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I have had the pleasure and privilege of being involved in one facet of Geoffrey Burnstock’s early career. I have reviewed this work together with more recent developments in the area. In 1968, the presence of non-adrenergic, non-cholinergic inhibitory nerves had been established but the identity of their neurotransmitter was unknown. Stimulation of these nerves in recycled perfused toad and guinea-pig stomachs caused release of adenosine and inosine. When ATP was added to recycled perfusates, it was broken down to adenosine and inosine. These findings together with information that AMP was released from stimulated, isolated turkey Auerbach’s plexus which was known to contain the nerves, suggested that ATP could be the neurotransmitter. This was supported by observations that ATP elicited responses similar to that of nerve stimulation in a variety of tissues. Developments from the early purinergic nerve hypothesis are considered including independence of extracellular actions of ATP from its intracellular actions, identification and cloning of purinoceptors and cotransmission of ATP with other substances.

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.

Origins and extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle were studied using fluorescence histochemical techniques. Bilateral superior cervical ganglionectomy caused a marked reduction in the adrenergic innervation of the extra-thoracic region, which suggests that these ganglia are a major source of adrenergic innervation to this muscle. Combined anterior and posterior transection of the recurrent laryngeal nerves also caused a marked reduction in the density of adrenergic fibres in the extra-thoracic trachealis muscle. Crushing of these nerves revealed adrenergic fibres running both anteriorly and posteriorly. The majority of these adrenergic nerves were lost after superior cervical ganglionectomy and thus the fibres running in both directions originate in the superior cervical ganglion. Antero-posteriorly directed fibres entered the recurrent laryngeal nerve from the superior cervical ganglion via an anastomosis at the level of the cricoid cartilage, while those running postero-anteriorly entered the recurrent laryngeal nerve posteriorly from the vagus nerve and these adrenergic fibres were lost after cervical vagotomy.

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.

The effect of alteration of the external-Ca 2+ level on 22 Na efflux from muscle and on respiration, glycolysis, and the “high-energy” phosphate compounds has been investigated. Ca 2+ depletion reversibly accelerated 22 Na efflux and increased the oxygen uptake of the tissue. Glycolysis was unaffected. Falls in creatine phosphate and ATP level were noted, suggesting an uncoupling action. This could not be related to spontaneous muscular activity, since it was augmented in EDTA solutions when muscles were inexcitable. Simultaneous depletion of Ca 2+ and Na + reduced or abolished the actions of Ca 2+ lack. Complicated interactions between Ca 2+ and K + were noted. The Na + flux was insensitive to alteration of the external K + level. However, respiration, which was increased either by low Ca 2+ or high K + , was extremely susceptible to variation of the ratio of K + /Ca 2+ . High-Ca 2+ solutions brought about increases in Na + efflux, respiration and glycolysis. Such changes were associated with movements of cell Na + and K + down their concentration gradients. The importance of ions and membrane potential in repiratory-control mechanisms is discussed.

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.

An unknown phosphate-containing compound has been isolated from the sartorius muscle of the toad. The compound was detected on paper chromatograms run to isolate the ademine nucleotides and creatine phosphate. Using solvent systems usually employed to separate ATP and ADP it was found to have an RF value identical to that of ADP, and consequently was routinely estimated with it. This finding accounts for the high values of ADP reported by us, taking account of the levels of creatine phosphate known to be present in muscle, and the activity of the creatine phosphoryltransferase system. The compound was found not to be a nucleotide, or a purine or pyrimidine derivative. Microanalysis showed it to contain C, N, H, orthophosphate and a large but constant proportion of non-combustible ash. Al was the principal metal contained in the ash. Evidence from infrared and ultraviolet spectra suggests that the compound contains an unsaturated heterocyclic ring. The Al may be present as a metal chelate with the phosphate moiety. The phosphate group of the compound failed to exchange with 32P-labelled orthophosphate under conditions when there was a substantial exchange of the other acid-soluble phosphate compounds of muscle. No information is available as to the possible biological function of the compound.

1. 1. Non-adrenergic, non-cholinergic excitatory postganglionic fibres in the splanchnic nerves supply the small intestine of the toad ( Bufo marinus ) and the lizard ( Tiliqua rugosa ). 2. 2. 5-Hydroxytryptamine, histamine, bradykinin, and prostaglandin E 1 have been rejected as the putative neurotransmitters in these nerves on the grounds that they do not have actions which parallel those of nerve stimulation or that drugs which antagonize their effects do not similarly antagonize the effects of nerve stimulation. 3. 3. ATP produces excitatory responses, which closely mimic those of non-adrenergic, non-cholinergic nerve stimulation. Quinidine causes parallel block of the responses to nerve stimulation and exogenously applied ATP, but this action may be non-specific. 4. 4. While the results do not provide direct evidence for purinergic excitatory nerves to the small intestine of lower vertebrates, they are consistent with the hypothesis.

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.

A study of the actions of adrenergic and non-adrenergic nerves which affect mammalian airways was carried out. The preparations studied included strips of lung from guinea-pig, rat, rabbit, monkey and human, tracheal strips from the first 4 animals and bronchial strips from the last 3. Relaxations to field stimulation of sympathetic nerves were found in the guinea-pig trachea only. Functional nonadrenergic inhibitory nerves were found in the larger airways of all species except rat. Lung strips from all the mammals failed to respond to sympathetic or nonadrenergic inhibitory nerve stimulation suggesting a lack of functional inhibitory nerves of either type in the fine airways. Studies on the distribution of adrenergic nerves showed that primary target of the nerves in all species appeared to be the vasculature, especially in lung. Occasional fibres were seen entering the smooth muscle of the fine airways in guinea-pig, rabbit, and rat, but not in monkey or human lung or in monkey trachea or bronchus or human bronchus. Guinea-pig and rabbit trachealis muscles received a significant innervation but only the guinea-pig tissue responded to sympathetic stimulation. Inhibitory β-adrenoceptors were demonstrated in the proximal airways of all species except rabbit. The fine airways of rat, monkey and human contained a mixed population of α-excitatory and β-inhibitory adrenoceptors. β-Adrenoceptors only were found in guinea-pig lung and α-adrenoceptors only in rabbit lung.

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.

When ATP or adenosine are applied as relaxants to isolated smooth muscle preparations in organ baths despite the presence of large volumes of nutrient solution containing these compounds it has been difficult to ascertain what compounds and what concentrations of compounds are indeed acting on the receptors of the muscle cell surface. For example it is quite possible that applied ATP is rapidly broken down in the extracellular space and that the metabolites themselves may act on muscle receptors in lieu of or in addition to the added substance. The understanding of what happens in this situation may be complicated by the formation of more than one compound, the uptake of metabolites, e.g. adenosine, into the cells and the diffusion of metabolites into the outside medium.

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.

1. 1. The effects of high levels of external K+ and fatty acid substrates have been studied on the isolated ventricle of the toad, Bufo marinus . 2. 2. High K+ levels reduced ATP levels and increased levels of ADP and inorganic phosphate. 3. 3. High K + had marked effects on carbohydrate utilization and oxidation in winter toad hearts but had little effect on summer toad hearts. 4. 4. Contrary to findings on mammalian heart, toad ventricle glycogen stores were depleted by media containing octanoate or butyrate. This phenomenon, seen in summer toads, could be related to the synthesis of triglyceride and phospholipid.

ATP causes relaxation of various smooth muscle containing preparations. ATP and related analogs were applied to strips of trachea and taenia coli from guinea-pigs in organ baths for 60 sec. The formation of adenosine was signalled by potentiation of relaxations in the presence of the adenosine transport inhibitor dipyridamole. The basis for this potentiation is that adenosine which is also a potent relaxant is rapidly taken up into muscle cells decreasing its effect. ATP caused relaxations of both taenia coli and tracheal strips and both responses were potentiated by dipyridamole signifying that adenosine was being formed during the contact times. In the taenia both the α,β and the β,γ-methylene isosteres of ATP caused relaxations. Dipyridamole potentiated responses to the latter, but failed to affect responses to the former. Since methylene subtitution renders the analog stable at that site it is presumed that the β,γ-methylene isostere rapidly formed adenosine due to the action of a pyrophosphohydrolase. In tracheal strips the β,γ-methylene isostere of ATP caused relaxations which were potentiated by dipyridamole, the α,β-methylene isostere was inactive either in the presence or absence of dipyridamole. This suggested that in contrast to the taenia, ATP is inactive in the trachea per se and must first be degraded to adenosine which in turn causes the relaxation.

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.

136 NUCLEOTIDE PYROPHOSPHATASE ANTAGONIZES CONTRACTIONS DUE TO THE TWITCH COMPONENT OF THE SYMPATHETIC NERVE RESPONSE AND TO APPLIED ATP IN THE GUINEA-PIG VAS DEFERENS