1/*M) (% of FS)
Before
After
First
Second
Third
Fourth
7 5 5 8 5
19__+ 1 20_+2 17_+2 I7±2 18_+3
21_+2 18+2 18+2 18_+2
136,-- 16 143+12 128_+19 91_+ 7a 40_+13 ~
3+ 3 38+10 a 57___13a 76__+14~ 58-+1D
NE NE 15± 3 33_+19 66-+ 6
NE NE NE NE 43_+7
desensitization was extended to the third and fourth (at 10/,M of RR) dose ofcapsaicin, despite the repeated washouts intervening between each application. In strips exposed to RR 10/tM the second and third application of capsaicin produced responses higher than that of the first dose and some response was observed up to a fifth or sixth exposure to capsaicin (data not shown). Despite repeated washouts, the tissues exposed to R R retained a pink to Violet color (depending on the RR concentration) throughout the experiment, suggesting that a given fraction of R R was tightly bound to the tissue. Ruthenium chloride (10/,M, n = 4 ) had no effect toward the capsaicin (1 /,M)induced contraction nor induced any protection toward capsaicin desensitization (data not shown). The cellular bases of capsaicin desensitization are unknown. In the rat bladder, A single exposure to capsaicin for a few minutes produced an almost complete unresponsiveness to a further challenge with the drug which is not attributable to SP depletion from sensory fibers [7]. Previous observations indicated that the presence of extracellular Ca is essential for the establishment of the desensitized state at the level of peripheral terminals of these sensory fibers [10], supporting the idea that an excess of intracellular Ca and/or inability of the sensory neuron/fiber to buffer it were involved in the desensitizing action of capsaicin [5]. The present findings indicate that RR up to 10 # M has a selective inhibitory action toward the effects of capsaicin involving both the acute response (depolarization of sensory fibers) and the subsequent desensitization. The present experiments do not provide any direct link between the action of RR toward capsaicin and its inhibitory action on transmembrane Ca movements. Although direct experiments are needed to assess this point, the assumption seems tenable for 3 reasons: (a) R R affects transmembrane Ca fluxes in a variety of tissues [2, 8]; (b) in low concentrations, RR was shown to block transmitter release from synaptosomes due to depolarization-coupled Ca entry [ 13]; and (c), a recent prelim±n-
205 a r y r e p o r t [l 5] i n d i c a t e s t h a t R R in a n a n o m o l a r induced
establishment
of the desensitized
Although a direct comparison
concentration inhibits the capsaicin-
state in sensory
neurons
in c u l t u r e .
b e t w e e n o u r f i n d i n g s a n d t h o s e o f W o o d [15] is diffi-
cult, the presence of extracellular Ca was shown to be essential for the establishment o f t h e d e s e n s i t i z e d s t a t e in b o t h c a s e s [10, 15]. T h u s it s e e m s c o n c e i v a b l e to a s s u m e t h a t t h e p r o t e c t i v e effect o f R R i n v o l v e d s o m e a n t a g o n i s m t o w a r d t h e a d v e r s e effect o f a ' h i g h ' C a i n f l u x in s e n s o r y n e u r o n / f i b e r . In t h i s s t u d y we a l s o o b s e r v e d t h a t R u t h e n i u m
i o n s p e r se a r e n o t i m p o r t a n t s i n c e
Ruthenium chloride was ineffective toward the actions ofcapsaicin. This observation p a r a l l e l s p r e v i o u s f i n d i n g s [8] i n d i c a t i n g t h a t t h e e n t i r e m o l e c u l e is r e q u i r e d to b l o c k Ca movements. The observation that at certain concentrations r e s p o n s e t o c a p s a i c i n w h i l e h a v i n g n o effect t o w a r d
R R r e d u c e d t h e first
SP suggests a reduced release
o f s e n s o r y n e u r o p e p t i d e s . T h i s p o i n t is c u r r e n t l y u n d e r i n v e s t i g a t i o n . I Gamse, R., Molnar, A. and Lembeck, F., Substance P release from spinal cord by capsaicin, Life Sci., 25 (1979) 629 636. 2 Greenberg, S., Long, J.P. and Diecke, F.P.J., Differentiation of calcium pools utilized in the contractile response of canine arterial and venous smooth muscle to norepinephrine, J. Pharmacol. Exp. Ther., 185 (1973)493 504. 3 Jancs6, G., Karcsfl, S., Kiraly, E., Szebeni, A., Toth, L., Bacsy, E., Jo6, F. and Parducz, A., Neurotoxin-induced nerve cell degeneration: possible involvement of calcium, Brain Res.. 295 (1984) 211 216. 4 Luft, J.H., Ruthenium red and violet. 1. Chemistry, purification, methods of use for electron microscopy and mechanism of action, Anat. Rec., 171 ( 1971 ) 347 368. 5 Maggi, C.A. and Meli, A., The sensory-efferent functions ofcapsaicin sensitive sensory neurons, Gen. Pharmacol., 19 (1988) 1~,3. 6 Maggi, C.A., Santicioli, P., Abelli, L., Parlani, M., Capasso, M., Con[e, B., Giuliani, S. and Meli, A., Regional differences in the effects of tachykinins on motor activity and vascular permeability in the rat lower urinary tract, Naunyn-Schmiedeberg's Arch. Pharmacol., 335 (1987)636 645. 7 Maggi, C.A., Santicioli, P., Geppetti, P., Furio, M., Frilli, S., Conte, B., Fanciullacci, M., Giuliani, S. and Mcli, A., The contribution of capsaicin-sensitive innervation to the activation of spinal vesicovesical reflex in rats: relationship between substance P levels in the urinary bladder and the sensoryefferent function of capsaicin-sensitive neurons, Brain Res., 415 (1987) 1 13. 8 Moore, C.L., Specific inhibition of mitochondrial calcium transport by ruthenium red, Biochcm. Biophys. Res. Commun., 42 (197[) 298 305. 9 Santicioli, P., Maggi, C.A. and Meli, A., Functional evidence for the existence of a capsaicin-sensitive innervation in the rat urinary bladder, J. Pharm. Pharmacol., 38 (1986) 446451. 10 Santicioli, P., Patacchini, R., Maggi, C.A. and Meli, A., Exposure to calcium-free medium protects sensory fibers by capsaicin desensitization, Neurosci. Let[., 80 (1987) 167 172. 11 Szolcsanyi, J., Capsaicin-sensitive chemoceptive neural system with dual sensory-afferent function. In L.A. Chahl, J. SzolcsS.nyi and F.Lcmbeck (Eds.), Antidromic Vasodilation and Neurogenic lnflammatit)n, Akademiai Kiado, Budapest, 1984, pp. 26-52. 12 Szolcstinyi, J., Jancs6-Gabor, A. and Jo6, F., Functional and fine structural characteristics of the sensory neuron blocking action of capsaicin, Naunyn-Schmiedeberg's Arch. Pharmacol., 28;7 (1975) 157 169. 13 Tapia, R.. Arias, C. and Morales, E.. Binding of lanthanum ions and ruthenium red to synaptosomes and its effects on neurotransmitter release, J. Neurochem., 45 (1985) 1464 1470. 14 Williams, J.T. and ZieglgS.nsberger, W,, The acute effects of capsaicin on rat primary afferents and spinal neurons, Brain Res., 253 (1982) 125 131. 15 Wood, J.N., Rerversible desensitization to capsaicin induced Ca uptake in rat sensory neurons in culture, Neuroscience, 22 (1987) $247.
2111
Elsevier Scientific Publishers Ireland Ltd.
NSL05315
Protective action of Ruthenium red toward capsaicin desensitization of sensory fibers Carlo Alberto Maggi l, Riccardo Patacchini l, Paolo Santicioli 1, Sandro Giuliani 1, Pierangelo Geppetti 2 and Alberto M eli l ~Pharmaeology Department, Smooth Muscle Division, Research Laboratories, 'A. Menarini' Pharmaceuticals, Florence (Italy) and "Department of Internal Medicine and Clinical PharmacMogy, University qf Florence, Florence (Italy) (Received 3 November 1987; Revised version received 28 January 1988; Accepted 4 February 1988) Key word~v Capsaicin; Desensitization; R u t h e n i u m red: Rat urinary bladder: Efferent function of sensory nerves In the rat isolated urinary bladder, exposure to capsaicin (1 tiM) produced a contraction thought to involve neuropeptide(s) release from sensory nerves. A second application of the drug had no motor effect indicating desensitization. The establishment of the desensitized state requires the presence of cxtraccllular calcium. In the presence of R u t h e n i u m red (RR, 30 nM-10/.tM) the first response to capsaicin was reduced and a concentration-dependent protection from capsaicin desensitization was observed. R R up to 10(I/tM had no inhibitory effect toward contractions produced by exogenous substance P nor by electrical stimulation of efferent nerves.
Capsaicin exerts a selective excitatory (depolarizing) action on certain sensory nerves [5, 11]. Although the ionic basis of depolarization of sensory terminals is still largely unexplored, some evidence indicates that an increase in conductance to calcium ions (Ca) may be involved [1, 5, 11], This event determines the secretion of stored transmitters from peripheral sensory terminals through a tetrodotoxin-resistant depolarization e.g. the sensory receptor potential-coupled 'effbrent'response [5]. Circumstantial evidence also supports the idea that Ca accumulation into sensory neurons and nerves [3, 10] due to the prolonged depolarization by capsaicin [14], is involved in both the desensitizing and neurotoxic effect ofcapsaicin on primary afterents [6, 11]. At the ultrastructural level, accumulation of radiolabeled Ca within mitochondria of sensory neurons was observed following systemic capsaicin desensitization and this effect was implicated in the neurodegenerative action of capsaicin [3]. Indeed alterations at mitochondrial level were evident also in sensory a x o n s of rats desensitized to capsaicin as adults [12]. We have shown previously that, in the ab-
Correspondence: C.A. Maggi, Pharmacology Department, Smooth Muscle Division, Research Laboratories, "A Menarini' Pharmaceuticals, Via Sette Santi 3, 1-50131 Florence, Italy. 0304-3940/88,,$ 03.50 ~) 1988 Elsevier Scientific Publishers Ireland Ltd.
202 sence of extracellular Ca, there is a protection toward the capsaicin desensitization phenomenon at the level of peripheral terminals of the rat urinary bladder [10]. We now present evidence that Ruthenium red (RR), a compound reported to inhibit Ca mobilization in various tissues [2, 4, 8] and Ca uptake from mitochondria [7], behaves, in a quite large concentration range (30 nM-10/,tM), as a selective antagonist of the 'efferent' response (contraction) mediated by capsaicin-sensitive nerves of the rat urinary bladder and protects sensory fibers from capsaicin desensitization. Male albino rats of the Wistar-Morini strain weighing 340-360 g were killed by cervical dislocation and exsanguinated. The whole urinary bladder was rapidly removed, a strip of detrusor muscle (about I cm long) was dissected out and placed in a 5 ml organ bath (at 37°C) containing a standard Krebs solution (Ca concentration 2.5 raM) gassed with 96% O2~4%CO2, as described previously [9, 10]. Tension was recorded by means of an isometric strain gauge connected to a Basile 7050 Unirecord. Field stimulation was obtained by means of two wire platinum electrodes placed at the top and the bottom of the organ bath. Square wave pulses (60 V, 0.5 ms) were delivered automatically at a frequency of 0.1 Hz by means of a Grass S11 stimulator. In each experiment, capsaicin (1/~M) was added to the organ bath 5 min after the interruption of field stimulation. RR was added to the organ bath 25 min before the first application of capsaicin. On each application, capsaicin was left in contact with the tissue for 5 min and the maximal contractile response observed during this period was expressed as % of the response to field stimulation (FS, 0.5 ms, 60 V, 0.1 Hz) obtained in the same tissue. After each application of capsaicin (at 25 rain intervals) the bathing solution was repeatedly renewed during the first 15 min, e.g. RR was present in the bath only during the first exposure to capsaicin. During the washout period the strips were superfused with capsaicin-free Krebs solution at 20 ml/min for 1 min and then at 2.5 ml/min for 14 min. All data in the text are mean_+S.E.M. Statistical analysis was made by means of Student's t-test or analysis of variance, when applicable. Drugs used were R R (Serva), Ruthenium chloride (Aldrich), substance P (SP, Peninsula), capsaicin (Sigma). RR (30 nM-300/~M) did not inhibit twitches amplitude up to 10/~M. At 30-300 /,M, a 38 + 4 and 9 6 _ 2% inhibition was observed, respectively. At lower concentrations (30 nM-10 ktM) either no effect or a small (10-15%) potentiation or inhibition (Fig. I) of twitches was observed. These latter effects were present in a 40% of cases, were concentration-independent and their nature was not investigated further. In the presence of RR a concentration-dependent inhibition of the response to capsaicin (1 /~M) was observed (Table I, Fig. l) but at 30-300 nM there was no significant inhibitory effect. In control experiments RR up to 10/~M had no inhibitory effect toward contractions produced by SP at a concentration (30 nM, n = 8) which produced a response similar to that induced by capsaicin (1 /~M). At higher concentrations, RR inhibited the SP-induced contraction and this effect was similar to that observed toward twitches (35 + 4 and 86+ 3% inhibition at 30 and 300/~M, respectively, n = 4). In control strips, the first exposure to capsaicin (1 /tM for 5 min) was sufficient to produce desensitization to further applications of the drug (Fig. 1, Table I). On the other hand, exposure to RR before administration of capsaicin resulted in a con-
203 5 rain 10 mN
CONTROL
B
¸¸¸¸¸¸I RUT.EN,UM REO ~3 r "
l
I., RUr.EN,UM REO 3rM
C
1 D
l
• RUTHENIUM
RED
• 10 }JM
1
• •
= CAPSAICIN
• 1 ~M
Fig. 1. Typical tracings showing the effect of increasing concentrations of RR (A, control; B, RR 0.3/~M; C, RR 3 izM; D, RR I0 ,uM) on the contractile response to capsaicin (1/LM at the dots) of the rat isolated urinary bladder and subsequent desensitization. The response to field stimulation is shown before cach application of capsaicin. In each preparation, capsaicin was applied at 25 min intervals with repeated renewals of the bathing solution between each application. After the first application ofcapsaicin the preparations were washed with RR-free Krebs solution.
c e n t r a t i o n - d e p e n d e n t p r o t e c t i o n f r o m c a p s a i c i n d e s e n s i t i z a t i o n ( T a b l e I). T h i s is well e v i d e n t in a t y p i c a l e x p e r i m e n t s h o w n in Fig. 1: at 300 n M o f R R a s e c o n d a p p l i c a t i o n o f c a p s a i c i n p r o d u c e d a large, s l o w l y d e v e l o p i n g c o n t r a c t i o n w h i l e a t h i r d o r f o u r t h a p p l i c a t i o n was ineffective. A t h i g h e r R R c o n c e n t r a t i o n s , the p r o t e c t i o n f r o m
204 TABLE I E F F E C T OF E X P O S U R E TO R U T H E N I U M R E D ON T H E C O N T R A C T I L E RESPONSE OF T H 1 RAT I S O L A T E D B L A D D E R TO R E P E A T E D A D M I N I S T R A T I O N OF C A P S A I C I N Each value is mean -+S.E.M. RR was added to the organ bath 25 rain before the first application ofcapsai cin. After each capsaicin administration there was a 30 min interval between doses during which the Krebs solution was repeatedly renewed. NE, no effect. ~Significantly different from controls, P<0.05. Treatment
Controls R R 0.03 ~M R R 0.3/.tM RR 3/tM R R 10/tM
No. of experiments
Response to FS (raN)
Response to capsaicin ( 1/*M) (% of FS)
Before
After
First
Second
Third
Fourth
7 5 5 8 5
19__+ 1 20_+2 17_+2 I7±2 18_+3
21_+2 18+2 18+2 18_+2
136,-- 16 143+12 128_+19 91_+ 7a 40_+13 ~
3+ 3 38+10 a 57___13a 76__+14~ 58-+1D
NE NE 15± 3 33_+19 66-+ 6
NE NE NE NE 43_+7
desensitization was extended to the third and fourth (at 10/,M of RR) dose ofcapsaicin, despite the repeated washouts intervening between each application. In strips exposed to RR 10/tM the second and third application of capsaicin produced responses higher than that of the first dose and some response was observed up to a fifth or sixth exposure to capsaicin (data not shown). Despite repeated washouts, the tissues exposed to R R retained a pink to Violet color (depending on the RR concentration) throughout the experiment, suggesting that a given fraction of R R was tightly bound to the tissue. Ruthenium chloride (10/,M, n = 4 ) had no effect toward the capsaicin (1 /,M)induced contraction nor induced any protection toward capsaicin desensitization (data not shown). The cellular bases of capsaicin desensitization are unknown. In the rat bladder, A single exposure to capsaicin for a few minutes produced an almost complete unresponsiveness to a further challenge with the drug which is not attributable to SP depletion from sensory fibers [7]. Previous observations indicated that the presence of extracellular Ca is essential for the establishment of the desensitized state at the level of peripheral terminals of these sensory fibers [10], supporting the idea that an excess of intracellular Ca and/or inability of the sensory neuron/fiber to buffer it were involved in the desensitizing action of capsaicin [5]. The present findings indicate that RR up to 10 # M has a selective inhibitory action toward the effects of capsaicin involving both the acute response (depolarization of sensory fibers) and the subsequent desensitization. The present experiments do not provide any direct link between the action of RR toward capsaicin and its inhibitory action on transmembrane Ca movements. Although direct experiments are needed to assess this point, the assumption seems tenable for 3 reasons: (a) R R affects transmembrane Ca fluxes in a variety of tissues [2, 8]; (b) in low concentrations, RR was shown to block transmitter release from synaptosomes due to depolarization-coupled Ca entry [ 13]; and (c), a recent prelim±n-
205 a r y r e p o r t [l 5] i n d i c a t e s t h a t R R in a n a n o m o l a r induced
establishment
of the desensitized
Although a direct comparison
concentration inhibits the capsaicin-
state in sensory
neurons
in c u l t u r e .
b e t w e e n o u r f i n d i n g s a n d t h o s e o f W o o d [15] is diffi-
cult, the presence of extracellular Ca was shown to be essential for the establishment o f t h e d e s e n s i t i z e d s t a t e in b o t h c a s e s [10, 15]. T h u s it s e e m s c o n c e i v a b l e to a s s u m e t h a t t h e p r o t e c t i v e effect o f R R i n v o l v e d s o m e a n t a g o n i s m t o w a r d t h e a d v e r s e effect o f a ' h i g h ' C a i n f l u x in s e n s o r y n e u r o n / f i b e r . In t h i s s t u d y we a l s o o b s e r v e d t h a t R u t h e n i u m
i o n s p e r se a r e n o t i m p o r t a n t s i n c e
Ruthenium chloride was ineffective toward the actions ofcapsaicin. This observation p a r a l l e l s p r e v i o u s f i n d i n g s [8] i n d i c a t i n g t h a t t h e e n t i r e m o l e c u l e is r e q u i r e d to b l o c k Ca movements. The observation that at certain concentrations r e s p o n s e t o c a p s a i c i n w h i l e h a v i n g n o effect t o w a r d
R R r e d u c e d t h e first
SP suggests a reduced release
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