Biochemical, anatomical and functional correlates of postnatal development of the capsaicin-sensitive innervation of the rat urinary bladder

Developmental Brain Research, 43 (1988) 183-190 Elsevier

183

BRD 50806

Biochemical, anatomical and functional correlates of postnatal development of the capsaicin-sensitive innervation of the rat urinary bladder Carlo Alberto Maggi 1, Paolo Santicioli 1, Pierangelo Geppetti 2, Stefania Frilli 2, Maria Grazia Spillantini 3, Chiara Nediani 2, Stephen P. Hunt 3 and Alberto Meli l 1Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Florence (Italy), 2Department of Internal Medicine and Clinical Pharmacology, Florence (Italy) and ~Department of Molecular Neurobiology, MRC Cambridge (U. K.) (Accepted l l May 1988)

Key words." Micturition reflex; Newborn rat; Capsaicin; Sensory innervation; Substance P-like immunoreactivity; Immunohistochemistry

The postnatal development of substance P-like immunoreactivity (SP-LI) in the urinary bladder (assayed by radioimmunoassay and immunohistochemistry) was investigated in rats and compared with changes in the contractile response to acetylcholine, SP or capsaicin. In adult rats, bladder SP-LI was depleted by systemic capsaicin desensitization or extrinsic bladder denervation indicating that it is completely stored in sensory nerves. Bladder SP-LI was not detected in rat fetuses nor in newborn rats until day 3 of postnatal life (P3). SP-LI increased thereafter to reach, at P20, values approaching 60% of the SP-LI observed in the adult rat. By immunohistochemistry, SP-LI positive varicose fibers were not observed until P13. The contractile response to capsaicin was absent at P0, both in vivo (topical application) and in vitro. In adult rats, the capsaicin-induced bladder contraction was abolished by extrinsic denervation and is produced by release of transmitters from sensory nerves. The amplitude of the capsaicin-induced contraction in the postnatal rat bladder was significantly correlated with SP-LI concentration in the organ. Bladders excised from newborn (P0) or adult rats were equally sensitive to exogenous SP which, in both cases, produced a concentration-related contraction. It is concluded that the postnatal development of the 'efferent' function mediated by capsaicin-sensitive nerves of the rat bladder is strictly related to development of peptidergic sensory innervation.

INTRODUCTION

ty of mechanoreceptive elements which detect bladder distension s'j2"l~. In recent years, much evidence has been presented 5.s.lc~,~4indicating that in rats, cer-

The rat urinary bladder undergoes marked developmental changes in the early postnatal period with regard to myogenic contractile activity and innervation ~2'~3. These changes are thought to be relevant for development of an adult-like micturition pattern, e.g. transition from a somato-vesical reflex, which determines voiding in newborns when the mother

cin. A n increased bladder capacity and selective impairment of the afferent arm of reflex micturition have been documented in capsaicin-desensitized rats s. In response to environmental stimuli, capsai-

licks the perineal area, to a vesico-vesical micturition reflex ~3. The ability of the rat bladder to contract reflexly in response to distension requires, as its primum movens, the anatomical and functional integri-

cin-sensitive sensory nerves of the rat bladder also mediate an 'efferent' functions'2° through the release of sensory neuropeptides which, in turn, can produce a wide range of visceromotor and inflammatory re-

tain bladder mechanoreceptors are sensitive to both acute excitatory and desensitizing action of capsai-

Correspondence: C.A. Maggi, Research Laboratories, A. Menarini Pharmaceuticals, Department of Pharmacology, Smooth Muscle Division, Via Sette Santi 3, 50131, Florence, Italy.

184 sponses 7~'t3-1517,

Previous findings d e m o n s t r a t e d

that both sensory and +efferent" functions induced by capsaicin develop postnatally 13, but the mechanisms underlying these postnatal changes were not elucidated. The aim of this study is to investigate the time course of d e v e l o p m e n t of the sensory SP innervation of the rat b l a d d e r and the relative role of pre- and postjunctional factors in the postnatal d e v e l o p m e n t of the 'efferent' response m e d i a t e d by capsaicin-sensitive sensory nerves in this organ.

MATERIALS AND METHODS

General D a t e d pregnant W i s t a r - M o r i n i female rats were obtained at least one week prior to parturition. The date of parturition of each litter was r e c o r d e d and the pups (postnatal days 0 - 2 0 , hereafter referred to as P 0 - P 2 0 ) r e m a i n e d with their mothers until the day of the experiment. T w o - m o n t h s old male rats of the same strain, were used for comparison.

Determination of substance P content of the urinary bladder The urinary b l a d d e r was r e m o v e d from rat fetuses on day 18 of gestational age (F18) or in postnatal rats at various ages ( P 0 - P 2 0 ) or from 2-month-old rats. The b l a d d e r s were h o m o g e n i z e d in 95 °C 2 N acetic acid (1/10; w/v), centrifuged at 20,000 g for 30 ntin and the supernatant was freeze-dried. A f t e r reconstitution in assay buffer (0.1 M, p H 7.4 p h o s p h a t e buffer containing 0.9% NaC1, (I.(t1% NaN 3 and 0.1(~ bovine serum albumin), samples were measured by r a d i o i m m u n o a s s a y (R1A) as described previously7'tl. H u n d r e d ul of samples or SP s t a n d a r d , 100 ul of [1251]Botton and H u n t e r c o n j u g a t e d SP ( A m e r s h a m . U . K . ) and 100 ~1 of 1/20,00(t 144 rabbit SP antiserum (kind gift of Dr. P. Pradelles) were incubated overnight. A f t e r addition of 1 ml of buffer containing 7.5% polyethylene glycol, and 1/200 goat antirabbit antiserum and 1/2000 n o r m a l rabbit serum, free antigen was s e p a r a t e d from bound by centrifugation at 2000 g for 30 rain at 4 °C. The coefficient of percentage variation was less than 10% for values between 15 and 300 pmol/1. The antiserum corssreacts by lC; with neurokinin A , 0.5% with neurokinin B and less than 0.1% with physalaemin and eledoisin.

lmmunohistochernistrv Newborn rats were killed by decapitation at different postnatal days ( P 0 - P 1 5 ) . For comparison, four adult rats (320-340 g) were anesthetized and perfused transcardially with 0.1 M p h o s p h a t e - b u f f e r e d saline (PBS). The bladders were removed, o p e n e d and pinned to balsa. Following fixation with Z a m b o ni's fixative at 4 °C for 3 days. the bladders were rinsed for 24 h in phosphate buffer (PB. (). 1 M, pH 7.4) containing 30% sucrose and frozen in liquid nitrogen for a few seconds and thawed. A f t e r having been washed in 80% ethanol to remove picric acid t h e specimens were d e h y d r a t e d m 95c'~ attd lt!l)+:; ethanol and then xylol (30 rain each ). They were then rehydrated through 100G;. Sl)(~f ~w;> ethanol <30 cain eachl to PBS and kept in PBS/3054 sucrose (1.04% NaN~ until processed for p c r o x i d a s e - a n t i p e roxidase ( P A P I staining as elsewhere indicated :~ The treatment of b l a d d e r whole mount preparations was carried out in the following o r d e r and at room t e m p e r a t u r e unless otherwise indicated: 15 mm m 1% NaBH~ m PBS {freshly p r e p m e d t : 3 x 10 rain wash; 5 min ut H , O < 3 x l{I toni wash: 1 it in 2 0 q normal sheep serum: 4 × 15 mm wash: 72 h at 4 J ( ' l n SP antiserum (SPI771 diluted i:l{/00 or in 1:1000 normal rabbit serum {control): 5 40 rain wash: overnight in sheep anti rabbit igG diluted 1:20 (Serotec): 3 × 40 rain wash: 3 h in PAP complex {Miles Scientific) diluted l : 1 0 0 : 3 x d-{I mm wash the last ot which in PBS. All the washes unless otherwise slated were p e r f o r m e d in PBS/0.2 ] r i t o n X100. The antisera were all diluted with PIES/1C,, 1 rtton X100 and in all the solutions 1% of normal -;heep serum was added. In o r d e r to reveal the staining of SP. bladders were p r e - m c u b a t e d for 1 h in I .(15r~ 3',3-diaminobenzidine tetra HCI (Sigma l dissolved in 0.05 M PBS p i t 7.4, followed bv 6 - t 0 mm m the same solution added to ql IJl
In vivo experiments The rats were anesthetized with s.c urethane: in newborn rats larger doses of urethane ( [ . 5 - 2 . 5 g/kg s.c.) were required according to age. Through a midline incision of the a b d o m e n the urinary b l a d d e r was

185 exposed and e m p t i e d of urine by application of a slight manual pressure. Recording of intravesical pressure and infusion of saline were made by the transurethral route by means of a polyethylene tubing, which was inserted into the b l a d d e r through a small incision into the proximal urethra as described previously12'~7; the tubing was secured in place by means of a silk ligature. The free end of the tubing was connected to a pressure transducer and the whole system filled with saline. Intraluminal pressure signals were delivered to an H.P. 8805B carrier amplifier and displayed on an H.P. 7754A four-channel polygraph. W a r m saline-soaked cotton wool swabs were laid around the exteriorized organ to maintain its t e m p e r a t u r e and keep it moist. Topical (in 100 ~tl of saline) application was done by dropping the test substance in 2 - 3 s on the b l a d d e r d o m e by means of a Hamilton microsyringe as described previously 13'14. In vitro experiments Either newborn (P0) or two-month-old adult rats were killed by cervical dislocation. The urinary bladder was rapidly removed and placed in oxygenated (96% O 2 and 4% CO2) standard Krebs solution as described previously j2`j7. The bladders were placed in a 5-ml organ bath maintained at 37 °C and connected under a resting tension of 0.5 g to an isometric transducer. Drugs (capsaicin, SP, acetylcholine) were a d d e d to the organ bath after a 30-rain equilibration period. C o n c e n t r a t i o n - r e s p o n s e curves to SP were obtained in a non-cumulative m a n n e r at 20rain intervals. Extrinsic bladder denervation and systemic capsaicin desensitization Some experiments were p e r f o r m e d in rats whose bladder had been extrinsically d e n e r v a t e d by bilateral removal of the pelvic ganglia, 72 h before, as described previously ~°,17. Some experiments were performed in bladder of two-month-old rats systemically p r e t r e a t e d with s.c. capsaicin 50 mg/kg either as newborns (2nd day of life) or as adults (4 days before), under light ether anesthesia, as described previously 111,17. Statistical analysis All data in the text, tables and figures are mean _+ S.E.M. Statistical analysis of the data was p e r f o r m e d

by means of Student's t-test for paired or unpaired data when applicable. The effect of age on various p a r a m e t e r s of b l a d d e r activity was d e t e r m i n e d by analysis of variance. Drugs Drugs used were: tetrodotoxin (Sankyo), capsaicin (Sigma), acetylcholine chloride (Merck), SP (Peninsula). RESULTS Postnatal development o f S P - L I in the rat urinary bladder The mean content of SP-LI of the urinary b l a d d e r in 2-month-old rats was 3.5 + 0.1 pmol/g (n = 16). H P L C analysis of b l a d d e r extracts indicated that more than 85% of SP-LI co-eluted with synthetic SP or its oxidized form. SP-LI of the rat b l a d d e r was almost completely depleted ( < 5 % of control) by extrinsic denervation achieved by bilateral removal of pelvic ganglia, 72 h before (n = 6) or systemic capsaicin pretreatment. Capsaicin p r e t r e a t m e n t also abolished the tetrodotoxin-resistant contraction p r o d u c e d by capsaicin "~'17. Bladder SP-LI was unaffected in vehicle-treated or s h a m - o p e r a t e d animals (n = 4 for each group, data not shown). SP-LI could not be detected in bladders from F18 or P0 rats. SP-LI was first detected in P3 rats and increased thereafter with age (Table I) to reach, at P20, 60% of levels observed in the bladders from 2-month-old rats.

TABLE I Postnatal development of bladder response to topical capsaicin or acetylcholine and bladder content of SP-like irnmunoreactivitv in rats Each value is the mean +_S.E.M. n, number of determinations; F, fetal preparation; P, postnatal preparation. The number after F or P indicates the gestational or postnatal age in days. NT, not tested; NR, no response. Age

F18 P0 P3 PlO P20

n

4 5 6 6 6

Contractile response (rnnrnHg) Capsaicin

Acetylcholine

NT NR 2 _+0.3 6 _+ 1 21 + 3

NT 27 _+2 22 _+3 38 _+5 35 + 6

Bladder SP-L1 (pmol/g)

not detectable not detectable 0.425 + 0.13 0.778 + 0.04 2.841 _+0.25

186

Fig, 1. Varicose fibers stained with anti SP antibody in whole m o u n t preparations of urinary bladder of rats at different agc; observed with interference contrast microscopy. A: fiber in urinary bladder of 13-day-old rat. B - D : fibers in the urinary bladder of 15-day-oR1 rat. E: fibers in the urinary bladder of adult rat. Bars = 10 t t m the bar of panel C also applies to panel~ A, B anc4 I

187

lmmunohistochemistry No staining was observed in control bladders from adult rats incubated with normal rabbit serum. When they were incubated with SP antibody, long varicose fibers were seen running along blood vessels and over bundles of smooth muscle (Fig. 1E). No SPstained fibers have been observed in urinary bladders from newborn rats at P0, P4 and P8. A small number of small diameter varicose fibers were seen in the urinary bladder at P13 (Fig. 1A) and their number increased at P15 (Fig. I B - D ) .

Postnatal development of the tetrodotoxin-resistant contraction induced by topical capsaicin in vivo Topical application of capsaicin on the serosal surface of the bladder produced a tetrodotoxin-resistant contraction, due to transmitter release from sensory fibers in the bladder wall H)'27. This response was almost absent at birth and increased thereafter in relation to the age of the animal (Fig. 2). The amplitude of the response to topical acetylcholine was not significantly related to age (Fig. 2). There was a highly significant relationship between the amplitude of capsaicin- (r = 0.997, n = 3, P < 0.05) but not acetylcholine (r = 0.468, n = 3, n.s.)-induced bladder contraction and development of SP-LI of the rat bladder.

Response to capsaicin or acetylcholine of the isolated urinary bladder from newborn rats The urinary bladders of newborn rats (P0-P1)

40ol Z

E E

Z O

were mechanically quiescent while those from adult animals exhibited a low amplitude, tetrodotoxin (1 ~M)-resistant spontaneous activity. This activity could also be observed in strips excised 48-72 h after extrinsic bladder denervation (n = 4) indicating its myogenic origin. Acetylcholine (1 raM) produced a contraction of the isolated bladder at all stages of postnatal development (P0-P20). The amplitude of this contraction was unrelated to age (r -- 0.679, n = 4, n.s.) or SP-LI bladder content (r = 0.701, n = 3, n.s.). On the other hand, the amplitude of the capsaicin (1 ~M)-induced contraction was significantly correlated to age (r = 0.996, n = 3, P < 0.05) and SP-LI of the bladder (r = 0.999, n = 3, P < 0.05). These doses of capsaicin and acetylcholine produced maximal responses in bladders from both newborn and adult animals.

Response to exogenous substance P in the isolated urinary bladder from newborn or adult rats Substance P (1 n M - 1 pM) produced a concentration-related contraction in bladder strips from newborn (P0) or adult animals. In newborn animals, low concentrations (3-100 nM) of SP activated a series of high-amplitude phasic contractions mimicking the pattern of spontaneous activity observed at a later developmental stage. Higher concentrations produced a phasic followed by a tonic contraction. Sensitivity to SP was slightly, but not significantly, lower in bladders from newborn as compared to adult ani-

CAPSAICIN

ACh

n=3

30~

r : 0.9979 20. •

n, lZ

o (j

~

n~4 r =0.7141

10-

n.$.

00

3

10

20 0 AGE (days)

3

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Fig. 2. Correlation between contractile response of the rat urinary bladder in vivo to topical capsaicin (left) or acetylcholine (right) in relation to age of the animals. Each value is mean _+S.E.M. of at least 5 experiments.

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NEWBORN

RATS

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Fig. 3. Contractile effect induced by SP on isolated bladder from newborn (P0) or adult rats. Each value is mean + S.E.M. of at least 5 experiments. Contractile responses were expressed as percentage of the maximal response obtained in each preparation.

mals, E C 50 and 95% c.l. (in brackets) being 90 (45-233) and 45 (26-94) nM, respectively (Fig. 3). DISCUSSION Capsaicin induces a tetrodotoxin-resistant contraction of the rat urinary b l a d d e r which is (a) antagonized by an SP antagonist15; (b) p r e v e n t e d by bladder denervation m'17 and (c) directly p r o p o r t i o n a l to b l a d d e r SP-LI v. This indicates its d e p e n d e n c y upon neuropeptide(s) release from sensory nerve endings in the b l a d d e r wall s'H, although a direct release of SP-LI capsaicin could not be d e m o n s t r a t e d in superfusates from the rat b l a d d e r , p r o b a b l y because of the low levels involved It. In this study, we l o o k e d at a possible correlation between d e v e l o p m e n t of SP innervation of the b l a d d e r and functional response to capsaicin. I n d e e d the capsaicin-sensitive nerves of the rat urinary b l a d d e r contain not only SP but also other tachykinins and calcitonin gene-related peptide 19 which might be involved in responses observed. A l t h o u g h a causal relationship b e t w e e n dev e l o p m e n t of SP innervation and functional response to capsaicin might be hypothesized, postnatal changes in b l a d d e r SP-LI should be m o r e cautiously interpreted as a biochemical and i m m u n o h i s t o c h e m i cal m a r k e r for the presence and d e v e l o p m e n t of t h e s e sensory nerves.

The capsaicin-induced contraction of the rat H a d der is almost absent m newborn rat~, and increases ~sith age. This postnatal d e v e l o p m c n t may involve essentially 4 mechanisms: i 1 ) postnatal d e v e l o p m e n t of a capsaicin r e c e p t o r on sensor\ nerves'", i2) postnatal d e v e l o p m e n t of b l a d d e r content of SP. (3) postnatal development of mechanisms inxolved in SP release from sensory nerves followine ~timutation by capsaicin {1-3 can be considered ~,, p r e l u n c t i o n a l factors) and (4l postnatal d e v e l o p m c n t of sensitivity to SP at the post junctional level I n u m b e r of receptors on muscle cclls, d e v e l o p m e n t ,,>t postreceptoriat transduction mechanismsl. lih~ ~ first hypothesis seems unlikely since the specific ncurotoxic effect c~t capsaicin on certain sensory nerve, can be casil~ observed following neonatal capsaicm administration 3'av~. Indeed, the intensity ol lhe neurotoxtc action of capsaJcin decreases rapidly with the increase of the age of the animal ~. Likewise. the 4th hypothesis seems untenable since urinarx bladders from newborn rats (at P0) were only slightly tess sensitive to SP than those from adult rats. Taken together, our findings indicate that increase in n e u r o p e p t i d e content in the peripheral terminals of capsaicin-sensitive fibers is a mare d e t e r m i n a n t for the postnatal d e v e l o p m e n t of the sensory-efferent response to capsaicln m the rat bladder. It should be noted that SP-LI was detectable by R I A in the rat bladder well before the SP-LI fibers could be observed by immunohistochemistrv l h i s suggests that a critical amount of peptide n3ust be present in the fibers to be visualized with the latter technique. Moreover, the parallel postnatal increase of b l a d d e r SP-LI seen by R I A and the functional response to capsatcin suggests that even m the presence of low peptide levels a fraction of total immunoreactivit~ is present m a releasable pool. Studies on the postnatal d e v e l o p m e n t of SP-LI in dorsal root ganglia indicate that peptide levels increase even ~tfter P20. at which IllllC It Call be estimated to be around 8()~ of that observed m 2month-old rats~L Likewise. the densits of fibers containing SP-LI in dorsal horns of the rat spinal cord increased postnatally and, at P S - P I 0 was lower than thai observed in adult rats ~. Likewise. SP-LI nerve fibers in the rat skin, although prcsem at P1. reached a staining pattern similar to the adult at P 8 - P 1 5 (ref. 1). As a whole, present observations fit in a more

189 general scheme which indicates p r o m i n e n t postnatal changes of SP-LI containing primary sensory neurons at both central and peripheral level. The sensory input from capsaicin-sensitive nerves of the rat bladder becomes functional around P 1 0 - P l l (appearance of a vesico-vesical micturition reflex) and devel-

bladder, post junctional sensitivity to SP is well developed at birth and postnatal development of prejunctional factors (peptide content in sensory fibers) seems the main d e t e r m i n a n t of postnatal changes in the 'efferent' response mediated by these sensory nerves.

ops thereafter in parallel to the ability of topically applied capsaicin to stimulate the micturition reflex 13.

ACKNOWLEDGEMENTS

Neurophysiological experiments indicate that in rats C fiber input to dorsal horn does not fully develop un-

We wish to thank Mr. L. Bigiarini for excellent

til the second postnatal week and this was ascribed to

technical assistance. This work was in part supported

late maturation of interneuronal pathways 2. There-

by IMI, Rome (Progetto di Ricerca: Farmaci per il

fore, the fact that the C-fiber-mediated input from rat skin and urinary bladder to the CNS becomes

trattamento a lungo termine della incontinenza urinaria: VES, G r a n t 46287) and CNR, Rome (Proget-

functional at a certain time after birth ( P 1 0 - P l l ) could be explained through maturation of sensory pathways at CNS level. On the other hand, in the rat

to Finalizzato Medicina Preventiva e Riabilitativa, Sottoprogetto Controllo del Dolore Grant 85.0059.56).

REFERENCES

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19() stance P, neurokinin A and calcitonin gene-related peptidc. In R. Hakanson and F. Sundler (Eds.), Tachykinirl Antagonists, Elservier/North Holland, Amsterdam. 1985, pp. 3-14. 20 Szolcs~inyi, J., Capsaiciu-seusitive chcmoceptivc neur~d system with dual sensory-efferent function. In L.A. Chahl, J. Szolcs~nyi and F, Lembeck (Eds./, Antidronlic Vczsodilet-

ration and Neurogenic Inflammation Akademiai Kiadn, Budapest, 1984, pp. 26-52. 21 Yokokawa, K., Sakanaka, M., Shiosak~, S., Tohyama, M., Shiotani, Y. and Sonoda, T. Three dimensional distribution of substance P-like immunoreactivity in the urinary bladder of rat, J. Neural Transrn., 63 (i98.6) 2('Pq-222.