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Characterization of a peripheral vanilloid (capsaicin) receptor in the urinary bladder of the rat
Life
Sciences,
Vol.
52,
pp. PL 221-226
Pergamon Press
Printed in the USA
PHAJWACOLOGY Accelerated
CHARACTERIZATION
Arpad
Szallasi,
LETTERS
Communication
0~ A PERIPHERAL VANILLOID (CAPSAICIN) THE URINARY BLADDER OF THE RAT Bruno
Conte, Cristina Goso, and Stefano Manzini
RECEPTOR
*Peter
M.
IN
Blumberg
(Roma), Menarini Ricerche Sud, Via Tit0 Speri 10, 00040 Pomezia Italy and *National Cancer Institute, Bethesda, MD 20892, U.S.A. (Submitted December 28, 1992; accepted February 2, 1993; received in final form March 8, 1993) Abstract. Specific binding of [3H]resiniferatoxin (RTX) is thought In the present to represent the vanilloid (capsaicin) receptor. have used this binding assay to identify for the first study, we time a vanilloid receptor in the periphery and to compare it to central vanilloid receptors present in dorsal root ganglia (DRG) as Rat urinary bladder membranes well as in spinal cord of the rat. bound [3~]~~~ with a Kd of 30 f 4 pM and a Bmax of 65 + 14 fmol/mg the corresponding values were 19 + 3 pM and 104 + 14 protein; fmol/mg protein in DRG, and 16 + 3 pM and 50 + 9 fmol/mg protein in Capsaicin inhibited [3~1~~~ binding to membranes from spinal cord. potency spinal cord, and DRG with similar urinary bladder, and 0.6 + 0.1 1:: 0.5 _t 0.1 PM, 0.3 _f_ 0.1, were values [3H]RTX bound to urinary bladder in Interestingly, respectively). a non-cooperative fashion in contrast with the apparent positive [3~1~~~ binding in both DRG and spinal cord cooperativity of (cooperativity index = 1.8 and 1.7, respectively). This finding suggests heterogeneity in the properties of the vanilloid receptors in the rat.
Introduction A major group of primary afferent neurons mediating nociception sensation (afferent function), subserving neurogenic and pain inflammation and triggering a variety of motor, vascular, and secretory actions (efferent function) is characterized by selective susceptibility to the excitatory, sensory blocking and neurotoxic actions of capsaicin, the pungent principle in hot peppers (1). selectivity of capsaicin actions and the fairly strict The structural requirements for capsaicin-like activity implied that capsaicin interacted at a specific recognition site to produce these effects Whilst the combination of relatively poor (2). potency and high lipophilicity prevented the use of radiolabeled Corresponding Department of (Roma), Italy
author: Arpad Pharmacology,
Szallasi, via Tito
Menarini Speri 10,
0024-3205/93 $6.00 + .OO
Ricerche Sud, 00040 Pomezia
PL-222
Vanilloid
Receptor
in Bladder
Vol.
52, No.
20,
1993
capsaicin to detect the vanilloid (capsaicin) receptor, by using [3H]resiniferatoxin (RTX), an ultrapotent capsaicin analog (31, we have recently been able to overcome these technical obstacles and to demonstrate saturable high affinity L3HlRTX binding by sensory ganglion membranes of the rat (4). The identification of peripheral vanilloid receptors was, however, hampered by the high level of nonspecific [~H]RTX binding (4,5). As part of our efforts to detect and characterize peripheral vanilloid receptors, in the present study we have used a modified L3HlRTX binding assay (6,7) in which the non-specific binding was reduced by adding alphal-acid glycoprotein, a plasma protein that binds RTX (6). Using this modified assay, we have recently shown that specific [3H]RTX binding by rat spinal cord and sensory ganglion membranes displays apparent positive cooperativity at low ligand concentrations (7,8), which could not be detected with the previous binding methodology (4,8). We confirm these findings here, report the detection of a vanilloid receptor in urinary bladder, and compare its properties to those present in the perikarya and central terminals of the vanilloid-sensitive primary afferent neurons. Methods Membranes were prepared as described (4,5). Briefly, Sprague-Dawley rats (males, 250-300 g) were sacrificed by cervical dislocation; the urinary bladder, the lumbar dorsal root ganglia (DRG), and the corresponding segment of the spinal cord were removed and disrupted with the aid of a Polytron tissue homogenizer in ice-cold buffer A (in mM) KC1 5, NaCl 5.8, MgC12 2, CaC12 0.75, (pH 7.4), containing sucrose 137, HEPES 10. The homogenates were centrifuged for 10 min at 2000 g (4 OC), the pellets were discarded, and the supernatants were then centrifuged at 35 000 g for 30 min. The resulting pellets were resuspended in buffer A and stored at -70 OC until assayed. Urinary bladders were also collected from rats 4 days aftersurgical bladder denervation (bilateral removal of the major pelvic ganglia as described by Santicioli et al.(g) 1 or sham-operation, as well as from adult rats that had received a single S.C. injection capsaicin (10:10:80 of (50 mg/kg) or vehicle Tween v/v 80:EtOH:saline) on the 2nd day of life, a procedure used to destroy vanilloid-sensitive nerve endings (101. 40-60 pg aliquots of the particulate fraction protein in 0.5 ml of buffer A containing 0.25 mg/ml bovine serum albumin (Cohn fraction protein RTX in aqueous included to stabilize V), a carrier with [3~1~~~ and solution, were incubated in triplicate nonradioactive ligands at 37 OC for 30 min. Nonspecific binding was nonradioactive RTX. After determined in the presence of 100 nM the binding reaction was terminated by chilling the assay mixture in 50 p1 of on ice, 100 pg bovine alphal-acid glycoprotein Dulbeccols phosphate buffered saline was added to each tube to reduce nonspecific binding (6). Bound and free [3H]RTX were then separated by pelleting the membranes in a Beckman 12 microfuge; a 200 1.11aliquot of the supernatant was removed to determine the free [3H]RTX concentration; the remainder of the supernatant was removed by aspiration; the pellet was dried with the tip of a rolled facial
Vol. 52, NO. 20, 1993
Vanilloid Receptor in Bladder
PL-223
tube containing the pelleted the tip of the microfuge tissue; with a razor blade; and the bound was cut off membranes radioactivity was determined by scintillation counting. using increasing from saturation experiments Binding data concentrations of L3HlRTX were analyzed by computer fit to the Hill equation (11) using the program Fit P (Biosoft, Cambridge, UK), or the curvilinear regression program LIGAND (Biosoft, by using RTX binding was also analyzed in the presence of a Cambridge, UK). using 1 : 3 dilutions of concentration of L3H]RTX fixed (30 pM) The concentration of nonradioactive ligand the competing ligand. was determined by a [3H]RTX binding by 50% inhibiting (1c50) computer program fitting a theoretical sigmoidal competition curve Ki values were calculated using the general equation to the data; Ki = IC50 / (1 + L/Kd). [3H]RTX (37 Ci/mmol) was synthesized by the Chemical Synthesis and NCI-FCRDC (Frederick, MD, Laboratory; U.S.A.). Analysis Nonradioactive RTX was purchased from LC Services (Woburn; MA, All the other chemicals used were from Sigma (St. Louis, U.S.A.). MO, U.S.A.). Results In accord with our previous observations (7,8), L3HlRTX displayed specific saturable binding to DRG and spinal cord membranes with sigmoidal saturation kinetics indicating positive cooperativity (not shown). The binding parameters were calculated by fitting the Hill equation to the measured data allosteric (11). In DRG the maximal binding capacity was 104 + 14 fmol/mq membranes, protein (mean + SEM; n = 4); the half maximal binding was attained index, also known as the Hill at 19 +. 3 pM; and the cooperativity was 1.8 + 0.1. In spinal cord membranes coefficient, the corresponding values were 50 L 9 fmol/mq protein, 16 + 3 pM, and (mean + SEM; 5 determinations). 1.7 + 0.2, respectively The Scatchard plot of the data demonstrates the dramatic effect of the apparent positive cooperativity on [ 3 H]RTX binding (Figure 1). In contrast, urinary bladder membranes bound [3~1~~~ with a Hill coefficient approaching 1; the curvilinear analysis of the data confirmed the one-site model suggested by the Scatchard plot (Figure 1) and gave final parameter estimates of Kd = 30 + 4 pM and Bmax = 65 + 14 fmol/mq protein (mean & SEM; 4 determinations). At the Kd value nonspecific binding represented approximately 20% of the total binding. The pharmacological specificity of L3H]RTX binding to urinary bladder membranes was confirmed by 2 approaches. First, no specific L3HlRTX binding could be detected in the urinary bladder of rats pretreated with capsaicin as neonates (chemical denervation), or in urinary bladders following surgical bladder denervation. Second, the [3~1~~~ binding by urinary bladder membranes was inhibited completely by capsaicin (Figure 2) whereas resiniferonol 9,13,14-orthophenylacetate, the C20-deesterified parent diterpene of RTX which is inactive as a vanilloid (12), did not inhibit L3H]RTX binding at a concentration (1 J1M) 30-fold higher than its Kd for protein kinase C (12). Capsaicin inhibited
PI_.-224
Vanilloid
Receptor
Vol.
in Bladder
52, No.
20,
1993
3.5 ii L
3.0
5
2.5
5 0 03
2.0
ST
1.5
k z 0 -
1.0 0.5 0.0 0
20
40
60
13HlRTX;
80
100
120
Bound
FIG. 1 Scatchard plot of specific [3Hlresiniferatoxin obtained from dorsal particulate preparations spinal cord bladder ( w (A ), and urinary
(RTX) binding by root ganglia ( o,, ) of the rat. Points
represent mean values from a single experiment; bound L3~]~TX is in expressed in fmol/mg protein, bound/free is given fmol/mg protein/pM; for dorsal root ganglia and spinal cord membranes, theoretical curves were calculated from the values for Bmax, Kd and Hill number determined by computer fit to the allosteric Hill the line was fitted using equation; for urinary bladder membranes, (dorsal root ganglion the LIGAND program. Replicated experiments 4 determinations; spinal membranes and urinary bladder membranes: cord membranes: 5 determinations) gave similar results.
a Ki value of [3H]RTX binding to urinary bladder membranes with this affinity agrees with 0.5 + 0.1 PM (mean k range; Figure 2); its affinity for DRG membranes (0.6 + 0.1 PM; mean + range; not shown) or spinal cord membranes (0.3 - 0.1 J1M, mean + SEM, n=3; not shown). Discussion Although irritant compounds of the capsaicinoid and resiniferanoid classes seem to share a common binding site, referred to as the vanilloid receptor (3), they show striking differences in relative potencies to provoke and then desensitize biological responses (3,13,14). A dramatic example of this phenomenon has been described on the one hand, by Maggi et al. (13) who found, RTX to be equipotent to capsaicin for contracting the isolated rat urinary
Vol. 52, No. 20, 1993
Vanilloid Receptor in Bladder
PL-225
80 60 40 20 0
Competitor;
log M
FIG. 2 Competition curves for inhibition of t3H]resiniferatoxin (RTX) binding to urinary bladder membranes of the rat by nonradioactive RTX 2 experiments; points are ( 0) and capsaicin (A ). Curves represent the mean of the 2 determinations; error bars indicate range.
bladder, and, on the other hand, lOOO-fold more potent than for desensitizing the same response. capsaicin To explain the divergence between the stimulatory and desensitizing potencies of vanilloids, two hypotheses have been put forward. One postulates the existence of receptor subclasses responsible for stimulation and desensitization, respectively, with distinct structure-activity relations (1,12,15); the other assumes a common site of action and proposes a crucial role for differences in pharmacokinetics (13). Our demonstration of a single, high affinity binding site both for RTX and capsaicin in the urinary bladder supports Magqifs conclusion (13) that a slower tissue penetration rate for RTX might be the predominant factor contributing to the divergence between stimulatory and desensitizing actions of RTX in the bladder. Nevertheless, our results do not rule out the existence of additional specific RTX binding site(s) that are either quantitatively minor or are of low affinity. Our finding that urinary bladder membranes, unlike DRG or spinal cord membranes of the same rats, bind i3HlRTX in a noncooperative manner may imply heterogeneity of the vanilloid receptor. The underlying mechanism of positive cooperativity of binding is ill understood. In general there are two basic models. The first model assumes at least two binding sites that interact: binding of a ligand to a binding site promotes the binding of an additional liqand (or several molecules of ligand) to another binding site (or several binding sites) (16; 17). Alternatively, a receptor monomer
PL-226
Vanilloid
Receptor
in Bladder
Vol.
52, No.
20,
1993
may exist
in multiple affinity states, and the ligand-receptor interaction may shift the receptor to a higher affinity state (18). Since the molecular target size of central vanilloid receptors -270 kDa in pig DRG and 280 kDa in pig spinal cord (19)- implies the existence of a receptor complex, we think that interaction among members of a receptor oligomer is the most likely explanation for positive cooperativity in RTX binding (7,8). In keeping with this the non-cooperative manner of RTX binding in urinary theory, bladder may indicate the existence of a monomeric vanilloid receptor. Whether the non-cooperative binding is a general feature of the peripheral vanilloid receptors or a unique feature of the vanilloid receptors in urinary bladder the remains to be established. At the methodological level, the specific L3HlRTX binding assay utilizing urinary bladder membranes represents a biochemical marker of capsaicin-sensitive nerves; this assay may reveal whether or not the capsaicin-sensitive sensory innervation of the bladder is altered in pathophysiological conditions. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
P. HOLZER, Pharmacol. Rev., 43 144-201 (1991). J. SZOLCSANYI and A. JANCSO-GABOR, Drug. Res., 25 1877-1881 (1975). A. SZALLASI and P.M. BLUMBERG, Life Sci., 47 1399-1408 (1990). A. SZALLASI and P.M. BLUMBERG, Brain Res., 524 106-111 (1990). A. SZALLASI and P.M. BLUMBERG, Methods, Vol. 8, P.M. Conn ted), 368-380, Academic Press, Orlando, FL (1992). A. SZALLASI, N.A. LEWIN and P.M. BLUMBERG, J. Pharmacol. Exp. Ther., 262 883-888 (1992). A. SZALLASI and P.M. BLUMBERG, Naunyn-Schmiedeberg's Arch. Pharmacol., in press. A. SZALLASI, N.A. LEWIN and P.M. BLUMBERG, J. Pharmacol. Exp. Ther., in press. P. SANTICIOLI, C.A. MAGGI and A. MELI, J. Pharm. Pharmacol., B 446-451 (1986). G. JANCSO, E. KIRALY and A. JANCSO-GABOR, Nature, 270 741-743 (1977). L. ENDRENYI, CS. FAJSZI and F.H.F. KWONG, Eur. J. Biochem., 51 317-328 (1975). A. SZALLASI, N.A. SHARKEY and P.M. BLUMBERG, Phytother. Res., 1 253-257 (1989). C.A. MAGGI, R. PATACCHINI, M. TRAMONTANA, R. AMANN, S. GIULIANI and P. SANTICIOLI, Neuroscience, 37 531-539 (1990). J. WINTER, A. DRAY, J.N. WOOD, J.C. YEATS and S. BEVAN, Brain Res., 520 131-140 (1990). A. SZALLASI and P.M. BLUMBERG, Neuroscience, 30 515-520 (1989). G. FELS, E.K. WOLFF and A. MAELICKE, Eur. J. Biochem., 127 3138 (1982). G.J. MOORE and M.N. SCANLON, Gen. Pharmacol., 20 193-198 (1989). T. COSTA, M. WUSTER, C. GRAMSCH and A. HERZ, Mol. Pharmacol. 2 146-154 (1985). A. SZALLASI and P.M. BLUMBERG, Life Sci., & 1863-1869 (1991).
Sciences,
Vol.
52,
pp. PL 221-226
Pergamon Press
Printed in the USA
PHAJWACOLOGY Accelerated
CHARACTERIZATION
Arpad
Szallasi,
LETTERS
Communication
0~ A PERIPHERAL VANILLOID (CAPSAICIN) THE URINARY BLADDER OF THE RAT Bruno
Conte, Cristina Goso, and Stefano Manzini
RECEPTOR
*Peter
M.
IN
Blumberg
(Roma), Menarini Ricerche Sud, Via Tit0 Speri 10, 00040 Pomezia Italy and *National Cancer Institute, Bethesda, MD 20892, U.S.A. (Submitted December 28, 1992; accepted February 2, 1993; received in final form March 8, 1993) Abstract. Specific binding of [3H]resiniferatoxin (RTX) is thought In the present to represent the vanilloid (capsaicin) receptor. have used this binding assay to identify for the first study, we time a vanilloid receptor in the periphery and to compare it to central vanilloid receptors present in dorsal root ganglia (DRG) as Rat urinary bladder membranes well as in spinal cord of the rat. bound [3~]~~~ with a Kd of 30 f 4 pM and a Bmax of 65 + 14 fmol/mg the corresponding values were 19 + 3 pM and 104 + 14 protein; fmol/mg protein in DRG, and 16 + 3 pM and 50 + 9 fmol/mg protein in Capsaicin inhibited [3~1~~~ binding to membranes from spinal cord. potency spinal cord, and DRG with similar urinary bladder, and 0.6 + 0.1 1:: 0.5 _t 0.1 PM, 0.3 _f_ 0.1, were values [3H]RTX bound to urinary bladder in Interestingly, respectively). a non-cooperative fashion in contrast with the apparent positive [3~1~~~ binding in both DRG and spinal cord cooperativity of (cooperativity index = 1.8 and 1.7, respectively). This finding suggests heterogeneity in the properties of the vanilloid receptors in the rat.
Introduction A major group of primary afferent neurons mediating nociception sensation (afferent function), subserving neurogenic and pain inflammation and triggering a variety of motor, vascular, and secretory actions (efferent function) is characterized by selective susceptibility to the excitatory, sensory blocking and neurotoxic actions of capsaicin, the pungent principle in hot peppers (1). selectivity of capsaicin actions and the fairly strict The structural requirements for capsaicin-like activity implied that capsaicin interacted at a specific recognition site to produce these effects Whilst the combination of relatively poor (2). potency and high lipophilicity prevented the use of radiolabeled Corresponding Department of (Roma), Italy
author: Arpad Pharmacology,
Szallasi, via Tito
Menarini Speri 10,
0024-3205/93 $6.00 + .OO
Ricerche Sud, 00040 Pomezia
PL-222
Vanilloid
Receptor
in Bladder
Vol.
52, No.
20,
1993
capsaicin to detect the vanilloid (capsaicin) receptor, by using [3H]resiniferatoxin (RTX), an ultrapotent capsaicin analog (31, we have recently been able to overcome these technical obstacles and to demonstrate saturable high affinity L3HlRTX binding by sensory ganglion membranes of the rat (4). The identification of peripheral vanilloid receptors was, however, hampered by the high level of nonspecific [~H]RTX binding (4,5). As part of our efforts to detect and characterize peripheral vanilloid receptors, in the present study we have used a modified L3HlRTX binding assay (6,7) in which the non-specific binding was reduced by adding alphal-acid glycoprotein, a plasma protein that binds RTX (6). Using this modified assay, we have recently shown that specific [3H]RTX binding by rat spinal cord and sensory ganglion membranes displays apparent positive cooperativity at low ligand concentrations (7,8), which could not be detected with the previous binding methodology (4,8). We confirm these findings here, report the detection of a vanilloid receptor in urinary bladder, and compare its properties to those present in the perikarya and central terminals of the vanilloid-sensitive primary afferent neurons. Methods Membranes were prepared as described (4,5). Briefly, Sprague-Dawley rats (males, 250-300 g) were sacrificed by cervical dislocation; the urinary bladder, the lumbar dorsal root ganglia (DRG), and the corresponding segment of the spinal cord were removed and disrupted with the aid of a Polytron tissue homogenizer in ice-cold buffer A (in mM) KC1 5, NaCl 5.8, MgC12 2, CaC12 0.75, (pH 7.4), containing sucrose 137, HEPES 10. The homogenates were centrifuged for 10 min at 2000 g (4 OC), the pellets were discarded, and the supernatants were then centrifuged at 35 000 g for 30 min. The resulting pellets were resuspended in buffer A and stored at -70 OC until assayed. Urinary bladders were also collected from rats 4 days aftersurgical bladder denervation (bilateral removal of the major pelvic ganglia as described by Santicioli et al.(g) 1 or sham-operation, as well as from adult rats that had received a single S.C. injection capsaicin (10:10:80 of (50 mg/kg) or vehicle Tween v/v 80:EtOH:saline) on the 2nd day of life, a procedure used to destroy vanilloid-sensitive nerve endings (101. 40-60 pg aliquots of the particulate fraction protein in 0.5 ml of buffer A containing 0.25 mg/ml bovine serum albumin (Cohn fraction protein RTX in aqueous included to stabilize V), a carrier with [3~1~~~ and solution, were incubated in triplicate nonradioactive ligands at 37 OC for 30 min. Nonspecific binding was nonradioactive RTX. After determined in the presence of 100 nM the binding reaction was terminated by chilling the assay mixture in 50 p1 of on ice, 100 pg bovine alphal-acid glycoprotein Dulbeccols phosphate buffered saline was added to each tube to reduce nonspecific binding (6). Bound and free [3H]RTX were then separated by pelleting the membranes in a Beckman 12 microfuge; a 200 1.11aliquot of the supernatant was removed to determine the free [3H]RTX concentration; the remainder of the supernatant was removed by aspiration; the pellet was dried with the tip of a rolled facial
Vol. 52, NO. 20, 1993
Vanilloid Receptor in Bladder
PL-223
tube containing the pelleted the tip of the microfuge tissue; with a razor blade; and the bound was cut off membranes radioactivity was determined by scintillation counting. using increasing from saturation experiments Binding data concentrations of L3HlRTX were analyzed by computer fit to the Hill equation (11) using the program Fit P (Biosoft, Cambridge, UK), or the curvilinear regression program LIGAND (Biosoft, by using RTX binding was also analyzed in the presence of a Cambridge, UK). using 1 : 3 dilutions of concentration of L3H]RTX fixed (30 pM) The concentration of nonradioactive ligand the competing ligand. was determined by a [3H]RTX binding by 50% inhibiting (1c50) computer program fitting a theoretical sigmoidal competition curve Ki values were calculated using the general equation to the data; Ki = IC50 / (1 + L/Kd). [3H]RTX (37 Ci/mmol) was synthesized by the Chemical Synthesis and NCI-FCRDC (Frederick, MD, Laboratory; U.S.A.). Analysis Nonradioactive RTX was purchased from LC Services (Woburn; MA, All the other chemicals used were from Sigma (St. Louis, U.S.A.). MO, U.S.A.). Results In accord with our previous observations (7,8), L3HlRTX displayed specific saturable binding to DRG and spinal cord membranes with sigmoidal saturation kinetics indicating positive cooperativity (not shown). The binding parameters were calculated by fitting the Hill equation to the measured data allosteric (11). In DRG the maximal binding capacity was 104 + 14 fmol/mq membranes, protein (mean + SEM; n = 4); the half maximal binding was attained index, also known as the Hill at 19 +. 3 pM; and the cooperativity was 1.8 + 0.1. In spinal cord membranes coefficient, the corresponding values were 50 L 9 fmol/mq protein, 16 + 3 pM, and (mean + SEM; 5 determinations). 1.7 + 0.2, respectively The Scatchard plot of the data demonstrates the dramatic effect of the apparent positive cooperativity on [ 3 H]RTX binding (Figure 1). In contrast, urinary bladder membranes bound [3~1~~~ with a Hill coefficient approaching 1; the curvilinear analysis of the data confirmed the one-site model suggested by the Scatchard plot (Figure 1) and gave final parameter estimates of Kd = 30 + 4 pM and Bmax = 65 + 14 fmol/mq protein (mean & SEM; 4 determinations). At the Kd value nonspecific binding represented approximately 20% of the total binding. The pharmacological specificity of L3H]RTX binding to urinary bladder membranes was confirmed by 2 approaches. First, no specific L3HlRTX binding could be detected in the urinary bladder of rats pretreated with capsaicin as neonates (chemical denervation), or in urinary bladders following surgical bladder denervation. Second, the [3~1~~~ binding by urinary bladder membranes was inhibited completely by capsaicin (Figure 2) whereas resiniferonol 9,13,14-orthophenylacetate, the C20-deesterified parent diterpene of RTX which is inactive as a vanilloid (12), did not inhibit L3H]RTX binding at a concentration (1 J1M) 30-fold higher than its Kd for protein kinase C (12). Capsaicin inhibited
PI_.-224
Vanilloid
Receptor
Vol.
in Bladder
52, No.
20,
1993
3.5 ii L
3.0
5
2.5
5 0 03
2.0
ST
1.5
k z 0 -
1.0 0.5 0.0 0
20
40
60
13HlRTX;
80
100
120
Bound
FIG. 1 Scatchard plot of specific [3Hlresiniferatoxin obtained from dorsal particulate preparations spinal cord bladder ( w (A ), and urinary
(RTX) binding by root ganglia ( o,, ) of the rat. Points
represent mean values from a single experiment; bound L3~]~TX is in expressed in fmol/mg protein, bound/free is given fmol/mg protein/pM; for dorsal root ganglia and spinal cord membranes, theoretical curves were calculated from the values for Bmax, Kd and Hill number determined by computer fit to the allosteric Hill the line was fitted using equation; for urinary bladder membranes, (dorsal root ganglion the LIGAND program. Replicated experiments 4 determinations; spinal membranes and urinary bladder membranes: cord membranes: 5 determinations) gave similar results.
a Ki value of [3H]RTX binding to urinary bladder membranes with this affinity agrees with 0.5 + 0.1 PM (mean k range; Figure 2); its affinity for DRG membranes (0.6 + 0.1 PM; mean + range; not shown) or spinal cord membranes (0.3 - 0.1 J1M, mean + SEM, n=3; not shown). Discussion Although irritant compounds of the capsaicinoid and resiniferanoid classes seem to share a common binding site, referred to as the vanilloid receptor (3), they show striking differences in relative potencies to provoke and then desensitize biological responses (3,13,14). A dramatic example of this phenomenon has been described on the one hand, by Maggi et al. (13) who found, RTX to be equipotent to capsaicin for contracting the isolated rat urinary
Vol. 52, No. 20, 1993
Vanilloid Receptor in Bladder
PL-225
80 60 40 20 0
Competitor;
log M
FIG. 2 Competition curves for inhibition of t3H]resiniferatoxin (RTX) binding to urinary bladder membranes of the rat by nonradioactive RTX 2 experiments; points are ( 0) and capsaicin (A ). Curves represent the mean of the 2 determinations; error bars indicate range.
bladder, and, on the other hand, lOOO-fold more potent than for desensitizing the same response. capsaicin To explain the divergence between the stimulatory and desensitizing potencies of vanilloids, two hypotheses have been put forward. One postulates the existence of receptor subclasses responsible for stimulation and desensitization, respectively, with distinct structure-activity relations (1,12,15); the other assumes a common site of action and proposes a crucial role for differences in pharmacokinetics (13). Our demonstration of a single, high affinity binding site both for RTX and capsaicin in the urinary bladder supports Magqifs conclusion (13) that a slower tissue penetration rate for RTX might be the predominant factor contributing to the divergence between stimulatory and desensitizing actions of RTX in the bladder. Nevertheless, our results do not rule out the existence of additional specific RTX binding site(s) that are either quantitatively minor or are of low affinity. Our finding that urinary bladder membranes, unlike DRG or spinal cord membranes of the same rats, bind i3HlRTX in a noncooperative manner may imply heterogeneity of the vanilloid receptor. The underlying mechanism of positive cooperativity of binding is ill understood. In general there are two basic models. The first model assumes at least two binding sites that interact: binding of a ligand to a binding site promotes the binding of an additional liqand (or several molecules of ligand) to another binding site (or several binding sites) (16; 17). Alternatively, a receptor monomer
PL-226
Vanilloid
Receptor
in Bladder
Vol.
52, No.
20,
1993
may exist
in multiple affinity states, and the ligand-receptor interaction may shift the receptor to a higher affinity state (18). Since the molecular target size of central vanilloid receptors -270 kDa in pig DRG and 280 kDa in pig spinal cord (19)- implies the existence of a receptor complex, we think that interaction among members of a receptor oligomer is the most likely explanation for positive cooperativity in RTX binding (7,8). In keeping with this the non-cooperative manner of RTX binding in urinary theory, bladder may indicate the existence of a monomeric vanilloid receptor. Whether the non-cooperative binding is a general feature of the peripheral vanilloid receptors or a unique feature of the vanilloid receptors in urinary bladder the remains to be established. At the methodological level, the specific L3HlRTX binding assay utilizing urinary bladder membranes represents a biochemical marker of capsaicin-sensitive nerves; this assay may reveal whether or not the capsaicin-sensitive sensory innervation of the bladder is altered in pathophysiological conditions. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
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