Chemistry and bioactivity of Flos Magnoliae, a Chinese herb for rhinitis and sinusitis.

Flos Magnoliae (FM, Chinese name: Xin-yi) is one of the most commonly used Chinese medicinal herbs. It has a long history of clinical use for managing rhinitis, sinusitis and headache. More than 20 different FM species have been used clinically, which makes species identification and evaluation of pharmacological effects of individual chemical ingredients difficult. In this review, we have summarized the current knowledge on FM phytochemistry and its bioactivity activities. The bioactive compounds in FM include both lipid and water-soluble components. More than 90% of the essential components of FM species are terpenoids, including monoterpenes and sesquiterpenes. Lignans and neolignans including tetrahydrofurofuran, tetrahydrofuran and aryltetralin are also present in FM species. A small number of water-soluble compounds have been isolated from Magnolia flower buds, including a benzylisoquinoline alkaloid magnoflorine, an ester ethyl-E-p-hydroxyl-cinnamate and a flavonoid biondnoid. A wide range of pharmacological actions of FM have been reported, including anti-allergy, anti-inflammation and anti-microbial activity. The structure-activity relationship analysis revealed the influence of methylation at position 5 on the 3,7-dioxabicyclo-(3,3,0)-octane backbone of six lignans in antagonistic activities against platelet-activating factor. In addition, the trans stereoisomer fargesin had a much lower bioactivity than the cis stereoisomer demethoxyaschantin. Recent studies have been directed towards the isolation of other bioactive compounds. Further studies on FM may help to develop new anti-inflammatory and anti-allergic drugs.

Efficacy, chemistry and pharmacology of chinese herbal medicine for allergic rhinitis.

An extensive literature search identified six randomized controlled clinical trials in which the efficacy of Chinese herbal medicine had been investigated for the treatment of allergic rhinitis. Although four of these trials had methodological flaws, the therapeutic outcomes of all six have been reviewed. One of two trials considered to be of high quality was concerned with the treatment of seasonal allergic rhinitis and the other with perennial allergic rhinitis. It is considered that all six studies demonstrated various degrees of alleviation of the symptoms of allergic rhinitis. No serious side effects were reported in any of the trials. A number of the herbs in the Chinese herbal formulae used in the trials, and/or their constituent compounds have been reported to possess anti-allergic, anti-inflammatory or immune modulation activity. Such actions include inhibition of the release or action of mast cell mediators such as histamine, inhibition of inflammation induced by chemical agents, and modulation of serum IgE levels or of lymphocyte and/or macrophage activity. An aqueous, unresolved extract of the herbal formula used in one of the six trials has been reported to exhibit a range of pharmacological actions relevant to the treatment of allergic rhinitis. Essential oils, lignans, flavonoids and saponins are chemical classes that are frequently represented in individual herbs of the six Chinese herbal formulae used in the trials. The chemical structures characterising these classes of compound and the pharmacological actions of these and other constituents of the herbs, relevant to allergic rhinitis, have been reviewed.

Electroacupuncture for tension-type headache on distal acupoints only: a randomized, controlled, crossover trial.

OBJECTIVE: To investigate the efficacy of electroacupuncture, applied to distal acupoints only, for tension-type headache. BACKGROUND: Electroacupuncture is commonly used for tension-type headache, but when applied to distal acupoints only, evidence of its efficacy is lacking. DESIGN: A randomized, single-blinded, sham-controlled, crossover clinical trial. Methods.-The trial had 5 stages: baseline (2 weeks), phases I and II (each 4 weeks), washout period (2 weeks), and follow-up (3 months after phase II). Forty patients were randomly assigned to either group A or group B. Group A received real electroacupuncture during phase I, then sham electroacupuncture in phase II. Group B received the treatments in reverse order. Outcome measures were headache frequency and duration, pain intensity using a visual analog scale, mechanical pain threshold, headache disability, and sickness impact. Data were analyzed by univariate 2-way analysis of variance. RESULTS: Thirty-seven patients completed the trial. There were no significant differences between the 2 groups at baseline. At the end of phase I, group A, but not group B, demonstrated significant improvement in mean (standard error of the mean [SEM]) headache frequency (3.0 per month [0.3] versus 12.0 per month [1.7]), duration (13.3 hours [3.5] versus 32.0 hours [6.2]), pain intensity (32.8 mm [4.1] versus 47.5 mm [2.7]), pain threshold (right side, 2.9 kg/second [0.1] versus 0.9 kg/second [0.1]; left side, 2.4 kg/second [0.1] versus 1.1 kg/second [0.1]), headache disability score (6.0 [1.0] versus 16.3 [1.6]), and sickness impact score (288.7 [48.0] versus 687.1 [77.2]). For each parameter, significant differences also were demonstrated for both groups between baseline and phase II, and baseline and follow-up. There were no significant differences between the groups at the end of follow-up (P >.05). CONCLUSION: Electroacupuncture to distal points alone is effective for short-term symptomatic relief of tension-type headache.

Influence of the epithelium on acetylcholine release from parasympathetic nerves of the rat trachea.

1. The present study was undertaken to investigate the influence of the airway epithelium on the release of acetylcholine (ACh) from parasympathetic nerves of the rat trachea. Epithelium-intact and epithelium-denuded preparations of rat trachea were incubated with [3H]-choline to incorporate [3H]-ACh into the cholinergic transmitter stores. Release of radiolabelled transmitter ACh was evoked by electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V). 2. Field stimulation both of epithelium-intact and epithelium-denuded radiolabelled tracheal preparations evoked an increase in the efflux of radioactivity; however, the mean stimulation-induced (S-I) efflux from epithelium-denuded preparations (2932 +/- 190 d.p.m., n = 9) was approximately 60% of that from epithelium-intact preparations (4802 +/- 820 d.p.m., n = 11). We have shown previously that, in epithelium-intact (but not epithelium-denuded) tracheal preparations, a substantial proportion of the S-I efflux is resistant to tetrodotoxin (1 microM) and to the removal of extracellular Ca2+, indicating that much of the S-I efflux is not caused by exocytotic release of neuronal [3H]-ACh. In epithelium-denuded tracheal preparations, superfused individually, phosphorylcholine (1 and 100 microM) did not alter S-I efflux. In epithelium-intact tracheal preparations, both in the absence and in the presence of atropine (1 microM), neither N(G)-nitro-L-arginine (100 microM), superoxide dismutase (100 units ml(-1)), indomethacin (10 microM), capsaicin (30 microM) nor alpha-chymotrypsin (1 unit ml(-1)) altered S-I efflux. 3. Experiments were also performed using two tracheal preparations superfused in series. When unlabelled epithelium-intact preparations were present in the upper chamber (superfused first), the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber (superfused second) did not differ significantly from radiolabelled epithelium-denuded preparations superfused individually. Moreover, there was no significant difference in the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber between experiments in which the upper chamber contained epithelium-intact or epithelium-denuded preparations. 4. Field stimulation of epithelium-intact tracheal preparations in the upper chamber with 90, 120 and 300-s periods (trains of 1 ms pulses, 5 Hz, 15 V) did not significantly alter the S-I efflux from radiolabelled epithelium-denuded tracheal preparations in the lower chamber. 5. When introduced into the upper (unlabelled epithelium-intact) and subsequently allowed to superfuse the lower (radiolabelled epithelium-denuded) tracheal preparations, the stable cholinomimetic carbachol (3 microM) markedly reduced the S-I efflux whereas ACh (0.1 and 1 microM) had no significant effect. However, in the presence of the anti-cholinesterase neostigmine (1 microM), ACh (1 microM) significantly reduced S-I efflux, indicating that ACh is subject to rapid hydrolysis by cholinesterase enzymes. When atropine (10 microM) was only exposed to radiolabelled epithelium-denuded preparations in the lower chamber, the inhibitory effects of ACh (1 microM) and carbachol (3 microM) on S-I efflux were prevented. 6. In conclusion, the findings of the present study do not support the notion that the airway epithelium exerts an inhibitory influence on ACh release from parasympathetic nerves of the rat trachea. Alternatively, if epithelium-dependent modulation of cholinergic transmission does occur in the rat trachea, then the mechanism does not appear to involve phosphorylcholine, nitric oxide, superoxide radicals, cyclo-oxygenase products of arachadonic acid, capsaicin-sensitive neuropeptides or vasoactive intestinal peptide. Moreover, the inhibitory effect of carbachol and ACh on transmitter ACh release in the rat trachea appears to be due solely to activation of prejunctional inhibitory muscarinic cholinoceptors on parasympathetic nerves and does not involve the liberation of a putative epithelium-derived inhibitory factor.

Interaction of the renin-angiotensin system, bradykinin and sympathetic nerves with cholinergic transmission in the rat isolated trachea.

1. The present study was undertaken to investigate the interaction of the renin-angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release. 2. In both epithelium-intact and epithelium-denuded tracheal preparations, the alpha 2-adrenoceptor agonist UK14304 (0.1 and 1 microM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 microM) was antagonized by the selective alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Idazoxan (0.3 microM) alone had no effect on the S-I efflux. 3. Angiotensin II (0.1 and 1 microM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 microM), angiotensin II (1 microM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 microM), to block prejunctional alpha 2-adrenoceptors, angiotensin II (0.1 and 1 microM) did not alter the S-I efflux. When added alone, perindoprilat (10 microM) did not alter the S-I efflux. 4. In epithelium-denuded preparations, bradykinin (0.01-1 microM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 microM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 microM) and phosphoramidon (1 microM), respectively, bradykinin (1 microM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 microM) and phosphoramidon (1 microM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 microM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM), in either epithelium-intact or epithelium-denuded preparations. 5. In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with alpha 2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.

Multiple prejunctional actions of angiotensin II on noradrenergic transmission in the caudal artery of the rat.

1. Angiotensin II produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The threshold concentrations of angiotensin II for enhancement of S-I efflux (between 0.03 and 0.1 microM) and of the stimulation-evoked vasoconstrictor responses (about 0.3 microM) were 10-1000 times higher than those that have been found for several other vascular preparations. 2. The AT1 angiotensin II receptor antagonist losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of S-I efflux by 1 and 3 microM angiotensin II and the enhancement of vasoconstrictor responses by 1 microM angiotensin II. Surprisingly, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced S-I efflux to a much greater extent than did 0.1 microM angiotensin II alone. Moreover, the combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced stimulation-evoked vasoconstrictor responses, in contrast to the lack of effect of 0.1 microM angiotensin II alone. 3. In a concentration of 0.01 microM, the angiotensin II AT2 receptor antagonist PD 123319 did not affect the enhancement of either S-I efflux or vasoconstrictor responses by angiotensin II. However, in a higher concentration (0.1 microM), PD 123319 antagonized the enhancement of both the S-I efflux and vasoconstrictor responses by angiotensin II. 4. In concentrations of 0.01 and 0.1 microM, PD 123319 prevented the marked enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses produced by the combination of 0.1 microM angiotensin II and 0.01 microM losartan. 5. The potentiation by losartan (0.01 microM) of the facilitatory effect of 0.1 microM angiotensin II on S-I efflux and on stimulation-evoked vasoconstriction was still observed in the presence of either the cyclooxygenase inhibitor indomethacin (3 microM), or the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 6. The findings confirm our previous suggestion that, in the rat caudal artery, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 7. The synergistic prejunctional interaction of 0.01 microM losartan and 0.1 microM angiotensin II may be due to either the unmasking by losartan of a latent population of angiotensin II receptors also subserving facilitation of transmitter noradrenaline release, or alternatively, losartan may block an inhibitory action of angiotensin II on transmitter noradrenaline release which normally opposes its facilitatory effect.

Angiotensin II receptors involved in the enhancement of noradrenergic transmission in the caudal artery of the spontaneously hypertensive rat.

1. The effects of the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD 123319, on actions of angiotensin II in isolated caudal arteries of spontaneously hypertensive (SH) and age-matched normotensive (Wistar-Kyoto) rats were compared. 2. Angiotensin II (0.1-3 microM) produced concentration-dependent increases in perfusion pressure in artery preparations from both SH and Wistar-Kyoto (WKY) rats, the maximal increase in the SH rat being significantly greater than the increase in WKY rats. The increase in perfusion pressure in preparations from both strains of rats was prevented by losartan (0.1 microM) and unaffected by PD 123319 (0.1 microM), indicating that the vasoconstrictor action of angiotensin II is subserved by AT1 receptors. 3. Angiotensin II (0.1-3 microM) produced concentration-dependent enhancement of both stimulation-induced (S-I) efflux of [3H]-noradrenaline and stimulation-evoked vasoconstrictor responses in isolated preparations of caudal artery from both SH and WKY rats, in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux produced by angiotensin II (1 microM) was significantly greater in artery preparations from WKY rats than in preparations from SH rats, whereas the maximum enhancement of stimulation-evoked vasoconstrictor responses was greater in preparations from SH rats than in those from WKY rats. 4. In artery preparations from both WKY and SH rats, the AT1 angiotensin II receptor antagonist, losartan (0.01 and 0.1 microM), reduced or abolished the enhancement of both S-I efflux and vasoconstrictor responses by 1 microM angiotensin II. 5. The combination of 0.01 microM losartan and 0.1 microM angiotensin II enhanced both the S-I efflux and stimulation-evoked vasoconstrictor response in caudal artery preparations from WKY rats, whereas 0.1 microM angiotensin alone was ineffective. The AT2 receptor antagonist PD 123319 (0.01 and 0.1 microM) prevented the enhancement of both S-I efflux and stimulation-evoked vasoconstrictor responses by the combination of angiotensin II and losartan. 6. In contrast to findings in WKY preparations and those previously obtained for arteries from another normotensive strain (Sprague-Dawley), in artery preparations from SH rats there was no synergistic interaction between losartan and angiotensin II. Rather, combinations of 0.1 microM angiotensin II and PD 123319 (both 0.01 and 0.1 microM) enhanced S-I [3H]-noradrenaline efflux, whereas 0.1 microM angiotensin II alone was without effect. Moreover, losartan (0.1 microM) prevented the enhancement of S-I efflux by the combination of angiotensin II and PD 123319. 7. The present findings indicate that in the caudal artery of WKY and SH rats, and as previously found in Sprague-Dawley preparations, angiotensin II receptors similar to the AT1B subtype subserve enhancement of transmitter noradrenaline release. 8. As previously suggested for Sprague-Dawley caudal artery preparations, the synergistic prejunctional interaction of losartan and 0.1 microM angiotensin II in caudal artery preparations from WKY rats may be due to either the unmasking by losartan of a latent population of angiotensin II receptors subserving facilitation of transmitter noradrenaline release, or blockade by losartan of an inhibitory action of angiotensin II on transmitter release. 9. The synergistic interaction of PD 123319 and 0.1 microM angiotensin II in caudal arteries of SH rats may also be explained by either of the mechanisms proposed for the normotensive strains, but the involvement of different receptor subtypes would need to be postulated for each of the proposed mechanisms.

Epithelium-dependent inhibition of cholinergic transmission in rat isolated trachea by potassium channel openers.

We have investigated the effects of the potassium channel openers, cromakalim and pinacidil, on cholinergic transmission in rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. In radiolabelled, epithelium-intact preparations, electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V) evoked an efflux of radioactivity that was unaffected by the removal of extracellular Ca2+ and, a large proportion of which was resistant to tetrodoxin (1 microM). In contrast, in epithelium-denuded preparations, both tetrodotoxin and Ca2+ withdrawal virtually abolished the stimulation-induced (S-I) efflux. Thus, with epithelium-denuded but not with epithelium-intact tracheal preparations, the S-1 efflux reflects the release of [3H]-acetylcholine from cholinergic nerves. Atropine (1 microM) markedly enhanced the S-I efflux in both epithelium-intact and epithelium-denuded preparations. In epithelium-intact preparations, the combination of atropine (1 microM) and tetrodotoxin (1 microM) reduced the S-I efflux to about the same level as did tetrodotoxin alone. Thus, in epithelium-intact tracheal preparations, when prejunctional muscarinic cholinoceptors subserving autoinhibition of transmitter release are blocked, S-I efflux may be taken as an index of transmitter acetylcholine release. Cromakalim (1 microM) had no effect on the S-I efflux from either epithelium-intact or epithelium-denuded tracheal preparations. However, in epithelium-intact preparations, when atropine (1 microM) was present, cromakalim (1 and 10 microM) and pinacidil (100 microM) significantly inhibited the S-I efflux. In epithelium-denuded preparations, in the presence of atropine (1 microM), cromakalim (1 microM) and pinacidil (100 microM) were without effect on S-I efflux. The inhibition of S-I efflux produced by cromakalim (1 microM) and pinacidil (100 microM) in epithelium-intact tracheal preparations (in the presence of atropine) was prevented by the ATP-sensitive potassium channel blocking drug glibenclamide (1 microM). Glibenclamide (1 microM) alone enhanced S-I efflux from epithelium-intact preparations in the absence but not in the presence of atropine (1 microM). Glibenclamide (1 microM) was without effect on S-I efflux in epithelium-denuded preparations both in the absence or presence of atropine (1 microM). In conclusion, the present study has provided additional evidence of an inhibitory action of the potassium channel openers, cromakalim and pinacidil, on the release of acetylcholine from parasympathetic nerves of the rat trachea which is dependent upon the functional integrity of the airway epithelium. The findings suggest that cromakalim and pinacidil may inhibit transmitter acetylcholine release by opening ATP-sensitive potassium channels, presumably on epithelial cells. In addition, the enhancement of S-I efflux from epithelium-intact tracheal preparations by glibenclamide may indicate that ATP-sensitive potassium channels on epithelial cells play a functional role in the modulation of transmitter acetylcholine release from parasympathetic cholinergic nerves of the airways.

Inhibition of sympathetic noradrenergic transmission by guanabenz and guanethidine in rat isolated mesenteric artery: involvement of neuronal potassium channels.

The present study investigated the effects of the alpha 2-adrenoceptor agonist guanabenz and the adrenergic neurone blocking drug guanethidine on the resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. The efflux of radioactivity evoked by electrical field stimulation of periarterial sympathetic nerves (60 s trains of 1 ms pulses, 2 Hz, 12 V) was taken as an index of transmitter noradrenaline release. Guanabenz (0.1-10 microM) decreased, in a concentration-dependent manner, both the resting and S-I effluxes of radioactivity. Guanethidine (0.1 and 1 microM) also decreased S-I efflux but increased resting efflux, both effects being concentration dependent. The inhibitory effects of guanabenz on both resting and S-I effluxes were reduced by blockade of the neuronal amine carrier with desipramine (1 microM). The inhibitory effect of guanabenz on resting efflux was prevented by inhibition of monoamine oxidase with pargyline (100 microM). The inhibitory effect of guanabenz on S-I efflux was not due to activation of prejunctional alpha 2-adrenoceptors since the inhibition was not blocked by the selective alpha 2-adrenoceptor antagonist idazoxan (0.1 microM). However, the inhibitory effect of guanabenz and guanethidine on S-I efflux was reduced by the inhibitor of Ca(2+)-activated potassium channels apamin (0.1 microM). The findings suggest that guanabenz, like guanethidine, enters noradrenergic nerve terminals by the neuronal amine carrier. The inhibition of resting efflux produced by guanabenz may be due to inhibition of neuronal monoamine oxidase. The enhancement of resting efflux produced by guanethidine is attributable to its indirect sympathomimetic action. Finally, guanabenz and guanethidine may inhibit transmitter noradrenaline release by activating potassium channels on sympathetic noradrenergic nerve terminals. These findings may be relevant to the mechanism of adrenergic neurone blockade.

Effects of cromakalim, pinacidil and glibenclamide on cholinergic transmission in rat isolated atria.

The effect of the potassium channel openers cromakalim and pinacidil, and the potassium channel blocking drug glibenclamide, were investigated on cholinergic transmission in rat isolated atrial preparations which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. The efflux of radioactivity evoked by electrical field stimulation of intrinsic parasympathetic nerves (pulses at 5 Hz frequency in trains of 60 s duration) was taken as an index of transmitter acetylcholine release. Stimulation-induced (S-I) efflux of radioactivity was virtually abolished by tetrodotoxin (1 mu M) and by the removal of Ca2+ from the atrial superfusion fluid. The muscarinic cholinoceptor antagonist atropine (0.3 mu M) and the alpha2-adrenoceptor antagonist idazoxan (0.3 mu M) each enhanced the S-I efflux. Cromakalim (1 and 10 mu M) produced concentration-dependent reductions in S-I efflux. Pinacidil (10 mu M) also reduced S-I efflux. The inhibition of S-I efflux produced by cromakalim (10 mu M) and pinacidil (10 mu M) was prevented by the ATP-sensitive potassium channel blocking drug glibenclamide (1 mu M). Moreover, glibenclamide (1 mu M) alone enhanced S-I efflux. The findings suggest that cromakalim and pinacidil may inhibit transmitter acetylcholine release from atrial parasympathetic nerves by activation of ATP-sensitive potassium channels. In addition, the finding that glibenclamide alone enhanced S-I efflux in radiolabelled atrial preparations suggests that ATP-sensitive potassium channels are activated under the experimental conditions employed. Taken together, the findings indicate that, in rat atria, ATP-sensitive potassium channels may play a functional role in the regulation of transmitter acetylcholine release from parasympathetic cholinergic nerve terminals.

Evidence for the involvement of different receptor subtypes in the pre- and postjunctional actions of angiotensin II at rat sympathetic neuroeffector sites.

1. The effects of the nonpeptide angiotensin II receptor (AT) antagonists losartan and PD 123319 on actions of angiotensin II in the rat caudal artery and rat vas deferens preparations were investigated. 2. Angiotensin II (1.0 microM) increased perfusion pressure in isolated segments of the rat caudal artery. This increase in perfusion pressure was prevented by the AT1-antagonist, losartan (0.1 microM) but was not affected by the AT2-antagonist, PD 123319 (0.1 microM). 3. Angiotensin II (0.1-3.0 microM) produced a concentration-dependent enhancement of the stimulation-induced (S-I) efflux of [3H]-noradrenaline from isolated segments of rat caudal artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline. The maximum enhancement of S-I efflux was approximately 60% with 1.0 microM angiotensin II. 4. Losartan (0.01 and 0.1 microM) reduced the enhancement of S-I efflux produced by 1.0 microM angiotensin II in the caudal artery. 5. PD 123319 (0.01 microM) did not affect the enhancement of S-I efflux produced by angiotensin II (1.0 microM) in the caudal artery. However, in a higher concentration (0.1 microM), PD 123319 reduced the enhancement of S-I efflux produced by 1.0 microM angiotensin II. 6. Angiotensin II produced concentration-dependent enhancement of the purinergic twitch responses (1 pulse/60 s) in the rat vas deferens. 7. Losartan (0.03 microM) and PD 123319 (0.03 microM) each reduced the angiotensin II-induced enhancement of the twitch responses in the rat vas deferens. 8. These findings indicate that the enhancement of sympathetic neuroeffector transmission in both the caudal artery and vas deferens of the rat involves angiotensin receptor subtype(s) sensitive to both losartan and PD 123319. In contrast, the direct vasoconstrictor effect of angiotensin II in the rat caudal artery involves activation of a receptor subtype sensitive only to losartan.

Preliminary studies on the inflammatory actions of the venoms of some Australian spiders.

Experiments were performed using an anaesthetised rat model to investigate the local inflammatory responses produced by intradermal injections of crude venom gland extracts from a number of Australian spiders, namely, Phonognatha graeffei, Delena cancerides, Isopeda montana, Badumna insignis, Lampona cylindrata, Steatoda grossa, S. capensis Hann. All of the venom gland extracts tested, with the exception of that from S. capensis, produced increases in vascular permeability consistent with acute inflammatory responses. The responses primarily involved the activation of 5-HT receptors, since they were markedly reduced by the nonselective 5-HT1/5-HT2-receptor antagonist methiothepin. Some of the venoms caused liberation of endogenous mediators of inflammation, and some had components that acted directly on the vasculature to increase vascular permeability. Histamine appeared to have little if any role in the observed increases in vascular permeability following intradermal injection of the spider venoms.

Prejunctional effects of cromakalim, nicorandil and pinacidil on noradrenergic transmission in rat isolated mesenteric artery.

The present study investigated the effects of cromakalim, nicorandil and pinacidil on resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. The efflux of radioactivity evoked by field stimulation of peri-arterial sympathetic nerves (pulses at 2 Hz frequency in trains of 60 s duration) was taken as an index of transmitter noradrenaline release. Cromakalim (1-100 microM) and nicorandil (1-1000 microM) produced minor effects on resting and S-I effluxes of radioactivity, but these did not exhibit concentration-dependency. Pinacidil (1-1000 microM) produced concentration-dependent increases, in both resting and S-I effluxes of radioactivity. With 1000 microM pinacidil, resting and S-I effluxes were increased to approximately 348% and 358% of their respective control values. The effects of pinacidil on resting and S-I effluxes were unaltered when the neuronal amine transport system was inhibited by desipramine (1 microM). Inhibition of monoamine oxidase with pargyline (100 microM) treatment markedly reduced the enhancement of resting efflux by 1000 microM pinacidil but did not alter its effect on S-I efflux. It is proposed that the enhanced resting efflux produced by pinacidil without pargyline treatment consists of deaminated [3H]-noradrenaline metabolites formed from [3H]-noradrenaline displaced from transmitter storage vesicles by pinacidil. The enhancement of S-I efflux by pinacidil does not appear to involve disruption of alpha 2-adrenoceptor auto-inhibition of transmitter release since equi-effective concentrations of phentolamine (1 microM) and pinacidil (1000 microM) produced additive effects on S-I efflux, whereas increasing the concentration of phentolamine from 1 to 2M produced no further increases in S-I efflux. In conclusion, this study has provided no evidence of a prejunctional inhibitory effect of the potassium channel openers cromakalim, nicorandil and pinacidil on transmitter noradrenaline release. However, the findings with pinacidil suggest that, in high concentrations, pinacidil displaces noradrenaline from transmitter stores, such that deaminated noradrenaline metabolites are released from the nerve terminals. Furthermore, pinacidil enhances S-I transmitter noradrenaline release, possibly by blocking neuronal potassium channels.

Effects of the venom of the theridiid spider, Steatoda capensis Hann, on autonomic transmission in rat isolated atria and caudal artery.

The possibility of alpha-latrotoxin-like activity in the crude venom gland extract (VGE) of a related Theridiid spider, Steatoda capensis Hann, was investigated. The VGE from female S. capensis Hann spiders produced vasoconstriction in isolated segments of rat caudal artery but was without effect in artery segments obtained from rats that had been pretreated with reserpine (2.5 mg/kg) 24 hr prior to experimentation, indicating that the vasoconstriction was due to the release of noradrenaline from periarterial sympathetic nerves. Steatoda capensis Hann VGE also increased the rate of beating of rat isolated atrial preparations. The positive chronotropic action of the VGE was partly due to the release of noradrenaline from atrial sympathetic nerves since it was reduced by the beta-adrenoceptor antagonist propranolol, and smaller increases in rate were observed in atria taken from rats pretreated with reserpine. The positive chronotropic effect of the VGE was enhanced by atropine, suggesting that the VGE also releases acetylcholine from atrial parasympathetic nerves. The VGE evoked release of radioactivity from rat atria in which the transmitter stores of the atrial intramural noradrenergic nerves had been labelled with [3H]noradrenaline. There appeared to be two components of the release, one involving omega-conotoxin GVIA-sensitive Ca2+ channels, and the other independent of extracellular Ca2+.

Alpha- and beta-adrenoceptor agonist activity in the venom of the Australian huntsman spiders, Delena cancerides and Isopeda montana.

1. Crude venom gland extracts (VGE) were prepared from female Delena cancerides and Isopeda montana spiders. The VGE were tested in isolated rat atrial, caudal artery and pithed rat preparations for pharmacological activity. 2. In rat isolated atrial preparations, D. cancerides and I. montana VGE, each in a concentration of 2 glands/mL, produced increases in atrial rate which were abolished by propranolol (1 mumol/L) but not by ketanserin (0.1 mumol/L) or reserpine pretreatment (2.5 mg/kg s.c. 24 h prior to experimentation) indicating a direct action on atrial beta-adrenoceptors. 3. In rat caudal artery preparations each VGE produced an increase in perfusion pressure, which was taken as an index of vasoconstriction. Pressor responses to D. cancerides VGE (1 gland/mL) were abolished in the presence of prazosin (1 mol/L) but not by reserpine pretreatment, indicating an action of the VGE on vascular alpha 1-adrenoceptors. Neither prazosin nor reserpine pretreatment had any effect on pressor responses of rat caudal artery preparations to I. montana VGE. Ketanserin (6 nmol/L) produced a small reduction in the degree of vasoconstriction produced by the VGE. This demonstrates a lack of alpha 1-adrenoceptor agonist activity of the VGE. 4. Both VGE produced dose-dependent increases in mean arterial pressure and heart rate in pithed rat preparations. The use of relatively selective receptor antagonists indicated that the increases in mean arterial pressure (MAP) produced by both VGE were mediated by an action on alpha 2-adrenoceptors.

Prejunctional actions of tacrine on autonomic neuroeffector transmission in rabbit isolated pulmonary artery and rat isolated atria.

1. This study investigated the effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) on the resting and stimulation-induced (SI) release of radioactive substances from isolated preparations of rat atria and rabbit pulmonary artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline, and from rat atrial preparations in which cholinergic transmitter stores had been labelled with [3H]-acetylcholine. In addition, the effect of tacrine on the uptake of [3H]-noradrenaline by noradrenergic nerves in rat atria was determined. 2. Tacrine produced concentration-dependent increases in the resting efflux of radioactivity from both the [3H]-noradrenaline-loaded artery and atrial preparations. Blockade of neuronal amine transport with desipramine reduced the release of radioactivity evoked by tacrine from atria but not that evoked from artery preparations. Inhibition of monoamine oxidase by pargyline pretreatment markedly reduced the tacrine-evoked release of radioactivity in both atrial and artery preparations. 3. The radioactivity released from [3H]-noradrenaline-labelled rat atrial preparations by 30 mumol/L tacrine consisted entirely of the deaminated metabolite [3H]-DOPEG. The evoked release of [3H]-DOPEG from atria was reduced by approximately 50% by desipramine (1 mumol/L). When atrial monoamine oxidase had been inhibited by pargyline treatment in vivo and in vitro, 30 mumol/L tacrine evoked the release of [3H]-noradrenaline instead of [3H]-DOPEG. However, the amounts of [3H]-noradrenaline released by tacrine when monoamine oxidase was inhibited were only about 25% of the amounts of [3H]-DOPEG released in untreated atria. 4. Tacrine, in concentrations of 1 and 10 mumol/L, enhanced the release of radioactivity evoked by field stimulation of [3H]-noradrenaline-loaded rabbit pulmonary artery preparations. This effect was unaltered by desipramine or pretreatment with pargyline. However, in artery preparations pretreated with pargyline, a high concentration of tacrine (100 mumol/L) markedly reduced SI efflux. In contrast to the findings with artery preparations, tacrine (1-30 mumol/L) did not alter SI efflux in rat atrial preparations. 5. It is concluded that tacrine displaces noradrenaline from intraneuronal transmitter stores of sympathetically-innervated tissues, and that the displaced amine is totally metabolized by monoamine oxidase before leaving the nerve terminals. When deamination of neuronal cytoplasmic noradrenaline is prevented, only a portion of the noradrenaline displaced from storage vesicles passes to the extracellular space. It is likely that the transfer of cytoplasmic noradrenaline out of the terminals is limited by the activity of the amine transport mechanism.

Angiotensin II generation in the rat vena cava: stimulation of local synthesis by beta-adrenoceptor activation.

There is considerable evidence to suggest that isolated tissues have the capacity to generate angiotensin II and that angiotensin II thus generated may enhance noradrenergic neurotransmission. In the present study the possibility that there may be local formation of angiotensin II within the rat vena cava and its consequence to noradrenergic transmission has been investigated. The angiotensin II precursors, angiotensin I and a synthetic tetradecapeptide renin substrate, each enhanced the stimulation-induced efflux of radioactivity from tissues previously incubated with 3H-noradrenaline. The effects of angiotensin I were blocked by the converting enzyme inhibitor captopril and the receptor antagonist saralasin, indicating local conversion to angiotensin II. The effect of the tetradecapeptide was blocked by saralasin, but not by captopril, suggesting either a direct effect of this peptide or conversion to angiotensin II by a pathway not involving angiotensin converting enzyme. The beta-adrenoceptor agonist isoprenaline enhanced noradrenergic transmission in the rat vena cava, relaxed guinea-pig trachea precontracted with carbachol and increased heart rate in rat isolated atria. Captopril and saralasin blocked the effect of isoprenaline in the vena cava, but did not alter its effects in the atria or trachea. This suggests that in the rat vena cava the facilitation of noradrenergic transmission by isoprenaline may involve stimulation of local angiotensin II production.

Differential sensitivities of avian and mammalian neuromuscular junctions to inhibition of cholinergic transmission by omega-conotoxin GVIA.

Nerve stimulation-induced contractions of the chick biventer cervicis muscle were slowly reduced by omega-conotoxin. However, omega-conotoxin had no effect on skeletal muscle function after i.v. injection in mice or on nerve stimulation-induced contractions of focally innervated muscle of the rat diaphragm or the rabbit proximal oesophagus, or the multiply innervated extra-ocular rectus muscle from rabbit. The lack of effect of omega-conotoxin on mammalian neuromuscular junctions was not due to the high safety factor in transmission or to a high local concentration of Ca2+ originating from the muscle, and could not be accounted for in terms of the operation of facilitatory or inhibitory feedback modulation of transmitter release from motoneurone terminals. It is concluded that the Ca2+ channels of mammalian motoneurone terminals differ from those of avian motoneurone terminals and other omega-conotoxin-sensitive nerve terminals.

Examination of the paradoxical lack of responsiveness of the guinea-pig vas deferens to tyramine.

1. The isolated superfused vas deferens from the guinea-pig was more sensitive to the contractile action of noradrenaline than that from the rat. Tyramine had about 10% of the potency of noradrenaline on the rat vas deferens, but had no contractile activity on the guinea-pig vas deferens (potency less than 0.1% of noradrenaline). 2. The response of the guinea-pig vas deferens to noradrenaline was not affected by tyramine in a concentration that produced a clear contractile response of the rat vas deferens. 3. Noradrenaline and tyramine released less radioactivity from guinea-pig than from rat vasa deferentia in which noradrenergic transmitter stores had been labelled with 3H-noradrenaline. In vasa deferentia from either species, tyramine released less radioactivity than noradrenaline. 4. Pretreatment with the monoamine oxidase inhibitor iproniazid increased the amount of radioactivity released by tyramine from vasa deferentia of both species, increased the contractile response of the rat vas deferens to tyramine, and resulted in the appearance of a contractile response to tyramine in the guinea-pig vas deferens. 5. alpha-methyltyramine, which is not a substrate for monoamine oxidase, had about the same potency as tyramine in releasing radioactivity from vasa deferentia of both species, and in contracting the rat vas deferens, but was virtually without contractile activity on the guinea-pig vas deferens. 6. Pretreatment with iproniazid resulted in the appearance of a contractile response to alpha-methyltyramine in the guinea-pig vas deferens. 7. Pretreatment with iproniazid resulted in change in the composition of the radioactivity released by tyramine, with an increase in the proportion of noradrenaline and a decrease in the proportion of deaminated products. There was also an increase in the amount of endogenous noradrenaline released by tyramine. 8. It was concluded that the main factor accounting for the lack of reactivity of the guinea-pig vas deferens to tyramine is intraneuronal metabolism of the noradrenaline displaced by it.