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Purinergic modulation of field stimulation responses of rat and human vas deferens smooth muscle
Gen. Pharmac. Vol. 22, No. 5, pp. 869-872, 1991 Printed in Great Britain. All fights reserved
0306-3623/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie
PURINERGIC MODULATION OF FIELD STIMULATION RESPONSES OF RAT A N D H U M A N VAS DEFERENS SMOOTH MUSCLE M. LYNCH and H. HUDDART* Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, England [Tel. (0524) 65201; Fax (0524) 843854] (Received 17 December 1990) Abstract--1. Guanethidine at 5 x 10-6 M strongly inhibited rat prostatic but not epididymal vas deferens, reflecting differences in innervation and the neurogenic field stimulation responses of these tissues. 2. Adenosine and ATP inhibited the field stimulation responses of rat prostatic vas deferens by 56 and 50% respectively. A 10-min pretreatment with 10-4 M caffeine partly reversed this inhibition, by 55% in the case of adenosine and 60% for ATP. 3. Pretreatment for 10 rain with 5 #M qulnidine failed to significantly alter the extent of either adenosine or ATP inhibition of the field stimulation responses of rat prostatic vas deferens. 4. 8-Phenyltheophylline, the selective blocker of the A, subtype of the P, receptor, partly reversed adenosine-induced inhibition of the vas deferens FS responses. NECA, the selective agonist of the A2 subtype of the Pl receptor, very strongly inhibited vas deferens FS responses. 5. Field stimulation responses of human vas deferens were also inhibited by both adenosine and ATP but to a lesser extent and more variably than in rat tissue. 6. Adenosine and ATP inhibition was reversed by caffeine pretreatment, but far more variably than in rat tissue, and quinidine was without significant effect on inhibition of the responses. 7. It is concluded that in these tissues adenosine and ATP may operate via a P~ type receptor of both A~ and A 2 subtypes and that a P2 type receptor may be lacking.
INTRODUCTION
Apart from the studies by Stone (1981) with adenosine and Meldrum and Burnstock (1983) and Sneddon and Burnstock (1984) with A T P on guineapig vas deferens, this latter tissue has been rather neglected. To extend these studies, here we report experiments on rat and human vas deferens which provide further evidence for purinergic modulation in those tissues.
Evidence that purine compounds had potent actions on excitable tissues came in the first half of this century (Drury and Szent-Gyorgi, 1929; G a d d u m and Holtz, 1933; Green and Stoner, 1950)' when adenosine and related purines were shown to slow heart rate and reduce arterial pressure. Such actions are not confined to mammals. Lennard and Huddart (1989a) demonstrated purinergic neuromodulation in the flounder heart, and adenosine compounds and purinoreceptors have been found in a number of invertebrate phyla (Hoyle and Greenberg, 1988; Hoyle et al., 1989). The evidence of non-cholinergic, non-adrenergic transmission in the gastrointestinal tract (Burnstock et al., 1966), the urinary bladder (Ambache and Zar, 1970; Burnstock et al., 1978) and the vascular system (Burnstock et al., 1979; De Mey et al., 1979) led to the formulation of purinergic transmission (Burnstock, 1975, 1981). C o m m o n l y emerging candidates from these studies were adenosine and ATP, and their various roles have been reviewed in recent years (Williams, 1984; Williams and Cusack, 1990). The identification of membrane receptors to adenosine c o m p o u n d s (see Stone, 1985), showed a PI type (now the A type) responding to adenosine and a Pz type responding to ATP. Sub-types, distinguished by their agonist and antagonist pharmacology, have now been characterized (Williams and Cusack, 1990).
MATERIALS AND METHODS
Male Wistar strain albino rats were killed by a sharp blow to the head and cervical dislocation. Vasa deferentia were dissected free and placed in aerated Krebs saline at 37°C containing (in mM): NaCI 120.7, KC1 5.9, CaC12 2.5, MgCI2 1.2, NaH2PO 4 1.2, NaHCO 3 15.5 and glucose 11.5. The pH was adjusted to 7.3 at 37°C using HC1. Adherent tissue was removed and each vas deferens was bisected at the junction of the epididymal and prostatic section to yield a thin epididymal and a thick prostatic preparation (see Langton and Huddart, 1987, 1988). The preparations were ligated at both ends with monofilament nylon thread and mounted in 25-ml glass-jacketed organ-baths maintained at 37°C. Tension was recorded isometerically using Grass FT 0.3 force displacement transducers connected to a four-channel Grass polygraph. Human vas deferens, obtained at elective vasectomy or orchidectomy, was maintained at 37°C during transport to the laboratory and was treated in the same way as rat tissue for ligation and tension recording. Each tissue was drawn through the rings of silver/silver chloride field stimulation electrodes before final mounting in the organ-baths. Field stimulation responses were induced by stimulus trains from a Grass $88 dual-channel stimulator connected via two SIU5 stimulus isolation units which could drive four preparations simultaneously (Huddart and Butler, 1986). Train parameters varied in different prep-
*To whom all correspondence should be addressed. 869
870
M. LYNCHand H. HUDDART
arations but a common regime consisted of a 200-msectrain of pulses at 50 Hz, each pulse of 2-5 msec at 40-50 V. Trains were automatically repeated at 100-see intervals. All drugs used in this study were obtained from Sigma Chemical Co. and were directly added with digital adjust pipettes to the organ-baths from concentrated 10 mM stock solutions freshly made up in distilled water.
(a)
(b)
RESULTSAND DISCUSSION In the sparsely innervated rat bladder detrusor the field stimulation responses result largely from direct depolarization (Huddart and Butler, 1986). Rat vas defvrens receives a dense adrenergic innervation so the nature of its field stimulation response was assessed using the anti-adrcnergic agent guancthidine. Figure 1 shows the effect of 5 x 10-6M guanethidine on rat prostatic and epididymal vas defcrens and is typical of the response patterns of 16 different preparations. Whereas the prostatic vas deferens was clearly and very strongly inhibited by guanethidine, no inhibition was seen in the epididymal vas deferens although the latter did consistently develop low-level spontaneous activity in the presence of guanethidine. This is consistent with the differences in innervation and pharmacology of these two separate portions of the rat vas deferens reported by Langton and Huddart (1987, 1988). It also indicated that the rat epididymal vas deferens, unlike human epididymal vas deferens, was not amenable to field stimulation studies of neurogenic responses.
The effect of adenosine and A TP Both adenosine and ATP were found to strongly inhibit the field stimulation responses of rat prostatic vas deferens at 50/~M (Fig. 2a, c). Inhibition was about 65% with adenosine and over 50% with ATP (n = 12) (Fig. 3). When the preparations were pre(a) Prostatic
~
'"'""'"'"'"'"111111 A Caff
Ad
A Ad
Jllllllllll|llllllllllllll A ATP
A Caff
A ATP
Fig. 2. Traces typical of 12 experiments showing the effect of 50/zM adenosine (a) and 50/~M ATP (c) on the FS responses of rat prostatic vas deferens. A 10-min prctreatment with l0 -4 M caffeine reversed this inhibition by about 55% for adenosine (b) and by over 60% for ATP (d), Tension scale applies to all traces. Time base, 1 rain marks. treated for 10min with 10-4M caffeine, which is a selective antagonist of the P] type receptor (Burnstock, 1981; Fredholm and Persson, 1982), a significant reversal of both adenosine and ATPinduced inhibition of field stimulation responses was seen (Fig. 2b, d). This finding was very similar to the reversal of adenosine inhibition of flounder ileum by theophyiline (Lennard and Huddart, 1989b). The reversal of inhibition of adenosine responses by caffeine was about 55%, that seen with ATP responses was over 60% (n = 12) (Fig. 3). This was a rather unexpected finding in the case of ATP inhibition as the purinergic transmission hypothesis suggests that the P2 receptor type is more sensitive to ATP and this receptor is less sensitive to methylxan70
IIIII . . . . . . . . . . . ' " . . . . . . . . . . ' " (b)
t Gu
60
0
Elaldldymal
=~ 8o
0
~
I0.5g
i
+ , i
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o
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Fig. 1. The effect of 5 x 10 - 6 M guanethidine (Gu) on the field stimulation (FS) responses of rat prostatic (a) and epididymal (b) vas deferens. Tension scale applies to both traces. Time base, l min marks.
+ °
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i
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O. Fig. 3. Summary of adenosine and ATP inhibition of FS responses of rat prostatic vas deferens and their reversal by caffeine. Each histogram is the mean of 12 experiments _+SEM.
Purinergic actions on vas deferens thines than the P] receptor but can be selectively non-competitively blocked by quinidine (Burnstock, 1981).
871
O.Sg
(a)
(c)
The effect of quinidine When rat prostatic vas deferens was exposed to 5 # M quinidine for 10 min before adenosine and ATP exposure, no significant reversal was seen in the extent of inhibition (Fig. 4). This was a most surprising finding and completely contrasts with the situation in flounder ileum and rectum where the P: receptor antagonist ~,/Lmethylerie ATP completely blocked responses to ATP (Lennard and Huddart, 1989b). Inevitably, the conclusion must be that adenosine and ATP actions in prostatic rat vas deferens must operate via the P] receptor and the likelihood is that this tissue lacks the P2 receptor. The P~ receptor of Burnstock terminology (Burnstock, 1978) has more recently been reclassified into A~ and A2 subtypes (Londos and Wolff, 1977; Williams and Cusack, 1990). In an attempt to define more clearly the potential receptor population in the rat vas deferens we employed two receptor-selective agents. The xanthine 8-phenyltheophylline has been shown to be a potent A] receptor blocker (Griffith et al., 1981). At 10#M this agent was found to reverse the adenosine-induced inhibition of vas deferens by about 50% (Fig. 5a, b). NECA (Y-ethyicarboxamidoadenosine) has been shown to be a selective agonist of the A2 receptor (Lohse et al., 1988). At concentrations as low as 5 x 10-TM we found that this agent virtually eliminated vas deferens field stimulation responses (Fig. 5c, d). The findings with both 8-phenyltheophylline and NECA would seem to strongly support the hypothesis that rat vas deferens possesses a population of Pm receptors of the A, and A2 subtypes.
(b)
(a)
///
(b)
Fig. 5. (a) Inhibition of FS responses of rat vas deferens induced by 50#M adenosine. (b) Partial reversal of adenosine-induced inhibition by a 10-minpretreatment with 10 #M 8-phenyltheophylline(first arrow, 8pt). (c), (d) Two examples, typical of eight such experiments, showing the inhibitory effect of 5 x l0 -7 M NECA on FS responses of rat vas deferens. Tension scale applies to all traces. Time base, 1 min marks.
Human vas deferens The human tissues proved to be slightly more variable in field stimulation response pattern than rat tissue. At 50/~M, both adenosine and ATP induced inhibition but to a variable extent. The mean inhibition seen in 12 experiments was about 30% (Fig. 6a, c). A 10-rain pretreatment with 10 - 4 M caffeine did reverse adenosine and ATP-induced inhibition, but again to a rather more variable extent to that seen in rat tissue, averaging 35% for both agents (Fig. 6c, d). When human vas deferens was pretreated with 5 # M quinidine the results (not shown) were very similar to those of rat tissue, there being no significant alteration to adenosine- or ATP-induced responses. This again suggests that a P] type receptor may be involved here and not a P2 receptor.
llll,,,,,, llill I o.5g
(al
\\\\\\\\\ A Ad
(el
Illl'""]"'"'"'""" A A (d)
Qu
Ad
A Ad
Ill/I/Ill,,,,,,
[cl
Qu
ATP
Fig. 4. Typical traces of 12 experiments showing the effect of 50 # M adenosine (a) and 50/~M ATP (c) on FS responses of rat prostatic vas deferens. A 10-min pretreatment with 5 #M quinidine had no significant effect on adenosine-induced inhibition (b) or ATP-induced inhibition (d). Tension scale applies to all traces. Time base, 1 min marks.
A Caff
A Ad
Idl
...........................
ATP
(bl
illllllr ....................
A
l
A
ATP
Coff
ATP
Fig. 6. Typical traces of 12 experiments showing the effect of 50 #M adenosine (a) and 50/zM ATP (c) on FS responses of human epididymal vas deferens. A lO-min pretreatment with 10-4 M cafeine reversed this inhibition by about 35% for both agents. Tension scale applies to all traces. Time bases, 1 rain marks also apply to all traces.
872
M. LYNCH and H. HUDDAKT
A major problem with all studies o f adenosine and A T P a c t i o n s / n vitro is the significance to the in vivo situation. T w o major factors have to be considered: the intimate interrelationships o f adenosine and A T P themselves, and the source of the in vivo supply o f either compound. Adenosine in both intra- and extracellular situations can be formed from A T P by hydrolysis and A T P can be formed from adenosine by energy-dependent phosphorylation. W h a t may seem clear in the organ-bath may be more problematic in the animal. In the guinea-pig vas deferens there is some evidence that A T P may act as a co-transmitter along with noradrenaline in the sympathetic supply (Meldrum and Burnstock, 1983). This view is strengthened as ~. B-methylene A T P inhibits excitatory junctional potentials (Sneddon and Burnstock, 1984). In the rat and h u m a n vas deferens the actions o f both adenosine and A T P are clear enough although we have no direct evidence as yet for co-transmission in those tissues.
Acknowledgements--This study was carried out while M.L. was in receipt of an SERC research studentship. The authors are most grateful to Mr W. G. Staff of the Urology Department and to the theatre staff of the Queen Victoria Hospital in Morecambe for their collaboration in obtaining human material for this study. REFERENCES
Ambache N. and Zar A. M. (1970) Non-cholinergic transmission by post-ganglionic motor neurones in the mammalian bladder. J. Physiol. (Lond.) 210, 761-783. Burnstoek G. (1975) Purinergic transmission. In Handbook of Psychopharmacology Iiol. 5, Synaptic Modulators (Edited by Iversen L. L., Iversen S. D. and Snyder S. H.), pp. 131-194. Plenum Press, New York. Burnstock G. (1978) A basis for distinguishing two types of purinerglc receptor. In Cell Membrane Receptors for Drugs and Hormones (Edited by Bolis L. and Straub R. W.), pp. 107-118. Raven Press, New York. Burnstoek, G. (1981) Neurotransmitters and trophic factors in the autonomic nervous system. J. Physiol. (Lond.) 313, 1-35. Burnstock G., Campbell G. and Rand M. J. (1966) The inhibitory innervation of the taenia of the gunea-pig caecum. J. Physiol. (Lond.) 182, 504-526. Burnstock G., Crowe R. and Wong H. K. (1979) Comparative pharmacological and histochemical evidence for purinergic inhibitory innervation of the portal vein of the rabbit, but not guinea-pig. Br. J. Pharmac. 65, 377-388. Burnstock G., Cocks T., Crowe R. and Kasakov L. (1978) Purinergic innervation of the guinea-pig urinary bladder. Br. J. Pharmac. 63, 125-138. De Mey J., Burnstock G. and Vanhoutte P. M. (1979) Modulation of the evoked release of noradrenaline in canine saphenous vein via presynaptic receptors for adenosine but not ATP. Fur. J. Pharmac. 55, 401--405. Drury A. N. and Szent-Gyorgi A. (1929) The physiological activity of adenosine compounds with special reference to their action upon mammalian heart. J. PhysioL (Lond.) 28, 213-237.
Fredholm B. B. and Persson C. G. A. (1982) Xanthine derivatives as adenosine receptor antragonists. Eur. J. Pharmac. 81, 673-676. Gaddum J. H. and Holtz P. (1933) The localization of the action of drugs on the pulmonary vessels of dogs and cats. J. Physiol. (Lond.) 77, 139-158. Green N. H. and Stoner H. B. (1950) Biological Actions of the Adenine Nucleotides. Lewis, London. Griffith S. G., Meghhgl P., Moody C. J. and Burnstock G. (1981) 8-Phenyltheophylfine: a potent Pz antagonist. Eur. J. Pharmac. 75, 61--64. Hoyle C. H. V. and Greenberg M. J. (1988) Actions of adenylyl compounds in invertebrates from several phyla, evidence for internal purinoceptors. Comp. Biochem. Physiol. 9012, 207-214. Hoyle C. H. V., Knight G. E. and Burnstock (3. (1989) Actions of adenylyl compounds in the pedal disc of the cnidarian Actinea equina. Comp. Biochem. Physiol. 94C, 111-114. Huddart H. and Butler D. J. (1986) Field stimulation responses of rat urinary bladder detrusor smooth muscle. Dependence upon slow calcium channel activity determined by K ÷ depolarization and calcium antagonists. Gen, Pharmac. 17, 695-703. Langton P. D. and Huddart H. (1987) The involvement of fast calcium channel activity in the selective activation of phasic contractions by partial depolarization in rat vas deferens smooth muscle. Gen. Pharmac. 18, 47-55. Langton P. D. and Huddart H. (1988) Voltage and time dependency of calcium mediated phasic and tonic responses in rat vas deferens smooth muscle---the effect of some calcium agonist and antagonist agents. Gen. Pharmac. 19, 775-787. Lennard R. and Huddart H. (1989a) Purinergic modulation of cardiac activity in the flounder during hypoxic stress. J. Comp. Physiol. 159B, 105-113. Lennard R. and Huddart H. (1989b) Purinergic modulation in the flounder gut. Gen. Pharmac. 20, 849--853. Lodos C. and Wolff J. (1977) Two distinct adenosine-sensitive sites on adenylate cyclase. Proc. Natl Acad. Sci. U.S.A 74, 5482-5486. Lohse M. J., Elger B., Lindenborn-Fotinos J., Klotz K. N. and Schwabe U. (1988) Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration. NaunynSchmiedeberg's Arch. Pharmac. 337, 64~8. Meldrum L. A. and Burnstock G. (1983) Evidence that ATP acts as a co-transmitter with noradrenaline in sympathetic nerves supplying the guinea pig vas deferens. Eur. J. Pharmac. 92, 161-163. Sneddon P. and Burnstoek G. (1984) Inhibition of excitatory junction potentials in guinea pig vas deferens by a, #-methylene ATP: further evidence for noradrenaline and ATP as co-transmitters. Fur. J. Pharmac. 100, 85-90. Stone T. W. (1981) The effect of 4-aminopyridine on isolated vas deferens and its effect on inhibitory properties of adenosine, morphine, noradrenaline and 8-aminobutyric acid. Br. J. Pharmac. 73, 791-976. Stone T. W. (1985) Classifications of adenosine receptors in the nervous system. In Methods in Pharmacology (Edited by Paton D. M.). Plenum Press, New York. Williams M. (1984) Neuromodulatory actions of adenosine. Neurotransmissions 1, 1-3. Williams M. and Cusack N. J. (1990) Neuromodulatory roles of purine nucleosides and nuclcotides: their receptors and ligands. Neurotransmissions 6, 1-6.
0306-3623/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie
PURINERGIC MODULATION OF FIELD STIMULATION RESPONSES OF RAT A N D H U M A N VAS DEFERENS SMOOTH MUSCLE M. LYNCH and H. HUDDART* Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, England [Tel. (0524) 65201; Fax (0524) 843854] (Received 17 December 1990) Abstract--1. Guanethidine at 5 x 10-6 M strongly inhibited rat prostatic but not epididymal vas deferens, reflecting differences in innervation and the neurogenic field stimulation responses of these tissues. 2. Adenosine and ATP inhibited the field stimulation responses of rat prostatic vas deferens by 56 and 50% respectively. A 10-min pretreatment with 10-4 M caffeine partly reversed this inhibition, by 55% in the case of adenosine and 60% for ATP. 3. Pretreatment for 10 rain with 5 #M qulnidine failed to significantly alter the extent of either adenosine or ATP inhibition of the field stimulation responses of rat prostatic vas deferens. 4. 8-Phenyltheophylline, the selective blocker of the A, subtype of the P, receptor, partly reversed adenosine-induced inhibition of the vas deferens FS responses. NECA, the selective agonist of the A2 subtype of the Pl receptor, very strongly inhibited vas deferens FS responses. 5. Field stimulation responses of human vas deferens were also inhibited by both adenosine and ATP but to a lesser extent and more variably than in rat tissue. 6. Adenosine and ATP inhibition was reversed by caffeine pretreatment, but far more variably than in rat tissue, and quinidine was without significant effect on inhibition of the responses. 7. It is concluded that in these tissues adenosine and ATP may operate via a P~ type receptor of both A~ and A 2 subtypes and that a P2 type receptor may be lacking.
INTRODUCTION
Apart from the studies by Stone (1981) with adenosine and Meldrum and Burnstock (1983) and Sneddon and Burnstock (1984) with A T P on guineapig vas deferens, this latter tissue has been rather neglected. To extend these studies, here we report experiments on rat and human vas deferens which provide further evidence for purinergic modulation in those tissues.
Evidence that purine compounds had potent actions on excitable tissues came in the first half of this century (Drury and Szent-Gyorgi, 1929; G a d d u m and Holtz, 1933; Green and Stoner, 1950)' when adenosine and related purines were shown to slow heart rate and reduce arterial pressure. Such actions are not confined to mammals. Lennard and Huddart (1989a) demonstrated purinergic neuromodulation in the flounder heart, and adenosine compounds and purinoreceptors have been found in a number of invertebrate phyla (Hoyle and Greenberg, 1988; Hoyle et al., 1989). The evidence of non-cholinergic, non-adrenergic transmission in the gastrointestinal tract (Burnstock et al., 1966), the urinary bladder (Ambache and Zar, 1970; Burnstock et al., 1978) and the vascular system (Burnstock et al., 1979; De Mey et al., 1979) led to the formulation of purinergic transmission (Burnstock, 1975, 1981). C o m m o n l y emerging candidates from these studies were adenosine and ATP, and their various roles have been reviewed in recent years (Williams, 1984; Williams and Cusack, 1990). The identification of membrane receptors to adenosine c o m p o u n d s (see Stone, 1985), showed a PI type (now the A type) responding to adenosine and a Pz type responding to ATP. Sub-types, distinguished by their agonist and antagonist pharmacology, have now been characterized (Williams and Cusack, 1990).
MATERIALS AND METHODS
Male Wistar strain albino rats were killed by a sharp blow to the head and cervical dislocation. Vasa deferentia were dissected free and placed in aerated Krebs saline at 37°C containing (in mM): NaCI 120.7, KC1 5.9, CaC12 2.5, MgCI2 1.2, NaH2PO 4 1.2, NaHCO 3 15.5 and glucose 11.5. The pH was adjusted to 7.3 at 37°C using HC1. Adherent tissue was removed and each vas deferens was bisected at the junction of the epididymal and prostatic section to yield a thin epididymal and a thick prostatic preparation (see Langton and Huddart, 1987, 1988). The preparations were ligated at both ends with monofilament nylon thread and mounted in 25-ml glass-jacketed organ-baths maintained at 37°C. Tension was recorded isometerically using Grass FT 0.3 force displacement transducers connected to a four-channel Grass polygraph. Human vas deferens, obtained at elective vasectomy or orchidectomy, was maintained at 37°C during transport to the laboratory and was treated in the same way as rat tissue for ligation and tension recording. Each tissue was drawn through the rings of silver/silver chloride field stimulation electrodes before final mounting in the organ-baths. Field stimulation responses were induced by stimulus trains from a Grass $88 dual-channel stimulator connected via two SIU5 stimulus isolation units which could drive four preparations simultaneously (Huddart and Butler, 1986). Train parameters varied in different prep-
*To whom all correspondence should be addressed. 869
870
M. LYNCHand H. HUDDART
arations but a common regime consisted of a 200-msectrain of pulses at 50 Hz, each pulse of 2-5 msec at 40-50 V. Trains were automatically repeated at 100-see intervals. All drugs used in this study were obtained from Sigma Chemical Co. and were directly added with digital adjust pipettes to the organ-baths from concentrated 10 mM stock solutions freshly made up in distilled water.
(a)
(b)
RESULTSAND DISCUSSION In the sparsely innervated rat bladder detrusor the field stimulation responses result largely from direct depolarization (Huddart and Butler, 1986). Rat vas defvrens receives a dense adrenergic innervation so the nature of its field stimulation response was assessed using the anti-adrcnergic agent guancthidine. Figure 1 shows the effect of 5 x 10-6M guanethidine on rat prostatic and epididymal vas defcrens and is typical of the response patterns of 16 different preparations. Whereas the prostatic vas deferens was clearly and very strongly inhibited by guanethidine, no inhibition was seen in the epididymal vas deferens although the latter did consistently develop low-level spontaneous activity in the presence of guanethidine. This is consistent with the differences in innervation and pharmacology of these two separate portions of the rat vas deferens reported by Langton and Huddart (1987, 1988). It also indicated that the rat epididymal vas deferens, unlike human epididymal vas deferens, was not amenable to field stimulation studies of neurogenic responses.
The effect of adenosine and A TP Both adenosine and ATP were found to strongly inhibit the field stimulation responses of rat prostatic vas deferens at 50/~M (Fig. 2a, c). Inhibition was about 65% with adenosine and over 50% with ATP (n = 12) (Fig. 3). When the preparations were pre(a) Prostatic
~
'"'""'"'"'"'"111111 A Caff
Ad
A Ad
Jllllllllll|llllllllllllll A ATP
A Caff
A ATP
Fig. 2. Traces typical of 12 experiments showing the effect of 50/zM adenosine (a) and 50/~M ATP (c) on the FS responses of rat prostatic vas deferens. A 10-min prctreatment with l0 -4 M caffeine reversed this inhibition by about 55% for adenosine (b) and by over 60% for ATP (d), Tension scale applies to all traces. Time base, 1 rain marks. treated for 10min with 10-4M caffeine, which is a selective antagonist of the P] type receptor (Burnstock, 1981; Fredholm and Persson, 1982), a significant reversal of both adenosine and ATPinduced inhibition of field stimulation responses was seen (Fig. 2b, d). This finding was very similar to the reversal of adenosine inhibition of flounder ileum by theophyiline (Lennard and Huddart, 1989b). The reversal of inhibition of adenosine responses by caffeine was about 55%, that seen with ATP responses was over 60% (n = 12) (Fig. 3). This was a rather unexpected finding in the case of ATP inhibition as the purinergic transmission hypothesis suggests that the P2 receptor type is more sensitive to ATP and this receptor is less sensitive to methylxan70
IIIII . . . . . . . . . . . ' " . . . . . . . . . . ' " (b)
t Gu
60
0
Elaldldymal
=~ 8o
0
~
I0.5g
i
+ , i
~ ao
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•
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0 0
20
10
Fig. 1. The effect of 5 x 10 - 6 M guanethidine (Gu) on the field stimulation (FS) responses of rat prostatic (a) and epididymal (b) vas deferens. Tension scale applies to both traces. Time base, l min marks.
+ °
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@
!
0
i
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O. Fig. 3. Summary of adenosine and ATP inhibition of FS responses of rat prostatic vas deferens and their reversal by caffeine. Each histogram is the mean of 12 experiments _+SEM.
Purinergic actions on vas deferens thines than the P] receptor but can be selectively non-competitively blocked by quinidine (Burnstock, 1981).
871
O.Sg
(a)
(c)
The effect of quinidine When rat prostatic vas deferens was exposed to 5 # M quinidine for 10 min before adenosine and ATP exposure, no significant reversal was seen in the extent of inhibition (Fig. 4). This was a most surprising finding and completely contrasts with the situation in flounder ileum and rectum where the P: receptor antagonist ~,/Lmethylerie ATP completely blocked responses to ATP (Lennard and Huddart, 1989b). Inevitably, the conclusion must be that adenosine and ATP actions in prostatic rat vas deferens must operate via the P] receptor and the likelihood is that this tissue lacks the P2 receptor. The P~ receptor of Burnstock terminology (Burnstock, 1978) has more recently been reclassified into A~ and A2 subtypes (Londos and Wolff, 1977; Williams and Cusack, 1990). In an attempt to define more clearly the potential receptor population in the rat vas deferens we employed two receptor-selective agents. The xanthine 8-phenyltheophylline has been shown to be a potent A] receptor blocker (Griffith et al., 1981). At 10#M this agent was found to reverse the adenosine-induced inhibition of vas deferens by about 50% (Fig. 5a, b). NECA (Y-ethyicarboxamidoadenosine) has been shown to be a selective agonist of the A2 receptor (Lohse et al., 1988). At concentrations as low as 5 x 10-TM we found that this agent virtually eliminated vas deferens field stimulation responses (Fig. 5c, d). The findings with both 8-phenyltheophylline and NECA would seem to strongly support the hypothesis that rat vas deferens possesses a population of Pm receptors of the A, and A2 subtypes.
(b)
(a)
///
(b)
Fig. 5. (a) Inhibition of FS responses of rat vas deferens induced by 50#M adenosine. (b) Partial reversal of adenosine-induced inhibition by a 10-minpretreatment with 10 #M 8-phenyltheophylline(first arrow, 8pt). (c), (d) Two examples, typical of eight such experiments, showing the inhibitory effect of 5 x l0 -7 M NECA on FS responses of rat vas deferens. Tension scale applies to all traces. Time base, 1 min marks.
Human vas deferens The human tissues proved to be slightly more variable in field stimulation response pattern than rat tissue. At 50/~M, both adenosine and ATP induced inhibition but to a variable extent. The mean inhibition seen in 12 experiments was about 30% (Fig. 6a, c). A 10-rain pretreatment with 10 - 4 M caffeine did reverse adenosine and ATP-induced inhibition, but again to a rather more variable extent to that seen in rat tissue, averaging 35% for both agents (Fig. 6c, d). When human vas deferens was pretreated with 5 # M quinidine the results (not shown) were very similar to those of rat tissue, there being no significant alteration to adenosine- or ATP-induced responses. This again suggests that a P] type receptor may be involved here and not a P2 receptor.
llll,,,,,, llill I o.5g
(al
\\\\\\\\\ A Ad
(el
Illl'""]"'"'"'""" A A (d)
Qu
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A Ad
Ill/I/Ill,,,,,,
[cl
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ATP
Fig. 4. Typical traces of 12 experiments showing the effect of 50 # M adenosine (a) and 50/~M ATP (c) on FS responses of rat prostatic vas deferens. A 10-min pretreatment with 5 #M quinidine had no significant effect on adenosine-induced inhibition (b) or ATP-induced inhibition (d). Tension scale applies to all traces. Time base, 1 min marks.
A Caff
A Ad
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...........................
ATP
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l
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Coff
ATP
Fig. 6. Typical traces of 12 experiments showing the effect of 50 #M adenosine (a) and 50/zM ATP (c) on FS responses of human epididymal vas deferens. A lO-min pretreatment with 10-4 M cafeine reversed this inhibition by about 35% for both agents. Tension scale applies to all traces. Time bases, 1 rain marks also apply to all traces.
872
M. LYNCH and H. HUDDAKT
A major problem with all studies o f adenosine and A T P a c t i o n s / n vitro is the significance to the in vivo situation. T w o major factors have to be considered: the intimate interrelationships o f adenosine and A T P themselves, and the source of the in vivo supply o f either compound. Adenosine in both intra- and extracellular situations can be formed from A T P by hydrolysis and A T P can be formed from adenosine by energy-dependent phosphorylation. W h a t may seem clear in the organ-bath may be more problematic in the animal. In the guinea-pig vas deferens there is some evidence that A T P may act as a co-transmitter along with noradrenaline in the sympathetic supply (Meldrum and Burnstock, 1983). This view is strengthened as ~. B-methylene A T P inhibits excitatory junctional potentials (Sneddon and Burnstock, 1984). In the rat and h u m a n vas deferens the actions o f both adenosine and A T P are clear enough although we have no direct evidence as yet for co-transmission in those tissues.
Acknowledgements--This study was carried out while M.L. was in receipt of an SERC research studentship. The authors are most grateful to Mr W. G. Staff of the Urology Department and to the theatre staff of the Queen Victoria Hospital in Morecambe for their collaboration in obtaining human material for this study. REFERENCES
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