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Non-Adrenergic Non-Cholinergic Excitatory Innervation of the Guinea-Pig Isolated Renal Pelvis: Involvement of Capsaicin-Sensitive Primary Afferent Neurons
0022-5347 /92/14 75-1394$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1992 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 147, 1394-1398, May 1992
Printed in U.S.A.
NON-ADRENERGIC NON-CHOLINERGIC EXCITATORY INNERVATION OF THE GUINEA-PIG ISOLATED RENAL PELVIS: INVOLVEMENT OF CAPSAICIN-SENSITIVE PRIMARY AFFERENT NEURONS CARLO ALBERTO MAGGI*
AND
SANDRO GIULIANI
From the Pharmacology Department, A. Menarini Pharmaceuticals, Florence, Italy
ABSTRACT
Circular muscle strips from the guinea-pig renal pelvis exhibit a spontaneous activity averaging about 50% of maximal contractile response at a frequency of about six contractions/min. This spontaneous activity is tetrodotoxin resistant. Acetylcholine and noradrenaline (one to 100 µM) produce a positive inotropic effect which was abolished or strongly inhibited by atropine and phentolamine, respectively. Electrical field stimulation (five to 10 Hz, 0.5 ms pulse width for 10 s, 60 V, maximal voltage) produces a transient positive inotropic response which is tetrodotoxinsensitive but unaffected by atropine and phentolamine alone or in combination, as well as by atropine plus guanethidine. Application of capsaicin produced a large positive inotropic response which was not reproduced upon a second application of the drug (desensitization). After in vitro capsaicin desensitization, the response to electrical field stimulation was abolished, indicating its dependence on activation of peripheral endings of sensory nerves. These findings demonstrate the existence of a non-adrenergic non-cholinergic functional innervation of the guinea-pig renal pelvis which is entirely dependent on sensory nerve activation. KEY
W OROS: kidney, nerve cells, neurotransmitters
Ureteral peristalsis is a primarily myogenic phenomenon initiated by pacemaker cells in the proximal part of the renal pelvis. 1-4 Neither pacemaker cell activity nor propagation of peristaltic waves along the ureter are affected by tetrodotoxin,5· 6 and ureteral peristalsis is still present after transplantation 7 or denervation. 8 As a whole, these observations indicate that neural influences are not an obligatory prerequisite for ureteral peristalsis to occur. The question about the existence of a neural modulation of ureteral peristalsis has been much debated. Cholinergic and adrenergic nerve terminals have been described in the renal pelvis and ureter, and exogenously administered acetylcholine or catecholamines reportedly affect ureteral motility. 9-13 However, firm evidence indicating the existence of a functional cholinergic or noradrenergic innervation of the renal pelvis-ureter is lacking. In recent years, the issue about neural modulation of ureteral motility has been enriched by the observation that a dense network of neuropeptide-containing sensory nerves exist in the renal pelvis-ureter of various species. 14-16 The importance of the afferent innervation of the ureter is well known from a clinical point of view, inasmuch as ureteral pain is one of the most common and vivid forms of visceral pain. It is conceivable that afferent impulses arising from the renal pelvis-ureter may affect ureteral peristalsis indirectly through reflex pathways, 17 · 18 but again firm evidence for such an arrangement is lacking. In certain species, such as rats or guinea-pigs, a large part of ureteral afferent fibers belong to that subset of sensory neurons which are capsaicin-sensitive. 14-16 A distinct feature of capsaicin-sensitive primary afferent neurons is that of exerting a dual, sensory and 'efferent' function, the latter being accomplished through transmitters (neuropeptides) released from their peripheral endings. 19· 20 In the middle portion of both the rat and guinea-pig 21 -25 ureter, transmural electrical field stimAccepted for publication October 30, 1991.
* Requests for reprints: Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131 Florence, Italy.
ulation has been shown to produce distinct local motor responses (inhibition of evoked motility) which are entirely determined by antidromic invasion of peripheral terminals of the capsaicin-sensitive primary afferent neurons. The aim of this study was to assess whether nervous stimulation affects motility of the guinea-pig isolated renal pelvis, and, in that case, ascertain which type of nerve is involved. METHODS
Male albino guinea-pigs (250 to 300 gm. b.w.) were stunned and bled. The whole kidney and attached ureter was removed and placed in oxygenated (96% 0 2 and 4% CO2, pH 7.4 at 37C) Krebs solution of the following composition (mM): NaCl 119, KC! 4.7, MgS0 4 1.5, CaClz 2.5, KH 2P04 1.2, NaHCOs 25 and glucose 11. The renal pelvis was carefully dissected from the renal parenchyma, separated from the ureter, cut and connected to threads to record motility along the circular axis. The preparation was suspended in a five ml. organ bath and mechanical activity recorded by means of an isotonic transducer (load one mN). Transmural electrical field stimulation was made by means of platinum wire electrodes placed at the top and the bottom of the organ bath and connected to a GRASS S 88 stimulator. Square wave pulses (pulse width 0.5 ms, 60 V, maximal voltage) were delivered in trains of 10 s duration at frequencies of five to 10 Hz. All experiments commenced after a 60 to 90 minute equilibration period after which amplitude and frequency of spontaneous activity had reached a steady state. In vitro capsaicin desensitization was made by exposure of the preparations to 10 µM for 15 minutes, followed by washing out and further equilibration for 30 to 60 minutes. All changes in mechanical activity were expressed as % of maximal shortening produced by application of potassium chloride (120 mM) which was added to the bath at the end of the experiment. Preliminary experiments showed that the response to KCl was unaffected by atropine, phentolamine or in vitro
1394
1395
J\/IAGGI AND GIULIAN[
desensitization. Contact time of the was 15 minutes. Concentration response curves to noradrenaline or acetylcholine were performed noncumulative addition to the bath at 20 minute intervals. All values in the text and figures are mean ± S.E. of the mean. Statistical analysis was performed by means of Student's t test for paired or unpaired data or means of analysis of variance, when applicable. Drugs used were: tetrodotoxin (Sankyo), atropine HC! (Serva), guanethidine sulphate (ICFI), acetylcholine chloride (Merck), noradrenaline, eserine hemisulphate and capsaicin (Sigma), phentolamine mesylate (Ciba Geigy). RESULTS
Circular strips of the guinea-pig isolated renal pelvis showed a spontaneous phasic contractile activity which had a mean frequency of 6.1±0.2 contractions/minute (n=l9, range four to seven contractions/minute). Amplitude of these contractions averaged 51±3% of the maximal response to KC! (range 29 to 64%). Tetrodotoxin (one µM) did not affect spontaneous contractions of the renal pelvis. The spontaneous activity was observed for several hours although after about three hours from setup it decreased in frequency and became irregular. Acetylcholine (one to 100 µM) or noradrenaline (one to 100 µM) produced a concentration dependent positive inotropic response and also a transient positive chronotropic effect in some preparations (figures 1, 2). The response to both agents was reproducible upon two consecutive challenges in the same preparation, 20 minutes apart from each other. Atropine (three µM, 15 minutes before) inhibited or abolished the effect of acetylcholine while phentolamine (three µM, 15 minutes before) abolished the effect of noradrenaline (figure 2). Electrical field stimulation (five to 10 Hz, 0.5 ms pulse width, 60 V, maximal voltage, train duration 10 s) evoked positive
responses which peaked in two to three minutes from application of the stimulus and slowly declined to baseline (figure 3). In about half of cases tested (n=32) a transient positive chronotropic and small inotropic response was observed during train delivery which preceded the late and more intense positive inotropic response. The 'fast' response observed during train delivery was not consistently reproducible between consecutive cycles of stimulation in the same strip while the late inotropic response did not significantly change over six stimulation cycles at five to 10 Hz performed at 20 minutes from each other. Unless otherwise stated, all drugs effects are described and discussed in relation to the 'late' inotropic response which amounted to about 20 and 30% of maximal shortening of the preparations at five and 10 Hz, respectively (quantitative data shown in figure 4). The inotropic responses to electrical field stimulation were abolished by tetrodotoxin (one µM) or previous application of capsaicin (10 µM for 15 minutes followed by washout, figures 3, 4). Capsaicin itself produced a large positive inotropic response which approached 100% of the response to potassium (figure 3). A second application of capsaicin 60 minutes after the first one had no further effect, indicating complete desensitization, a hallmark of the specific action of this substance on sensory nerves. Neither atropine (three µM) nor phentolamine (three µM) alone or in combination did significantly affect the neurogenic inotropic response to field stimulation (figure 4). Likewise the positive inotropic response to field stimulation was unaffected by atropine plus guanethidine (three µM each, n=4, data not shown). To further test the possible involvement of cholinergic nerves, the effect of the acetylcholinesterase inhibitor, eserine (0.3 µM, 15 minutes before) was also investigated. Eserine (n=6) did not change amplitude of spontaneous contractions of the renal pelvis nor affected the amplitude of the inotropic response to electrical field stimulation which averaged 19±3 and 25±4 and to 20±4 and 27±5% of maximal response to KC!
1 min
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ACETYLCHOLINE 100 µM
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FIG. 1. Typical tracings illustrating positive inotropic effect produced by acetylcholine or noradrenaline in circular muscle of guinea-pig renal pelvis. Vertical bars indicate maximal contraction for each preparation.
1396
INNERVATION OF GUINEA PIG RENAL PELVIS
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NORADRENALINE FIG. 2. Concentration-dependent positive inotropic effect produced by acetylcholine or noradrenaline on circular muscle of guinea-pig renal pelvis and effect of atropine and phentolamine. Each value is mean ± S.E.M. of at least six experiments. Responses to acetylcholine and noradrenaline are expressed as % of maximal possible shortening of preparation in response to KC!. * Significantly different from controls, p <0.05.
before and after addition of eserine, at five and 10 Hz, respectively. In these experiments, a 'fast' chronotropic response to electrical stimulation was observed in three out of six strips before addition of eserine which was apparently unaffected by the drug. DISCUSSION
The present findings demonstrate the existence of a functional innervation of the circular muscle of the guinea-pig renal pelvis which is entirely dependent upon transmitter release from peripheral endings of capsaicin-sensitive primary afferent neurons. The existence of a neural control of ureteral motility has been a matter of debate for a long time. Available data indicate that ureteral peristalsis is a primarily myogenic phenomenon i-s initiated by pacemakers cells in the renal pelvis region and which propel urine along the ureter to the bladder. Noradrenaline release from sympathetic nerves is usually thought to play some modulatory role on this process 2• :i, 10• 12• rn although the physiological evidence supporting this statement is scanty, Cholinergic nerves have also been described at this level 9 - 11 but there is no firm indication for a role of acetylcholine
in modulating ureteral peristalsis. In this study we found that atropine plus phentolamine or atropine plus guanethidine had no effect on the nerve-mediated contractions produced by transmural electrical stimulation of the guinea-pig renal pelvis; in effect no evidence for cholinergic or adrenergic local modu lation ofureteral motility. Similar observations have been made previously in the middle portion of the rat or guinea-pig ureter. 21 • 2'1• 25 The possibility that cholinergic or noradrenergic nerves contribute to the 'fast' chronotropic response observed in some strips during train delivery cannot be completely ruled out. The poor reproducibility of this fast response does not allow us to draw firm conclusions about its origin, It is equally evident however, that the role of capsaicin-sensitive primary afferent neurons at this level is quantitatively much more important than any putative cholinergic or noradrenergic innervation. The positive inotropic response to electrical field stimulation in the renal pelvis is totally ascribable to antidromic invasion of capsaicin-sensitive afferent fibers and consequent transmitter release, being abolished by tetrodotoxin and capsaicin pretreatment. Our present and previous 25 findings indicate that activation of capsaicin-sensitive primary afferent neurons accounts for all the local motor responses produced by transmural nerve stim ulation in the guinea-pig renal pelvis and ureter. In the middle portion of the guinea-pig ureter, the only type of neurogenic response to electrical field stimulation is inhibition of motility, suppression of rhythmic contractions evoked by pharmacological stimulation of latent pacemakers, 25 The present findings indicate that the same type of primary afferent fibers, the capsaicin-sensitive ones, which inhibits motility in the middle ureter 25 exert a positive inotropic effect in the renal pelvis. Which could be the physiological significance of the opposite local motor effects produced by sensory nerves activation in the renal pelvis and middle ureter? Any speculation on this matter requires information about the adequate stimuli which activate the capsaicin-sensitive primary afferents innervating the ureter. Inasmuch as the capsaicin-sensitive primary afferent neurons play a dual sensory and 'efferent' function, both activated by stimulation of their peripheral receptive fields, the results of neurophysiological experiments on ureteral sensory nerves can be used for this purpose. Cervera and Sann 18 reported the presence of two types of unmyelinated mechanosen sitive afferent fibers in the guinea-pig ureter: Ul units (about 9% of all mechanosensitive units) which monitor the normal peristalsis and U2 units (91 % of all mechanosensitive units) which are likely to be involved in signalling noxious events, U2 units are chemosensitive as well and are excited by capsaicin. 18 Furthermore, neurochemical evidence indicates that local peptide release from capsaicin-sensitive nerves is elicited by stimuli which are normally present in urine such as KC!, hyperosmolarity and protons (low pH), 2G· 27 as well as by mediators of inflammation (bradykinin, prostanoids) 28 or foreign chemicals produced during bacterial infections, 29 Bearing in mind these results, we speculate that noxious stimuli (chemical or mechanical) produced in the presence of stones or bacterial infection determine, through a local peptide release from sensory nerves, a neurogenic reinforcement of renal pelvis activity and neurogenic suppression of latent pacemakers in the middle portion of the ureter. The overall effect would be that of preventing or minimizing the occurrence of antiperistaltic waves and backflow of material present in the ureteral lumen toward the kidney. In perspective, the local motor control of pyeloureteral motility produced by capsaicinsensitive primary afferents nerves could be interpreted as a braking system or safety factor to protect the kidney from material of foreign origin. Further studies are needed to identify the transmitter(s) responsible for local modulation of renal pelvis motility by
1397
MAGGI AND GIULIANI
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FIG. 3. Typical tracings illustrating positive inotropic effect produced by electrical field stimulation in circular muscle of guinea-pig renal pelvis. Response is completely unaffected by atropine and phentolamine but is tetrodotoxin-sensitive. In lower panel (atropine and phentolamine were not present in this case), positive inotropic response produced by application of capsaicin is shown. After capsaicin desensitization response to electrical field stimulation is abolished indicating its dependence from sensory nerves activation. Vertical bars indicate maximal contraction for each preparation.
sensory nerves and establish the full pathophysiological significance of this arrangement.
11. Del Tacca, M.: Acetylcholine content and release from isolated pelviureteral tract. Naun. Schmied. Arch. Pharmacol., 302: 293, 1978. 12. Hannappel, J. and Golenhofen, K.: The effect of catecholamines
REFERENCES
on ureteral peristalsis in different species (dog, guinea-pig and rat). Pflugers Archiv, 350: 55, 1974. 13. Morita, T., Wada, I., Saeki, H., Tsuchida, S. and Weiss, R. M.: Ureteral urine transport: changes in bolus volume, peristaltic frequency, intraluminal pressure and volume of flow resulting from autonomic drugs. J. Urol., 137: 132, 1987. 14. Hua, X. Y., Saria, A., Gamse, R., Theodorsson-Norheim, E., Brodin, E. and Lundberg, .J. M.: Capsaicin induced release of multiple tachykinins (substance P, neurokinin A and eledoisin-like material) from guinea-pig spinal cord and ureter. Neuroscience,
1. Engelmann, T. W.: Zur physiologie der ureter. Arch. Gesamt. Physiol., 2: 243, 1869. 2. Kiil, F. and Setekleiv, J.: Physiology of ureter and renal pelvis. In: Handbook of Physiology: Renal physiology. Edited by J. Orloff and R. W. Berliner. American Physiological Society, Washington DC USA, pp. 1033-1057, 1973.
3. Boyarsky, S. and Labay, P.: Ureteral motility. Ann. Rev. Med., 20: 383, 1969.
4. Constantinou, C. E.: Renal pelvic pacemaker control of ureteral peristaltic rate. Am. J. Physiol., 226: 1413, 1974. 5. Washizu, Y.: Grouped discharges in ureter muscle. Comp. Biochem. Physiol., 19: 713, 1966. 6. Golenhofen, K. and Hannappel, J.: Normal spontaneous activity of the pyeloureteral system in the guinea-pig. Pflugers Archiv, 341: 257, 1973. 7. O'Connor, V. J. and Dawson-Edwards, P.: Role of the ureter in renal transplantation. I. Studies of denervated ureter with particular reference of the ureteroureteral anastomosis. J. Urol., 82:
19: 313, 1986. 15. Su, H. C., Wharton, ,J., Polak, J. M., Mulderry, P. K., Ghatei, M. A., Gibson, S. J., Terenghi, G., Morrison, J. F. B., Ballesta, J. and Bloom, S. R.: CGRP immunoreactivity in afferent neurons
16.
566, 1959.
8. Wharton, L. R.: The innervation of the ureter, with respect to denervation. J. Urol., 28: 639, 1932. 9. Dixon, J. S. and Gosling, J. A.: Histochemical and electron microscopy observations on the innervation of the upper segment of the mammalian ureter. J. Anat., 110: 57, 1971. 10. Schulman, C.: Ultrastructural evidence for adrenergic and cholinergic innervation of the human ureter. J. Urol., 113: 765, 1975.
19.
18.
19.
supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neuroscience, 3: 727, 1986. Hua, X. Y., Theodorsson-Norheim, E., Lundberg, J. M., Kinn, A. C., Hokfelt, T. and Cuello, A. C.: Co-localization of tachykinins and calcitonin gene-related peptide in capsaicin-sensitive afferents in relation to motility effects on the human ureter in vitro. Neuroscience, 23: 693, 1987. Amann, R., Dray, A. and Hankins, M. W.: Stimulation of afferent fibres of the guinea-pig ureter evokes potentials in inferior mesenteric ganglion neurones. J. Physiol. (London), 402: 543, 1988. Cervero, F. and Sann, H.: Mechanically evoked responses of afferent fibres innervating the guinea-pig's ureter: an in vitro study. J. Physiol. (London), 412: 245, 1989. Maggi, C. A. and Meli, A.: The sensory-efferent function of cap-
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INNERVATION OF GUINEA PIG RENAL PELVIS saicin-sensitive nerves. Gen. Pharmacol., 19: 1, 1988. 20. Szolcsanyi, J.: Capsaicin-sensitive chemoceptive neural system
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FIG. 4. Effect of capsaicin pretreatment, atropine (three µM), phentolamine (three µM) or atropine plus phentolamine (three µM each) on positive inotropic response of circular muscle of guinea-pig renal pelvis produced by electrical field stimulation at five and 10 Hz. Each value is mean ± S.E.M. of at least six experiments. Response to electrical field stimulation is expressed as % of maximal possible shortening in response to KC!. * Significantly different from control p <0.05.
28.
29.
with dual sensory-efferent function. In: Antidromic Vasodilation and Neurogenic Inflammation. Edited by L. A. Chahl, J. Szolcsanyi and F. Lembeck. Akademiai Kiado, Budapest, pp. 2652, 1984. Maggi, C. A., Giuliani, S., Santicioli, P., Abelli, L. and Meli, A.: Visceromotor responses to CGRP in the rat lower urinary tract: evidence for a transmitter role in the capsaicin-sensitive nerves of the ureter. Eur. J. Pharmacol., 143: 73, 1987. Maggi, C. A., Santicioli, P., Abelli, L., Parlani, M., Capasso, M., Conte, B., Giuliani, S. and Meli, A.: Regional differences in the effects of capsaicin and tachykinins on motor activity and vascular permeability of the rat lower urinary tract. Naun. Schmied. Arch. Pharmacol., 335: 636, 1987. Maggi, C. A., Santicioli, P., Giuliani, L., Abelli, L. and Meli, A.: The.motor effect of the..capsaicin-sensiti~ inhibitory innervation of the rat ureter. Eur. J. Pharmacol., 126: 333, 1986. Amann, R., Skofitsch, G. and Lembeck, F.: Species-related differences in the capsaicin-sensitive innervation of the rat and guinea-pig ureter. Naun. Schmied. Arch. Pharmacol., 338: 407, 1988. Maggi, C. A. and Giuliani, S.: The neurotransmitter role of calcitonin gene-related peptide (CGRP) in the rat and guinea-pig ureter: effect of a CGRP antagonist and species-related differences in the action of omega conotoxin on CGRP release from primary afferents. Neuroscience, 43: 261, 1991. Tramontana, M., Cecconi, R., Del Bianco, E., Santicioli, P., Maggi, C. A., Alessandri, M. and Geppetti, P .: Hypertonic media produce calcium dependent release of CGRP from capsaicin-sensitive nerve fibres in the rat urinary bladder. Neurosci. Letters, 124: 79, 1991. Geppetti, P., Del Bianco, E., Patacchini, R., Santicioli, P., Maggi, C. A. and Tramontana, M.: Low pH-induced release of calcitonin gene-related peptide from capsaicin-sensitive sensory nervesmechanism of action and biological response. Neuroscience, 41: 295, 1991. Maggi, C. A., Patacchini, R., Santicioli, P., Geppetti, P., Cecconi, R., Giuliani, S. and Meli, A.: Multiple mechanisms in the motor responses of the guinea-pig urinary bladder to bradykinin. Br. J. Pharmacol., 98: 619, 1989. Giuliani, S., Santicioli, P., Tramontana, M., Geppetti, P. and Maggi, C. A.: Peptide N-formyl-leucyl-phenylalanine (FMLP) activates capsaicin-sensitive primary afferent nerves in guineapig atria and urinary bladder. Br. J. Pharmacol., 102: 730, 1991.
Vol. 147, 1394-1398, May 1992
Printed in U.S.A.
NON-ADRENERGIC NON-CHOLINERGIC EXCITATORY INNERVATION OF THE GUINEA-PIG ISOLATED RENAL PELVIS: INVOLVEMENT OF CAPSAICIN-SENSITIVE PRIMARY AFFERENT NEURONS CARLO ALBERTO MAGGI*
AND
SANDRO GIULIANI
From the Pharmacology Department, A. Menarini Pharmaceuticals, Florence, Italy
ABSTRACT
Circular muscle strips from the guinea-pig renal pelvis exhibit a spontaneous activity averaging about 50% of maximal contractile response at a frequency of about six contractions/min. This spontaneous activity is tetrodotoxin resistant. Acetylcholine and noradrenaline (one to 100 µM) produce a positive inotropic effect which was abolished or strongly inhibited by atropine and phentolamine, respectively. Electrical field stimulation (five to 10 Hz, 0.5 ms pulse width for 10 s, 60 V, maximal voltage) produces a transient positive inotropic response which is tetrodotoxinsensitive but unaffected by atropine and phentolamine alone or in combination, as well as by atropine plus guanethidine. Application of capsaicin produced a large positive inotropic response which was not reproduced upon a second application of the drug (desensitization). After in vitro capsaicin desensitization, the response to electrical field stimulation was abolished, indicating its dependence on activation of peripheral endings of sensory nerves. These findings demonstrate the existence of a non-adrenergic non-cholinergic functional innervation of the guinea-pig renal pelvis which is entirely dependent on sensory nerve activation. KEY
W OROS: kidney, nerve cells, neurotransmitters
Ureteral peristalsis is a primarily myogenic phenomenon initiated by pacemaker cells in the proximal part of the renal pelvis. 1-4 Neither pacemaker cell activity nor propagation of peristaltic waves along the ureter are affected by tetrodotoxin,5· 6 and ureteral peristalsis is still present after transplantation 7 or denervation. 8 As a whole, these observations indicate that neural influences are not an obligatory prerequisite for ureteral peristalsis to occur. The question about the existence of a neural modulation of ureteral peristalsis has been much debated. Cholinergic and adrenergic nerve terminals have been described in the renal pelvis and ureter, and exogenously administered acetylcholine or catecholamines reportedly affect ureteral motility. 9-13 However, firm evidence indicating the existence of a functional cholinergic or noradrenergic innervation of the renal pelvis-ureter is lacking. In recent years, the issue about neural modulation of ureteral motility has been enriched by the observation that a dense network of neuropeptide-containing sensory nerves exist in the renal pelvis-ureter of various species. 14-16 The importance of the afferent innervation of the ureter is well known from a clinical point of view, inasmuch as ureteral pain is one of the most common and vivid forms of visceral pain. It is conceivable that afferent impulses arising from the renal pelvis-ureter may affect ureteral peristalsis indirectly through reflex pathways, 17 · 18 but again firm evidence for such an arrangement is lacking. In certain species, such as rats or guinea-pigs, a large part of ureteral afferent fibers belong to that subset of sensory neurons which are capsaicin-sensitive. 14-16 A distinct feature of capsaicin-sensitive primary afferent neurons is that of exerting a dual, sensory and 'efferent' function, the latter being accomplished through transmitters (neuropeptides) released from their peripheral endings. 19· 20 In the middle portion of both the rat and guinea-pig 21 -25 ureter, transmural electrical field stimAccepted for publication October 30, 1991.
* Requests for reprints: Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131 Florence, Italy.
ulation has been shown to produce distinct local motor responses (inhibition of evoked motility) which are entirely determined by antidromic invasion of peripheral terminals of the capsaicin-sensitive primary afferent neurons. The aim of this study was to assess whether nervous stimulation affects motility of the guinea-pig isolated renal pelvis, and, in that case, ascertain which type of nerve is involved. METHODS
Male albino guinea-pigs (250 to 300 gm. b.w.) were stunned and bled. The whole kidney and attached ureter was removed and placed in oxygenated (96% 0 2 and 4% CO2, pH 7.4 at 37C) Krebs solution of the following composition (mM): NaCl 119, KC! 4.7, MgS0 4 1.5, CaClz 2.5, KH 2P04 1.2, NaHCOs 25 and glucose 11. The renal pelvis was carefully dissected from the renal parenchyma, separated from the ureter, cut and connected to threads to record motility along the circular axis. The preparation was suspended in a five ml. organ bath and mechanical activity recorded by means of an isotonic transducer (load one mN). Transmural electrical field stimulation was made by means of platinum wire electrodes placed at the top and the bottom of the organ bath and connected to a GRASS S 88 stimulator. Square wave pulses (pulse width 0.5 ms, 60 V, maximal voltage) were delivered in trains of 10 s duration at frequencies of five to 10 Hz. All experiments commenced after a 60 to 90 minute equilibration period after which amplitude and frequency of spontaneous activity had reached a steady state. In vitro capsaicin desensitization was made by exposure of the preparations to 10 µM for 15 minutes, followed by washing out and further equilibration for 30 to 60 minutes. All changes in mechanical activity were expressed as % of maximal shortening produced by application of potassium chloride (120 mM) which was added to the bath at the end of the experiment. Preliminary experiments showed that the response to KCl was unaffected by atropine, phentolamine or in vitro
1394
1395
J\/IAGGI AND GIULIAN[
desensitization. Contact time of the was 15 minutes. Concentration response curves to noradrenaline or acetylcholine were performed noncumulative addition to the bath at 20 minute intervals. All values in the text and figures are mean ± S.E. of the mean. Statistical analysis was performed by means of Student's t test for paired or unpaired data or means of analysis of variance, when applicable. Drugs used were: tetrodotoxin (Sankyo), atropine HC! (Serva), guanethidine sulphate (ICFI), acetylcholine chloride (Merck), noradrenaline, eserine hemisulphate and capsaicin (Sigma), phentolamine mesylate (Ciba Geigy). RESULTS
Circular strips of the guinea-pig isolated renal pelvis showed a spontaneous phasic contractile activity which had a mean frequency of 6.1±0.2 contractions/minute (n=l9, range four to seven contractions/minute). Amplitude of these contractions averaged 51±3% of the maximal response to KC! (range 29 to 64%). Tetrodotoxin (one µM) did not affect spontaneous contractions of the renal pelvis. The spontaneous activity was observed for several hours although after about three hours from setup it decreased in frequency and became irregular. Acetylcholine (one to 100 µM) or noradrenaline (one to 100 µM) produced a concentration dependent positive inotropic response and also a transient positive chronotropic effect in some preparations (figures 1, 2). The response to both agents was reproducible upon two consecutive challenges in the same preparation, 20 minutes apart from each other. Atropine (three µM, 15 minutes before) inhibited or abolished the effect of acetylcholine while phentolamine (three µM, 15 minutes before) abolished the effect of noradrenaline (figure 2). Electrical field stimulation (five to 10 Hz, 0.5 ms pulse width, 60 V, maximal voltage, train duration 10 s) evoked positive
responses which peaked in two to three minutes from application of the stimulus and slowly declined to baseline (figure 3). In about half of cases tested (n=32) a transient positive chronotropic and small inotropic response was observed during train delivery which preceded the late and more intense positive inotropic response. The 'fast' response observed during train delivery was not consistently reproducible between consecutive cycles of stimulation in the same strip while the late inotropic response did not significantly change over six stimulation cycles at five to 10 Hz performed at 20 minutes from each other. Unless otherwise stated, all drugs effects are described and discussed in relation to the 'late' inotropic response which amounted to about 20 and 30% of maximal shortening of the preparations at five and 10 Hz, respectively (quantitative data shown in figure 4). The inotropic responses to electrical field stimulation were abolished by tetrodotoxin (one µM) or previous application of capsaicin (10 µM for 15 minutes followed by washout, figures 3, 4). Capsaicin itself produced a large positive inotropic response which approached 100% of the response to potassium (figure 3). A second application of capsaicin 60 minutes after the first one had no further effect, indicating complete desensitization, a hallmark of the specific action of this substance on sensory nerves. Neither atropine (three µM) nor phentolamine (three µM) alone or in combination did significantly affect the neurogenic inotropic response to field stimulation (figure 4). Likewise the positive inotropic response to field stimulation was unaffected by atropine plus guanethidine (three µM each, n=4, data not shown). To further test the possible involvement of cholinergic nerves, the effect of the acetylcholinesterase inhibitor, eserine (0.3 µM, 15 minutes before) was also investigated. Eserine (n=6) did not change amplitude of spontaneous contractions of the renal pelvis nor affected the amplitude of the inotropic response to electrical field stimulation which averaged 19±3 and 25±4 and to 20±4 and 27±5% of maximal response to KC!
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FIG. 1. Typical tracings illustrating positive inotropic effect produced by acetylcholine or noradrenaline in circular muscle of guinea-pig renal pelvis. Vertical bars indicate maximal contraction for each preparation.
1396
INNERVATION OF GUINEA PIG RENAL PELVIS
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NORADRENALINE FIG. 2. Concentration-dependent positive inotropic effect produced by acetylcholine or noradrenaline on circular muscle of guinea-pig renal pelvis and effect of atropine and phentolamine. Each value is mean ± S.E.M. of at least six experiments. Responses to acetylcholine and noradrenaline are expressed as % of maximal possible shortening of preparation in response to KC!. * Significantly different from controls, p <0.05.
before and after addition of eserine, at five and 10 Hz, respectively. In these experiments, a 'fast' chronotropic response to electrical stimulation was observed in three out of six strips before addition of eserine which was apparently unaffected by the drug. DISCUSSION
The present findings demonstrate the existence of a functional innervation of the circular muscle of the guinea-pig renal pelvis which is entirely dependent upon transmitter release from peripheral endings of capsaicin-sensitive primary afferent neurons. The existence of a neural control of ureteral motility has been a matter of debate for a long time. Available data indicate that ureteral peristalsis is a primarily myogenic phenomenon i-s initiated by pacemakers cells in the renal pelvis region and which propel urine along the ureter to the bladder. Noradrenaline release from sympathetic nerves is usually thought to play some modulatory role on this process 2• :i, 10• 12• rn although the physiological evidence supporting this statement is scanty, Cholinergic nerves have also been described at this level 9 - 11 but there is no firm indication for a role of acetylcholine
in modulating ureteral peristalsis. In this study we found that atropine plus phentolamine or atropine plus guanethidine had no effect on the nerve-mediated contractions produced by transmural electrical stimulation of the guinea-pig renal pelvis; in effect no evidence for cholinergic or adrenergic local modu lation ofureteral motility. Similar observations have been made previously in the middle portion of the rat or guinea-pig ureter. 21 • 2'1• 25 The possibility that cholinergic or noradrenergic nerves contribute to the 'fast' chronotropic response observed in some strips during train delivery cannot be completely ruled out. The poor reproducibility of this fast response does not allow us to draw firm conclusions about its origin, It is equally evident however, that the role of capsaicin-sensitive primary afferent neurons at this level is quantitatively much more important than any putative cholinergic or noradrenergic innervation. The positive inotropic response to electrical field stimulation in the renal pelvis is totally ascribable to antidromic invasion of capsaicin-sensitive afferent fibers and consequent transmitter release, being abolished by tetrodotoxin and capsaicin pretreatment. Our present and previous 25 findings indicate that activation of capsaicin-sensitive primary afferent neurons accounts for all the local motor responses produced by transmural nerve stim ulation in the guinea-pig renal pelvis and ureter. In the middle portion of the guinea-pig ureter, the only type of neurogenic response to electrical field stimulation is inhibition of motility, suppression of rhythmic contractions evoked by pharmacological stimulation of latent pacemakers, 25 The present findings indicate that the same type of primary afferent fibers, the capsaicin-sensitive ones, which inhibits motility in the middle ureter 25 exert a positive inotropic effect in the renal pelvis. Which could be the physiological significance of the opposite local motor effects produced by sensory nerves activation in the renal pelvis and middle ureter? Any speculation on this matter requires information about the adequate stimuli which activate the capsaicin-sensitive primary afferents innervating the ureter. Inasmuch as the capsaicin-sensitive primary afferent neurons play a dual sensory and 'efferent' function, both activated by stimulation of their peripheral receptive fields, the results of neurophysiological experiments on ureteral sensory nerves can be used for this purpose. Cervera and Sann 18 reported the presence of two types of unmyelinated mechanosen sitive afferent fibers in the guinea-pig ureter: Ul units (about 9% of all mechanosensitive units) which monitor the normal peristalsis and U2 units (91 % of all mechanosensitive units) which are likely to be involved in signalling noxious events, U2 units are chemosensitive as well and are excited by capsaicin. 18 Furthermore, neurochemical evidence indicates that local peptide release from capsaicin-sensitive nerves is elicited by stimuli which are normally present in urine such as KC!, hyperosmolarity and protons (low pH), 2G· 27 as well as by mediators of inflammation (bradykinin, prostanoids) 28 or foreign chemicals produced during bacterial infections, 29 Bearing in mind these results, we speculate that noxious stimuli (chemical or mechanical) produced in the presence of stones or bacterial infection determine, through a local peptide release from sensory nerves, a neurogenic reinforcement of renal pelvis activity and neurogenic suppression of latent pacemakers in the middle portion of the ureter. The overall effect would be that of preventing or minimizing the occurrence of antiperistaltic waves and backflow of material present in the ureteral lumen toward the kidney. In perspective, the local motor control of pyeloureteral motility produced by capsaicinsensitive primary afferents nerves could be interpreted as a braking system or safety factor to protect the kidney from material of foreign origin. Further studies are needed to identify the transmitter(s) responsible for local modulation of renal pelvis motility by
1397
MAGGI AND GIULIANI
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FIG. 3. Typical tracings illustrating positive inotropic effect produced by electrical field stimulation in circular muscle of guinea-pig renal pelvis. Response is completely unaffected by atropine and phentolamine but is tetrodotoxin-sensitive. In lower panel (atropine and phentolamine were not present in this case), positive inotropic response produced by application of capsaicin is shown. After capsaicin desensitization response to electrical field stimulation is abolished indicating its dependence from sensory nerves activation. Vertical bars indicate maximal contraction for each preparation.
sensory nerves and establish the full pathophysiological significance of this arrangement.
11. Del Tacca, M.: Acetylcholine content and release from isolated pelviureteral tract. Naun. Schmied. Arch. Pharmacol., 302: 293, 1978. 12. Hannappel, J. and Golenhofen, K.: The effect of catecholamines
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19: 313, 1986. 15. Su, H. C., Wharton, ,J., Polak, J. M., Mulderry, P. K., Ghatei, M. A., Gibson, S. J., Terenghi, G., Morrison, J. F. B., Ballesta, J. and Bloom, S. R.: CGRP immunoreactivity in afferent neurons
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8. Wharton, L. R.: The innervation of the ureter, with respect to denervation. J. Urol., 28: 639, 1932. 9. Dixon, J. S. and Gosling, J. A.: Histochemical and electron microscopy observations on the innervation of the upper segment of the mammalian ureter. J. Anat., 110: 57, 1971. 10. Schulman, C.: Ultrastructural evidence for adrenergic and cholinergic innervation of the human ureter. J. Urol., 113: 765, 1975.
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supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neuroscience, 3: 727, 1986. Hua, X. Y., Theodorsson-Norheim, E., Lundberg, J. M., Kinn, A. C., Hokfelt, T. and Cuello, A. C.: Co-localization of tachykinins and calcitonin gene-related peptide in capsaicin-sensitive afferents in relation to motility effects on the human ureter in vitro. Neuroscience, 23: 693, 1987. Amann, R., Dray, A. and Hankins, M. W.: Stimulation of afferent fibres of the guinea-pig ureter evokes potentials in inferior mesenteric ganglion neurones. J. Physiol. (London), 402: 543, 1988. Cervero, F. and Sann, H.: Mechanically evoked responses of afferent fibres innervating the guinea-pig's ureter: an in vitro study. J. Physiol. (London), 412: 245, 1989. Maggi, C. A. and Meli, A.: The sensory-efferent function of cap-
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INNERVATION OF GUINEA PIG RENAL PELVIS saicin-sensitive nerves. Gen. Pharmacol., 19: 1, 1988. 20. Szolcsanyi, J.: Capsaicin-sensitive chemoceptive neural system
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FIG. 4. Effect of capsaicin pretreatment, atropine (three µM), phentolamine (three µM) or atropine plus phentolamine (three µM each) on positive inotropic response of circular muscle of guinea-pig renal pelvis produced by electrical field stimulation at five and 10 Hz. Each value is mean ± S.E.M. of at least six experiments. Response to electrical field stimulation is expressed as % of maximal possible shortening in response to KC!. * Significantly different from control p <0.05.
28.
29.
with dual sensory-efferent function. In: Antidromic Vasodilation and Neurogenic Inflammation. Edited by L. A. Chahl, J. Szolcsanyi and F. Lembeck. Akademiai Kiado, Budapest, pp. 2652, 1984. Maggi, C. A., Giuliani, S., Santicioli, P., Abelli, L. and Meli, A.: Visceromotor responses to CGRP in the rat lower urinary tract: evidence for a transmitter role in the capsaicin-sensitive nerves of the ureter. Eur. J. Pharmacol., 143: 73, 1987. Maggi, C. A., Santicioli, P., Abelli, L., Parlani, M., Capasso, M., Conte, B., Giuliani, S. and Meli, A.: Regional differences in the effects of capsaicin and tachykinins on motor activity and vascular permeability of the rat lower urinary tract. Naun. Schmied. Arch. Pharmacol., 335: 636, 1987. Maggi, C. A., Santicioli, P., Giuliani, L., Abelli, L. and Meli, A.: The.motor effect of the..capsaicin-sensiti~ inhibitory innervation of the rat ureter. Eur. J. Pharmacol., 126: 333, 1986. Amann, R., Skofitsch, G. and Lembeck, F.: Species-related differences in the capsaicin-sensitive innervation of the rat and guinea-pig ureter. Naun. Schmied. Arch. Pharmacol., 338: 407, 1988. Maggi, C. A. and Giuliani, S.: The neurotransmitter role of calcitonin gene-related peptide (CGRP) in the rat and guinea-pig ureter: effect of a CGRP antagonist and species-related differences in the action of omega conotoxin on CGRP release from primary afferents. Neuroscience, 43: 261, 1991. Tramontana, M., Cecconi, R., Del Bianco, E., Santicioli, P., Maggi, C. A., Alessandri, M. and Geppetti, P .: Hypertonic media produce calcium dependent release of CGRP from capsaicin-sensitive nerve fibres in the rat urinary bladder. Neurosci. Letters, 124: 79, 1991. Geppetti, P., Del Bianco, E., Patacchini, R., Santicioli, P., Maggi, C. A. and Tramontana, M.: Low pH-induced release of calcitonin gene-related peptide from capsaicin-sensitive sensory nervesmechanism of action and biological response. Neuroscience, 41: 295, 1991. Maggi, C. A., Patacchini, R., Santicioli, P., Geppetti, P., Cecconi, R., Giuliani, S. and Meli, A.: Multiple mechanisms in the motor responses of the guinea-pig urinary bladder to bradykinin. Br. J. Pharmacol., 98: 619, 1989. Giuliani, S., Santicioli, P., Tramontana, M., Geppetti, P. and Maggi, C. A.: Peptide N-formyl-leucyl-phenylalanine (FMLP) activates capsaicin-sensitive primary afferent nerves in guineapig atria and urinary bladder. Br. J. Pharmacol., 102: 730, 1991.