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1. The relative contribution of the putative transmitters, nitric oxide (NO) and an apamin-sensitive factor, possibly ATP, to inhibitory responses evoked by electrical field stimulation (EFS; 0.2-5 Hz, 0.2 ms duration, supra-maximal voltage for 10 s) of non-adrenergic, non-cholinergic (NANC) nerves was investigated in the guinea-pig isolated taenia coli contracted with histamine (1 microM). 2. Peak relaxations to EFS (0.2-5 Hz) were tetrodotoxin (1 microM)-sensitive, maximal at 0.2 Hz and completely resistant to the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG; 100 microM) in either the presence or absence of atropine (1 microM). Furthermore, the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ; 10 microM), the cytochrome P450 inhibitor and free radical generator, 7-ethoxyresorufin (7-ER; 10 microM) and the NO scavenger, oxyhaemoglobin (HbO; 30 microM) had no effect on EFS-induced relaxations alone and in combination with L-NOARG (100 microM). 3. Maximum relaxation to the NO donor, sodium nitroprusside (SNP; 1 microM) was significantly reduced by HbO (30 microM), abolished by 7-ER (10 microM) and ODQ (10 microM) but was unaffected by apamin (0.1 microM), an inhibitor of small conductance Ca(2+)-activated K+ channels. 4. The relaxation to EFS at 0.2 Hz was resistant to apamin but those to 0.5 and 5 Hz were significantly reduced. EFS (0.2-5 Hz)-evoked relaxations that persisted in the presence of apamin were further significantly inhibited by L-NOARG (100 microM) or ODQ (10 microM), but not by HbO (30 microM) or 7-ER (10 microM). 5. ATP (1-30 microM) produced concentration-dependent relaxations that were abolished by apamin (0.1 microM), unaffected by ODQ (10 microM) but only significantly reduced by L-NOARG (100 microM) at the lowest concentration of ATP (1 microM) used. 6. Nifedipine (0.3 microM), abolished contractions to 67 mM KCl, histamine (10 microM), endothelin-1 (0.03 microM), 5-hydroxytryptamine (5-HT; 10 microM) and the thromboxane-mimetic, 9-11-dideoxy-9 alpha, 11 alpha-methano-epoxy-prostaglandin F2 alpha (U46619; 0.1 microM). 7. The findings of the present study suggest that NO is released during NANC nerve stimulation, but plays no role in NANC relaxations in the guinea-pig taenia coli unless the effects of another apamin-sensitive, nerve-derived hyperpolarizing factor (NDHF) are blocked. Thus, we propose that in this tissue, NO acts as a 'backup' or redundant NANC nerve inhibitory transmitter and like NDHF mediates relaxation via hyperpolarization.

Summary 1 . Stimulation of the vagal non-adrenergic inhibitory innervation caused the release of adenosine and inosine into vascular perfusates from the stomachs of guinea-pigs and toads. 2 . Stimulation of portions of Auerbach's plexus isolated from turkey gizzard caused the release of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). 3 . ATP, added to solutions perfused through the toad stomach vasculature, was broken down to adenosine, inosine and adenine. 4 . Of a series of purine and pyrimidine derivatives tested for inhibitory activity on the guinea-pig isolated taenia coli, ATP and ADP were the most potent. 5 . ATP caused inhibition of twelve other gut preparations previously shown to contain non-adrenergic inhibitory nerves. The inhibitory action of ATP was not prevented by tetrodotoxin. 6 . Quinidine antagonized relaxations of the guinea-pig taenia coli caused by catecholamines or adrenergic nerve stimulation. Higher concentrations of quinidine antagonized relaxations caused by ATP or non-adrenergic inhibitory nerve stimulation. 7 . When tachyphylaxis to ATP had been produced in the rabbit ileum, there was a consistent depression of the responses to non-adrenergic inhibitory nerve stimulation but not of responses to adrenergic nerve stimulation. 8 . It is suggested that ATP or a related nucleotide is the transmitter substance released by the non-adrenergic inhibitory innervation of the gut.

Although the effects of adenine nucleotides on smooth muscle were observed as long ago as the 1920s, it is only relatively recently that the study of the pharmacology of these compounds has been seriously undertaken. In reviewing the effect of these purines and their derivatives on smooth muscle, David Satchell describes the classification of purine receptors, and considers whether they have a true physiological role. The major receptor types. P 1 and P 2 , which bind adenosine and ATP respectively, mediate opposing responses within the same tissue and have very specific structural requirements.

1. 1. Small quantities of sea water were recyclically perfused over the surface of paired anterior byssus retractor muscles of Mytilus californianus . 2. 2. Dopamine was identified in the perfusate by thin-layer chromatography. 3. 3. Stimulation of the pedal ganglion caused the dopamine content of the perfusate to increase. 4. 4. A significant increment of release of dopamine was detected at stimulation frequencies above 3 Hz and increased progressively with increase in stimulation frequency. 5. 5. The possibility of a role for dopamine as a relaxing or inhibitory neurotransmitter in Mytilus is considered in relation to the present and related evidence and to the actions of 5-HT, the probable relaxing transmitter.

Field stimulation of isolated rat vas deferens preparations caused a twitch contraction followed by a slower phasic contraction. Exogenously applied ATP and noradrenaline caused rapid contractions and slowly developing contractions respectively. Pretreatment with phentolamine antagonised the phasic response to stimulation and the contraction to noradrenaline. Pretreatment with AMP antagonized the twitch response to stimulation and concentrations to ATP. The results support the view that ATP is released as a co-transmitter with noradrenaline in sympathetic nerves.

Inhibitory actions of three pairs of congeneric ATP and adenosine analogues on the isolated guinea-pig taenia coli were compared with the actions of ATP and adenosine. 8-Bromoadenosine, and 9-β- D -arabinofuranosyladenosine (ara-adenosine) were inactive; 2′-deoxyadenosine was a weak partial agonist. 8-Bromo-ATP, ara-ATP and 2′-deoxy-ATP behaved like ATP and elicited rapid relaxations of the taenia but were not potentiated by dipyridamole. The divergent effects of identical structural modifications to ATP and adenosine provide for separate adenosine and ATP receptors in the taenia coli.

Guinea-pig tracheae were cut into transverse strips or spirals and although there is only a minor difference in the angle of cut, the former preparations relaxed in response to ATP and adenosine while the latter contracted. Theophylline antagonised both types of response suggesting involvement of P 1 -purinoceptors. Dipyridamole potentiated inhibitory responses to ATP and adenosine in transverse strips but reversed contractile responses to ATP and adenosine in spiral strips. Antagonism of contractions to ATP and adenosine in spiral strips by hyoscine is consistent with an action of adenosine on cholinergic nerves. The likelihood that ATP rapidly forms adenosine by which it acts is discussed.

1. Small quantities of artificial seawater were recyclically perfused over the surface of paired anterior byssus retractor muscles of Mytilus. 2. Perfusates were found to contain 5-HT. 3. Stimulation of the pedal ganglion caused a marked qualitative enhancement of release of 5-HT (serotonin) to the perfusate. 4. 5-HT was detected in the pedal ganglion. 5. Evidence is considered for a role of 5-HT as a neurotransmitter in this tissue. 6. Release of dopamine to perfusates was not observed although low concentrations may have remained undetected.

2-Substituted imidazolines and structurally related compounds, have been tested as possible antagonists to the inhibitory actions of ATP and purinergic nerve stimulation in the guinea-pig taenia coli. Imidazole at neutral pH had no effect on these responses. High concentrations of phentolamine, antazoline, tolazoline and yohimbine strongly reduced the inhibitory responses to ATP and purinergic nerve stimulation but not the inhibitory responses to amyl nitrite. These results are consistent with the purinergic nerve hypothesis. However, it is unlikely that the imidazoline drugs are causing specific blockade of purinergic transmission since they are known to have a wide range of pharmacological actions.

Dipyridamole and hexobendine potentiate the inhibitory responses of the guinea-pig isolated taenia coli to both stimulation of non-adrenergic (‘purinergic’) inhibitory nerves and to exogenously applied ATP, while the inhibitory responses to stimulation of the adrenergic perivascular nerves and to exogenously applied noradrenaline remained the same or were reduced. Dipyridamole and hexobendine are potent coronary vasodilators and it has been suggested that they act by inhibiting the uptake of adenosine, another coronary vasodilating compound. Both dipyridamole and hexobendine inhibit the uptake of labelled adenosine by the guinea-pig taenia coli. These results are consistent with the hypothesis that ATP is the transmitter released from purinergic inhibitory nerves in the gut, and can be considered as support for the view that during purinergic transmission, adenosine is taken up by the nerves for conversion to ATP and reincorporation into physiological stores.

In guinea-pig isolated tracheal tube preparations treated with dipyridamole, adenosine deaminase antagonized inhibitory responses to adenosine and non-adrenergic, non-cholinergic nerve stimulation. Inhibitory responses to vasoactive intestinal peptide (VIP) were unaffected and hyoscine-sensitive excitatory responses to field stimulation were not reduced. The evidence supports a role of adenosine as an inhibitory neurotransmitter in non-adrenergic, non-cholinergic nerves in trachea.

1. The innervation of the smooth muscle of the lung of the Australian snake-necked tortoise. Chelodina longicollis, was investigated using in vitro pharmacological techniques. 2. The smooth muscle bands of the lung were innervated by cholinergic excitatory fibres and non-cholinergic, non-adrenergic, excitatory fibres. 3. Inhibitory responses of the smooth muscle bands to transmural stimulation occurred infrequently following excitatory responses and the neurotransmitter(s) involved were not determined. Noradrenaline caused a beta-adrenoceptor mediated relaxation of the smooth muscle bands. 4. The excitatory response caused by cholinergic and non-cholinergic, non-adrenergic fibres was tetrodotoxin resistant.

The enzyme nucleotide pyrophosphatase converted adenosine 5'-triphosphate (ATP) to adenosine 5'-monophosphate (AMP). In the isolated taenia coli of the guinea-pig it reduced the inhibitory responses to exogenously applied ATP. This could be explained on the basis that the ATP was rapidly converted to AMP which is less potent. The enzyme also reduced inhibitory responses to stimulation of non-adrenergic, non-cholinergic nerves but failed to reduce inhibitory responses to either perivascular sympathetic nerve stimulation or to noradrenaline. The results support the hypothesis that ATP is the transmitter released by non-adrenergic, non-cholinergic ("purinergic') inhibitory nerves.

Dipyridamole potentiated the inhibitory responses to ATP and ADP and in particular responses to AMP and adenosine. The results are discussed in relation to the known actions of dipyridamole and suggest that adenine nucleotides are metabolized to adenosine during contact with the preparation; and that previous observations of the relative potencies of adenine nucleotides and nucleosides do not represent the actual receptor agonist potencies because of rapid uptake of adenosine into the tissues.

ATP, adenosine, adenine and inosine exhibited similar potency in relaxing transverse strips of guinea-pig trachea. Dipyridamole potentiated responses to ATP and adenosine equally but antagonized responses to inosine and failed to affect responses to adenine. In dipyridamole-treated preparations, adenosine deaminase markedly reduced responses to ATP and adenosine; responses to adenine and inosine were unaffected. The results suggest that ATP acts via its adenosine metabolite and that inhibitory ATP receptors are absent.

1. The effects of adrenaline and adrenergic nerve stimulation have been studied on tension in preparations of guinea-pig and rabbit airways and related to fluorescence histochemical studies of the distribution of the nerves. 2. Guinea-pig trachea and lung were found to contain inhibitory adrenoceptors. Functional adrenergic nerves were found only in the trachea. 3. In rabbit trachea, bronchus and lung inhibitory adrenoceptors were not demonstrated. Catecholamines caused a contraction in lung strips. Adrenergic nerves were present in the airways although sparse in the fine airways and failed to respond to field stimulation in the three preparations. 4. It is suggested that excitatory responses of rabbit lung strips to catecholamines are mediated via alpha receptors in the fine airways although the possibility that the blood vessels in the tissue caused the response cannot be discounted. 5. An evolutionary basis for the species differences is considered.

In guinea-pig trachea adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-phosphate (AMP), adenosine and adenine were similarly potent in causing relaxation of the smooth muscle. This is in contrast to gut where ATP and ADP are 30 times more potent than adenosine. Studies with dipyridamole suggest that in trachea, as in gut, nucleotides are rapidly metabolized to adenosine. A polyphosphate modified analogue of ATP, the alpha,beta-methylene isostere, which resists degradation to adenosine was inactive in trachea although it is a potent relaxant in gut. This result may suggest that the intact ATP molecule is also inactive in the tracheal preparation: i.e. ATP acts only via its adenosine metabolite implying that receptors for adenosine but not ATP are present in the tissue.

1. Relaxations to field stimulation of sympathetic nerves were found in the guinea-pig trachea only. 2. Relaxations to field stimulation of non-adrenergic inhibitory nerves were found in the larger airways of human, monkey, guinea-pig and rabbit, but not rat. 3. Lung strips from all the mammals failed to respond to sympathetic or non-adrenergic inhibitory nerve stimulation. 4. Inhibitory beta-adrenoceptors were demonstrated in the proximal airways of all species except rabbit. 5. Of the fine airways examined, beta-adrenoceptors only were found in guinea-pig and alpha-adrenoceptors only in rabbit while rat, monkey and human tissues contained mixed populations of both receptors. 6. Adenosine is a likely candidate for the neurotransmitter in the non-adrenergic inhibitory nerves. Inhibitory receptors for adenosine were present in guinea-pig trachea although receptors for ATP are probably lacking.

1. The responses to non-adrenergic, non-cholinergic nerve stimulation have been compared with those to exogenously applied ATP on seventeen different tissues from a number of vertebrate classes.2. Stimulation of all the mammalian gut preparations studied (with the exception of the guinea-pig ileum) after blockade of the effects of adrenergic and cholinergic nerve stimulation by guanethidine (3.5 muM) and hyoscine (1.3 muM) caused inhibition; exogenously applied ATP mimicked this inhibitory response.3. Stimulation of the guinea-pig ileum in the presence of hyoscine and guanethidine, usually caused a diphasic response, relaxation followed by contraction; exogenously applied ATP mimicked this response, in contrast to acetylcholine and noradrenaline which caused excitation and relaxation respectively.4. Stimulation of preparations of lower vertebrate gut and guinea-pig bladder in the presence of hyoscine and guanethidine caused contraction; exogenously applied ATP mimicked this contractile response.5. In each preparation the time course of the response to ATP was similar or identical to the response to non-adrenergic, non-cholinergic nerve stimulation.6. The results are consistent with the hypothesis that a purine nucleotide may be the transmitter substance released from non-adrenergic, non-cholinergic nerves supplying smooth muscle preparations from a number of vertebrate classes.

It is likely that an exogenously applied enzyme can access the synaptic region and can be used to examine the role of its substrate as a neurotransmitter provided that the end product has reduced activity in the tissue. In guinea-pig ATP has been suggested as the neurotransmitter mediating relaxation by the non-adrenergic, non-cholinergic (NANC) nerves in the taenia coli and as a cotransmitter mediating the twitch component of the contraction in response to sympathetic nerve stimulation in the vas deferens. Nucleotide pyrophosphatase converts ATP to a much less active AMP and caused a significant reduction in the relaxations to NANC nerve stimulation in the taenia and to the twitch components of contractions to sympathetic nerve stimulation in the vas deferens. The enzyme failed to affect responses to noradrenaline and adrenergic neurotransmission in each tissue demonstrating its specificity. In the guinea-pig trachea adenosine has been suggested as the neurotransmitter in NANC inhibitory nerves. In dipyridamole treated preparations; adenosine deaminase caused a significant reduction in inhibitory responses to NANC nerve stimulation and to adenosine wherease responses to VIP and cholinergic nerve stimulation were unaffected. Thus studies with nucleoside and nucleotide metabolising enzymes have provided support for purinergic transmission in the guinea-pig.