E L S E V I E R Neuroscience Letters 169 (1994) 59 62

N[UROSCI[NC[ LETTERS

Adenosine 5'-triphosphate (ATP), the neurotransmitter in the prostatic portion of the longitudinal muscle layer of the rat vas deferens

M. Ver6nica Donoso, Felipe Bates, Jaime Montiel, J. Pablo Huidobro-Toro*

Unidad de Regulaci6n Neurohumoral, Departamento de Fisiologia, Facultad de Ciencias Bioldgicas, Pont(h'cia Univer~'idad ('ut~Jlica de ('bile, Casilla 114-D, Santiago l, Chile

Received 24 September 1993; Revised version received 3 January 1994: Accepted 3 January 1994

Abstract Suramin (l 100/aM) and ~,fl-methylene adenosine 5'-triphosphate (AMPCPP, 39/aM), antagonized the motor activity induced

by exogenous adenosine 5'-triphosphate (ATP) but not exogenous noradrenaline (NA) in the longitudinal musculature of prostatic (P) and epididymal (E) segments of the rat vas deferens. Likewise, application of these drugs reduced the last component of the nerve-stimulated contraction in response to a single transmural electrical pulse in E and P. Suramin also blocked in a concentration- dependent fashion, the contractile responses to trains of 1.5, 5, 15 or 30 Hz transmural electrical pulses in P, while it did not affect those in E. AMPCPP obliterated responses to trains of 1.5, 5, and 15 Hz in R while reducing these responses in E to a significantly lesser extent. Present results strongly support that ATP is the motor transmitter in R while in E, ATP and NA are likely the co-transmitters responsible for the motor tone.

Key words. Suramin: AMPCPP: Purinoceptor desensitization: P2x-purinoceptor; Purinergic neurotransmission: Rat vas deferens

The fact that more than one synaptically active sub- stance is synthesized, stored and released from nerve terminals originated the concept of co-transmission. In sympathetic neurons, ATP may act as a co-transmitter together with NA [3,5,14]. The best studied systems where ATP may play a role in neurotransmission, acting either as transmitter or as a co-transmitter are the blad- der [4,13], perivascular nerves [8,20], and the vas deferens [9,19,23]. Data supporting the role of ATP in the central nervous system are beginning to emerge [7].

The transmural electrical stimulation of the nerve fi- bers of the rat vas deferens gives rise to compounded contractions of its longitudinal musculature. A part of the response is attributed to the neurotransmitter action of NA, while the non-adrenergic component is suspect- edly due to ATP [1,9,10,17]. The adrenergic component of neurotransmission is restrained essentially to the epi- didymal (E) portion; whereas the prostatic segment (P)

*Corresponding author.

0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 0 2 3 - 4

is apparently of purinergic nature [2,21]. The role of ATP as a neurotransmitter has been characterized pharmacol- ogically using AMPCPP to desensitize purinoceptors, by a mechanism which is as yet unclear [13,19,21]. The pres- ent experiments were aimed at comparing the effect of suramin, as a competitive ATP antagonist [6,11,18,22], with purinoceptor desensitization in P and E neurotrans- mission.

Adult Sprague Dawley rats (250 300 g) were sacri- ficed by cervical dislocation. The abdomen was opened, the vas deferens was carefully dissected free from adipose and connective tissue and bissected into P and E seg- ments which were placed in Krebs Ringer solution gassed with 95% 0 4 5 % CO~ at 37 °C. Tissues were given 0.8 1 g of basal tension. Isometric contractions from the longitudinal muscle layer of P and E segments were re- corded with a Grass force displacement transducer con- nected to a Grass oscillograph. Paired P and E portions were stimulated either by application of non-cumulative concentrations of exogenous ATP and NA, or transmu- ral field stimulation (70 V, 1 ms duration) delivered fol-

60 '~1. K Donosv ~t a/./,V(,urosctem'c Letters 169 (1994) 5962

PROSTATIC EPIDIDYHAL

100JJM SURAMIN 100.1JM SURAMtN

[ 03 M...z0s,.

Fig. 1. Single pulse nerve-evoked contractions in paired prostatic (P) and epididymal (E) segments of rat vas deferens. In separate and inde- pendent protocols, recordings of control responses to transmural nerve stimulation in P and E segments were obtained prior to and following 15 min of incubation with either 100/JM suramin or 0.3/tM prazosin. Purinoceptor desensitization was achieved after 3 consecutive applica- tions of 13/,tM 0~,fl-methylene ATP (AMPCPP) spaced each 3 min apart. Similar results were obtained in at least 6 separate preparations. The magnitude of the motor responses, exhibited more variability in P than in E. Calibration scales valid for all recordings refer to I g and 1 s.

lowing either a single pulse, or 30 s trains o f 1.5, 5, 15 and 30 Hz. Nerve-evoked contract ion experiments were per formed in the absence or in the presence o f 10, 30 or 100 g M suramin, 39 g M A M P C P P , or 0.3 ~tM prazosin, a selective ~ l - ad renocep to r antagonist . P2x-purinocep- tor desensitization was as described by Rohde et al. [21 ].

The contract ions elicited in P and E following a single t ransmural electrical pulse are compounded . The magni- tude o f each c o m p o n e n t is characteristically different in P and E, but the delay and dura t ion o f each element is a lmost identical. In P, the first componen t peaked 240 to 260 ms after stimuli and developed 1.4 + 0.2 g o f tension (n = 18); the second, developed 0.7 + 0.1 g (n = 18), and was observed at 640 to 660 ms, as a small shoulder in the decaying phase o f the first. In E, the first contracti le componen t generated 0.4 + 0.1 g (n = 18) and peaked at about 250 ms. The second, peaked at 650 to 660 ms and generated 0.8 + 0.2 g (n = 18). Fig. 1 shows separate and representative sets o f 3 paired recordings; similar results were obtained in all cases. These nerve-evoked contrac- t ions were abolished by 100 nM tetrodotoxin. To assess which o f the two components is due to pur inoceptor activity, we characterized the pha rmacodynamics o f suramin as an ATP antagonist and next ascertained the effect o f suramin, A M P C P P and prazosin in P and E neurotransmission.

In P, 10 and 100 g M suramin displaced to the right, in a parallel fashion, the exogenous ATP concentration-- response curves, 10- and 22-fold (F~.53 = 17.7, P < 0.005; FL49 = 13, P < 0.005, respectively). In E, 10 and 100/,tM

suramin, shifted the exogenous ATP concent ra t ion-re- sponse curves to the right 3.6- and 5-fold, respectively (FL53 = 7.4, P < 0 . 0 2 5 ; FI.49 = 8.8, P < 0.005). The suramin- induced pur inoceptor blockade was reversible. 100 ~tM suramin did not antagonize the exogenous N A contract ions in P nor in E (FL2 ~ = 1.3, P > 0 . 0 5 , Ft2~ = 0.03, P > 0.05, respectively). Following P2x- pur inoceptor desensitization, exogenous ATP moto r re- sponses were abolished flattening the ATP concentra- t ion-response curve.

Applicat ion o f 100 g M suramin obliterated the moto r response elicited by a single t ransmural nerve electrical pulse in P; the effect being concentra t ion dependent. In E, while the first mo to r componen t dissapeared, the sec- ond was essentially maintained. Likewise, following A M P C P E single pulse nerve-evoked mo to r contract ions were abolished in P but not in E. While the first mo to r componen t in E was impaired, the magni tude o f the second was preserved. Incuba t ion with 0.3 ~tM prazosin caused a strikingly different result since the first mo to r componen t was only slightly reduced in P or E, whereas the second componen t was obliterated in both P and E. Full b lockade of neurotransmission in P and E was

PROSTATIC EPIDID~AL

I mm m l I l l mm BIB

CONTROL SURAMIN PZ CONTROL SURAMIN PZ

4 ~ PHASIC - 4

I / -.- i ,, -o-+30 ,M s(3) | so3, 12

so.. ,s s ,s o

| i i i 1 i

1.5 S 15 50Hz 1.5 5 15 SOHz

Fig. 2. Effect of suramin (S) and prazosin (PZ) on neuromuscular transmission in paired prostatic (P) and epididymal (E) portions of the rat vas deferens following transmural electrical stimulation. Upper panel: polygraphic recordings of the P and E tetanic response to 30 s trains of 15 Hz prior to (control) and 15 rain after application of either 100 gM S or 0.3 pM PZ. Calibration valid for both segments refers to 1 g and 1 min. Lower panel: phasic and tonic components following 30 s trains of pulses of varying frequencies in paired P and E segments prior to (control) and after a 15 min incubation with either 30, or 100 ,uM S. Symbols indicate mean values; bars S.E.M.

M. ~ Donoso et aL / Neuroscience Letter.~ 169 (1994) 59 62 61

PROSTATIC EPIDIDYMA L

PHASIC

' 11 "~ +39j~M . , , A ' AMPCPP (6)

.of 1.5 5 15 50Hz 1.5 5 15 50Hz

Z

I--

o o | i

1.5 5 15 50Hz 1.5 5 15 50Hz

Fig. 3. Effect of P2x-purinoceptor desensitization in the motor activity of paired prostatic (P) and epididymal (E) portions of the rat vas deferens transmurally stimulated with 30 s trains of pulses of wlrying frequencies. Phasic and tonic components following 30 s trains of 1.5 30 Hz pulses prior to (control), and after P2x-purinoceptor desensitiza- tion achieved by 3 consecutive 13 ,uM AMPCPP applications at 3 min intervals. Symbols refer to mean values, bars S.E.M.

achieved by the simultaneous incubation of 0.3/aM pra- zosin plus either suramin or AM P C P P (Fig. 1).

Consistent with the magnitude of the first component of the nerve-mediated motor response, exogenous ATP concentrat ion-response curves showed that the nucleo- tide generated more tension in P than in E (data not shown). The ATP contractions were not maintained; the peaks were observed in less than 20 s and declined rap- idly thereafter. The acute motor responses elicited with AMPCPP also faded. Furthermore, in homology to the second component, of the nerve-evoked contraction, E developed at least 3-fold more tension to exogenous NA than P: the contractions being slower than those of ATE confirming the results of Rohde et al. [21].

A parsimonious interpretation of these results suggests that blockade of P2x-receptors either by reversible com- petitive antagonism, or desensitization, leads to an im- pairement or neuroeffector transmission in P but not in E. To further test this working hypothesis, a series of experiments were performed following trains of 1.5 30 Hz transmural electrical stimulation under purinoceptor blockade or purinoceptor desensitization. The com- pounded nature of the tetanic responses showed charac- teristics proper in P and E (Fig. 2). In R the phasic component generated, at all frequencies, 2- to 3-fold more tension than its tonic element. In E, the phasic component, generated as much tension as in P: its tonic element being quantitatively similar to the phasic (Figs. 2 and 3). While suramin blocked, in a concentration- dependent manner both components of the tetanic re- sponses in P, it slightly potentiated E contractions (Fig.

2). Consonant with these results, P2x-purinoceptor de- sensitization, obliterated the 1.5 15 Hz nerve-evoked te- tanic contractions in P; only at the 30 Hz stimuli, a partial recuperation or the tetanus was observed (Fig. 3). E was considerable less sensitive than P to purinoceptor desensitization; the 30 Hz tetanic response in E was es- sentially unaltered (Fig. 3). In addition, preincubation of E segments with 0.3/aM prazosin resulted in 85 + 7.2% (n = 6) suppression of the tonic and phasic response elic- ited by the 15 Hz train stimuli. In P, the phasic compo- nent of the response to a 15 Hz train of stimuli was essentially unaltered; while its tonic element was reduced by prazosin in 53 _+ 8% (n = 5, Fig. 2).

Present results show great similitude in the pharmacol- ogy of suramin and AMPCPP in this neuroeffector junc- tion. Application of 100/aM suramin causes substantial purinoceptor blockade comparable to that likely achieved by desensitization. The composite of these re- sults lend support to our notion that ATP has a prepon- derant role as the motor transmitter in the longitudinal muscles of R In contrast, ATP participates as a co- transmitter in E. Furthermore, while the last, phasic motor component of the nerve-induced contractions, is due, or originated by action of ATE the tonic compo- nent is c~-adrenergic ill nature. The fast excitatory action of ATP might be related to the opening of receptor- operated Na+/Ca TM channels, which likely trigger excita- tory junction potentials [23] which lead to the contrac- tion. In E, the ATP-induced depolarization likely facili- tates the late secondary metabolotropic response of NA, thereby cooperating in the completion of the motor re- sponse [12].

The uneven contribution of ATP to the motor tone of the rat vas deferens could perhaps be related, among other reasons, to a different nerve population supplying its longitudinal muscles in P and E. The neuronal effer- ents of the rat vas deferens arise from the ipsilateral pelvic accessory ganglion, the major pelvic ganglion, the mesenteric and the sympathetic chain ganglia [16]. It is possible that this heterogeneity of sympathetic neurons might be unevenly distributed along the vas deferens and that in addition, they might have varying proportions of ATP/NA, in such a way that, the P segment is supplied with a more abundant 'purinergic' nerve population. Al- ternatively, the P2x-purinoceptors or the c~l-adrenocep- tor population might be asymmetrically distributed along the vas deferens smooth muscle, predominating purinoceptors towards the P segment of the longitudinal muscle layer. In support for this notion, several investi- gations show regional variations in the distribution of the c~l-adrenoceptor responses which in all cases pre- dominante in E [15,21,24]. The elucidation of these hy- potheses warrants the further experimentation.

Acknowledgements to Prof. G. Burnstock for a stimu- lating discussion, E. Moiler (Bayer) for suramina and R.

62 M. V Donoso et al./ Neuroscience Letters 169 (1994) 59-62

M i r a n d a fo r t e c h n i c a l a n d a r t i s t i c c o n t r i b u t i o n . P a r t i a l l y

s u p p o r t e d b y F o n d e c y t G r a n t 0658 /92 . M . V . D . is a re-

c i p i e n t o f D I U C t r a i n i n g f e l l ow s h i p .

[1] Anton, P.G., Duncan, M.E. and McGrath, J.C., An analysis of the anatomical basis for the mechanical response to motor nerve stim- ulation of the rat vas deferens, J. Physiol., 273 (1977) 23-43.

[2] Brown, C.M., McGrath, J.C. and Summers, R.J., The effect of alpha-adrenoceptor agonists and antagonists on response of trans- murally stimulated prostatic and epididymal portions of the iso- lated vas deferens of the rat, Br. J. Pharmacol., 66 (1979) 553-564.

[3] Burnstock, G., Co-transmission, Arch. Int. Pharmacodyn., 304 (1990) 7-33.

[4] Burnstock, G., Cocks, T., Kasakov, L. and Wong, H.K., Direct evidence for ATP release from non-adrenergic, non-cholinergic nerves in the guinea-pig taenia coli and bladder, Eur. J. Pharma- col., 49 (1978) 145 149.

[5] Campbell, G., Co-transmission, Annu. Rev. Pharmacol., 27 (1987) 51 70.

[6] Dunn, P.M. and Blakeley, A.G.H., Suramin: a reversible P2- purinoceptor antagonist in the mouse vas deferens, Br. J. Pharma- col., 93 (1988) 243-245.

[7] Edwards, F.A., Gibb, A.J. and Colquhoun, D., ATP receptor- mediated synaptic currents in the central nervous system, Nature, 359 (1992) 144-146.

[8] Evans, R.J. and Surprenant, A., Vasoconstriction of guinea-pig submucosal arteries following sympathetic nerve stimulation is mediated by the release of ATE Br. J. Pharmacol., 106 (1992) 242 249.

[9] Fedan, J.S., Hogaboom, G.K., O'Donnell, J.P., Colby, J. and Westfall, D.P., Contribution of purines to the neurogenic response of the vas deferens of the guinea-pig, Eur. J. Pharmacol., 69 (1982) 41 53.

[10] French, A.M. and Scott, N.C., Evidence to support the hypothesis that ATP is a co-transmitter in the rat vas deferens, Experientia, 39 (1983) 264-266.

[11] Hoyle, C.H.V., Knight, G.E. and Burnstock, G., Suramin antago- nizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli, Br. J. Pharmacol., 99 (1990) 617 621.

[12] Huidobro-Toro, J.P. and Parada, S., Co-transmission in the rat vas deferens: postjunctional synergism of noradrenaline and ade- nosine 5"-triphosphate, Neurosci. Lett., 85 (1988) 339-344.

[13[ Kasakov, L. and Burnstock, G., The use of the slowly degradable analog, c~,fl-methylene ATP, to produce desensitisation of the P2- purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder, Eur. J. Pharmacol., 86 (1983) 291 294.

[14] Kasakov, L., Ellis, J., Kirkpatrick, K., Milner, P. and Burnstock, G., Direct evidence for concomitant release of noradrenaline, ATP and neuropeptide Y from sympathetic nerves supplying the guinea-pig vas deferens, J. Auton. Nerv. Syst., 22 (1988) 75 82.

[15] Kasuya, Y. and Sizuki, N., Variation of postjunctional natures along the length of the rat vas deferens as a cause of regional differences in the sensitivity to norepinephrine, Arch. Int. Pharma- codyn. Ther., 241 (1979) 24-31.

[16] Kolbeck, S.C. and Steers, W.D., Origin of neurons supplying the vas deferens of the rat, J. Urol., 149 (1992) 918-921.

[17] McGrath, J.C., Adrenergic and 'non-adrenergic" components in the contractile response of the vas deferens to a single stimulus, J. Physiol., 283 (1978) 23-39.

[18] Mallard, N., MarshalL, R., Sithers, A. and Spriggs, B., Suramin: a selective inhibitor of purinergic neurotransmission in the rat isolated vas deferens, Eur. J. Pharmacol., 220 (1992) 1-10.

[19] Meldrum, L.A. and Burnstock, G., Evidence that ATP acts as a co-transmitter with noradrenaline in sympathetic nerves supplying the guinea-pig vas deferens, Eur. J. Pharmacol., 92 (1983) 161 163.

[20] Ramme, D., Regenold, J.T., Starke, K., Busse, R. and Illes, P., Identification of ATP as a neuroeffector transmitter in jejunal branches of the rabbit mesenteric artery, Naunyn Schmiedeberg's Arch. Pharmacol., 336 (1987) 267-273.

[21] Rohde, G.C., Venezian, E. and Huidobro-Toro, J.P., Asymmetric distribution of purinergic and adrenergic neurotransmission coop- erates in the motor activity along the rat vas deferens, Neurosei. Lett., 71 (1986) 197-202.

[22] Sneddon, P., Suramin inhibits excitatory junction potentials in guinea-pig isolated vas deferens, Br. J. Pharmacol., 107 (1992) 101 103.

[23] Sneddon, P. and Westfall, D.P., Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens, J. Physiol., 347 (1984) 561-578.

[24] Vardolov, L. and Pennefather, J.N., Regional variations in the distributions of alpha-adrenergic receptors in the vas deferens of the rat, Arch. Int. Pharmacodyn. Ther., 221 (1976) 212-222.