Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells.

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.

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

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.

Evidence for a novel P2X purinoceptor in human placental chorionic surface arteries.

To characterize the P2x purinoceptor of arteries of human term placenta, a non-innervated organ, actions of ATP, alpha, beta-methylene-ATP and UTP on de-endothelialized chorionic surface artery segments were compared. ATP and alpha,beta-methylene-ATP caused reversible concentration-dependent contractions, but UTP elicited little or no contraction up to 517 microM. Concentration-effect curves to ATP and alpha,beta-methylene-ATP were parallel, and alpha,beta-methylene-ATP, EC50 4.2 +/- 1.2 microM, was 28-times as potent as ATP. At a saturating concentration, 103 microM, alpha,beta-methylene-ATP did not desensitize the ATP receptor. Contractions to ATP and alpha,beta-methylene-ATP were antagonized by 300 microM suramin. These findings indicate that P2X purinoceptors are present in placental chorionic surface arteries and that they differ from P2X purinoceptors in arteries of other tissues.

Darmstoff analogues. 3. Actions of choline esters of acetal phosphatidic acids on visceral smooth muscle.

A number of naturally occurring phospholipids, e.g. the acetal phosphatidic acid derivatives that comprise Darmstoff (1) and the phosphatidylcholine derivative platelet activating factor (PAF), cause contraction of certain visceral smooth muscles and cause platelet activation. Because the Darmstoff phosphatidic acids and PAF are structurally similar, it was of interest to compare the biological actions of choline esters of Darmstoff with those of PAF and of the parent Darmstoff phosphatidic acids. To this end, [(2-pentadecyl-1,3-dioxolan-4-yl)methyl]phosphocholine (3a), [[2-(cis-8-heptadecenyl)-1,3-dioxolan-4-yl]methyl]phosphocho line (3b), and [[2-(cis-8-pentadecenyl)-1,3-dioxolan-4-yl]methyl]phosphocho line (3c) were synthesized. Compounds 3a, 3b, 3c, and PAF caused dose-dependent relaxation of taenia coli strips. In contrast, the unesterified materials 1a and 1b, as well as lyso-PAF, caused contraction in taenia coli strips. Thus, the contractile effect of Darmstoff is reversed on esterification with choline. In preparations of whole trachea, both 1a and 3a had contractile effects similar to those of PAF.

Evidence that tracheal smooth muscle tone in guinea-pig is unaffected by overflow of noradrenaline released by perivascular nerve fibres.

1. Fluorescence histochemical techniques demonstrated that in the guinea-pig the cervical common carotid, cranial thyroid and caudal thyroid arteries were surrounded by a rich plexus of adrenergic nerve fibres. 2. The superior cervical ganglia were the source of the perivascular adrenergic fibres of the cranial thyroid arteries and the anterior half of the cervical common carotid arteries. These ganglia may also provide some of the perivascular adrenergic fibres of the caudal thyroid arteries. 3. Perivascular stimulation of the cranial thyroid and caudal thyroid arteries failed to elicit a response from isolated extrathoracic tracheal tube preparations. This suggests that it is unlikely that the guinea-pig extrathoracic trachealis muscle is innervated by adrenergic fibres arising from these perivascular plexuses or that overflow of noradrenaline from perivascular nerves causes dilatation of the trachealis muscle.

Innervation of the lung of the Australian snake-necked tortoise, Chelodina longicollis.

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.

Histochemistry of the lung of the Australian snake-necked tortoise chelodina longicollis.

The innervation and structure of the lung of the Australian snake-necked tortoise, Chelodina longicollis, was examined by using light microscopy including fluorescence histochemical techniques. The anterior lung was divided into a number of compartments with numerous alveolar spaces. The posterior lung was simpler and saclike in structure and alveolar spaces were absent. Smooth muscle fibers occurred in discrete muscle bands and in the walls of the septal bands. Ganglion cells occurred along nerve trunks throughout the lung but were more numerous in the posterior lung. Smooth muscle bands, the extrinsic pulmonary artery, and the arteries within the lung were sparsely innervated by adrenergic fibers. Substance P-containing sensory fibers were not demonstrated. The innervation and structure of the lung are compared to published work on other reptiles.

Determination of extrinsic pathways of adrenergic nerves to the guinea-pig trachealis muscle using surgical denervation and organ-bath pharmacology.

Intraluminal pressure changes were recorded in an isolated tracheal tube during electrical stimulation of the recurrent laryngeal nerve or transmural stimulation of the extra-thoracic trachealis muscle. The recurrent laryngeal nerve contained adrenergic nerve fibres running both anteriorly and posteriorly along this nerve and they caused an adrenergic inhibitory response of the trachealis muscle. The superior cervical ganglion was the source of the majority of the adrenergic fibres running both anteriorly and posteriorly within the recurrent laryngeal nerve. The antero-posterior adrenergic fibres reached the recurrent laryngeal nerve via an anastomosis with the superior cervical ganglion. The postero-anterior adrenergic fibres reached the recurrent laryngeal nerve via the vagus nerve, as ipsilateral vagotomy markedly reduced the inhibitory response of the trachealis muscle which had been due to the postero-anterior adrenergic fibres. Superior cervical ganglionectomy caused a significant reduction in the adrenergic response as a percentage of the total inhibitory response of the extra-thoracic trachealis muscle following transmural stimulation. The remaining response was due to stimulation of non-adrenergic non-cholinergic fibres and adrenergic fibres from another source.

Extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle.

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.

Adenosine deaminase antagonizes inhibitory responses to adenosine and non-adrenergic, non-cholinergic inhibitory nerve stimulation in isolated preparations of guinea-pig trachea.

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.

Evidence for separate receptors for ATP and adenosine in the guinea-pig taenia coli.

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-beta-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 evidence for separate adenosine and ATP receptors in the taenia coli.

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.

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.

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 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.

The inhibitory innervation of the guinea-pig trachea: a study of its adrenergic and non-adrenergic components.

(1) Inhibitory responses to field stimulation have been determined in strip preparations from the thoracic, middle and cervical regions of the trachea and in the tracheal tube preparation. (2) The adrenergic neurone blocking drug guanethidine was found to cause a partial reduction of the amplitudes of the responses in all preparations. (3) The guanethidine-resistant inhibitory responses were resistant to pentolinium but were abolished by tetrodotoxin. The data favours the existence of non-adrenergic inhibitory intramural nerves in the guinea-pig trachea. (4) Inhibitory responses have been determined in the presence and absence of guanethidine at frequencies ranging from 2 to 60 Hz. This has enabled the combined responses to stimulation of both adrenergic and non-adrenergic nerves to be compared with the response to stimulation of non-adrenergic nerves alone at each frequency and in each preparation. (5) The contribution of each innervation to the combined inhibitory response was frequency dependent. The adrenergic innervation was more effective at lower frequencies and the non-adrenaration is discussed. Its origin is considered.

Inhibitory effects of adenine nucleotide analogs on the isolated guinea-pig taenia coli.

The inhibitory actions of adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine and 16 adenine nucleotide and nucleoside analogs on the isolated guinea-pig taenia coli preparation were compared with those of adenosine triphosphate (ATP). Responses were quantitated as magnitude of maximal relaxation, time taken to reach maximal relaxation and activity relative to that of ATP. Inhibitory responses induced by the 5'-di- and triphosphates of 2-chloroadenosine and 2-methylthioadenosine resembled those elicited by ADP and ATP, but the 2-substituted analogs were markedly more potent. AMP and adenosine were less active than ATP; their activities were enhanced by 2-chloro substitution but not by 2-methylthio substitution. 2-Methylthio-AMP and 2-methylthioadenosine were the only analogs which did not elicit maximal relaxation of the taenia coli. 6'-Deoxyhomoadenosine 6'-phosphonic acid was inactive. Adenine nucleotide analogs in which the polyphosphate moiety was modified had steeper log dose-response curves than ATP and induced greater maximal responses than ATP. Analogs in which the polyphosphate alpha, beta-anhydride oxygen was replaced by methylene took up to 5 times longer than ATP to cause maximal relaxation. Other analogs with modified or unmodified polyphosphate side chains caused rapid relaxation of the taenia coli. There was no apparent correlation between relative activity and time to reach maximal response. The findings obtained indicate that di- or triphosphate groupings are of prime importance in binding adenine nucleotides to the putative smooth muscle receptor which mediates their inhibitory responses, and that hydrolysis of the terminal phosphates of adenosine 5'-polyphosphates is not a requirement for inhibitory activity. Reasons for the distinctive inhibitory actions of the phosphate-modified adenine nucleotide analogs are considered.

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.