D.G. Satchell

Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non‐adrenergic inhibitory nerves in the gut

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 Auerbachs 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.

A comparison of the excitatory and inhibitory effects of non‐adrenergic, non‐cholinergic nerve stimulation and exogenously applied ATP on a variety of smooth muscle preparations from different vertebrate species

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 μm) and hyoscine (1–3 μm) 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.

Adrenergic and non-adrenergic inhibitory nerves in mammalian airways.

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 beta-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 alpha-excitatory and beta-inhibitory adrenoceptors only were found in guinea-pig lung and alpha-adrenoceptors only in rabbit lung.

Potentiation of the effects of exogenously applied ATP and purinergic nerve stimulation on the guinea-pig taenia coli by dipyridamole and hexobendine.

Abstract 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.

Comparison of the inhibitory effects on the guinea-pig taenia coli of adenine nucleotides and adenosine in the presence and absence of dipyridamole

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.

PURINE RECEPTORS IN THE TRACHEA: IS THERE A RECEPTOR FOR ATP?

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 α,β‐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.

Antagonism of the effects of purinergic nerve stimulation and exogenously applied ATP on the guinea-pig taenia coli by 2-substituted imidazolines and related compounds

Abstract 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.

NUCLEOTIDE PYROPHOSPHATASE ANTAGONIZES RESPONSES TO ADENOSINE 5′ ‐TRIPHOSPHATE AND NON‐ADRENERGIC, NON‐CHOLINERGIC INHIBITORY NERVE STIMULATION IN THE GUINEA‐PIG ISOLATED TAENIA COLI

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.

Evidence that NO acts as a redundant NANC inhibitory neurotransmitter in the guinea-pig isolated taenia coli.

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 μM). Peak relaxations to EFS (0.2–5 Hz) were tetrodotoxin (1 μM)‐sensitive, maximal at 0.2 Hz and completely resistant to the nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG; 100 μM) in either the presence or absence of atropine (1 μM). Furthermore, the specific inhibitor of soluble guanylyl cyclase, 1H‐[1,2,4] oxadiazolo [4,3‐a] quinoxaline‐1‐one (ODQ; 10 μM), the cytochrome P450 inhibitor and free radical generator, 7‐ethoxyresorufin (7‐ER; 10 μM) and the NO scavenger, oxyhaemoglobin (HbO; 30 μM) had no effect on EFS‐induced relaxations alone and in combination with L‐NOARG (100 μM). Maximum relaxation to the NO donor, sodium nitroprusside (SNP; 1 μM) was significantly reduced by HbO (30 μM), abolished by 7‐ER (10 μM) and ODQ (10 μM) but was unaffected by apamin (0.1 μM), an inhibitor of small conductance Ca2+‐activated K+ channels. 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 μM) or ODQ (10 μM), but not by HbO (30 μM) or 7‐ER (10 μM). ATP (1–30 μM) produced concentration‐dependent relaxations that were abolished by apamin (0.1 μM), unaffected by ODQ (10 μM) but only significantly reduced by L‐NOARG (100 μM) at the lowest concentration of ATP (1 μM) used. Nifedipine (0.3 μM), abolished contractions to 67 mM KCl, histamine (10 μM), endothelin‐1 (0.03 μM), 5‐hydroxytryptamine (5‐HT; 10 μM) and the thromboxane‐mimetic, 9‐11‐dideoxy‐9α, 11α‐methano‐epoxy‐prostaglandin F2α (U46619; 0.1 μM). 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.

An investigation of the identity of the transmitter substance released by non-adrenergic, non-cholinergic excitatory nerves supplying the small intestine of some lower vertebrates

Abstract 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.