L-Ornithine decarboxylase:an essential role in early mammalian embryogenesis.

The highly selective, enzyme-activated, irreversible inhibitor of L-ornithine decarboxylase, DL-alpha-difluoromethylornithine, suppresses the increase in uterine L-ornithine decarboxylase activity associated with early embryogenesis in the mouse and arrests embryonic development at that stage. Contragestational effects were confirmed in the rat and rabbit. An increase in L-ornithine decarboxylase activity that leads to a rapid increase in putrescine concentration appears to be essential during a critical period after implantation for continued mammalian embryonal growth.

In vivo pharmacological evaluation of compound 48/80-induced airways oedema by MRI.

BACKGROUND AND PURPOSE: Allergen-induced airways oedema in actively sensitized rats has been studied earlier by magnetic resonance imaging (MRI). We used MRI to follow the consequences of non-immunological mast cell activation induced by compound 48/80 in the rat lungs in vivo. EXPERIMENTAL APPROACH: Male naïve rats were scanned by MRI prior to and at several time points following intratracheal administration of the mast cell secretagogue, compound 48/80. The effects of a range of drugs on the response induced by compound 48/80 were studied. KEY RESULTS: Strong fluid signals were detected by MRI in the lungs at 24 h after compound 48/80, correlating with increased protein concentration and inflammatory cell infiltration in bronchoalveolar lavage, and with perivascular oedema observed histologically. Pharmacological intervention demonstrated that the increase in MRI signal volume induced by compound 48/80 24 h after challenge was blocked by disodium cromoglycate and the glucocorticoid, budesonide. Pretreatment with wortmannin, capsazepine, DNK333 (a dual neurokinin (NK) 1 and NK2 antagonist) or the anti-allergy drug CGS8515, but not indomethacin, resulted in partial inhibition. CONCLUSIONS AND IMPLICATIONS: Compound 48/80 induced a complex inflammatory reaction which did not solely involve mast cell degranulation but also activation of sensory nerves and was qualitatively similar to allergen challenge. Changes observed by MRI correlated with decreases in protein concentration in BAL fluid. However, the magnitude of the changes detected was greater using MRI. Our results demonstrate that MRI is a sensitive and efficient tool to assess the effects of drugs on lung inflammation.

Capsaicin-induced mucus secretion in rat airways assessed in vivo and non-invasively by magnetic resonance imaging.

BACKGROUND AND PURPOSE: An up-regulation of the sensory neural pathways in the lung has been implicated in asthma and chronic obstructive pulmonary disease (COPD) and is thought to contribute to mucus hypersecretion, an essential feature of both diseases. The aim of this study was to assess non-invasively the acute effects (up to 60 min) of sensory nerve stimulation by capsaicin in the lung, using magnetic resonance imaging (MRI). EXPERIMENTAL APPROACH: Male Brown Norway rats were imaged prior to and 10, 30 and 60 min after intra-tracheal challenge with capsaicin (30 microg kg(-1)) or vehicle (0.5% ethanol solution). In subsequent studies, pre-treatment with the transient receptor potential vanilloid (TRPV)-1 antagonist, capsazepine; the dual neurokinin (NK) 1 and NK2 receptor antagonist, DNK333 and the mast cell stabilizer, di-sodium cromoglycate (DSCG) was used to modulate the effects of capsaicin. KEY RESULTS: Diffuse fluid signals were detected by MRI in the lung as early as 10 min after capsaicin, remaining constant 30 and 60 min after treatment. Broncho-alveolar lavage (BAL) fluid analysis performed 60 min after capsaicin revealed increased mucin concentration. Capsazepine (3.5 mg kg(-1)), DNK333 (10 mg kg(-1)) but not DSCG (10 mg kg(-1)) administered prophylactically were able to block the effect of capsaicin in the airways. CONCLUSIONS AND IMPLICATIONS: These observations suggest that the fluid signals detected by MRI after capsaicin administration reflected predominantly the release of mucus following activation of sensory nerves. They point to the opportunity of non-invasively assessing with MRI the influence of neuronal mechanisms in animal models of asthma and COPD.

Subtypes of the type 4 cAMP phosphodiesterases: structure, regulation and selective inhibition.

The 'famille nombreuse' of cyclic nucleotide phosphodiesterases, responsible for degrading the ubiquitous second messenger molecules, cAMP and cGMP, maintains its place as a major focus of interest for many research laboratories in both academia and industry. The increase in knowledge of the primary sequences, plus the availability of selective inhibitors, are rapidly improving our insight into the structure, regulation and function of these pivotal enzymes of cellular homeostasis. Here, Thomas Müller, Peter Engels and John Fozard focus on family 4 of the phosphodiesterases, which is of particular interest owing to both the number of genes (and splice variants) and the emergence of selective inhibitors, which are enabling the functional significance of these enzymes to be defined.

Effects of endotoxin and allergen alone and in combination on the sensitivity of the rat airways to adenosine.

1 The airways of patients with asthma are hyperresponsive to adenosine. The phenomenon can be mimicked in the actively sensitized Brown Norway rat by exposure to allergen or lipopolysaccharide (LPS). We wondered whether combined treatment with allergen and endotoxin would result in additive effects or synergism with respect to increasing the sensitivity of the airways of the Brown Norway rat to adenosine. 2 Animals actively sensitized to ovalbumin and challenged intratracheally with allergen or endotoxin manifested increased bronchoconstrictor responses to adenosine. A combination of ovalbumin and endotoxin also increased the response to adenosine but the effects were at best additive. 3 Changes in the response to adenosine were selective as responses to 5-hydroxytryptamine were unaltered following ovalbumin or LPS either alone or in combination. 4 Thus, endotoxin and allergen acting together could play a role in up-regulating the response of the human asthmatic airway to adenosine. However, our data suggest that the interaction would be additive rather than synergistic.

Neuronal 5-HT receptors in the periphery.

5-Hydroxytryptamine (5-HT) induces responses in neurones from all branches of the mammalian peripheral nervous system. Responses may be excitatory or inhibitory and are mediated through at least four distinct receptor sites. One receptor mediates excitation in motoneurones and preganglionic sympathetic neurones and can be designated a D (or possibly 5-HT2) receptor since "classical" antagonists such as methysergide, metergoline or cinanserin are potent and selective antagonists at this site. A second receptor mediating neuronal excitation can be positively identified on the basis of susceptibility to blockade by small concentrations of 1 alpha H,3 alpha,5 alpha H-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222) and the weak or negligible affinity, relative to 5-HT, of certain agonists such as 5-methoxytryptamine. Such sites mediate depolarization of sympathetic and parasympathetic neurones and excitation of both the cell bodies and terminals of primary afferent fibres. A third receptor, mediating neuronal excitation, is the classical M-receptor of Gaddum and Picarelli, at this stage clearly identified only on postganglionic parasympathetic neurones of the guinea-pig myenteric plexus. These sites can be differentiated from other excitatory 5-HT receptors since MDL 72222 is neither potent nor selective as an antagonist and 5-methoxytryptamine approaches the potency of 5-HT as an agonist. (3 alpha-Homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid is a potent, surmountable antagonist of 5-HT at the M-receptor of the ileum, but is non-selective. Neuronal inhibitory responses have been observed using electrophysiological techniques or by monitoring the decrease in depolarization-evoked release of transmitter in enteric, parasympathetic and sympathetic neurones. Largely negative results, using selective agonists and antagonists, allow the receptor(s) mediating inhibition to be clearly differentiated from the three neuronal excitatory receptors for 5-HT. Comparison of relative potencies of agonists suggests similarities with the 5-HT1 recognition site of the central nervous system; no selective antagonist has yet emerged to permit their positive identification.

Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine.

As a result of controversy in the literature regarding the classification and nomenclature of functional receptors for 5-hydroxytryptamine (5-HT), a framework for classification is proposed. The formulation of these proposals has only been made possible by the recent advent of new drug tools. It is considered that there are three main types of 5-HT receptor, two of which have been well characterised pharmacologically, using selective antagonists, and which it is proposed to name 5-HT2 and 5-HT3. These two groups broadly encompass the "D" and "M" receptors, respectively, which Gaddum identified in the guinea-pig ileum (Gaddum and Picarelli, 1957). The 5-HT2 receptor, which mediates a variety of actions of 5-HT, has been definitively shown to correlate with the 5-HT2 binding site in the brain. No binding studies in brain tissue have yet been published with radiolabelled ligands specific for 5-HT3 receptors. A number of other actions of 5-HT appear to be mediated via receptors distinct from 5-HT2 or 5-HT3 receptors. Since selective antagonists are not yet available, these receptors cannot be definitively characterised, although in many cases they do have some similarities with 5-HT1 binding sites, which are a heterogeneous entity. Criteria are proposed for tentatively classifying these receptors as "5-HT1-like" (Table 1). Definitive characterisation of these receptors will await the identification of specific antagonists. This classification of 5-HT receptors into three main groups (Table 1) is based largely, but not exclusively, on data from studies in isolated peripheral tissues where definitive classification is possible. However, it is believed that this working classification will be relevant to functional responses to 5-HT in the central nervous system.

Graphics computer-aided receptor mapping as a predictive tool for drug design: development of potent, selective, and stereospecific ligands for the 5-HT1A receptor.

A conformational study of four 5-HT1A (serotonin) receptor ligands ((R-(-)-methiothepin, spiperone, (S)-(-)-propranolol, and buspirone) led to the definition of a pharmacophore and a three-dimensional map of the 5-HT1A antagonist recognition site. These models were used to design new compounds and successfully predict their potency, stereospecificity, and selectivity. For example, 8-[4-[(1,4-benzodioxan-2-ylmethyl)amino] butyl]-8-azaspiro[4.5]decane-7,9-dione (1, MDL 72832) has nanomolar affinity (pIC50 = 9.14) for the 5-HT1A binding site in rat frontal cortex. As predicted, the S-(-) enantiomer of 1 was more active than its R-(+) enantiomer (pIC50 = 9.21 and 7.66, respectively) and a naphthalene analogue of 1 displayed the expected improved selectivity.

Effects of progesterone on cardiovascular responses to amines and to sympathetic stimulation in the pithed rat.

1. Blood pressure and heart rate responses to adrenaline, noradrenaline, tyramine, 5-hydroxytryptamine and stimulation of the spinal sympathetic outflow were measured in pithed rats pretreated either with progesterone (20 mg/kg daily for 14 days) or the vehicle solution of ethyl oleate.2. Pretreatment with progesterone increased the durations but not the magnitudes of the blood pressure and heart rate responses to adrenaline and that phase of the response to sympathetic stimulation attributable to amine release from the adrenal medulla.3. Responses to noradrenaline, tyramine, 5-hydroxytryptamine and that phase of the response to sympathetic stimulation associated with amine release from the sympathetic nerves were not significantly different in the two groups.4. Pyrogallol (5 mg/kg) increased the duration but not the magnitude of responses to adrenaline, noradrenaline and sympathetic stimulation in both experimental groups. The increases in duration were consistently less in animals pretreated with progesterone than in controls.5. Pretreatment with progesterone did not affect the total amount of radioactivity nor the proportion of catechol to non-catechol metabolites excreted in the urine during a period of 7.25 h following an intraperitoneal injection of (+/-) isoprenaline-7-(3)H.6. It is concluded that the effects of progesterone may result from a localized decrease in catechol O-methyl transferase activity within the cardiovascular system.

Adenosine A3 receptors mediate hypotension in the angiotensin II-supported circulation of the pithed rat.

The cardiovascular effects of N6-2-(4-aminophenyl)ethyladenosine (APNEA), which when radiolabelled with 125I shows high affinity for the newly described adenosine A3 receptor, have been investigated in the angiotensin II-supported circulation of the pithed rat. APNEA induces hypotensive responses which are unaffected by high doses (20-40 mg kg-1) of the broad spectrum, adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT). 8-SPT-resistant falls in blood pressure are also seen, in the absence of bradycardia, with 5'-N-ethylcarboxamidoadenosine (NECA) and the R- and S-enantiomers of N6-phenylisopropyladenosine (PIA). Xanthine insensitivity, high potencies of APNEA, NECA and R-PIA, and an enantiomeric selectivity favouring R- over S-PIA are distinguishing features of the adenosine A3 receptor. We suggest that hypotension in the pithed rat may be a functional correlate of this site.

Haemodynamic responses to NG-monomethyl-L-arginine in spontaneously hypertensive and normotensive Wistar-Kyoto rats.

1. Nitric oxide (NO) is a major component of endothelium-derived relaxing factor (EDRF) the synthesis of which from L-arginine can be inhibited by NG-monomethyl-L-arginine (L-NMMA). To assess whether basal NO tone is different in experimental hypertension, the haemodynamic effects of L-NMMA have been compared in anaesthetized spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats in which autonomic reflexes were blocked by ganglion blockade. 2. Bolus intravenous injections of L-NMMA, 1-30 mg kg-1, but not D-NMMA, 1-30 mg kg-1, induced dose-related increases in mean arterial pressure and decreases in conductances in the renal, carotid, hindquarters and mesenteric vascular beds in both SH and WKY rats. Although the different vascular beds varied in their maximum responses to L-NMMA, there were neither qualitative nor quantitative differences between the two rat strains in this respect. 3. The effects of L-NMMA, 30 mg kg-1, i.v. on all parameters were rapidly and completely reversed by L-arginine, 30 mg kg-1, i.v., in both SH and WKY rats. 4. The results indicate that NO derived from L-arginine exerts a powerful vasodilator tone in both anaesthetized, ganglion-blocked SH and WKY rats. Although NO appears to contribute differentially to tone in the different vascular beds, there were no major differences between the two rat strains in this respect. Hence a reduced NO tone to the vasculature is unlikely to be a major factor contributing to the elevated blood pressure in the adult SH rat.

Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation.

1. A series of adenosine receptor agonists of varying degrees of selectivity induced concentration-dependent contraction of the rat isolated spleen. With the exception of the response to the selective A2A receptor agonist, 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS 21680), responses to each ligand were blocked surmountably and to a broadly similar extent by 8-p-sulphophenyltheophylline (10(-5) M). 2. There was a significant correlation between the pEC50 values obtained on the spleen and the binding affinities (pKD; measured with [3H]-NECA) for the A1 receptor of pig striatum (r = 0.98, P < 0.001) but not the A2A receptor (r = 0.14, NS). 3. The antagonist potencies of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and 9-chloro-2-furyl [1,2,3]triazolo[1,5-C]quinazoline-5-amine (CGS 15943) were measured against the prototype selective A1 receptor agonist, R-N6-phenylisopropyladenosine (R-PIA). The resulting pKB values of 8.67 and 7.70, respectively are consistent with the A1 receptor subtype mediating splenic contraction. 4. The response to R-PIA was unaltered in the presence of a concentration (10(-7) M) of CGS 21680 which is 6 fold its KD concentration at the A2A binding site in pig striatum but below the threshold for causing contraction per se; thus, A2A receptors inhibitory to contraction appear to be absent. 5. The response to R-PIA was resistant to blockade by prazosin (10(-7) M) and by nifedipine (10(-6) M) but partially blocked by indomethacin (10(-6) M). 6. The results show that the rat isolated spleen responds to adenosine receptor agonists with contraction. Both the relative potencies of agonists and the effects of antagonists indicate mediation by the A1 receptor subtype. alpha1-Adrenoceptor activation is not involved in contraction but a role for products of cyclo-oxygenase and calcium from a source not dependent on entry through L-channels is implicated.

8-OH-DPAT, flesinoxan and guanfacine: systemic and regional haemodynamic effects of centrally acting antihypertensive agents in anaesthetized rabbits.

1. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and flesinoxan, agents which show high affinity and selectivity for 5-HT1A receptors, were administered intravenously in doses of 0.003 to 0.1 and 0.01 to 0.3 mg kg-1 respectively to 5 rabbits each. Their effects were compared with those of the centrally acting agent and alpha 2-adrenoceptor agonist, guanfacine, 0.01-0.3 mg kg-1, administered to a group of 5 rabbits. Five further rabbits were used as controls and treated with the vehicle of the active agents. 2. Both flesinoxan and 8-OH-DPAT induced similar systemic and regional haemodynamic changes. Both lowered mean arterial pressure and heart rate. The principal blood pressure lowering mechanism was vasodilatation; cardiac output changed minimally despite the falls in heart rate and myocardial contractile force. 3. With guanfacine the maximal fall of blood pressure was comparable to that obtained with the 5-HT1A receptor ligands; however, in contrast to the latter, the dose-response curve was U-shaped, the highest dose eliciting a pressor effect with reversal of the vasodilatation. 4. Widespread peripheral vasodilatation was found with all the agents in the splanchnic circulation and also in the brain and skeletal muscle. A weak tendency towards vasodilatation was found in the kidneys where the dose-response curve was bell-shaped for guanfacine. 5. This spectrum of activity is different from that of peripheral vasodilators, such as calcium antagonists, potassium channel activating agents or hydralazine; it is, however, consistent with the putative mechanism of action of these compounds to reduce peripheral sympathetic tone by a central mechanism of action.

The role of nitric oxide in the regional vasodilator effects of endothelin-1 in the rat.

1. The role of nitic oxide (NO) derived from L-arginine in the regional vasodilator effects of endothelin-1 has been investigated in anaesthetized, spontaneously hypertensive (SH) rats in which autonomic reflexes were abolished by ganglion blockade. The experimental design incorporated animals infused with phenylephrine to mimic the peripheral vasconstrictor effects of the NO biosynthesis inhibitors and a single dose per animal paradigm to obviate problems of tachyphylaxis to the vasodilator effects of endothelin-1. 2. Infusion of the inhibitor of NO synthase, N-monomethyl-L-arginine (L-NMMA) at a dose (5 mg kg-1 min-1) which maximally raised blood pressure did not influence either the fall in blood pressure or the vasodilator responses induced in the hindquarters and carotid vascular beds by endothelin-1 (1 nmol kg-1, i.v.) The duration (but not the initial magnitude) of the vasodepressor response to endothelin-1 was however significantly attenuated (by 49%) during infusion of the more potent inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), 2 mg kg-1 min-1. 3. Increasing the dose of L-NAME to 10 and 25 mg kg-1min-1 significantly attenuated, but did not abolish, the falls in blood pressure and hindquarters vasodilator responses to acetylcholine, 1 microgram kg-1, and endothelin-1, 1 nmol kg-1 min-1. The effects were selective in that vasodepressor responses to the endothelium-independent vasodilator, sodium nitroprusside, 1-10 micrograms kg-1 min-1, were unaltered. The effects were selective in that vasodepressor responses to the endothelium-independent vasodilator, sodium nitroprusside, 1-l0mg kg min , were unaltered.4. The data indicate that NO generated de novo from L-arginine mediates a significant component of the vasodilator effect of endothelin-1 in the anaesthetized, ganglion-blocked SH rat. However, a major component of the vasodepressor effects of both endothelin-1 and acetylcholine may occur independently of this mechanism.

SCA 40: studies of the relaxant effects on cryopreserved human airway and vascular smooth muscle.

1. 6-Bromo-8-methylaminoimidazol[1,2-a]pyrazine-2carbonitrile (SCA 40) has been claimed to induce relaxation in guinea-pig trachea by opening high conductance, calcium-activated potassium (BKCa) channels. The mechanism of action of SCA 40 has now been further investigated in ring preparations from cryopreserved human airway and vascular smooth muscle preparations in vitro. 2. Human bronchi with spontaneous tone relaxed in response to SCA 40 in a biphasic way. A high affinity component (pD2 8.61 +/- 0.21; mean +/- s.e.mean) accounted for 30% of the response and a low affinity component (pD2 6.53 +/- 0.14) for the remaining 70%. In contrast, in bronchi contracted with carbachol, 1 microM, the concentration-response curve to SCA 40 was monophasic and yielded a pD2 of 6.31 +/- 0.29. 3. SCA 40 relaxed pulmonary and mesenteric arteries and peripheral veins which had been precontracted by 10 nM U46619 nearly completely and in a monophasic way; the pD2 values were 6.37 +/- 0.08, 6.17 +/- 0.15 and 5.45 +/- 0.25, respectively. 4. Lemakalim, an opener of ATP-dependent potassium (KATP) channels, also relaxed human bronchi under spontaneous tone and the vascular tissues. NS 1619, a recognised opener of BKca channels, was inactive up to 10 microM on bronchial and vascular tissues. 5. The SCA 40-induced relaxation of human bronchi was reduced concentration-dependently in the presence of high potassium chloride (20 and 80 mM). However, in the presence of 80 mM KCl and nifedipine, 30 nM, SCA 40 fully relaxed the remaining contractile response with pD2 values of 8.08 +/- 0.13 and 5.27 +/- 0.13 for the high and low affinity component, respectively. 6. Relaxation responses to SCA 40 in human bronchi were resistant to blockade by glibenclamide at concentrations up to 10 microM (which blocked the relaxant response to lemakalim), quinine (30 microM), apamin (100 nM), tetraethylammonium (0.1-1 mM) and charybdotoxin (10-100 nM), thus excluding the involvement of a variety of K+ channels including KATP and KCa channels. 7. In bronchi contracted with carbachol, 1 microM, the nature of the interaction between SCA 40 and the beta 2-adrenoceptor agonist, salbutamol, was synergistic. 8. These experiments establish that SCA 40 is a potent relaxant of human bronchial smooth muscle manifesting spontaneous tone. A low affinity relaxant component has its counterpart in the relaxation seen in both human arterial and venous smooth muscle. The consensus of the evidence suggests that K+ channel opening is not the basis of the relaxant response to SCA 40. Furthermore, BKCa channels appear to be of minor importance in the regulation of human airway smooth muscle tone. Our data suggest that inhibition of an adenosine 3':5'-cyclic monophosphate phosphodiesterase may contribute, at least to the low affinity relaxant component of SCA 40. However, the exact mechanism mediating the SCA 40-induced relaxation of human airways remains to be defined.

Atropine-resistant effects of the muscarinic agonists McN-A-343 and AHR 602 on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart.

1 The effects of 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) and N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602) on cardiac performance and noradrenaline release from terminal sympathetic fibres were measured in isolated perfused hearts of rabbits.2 In the presence of sufficient atropine to block muscarinic receptors, high concentrations of McN-A-343 and AHR 602 caused no cardiac stimulation and there was no increase in the resting output of noradrenaline into the perfusates.3 McN-A-343 and AHR 602 increased both the mechanical responses and the transmitter overflow evoked by electrical stimulation of the sympathetic nerves (SNS) but inhibited both parameters during perfusion with 1,1-dimethyl-4-phenylpiperazinium (DMPP). The effects were atropine-resistant and qualitatively similar to those seen with cocaine. Hexamethonium inhibited DMPP, but affected neither SNS per se nor the facilitatory effects of McN-A-343 and AHR 602 on SNS.4 McN-A-343, cocaine and desipramine (but not AHR 602 or hexamethonium) blocked the net cardiac noradrenaline uptake and increased the positive chronotropic effect of noradrenaline.5 Prior perfusion with concentrations of cocaine and desipramine sufficient to block uptake reduced or abolished the facilitatory effects of both McN-A-343 and AHR 602 on SNS.6 Cocaine, McN-A-343 and AHR 602 displayed local anaesthetic properties on the guinea-pig wheal and frog nerve plexus tests, and their relative potencies in this respect were similar to those for inhibition of DMPP-evoked transmitter overflow. Hexamethonium did not produce local anaesthesia.7 The results indicate that the facilitated release of noradrenaline after SNS and the inhibition of release after DMPP produced by McN-A-343 and AHR 602 are the result of their combined local anaesthetic action and inhibition of amine uptake.

Effects of several muscarinic agonists on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart.

1. The effects of several muscarinic agonists on atrial tension development, ventricular rate and noradrenaline release from terminal sympathetic fibres evoked by electrical nerve stimulation (SNS) and 1,1-dimethyl-4-phenylpiperazinium (DMPP) were measured in isolated perfused rabbit hearts.2. Hexamethonium, in a concentration which almost abolished the release of noradrenaline by DMPP, had no effect on the release produced by SNS, confirming that the stimulation was postganglionic.3. The order of potency for inhibition of atrial tension development was N-methyl-1,2,5,6, tetrahydro-nicotinic acid prop-2-yne ester (MH-1)>oxotremorine > acetylcholine > methacholine > carbachol > furtrethonium > pilocarpine>4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343)>N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602). All effects were abolished by atropine (1.4 x 10(-6)M).4. Each compound was more potent relative to acetylcholine in inhibiting ventricular rate than atrial tension. With the exception of carbachol, the order of potency was the same.5. Both AHR 602 and McN-A-343 facilitated the release of noradrenaline by SNS and inhibited that by DMPP. The effects were atropine-resistant and hence non-muscarinic.6. The muscarinic compounds (except AHR 602 and McN-A-343) each produce atropine-sensitive inhibition of noradrenaline release evoked both by SNS and DMPP although it is likely that furtrethonium and pilocarpine have additional non-muscarinic inhibitory activity against DMPP. The order of potency on both parameters and the potencies relative to acetylcholine were in good agreement with those for inhibition of atrial tension.7. The results suggest that similar muscarinic receptors mediate inhibition of atrial tension development, ventricular rate and neuronal noradrenaline release caused by SNS and DMPP.8. In terms of the two muscarinic sites known to be present in the superior cervical ganglion, the receptors of the terminal fibres mediating inhibition of noradrenaline release are more likely to correspond to those mediating hyperpolarization than to those mediating depolarization, for which AHR 602 and McN-A-343 show specificity.

Mechanism of the indirect sympathomimetic effect of 5-hydroxytrypt-amine on the isolated heart of the rabbit.

1 Rabbit isolated hearts, perfused by the Langendorff technique, were used to investigate the indirect sympathomimetic effects of 5-hydroxytryptamine (5-HT). Comparisons were made with noradrenaline and with two indirectly acting sympathomimetic agents with entirely different mechanisms of action, tyramine and dimethylphenylpiperazinium (DMPP). 2 The cardiac stimulant effects of 5-HT, tyramine and DMPP were inhibited by propranolol and practolol and the pA2 values obtained were similar to those obtained with noradrenaline as the agonist. 3 Responses to 5-HT, tyramine and DMPP were greatly reduced on hearts from rabbits pretreated with 6-hydroxydopamine. Such hearts had less than 7% of their normal catecholamine concentration and no fluorescence characteristic of noradrenaline in the cardiac sympathetic nerves could be demonstrated. 4 Rapid, reversible and selective tachyphylaxis to 5-HT was demonstrated during perfusion with 5-HT. In hearts desensitized to DMPP by perfusion with DMPP, responses to 5-HT were also reduced. 5 Perfusion of hearts with colchicine inhibited stimulant responses to 5-HT and DMPP but had little effect on responses to noradrenaline or tyramine. 6 Desmethylimipramine enhanced cardiac stimulant responses to noradrenaline and to a lesser extent, those to 5-HT and DMPP. Responses to tyramine were consistently inhibited by desmethylimipramine. 7 Tetrodotoxin abolished responses of the heart to electrical nerve stimulation but left responses to noradrenaline, 5-HT and DMPP unaffected. 8 5-HT, tyramine and DMPP evoked 3H-release from hearts whose neuronal noradrenaline stores had been labelled by perfusion with [3H]-(-)-noradrenaline. The pattern of release evoked by 5-HT was similar to that of DMPP but differed from that of tyramine. 9 Reducing the calcium concentration in the Tyrode solution from 3.6 to 0.2 mEq/1 did not affect 3H-overflow after tyramine but greatly inhibited that evoked by 5-HT and DMPP. 10 The results confirm that the stimulatn effects of 5-HT on the rabbit isolated heart are the result of noradrenaline release. They further suggest that the site of the release is the terminal sympathetic nerve network. The mechanism of release shows more similarities to that of DMPP (calcium-dependent depolarization and exocytosis) than to that of tyramine (neuronal uptake and stoichiometric displacement).

Differences in regional vascular sensitivity to endothelin-1 between spontaneously hypertensive and normotensive Wistar-Kyoto rats.

1. The systemic and regional haemodynamic effects of porcine endothelin-1 (endothelin) have been measured in anaesthetized spontaneously hypertensive (SH) rats rendered areflexic by ganglion blockade; comparisons were made with age-matched Wistar-Kyoto (WKY) control animals. 2. In both SH and WKY rats endothelin (0.1-1 nmol kg-1 i.v.) elicited an initial, short-lived (less than 2 min), fall in blood pressure which was associated with substantial increases in hindquarter and carotid vascular conductances. Both the blood pressure falls and the peripheral vasodilator responses were greater in SH than in WKY rats. 3. The initial depressor effects of endothelin were followed by marked and sustained increases in blood pressure associated with constriction in carotid, hindquarter, renal and mesenteric vascular beds. Vasoconstrictor responses were quantitatively similar in the two rat strains. 4. Pretreatment with indomethacin (5 mg kg-1 i.p. or i.v.) did not alter the responses to endothelin, 1 nmol kg-1, in SH rats. 5. The regional haemodynamic effects of intravenously administered acetylcholine (0.01-1 microgram kg-1), nitroprusside (0.3-10 micrograms kg-1) and angiotensin II (0.01-0.1 microgram kg-1) were similar in SH and WKY rats. 6. Endothelin (10(-10)-3 x 10(-8) M) contracted aortic rings from both SH rats and WKY control animals. Removal of the endothelium enhanced significantly the sensitivity of tissues from both WKY and SH rats to endothelin; the increase in sensitivity was greater in tissues from SH than WKY rats. 7. The results demonstrate qualitative similarities in the complex haemodynamic effects of endothelin in SH rats and WKY control animals. However, the SH rats display substantially greater vasodilator responses to endothelin than WKY. Eicosanoid generation is not the mechanism of the vasodilator action of endothelin in SH rats under the conditions of our experiments.

Role of endogenous adenosine in the acute and late response to allergen challenge in actively sensitized Brown Norway rats.

1. Endogenous adenosine has been suggested to amplify the response of airway mast cells to allergen in vivo. We have sought evidence for this by monitoring the acute and late-phase response to allergen in Brown Norway (BN) rats actively sensitised to ovalbumin (OA) and treated either with adenosine deaminase (ADA) linked covalently to polyethylene glycol (PEG-ADA; Adagen) to decrease adenosine availability or with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an inhibitor of ADA, plus S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of facilitated adenosine transport, to increase adenosine availability. 2. The cardiovascular effects of adenosine (0.01-3 mg kg(-1) i.v.) were significantly reduced in PEG-ADA-treated animals and augmented in EHNA/NBTI-treated animals. The difference in sensitivity to adenosine in the treated groups was 33- and 15-fold, at the level of 30% reduction in blood pressure and heart rate, respectively. 3. The acute response to allergen, given either intravenously or intratracheally, was quantified as bronchoconstriction. The late phase to allergen was measured as the influx and activation of immunoinflammatory cells into the bronchoalveolar lavage fluid 24 h after challenge. 4. Despite evidence of a substantial difference in adenosine availability following pretreatment with PEG-ADA or EHNA/NBTI, there were no differences in either the acute or late response to allergen in the actively sensitised BN rat. 5. Our data suggest no role for endogenous adenosine in determining the response to allergen under our experimental conditions.