Etiropean Journal of Pharmacology, 203 (1991) 267-274 0 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50 ADONlS 0014299991006~8V

267

EJP 52075

Marcela Bitran, Gonzalo Torres, Alain Fournier ‘, Serge St Pierre ’ and J. Pablo Huidobro-Toro Neurohumoral Regulation Unit, Department of Physiology, Faculty of Biological Sciences, P. Catholic lfnicersity of Chile, P.0. Box 114-D. Santiago,

Chile and ’ ~n~i~ersit~ du Qu&ec, CARS-Santa, 24.5 BouLcard Hymus, Pointe Claire, Q.&bee; Canada H9R IG6

Received 5 June 1991, revised MS received 17 July 1991, accepted 23 July 1991

The potency of ilemopeptide Y (NPY) to inhibit the electricaliy induced contractions of the epididymal half of the vas deferens diminishes markedly with age, being at least 20 times lower in the adult than in the 26day-old rat. Castration sensitizes the epididymal segment to NPY in a testosterone-reversible manner. [Pro”4]NPY was 3 times less pntent than NPY in prepubertal rats and inactive in castrated adults, while NPY-(13-36) had no effect in either group. In the prostatic half, NPY and its analogs were active in rats from all ages studied; the order of potency heing NPY > fPros41NPY > NPf-(f3-35). The sensitivity of the prostatic segment from adult rats to NPY was unchanged by castration or testosterone replacement therapy. The NPY content of the ductus increases during development being higher in the prostatic than in the epididymal half at all ages studied. Castration decreases the peptide content in the two segments and the effect is prevented by testosterone administration. The present investigation demonstrated that the effect of NPY on vas deferens neurotransmission is subject to regulation by sex steroids, which affects differently the response of the two segments of the ductus.

Neuropeptide Y (NPY); Vas deferens; Neurotransmission; Hormonal regulation; Castration/neurotransmission; (Age and castration); (Rat>

Neuropeptide Y (NPY) is a highly conserved pep- tide isolated from porcine brain (Tatemoto, 1982a; Tatemoto et al., 19821. It is a neural member of the pancreatic polypeptide famity, which includes the gut hormone, peptide YY (Tatemoto, 1982b) and paacre- atic polypeptide (PP) (Kimme! et a!., 19751. One aspect of the neurobiolo~ of NPY that has attracted much attention is its frequent, though not exclusive, associa- tion with noradrenaline (NA) both in the CNS (Hokfelt et a!., 1983; Everitt et a!., 1984) and in the periphery (Lundberg et al., 1982; Ekblad et a!., 19841. High levels of NPY-like immunoreactivity (NPY-LI) are found in ganglia and tissues with a dense sympathetic innerva- tion, such as the vas deferens, heart atrium, blood vessels and spleen (Lundberg et a!., 19831. Further- more, NPY and dopamine p-hydroxylase (DPH) im- munoreactive fibers have a parallel occurrence in the heart, spieen, kidney, respiratory and urogenital tracts,

Correspondence IO: J.P. Huidobro-Tare, Nelirohumor~i Regulation Unit, Department of Physi(~l(~6y. Faculty of Biolo~icill Scicnccs, P. Cutholic University of Chile, P.O. Box 114-D. Santiago, Chile.

around blood vessels and within viscera! smooth mus- cle (Sundler et d!., 19861, suggesting that NPY is a major peptide of the sympathetic nervous system. In nerve terminals of the vas deferens, NPY is co-stored with NA and adenosine S’-triphosphate (ATP) (Fried et al., 1985; StjHrne et a!., 1986); NA, ATP and NI’Y may be co-released following high-frequency nerve stimu!a:‘bn (Kasakov et a!., 19881, supporting the con- cept of co-transmission.

It has been known for some time that sex hormones effect profound changes in the organization and func- tion of the nervous system (Sjostrand and Swedin, 1976; Hamill and Guernsey, 1983). Short axon sympa- thetic neurons, such as those projecting From the hy- pogastric ganglion to the vas deferens (Wakade and Kirpekar, 19711, are particularly sensitive to steroid action. Several indicators of adrenergic function, such as the levels of NA and the activities of DPH and tyrosine hydroxylase (TH) in the hypogastric ganglion and vas deferens are influenced by age and by changes in hormonal status (Hami!! et a!., 1984, Lara et a!, 1985; Bustamante et a!., 19891. It has been shown recently that the NPY content in the hypogastric gan- glion decreases in paralie! with the reduction in TH activity after castration (Hamill and Schroeder, 1990).

These results suggest that hormonal influences on these neurons may also extend to NPY, the sympathetic co-transmitter.

1n the neuroeffector junction of the vas deferens, NPY induces a concentration-dependent inhibition of electrically induced contractions (Allen et al., 1982; Huidobro-Toro, 1985; Huidobro-Toro et al., 1985; Donoso et al., 1988), which is mediated through the activation of specific presynaptic NPY receptor(s). The peptide operates a negative feedback mechanism regu- lating the release of NA and ATP (Lundberg and StjGme, 1984; Donoso et al., 1988; Ellis and Burnstock, 1990). Though still hypothetical, these neuronal NPY receptors have been characterized as belonging to the NPY Y, receptor subtype (Wahlestedt et al., 1986). The epididymal and prostatic ends of the vas deferens differ with respect to the mode of response to electri- cal stimulation as well as regarding sensitivity to NPY. Whereas NPY blocks the twitch response of the pro- static segment with a potency of about 20 nM, the epididymal half is virtually not sensitive (Donoso et al., 1988). The reason for this difference is not known. In an attempt to ascertain whether the inhibitory effect of NPY on neurotransmission in the rat vas deferens is subject to the influence of sex steroids, the present investigation was aimed at studying the effect of devel- opment and castration on the modulatory action of NPY, and on its content in the vas deferens. These questions were addressed separately in the prostatic and epididymal segments of the rat ductus so as to obtain further insight into the mechanism(s) underlying the regional differences of this tissue in relation to the functional properties of NPY. The results indicate that the inhibitory action of NPY in the epididymal hut not in the prostatic segment of the vas deferens is pro- foundly affected by age and castration, suggesting the involvement of different mechanisms regulating the sensitivity of tl>e nerve terminals to NPY.

2, Materials and methods

2.1. Animals and drug sources

Male Sprague-Dawley rats of different postnatal ages (26, 37 and 60-65 days old) were obtained from our local animal reproduction facilities at the Catholic University. The animals were housed under standard conditions (i.e. 25 o C and a controlled 12 : 12 light-dark cycle) and had free access to food and water. The rats were killed by decapitation: after blood collection, the abdomen was opened by a midline incision and the vasa deferentia were carefully dissected free from adi- pose and connective tissue and placed in Tyrode solu- tion.

A sample of NPY was kindly donated by Dr. A. Daniels (Burroughs Wellcome Co, USA). NPY and NPY-(13-36) were synthesized by Drs. St-Pierre and Fournier (INRS-Sante, Quebec, Canada) and [Proj4] NPY was generously provided by Dr. T.W. Schwartz (Rigshospitalet, Copenhagen, Denmark).

2.2. Castration

Adult male rats (60-65 days old) were castrated bilaterally under pentobarbital anaesthesia, taking care to preserve the vas deferens and the entire epididymis. The castrated animals were subdivided into two groups: one that received S.C. injections of testosterone enan- tate 0.5 mg/rat daily (treatment started 3-4 days after castration) and a second group that received vehicle (sunflower oil) injections. The rats were killed 3 weeks after castration, and 24 h after the last testosterone or vehicle injection. In a subsequent series of experi- ments, groups of rats were castrated as described above and killed either 1 or 2 weeks after surgery. This particular protocol did not include the testosterone repla::ement therapy groups.

2.3. Vus deferens bioassay

The vasa deferentia were dissected into epididymal and prostatic segments which correspond roughly to the tissue halves (Donoso et al., 1988). These segments of the ductus were mounted in a 30-ml dtiuble-jacketed bath chamber, maintained at 37” C and superfused with Tyrode buffer (pH 7.4, continuously aerated). The composition of the buffer in mmol/l was: 118 NaCI, 5.4 KCI, 2.5 CaCl,, 1.2 KH,P04, 1.2 MgSO,, 23.8 NaHCO,, 11.1 glucose and 20 L-tyrosine. The tissues were adjusted initially with 1.0 g of tension, which was maintained throughout the experiment, as detailed by Donoso et al. (1988). The ductus were transmurally stimulated at supramaximal voltage, with square elec- trical pulses (0.15 Hz, 1 ms), delivered via a Grass S 44 stimulator, conditions known to produce neurogenlc responses (Lundberg and Stj&-ne, 1984; Donoso et al., 1988). Isometric contractions were recorded using force-displacement transducers connected to a Grass recording osciliograph. The tissues were rinsed every 15 min during the first hour of incubation, prior to the addition of drugs.

2.4. Concentration-response studies

Once the tissues had equilibrated and responded with regular muscle twitches to the 0.15 Hz stimula- tion, NPY and. related analogs were added non-cumu- latively to the bath chamber. The peptides were left in contact with the tissues for 6 min, the time required to

269

reach a maximal effect. Each concentration of peptide was tested at least 30 min apart to allow for the twitching to return to the control pre-drug values. The results were plotted as concentration-res~nse curves. Median inhibitor concentrations iIC,) were obtained by interpolation from the respective concentration-re- sponse curves using linear regression analysis. IC& values are expressed as mean values & S.E.M.

2.5. NPY radjoimmunoassay @&I~

2.5.1. NPY extraction Epididymal and prostatic segments were diced and

extracted in lo-20 volumes of 1 M acetic acid for 10 min at 100 “C. After sedimentation of the tissue (5 min, 10000 rpm), a lo-20 ~1 aliquot of the super- natant was diluted to a final volume of 100 ~1 in RIA buffer for NPY determination.

2.5.2. Reagents and procedure Radiolabelled NPY {‘*‘I-NPY) was prepared by iod-

ination of synthetic NPY, using the Chloramine-T method (Greenwood et al., 1963). NPY antiserum was produced in our unit by immunization of rabbits with synthetic NPY coupled to albumin. On day 1 of RIA, a 100 p! aliquot of NPY standard solutions (10-400 pg) or biological samples was incubated with an equivalent volume of NY antiserum (dilution 1: 30000) at 4 ’ C for 24 h. Both the standard solutions and the antiserum were prepared in a buffer of the following composition (mM); 19 NaH,PO,, 81 ,Na,HPO,, 50 NaCI, 0.1% bovine serum albumin, 0.1% Triton X-100 and 0.01% NaN,. ‘=I-NPY (10000 cpm/tube) was added to the reaction mixture in 100 ~1 of RIA buffer containing 0.1% normal rabbit serum and the incubation was continued for the next 24 h at 4’ C. After this period was completed, the second antibody (goat-antirabbit IgG, 3 f&I undiluted antiserum/tub~} was added in 500 ~1 of the following buffer: 18 mM NaH,PG,, 81 mM Na,HPO,, 50 mM NaCl, 0.1% Triton X-100, 0.01% NaN,, 8% polyethyleneglycol. The precipitates were allowed to form for 2 h at 4°C. After sedimentatton of the pellets in a Sorvall refrigerated centrifuge (20 min, 7000 rpm), the supernatants were discarded and the pellets were counted in an LK5 gamma counter. The semitivity limit of this assay was between 10 and 30 pg (9C% displacement of bound ‘2sIdNPY), depending on the batch of unlabeled NPY used for calibration. This implied some degree of variabili~ in the levels of i~munorea~tive NPY detected in biological samples assayed at different times. Parallel assays run with our NPY antiserum and one commercially available from Peninsula Lab. lnc, detected virtually identical levels of NPY-LI in the two halves of the rat vds deferens (data not shown).

2.6. Radioimmunoassay for testosterone

RIA for testosterone was carried out according to the WHO Programme for the Provision of Matched Assay Reagents, as previously reported (Forcelledo and Croxatto, 1988). Serum testosterone was assayed using a monoclonal antiserum raised in mice against testosterone conjugated with bovine serum albumin. Serum (SO ~1) was extracted with 1.5 ml diethylether with a tracer amount of ~3Hltestosterone to monitor procedural losses. Recovery was 93%. The antiserum was used at a final dilution of 1:210000 and the sensitivity was 5.9 pmol/tube.

2.7. ~tat~sticai analysis

Differences between group means were evaluated by non-paired Student’s t-test; Dunnett’s tables were used for multiple comparisons with a single control. In all cases, significance was set at a P value less than 0.05. The number of separate preparations used in each experimental pratoco1 is indicated as ‘n’ in the legend to the figures.

3. RemIts

3.1. Zn~~~~to~ a&n of NPY and st~cruraf~y related analogs

In adult rats, nanomolar concentrations of NPY significantly decreased the two components of the neu- rogenic contraptions in the prostatic segment, but not in the epididymal half (fig. 1). The effect of NPY was reversible upon washout of the peptide from the incu- bation medium.

PROSTATIC

Fig. 1. Effect of NPY on vas deferens nzurotransmission in the adult rat. Polygraph recordings show the muscle contractions of the pro- static and epididymal segments of the ductus following a single pulse of transmural electrical stimulation. Note the compound nature of the responses and the differences in magnitude between the first and second components of the contractions in the epididymal and pro- static segments. The arrows indicate the aplication of 40 nM NPY; NPY affects neurotransmission mainly in the prostatic segment,

reducing both elements of the contractile response.

The potency of NPY in the epididymal segment diminishes markedly with age. In 26-day-old rats, the ~~ect~~~a~~~ driven twitches were inhibited in a concen- tratio~-dependent manner with an lCsFtl of 41.8 It 6.7 nM fn = 4). The peptide was significantly less potent in 37-day-old rats UC,,: 251+ 49 nM, n = 6). In 60-day old rats, the epididymal segment was markedly resis- tant; the extrapolated fC,, was estimated to be greater than 1000 nM (n = 12). Concentration-respo~~se curves are presented in fig. 2. The magnitude of the contrac- tile response to the 0.15 Hz stimuli increased signifi- cantly with age; it was 0.28 f 0.03 g In = 36) in 26-day- old rats; 0.41 + 0.06 g (n = 22) in 37-day-old rats and 0.81 + 0.05 g (a = 53) in 60-day-old rats.

The putative NPY Y, specific agonist, ~Pro3~]NPY~ was 3-fotd less potent than NPY to inhibit the muscu- lar contractions of the epididymal segment of 26day- old rats. In contrast, the NPY fragment NPY-(13-361, a purported NPY Yz selective agonist, was almost inactive (fig. 3, left pane!).

On the other hand, the potency of NPY in the prostatic segment wab less affected by development, showing a 3-fold decrease in the age range studied. The IC,, in 26-day-old rats was 4.7 + 0.3 nM (n = 61, as compared to 9.2 & 0.8 nM in 37-day-old rats (n = 123, and 15.3 + 1.6 nM in adult rats (n = 12). The concen- trat~on-res~~s~ curves were paraBel (fig, 2, right panel). The tension generated by the contractile rc- sponse to the 0.15 Hz stimulation increased signifi- cantly with age. It was 0.62 f 0.05 g (n = 26) in 26-day- oId rats as compared to 1.01 rfi 0.09 g (n = 17) in 37- day-old and 1.24 + 0.05 g (n = 72) in 6Q-day-old rats. Note that the tension generated in the prostatic seg- ment was significantly greater than that of the epididy- ma1 segment at all postnatal ages studied; thus, as animals grow older, the difference shows a tendency to decrease. It is likely that the age-dependent increase in

EPMlOYtlAL PROSTATIC

100 - - 100

2 eo- - 80 2 B E 60-

z3

g - 60 iifi

P - 40- 3

-40 t -c 2 2 zo- - 20 E

oc

7--F-z 9876

-0

-O- NPY

oLt , , ( ,

98765 98765 -Log lP~PltDE~.M.

Fig. 2. Effect of NPY on rat vas deferens neurotransmissiort at different postnatal ages. Concentration-response curves were made with the epididymal and prostatic halves of the vasa dcferentia of 26-, 3% and 60-day.old rats. Symbols represent the mean values and

Fig. 4. Effect of NPY and structurally related analogs on the neuro- genie contractions of the prostatic segment of the rat vas deferens. Concentration-response curves for NPY, [Pro34]NPY and NPY-(13- 36) were made with tissues obtained from 26. and 60-day-old rats. Symbols indicate the mean values and bars denote the S.E.M.

bars denote the S.E.M. (n = 4-121. In = 7-12).

26 DAY-5 OLD 60 DAYS OLD

98755 I 9 1 1 8

98 7 65,

-Log [ PEPTIDE 1.M

Fig. 3. Effect of NPY and structurally related analogs on the neuro- grnic contractions of the epididymal segment of the rat vas deferens. Concentration-response curves for NPY, [P~o~~]NPY and NPYf 1% 361 were made with tissues from 24-day&d rats and 60-day-old rats, 3 weeks after castration. Symbols indicate the mean values and bars

denote the S.E.M. (n = 7-12).

muscular activity is related at least in part to the gain in tissue weight (table 2). On the other hand, the changes in NPY potency are not related to the magni- tude of the twitch tension generated by the tissues at the different ages.

The relative rank order of potency of the NPY analogs in the 26- and 6%day-old rats was NPY > [Pro34]NPY > NPY-(13-36) (fig. 41. The vasa deferentia from 26-day-aid rats were 4-5 times more sensitive to the action of these peptides than those of the adult population (fig. 4).

3 1.2. Effect of castration Castration of adult rats resulted in a marked sensiti-

zation of the normally resistant epididymai segment to the inhibitory action of NPY. As illustrated in fig. 5 (left panel), NPY produced a concentration-dependent inhibition of the muscle twitching of the epididymal

PROSTATIC

60 DAY5 OLD

- 100

100 r

s ao- E i 60- % z r 40- u c

2 20-

EPlOlDYMAL PROSTATIC

- 100

- 00 is s

- 60 $

I - - 40 B t

- 20 2

.J 0 .

9 0 7 6 9 0 1 6

-Log[NPYI,M.

Fig. 5. Effect of castration and testosterone replacement therapy on the potency of NPY to inhibit vas deferens neurotransmission. Con- centration-response curves for NPY were made with the epididymal and prostatic segments of the vasa deferentia obtained from control adult rats (80-85 days old), and 3 week-castrated rats of the same age, with or without testosterone replacement therapy. Symbols

indicate the mean values and bars denote the S.E.M. (n = 10-13).

segment in 3-weeks castrated rats with an IC,, of 33.7 f 4.1 nM, (n = 13). The NPY concentration-re- sponse curve in the epididymal segment of castrated rats was parallel to that for NPY in the prostatic segment of castrated or non-castrated rats (fig. 5). Testosterone replacement therapy reduced the sensitiv- ity of the epididymal segment to NPY, displacing the concentration-response curve towards that of the con- trol group (fig. 5). Castration affected the magnitude of the contractile response to the 0.15 Hz stimuli. The tension generated was 0.61 f 0.05 g (n = 37) in cas- trated rats; 1.03 + 0.16 g (n = 9) in castrated rats given testosterone and 0.81 f 0.05 g (n = 72) in control rats.

In castrated rats, the complete and original amino acid sequence was required to evidence peptide in- hibitory activity. [Pro34]NPY and NPY-(13-36) were inactive (fig. 3, right panel). The sensitization of the epididymal segment to the action of NPY was evident 2 weeks after castration and reached its maximum 3

TABLE 2

271

TABLE 1

Effect of castration on the potency of NPY on rat vas defercns neurotransmission.

NPY IC,,, x f S.E.M. (nM) Serum

Prostatic Epididymal iestosterone

X + S.E.M. (nM)

Control a 15.3&1.6(12) > 1000 (13) 7.60?1.70(11) 1 week 20.5k2.0 (7) > 1000 (4) 0.68+0.14 (7) b 2 week 16.8 * 3.1 (7) 71.0+26 (4) ND 3 week 26.lk4.4 (10) 33.7* 4.1 (13) 0.51+0.06 (8) b

a Unoperated rats aged 80-85 days. b P < 0.01 as compared to the control value (Student’s t-test). ND: not determined.

weeks after surgery (table 1). The epididymal segments were markedly resistant to NPY 1 week after castra- tion, when serum testosterone levels were maximally depressed and did not differ significantly from those found 3 weeks after castration (table 1).

The response of the prostatic segment to NPY was less affected by castration. NPY inhibited the electri- cally induced contraction of the prostatic segment in all experimental groups. The IC,, values for NPY were 15.3 f 1.6 (n = 12) in control rats; 26.1 + 4.4 (n = 10) in castrated rats and 18.2 f 2.1 nM (n = 13) in castrated rats that received testosterone replacement therapy (fig. 5, right panel). Moreover, the potency of NPY in this segment was not significantly affected by the time of castration. These results are summarized in table 1.

3.2. NPY content and serum testosterone 1eueIs

3.2.1. Effect of age The NPY content and wet tissue weight of the

epididymal and prostatic segments of the ductus in- creased significantly and proportionally with age. Both tissue weight and NPY content were significantly higher in the prostatic than in the epididymal segment at all ages studied. In contrast, the NPY concentration (ex-

Effect of age on tissue weight and on the content and concentration of NPY in the rat vas deferens. Values are given as %* S.E.M. Eight separate preparations were used from each experimental group (n = 8), except for the determination of testosterone in 60-day-old rats, done with n = 11.

Davs of aae

26 37 60

- Wet weight (mg) Prostatic 9.0* 0.3 b.c! 20.7+ 0 6 aqJ 42.9+ 2.8 ’ Epididymal 5.0* 0.5 d 13.9f 0:8 d 30.5+ 1.5

NPY content (pmol) Prostatic 8.2* 0.4 b*d 24.5 f 0 9 a.d 49.3+ 3.2 ’

Epididymal 4.3&- 0.3 d ll.O* 0:8 d 20.6* 0.9

NPY (pmol/g) Prostatic 907 *49 cs 1185 f45” 1149 rt75”

Epididymal 727 k51 792 *61 675 *30

Serum testosterone (nmol/l) 0.8f 0.1 d 1.4* 0.3’ 7.6+ I.7

” P < 0.001, b P < 0.01, c P < 0.05 relative to the corresponding value for the epididymal segments. d P < 0.001, ’ P < 0.01, ’ P < 0.05 as compared to the values for the 60-day-old group (Student’s t-test).

TXBEE 3

Etfect of castration on the NPY content and wet tissue weight of rat vas deferens. Values are given as xi+S.E.M. Eight separate preparations wete studied from each experimental group (n = 8).

Control Castrated Castrated + testosterone

Wet we&t (mg) Pntstatic 55.4 *3.1 a 16.2 + 0.Q ‘,= 42 1 f 1 3 il.d . . Epididymal 23.5 f 2.3 10.5 i 1.5 c 14.6rtO.7 ’

NPY content tpmol) Prostatic IS.3 * 1.4 il 8.1 5 0.6 WJ 19.1T1.7C

Epididymal 5.2 io.4 3.9 kO.4 4.9kO.6

Serum testosterone (ttmol/ 1 f 7.60 ?7 1.71) 0.51 + 0.06 d 68.5+11.0d --- _

.l P < 0.001, ’ P < 0.01 as compared to the corresponding value for the epididymai segments. ’ P < 0.001, d P < 0.01, E P <: 0.05 as compared to the values in the control group (Student’s t-test).

pressed as pmol/g wet tissue) did not vary significantly with age, but was consistently larger in the prostatic thm in the epididymal segment at all postnatal ages studied (table 2). Circulating testosterone levels in the adults were about IO-fold higher than in the 26-day-old rats (table 2).

3.22. Effect of cwra tion Endogenous NPY content and wet tissue weight of

the epididymal and prostatic segments were signifi- cantly decreased by castration. These changes were more marked in the prostatic than in the epididym~l ends. Castration resulted in a 70% reduction of wet weight and a 55% decrease of the endogenous NPY content In the prostatic segment. On the other hand, the wet weight of the epididymal segment was reduced by 47% by castration, while the NPY content de- creased by 25%, relative to control values (table 3). Castration reduced serum testosterone levels 93%. Testosterone administration to castrated rats increased serum hormone levels to above control values. How- eve:, this treatment resulted in a partial recovery of the weight of the two segments while preventing the reduc- tion of the NPY content in the epididyma1 and pro- static segments of the ductus (table 3).

4. Discussion

There is ample evidence favonring the interpreta- tion that the NPY-induced inhibition. of neurotransmis- sion in the vas deferens Is due to a presynaptic mecha- nism (Lundberg and StjIme, 1984; Donoso et al., 1988; Ellis and Burnstock, 1990). Consistent with this notion, the present results demonstrated that, in adult rats, NPY reduces the magnitude of both the purinergic and adrenergic components of the electrically induced mus- cle twitching only in the prostatic segment, thereby establishing that the target of NPY are the nerve terminals located in the prostatic but not in the epi- didymal segment of the ductus. The results clearly

demonstrated that the presynapti~ effect of NPY in the cpididymal segment is subject to endocrine regulation. Support for this proposal is based on the finding that the sensitivity of the epididymal segment to NPY, unlike that of the prostatic half, decreases sharply with age. Furthermore, castration profoLnd& affects the _.tino&kty of the epididymal segment to NPY, while it esseni ‘*JI!y does not modify the peptide potency in the prostatic segment.

The most interesting finding from this investigation is that the low sensitivity of the epididymal segment to NPY is a characteristic of sexually mature rats. NPY is a powerful inhibitor of n~urotransmission in prepuber- tal rats, its potency in the epididymal segment is re- duced with age, concomitantly lrlith the increase in circulating testosterone levels. These results Ied us to the hypothesis that the increase in circulating testos- terone associated to sexual maturation might exert a negative influence on the cellular mechanisms mediat- ing the action of NPY on the sympathetic nerve termi- nals in the epididymal segment. Consistent with this notion, castration of adult rats sensitized the epididy- ma1 segment to the action of NPY in a testosterone-re- versible manner. The mechanism by which testos- terone, putatively, preyrents the action of NPY is far from clear. The hormone-mediated effects can take place at the receptor level, affecting any of the events that mediate between the interaction of NPY with its binding sites and the activation of intracellular trans- duction mechanisms. ~ternatively, the hormone may modify post-receptor events, altering cellular signailing, ultimately leading to decreased neurotransmission. Ir- respective of the mechanism(s) involved, it is clear that the reduction in testosterone, whiIe probabIy neces- sary, is not sufficient to account for the regulatory changes, as evidenced by the fact that one week after castration the epididymai segment is resistant to NPY. It is of interest to mention that the circulating level of testosterone one week after castration is about the same as that found in the 26-day-old rats; however, the bioIogi~a1 response to NPY in these castrated rats is

273

not yet sensitized. Other cellular mediators or hor- mone-related adaptive changes occurring pre- or postjunctionally must therefore be operating.

Unlike the situation in the epididymal half, the nerve terminals present in the prostatic ,;gment are not a target of testosterone regulation, in so far as the modulatory effect of NPY on these neurons occurs independently of the changes in testosterone levels. This study is the first to indicate that the sympathetic nerve terminals supplying the epididymal and the pro- static segments of the rat vas deferens - besides the uneven sensitivity to NPY - respond differently to steroids. It is highly unlikely that the resistance of the epididymal segment to NPY in the adult rat might result from a pharmacokinetic variable related to a decreased access of NPY to its site of action. p-En- dorphin, 2 peptide of a molecular size similar to that of NPY, which also acts at a presynaptic site, has an almost identical potency to inhibit neurotransmission in the epididymal and prostatic segments (Torres, 1990). It is still unknown whether the ductus has an asymetric distribution of NPY receptor(s). Likewise, it is not clear whether the mechanism(s) of intracellular NPY signalling in the epididymal and prostatic segment dif- fer. The possibility that the inhibitory activity of NPY is related to the inhibition of adenylyl cyclase is cur- rzntly being explored thoroughly (Torres; 1990).

An interesting issue relates to the classification of the NPY receptors in the rat vas deferens. Assuming that the peptides with alleged selectivity for the NPY Y, and NPY YZ receptors subtypes effectively discrimi- nate between these sites of action (Fuhlendorff et al., 1990; Sheikh and Williams, 19901, our results might be interpreted to indicate that the inhibitory action of NPY in the prostatic segment is mediated by both NPY Y, and NPY YZ receptors. The participation of NPY Y2 receptors in the epididymal. segment seems unlikely, while the activity of [Pro34]NPY favours the involve- ment of NPY Y, receptors. At present we cannot rule out the possibility that the inhibitory activity of [Pro34]NPY may result from the activation of PP re- ceptors, the presence of which has been recently re- ported for the rat vas deferens (Jorgensen et al., 1990). Interestingly, in castrated rats, the epididymal segment responded to NPY but neither to [Pro”]NPY nor to NPY-(13-36). This might indicate that the NPY recep- tors expressed in the epididymal segment after castra- tion might have an altered conformation or belong to stih undescribed subclass. If a clearer picture of the function and distribution of NI-Y receptor subtypes is to emerge more specific agonists and the long awaited NPY receptor antagonis; would need to be synthe- sized.

Our results confirm and extend the results of previ- ous studies indicating that the effects of castration, classically known to affect noradrenergic ncurotrans-

mission (Sjiistrand and Swedin, 1976; Lara et al., 1985; Bustamante et al., 1989), also apply to the co-trans- mitter, NPY. Castration not only reduces the content of NPY in the cell perikarya (Hamill and Schroeder, 19901, but, as shown here, also decreases the peptide content in the nerve terminals present in the vas defer- ens. Whether the latter change is secondary to a dimin- ished synthesis of NPY in the cell body or reflects independent events related to an increased rate of NPY degradation is not known. Nevertheless, it seems clear that testosterone exerts a regulatory influence on several aspects of sympathetic neurotransmission that encompass the neurochemistry of NA and NPY. In this respect, and considering that sympathetic transmission is a finely regulated process that involves the concerted action of several co-transmitters, it would not be un- likely that the content of ATP - the third participant in this neuroeffector junction - and its effects would also be regulated by the hormonal status.

In conclusion, the present results demonstrate that the nerve terminals in the epididymal segment of the rat vas deferens are a target of sex steroids, which decrease the potency of NPY to inhibit neurotransmis- sion. In contrast, the sensitivity to NPY in the prostatic segment is essentially not altered by these hormones. These results establish a difference in regulatory prop- erties between the nerve terminals of the epididymal and prostatic segments which profoundly affects the modulatory action of NPY on neurotransmission in the rat vas deferens. Since these steroids also modify the synthesis of NPY in central neurons (Sahu et al., 19891, the present results allow the working hypothesis that sex hormones modulate centra! and/or peripheral sympathetic neurons modifying the synthesis of co- transmitter substances, as well as the neuronal sensitiv- ity to neurotrasmitters and regulatory peptides. Testing of this hypothesis as well as the elucidation of the cellular actions of NPY in these neurons needs further investigation.

Acknowledgements

To Dr. J. Szecowka for the NPY antiserum, to Drs. A. Daniels for samples of NPY and advice cn the RIA and T. Schwartz for a sample of [Pro”“]NPY. To Prof. J. Belmar for critical discussion during the performance of the investigation. To R. Miranda for surgical help with castration. Funded in part with CONICYT Grants p767/90, and 0748/91, the Medical Research Council of Canada and the Quebec Heart Foundation.

References

Allen, J.. T. Adrian, K. Tatemoto, J. Polak. J. Hughes and S. Bloom, 19X2. Two novel related peptides, neuropeptide Y (NPYl and peptide YY (PYY) inhibit the contraction of the electrically stimulated mouse vas deferens, Neuropeptides 3, 71.

Bustam;bnte. D.. H. Lara and J. Belmnr. 19X9. Changes of nore-

pinephrine ?evels. tyrosine bydroxyluse and dopamine-be- t,,bydr&ase activitie.. after castration and lrsloslerone lreal-

mrnt in vas deferens of adult ruts. Biol. Rrprod. 40. 54 I. h\noso. V.. M. Silva. S. St-Pierre and J.P. Huidobro-Tom. 19x8.

&uropepti& Y (NPY). an endogenous presynaplic modulator of

adrenergic neurotransmission in the rat vas deferens: Structural and functional studies. Peptides 9. 545.

Ekblad. E.. L. Edvinsson. C. Wahlestedt. R. Uddman. R. Hakilson

and F. Sundler, 1984. Neuropeptide Y co-exists and cooperates with noradrenaline in perivascular nerve fibers. Reg. Pepl. 8, 225.

Ellis. J. and G. Burnstock. 1990. Neuropeptide Y neuromodulation

of sympathetic co-transmission m the guinea-pig vas deferens. Br.

J. Pharmacol. 100. 157.

Everitt. B.. T. tiiikfelt. L. Terenius. K. Tatemoto. V. Mutt and M. Goldstein. 1984. Differential co-existence of ceuropeptide Y

like-immunoreacrivity with catecholamines in the central nervous

system of the rat. Neuroscience 1 I. 443. Forcelledo. M.L. and H.B. Croxatto. 198X. Effects of -I-hydroxy-

androstenodione and exogenous testosterone on blood concenlra-

lions of oestradiol and oviducal embryo transport. J. Endocrinol. 11s. 93.

Fried. G.. L. Trrenius. T. Hiikfelt and M. Goldstein. 19X5. Evidenci for differential localization of noradrenaline and nruroprptide Y in neuronal storage vesicles isolated from rat vas defercns, J.

Nrurosci. 5. 450. Fuhlrndorff. J.. U. Gether. L. Aakerlund. N. Langeland-Johansen,

H. Thogersen, S. Mrlberg. U. Olsen, 0. Thastrup and T. Schwartz, IYYO, [Leu”‘,Pro”]Nruropeptide Y: A specific Y I receptor ago- niqt. Proc. Natl. Acad. Sci. 87. 182.

Greenwood. F.C.. W.M. Hunter and J.S. Glower. 1963. The prepara- tion of ‘?‘I labelled human growth hormone of high specific activity, Biochem. J. 89. 114.

H:l.& R.. C. Earley and L.A. Guernsey. lY84. Hormonal regulation of adult sympathetic neurons: the effects of castration on tyrosine hydroxylase activity, Brain Res. 299. 331.

Hamill, R. and L.A. Guernsey, 1983. Hormonal regulation of sympa- thetic neuron deveiopment: the effects of neonatal castration, Dev. Brain Res. II, 303.

Hamill. R.W. and B. Schroeder, 1990. Hcrmonal regulation of adult sympathetic neurons: The effects of castration on neutopeptide Y. norepinephrine. and tyrc~me hydrci,ylase activity. J. Neuro- biol. 21, 731.

Hokf.“. T.. J.M. Lundberg, H. Lagercrantz, K. Tatemoto, V. Mutt, J. Lindberg. L. Terenius. B.J. Everitt, K. Fuxe. L. Agnati and M. Goldstein, 1983. Gcurrence of neuropeptide Y (NPY)-like im- munoreaclivity in catecholamine neurons in the human medulla oblongata, Neurosci. Lett. 36, 217.

Huidobro-Tore, J.P.. 1985, Reserpine-induced potentiation of the inhibitory action of neuropeptide Y (NPY) on the rat vas defer- ens, Neurosci. Lett. 59. 247.

Huidobro-Toro. J.P.. G.C. Rohde and K. Tatemoto, l9b5, Neuropep- tide Y (NPY): an endogenous modulator of the nl>n-adrenergic transmission of the rat vas deferens?, Eur. J. Pharmacol. 109,317.

Jorgensen. J., J. Fuhleqdorff and T. Schwartz, 1990, Structure-func-

lion studies on neuropeptide Y and pancreatic po,ypcptide-evi- dence for two PP-fold receptors in vas deferens, Eur. J. Pharma- col. 186, 10.5.

Kasakov, L.. J. Ellis, K. Kirkpdtrick, P. Milner and G. Burnstock, 1988. Direct evidence for concomitant release of noradrenaline,

adenosine S’-triphosphale and neuropeptide Y from sympathetic nerves supplying the guinea-pig vas deferens, J. Auton. Nerv. Syst. 22. 75.

Kimmel. J.R., L.J. Hayden and H.G. Pollock, 1975, Isolation and characterization of a new pancreatic polypeptide hormone, J. Biol. Chem. 250. 9369.

Lard. H., X. Galleguillos, J. Arrau and J. Belmar, 1985, Effect of castration and testosterone on norepinephrine storage and on the release of (‘Hjnorepinephrine from rat vas deferens, Neu- rochem. Int. 7, 667.

Lundberg, J.M. and L. StjPrne, 1984, Neuropeptide Y depresses the secretion of ‘H-noradrenaline and the contractile response evoked by field stimulation, in rat vas deferens, Acta Physiol. Stand. 120, 477.

Lundberg, J.M., L. Terenius, T. Hakfelt and M. Goldstein, 1983, High levels of neuropeptide Y in peripheral noradrenergic neu- rons in various mammals including man, Neurosci. Lett. 42, 167.

Lundberg, J.M., L. Terenius, T. Hijkfelt, C.R. Martling, K. Tate- moto. V. Mutt. J.M. Polak, S. Bloom and M. Goldstein, 1982, Neuropeptide Y-like immunoreactivity in peripheral noradrener- gic neurones and effects of NPY on sympathetic function, Acta Physiol. Stand. 116, 474.

Sahu, A., S.P. Kalra. W.R. Crowlcy and P.S. K?lra, 1984, Testos- terone raises neuropeptide Y concentrations in selected hypotha- lamic sites ar:d in vitro release from the medial basal hypothala- mus of castrated male rats, Endocrinology 124, 410.

Fheikh, P. and J.A. Williams, 1990, Slructura! characterization of Y 1 and Y2 receptors for neuropeptide Y and peptide YY by affinity cross-linking, J. Biol. Chem. 265, 8304.

Sjiistrand, N. and G. Swedin. 1976, Influence of age, growth, caslra- lion and testosterone treatment on the noradrenaline levels of the ductus deferens and Ihe auxiliary male reproductive glands of the rat, Acta Physiol. Stand. 98, 338.

Stjiirne, L., J. Lundberg and P. Astrand, 1986, Necropeptide Y-a cotransmitter with noradrenaline and adenosine 5’-triphosphate in the sympathetic nerves of the mouse vas deferens? A biochem- ical, physiological and electropharmacological study, Neuro- science 18, 151.

Sundler, F.. R. Hakinson. E. Ekblad, R. Uddman and C. Wahlest- edt, 1986, Neuropeptide Y in the peripheral adrenergic and enteric nervous systems, Int. Rev. Cytol. 103, 243.

Talemoto, K., l982a, Neuropeptide Y: complete amino acid se- quence of the brain peptide, Proc. Natl. Acad. Sci. U.S.A. 79, 5485.

Tatemoto, K., 1982b, Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion, Proc. Natl. Acad. Sci. U.S.A. 79, 2594.

Tatemoto, K., M. Carlquist and V. Mutt, 1982, Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature 296, 659.

Torres, G., 1990, Pharmacodynamic aspects of neuropeptide Y (NPY) in the rat vas deferens neurotransmission, Arch. Biol. Med. Exp. 23, R220.

Wakade, A.R. and S.M. Kirpekar, 1971, Chemical and hystochemical studies of the sympathetic innervation of the vas deferens and seminal c-sicle of the guinea pig, J. Pharmacol. Exp. Ther. 178,

432.

Wahlestedt. C., N. Yanaihara and R. Haklnson, 1986. Evidence for

different pre and post-junctional receptors for neuropeptide Y

and related peptides, Reg. Pept. 13, 307.