Pre- and postjunctional modulation of cholinergic neuroeffector transmission by adenine nucleotides. Experiments with agonist and antagonist

Adenosine, 2-chloroadenosine, AMP, AMPNH2, ADP, ATP, AMPPNP and beta, gamma meATP dose-dependently and reversibly inhibited contractile responses to nerve stimulation in longitudinal plexus-containing muscle preparations of guinea pig ileum. All the purines, except for 2-chloroadenosine, were virtually equipotent and elicited maximal inhibition within 2 min after application. Adenosine deaminase abolished inhibition by adenosine but did not block the effect of 2-chloroadenosine or the adenine nucleotides. During transmural nerve stimulation, the nucleotidase-resistant analogues alpha, beta meADP and AMPPNP significantly depressed acetylcholine release measured by gas chromatography-mass spectrometry. The inhibitory effect of all the purines was competitively, reversibly and at comparable pA2 values antagonized by 8-p-sulphophenyltheophylline. The results indicate that nucleotides per se can inhibit neurotransmission via a prejunctional receptor common to nucleosides and nucleotides. An excitatory effect by all the di- and triphosphate nucleotides was also observed. The excitation was unaltered by atropine, tetrodotoxin and 8-p-sulphophenyltheophylline, thus suggesting an action at postjunctional ADP- and ATP-like receptive sites.     

Agonist and antagonist characterization of the P2-purinoceptors in the guinea pig ileum

When adenine nucleotides were administered to isolated guinea pig ileum longitudinal muscle, two immediate effects were observed: a contractile effect and a concomitant inhibition of the responses elicited by transmural nerve stimulation. At concentrations up to 10^-4m the order of potency for the contractile effect was α,β-MeADP =α,β -MeATP > ADP = ATP = AMPPNP =β,t -MeATP > 2′-deoxy AMP = 2′-deoxy ADP. AMP and adenosine did not show any contractile effect, whereas both compounds dose-dependently and reversibly inhibited the nerve-induced contractile responses. ADP, ATP, β,t -MeATP and AMPPNP also inhibited contractile responses to transmural nerve stimulation, whereas 2′-deoxy AMP, 2′-deoxy ADP, α,β -MeADP and α,β -MeATP showed but weak inhibitory effects, 2′-deoxyadenosine, IMP, IDP, ITP, 8-BrATP and TDP lacked significant contractile effects and did not exert a significant inhibition on nerve-induced contractions. p-chloromercuribenzene sulphonic acid (PCMBS) irreversibly antagonized the contractile effects of ADP, ATP and the α,β -methylene derivatives, whereas dantrolene sodium, tetrodotoxin, scopolamine and 8-p-sulphophenyltheophylline were without effect on nucleotide-induced contractions. The contractile effect of ADP or ATP was unaffected by indomethacin, whereas the contractile effect by α,β -methylene derivatives was abolished by indomethacin. ADP, ATP and α,β -MeADP enhanced contractile responses to exogenous acetylcholine, α,β-MeADP being most effective. This enhancement was blocked by indomethacin. We suggest that ADP and ATP contracted the guinea pig ileum by an action at postjunctional P₂-purinoceptors with different characteristics from prejunctional P₁-purinoceptors. The α,β-methylene analogues seemed to act at least at one different excitatory site and promotion of prostaglandin biosynthesis was critical for their effect.     

Adenosine modulation of neuroeffector transmission in guinea-pig uterine smooth muscle.

Effects of adenosine and adenosine analogues on neuroeffector transmission in separate layers of the guinea-pig uterine smooth muscle during different phases of the oestrus cycle were studied. Adenosine (ADO), N6-[(R)-1-methyl-2-phenylethyl]- adenosine (R-PIA) and 5'-N-ethylcarboxamideadenosine (NECA) enhanced spontaneous contractile activity as well as contractile responses to nerve stimulation or direct muscle stimulation in the longitudinal and circular muscle layers both at the time of ovulation and implantation. The agonist potency order was R-PIA greater than or equal to NECA greater than ADO. Furthermore, in the circular muscle layer inhibitory effects were seen at time of implantation and the agonist potency for this inhibitory effect was NECA greater than R-PIA greater than or equal to ADO. 8-p-sulphophenyltheophylline (8-PSOT) antagonized both the stimulatory and inhibitory effects of the purines and 8-p-sulphophenyltheophylline applied to untreated preparations inhibited nerve-induced contractile activity. NECA inhibited stimulation-induced release of 3H in preparations incubated with [3H]noradrenaline. We suggest that adenosine and its analogues can modulate adrenergic neuroeffector transmission in guinea-pig uterine smooth muscle via action at postjunctional receptors stimulating contractile activity and prejunctional receptors inhibiting transmitter release. In the circular muscle layer postjunctional receptors inhibiting contractile activity were evident at time of implantation. Furthermore, endogenous purines exerting a stimulatory action on the neuroeffector transmission were likely formed during the present experimental conditions.     

Dual effects of adenosine and adenosine analogues on motor activity of the human fallopian tube

Effects of adenosine and adenosine analogues on spontaneous contractility of the human fallopian tube during different phases of the menstrual cycle were studied. In isthmic preparations, a stimulatory effect by L-N6-phenylisopropyladenosine (L-PIA), with preference for adenosine A1-receptors, was seen mainly during the proliferative phase. In ampullary preparations, stimulation by L-PIA was seen both in the secretory and proliferative phases. Adenosine and 2-chloroadenosine exerted similar stimulatory effects. 5'-N-ethylcarboxamide-adenosine (NECA), with selectivity for adenosine A2-receptors, or D-PIA never showed a stimulatory effect. At concentrations above those needed for stimulation, adenosine, 2-chloroadenosine and L-PIA inhibited spontaneous contractions, in common with NECA and D-PIA. Here, NECA was more potent than L-PIA. The D-PIA and L-PIA were equipotent. The inhibition was seen during the whole menstrual cycle. The competitive adenosine antagonist 8-p-sulphophenyltheophylline (PS?T) reversibly antagonized the stimulatory and inhibitory effects elicited by adenosine and the analogues. The PS?T alone could exert a stimulatory or an inhibitory action on spontaneous contractility. We suggest that adenosine can modulate contractile activity in the human fallopian tube via stimulatory A1-and inhibitory A2-receptors. These receptors are located on the smooth muscle cells, and might act via cAMP. The relative receptor dominance may be influenced by cyclic hormonal changes.     

Theophylline interferes with the modulatory role of endogenous adenosine on cholinergic neurotransmission in guinea pig ileum

The ability of theophylline and other phosphodiesterase inhibitors to alter contractile responses to cholinergic nerve stimulation was investigated in isolated longitudinal muscle of the guinea pig ileum. Theophylline in low concentrations (10–100 μM), having no or little effect on measured phosphodiesterase activity, antagonized inhibitory effects of exogenous adenosine. In higher concentrations (0.1–10 mM), shown to be effective in inhibiting phosphodiesterase, theophylline as well as a “pure” cAMP phosphodiesterase inhibitor, ZK 62, 711, inhibited contractile responses. Dipyridamole and dilazep, inhibitors of adenosine inactivation, and also selective inhibitors of cAMP and cGMP phosphodiesterase, respectively, were found to enhance effects of exogenous adenosine and to cause a marked leftward shift of adenosine threshold dose. When dipyridamole and dilazep by themselves had inhibitory effects these could be antagonized by theophylline, suggesting an action through increased levels of endogenous adenosine. As a further indication of endogenous adenosine modulating neurotransmission low concentrations of theophylline enhanced responses to transmural stimulation. Endogenous purine concentrations in tissues and bath media were measured by HPLC. Because of tissue and microbial adenosine inactivation direct estimates of extracellular adenosine concentration could not be obtained. However, adenosine levels increased during transmural stimulation, and during inhibition of adenosine inactivation were sufficient, even in the bath medium, to interfere with the cholinergic neurotransmission.     

Characterization of pre- and post-junctional adenosine receptors in guinea-pig ileum

The receptors involved in adenosine-induced modulation of cholinergic neuroeffector transmission in guinea-pig ileum were explored by means of the non-selective stable analogue, 2-chloroadenosine and analogues with preference for AI receptors, L-N6-phenylisopropyladenosine (L-PIA), and A2 receptors, 5'-N-ethylcarboxamideadenosine (NECA) and D-N6-phenylisopropyladenosine (D-PIA). 2-chloroadenosine, L-PIA and NECA were equipotent in inhibiting contractile responses to nerve stimulation, whereas D-PIA exerted a similar activity only in high concentrations. The release of acetylcholine induced by nerve stimulation was inhibited to a similar degree by NECA, L-PIA and D-PIA. The phosphodiesterase inhibitor, ZK 62.7II, and the activator of adenylate cyclase, forskolin, enhanced the inhibitory effect of NECA, but not that of L-PIA, on contractile responses to nerve stimulation. Only NECA inhibited contractions induced by direct muscle stimulation and ZK 62.7II enhanced this inhibition. It is concluded that adenosine inhibits the neuroeffector transmission in guinea-pig ileum mainly by a prejunctional, cAMP-independent, mechanism, involving AI receptors and a supplementary activation of post-junctional A2 receptors. In addition there may be a prejunctional inhibitory effect of high agonist concentrations, exerted via A2 receptors and influenced by the prevailing levels of cAMP.     

Neuromuscular actions of endothelin on smooth, cardiac and skeletal muscle from guinea-pig, rat and rabbit

The peptide endothelin (human, porcine) was investigated for effects on basal muscle tone and on responses to transmural nerve stimulation in a series of smooth muscle preparations, as well as in guinea-pig atrium and rat and guinea-pig diaphragm. Endothelin lacked effect on basal tone or on spontaneous and electrically driven contractions in skeletal and atrial muscle. It contracted guinea-pig ileum, pulmonary and femoral arteries, rat anococcygeus, vas deferens and urinary bladder and rabbit taenia coli, whereas guinea-pig taenia was relaxed. Guinea-pig urinary bladder and vas deferens and rabbit iris sphincter were unaffected up to 3 x 10(-8) M. Endothelin thus has a unique pattern of smooth muscle effects, exhibiting mostly contractile but also relaxing effects. Endothelin modified contractile responses to transmural nerve stimulation, yielding marked and persistent enhancement, in guinea-pig and rat vas deferens, and enhancement also in guinea-pig pulmonary artery. In guinea-pig and rat vas deferens the response to exogenous ATP was increased by endothelin, thus suggesting a strong post-junctional enhancement of neurotransmission. In guinea-pig ileum nerve-induced responses were inhibited by endothelin, whereas exogeneous acetylcholine was enhanced, an effect suggesting a simultaneous pre-junctional inhibition and post-junctional enhancement. The Ca2+ channel blocker felodipine counteracted the stimulatory effects of endothelin on tone and transmurally induced contractions. Tachyphylaxis to endothelin action was sometimes evident, but the anococcygeus being less prone to this might be useful for studies on endothelin antagonism. Endothelin thus has prominent post-junctional, and also probably pre-junctional, effects, lending further support for a distinct biological role of this peptide.     

Cholinergic neuromodulation by endothelin in guinea pig ileum

The effect of endothelin on cholinergic neuroeffector transmission in guinea pig ileum was investigated. Endothelin was shown to inhibit the nerve-induced contractions and concomitantly to increase the basal muscle tone. Furthermore, endothelin inhibited the nerve-induced release of [3H]acetylcholine whereas the contractile response to exogenous acetylcholine was enhanced. In conclusion, our findings suggest that endothelin is a modulator of cholinergic neuroeffector transmission in guinea pig ileum with possible action via both inhibitory prejunctional and stimulatory postjunctional mechanisms.     

Neuromodulation by adenine nucleotides, as indicated by experiments with inhibitors of nucleotide inactivation

The adenine nucleotides AMP, ADP and ATP (3 X 10(-7) M and above) inhibited contractile responses to transmural nerve stimulation in guinea-pig ileum longitudinal muscle via a prejunctional action. Nucleotides assumed to inhibit the degradation of adenine nucleotides were employed to determine whether inhibition of contractile responses was elicited by adenine nucleotides per se, or required breakdown to adenosine. The IMP or 2'-deoxy AMP enhanced the prejunctional inhibitory effect elicited by AMP. A similar enhancement of the inhibitory effect of ADP and ATP was seen after administration of IDP and ITP, respectively. The inhibitory effect of adenosine was not enhanced by inosine, IMP or IDP. The 5'-nucleotidase inhibitor, TDP enhanced inhibition elicited by ADP. In contrast, alpha, beta-meADP did not influence the prejunctional inhibitory effect elicited by the adenine nucleotides. However, the combination of alpha, beta-meADP and IMP enhanced the inhibitory effect of ATP. The postjunctional contractile effect elicited by ADP and ATP was enhanced by pretreatment with inosine nucleotides, alpha, beta-meADP or TDP, indicating decreased inactivation of ADP and ATP during concurrent nucleotide administration. The fact that the prejunctional effect of adenine nucleotides can be enhanced by forms of pretreatment known to antagonize the breakdown of adenine nucleotides, constitutes strong evidence for prejunctional action per se by adenine nucleotides.     

Endogenous purines may modulate adrenergic neurotransmission in the human fallopian tube

In the present investigation we have examined the possibility that endogenous purines may act as neuromodulatory agents in the human fallopian tube, using the selective adenosine receptor antagonist 8-p-sulfophenyltheophylline (PSOT). PSOT applied to untreated preparations enhanced the contractile responses to transmural nerve stimulation in the human fallopian tube, both in the secretory and proliferative hormonal phases. Furthermore, PSOT enhanced nerve-induced [3H]noradrenaline release during both hormonal phases. This can be taken as indirect evidence that endogenous purines inhibited the adrenergic neuroeffector transmission under the experimental conditions. The inhibitory effect presumably was exerted at both pre- and postjunctional adenosine receptors.     

Adenosine and ATP effects on isolated guinea pig gallbladder

The effects of adenosine, ATP and several derivatives of adenosine were measured in isolated strips of guinea pig gallbladder. Adenosine caused relaxations which were antagonized by theophylline and potentiated by an inhibitor of adenosine uptake, 6-(1-hydroxy-5-nitrobenzylthio)-guanosine (HNBTG). Among several adenosine derivatives, 2-chloroadenosine and 5′-N-ethylcarboxymidoadenosine were similarly effective while 1-N⁶-phenylisopropyladenosine was only a weak relaxant. None of the derivatives caused maximal relaxations at 100 μM, and thus absolute potencies could not be determined. ATP caused predominantly contractile effects, with relaxations sometimes being evident at high concentrations. Indomethacin abolished contractile effects of ATP, suggesting prostaglandin involvement, and only relaxations were evident in its presence. Adenosine deaminase abolished the effects of adenosine and partly reduced the relaxant effects of ATP in the presence of indomethacin. In view of the low potency of adenosine and ATP, physiological roles for these compounds in gallbladder motility are not readily evident.     

Enhancement of transmission at the frog neuromuscular junction by adenosine deaminase: evidence for an inhibitory role of endogenous adenosine on neuromuscular transmission

Adenosine deaminase reversibly increased the amplitude and the quantum content of the end-plate potentials (EPPs) recorded from superficial muscle fibers of frog sartorius preparations in which twitches have been prevented with high-magnesium solutions. Adenosine deaminase prevented the inhibitory effect of exogenously applied adenosine but not that of 2-chloroadenosine on the amplitude of EPPs. The effect of adenosine deaminase was abolished by erythro-9(2-hydroxy-3-nonyl)adenine (EHNA). The results suggest that endogenous adenosine exerts an inhibitory 'tone' over neuromuscular transmission.     

Influence of adenosine on responses to vagal nerve stimulation in the anesthetized rabbit

The influence of local adenosine infusion into the celiac artery on the gastric contractile responses to centrifugal vagal nerve stimulation was studied in anesthetized rabbits, and was compared with the effects of systemic administration of equivalent amounts of adenosine. Close arterial infusion of adenosine caused a marked reduction of gastric contractions induced by nerve stimulation, whereas corresponding responses to close arterial infusions of acetylcholine were enhanced during adenosine. The comparison with systemic adenosine administration revealed that the influence on gastric neurotransmission was not related to the hypotensive effect of the compound. No effects of adenosine were seen on bronchial activity as measured by insufflation pressure. Variable effects were obtained on cardiac responses to vagal stimulation. Gastric smooth muscle contractions elicited in vitro by transmural nerve stimulation were affected by adenosine in a biphasic manner, initial inhibition followed by potentiation of the apparently cholinergic responses. It is suggested that adenosine may modulate cholinergic neurotransmission in vivo by a dual effect, prejunctional inhibition and postjunctional enhancement.     

Inhibition of acetylcholine release in guinea pig ileum by adenosine.

The effect of adenosine on cholinergic neuroeffector transmission was studied in the isolated guinea pig ileum. Adenosine caused a dose-dependent and inverse frequency-dependent inhibition of contraction responses to transmural nerve stimulation. Blockade of adrenergic neurotransmission did not alter the inhibitory effect of adenosine. Adenosine also inhibited contraction responses to serotonin, angiotensin and high potassium, but not the responses to acetylcholine, histamine or direct electrical stimulation of the smooth muscle cells. Adenosine had little effect on basal outflow of acetylcholine but inhibited markedly and reversibly the release of acetylcholine induced by nerve stimulation. Acetylcholine was determined with gas chromatography-mass spectrometry. The results provide direct evidence that adenosine inhibits cholinergic neuroeffector transmission in the gut by a prejunctional action on acetylcholine release. This may be of functional importance since adenine compounds are released during stimulation of intestinal nerves.     

Adenosine reduces catecholamine contractile responses in oxygenated and hypoxic atria

The properties of adenosine attenuation of catecholamine-elicited increases in peak contractile force, rate of force development, and rate of relaxation were studied in isolated rat atria. Adenosine, at a concentration that did not cause a direct depressant effect by itself, was capable of reducing by approximately 15% the increase in the contractile parameters elicited by isoproterenol. This reduction was not overcome by elevating the catecholamine concentration. The adenosine reduction was prevented by theophylline or the presence of adenosine deaminase. The reduction appears to be independent of the acetylcholine-mediated reduction of catecholamine responses. Adenosine reduced the positive inotropic responses elicited by norepinephrine and epinephrine but not phenylephrine. Adenosine deaminase in oxygenated atria potentiated the catecholamine-elicited contractile responses and reduced the progressive fall of the elevated contractile responses observed with continual catecholamine stimulation. In hypoxic atria adenosine deaminase potentiated the positive inotropic responses observed with catecholamine stimulation. The results suggest that an adenosine-specific mechanism is capable of attenuating the elevation in contractility elicited by beta-adrenergic stimulation. In addition, endogenous adenosine may be responsible, in part, for the reduction of catecholamine-mediated contractile responses in oxygenated and hypoxic myocardial tissue.     

Presynaptic inhibitory actions of adenine nucleotides and adenosine on neurotransmission in the rat vas deferens.

Adenine nucleotides and adenosine inhibited the isometric contractions of the rat vas deferens in vitro in response to field stimulation but had no effect on the responses to exogenous noradrenaline. The inhibitions were potentiated by dipyridamole and compound 555, antagonized by theophylline and unchanged by indomethacin, 2-2′-pyridylistogen, phenoxybenzamine and atropine. Adenosine and adenosine 5′-triphosphate inhibited the release of [³H]noradrenaline produced by field stimulation.These results indicate that adenine nucleotides, probably acting via the common metabolite adenosine, inhibit adrenergic neurotransmission at a presynaptic site. Their antagonism by theophylline suggests that a presynaptic ‘purinergic’ receptor system could be involved.     

The inhibitory effect of adenosine and related nucleotides on the release of acetylcholine

Isolated Auerbach's plexus-longitudinal muscle preparations from guinea-pig ileum and slices of the rat cerebral cortex have been used to study the effect of adenine nucleotides on the release of acetylcholine. The release of acetylcholine evoked by cholecystokinin was completely inhibited by adenosine. The effect of nucleotides on neuro-effector transmission of electrically stimulated longitudinal muscle strip was also studied. Adenosine and adenosine triphosphate reduced the release of acetylcholine provided a low frequency of stimulation was applied. While the three adenine nucleotides (adenosine mono-, di- and triphosphate) dose-dependently reduced neuro-effector transmission in Auerbach's plexus-longitudinal muscle preparation, adenine, guanosine triphosphate and dibutyryl-cyclic AMP had no effect. Theophylline, an adenosine receptor antagonist, prevented the inhibitory effect of the nucleotides. In addition, theophylline alone enhanced the release of acetylcholine both from Auerbach's plexus and from the nerve terminals of the cortical slice. This indicates that there might be a continuous control of ACh release by an adenine nucleotide.These results are discussed in relation to the release of adenosine triphosphate from purinergic nerves in the intestine and of adenosine from slices of cerebral cortex; the possibility is raised that adenine nucleotides released from nerves might act as a type of presynaptic inhibitory transmitter on cholinergic neurons. Furthermore, if some of the released nucleotide originates from the synaptic vesicles of cholinergic neurons, it might serve as a negative feed-back transmitter for acetylcholine release.The inhibitory effect of adenosine and related nucleotides on the cholecystokinin-induced release of acetylcholine from the gut might be of physiological importance since gastrointestinal polypeptides play a very important role in maintaining gastrointestinal motility.     

An examination of nerve-mediated, hyoscine-resistant excitation of the guinea-pig colon

1. The mechanical and electrical activity of the smooth muscle of the distal colon of the guinea-pig has been recorded in experiments designed to determine the nature of the nerve-mediated excitation of the muscle.2. The spontaneous contractions of the colon, normally observed in vitro, were similarly antagonized by hyoscine or tetrodotoxin. However, neither drug caused complete cessation of the spontaneous activity.3. The contractile responses of the colon to repetitive stimulation of intramural nerve fibres were of two types, a primary contraction which occurred within 1 sec of the beginning of stimulation and a secondary contraction which occurred after stimulation. The primary contraction was blocked by hyoscine, usually revealing an inhibitory response to stimulation, but the secondary contraction persisted. Similar primary contractions were observed in response to stimulation of the pelvic and sometimes of the periarterial nerves.4. An initial relaxation during, and a secondary contraction after, stimulation were sometimes obtained when the periarterial nerves were stimulated. Propranolol completely blocked both the initial relaxation and the secondary contraction in response to sympathetic stimulation but did not affect either the relaxation or the secondary contraction in response to transmural stimulation.5. The direct effects of noradrenaline and adenosine triphosphate on the mechanical activity of the colon were studied. Both drugs caused a relaxation of the colon. Washout of either drug after a short exposure was followed by a period of increased activity. Especially in preparations of low tone, the initial relaxation and the secondary contraction in response to transmural stimulation could be mimicked by the application and washout of either noradrenaline or adenosine triphosphate.6. Two types of potential change were evoked in the muscle cells of the colon, either separately or in combination, when the intramural nerve fibres were stimulated: excitatory junction potentials (EJPs), which were blocked by hyoscine, and inhibitory junction potentials (IJPs). When the membrane potential recovered following an IJP, it was common to observe action potentials in muscle cells that were initially quiescent. A similar secondary firing of action potentials was initiated by hyperpolarizing the muscle cells with anodal current pulses in the presence of tetrodotoxin in sufficient concentration to block nerve-mediated responses.7. The results reported in this paper lead to the conclusion that the muscle cells of the distal colon of the guinea-pig are influenced by three sets of nerves: cholinergic excitatory, adrenergic inhibitory and intrinsic inhibitory fibres releasing a non-adrenergic transmitter substance. The secondary non-cholinergic excitation arises from a non-specific reaction of the muscle cells to a preceding inhibition.     

Interaction of adenosine and its phosphorylated derivatives with putative purinergic receptors in the gill vascular bed of rainbow trout

1. The haemodynamic responses of trout gill to pulses of adenosine and related nucleotides were recorded in isolated trout head preparations. 2. Pulses of adenosine and related nucleotides induced a vasoconstriction of arterial gill vessels. Theophylline antagonized the resonse to adenosine but had not influence on its metabolism. 3. Dipyridamole and two adenosine deaminase inhibitors [deoxycoformycin and erythro-9(2-hydroxy-3-nonyl) adenine] had no effect on either the haemodynamic response of adenosine or its deamination and its uptake by gill tissues. 4. The adenosine response was neither mediated by cholinergic nor adrenergic receptors. 5. These results suggest the existence of extracellular "purinergic receptors" in the gills of trout.     

Estimation of endogenous adenosine activity at adenosine receptors in guinea-pig ileum using a new pharmacological method

Aim: Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. Methods: Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p-sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. Results: Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10⁻⁶ m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). Conclusion: Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose–response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems.