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Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In [3H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1-100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium: evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [3H]-noradrenaline-pretreated tissues exposed to both prazosin (0.3 microM) and suramin (300 microM), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1-100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 microM), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1-100 nM). In some tissues not pretreated with [3H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 microM) and suramin (300 microM), respectively. Isoprenaline (1-100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 microM) were not changed and contractions elicited by noradrenaline (100 microM) were slightly increased by isoprenaline (1-100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 microM) by pieces of the vas deferens. It is concluded that, in the guinea-pig vas deferens, activation of prejunctional beta-adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.

Contractions, release of noradrenaline and release of ATP elicited by the indirectly acting sympathomimetic amine tyramine and responses elicited by exogenous noradrenaline were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In tissues pretreated with pargyline 1 mM, tyramine 300 microM, when added to the superfusion medium for 2 min, elicited contraction and an overflow of tritium (mainly [3H]-noradrenaline) and ATP. Contraction and ATP overflow responses were prevented and tritium overflow was greatly reduced by desipramine 10 microM. Prazosin 0.3 microM abolished contractions and evoked ATP overflow without changing tritium overflow. Blockade of postjunctional P2-purinoceptors by suramin 300 microM caused a marked decrease of tyramine-evoked contractions and a slight reduction of tritium overflow whereas evoked ATP overflow was markedly increased. The effect on contraction was not shared by two other P2-purinoceptor antagonists, namely pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) 32 microM and diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) 32 microM: PPADS increased contractions about fourfold, whilst DIDS had no effect at all. When the vas deferens was superfused for 24 min with medium containing tyramine 300 microM, evoked contractions and tritium overflow continued throughout whereas ATP overflow faded rapidly to basal values. In the presence of prazosin 0.3 microM, tyramine 300 microM again failed to elicit contractions as well as an overflow of ATP. Application of noradrenaline 10 microM instead of tyramine also resulted in prolonged contraction and an overflow of ATP that declined rapidly. It is concluded that all ATP released by tyramine is non-neuronal in origin, secondary to the activation of postjunctional alpha 1-adrenoceptors by released noradrenaline. The non-neural ATP does not seem to play a functional role in smooth muscle contraction and derives from a postjunctional source which is subject to a rapid depletion upon sustained alpha 1-adrenoceptor activation.

Contractions and overflow of tritium and ATP elicited by hypogastric nerve stimulation (HNS) and field stimulation (FS) were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. HNS and FS elicited contraction, tritium overflow and ATP overflow. HNS at supramaximal current strength produced smaller responses than did FS at supramaximal current strength (210 pulses/7 Hz). Supramaximal HNS and submaximal FS were used in the remainder of the study. Prazosin (0.3 mumol/l) reduced contractions and the overflow of ATP elicited by both HNS and FS; the evoked overflow of tritium was not changed (210 pulses/7 Hz). Combined administration of prazosin (0.3 mumol/l) and suramin (300 mumol/l) abolished contractions and reduced the overflow of ATP elicited by both HNS and FS slightly more than did prazosin alone; tritium overflow again was not changed (210 pulses/7 Hz). Contractions, tritium overflow and ATP overflow increased with the frequency of both HNS and FS (from 7 to 25 Hz; 210 pulses); the increase in ATP overflow with frequency was more marked than the increase in tritium overflow. The preferential increase of ATP overflow with the frequency of HNS and FS persisted in the combined presence of prazosin (0.3 mumol/l) and suramin (300 mumol/l). The study confirms for HNS, a more physiologic way of sympathetic nerve stimulation, several observations previously obtained with FS. First, HNS-evoked ATP release is detectable as an overflow of ATP into the superfusion fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [3H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methyl-thio ATP (all 100 microM) over 30 min. Evans blue (100 microM) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order alpha,beta-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 microM) shifted the concentration-response curve of alpha, beta-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > alpha, beta-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 microM) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an-albeit impure-ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.

Effects of angiotensin II and prostaglandin E2 on contractions, release of noradrenaline and release of ATP elicited by electrical stimulation (210 pulses, 7 Hz) were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In some experiments postsynaptic alpha 1-adrenoceptors and P2X-purinoceptors were blocked by prazosin and suramin, respectively, to isolate the neural fraction of the overflow of ATP. Electrical stimulation elicited an overflow of tritium and ATP and, in the absence of prazosin and suramin, contraction. In the absence of prazosin and suramin, angiotensin II (1-100 nM) enhanced contractions as well as the evoked overflow of tritium and ATP. All parameters were increased by about the same percentage for a given concentration of angiotensin II. The effect of prostaglandin E2 (1-100 nM) was complex. Contractions were mainly enhanced, the evoked overflow of tritium was reduced, whereas the evoked overflow of ATP was predominantly increased. No or almost no contraction remained in the presence of prazosin and suramin, and the evoked overflow of ATP was decreased to about 16%. Angiotensin II (1-100 nM) again enhanced the evoked overflow of tritium and ATP. Both were increased by about the same percentage for a given concentration of angiotensin II and also were increased by about the same percentage as obtained in the absence of prazosin and suramin.(ABSTRACT TRUNCATED AT 250 WORDS)

The purinergic response of the guinea-pig vas deferens to long trains of pulses at high frequency consists of an initial twitch followed by a much lower plateau. Mechanical, neurochemical and electrophysiological techniques were used to examine the reason for the fade. Mechanical measurements. In tissues stimulated by trains of 180 pulses/10 Hz and treated with prazosin to suppress the noradrenergic contraction component, the response to alpha, beta-methylene ATP and to exogenous ATP was as high during the secondary plateau of the purinergic neurogenic contraction as it was outside electrical stimulation periods; the response to 50 pulses/100 Hz was also unchanged during the low plateau. The plateau was not increased by reactive blue 2,8-(p-sulphophenyl)theophylline, propranolol or capsaicin. Neurochemical measurements. In tissues preincubated with [3H]-noradrenaline, electrical stimulation elicited an overflow of tritium and of ATP. In the absence of drugs as well as in the presence of prazosin and suramin to suppress contractions, the overflow of tritium per pulse decreased slightly in the course of trains of 90 pulses/10 Hz; the overflow of ATP per pulse decreased to a greater extent on average, but the decrease was not statistically significant. In the presence of prazosin and nifedipine, also to suppress contractions, the overflow of tritium per pulse again decreased slightly in the course of trains of 105 pulses/10 Hz, but the overflow of ATP per pulse if anything tended to increase. Electrophysiological measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release. In tissues preincubated with [3H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 microM and even more so by prazosin 0.3 microM combined with suramin 300 microM. They were also reduced by nifedipine 10 microM and even more so by nifedipine 10 microM combined with ryanodine 20 microM (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [3H]-noradrenaline, exogenous noradrenaline 10 microM and alpha,beta-methylene ATP 10 microM elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 microM but not suramin 300 microM and were greatly reduced by nifedipine 10 microM and in Ca(2+)-free medium. Responses to alpha,beta-methylene ATP were blocked by suramin 300 microM but not prazosin 0.3 microM, were reduced by nifedipine 10 microM (effect on ATP overflow not significant) and were reduced even more in Ca(2+)-free medium. Neuropeptide Y 0.3 microM caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of P1-purinoceptor activation on contractions, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) was studied in the superfused vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [3H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. In the absence of other drugs, adenosine (10-100 microM) did not change evoked contractions but reduced the evoked overflow of tritium and ATP. In subsequent experiments alpha 1-adrenoceptors were blocked by prazosin, P2-purinoceptors by suramin and alpha 2-adrenoceptors by rauwolscine. No or almost no contraction remained under these conditions. The evoked overflow of tritium was 505% and the evoked overflow of ATP 34% of that observed in the absence of prazosin, suramin and rauwolscine. Adenosine (1-100 microM) again reduced the evoked overflow of tritium and ATP, and so did the A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.032-0.32 microM). Adenosine and CCPA decreased the evoked overflow of ATP to a greater extent than the evoked overflow of tritium. It is concluded that neural release of both postganglionic sympathetic cotransmitters, noradrenaline and ATP, is decreased upon activation of prejunctional P1- (A1-) purinoceptors in guinea-pig vas deferens. The A1-receptor-mediated inhibition of the release of ATP is more marked than the inhibition of the release of noradrenaline, a pattern opposite to the inhibition produced by activation of prejunctional alpha 2-autoreceptors.

Contractions, release of previously stored [3H]-noradrenaline (measured as overflow of total tritiated compounds) and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) were studied in the superfused vas deferens of the guinea pig. Prazosin and suramin were used to suppress non-neural ATP release, and effects of bromoxidine and rauwolscine on the neural release thus isolated were examined. Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. Both prazosin (0.03-3 microM) and suramin (30-300 microM) reduced contractions as well as the evoked overflow of ATP. No visible contraction remained in 21 of 28 tissues exposed to prazosin 0.3 microM combined with suramin 300 microM. The evoked overflow of ATP under these conditions was about 17% of that observed in the absence of drugs. In the presence of prazosin 0.3 microM and suramin 300 microM, bromoxidine (0.01-1 microM) decreased and rauwolscine (0.1-10 microM) increased the evoked overflow of both tritium and ATP. Rauwolscine increased the evoked overflow of tritium to a significantly greater extent than the overflow of ATP. It is concluded that the overflow of ATP elicited by electrical (neural) stimulation in the presence of prazosin 0.3 microM and suramin 300 microM reflects purely neural release of ATP. This release of ATP, like the release of noradrenaline, is modulated through prejunctional alpha 2-adrenoceptors. The alpha 2-adrenoceptor modulation of the release of noradrenaline seems to be more marked than the modulation of the release of ATP.