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Effects of drugs acting at P2-purinoceptors on the release of newly taken up [3H]-noradrenaline were studied in slices of mouse and rat vas deferens. The slices were superfused and stimulated electrically, in most experiments by trains of 60 pulses/8 Hz. In mouse vas deferens, the P2-purinoceptor antagonists reactive blue 2 (1.8-100 microM) and brilliant blue G (10-300 microM) increased the stimulation-evoked overflow of tritium in a concentration-dependent manner as shown previously for suramin. Reactive blue 2, which preferentially blocks the P2Y-subtype, was the most potent compound and the compound with highest maximal effect, an increase by 104%. Pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in contrast, caused a small increase only at a single concentration (30 microM). The effects of reactive blue 2, brilliant blue G and suramin were not additive. The P2 agonist adenosine 5'-O-(3-thio)-triphosphate (ATP gamma S) reduced the evoked overflow of tritium. As shown previously for suramin, reactive blue 2 30 microM and brilliant blue G 100 microM antagonized the effect of ATP gamma S. From the shift of the ATP gamma S concentration-response curve to the right, an apparent pKB value of 5.3 was estimated for reactive blue 2 and an apparent pKB of 4.5 for brilliant blue G. In rat vas deferens, reactive blue 2 (3-30 microM), brilliant blue G (10 microM) and suramin (30-300 microM) also increased the evoked overflow of tritium. As in the mouse, reactive blue 2 was the most potent compound and the compound with highest maximal effect, an increase by 90%.(ABSTRACT TRUNCATED AT 250 WORDS)

The release of both sympathetic co-transmitters, noradrenaline and ATP, is modulated via presynaptic receptors. However, the degree of the modulation may differ indicating that the ratio of the released co-transmitters changes upon presynaptic receptor activation. For example, alpha 2-autoinhibition affects the release of noradrenaline more markedly than the release of ATP. Some sympathetic axon terminals possess presynaptic P2-purinoceptors which are activated by endogenous ATP. These receptors are a novel kind of auto-receptor: they mediate a presynaptic negative feedback mechanism in which released ATP inhibits subsequent co-transmitter release.

1. Strips of human saphenous veins were superfused with Krebs-Henseleit solution at either 25 degrees C or 37 degrees C. Constrictor responses to electrical stimulation (10 Hz, 40 s) but not to exogenous noradrenaline (0.1, 1 microM) were abolished by guanethidine (10 microM) and tetrodotoxin (1 microM). Hence, responses to electrical stimulation are due to action potential-induced release of sympathetic neurotransmitters. 2. Constrictor responses to electrical stimulation and noradrenaline were reduced by the alpha 1-adrenoceptor antagonist, prazosin (0.3 microM) as well as by the alpha 2-adrenoceptor antagonist, rauwolscine (1 microM). The combination of prazosin and rauwolscine abolished constrictor responses to noradrenaline at 25 degrees C and 37 degrees C. However, constrictor responses to electrical stimulation were partly resistant to alpha-adrenoceptor blockade by prazosin and rauwolscine (at 25 degrees C about 30%). Residual constrictor responses to electrical stimulation were also observed in the presence of the combination of prazosin (3 microM) and rauwolscine (10 microM) as well as in the presence of phenoxybenzamine (10 microM). 3. Veins, incubated with [3H]-noradrenaline, released tritium upon electrical stimulation (10 Hz, 40 s). Moreover, electrical stimulation also induced an overflow of ATP amounting to 4.8 +/- 1.5 pmol g-1 at 25 degrees C and 2.0 +/- 0.5 pmol g-1 at 37 degrees C. Both tritium and ATP overflow were abolished by tetrodotoxin (0.5 microM). The combination of prazosin (0.3 microM) and rauwolscine (1 microM) increased tritium overflow at either 25 degrees C or 37 degrees C by about 120%, but reduced ATP overflow by about 70%. Hence, a significant percentage of the electrically evoked ATP overflow seems to be released from non-neuronal cells upon activation of alpha-adrenoceptors by endogenous noradrenaline. The remaining ATP overflow, which was resistant to alpha-adrenoceptor blockade, may reflect neuronally released ATP.4. ATP (300 MicroM) and alpha,Beta-methylene-ATP (1, 10 MicroM), both induced constrictor responses. The P2-purinoceptor antagonist, suramin (300 MicroM) markedly inhibited constrictor responses to ATP and alpha, beta-methylene-ATP, but not those to electrical stimulation and to noradrenaline. Moreover, suramin(300 MicroM) failed to diminish the alpha-adrenoceptor blockade-resistant constrictor response to 10 Hz.5. In conclusion, constrictor responses to sympathetic nerve stimulation in human saphenous veins are mainly but not exclusively mediated by neuronally released noradrenaline. There is a concomitant release of ATP and noradrenaline. P2-purinoceptors which mediate vasoconstriction are present; however,a role of neuronally released ATP in constrictor responses to electrical stimulation could not be established. Therefore, the nature of the sympathetic transmitter responsible for alpha-adrenoceptor blockade-resistant constrictor responses remains unknown.

1. Prejunctional purinoceptors modulating the release of noradrenaline were compared in mouse and rat vas deferens. Tissue slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 60 pulses, 1 Hz. 2. In mouse vas deferens, 2-chloroadenosine (IC50 0.24 microM), beta,gamma-methylene-ATP (IC50 3.8 microM), alpha,beta-methylene-ATP (IC50 2.9 microM) and 2-methylthio-ATP (only 30 microM tested) reduced the evoked overflow of tritium. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), 10 nM, antagonized the effect of 2-chloro-adenosine (apparent pKB 10.2) as well as of beta,gamma-methylene-ATP (apparent pKB 9.6) and alpha,beta-methylene-ATP. Suramin, 300 microM, attenuated the effect of 2-chloroadenosine at best very slightly, antagonized the effect of beta,gamma-methylene-ATP (apparent pKB 4.5) and, when combined with DPCPX 10 nM, caused a further marked shift to the right of the concentration-response curve of beta,gamma-methylene-ATP beyond the shift produced by DPCPX alone. 3. In rat vas deferens, 2-chloroadenosine (IC50 0.20 microM), beta,gamma-methylene-ATP (IC50 4.8 microM), alpha,beta-methylene-ATP (IC50 3.0 microM) and 2-methylthio-ATP (only 30 microM tested) also reduced the evoked overflow of tritium. DPCPX, 10 nM, antagonized the effect of 2-chloroadenosine (apparent pKB 9.7) as well as of beta,gamma-methylene-ATP (apparent pKB 9.6) and alpha,beta-methylene-ATP. Suramin, 300 microM, did not change the effect of 2-chloroadenosine, attenuated the effect of beta,gamma-methylene-ATP at best very slightly and, when combined with DPCPX, caused at best a very small shift to the right of the concentration-response curve of beta,gamma-methylene-ATP beyond the shift produced by DPCPX alone.4. It is concluded that prejunctional purinoceptor mechanisms in mouse and rat vas deferens are similar. In either species, both nucleosides such as adenosine and nucleotides such as beta,gamma-methylene-ATP activate a common release-inhibiting receptor which is a Pl- or, more specifically, A1-purinoceptor.There seems to be no need to postulate the existence of a novel prejunctional P3-purinoceptor.Moreover, the sympathetic terminal axons possess an additional P2-purinoceptor in both species which is activated by some nucleotides such as beta,gamma-methylene-ATP and 2-methylthio-ATP, although the activation of the P2-purinoceptor by beta,gamma-methylene-ATP is difficult to demonstrate in the rat.

1. Glutamate inhibits the electrically evoked release of noradrenaline in rabbit brain cortex slices; the inhibition is mediated by adenyl compounds, presumably adenosine. The aim of the present study was to identify the receptors involved in this indirect inhibitory effect of glutamate. Slices of the occipitoparietal cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated by trains of 6 pulses, 100 Hz. 2. The ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AM-PA; 10-100 microM), kainate (10-100 microM) and N-methyl-D-aspartate (NMDA; 30-300 microM) but not the metabotropic glutamate receptor agonist, 1-amino-1,3-cyclopentanedicarboxylate (ACPD; 10-100 microM) reduced the electrically evoked overflow of tritium. 3. The effects of AMPA, kainate and NMDA were attenuated or abolished by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine deaminase but not by the alpha 2-adrenoceptor antagonist yohimbine, the gamma-aminobutyric acid (GABA) receptor antagonists, bicuculline and 2-hydroxysaclofen and the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). 4. The NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5) blocked the inhibitory effect of NMDA but not that of AMPA and kainate. The non-NMDA-receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effect of AMPA but not of kainate and NMDA. 5. In addition to decreasing the electrically evoked overflow of tritium, AMPA, kainate and NMDA but not ACPD caused a steep but transient rise of basal tritium efflux. This immediate releasing effect was not significantly changed by DPCPX, adenosine deaminase, yohimbine, bicuculline, 2-hydroxysaclofen and L-NAME (except that L-NAME enhanced the effect of kainate). AP5 and CNQX antagonized the immediate releasing effects in the same way that they antagonized the inhibition by AMPA, kainate and NMDA of the electrically evoked overflow of tritium.6. It is concluded that AMPA, kainate and NMDA, like glutamate, reduce the electrically evoked release of noradrenaline by releasing adenosine or an adenine nucleotide which is then degraded to adenosine. Activation of each of the three ionotropic glutamate receptors, AMPA, kainate and NMDA receptors, but not activation of metabotropic glutamate receptors can initiate this indirect inhibitory effect on the release of noradrenaline (as well as the known noradrenaline releasing effect).

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.

1. The effects of nifedipine and ryanodine on the adrenergic component of neurogenic contractions of the rat isolated vas deferens were studied in an attempt to identify the sources of Ca2+ mediating the contraction. The tissue was electrically stimulated by single pulses or pairs of widely spaced pulses. The purinergic component of contraction was suppressed by the presence of 300 microM suramin. 2. In Mg(2+)-free medium, nifedipine (0.01-10 microM) reduced the first and, to a greater extent, the second twitch elicited by two pulses 3 s apart. This pattern of inhibition was observed both in the absence of rauwolscine (when twitch 2 was smaller than twitch 1) and in the presence of 0.1 microM rauwolscine (when, due to interruption of prejunctional alpha 2-adrenoceptor-mediated autoinhibition, twitch 2 was of similar height to twitch 1). Nifedipine reduced only twitch 2 but not twitch 1 in medium containing 1.2 mM Mg2+. 3. Single pulses of increasing current strength elicited increasing contraction. Nifedipine reduced contractions by about the same absolute extent at all current strengths, so that the relative contribution of the nifedipine-sensitive component decreased with increasing current strength. 4. When the pulse interval in a pair was increased from 5 to 60 s, the inhibition by nifedipine of the second twitch was most marked at an interval of 5 s and declined as the interval increased. 5. In contrast to nifedipine, 20 microM ryanodine reduced the first twitch of a pair to a greater extent than a second twitch 5 s later, so that twitch 2 became greater than twitch 1. The inhibition by ryanodine of twitch 2 increased with increasing pulse interval.6. In vasa deferentia preincubated with [3H]-noradrenaline, I microM nifedipine and 20 microM ryanodine did not change the electrically evoked overflow of tritium, whereas 10 microM nifedipine increased it.7. It is concluded that, when the sympathetic axons of the vas deferens are stimulated by a single pulse(or the first pulse of a pair) in Mg2+-free medium, both Ca2+ mobilization inside the smooth muscle cells and Ca2+ entry contribute to the ensuing adrenergic contraction. The relative contribution of Ca2+ entry is small at maximal stimulus strength but increases with decreasing stimulus strength. When a second pulse follows the first after an appropriate interval, a switch of Ca2+ sources occurs: intracellular Ca2+mobilization is decreased during twitch 2, whereas Ca2+ entry is increased.

1. Effects of opioid agonists on the purinergic and adrenergic components of neurogenic contractions and in some experiments on transmitter overflow were studied in the mouse isolated vas deferens. 2. When the vas deferens was stimulated every 2 min by pairs of pulses 2 s apart in the presence of prazosin 0.3 microM (to isolate the purinergic component) or alpha,beta-methylene-ATP 3 microM (to isolate the adrenergic component), each pulse elicited a separate twitch. The opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO, mu-receptor-selective), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-selective) and trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide (U-50488, kappa-selective) concentration-dependently reduced both purinergic and adrenergic contractions. For each agonist, maximal effects and concentrations causing half-maximal effects were very similar for inhibition of the purinergic component on the one hand and for inhibition of the adrenergic component on the other hand, although the adrenergic component was inhibited with a slight preference. Moreover, effects on contractions elicited by the first and the second pulse of the pairs were very similar. 3. When vasa deferentia preincubated with [3H]-noradrenaline were stimulated with trains of 100 pulses delivered at 20 Hz, morphine 10 microM reduced significantly both evoked tritium overflow and evoked contractions. Its effect was antagonized by naloxone. 4. It is concluded that, in contrast to drugs acting at some other presynaptic receptors, opioid mu-, delta- and kappa-agonists inhibit purinergic and adrenergic neurogenic contractions of the mouse vas deferens in a similar manner. In contrast to a previous report, no enhancement by morphine of the release of noradrenaline elicited by high frequency pulse trains was observed.

A possible contribution of adenine nucleotides to the endogenous purinergic, A1-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1-100 nmol/l) as well as the enzyme adenosine deaminase (0.1-10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The alpha 1-adrenoceptor-selective agonist methoxamine (10 but not 1 mumol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mumol/l but then not attenuated further by DPCPX 100 nmol/l. L-Glutamate (300 mumol/l-2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mumol/l but greatly, and to the same extent, attenuated by DPCPX 100 nmol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg++ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mumol/l and 1 mmol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg++).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz. Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADP beta S, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine greater than ATP approximately ATP gamma S approximately beta,gamma-imido-ATP approximately ADP greater than beta,gamma-methylene-ATP. AMP 30 mumol/l and AMPS 30 mumol/l were approximately equieffective with 30 mumol/l of adenosine and ATP gamma S, and ADP beta S 30 mumol/l was approximately equieffective with 30 mumol/l of ADP. alpha,beta-Methylene-ADP, 2-methylthio-ATP, UTP and GTP gamma S did not change the evoked overflow of tritium. alpha,beta-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to alpha,beta-methylene-ATP or when the slices were stimulated by 30 pulses, 10 Hz. Neither adenosine deaminase (100 U/l) nor the blocker of 5'-nucleotidase, alpha,beta-methylene-ADP (10 mumol/l), attenuated the inhibition caused by ATP, ATP gamma S and beta,gamma-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP to the right by very similar degrees. 8-(p-Sulphophenyl)-theophylline (30 and 300 mumol/l) also markedly antagonized the inhibition produced by ATP gamma S. alpha,beta-Methylene-ATP (10 and 30 mumol/l) and suramin (100 mumol/l) did not modify the effects of adenosine, ATP gamma S and beta,gamma-methylene-ATP. It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A1 subtype of the P1-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2x-, P2y- or P3-purinoceptors.

Effects of electrical stimulation and nicotine on ATP and tritium outflow and smooth muscle tension were studied in the guinea-pig isolated vas deferens preincubated with [3H]-noradrenaline. ATP was measured using the luciferase technique. Electrical stimulation caused biphasic contractions and an acceleration of ATP and tritium outflow. The contraction amplitude and the overflow of ATP increased markedly, whereas the overflow of tritium increased only slightly with the frequency of stimulation (1-10 Hz; constant number of 60 pulses). The contraction amplitude did not increase with an increase in pulse number (20-540 pulses; constant frequency of 5 Hz), whereas the overflow of ATP increased slightly, and that of tritium markedly. Nicotine caused monophasic, transient contractions and, again, an acceleration of ATP and tritium outflow. Contractions, ATP and tritium overflow increased with the concentration of nicotine (56-320 mumol/l) in an approximately parallel manner. The influence of some drugs on responses to electrical stimulation (60 pulses, 5 Hz) and nicotine (180 mumol/l) was investigated. Tetrodotoxin blocked all effects of electrical stimulation but did not change those of nicotine. The reverse was true for hexamethonium. Neither electrical stimulation nor nicotine caused contraction or an increase in ATP outflow after pretreatment with 6-hydroxydopamine. The main effects of prazosin 0.3 mumol/l were to reduce electrically evoked contractions (above all second phase) as well as nicotine-evoked contractions and the nicotine-evoked overflow of ATP (the latter by about 81%). Prazosin also tended to diminish the electrically evoked overflow of ATP. alpha,beta-Methylene-ATP 10 mumol/l elicited a transient contraction and ATP overflow on its own. The main change in the subsequent state of desensitization was a decrease of the first phase of electrically evoked contractions. The main effects of prazosin combined with desensitization by alpha,beta-methylene-ATP were marked decreases of electrically evoked contractions (by 94%), the electrically evoked overflow ATP (by 66%), nicotine-evoked contractions (by 97%) and the nicotine-evoked overflow of ATP (by 70%). It is concluded that both electrical stimulation and nicotine release noradrenaline and ATP in guinea-pig vas deferens. Only part of the evoked overflow of ATP (about 32%) is neural in origin. Another part probably originates from smooth muscle cells where it is released by neurogenic noradrenaline acting at alpha 1-adrenoceptors. Corelease leads to cotransmission: electrically as well as nicotine-evoked contractions consist of adrenergic and purinergic components. Varying types of stimulation release cotransmitter mixtures of varying composition.(ABSTRACT TRUNCATED AT 400 WORDS)

The question of the existence of postjunctional, contraction-mediating alpha 2-adrenoceptors, in addition to the known alpha 1-adrenoceptors, was studied in the mouse isolated vas deferens. Both the alpha 1-selective agonist phenylephrine and the alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) caused contraction of the vas deferens. In the presence of the alpha 1-selective antagonist prazosin (added in order to prevent an alpha 1 component in the effect of high concentrations of UK 14,304), the alpha 2-selective antagonist yohimbine and idazoxan shifted the concentration-response curve of UK 14,304 to the right in a manner compatible with competitive antagonism and with dissociation constants KB indicating the involvement of alpha 2-adrenoceptors. The maximal contraction elicited by UK 14,304 (in the presence of prazosin) was much lower than the maximal contraction elicited by phenylephrine. The effect of UK 14,304 was not changed by the P2-purinoceptor agonist alpha,beta-methylene-ATP and was reduced by neuropeptide Y, but was markedly enhanced by relatively low concentrations of phenylephrine. When the sympathetic fibres of the vas deferens were stimulated by trains of ten widely spaced (0.5 Hz) electric pulses, the tissue responded with ten separate twitches in which purinergic and adrenergic components were isolated by prazosin and suramin, respectively. Prazosin reduced the first adrenergic twitch in these trains at concentrations close to its KB value at alpha 1-adrenoceptors, whereas yohimbine and idazoxan reduced the first adrenergic twitch at concentrations far lower than their KB values at alpha 1-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Postganglionic sympathetic cotransmission by noradrenaline (NA) and adenosine 5'-triphosphate (ATP) was studied in isolated arteries from rabbits using as tools alpha-adrenoceptor antagonists and alpha, beta-methylene-ATP which first activates and then desensitizes purine P2X receptors. In the pulmonary artery, NA was the only chemical signal responsible for neurogenic vasoconstriction. In sharp contrast, ATP was the only signal eliciting electric as well as mechanical postjunctional responses in small jejunal arteries. Mixed adrenergic and purinergic transmission was found in the largest ramus caecalis of the ileocolic artery. The purinergic component prevailed in short pulse trains and early in long trains, whereas the adrenergic component prevailed in the late phases of long (20 s) trains. Prejunctional alpha 2-adrenergic autoinhibition markedly depressed purinergic as well as adrenergic transmission as soon as a latency of about 2 s was exceeded.

Effects of various nucleotides, nucleosides and noradrenaline on smooth muscle tension were studied in the isolated mouse vas deferens. alpha, beta-Methylene-ATP, ATP gamma S, noradrenaline, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine or uridine (up to 100 mumol/l). Prolonged incubation with alpha, beta-methylene-ATP (concentration increased stepwise from 0 to 15 mumol/l) selectively reduced contractions induced by ATP and UTP but not those induced by noradrenaline, and there was cross-tachyphylaxis between ATP and UTP. Suramin (10-300 mumol/l) did not alter the response to noradrenaline but shifted the concentration-response curves for alpha, beta-methylene-ATP, ATP gamma S, UTP and lower concentrations of ATP (0.1-1 mumol/l) to the right. The pA2-values of suramin were 5.2 against alpha, beta-methylene-ATP, 4.8 against ATP gamma S, 5.1 against UTP and 5.4 against lower concentrations of ATP. The effects of higher concentrations of ATP were largely resistant to suramin. The results indicate that the mouse vas deferens possesses contraction-mediating smooth muscle P2x-receptors. UTP also acts at this receptor, and there is no evidence for a separate UTP receptor. The selective inhibition of nucleotide- but not noradrenaline-induced contractions by suramin confirms the view that suramin is a selective P2-antagonist. The resistance against suramin of part of the effect of ATP suggests that ATP activates a suramin-insensitive site in addition to the P2x-receptor.

Uridine 5'-triphosphate- (UTP-) and adenosine 5'-triphosphate- (ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure. Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5'-O-(3-thio)-triphosphate (ATP gamma S), and beta,gamma-imido adenosine 5'-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, alpha,beta-methylene-ATP and beta,gamma-methylene-ATP up to 10(-3) mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP much greater than UMP = CTP; that of the purine nucleotides was: ATP gamma S greater than AMP-PNP greater than ATP greater than ADP greater than 2-methylthio-ATP = alpha,beta-methylene-ATP = beta,gamma-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indomethacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to alpha,beta-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATP gamma S elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

The largest rami caecales of the ileocolic artery (a branch of the mesenteric artery) were perfused at a constant rate of flow. Either vasoconstriction or the release of previously incorporated [3H]noradrenaline was measured. The following opioid agonists inhibited the vasoconstriction elicited by electrical field pulses (5 pulses at 10 Hz; EC50 values in brackets): [Leu5]enkephalin (596 nmol/l), [D-Ala2,D-Leu5]enkephalin (69 nmol/l), dynorphin-(1-13) (366 nmol/l) and ethylketocyclazocine (668 nmol/l). Fentanyl (up to 30 mumol/l) and normorphine (up to 100 mumol/l) caused at best minimal inhibition. The effects of [Leu5]enkephalin and dynorphin-(1-13) were antagonized by naloxone. Only the effect of [Leu5]enkephalin but not that of dynorphin-(1-13) was antagonized by the delta-selective antagonist ICI 154129. [Leu5]Enkephalin and dynorphin-(1-13) did not decrease the vasoconstrictor response to exogenous noradrenaline or ATP. In arteries preincubated with [3H]noradrenaline, electrical stimulation (50 pulses at 1 Hz) increased the outflow of tritium. The stimulation-evoked overflow was reduced by [Leu5]enkephalin and dynorphin-(1-13), and the effect of [Leu5]enkephalin was antagonized by naloxone. It is concluded that the postganglionic sympathetic neurons of the ramus caecalis possess presynaptic opioid receptors which, when activated, inhibited transmitter release. The receptors appear to be of the delta- and kappa- but not the mu-type.

Effects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [3H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S1, S2). alpha,beta-Methylene-ATP, ATP gamma S, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 mumol/l) and uridine (up to 1 mmol/l). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATP gamma S greater than ATP = adenosine greater than UTP; alpha,beta-methylene-ATP (up to 10 mumol/l) and uridine (up to 1 mmol/l) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATP gamma S were not significantly attenuated. After prolonged exposure to alpha,beta-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2. It is concluded that ATP, ATP gamma S, alpha,beta-methylene-ATP and UTP produce contraction of the vas deferens by activation of P2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P2-receptor which differs from the P2x-receptor and may be a reactive blue 2-resistant "P2y-like" receptor.

1. The mechanism of uridine 5'-triphosphate-(UTP-)induced vasoconstriction was studied in the rabbit ear artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure. 2. Noradrenaline, adenosine 5'-triphosphate (ATP) and UTP caused concentration-dependent vasoconstriction. ATP and UTP were approximately equipotent. 3. The vasoconstrictor effect of UTP 300 mumol/l was enhanced by a mixture of atropine, diphenhydramine and methysergide (1 mumol/l each) and not affected by indomethacin 10 mumol/l. 4. Prazosin (0.01-1 mumol/l) and phentolamine (1-10 mumol/l) reduced the vasoconstrictor effect of UTP 300 mumol/l by up to 34%. Prazosin 1 mumol/l failed to diminish the vasoconstrictor effect of UTP 300 mumol/l after the sympathetic nerves had been destroyed with 6-hydroxydopamine. 5. alpha, beta-Methylene-ATP (10-50 mumol/l) elicited transient vasoconstriction. Subsequently, vasoconstrictor responses to ATP 100 or 300 mumol/l were reduced by 88%, whereas responses to UTP 100 mumol/l were enhanced, responses to UTP 300 mumol/l decreased by only 32% and responses to UTP 1000 mumol/l reduced by 74%. After in vitro-denervation with 6-hydroxydopamine or in the presence of phentolamine 1 mumol/l throughout, alpha, beta-methylene-ATP (10-50 mumol/l) reduced the vasoconstrictor effect of UTP 300 mumol/l by 44% and 43%, respectively. 6. We suggest that, in the rabbit ear artery, the non-adrenergic and alpha, beta-methylene-ATP-resistant vasoconstrictor response to UTP is mediated by a separate receptor mechanism, distinct from the P2 purinoceptor.

Vasa deferentia from mice were field-stimulated by trains of 10 pulses delivered at 0.5 Hz. The pulses elicited separate twitches, the first of which (corresponding to a single pulse) exceeded the following ones in height and width and often was clearly biphasic. alpha, beta-Methylene-ATP 1 mumol/l and suramin 100 mumol/l caused almost identical changes. They reduced the height of the first twitch in the train by about one half and also reduced its width in a manner indicating that only the second, slow phase remained, but reduced much more markedly the following twitches in which now a small second, slow phase also became detectable. Idazoxan 0.1 mumol/l or yohimbine 0.1 mumol/l, when added in the presence of alpha, beta-methylene-ATP or suramin, further decreased the first twitch but enhanced twitches No. 2 to 10. These responses were then almost abolished by prazosin 0.1 mumol/l. Successive addition of prazosin 0.1 mumol/l and idazoxan 0.1 mumol/l to previously untreated vasa deferentia depressed the response to the first pulse by about one half in a manner indicating that only the first, rapid phase remained, but had comparatively little effect on the responses to the subsequent pulses. Suramin 100 mumol/l almost abolished the contractions remaining in the presence of prazosin and idazoxan. The results indicate that the first, rapid phase of the neurogenic contractions elicited by single or low frequency pulses is mediated by ATP which substantially contributes to all responses in a train. The second, slow phase is mediated by noradrenaline which substantially contributes to the response to the first pulse only.(ABSTRACT TRUNCATED AT 250 WORDS)