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Effect of omega conotoxin on reflex responses mediated by activation of capsaicin-sensitive nerves of the rat urinary bladder and peptide release from the rat spinal cord
h’euroscience Vol. 34,No. 1,pp. 243-250,1990 Printedin GreatBritain
0306-4522/90 $3.00+ 0.00 PergamonPressplc 0 1990IBRO
EFFECT OF OMEGA CONOTOXIN ON REFLEX RESPONSES MEDIATED BY ACTIVATION OF CAPSAICIN-SENSITIVE NERVES OF THE RAT URINARY BLADDER AND PEPTIDE RELEASE FROM THE RAT SPINAL CORD C. A. MAGGI,* S. GIULIANI,* P. SANTICIOLI,* M. TRAMONTANA~ and A. MELI* *Pharmacology Department, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131 Florence, Italy tInstitute of Internal Medicine IV, University of Florence, Italy Abstract-The aim of this study was to assess whether omega conotoxin fraction GVIA, a potent blocker of N- and L-type voltage-sensitive calcium channels, might interfere with reflex responses (micturition, blood pressure rise in spinal rats) produced by activation of capsaicin-sensitive sensory nerves of the rat urinary bladder. The effect of conotoxin was also investigated on reflex micturition persisting after capsaicin pretreatment. Following topical application onto the bladder, conotoxin did not affect the volume threshold to elicit micturition although it reduced the amplitude of volume-evoked bladder contractions. Likewise, topical conotoxin did not prevent the reflex rise in blood pressure elicited by sudden bladder distension or topical application of capsaicin onto the bladder. In contrast, topical lidocaine strongly prevented both reflex responses. After intrathecal administration, conotoxin produced a dose-dependent inhibition of volume-evoked bladder contractions and the cardiovascular reflex produced by mechanical or chemical stimulation of bladder nerves. Intrathecal conotoxin inhibited micturition also in rats pretreated with capsaicin (50 mg/kg s.c., 4 days before). Depolarization by high potassium (80mM) produced release of both substance P- and calcitonin gene-related peptide-like immunoreactivity from superfused slices of the dorsal half of rat spinal cord. Capsaicin (1 PM) produced a similar effect, and a previous exposure to capsaicin prevented the effect of potassium. Conotoxin (0.1 PM) significantly reduced (about 50%) the potassium-induced release of neuropeptides from the dorsal half of the rat spinal cord. These findings indicate that conotoxin-sensitive calcium channels in the rat spinal cord play a role in the neurotransmission along reflex pathways activated by stimulation of capsaicin-sensitive nerves in the urinary bladder. Inhibition of sensory neuropeptide release from primary afferents in the spinal cord is a likely target for conotoxin. These findings do not support the hypothesis that conotoxin-sensitive calcium channels are implicated in the genesis of sensory impulses at the peripheral level, although clarification of this point needs further investigation.
Capsaicin-sensitive primary afferent neurons release nerve endings. 23 CTX is a potent blocker of both their transmitter content from both central and perL- and N-type voltage-sensitive Ca channels in ipheral endings thereby producing a dual, sensory chick sensory neurons’ and is gaining wide use as a and “efferent” function.‘3,22,37 Transmitter release tool for studying transmitter secretion in the central from both central and peripheral endings of these and peripheral nervous system.3~4*‘2+‘7,23,24 sensory nerves is a Ca-dependent process.7-9,26,27,30~34 Some information is available about the ionic bases Voltage-dependent Ca channels are present on perfor sensory impulse generation by capsaicin. At nerve ipheral endings of capsaicin-sensitive afferents, but trunk level, there is evidence that depolarization of they do not apparently play a role in the action of primary afferents produced by capsaicin depends capsaicin at this level.24~26~30 upon an increased permeability to both sodium Ca channels sensitive to the blocking action of and Ca ions.” A similar mechanism might operate omega conotoxin fraction GVIA (CTX) are apparat neuronal body level, as capsaicin was shown to ently involved in transmitter secretion from sensory induce influx of both sodium and Ca in primary nerves produced when an action potential invades the sensory neurons in culture.39 The ionic bases for sensory impulse generation produced by natural stimuli in capsaicin-sensitive sensory nerve endings (polyAbbreviations: CGRP-LI, calcitonin gene-related peptidemodal nociceptors) have not been investigated. In like immunoreactivity; CTX, omega conotoxin fraction many types of sensory nerve endings there is evidence GVIA; IPHFO, high frequency oscillations of intrathat adequate stimuli produce a local response luminal pressure; RIA, radioimmunoassay; SP-LI, substance P-like immunoreactivity. (generator potential) which generates the conducted 243
C. A. MAGGI et al
244
impulse (spike). The spike involves activation of tetrodotoxin-sensitive fast sodium channels which do not affect the local response.“*‘4 ‘6~18.32 Preliminary evidence has been presented suggesting that CTXsensitive Ca channels might be involved in sensory impulse generation in certain mechanoreceptors.35 The aim of this study was to assess whether CTX might affect reflex responses produced by stimulation of capsaicin-sensitive nerves in the rat urinary bladder20.22.2S.2”when administered by routes through which it could gain access to peripheral (topical application on the bladder) and central (intrathecal administration) endings of primary sensory neurons. Further, the effect of CTX on high K-induced release of sensory neuropeptides was investigated on superfused slices from the dorsal half of the rat spinal cord. EXPERIMENTAL PROCEDURES
Experiments on functions Male albino Wistar-Morini rats (S. Polo d’Enza, Italy) weighing 340-360 g were anaesthetized with subcutaneous urethane (1.2 g/kg). The left jugular vein was cannulated for drug injection. The left carotid artery was cannulated for blood pressure recording. The blood pressure signal was used for heart rate recording. Body temperature was kept constant at 37°C by means of a heating pad. The trachea was cannulated. The urinary bladder was prepared for simultaneous saline infusion and pressure recording by either the transurethral or transvesical route, as described previously.2’~25~2829 Intravesical pressure signals were delivered to a Hewlett Packard (H.P.) 8805B carrier amplifier and displayed on an H.P. four channel polygraph. In some experiments the bladder was filled by the transurethral route with an amount of saline (0.5 ml) sufficient to elicit regular rhythmic contractions determined by repetitive activation of the supraspinal vesicovesical micturition reflex.*’ In other experiments, transvesical bladder filling was made at a low, physiological rate (0.046 ml/min) in order to measure the volume threshold for eliciting the reflex** or at a high rate (0.25 ml/min) in order to get a regular series of bladder voiding cycles. ‘9.25,2qIn these latter experiments, three parameters were calculated: intercontraction interval, maximal amplitude of micturition contraction and resting pressure, as described previously.‘9.25,29 Some experiments were performed in rats pretreated with capsaicin, 50 mg/kg S.C. under ether anaethesia, 4 days before as compared to vehicle-treated rats. This capsaicin pretreatment has been repeatedly shown to produce depletion of several neuropeptides in the rat urinary bladder.‘,*’ The effectiveness of the capsaicin treatment was assessed in each animal by instillation of one drop of 0.01% capsaicin into one eye, as described previous1y.25 Drugs were administered by the intravenous or intrathecal route or applied topically onto the serosal surface of the urinary bladder. For the intrathecal administration, a polyethylene tubing (PE 10, Clay Adams) was inserted through the atlanto-occipital membrane and passed caudally for 8.5 cm in such a way that the catheter tip was located just below the lumbosacral enlargement.‘* Each dose was administered in 10~1 saline and washed with 10~1 saline. At the end of the experiment the position of the catheter was verified in each animal. For topical application, the urinary bladder was wrapped in a gauze moistened in a solution containing a stated concentration of CTX in saline or saline alone. The effect of CTX on the systolic blood pressure rise produced by topical application of capsaicin (2 bg in 25 ~1) onto the bladder or sudden bladder distension (2 ml saline)
were made in acute spinal urethane-anaesthetized
rats, as
described previously.‘0 Experiments on release Male albino rats weighing 240-300 g were stunned and bled. The spinal cord was rapidly removed and placed in cold oxygenated standard Krebs’ solution. The dorsal half of the cord, taken at the level of the lumbosacral enlargement was excised, cut in slices (thickness 0.5 mm), placed in 2 ml organ baths maintained at 37°C and superfused (2 ml/min) with oxygenated (96% 0, and 4% CO,) Krebs’ solution containing 0.1% bovine albumin and IO p M Thiorphan (Sigma). A 60 min equilibration period was allowed before drug administration. Fractions (2 ml) were collected every minute in tubes containing acetic acid to a 2 N final concentration. At the end of the experiments the tissues were blotted two to three times on filter paper and weighed. Superfusates were freeze-dried, reconstituted with the assay buffer (0.1 M, pH 7.4 phosphate buffer containing 0.9% NaCI, 0.01% NaN, and 0.1% bovine serum albumin) and substance P-like immunoreactivity (SP-LI) and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) content measured by radioimmunoassay (RIA). For SPLI assay, 100~1 of samples or SP standard, 100~1 of 1’251]Bolton-Hunter-SP (Amersham, U.K.) and 100 bl of i/20,000 anti SP 144 serum (kind gift of Dr P. Pradelles, SPI-LERI. CENiSaclav. Gif-Sur-Yvette. France) were incubated for 24 h. Separaiion of free from ‘bound antigen was made by the double antibody precipitation, as described previously.‘.20~24 The coefficient of percentage variation was less than 10% for values between 15 and 300 pmol/l. The sensitivity of the RIA was 1.1 fmol/tube. The antiserum cross-reacts by 1% with neurokinin A. 0.5% with neurokinin B and less than 0.1% with physalaemin and eledoisin. For CGRP-LI assay, 100~1 of rat CGRP (Peninsula) or samples were incubated for 48 h at 4°C with an identical volume of anti CGRP serum (raised in rabbits against human CGRP II, Peninsula). One hundred microlitres of [‘r51]iodohistidyl-human CGRP (Amersham, U.K.) were added and incubated for a further 48 h at 4°C. Separation of free from bound antigen was obtained as described previously. *‘Jo The coefficient of variation was less than 10% for values between 20 and 300 pmol/l. The sensitivity of the RIA was 2.5 fmol/tube. The anti human CGRP II serum cross-reacts to 100% with both rat and human CGRP. Statistical analysis All data are means + standard errors of the mean. Statistical analysis of the data was performed by means of Student’s t-test for paired or unpaired data or by means of analysis of variance, when applicable. Non parametric data in Table 3 were analysed by the Fisher exact test. Drugs Drugs used were: omega conotoxin and neurokinin A (Peninsula), Thiorphan, capsaicin and lidocaine (Sigma).
RESULTS
EfSect of topical application
of omega conotoxin
on the
volume -evoked micturition reflex Topically applied CTX did not affect the volume threshold for reflex micturition but significantly decreased (about 30%) the amplitude of the volumeevoked bladder contractions (Table 1). In contrast, the local anaesthetic lidocaine (42mM) produced a five-fold increase of bladder capacity and about 60% reduction of the volume-evoked bladder contractions (Table 1).
Effect of omega conotoxin on reflex responses Table 1. Effect of topical omega conotoxin or lidocaine on volume-evoked micturition reflex in urethaneanaesthethetized rats
Treatment
No. of animals
Volume threshold (ml)
Controls CTX Lidocaine
I 7 5
0.26 f 0.03 0.22 * 0.03 1.01 +0.15*
Amplitude of micturition contractions (mmHg) 27 + 2 19+2* 11*3*
Table 2. Effect of topical or intrathecal omega conotoxin or topical lidocaine on the systolic blood pressure increase produced by bladder distension or topical application of capsaicin onto the bladder in urethane-anaesthetized-spinal rats SBPt increase (mmHg) in response to No. of Distension Capsaicin animals (2ml) (2pg in 25~1)
Treatment
Each value is meankS.E. of the mean. The bladders were wrapped with a gauze moistened with CTX (IOpM) of lidocaine (42 mM) for 30min before start of saline filling. *Significantly different from controls, P < 0.01. E#ect of topical application of omega conotoxin on the blood pressure response to topical capsaicin or bladder distension
Topical application of CTX did not prevent the systolic blood pressure rise produced by sudden bladder distension (2 ml) or topical application of capsaicin onto the urinary bladder in spinal rats (Table 2). In contrast, topical lidocaine virtually abolished the blood pressure rise induced by both stimulants (Table 2). Effect of intrathecal omega conotoxin on volumeevoked bladder contractions
Intrathecal CTX produced tremors and movements of the hindlimbs in seven out of 12 and seven out of 10 rats at 0.3 and 3 pgg/rat, respectively. These effects ensued within l&20 min from administration. Resting values of heart rate, systolic and
245
Control Topical CTX Topical lidocaine Control Intrathecal CTX
6 8 6 7 7
19+2 16+3 4+2* 12k3 2* 1*
20 + 4 16 rf: 3 5 + 3* 18k3 3* 1*
Each value is mean+S.E. For topical application, the bladders were wrapped with a gauze moistened with CTX (10 PM) or lidocaine (42 mM) for 30 min before sudden bladder distension or topical application of capsaicin. For intrathecal administration the animal received CTX (3 pg/rat) in saline or the vehicle, 15 min before application of the stimuli. *Significantly different from controls, P < 0.01. tSBP, systolic blood pressure.
diastolic blood pressure were 344 f 16 beats/min, 146 + 7mm Hg and 95 f 7mm Hg, respectively. Intrathecal CTX produced a modest tachycardia and hypertension (+ 5 f 4 beats/min, + 16 + 3 mm Hg and + 19 + 4 mm Hg for systolic and diastolic blood pressure at a dose of 3 pg/rat, respectively, n = 6) which lasted for 26 f 5 min. Intrathecally administered CTX produced a dosedependent inhibition (Fig. 1, Table 3) of distensioninduced rhythmic bladder contractions in urethra-ligated rats which have been shown previously to depend upon activation of a supraspinal vesicovesical micturition reflex.*r Intrathecal injection
5 min BLADDER
DISTENSION
0.5 ml
SALINE
WITHDRAWAL
-0.3ml
-40 mmHg
-0
t
i.t. SALINE 10 pl
i.v. NKA 3 ys/Ke
mmHg
i.t. CTX 3Pg
i.v. NKA 3 PdKe
Fig. 1. Typical tracings showing the effect of intrathecal CTX (lower panel) or the vehicle (upper panel) on distension-induced rhythmic bladder contractions in urethane-anaesthetized rats. In the upper panel, an amount of saline was withdrawn from the bladder in order to stop the reflex contractions. Intravenous injection of neurokinin A produced an immediate myogenic contraction followed by rhythmic bladder contractions which are due to repetitive activation of a micturition reflex. This latter component was prevented in the CTX-treated animal (lower panel).
C. A. MAGGI et al.
246 Table 3. Effect of intrathecal omega conotoxin volume-evoked rhythmic contractions in urethane thetized rats No.
Inhibition of bladder motility
Controls (saline) CTX 0.03 ng/rat 0.3 ng/rat 3 p g/rat
6 5 9 6
l/6 215 8/9* 6/6*
*Significantly
from controls,
Treatment
of saline assess
had
whether
different
no significant this
inhibitory
effect
on the anaes-
1 min CONTROL IPHFO PHASE
40
I
Duration (min)
mmHg
3 11 +I 2-l + 6+ 43 * 5*
0
P < 0.01. (n = 4, Fig.
effect
of CTX
r40
yJlfvJL;_
1). To might
have been due to an unspecific depressant effect on bladder contractility, we studied the effect of intravenous neurokinin A (3 pg/kg). As shown previously, 25.28intravenous injection of tachykinins produces two distinct types of motor response in the rat urinary bladder in vivo, that is, an immediate myogenie contraction and a series of long lasting rhythmic bladder contractions, due to activation of a supraspinal micturition reflex. In order to appreciate this latter effect, an amount of saline was withdrawn from the bladder of vehicle-treated animals (Fig. 1, upper panel) which produced disappearance of the distension-induced rhythmic contractions. In vehicletreated rats, neurokinin A produced an immediate contraction (29 + 4mm Hg, n = 6) and activated the rhythmic contractions in all cases. In CTXtreated animals (3 pg intrathecally, 3WO min before), the immediate contraction to neurokinin A was unaffected (32 + 4mm Hg, n = 6) but in no instance was there activation of the rhythmic bladder contractions (Fig. 1). In other experiments, the effect of the intrathecal CTX on the micturition reflex was assessed in a more physiological model in which the bladder was filled via a needle inserted into the bladder (transvesical filling and pressure recording).2’.29 This procedure allowed bladder voiding which occurred in a repetitive fashion during non-stop bladder filling. The effects of intrathecal CTX were investigated in both vehicleand capsaicin-pretreated rats (SOmg/kg S.C. 4 days before). At a filling rate of 0.25 ml/min a regular series of voiding cycles was elicited in both vehicleand capsaicin-treated animals: intercontraction interval (related to bladder capacity) was 170 f 32 and 210 f 44s and the amplitude of the micturition contractions were 29 f 3 and 26 + 3mm Hg in vehicle- and capsaicin-treated animals (n = 6 and 7, respectively). Intrathecal CTX suppressed reflex micturition in three out of six vehicle-treated rats and in three out of seven capsaicin-treated rats at a dose of 0.3 pg/rat, respectively. A higher dose of CTX (3 pg/rat) suppressed micturition in all animals, either vehicle- or capsaicin-pretreated. A typical tracing showing the inhibitory effect of intrathecal CTX is shown in Fig. 2. As described previously,‘*,‘9,2’ a main determinant of voiding efficiency in this species is a series of high frequency oscillations of intraluminal pressure (hereafter referred to as IPHFO phase)
mmHg
LO t 11 min
from
i.t.
OVERFLOW
CTX
INCONTINENCE I0
t
20
min
from
i.t.
CTX
Fig. 2. Typical tracings showing the effect of intrathecal CTX on the voiding cycle of the urinary bladder in urethane-anaesthetized rats. Transvesical saline filling at 0.25 ml/min produced a regular series of voiding cycles (upper panel). Note that in the voiding cycle of rat bladder a series of high frequency oscillations of the intraluminal pressure occur (IPHFO phase) which have been shown to play a crucial role for efficient bladder voiding. After intrathecal CTX, voiding was impaired mainly because of a profound inhibitory action on the IPHFO phase of the voiding cycle (middle panel). As a consequence, the intracontraction interval was shortened (middle panel) and progressive failure of the bladder to void led to overflow incontinence (lower panel).
which accelerate urine flow during voiding. Activation of this mechanism occurs independently from capsaicin-sensitive nerves. Thus, IPHFO amplitude was 8.2 k 1 and 7.4 f 1.1 mm Hg in vehicle- and capsaicin-pretreated rats (n = 6 and 7, respectively, not significant). As shown in Fig. 2, CTX had a marked inhibitory effect on this component of the bladder voiding cycle: inhibition of the IPHFO phase was almost complete at a time when the detrusor muscle was still able to generate high amplitude pressure waves (middle panel in Fig. 2) but, in the absence of the IPHFO phase, voiding was incomplete. As a consequence resting pressure increased progressively and intercontraction interval decreased because of a larger residual volume. This progressive failure of bladder voiding resulted in a pattern of overflow incontinence (lower panel of Fig. 2) and urine dripping which persisted for > 30 min in both vehicle- and capsaicin-pretreated rats. Effect of intrathecal omega conotoxin on the blood pressure response to topical capsaicin or bladder distension Intrathecally-administered before) completely prevented
CTX (3 pg/rat, 15 min the blood pressure rise
Effect of omega conotoxin on reflex responses SP-LI
5 min
2500
24-l
CGRP-LI
*
25000
6 min
14
2000.
* *
.;z ii
7
1500~
%
*
Ir
1000~
<
2
500,
Fig. 3. SP-LI and CGRP-LI release by high K depolarizing medium from superfused slices from the dorsal half of the rat spinal cord. Each value is mean k SE. of four experiments. *Significantly different from control, P < 0.05.
produced by sudden bladder distension or by topical administration of capsaicin onto the bladder of spinal rats (Table 2). Effect of omega conotoxin on high K-induced release from the dorsal half of the rat spinal cord Exposure to high K (80mM, Fig. 3) or capsaicin (1 p M, Fig. 4, left panels) induced a prompt increase in SP-LI and CGRP-LI outflow from the superfused dorsal half of the rat spinal cord. A previous application of capsaicin (1 PM, 60 min before) completely prevented the response to the high K medium
(Fig. 4), indicating that both peptides are released from primary afferents. In contrast, a second application of high K elicited a consistent peptide release, although somehow less than that produced by the first one (n = 3, data not shown). As shown in Table 4, both SP-LI and CGRP-LI release by high K medium was significantly reduced (about 50% for both peptides) by CTX. DISCUSSION
In previous studies we showed that the action of capsaicin on sensory nerve terminals (involving both SP-LI
5
._ 4d s h
\m \
:
5 min
01 t HIGH K’(80mrd 25000.
l
*
CGRP- LI
20000.
Fig. 4. SP-LI and CGRP-LI release by capsaicin (1 PM) from superfused slices from the dorsal half of the rat spinal cord. Note that after exposure to capsaicin, high K medium failed to induce a significant release. Each value is mean f SE. of four experiments. *Significantly different from control, P -e0.05.
248
C. A.
MAGGI et al.
Table 4. Effect of omega conotoxin (0.1 PM for 30min) on high K (80mM)-induced release of substance P-like immunoreactivity and calcitonin gene-related peptide-like immunoreactivity the dorsal half of the rat spinal cord Total evoked release (pg/g/min) SP-LI CGRP-LI Control CTX
925 f 189 434 * 72*
12.320 + 2100 6.283 k 989*
Each value is mean+S.E. of four experiments. *Significantly different from controls, P < 0.05.
release of sensory neuropeptides
and establishment of desensitization) are not modified significantly by CTX.24,30 These observations are in line with the notion that CTX does not block Ca accumulation
induced by capsaicin in primary sensory neurons in culture.39 However, activation of CTX-sensitive Ca channels mediates transmitter release evoked from peripheral endings of these primary afferents when they are activated by electrical field stimulation.23 CTX-sensitive Ca channels could be involved in sensory impulse generation in certain mechanoreceptors3’ and we aimed to determine whether application of CTX on the receptive field of the capsaicin-sensitive sensory neurons (topical application onto the bladder) might have been able to block sensory impulse generation produced by a natural stimulus (distension). No evidence for this was found using two different models, e.g. volume-evoked reflex bladder contractions and blood pressure rise in spinal rats. In both paradigms, distension activates capsaicin-sensitive nerves of the bladder.‘0,20,25We cannot exclude that CTX, applied topically on the outer surface of the viscus, did not gain access in sufficient amounts to sensory nerve endings of the bladder. However, topical CTX reduced the amplitude of the volume-evoked bladder contractions, an effect probably related to the ability of the toxin to reduce transmitter release from postganglionic parasympathetic excitatory nerves in the rat bladder.24 A direct depressant action of CTX on muscle cells or blood vessels in the bladder seems unlikely, because the toxin has been shown to be remarkably selective in blocking nerve-mediated responses at the peripheral leve1.24.33 Reduction of the volume-evoked bladder contractions after topical application, indicates that CTX had the ability to gain access to intramural nerves. This suggests that CTX-sensitive Ca channels do not play a major role in the sensory impulse generation at this level, although further studies are needed to
assess this point firmly. It appears unlikely that, after topical administration onto the bladder, CTX might have affected bladder motility at a central site of action because this treatment did not affect the capsaicin- or distension-evoked blood pressure rise, while intrathecal CTX effectively blocked these responses. After intrathecal administration, CTX blocked reflexly-activated bladder motility. This action was observed in both vehicle- and capsaicin-pretreated rats, indicating that CTX can block reflex pathways in the spinal cord by acting at sites other than capsaicin-sensitive primary afferents. This might involve blockade of transmission from capsaicin-insensitive afferents to second order sensory neurons and/or other sites along the reflex pathway. We also have shown that CTX produced a marked inhibition of both SP-LI and CGRP-LI release induced by high K in superfused slices from the dorsal half of the rat spinal cord. A previous exposure to capsaicin prevented the response to high K, indicating that both peptides originate from primary afferents,36 a finding consistent with the notion that both SP and CGRP are present in central projections of capsaicin-sensitive sensory neurons.26,38 Thus, reduction of sensory neuropeptide release from central endings of capsaicin-sensitive neurons is a likely site of action for CTX. This is in accordance with data showing the presence of specific CTX-binding sites in the dorsal half of rat spinal cord.3,‘7 Further, CTX was shown to block the electrically-evoked release of SP from chick dorsal root ganglion neurons.” It therefore appears conceivable that the blocking action of CTX toward the blood pressure rise induced by capsaicin application onto the bladder or sudden bladder distension, might have involved blockade of sensory neuropeptides released from capsaicinsensitive nerves.
CONCLUSION
These findings provide further support to the notion that CTX is a potent pharmacological tool for studying the properties and function of capsaicinsensitive primary sensory neurons, with particular regard to mechanisms regulating transmitter release from both central and peripheral terminals. Acknowledgements-This
work was supported in part by IMI, Rome, Italy, Progetto di Ricerca: Farmaci per il trattamento a lungo termine della incontinenza urinaria, VES grant no. 46287.
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249
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Meli A. (1988) The effect of omega conotoxin GVIA a peptide modulator of the N-type voltage sensitive calcium channels, on motor responses produced by activation of efferent and sensory nerves in mammalian smooth muscles. Naunyn-Schmiedeberg’s Arch. Pharmac. 338, 107-l 13. Maggi C. A., Santicioli P., Borsini F., Giuliani S. and Meli A. (1986) The role of the capsaicin-sensitive innervation of the rat urinary bladder in the activation of micturition reflex. Naunyn-Schmiedeberg’s Arch. Pharmac. 332,276283. Maagi C. A., Santicioli P.. Gennetti P.. Parlani M.. Astolfi M.. Del Bianco E.. Patacchini R.. Giuliani S. and Meli A. @89) The effect of calcium free medium and nifedipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig and rat bladder. Gen. Pharmac. 20, 445-456. Maggi C. A., Santicioli P., Geppetti P., Parlani M., Astolfi A., Pradelles P., Patacchini R. and Meli A. (1988) The antagonism induced by Ruthenium Red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies. Eur. J. Pharmac. 154, I-10. Maggi C. A., Santicioli P. and Meli A. (1984) The effects of capsaicin on rat urinary bladder motility in uiuo. Eur. J.
Pharmac. 103, 41-50. 29. Maggi C. A., Santicioli P. and Meli A. (1986) The non-stop transvesical cystometrogram
in urethane anaesthetized rats: a simple procedure for quantitative studies on the various phases of the urinary bladder voiding cycle. J. Pharmac.
Meth. 15, 1577167. 30. Maggi C. A., Santicioli P., Patacchini R., Geppetti P., Del Bianco E. and Meli A. (1988) Calcium and capsaicin-induced substance P release from peripheral terminals of primary sensory neurons. Regul. Pept. 22, 117.
31. Marsh S. J., Stansfeld C. E., Brown D. A., Davey R. and McCarthy D. (1987) The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro. Neuroscience 23, 275-289. 32. Matsuura S. (1973) Depolarization of sensory nerve endings and impulse initiation in common carotid baroreceptors. J. Physiol., Lond. 235, 31-56. 33. Mohy El Din M. M. and Malik K. U. (1988) Differential effect of omega conotoxin
transmitter
and the vasoconstrictor
response to noradrenaline
on release of the adrenergic in the rat isolated kidney. Rr. J. Pharmac. 94,
355-361. 34. Moskowitz M. A., Brody M. and Liu-Chen L. Y. (1983) In vitro release of immunoreactive substance P from putative afferent nerve endings in bovine pia arachnoid. Neuroscience 9, 8099814.
N.S.C. 3411-I
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35. Ogawa H. and Yamashita Y. (1988) Mechano-electric transduction in the slowly adapting cutaneous afferent units of frogs. In Transduction and Cellular Mechanisms in Sensory Receptors (eds Hamann W. and Iggo A.). Prog. Brain Res. 74, 6348. 36. Saria A., Gamse R., Petermann J., Fischer J. A., Theodorsson-Norheim
E. and Lundberg J. M. (1986) Simultaneous release of several tachykinins and CGRP from rat spinal cord slices. Neurosci. L.ert. 63, 310-314. 37. Szolcsanyi J. (1984) Capsaicin-sensitive chemoceptive neural system with dual sensory-efferent function. In Antidromic Vasodilation and Neurogenic Inflammation (eds Chahl L. A., Szolcsanyi J. and Lembeck F.), pp. 2652. Akademiai Kiado, Budapest. 38. Wiesenfeld-Hallin Z., Hdkfelt T., Lundberg J. M., Forssmann W. G., Reinecke M., Tscbopp F. A. and Fischer J. A. (1984) Immunoreactive CGRP and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the cat. Neurosci. Letr. 52, 199-204. 39. Wood J. N., Winter J., James I. F., Rang H. P., Yeats J. and Bevan S. (1988) Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture. J. Neurosci. 8, 3209-3220. (Accepled 2 August 1989)
0306-4522/90 $3.00+ 0.00 PergamonPressplc 0 1990IBRO
EFFECT OF OMEGA CONOTOXIN ON REFLEX RESPONSES MEDIATED BY ACTIVATION OF CAPSAICIN-SENSITIVE NERVES OF THE RAT URINARY BLADDER AND PEPTIDE RELEASE FROM THE RAT SPINAL CORD C. A. MAGGI,* S. GIULIANI,* P. SANTICIOLI,* M. TRAMONTANA~ and A. MELI* *Pharmacology Department, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131 Florence, Italy tInstitute of Internal Medicine IV, University of Florence, Italy Abstract-The aim of this study was to assess whether omega conotoxin fraction GVIA, a potent blocker of N- and L-type voltage-sensitive calcium channels, might interfere with reflex responses (micturition, blood pressure rise in spinal rats) produced by activation of capsaicin-sensitive sensory nerves of the rat urinary bladder. The effect of conotoxin was also investigated on reflex micturition persisting after capsaicin pretreatment. Following topical application onto the bladder, conotoxin did not affect the volume threshold to elicit micturition although it reduced the amplitude of volume-evoked bladder contractions. Likewise, topical conotoxin did not prevent the reflex rise in blood pressure elicited by sudden bladder distension or topical application of capsaicin onto the bladder. In contrast, topical lidocaine strongly prevented both reflex responses. After intrathecal administration, conotoxin produced a dose-dependent inhibition of volume-evoked bladder contractions and the cardiovascular reflex produced by mechanical or chemical stimulation of bladder nerves. Intrathecal conotoxin inhibited micturition also in rats pretreated with capsaicin (50 mg/kg s.c., 4 days before). Depolarization by high potassium (80mM) produced release of both substance P- and calcitonin gene-related peptide-like immunoreactivity from superfused slices of the dorsal half of rat spinal cord. Capsaicin (1 PM) produced a similar effect, and a previous exposure to capsaicin prevented the effect of potassium. Conotoxin (0.1 PM) significantly reduced (about 50%) the potassium-induced release of neuropeptides from the dorsal half of the rat spinal cord. These findings indicate that conotoxin-sensitive calcium channels in the rat spinal cord play a role in the neurotransmission along reflex pathways activated by stimulation of capsaicin-sensitive nerves in the urinary bladder. Inhibition of sensory neuropeptide release from primary afferents in the spinal cord is a likely target for conotoxin. These findings do not support the hypothesis that conotoxin-sensitive calcium channels are implicated in the genesis of sensory impulses at the peripheral level, although clarification of this point needs further investigation.
Capsaicin-sensitive primary afferent neurons release nerve endings. 23 CTX is a potent blocker of both their transmitter content from both central and perL- and N-type voltage-sensitive Ca channels in ipheral endings thereby producing a dual, sensory chick sensory neurons’ and is gaining wide use as a and “efferent” function.‘3,22,37 Transmitter release tool for studying transmitter secretion in the central from both central and peripheral endings of these and peripheral nervous system.3~4*‘2+‘7,23,24 sensory nerves is a Ca-dependent process.7-9,26,27,30~34 Some information is available about the ionic bases Voltage-dependent Ca channels are present on perfor sensory impulse generation by capsaicin. At nerve ipheral endings of capsaicin-sensitive afferents, but trunk level, there is evidence that depolarization of they do not apparently play a role in the action of primary afferents produced by capsaicin depends capsaicin at this level.24~26~30 upon an increased permeability to both sodium Ca channels sensitive to the blocking action of and Ca ions.” A similar mechanism might operate omega conotoxin fraction GVIA (CTX) are apparat neuronal body level, as capsaicin was shown to ently involved in transmitter secretion from sensory induce influx of both sodium and Ca in primary nerves produced when an action potential invades the sensory neurons in culture.39 The ionic bases for sensory impulse generation produced by natural stimuli in capsaicin-sensitive sensory nerve endings (polyAbbreviations: CGRP-LI, calcitonin gene-related peptidemodal nociceptors) have not been investigated. In like immunoreactivity; CTX, omega conotoxin fraction many types of sensory nerve endings there is evidence GVIA; IPHFO, high frequency oscillations of intrathat adequate stimuli produce a local response luminal pressure; RIA, radioimmunoassay; SP-LI, substance P-like immunoreactivity. (generator potential) which generates the conducted 243
C. A. MAGGI et al
244
impulse (spike). The spike involves activation of tetrodotoxin-sensitive fast sodium channels which do not affect the local response.“*‘4 ‘6~18.32 Preliminary evidence has been presented suggesting that CTXsensitive Ca channels might be involved in sensory impulse generation in certain mechanoreceptors.35 The aim of this study was to assess whether CTX might affect reflex responses produced by stimulation of capsaicin-sensitive nerves in the rat urinary bladder20.22.2S.2”when administered by routes through which it could gain access to peripheral (topical application on the bladder) and central (intrathecal administration) endings of primary sensory neurons. Further, the effect of CTX on high K-induced release of sensory neuropeptides was investigated on superfused slices from the dorsal half of the rat spinal cord. EXPERIMENTAL PROCEDURES
Experiments on functions Male albino Wistar-Morini rats (S. Polo d’Enza, Italy) weighing 340-360 g were anaesthetized with subcutaneous urethane (1.2 g/kg). The left jugular vein was cannulated for drug injection. The left carotid artery was cannulated for blood pressure recording. The blood pressure signal was used for heart rate recording. Body temperature was kept constant at 37°C by means of a heating pad. The trachea was cannulated. The urinary bladder was prepared for simultaneous saline infusion and pressure recording by either the transurethral or transvesical route, as described previously.2’~25~2829 Intravesical pressure signals were delivered to a Hewlett Packard (H.P.) 8805B carrier amplifier and displayed on an H.P. four channel polygraph. In some experiments the bladder was filled by the transurethral route with an amount of saline (0.5 ml) sufficient to elicit regular rhythmic contractions determined by repetitive activation of the supraspinal vesicovesical micturition reflex.*’ In other experiments, transvesical bladder filling was made at a low, physiological rate (0.046 ml/min) in order to measure the volume threshold for eliciting the reflex** or at a high rate (0.25 ml/min) in order to get a regular series of bladder voiding cycles. ‘9.25,2qIn these latter experiments, three parameters were calculated: intercontraction interval, maximal amplitude of micturition contraction and resting pressure, as described previously.‘9.25,29 Some experiments were performed in rats pretreated with capsaicin, 50 mg/kg S.C. under ether anaethesia, 4 days before as compared to vehicle-treated rats. This capsaicin pretreatment has been repeatedly shown to produce depletion of several neuropeptides in the rat urinary bladder.‘,*’ The effectiveness of the capsaicin treatment was assessed in each animal by instillation of one drop of 0.01% capsaicin into one eye, as described previous1y.25 Drugs were administered by the intravenous or intrathecal route or applied topically onto the serosal surface of the urinary bladder. For the intrathecal administration, a polyethylene tubing (PE 10, Clay Adams) was inserted through the atlanto-occipital membrane and passed caudally for 8.5 cm in such a way that the catheter tip was located just below the lumbosacral enlargement.‘* Each dose was administered in 10~1 saline and washed with 10~1 saline. At the end of the experiment the position of the catheter was verified in each animal. For topical application, the urinary bladder was wrapped in a gauze moistened in a solution containing a stated concentration of CTX in saline or saline alone. The effect of CTX on the systolic blood pressure rise produced by topical application of capsaicin (2 bg in 25 ~1) onto the bladder or sudden bladder distension (2 ml saline)
were made in acute spinal urethane-anaesthetized
rats, as
described previously.‘0 Experiments on release Male albino rats weighing 240-300 g were stunned and bled. The spinal cord was rapidly removed and placed in cold oxygenated standard Krebs’ solution. The dorsal half of the cord, taken at the level of the lumbosacral enlargement was excised, cut in slices (thickness 0.5 mm), placed in 2 ml organ baths maintained at 37°C and superfused (2 ml/min) with oxygenated (96% 0, and 4% CO,) Krebs’ solution containing 0.1% bovine albumin and IO p M Thiorphan (Sigma). A 60 min equilibration period was allowed before drug administration. Fractions (2 ml) were collected every minute in tubes containing acetic acid to a 2 N final concentration. At the end of the experiments the tissues were blotted two to three times on filter paper and weighed. Superfusates were freeze-dried, reconstituted with the assay buffer (0.1 M, pH 7.4 phosphate buffer containing 0.9% NaCI, 0.01% NaN, and 0.1% bovine serum albumin) and substance P-like immunoreactivity (SP-LI) and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) content measured by radioimmunoassay (RIA). For SPLI assay, 100~1 of samples or SP standard, 100~1 of 1’251]Bolton-Hunter-SP (Amersham, U.K.) and 100 bl of i/20,000 anti SP 144 serum (kind gift of Dr P. Pradelles, SPI-LERI. CENiSaclav. Gif-Sur-Yvette. France) were incubated for 24 h. Separaiion of free from ‘bound antigen was made by the double antibody precipitation, as described previously.‘.20~24 The coefficient of percentage variation was less than 10% for values between 15 and 300 pmol/l. The sensitivity of the RIA was 1.1 fmol/tube. The antiserum cross-reacts by 1% with neurokinin A. 0.5% with neurokinin B and less than 0.1% with physalaemin and eledoisin. For CGRP-LI assay, 100~1 of rat CGRP (Peninsula) or samples were incubated for 48 h at 4°C with an identical volume of anti CGRP serum (raised in rabbits against human CGRP II, Peninsula). One hundred microlitres of [‘r51]iodohistidyl-human CGRP (Amersham, U.K.) were added and incubated for a further 48 h at 4°C. Separation of free from bound antigen was obtained as described previously. *‘Jo The coefficient of variation was less than 10% for values between 20 and 300 pmol/l. The sensitivity of the RIA was 2.5 fmol/tube. The anti human CGRP II serum cross-reacts to 100% with both rat and human CGRP. Statistical analysis All data are means + standard errors of the mean. Statistical analysis of the data was performed by means of Student’s t-test for paired or unpaired data or by means of analysis of variance, when applicable. Non parametric data in Table 3 were analysed by the Fisher exact test. Drugs Drugs used were: omega conotoxin and neurokinin A (Peninsula), Thiorphan, capsaicin and lidocaine (Sigma).
RESULTS
EfSect of topical application
of omega conotoxin
on the
volume -evoked micturition reflex Topically applied CTX did not affect the volume threshold for reflex micturition but significantly decreased (about 30%) the amplitude of the volumeevoked bladder contractions (Table 1). In contrast, the local anaesthetic lidocaine (42mM) produced a five-fold increase of bladder capacity and about 60% reduction of the volume-evoked bladder contractions (Table 1).
Effect of omega conotoxin on reflex responses Table 1. Effect of topical omega conotoxin or lidocaine on volume-evoked micturition reflex in urethaneanaesthethetized rats
Treatment
No. of animals
Volume threshold (ml)
Controls CTX Lidocaine
I 7 5
0.26 f 0.03 0.22 * 0.03 1.01 +0.15*
Amplitude of micturition contractions (mmHg) 27 + 2 19+2* 11*3*
Table 2. Effect of topical or intrathecal omega conotoxin or topical lidocaine on the systolic blood pressure increase produced by bladder distension or topical application of capsaicin onto the bladder in urethane-anaesthetized-spinal rats SBPt increase (mmHg) in response to No. of Distension Capsaicin animals (2ml) (2pg in 25~1)
Treatment
Each value is meankS.E. of the mean. The bladders were wrapped with a gauze moistened with CTX (IOpM) of lidocaine (42 mM) for 30min before start of saline filling. *Significantly different from controls, P < 0.01. E#ect of topical application of omega conotoxin on the blood pressure response to topical capsaicin or bladder distension
Topical application of CTX did not prevent the systolic blood pressure rise produced by sudden bladder distension (2 ml) or topical application of capsaicin onto the urinary bladder in spinal rats (Table 2). In contrast, topical lidocaine virtually abolished the blood pressure rise induced by both stimulants (Table 2). Effect of intrathecal omega conotoxin on volumeevoked bladder contractions
Intrathecal CTX produced tremors and movements of the hindlimbs in seven out of 12 and seven out of 10 rats at 0.3 and 3 pgg/rat, respectively. These effects ensued within l&20 min from administration. Resting values of heart rate, systolic and
245
Control Topical CTX Topical lidocaine Control Intrathecal CTX
6 8 6 7 7
19+2 16+3 4+2* 12k3 2* 1*
20 + 4 16 rf: 3 5 + 3* 18k3 3* 1*
Each value is mean+S.E. For topical application, the bladders were wrapped with a gauze moistened with CTX (10 PM) or lidocaine (42 mM) for 30 min before sudden bladder distension or topical application of capsaicin. For intrathecal administration the animal received CTX (3 pg/rat) in saline or the vehicle, 15 min before application of the stimuli. *Significantly different from controls, P < 0.01. tSBP, systolic blood pressure.
diastolic blood pressure were 344 f 16 beats/min, 146 + 7mm Hg and 95 f 7mm Hg, respectively. Intrathecal CTX produced a modest tachycardia and hypertension (+ 5 f 4 beats/min, + 16 + 3 mm Hg and + 19 + 4 mm Hg for systolic and diastolic blood pressure at a dose of 3 pg/rat, respectively, n = 6) which lasted for 26 f 5 min. Intrathecally administered CTX produced a dosedependent inhibition (Fig. 1, Table 3) of distensioninduced rhythmic bladder contractions in urethra-ligated rats which have been shown previously to depend upon activation of a supraspinal vesicovesical micturition reflex.*r Intrathecal injection
5 min BLADDER
DISTENSION
0.5 ml
SALINE
WITHDRAWAL
-0.3ml
-40 mmHg
-0
t
i.t. SALINE 10 pl
i.v. NKA 3 ys/Ke
mmHg
i.t. CTX 3Pg
i.v. NKA 3 PdKe
Fig. 1. Typical tracings showing the effect of intrathecal CTX (lower panel) or the vehicle (upper panel) on distension-induced rhythmic bladder contractions in urethane-anaesthetized rats. In the upper panel, an amount of saline was withdrawn from the bladder in order to stop the reflex contractions. Intravenous injection of neurokinin A produced an immediate myogenic contraction followed by rhythmic bladder contractions which are due to repetitive activation of a micturition reflex. This latter component was prevented in the CTX-treated animal (lower panel).
C. A. MAGGI et al.
246 Table 3. Effect of intrathecal omega conotoxin volume-evoked rhythmic contractions in urethane thetized rats No.
Inhibition of bladder motility
Controls (saline) CTX 0.03 ng/rat 0.3 ng/rat 3 p g/rat
6 5 9 6
l/6 215 8/9* 6/6*
*Significantly
from controls,
Treatment
of saline assess
had
whether
different
no significant this
inhibitory
effect
on the anaes-
1 min CONTROL IPHFO PHASE
40
I
Duration (min)
mmHg
3 11 +I 2-l + 6+ 43 * 5*
0
P < 0.01. (n = 4, Fig.
effect
of CTX
r40
yJlfvJL;_
1). To might
have been due to an unspecific depressant effect on bladder contractility, we studied the effect of intravenous neurokinin A (3 pg/kg). As shown previously, 25.28intravenous injection of tachykinins produces two distinct types of motor response in the rat urinary bladder in vivo, that is, an immediate myogenie contraction and a series of long lasting rhythmic bladder contractions, due to activation of a supraspinal micturition reflex. In order to appreciate this latter effect, an amount of saline was withdrawn from the bladder of vehicle-treated animals (Fig. 1, upper panel) which produced disappearance of the distension-induced rhythmic contractions. In vehicletreated rats, neurokinin A produced an immediate contraction (29 + 4mm Hg, n = 6) and activated the rhythmic contractions in all cases. In CTXtreated animals (3 pg intrathecally, 3WO min before), the immediate contraction to neurokinin A was unaffected (32 + 4mm Hg, n = 6) but in no instance was there activation of the rhythmic bladder contractions (Fig. 1). In other experiments, the effect of the intrathecal CTX on the micturition reflex was assessed in a more physiological model in which the bladder was filled via a needle inserted into the bladder (transvesical filling and pressure recording).2’.29 This procedure allowed bladder voiding which occurred in a repetitive fashion during non-stop bladder filling. The effects of intrathecal CTX were investigated in both vehicleand capsaicin-pretreated rats (SOmg/kg S.C. 4 days before). At a filling rate of 0.25 ml/min a regular series of voiding cycles was elicited in both vehicleand capsaicin-treated animals: intercontraction interval (related to bladder capacity) was 170 f 32 and 210 f 44s and the amplitude of the micturition contractions were 29 f 3 and 26 + 3mm Hg in vehicle- and capsaicin-treated animals (n = 6 and 7, respectively). Intrathecal CTX suppressed reflex micturition in three out of six vehicle-treated rats and in three out of seven capsaicin-treated rats at a dose of 0.3 pg/rat, respectively. A higher dose of CTX (3 pg/rat) suppressed micturition in all animals, either vehicle- or capsaicin-pretreated. A typical tracing showing the inhibitory effect of intrathecal CTX is shown in Fig. 2. As described previously,‘*,‘9,2’ a main determinant of voiding efficiency in this species is a series of high frequency oscillations of intraluminal pressure (hereafter referred to as IPHFO phase)
mmHg
LO t 11 min
from
i.t.
OVERFLOW
CTX
INCONTINENCE I0
t
20
min
from
i.t.
CTX
Fig. 2. Typical tracings showing the effect of intrathecal CTX on the voiding cycle of the urinary bladder in urethane-anaesthetized rats. Transvesical saline filling at 0.25 ml/min produced a regular series of voiding cycles (upper panel). Note that in the voiding cycle of rat bladder a series of high frequency oscillations of the intraluminal pressure occur (IPHFO phase) which have been shown to play a crucial role for efficient bladder voiding. After intrathecal CTX, voiding was impaired mainly because of a profound inhibitory action on the IPHFO phase of the voiding cycle (middle panel). As a consequence, the intracontraction interval was shortened (middle panel) and progressive failure of the bladder to void led to overflow incontinence (lower panel).
which accelerate urine flow during voiding. Activation of this mechanism occurs independently from capsaicin-sensitive nerves. Thus, IPHFO amplitude was 8.2 k 1 and 7.4 f 1.1 mm Hg in vehicle- and capsaicin-pretreated rats (n = 6 and 7, respectively, not significant). As shown in Fig. 2, CTX had a marked inhibitory effect on this component of the bladder voiding cycle: inhibition of the IPHFO phase was almost complete at a time when the detrusor muscle was still able to generate high amplitude pressure waves (middle panel in Fig. 2) but, in the absence of the IPHFO phase, voiding was incomplete. As a consequence resting pressure increased progressively and intercontraction interval decreased because of a larger residual volume. This progressive failure of bladder voiding resulted in a pattern of overflow incontinence (lower panel of Fig. 2) and urine dripping which persisted for > 30 min in both vehicle- and capsaicin-pretreated rats. Effect of intrathecal omega conotoxin on the blood pressure response to topical capsaicin or bladder distension Intrathecally-administered before) completely prevented
CTX (3 pg/rat, 15 min the blood pressure rise
Effect of omega conotoxin on reflex responses SP-LI
5 min
2500
24-l
CGRP-LI
*
25000
6 min
14
2000.
* *
.;z ii
7
1500~
%
*
Ir
1000~
<
2
500,
Fig. 3. SP-LI and CGRP-LI release by high K depolarizing medium from superfused slices from the dorsal half of the rat spinal cord. Each value is mean k SE. of four experiments. *Significantly different from control, P < 0.05.
produced by sudden bladder distension or by topical administration of capsaicin onto the bladder of spinal rats (Table 2). Effect of omega conotoxin on high K-induced release from the dorsal half of the rat spinal cord Exposure to high K (80mM, Fig. 3) or capsaicin (1 p M, Fig. 4, left panels) induced a prompt increase in SP-LI and CGRP-LI outflow from the superfused dorsal half of the rat spinal cord. A previous application of capsaicin (1 PM, 60 min before) completely prevented the response to the high K medium
(Fig. 4), indicating that both peptides are released from primary afferents. In contrast, a second application of high K elicited a consistent peptide release, although somehow less than that produced by the first one (n = 3, data not shown). As shown in Table 4, both SP-LI and CGRP-LI release by high K medium was significantly reduced (about 50% for both peptides) by CTX. DISCUSSION
In previous studies we showed that the action of capsaicin on sensory nerve terminals (involving both SP-LI
5
._ 4d s h
\m \
:
5 min
01 t HIGH K’(80mrd 25000.
l
*
CGRP- LI
20000.
Fig. 4. SP-LI and CGRP-LI release by capsaicin (1 PM) from superfused slices from the dorsal half of the rat spinal cord. Note that after exposure to capsaicin, high K medium failed to induce a significant release. Each value is mean f SE. of four experiments. *Significantly different from control, P -e0.05.
248
C. A.
MAGGI et al.
Table 4. Effect of omega conotoxin (0.1 PM for 30min) on high K (80mM)-induced release of substance P-like immunoreactivity and calcitonin gene-related peptide-like immunoreactivity the dorsal half of the rat spinal cord Total evoked release (pg/g/min) SP-LI CGRP-LI Control CTX
925 f 189 434 * 72*
12.320 + 2100 6.283 k 989*
Each value is mean+S.E. of four experiments. *Significantly different from controls, P < 0.05.
release of sensory neuropeptides
and establishment of desensitization) are not modified significantly by CTX.24,30 These observations are in line with the notion that CTX does not block Ca accumulation
induced by capsaicin in primary sensory neurons in culture.39 However, activation of CTX-sensitive Ca channels mediates transmitter release evoked from peripheral endings of these primary afferents when they are activated by electrical field stimulation.23 CTX-sensitive Ca channels could be involved in sensory impulse generation in certain mechanoreceptors3’ and we aimed to determine whether application of CTX on the receptive field of the capsaicin-sensitive sensory neurons (topical application onto the bladder) might have been able to block sensory impulse generation produced by a natural stimulus (distension). No evidence for this was found using two different models, e.g. volume-evoked reflex bladder contractions and blood pressure rise in spinal rats. In both paradigms, distension activates capsaicin-sensitive nerves of the bladder.‘0,20,25We cannot exclude that CTX, applied topically on the outer surface of the viscus, did not gain access in sufficient amounts to sensory nerve endings of the bladder. However, topical CTX reduced the amplitude of the volume-evoked bladder contractions, an effect probably related to the ability of the toxin to reduce transmitter release from postganglionic parasympathetic excitatory nerves in the rat bladder.24 A direct depressant action of CTX on muscle cells or blood vessels in the bladder seems unlikely, because the toxin has been shown to be remarkably selective in blocking nerve-mediated responses at the peripheral leve1.24.33 Reduction of the volume-evoked bladder contractions after topical application, indicates that CTX had the ability to gain access to intramural nerves. This suggests that CTX-sensitive Ca channels do not play a major role in the sensory impulse generation at this level, although further studies are needed to
assess this point firmly. It appears unlikely that, after topical administration onto the bladder, CTX might have affected bladder motility at a central site of action because this treatment did not affect the capsaicin- or distension-evoked blood pressure rise, while intrathecal CTX effectively blocked these responses. After intrathecal administration, CTX blocked reflexly-activated bladder motility. This action was observed in both vehicle- and capsaicin-pretreated rats, indicating that CTX can block reflex pathways in the spinal cord by acting at sites other than capsaicin-sensitive primary afferents. This might involve blockade of transmission from capsaicin-insensitive afferents to second order sensory neurons and/or other sites along the reflex pathway. We also have shown that CTX produced a marked inhibition of both SP-LI and CGRP-LI release induced by high K in superfused slices from the dorsal half of the rat spinal cord. A previous exposure to capsaicin prevented the response to high K, indicating that both peptides originate from primary afferents,36 a finding consistent with the notion that both SP and CGRP are present in central projections of capsaicin-sensitive sensory neurons.26,38 Thus, reduction of sensory neuropeptide release from central endings of capsaicin-sensitive neurons is a likely site of action for CTX. This is in accordance with data showing the presence of specific CTX-binding sites in the dorsal half of rat spinal cord.3,‘7 Further, CTX was shown to block the electrically-evoked release of SP from chick dorsal root ganglion neurons.” It therefore appears conceivable that the blocking action of CTX toward the blood pressure rise induced by capsaicin application onto the bladder or sudden bladder distension, might have involved blockade of sensory neuropeptides released from capsaicinsensitive nerves.
CONCLUSION
These findings provide further support to the notion that CTX is a potent pharmacological tool for studying the properties and function of capsaicinsensitive primary sensory neurons, with particular regard to mechanisms regulating transmitter release from both central and peripheral terminals. Acknowledgements-This
work was supported in part by IMI, Rome, Italy, Progetto di Ricerca: Farmaci per il trattamento a lungo termine della incontinenza urinaria, VES grant no. 46287.
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Effect of omega conotoxin on reflex responses
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