Stimulation-induced Efflux Research Articles

Prostanoids and adrenaline release: a study of [3H]adrenaline efflux from the rabbit isolated, perfused, adrenal gland.

[3H]Adrenaline was incorporated in an isolated perfused preparation of the rabbit adrenal gland and the effects of indomethacin, PGE2 and PGI2 on its release were investigated. Efflux of [3H]adrenaline was elicited by electrical stimulation of the splanchnic nerve (60 s at 5 Hz). Indomethacin (3 and 30 microM) had no effect on stimulation-induced efflux. PGE2 (30, 90 and 300 nM) reduced the efflux; with 90 nM PGE2 the inhibition amounted to approximately 30%. PGI2, in concentrations from 90 to 600 nM, was without effect. These findings indicate that release of [3H]adrenaline from the rabbit adrenal gland is not subject to modulation by endogenous adrenal prostaglandins; however, PGE2 may play a role in some pathological situations.

Facilitatory effect of nicotine on adrenergic neuroeffector transmission in the isolated ear artery of the rabbit.

The effects of nicotine were studied on perfusion pressure and vasoconstrictor responses to sympathetic nerve stimulation in the isolated ear artery of the rabbit. Infusions of nicotine (50 micro M) produced a transient increase in perfusion pressure and potentiated responses to nerve stimulation; these effects of nicotine were unaffected by atropine (0.3 micro M) and abolished or significantly reduced respectively by hexamethonium (300 micro M) or mecamylamine (1 micro M). In experiments with ear arteries previously labelled with [3H] noradrenaline an infusion of nicotine (50 micro M) produced a transient increase in tritium efflux and the potentiation of responses to nerve stimulation in the presence of nicotine was accompanied by a statistically significant increase in stimulation-induced tritium efflux; these effects of nicotine were abolished by hexamethonium (300 micro M) or mecamylamine (1 micro M).

AT1 and AT2-receptor antagonists inhibit Ang II-mediated facilitation of noradrenaline release in human atria.

It is generally accepted that regulation of blood pressure and sympathetic neurotransmission by angiotensin (Ang) II is brought about through activation of AT1-receptors. Since recent studies demonstrated a high proportion of AT2-receptors in the human heart, the aim of our study was to investigate whether Ang II modulates noradrenaline release also through activation of AT2-receptors in this tissue. Human atrial appendages were prelabeled with [3H]-noradrenaline and electrically field-stimulated. Stimulation-induced outflow of radioactivity was taken as an index of endogenous noradrenaline release. Ang I and II enhanced noradrenaline release in a dose-dependent manner up to 55 and 72%, respectively. These effects were blocked by the selective AT1-receptor antagonists EXP3174 and irbesartan (10 nmol/L). Moreover, the selective AT2-receptor antagonists PD123319 and CGP42112A (0.1 and 1 micromol/L) also inhibited Ang II-induced facilitation of noradrenaline release. Captopril (5 micromol/L) shifted the dose response curve for Ang I less potent to the right than EXP3174 (10 nmol/L). Ang I and II enhanced the stimulation-induced noradrenaline release significantly more potent in tissues of patients pretreated with ACE inhibitors than without. In conclusion, both AT1- and AT2-receptors seem to play a role in Ang II-mediated facilitation of noradrenaline release in the human heart. Chronic treatment with ACE inhibitors appears to affect cardiac sympathetic neurotransmission possibly by upregulation of presynaptic Ang II receptors.

Effect of phorbol esters and polymyxin B on modulation of noradrenaline release in mouse atria

Phorbol 12-myristate 13-acetate (PMA; 0.03, 0.1 and 1.0 mumol/l), a protein kinase C activating phorbol ester, significantly enhanced the stimulation-induced (S-I) outflow of radioactivity at 5 Hz stimulation in mouse atria preincubated with [3H]-noradrenaline, whereas a phorbol ester which does not activate protein kinase C, phorbol 13-acetate (0.1 mumol/l), had no effect. This suggests that protein kinase C may have a role in modulating sympathetic neurotransmission. Polymyxin B (7 and 21 mumol/l), an inhibitor of protein kinase C, had no effect on the S-I outflow of radioactivity. However, it had a significant inhibitory effect in a concentration of 70 mumol/l. Polymyxin B (21 mumol/l) reduced the facilitation of the S-I outflow of radioactivity produced by PMA (0.03 mumol/l), 8-bromo-cyclic AMP (90 mumol/l), tetraethylammonium chloride (300 mumol/l), and idazoxan (0.1 mumol/l). Furthermore, when a higher frequency of stimulation was applied (10 Hz rather than 5 Hz), polymyxin B (21 mumol/l) by itself inhibited the S-I outflow of radioactivity. In the presence of a concentration of PMA (0.1 mumol/l) that was maximally effective in enhancing the S-I outflow of radioactivity, both idazoxan (0.1 mumol/l) and 8-bromo-cyclic AMP (90 mumol/l) still enhanced the S-I outflow. This suggests that these agents are not operating through protein kinase C and further suggests that the inhibitory effect of polymyxin B on these agents cannot be due to inhibition of protein kinase C. The effects of clonidine on the S-I outflow were not affected by a maximally effective concentration of PMA (0.1 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Cromakalim inhibits transmitter acetylcholine release in rat trachea by an action on epithelial cells and a diffusible factor.

The present study was undertaken to investigate further the effects of the potassium channel opening drug cromakalim on the release of transmitter acetylcholine from cholinergic nerves of rat isolated trachea by using two tracheal preparations superfused in series. In all experiments, the lower chamber contained an epithelium-denuded preparation which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into cholinergic transmitter stores, whereas the upper chamber contained an unlabelled, epithelium-intact or epithelium-denuded preparation. When the upper chamber contained an epithelium-intact tracheal preparation, cromakalim (1-100 micro M) significantly reduced the stimulation-induced (S-I) efflux of [3H]-acetylcholine from the radiolabelled, epithelium-denuded tracheal preparation in the lower flow chamber. In contrast, when the upper flow chamber contained an epithelium-denuded preparation, cromakalim (10 micro M) was without effect on the S-I efflux. Glibenclamide (1 micro M), an ATP-sensitive potassium channel blocker, was without effect on the S-I efflux when the upper chamber contained an unlabelled, epithelium-intact tracheal preparation. However, glibenclamide (1 micro M) prevented the inhibition of the S-I efflux by cromakalim (10 micro M). When the upper chamber contained an epithelium-intact tracheal preparation and the lower chamber contained an epithelium-denuded tracheal preparation, cromakalim (10 micro M), when infused through the side-arm of the T-piece, such that only the lower radiolabelled epithelium-denuded tracheal preparation was exposed to the drug, was without effect on the S-I efflux. The findings of the present study have provided evidence of an inhibitory action of the potassium channel opener cromakalim on transmitter acetylcholine release in rat trachea which is dependent on the functional integrity of the tracheal epithelium. The findings suggest that cromakalim may inhibit transmitter acetylcholine release by opening ATP-sensitive potassium channels, probably, on cells in the epithelial layer to release a putative epithelial factor, which in turn acts prejunctionally to mediate the inhibitory effect of cromakalim.

Neurotransmitter release in an arterial preparation and the action of alpha-adrenoceptor antagonists.

1 This study examined the potentiating effects of competitive antagonists of the adrenergic alpha2 receptors and of phenoxybenzamine (POB) an irreversible antagonist, on the stimulation-induced efflux of [3H]-noradrenaline in arterial tissue of rabbit. This was done to determine if the lack of concordance of efflux potentiation by antagonists with the expectations of presynaptic negative feedback theory can be attributed to increasingly successful competition from rising perineuronal transmitter concentrations, when stimulation parameters are increased, in the presence of a fixed concentration of competitive antagonist. 2 Tissues were stimulated with a fixed pulse number and frequency, to rule out confounding factors, and major alterations in the concentration of released transmitter were achieved through variations in the pulse duration. 3 Rauwolscine potentiated transmitter release less at the longest, rather than at the shortest pulse duration, and showed a potentiation of release that was indifferent to the quantities of released transmitter. This was also seen with POB although it binds covalently to the presynaptic receptor. 4 Noradrenaline inhibited stimulation-induced transmitter release confirming the presence of presynaptic alpha inhibitory sites. 5 Yohimbine potentiated transmitter release the same as did rauwolscine and POB, and protected the relevant sites against POB potentiation, confirming site identity. The combination of POB and rauwolscine had no greater effect than did either alone certifying that they acted similarly and that maximally effective concentrations of each were used. 6 Consequently, noradrenaline breakthrough of presynaptic receptor blockade does not explain the non-conforming observations made with competitive antagonists in tests of presynaptic theory.

A morpholinocatechol compound (UK42620) with clonidine- and tyramine-like actions.

1. The actions of a morpholinocatechol (UK42620) were studied in rat isolated atria preparations consisting of spontaneously beating left and right atrial pairs. 2. UK42620 produced positive inotropic and chronotropic responses and, in atria that were incubated with [(3)H]-noradrenaline, it also produced a massive increase in the release of radioactivity. 3. These actions of UK42620 were similar to those of tyramine and were blocked by the beta-adrenoceptor antagonist propranolol (0.3 microM) and by the neuronal uptake blocker desipramine (1 microM). 4. In the presence of desipramine, UK42620 but not tyramine produced a decrease in the stimulation-induced efflux of radioactivity that was antagonized by idazoxan. 5. Thus, UK42620 had prejunctional alpha(2)-adrenoceptor activity like that of clonidine- and tyramine-like activity releasing large amounts of noradrenaline.

H3 receptor-mediated inhibition of intestinal acetylcholine release: pharmacological characterization of signal transduction pathways.

The present study investigates the mechanisms through which prejunctional histamine H3 receptors modulate intestinal cholinergic neurotransmission. The experiments were performed on longitudinal muscle-myenteric plexus preparations of guinea pig ileum, preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation. The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. The electrically induced [3H]acetylcholine release was inhibited by histamine (EC50)=33.5 nM) or the H3 receptor agonist R-alpha-methylhistamine (EC50=41.6 nM), whereas it was not affected by pyridylethylamine (H1 agonist), impromidine (H2 agonist), pyrilamine (H1 antagonist), cimetidine (H2 antagonist), thioperamide or clobenpropit (H3 antagonists). The inhibitory effects of histamine or R-alpha-methylhistamine were antagonized by thioperamide (pKd= 8.31 and 8.53, respectively) or clobenpropit (pKd=9.44 and 9.32, respectively), but not by pyrilamine or cimetidine. The modulatory action of histamine on the evoked tritium outflow was attenuated by pertussis toxin and abolished by N-ethylmaleimide, two selective blockers of Gi/Go proteins. Tetraethylammonium or 4-aminopyridine, acting as inhibitors of voltage-dependent K+ channels, enhanced the evoked tritium outflow when tested alone, and apparently counteracted the inhibitory effect of histamine. However, the blocking actions of tetraethylammonium and 4-aminopyridine were no longer evident when their enhancing actions were compensated by appropriate reductions of Ca2+ concentration in the superfusion medium. Histamine-induced inhibition of evoked tritium output was enhanced by omega-conotoxin, a selective blocker of N-type Ca2+ channels, or low Ca2+ concentration, whereas it was not modified by nifedipine, an antagonist of L-type Ca2+ channels. In addition, the inhibitory effect of histamine was not significantly affected by forskolin (activator of adenylyl cyclase), 8-bromo-cyclic AMP (a stable analog of cyclic AMP), rolipram (a selective blocker of type IV phosphodiesterase), phorbol myristate acetate (activator of protein kinase C), H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide, inhibitor of protein kinase A), Ro-31-8220 (2-(1-[3-(amidinothio)propyl]-1H-indol-3-yl)-3-(1-methylindol-3-yl)-maleimide, inhibitor of protein kinase C), KT5823 (N-methyl-(8R*,9S*,11S*)-(-)-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo [a,g]cycloocta[c,d,e]-trinden-1-one, inhibitor of protein kinase G), or lavendustin A (inhibitor of tyrosine kinase). The present results indicate that histamine inhibits intestinal cholinergic neurotransmission through presynaptic H3 receptors coupled to Gi/Go proteins. It is suggested that adenylyl cyclase, serine-threonine protein kinase and tyrosine kinase pathways are not implicated in this regulatory action, and that Gi/Go proteins modulate the activity of N-type Ca2+ channels through a direct link, thus causing a reduced availability of extracellular Ca2+ at the level of ileal cholinergic nerve terminals.

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.

No involvement of nicotinic receptors in the facilitation of acetylcholine outflow in mouse cortex in the presence of neostigmine and atropine

The role of nicotinic and muscarinic receptors in the modulation of acetylcholine release was studied using field stimulated mouse cortex slices incubated with [(3)H]-choline. Both acetylcholine (100 microM) and the cholinesterase inhibitor neostigmine (100 microM) inhibited the stimulation-induced (S-I) outflow of radioactivity but in the presence of atropine (0.3 microM) an enhancement was seen, which may be indicative of facilitatory nicotinic receptors. Mecamylamine (100 microM) was unable to antagonize the enhancement seen in the presence of acetylcholine and atropine. The nicotinic agonist dimethylphenylpiperazinium (30 microM) did not facilitate S-I outflow of radioactivity. A range of nicotinic blockers had no effect on the enhancement seen in the presence of neostigmine and atropine, nor did indomethacin, the 5HT(3) antagonist MDL 7222 nor the NMDA antagonist MK-801. The inability to block this effect suggests that nicotinic receptors are not involved. We postulate, at least for neostigmine, that the facilitation is an artefact because of the use of [(3)H]-choline as a radiotracer whereby the efflux of radioactivity is enhanced because the radiolabelled acetylcholine is not metabolized to choline and therefore flows out of the tissue more readily.

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic alpha2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for alpha2-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. Alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC50 from 0.13 to 1.05 microM; Emax from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC50=44.9 microM; Emax=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl] -4,5-dihydroimidazoline), whereas prazosin (10 microM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl- 1,3-(2H,4H)-isoquinolindione; 10 microM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]a zepine; 10 microM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of alpha2-adrenoceptor blockade by rauwolscine (3 microM) or RX 821002 (1 microM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the alpha2A and alpha2D binding sites as well as at previously classified alpha2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I1 or I2 binding sites. When tested on the electrically induced outflow of tritium, alpha-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 microM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 microM), whereas idazoxan (up to 100 microM) was without effect. When RX 821002 (1 microM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic alpha2-adrenoceptors belonging to the alpha2D subtype.

Modulatory effect of bradykinin on noradrenaline release in isolated atria from normal and B 2 knockout transgenic mice

The modulatory effect of bradykinin on electrically-induced noradrenaline release was assessed in isolated atria from normal and B 2 knockout transgenic mice preincubated with [ 3H]noradrenaline. Concentrations of 1, 3 and 10 nM of bradykinin did not significantly alter the outflow of radioactivity whereas higher concentrations of bradykinin (30 and 100 nM) enhanced it. The facilitatory effect of 30 nM bradykinin was inhibited by a selective bradykinin B 2 receptor antagonist, Hoe 140 ( d-Arg-[Hyp 3,Thi 5, d-Tic 7,Oic 8]bradykinin, 30 nM), and by a protein kinase C inhibitor, bisindolylmaleimide (1 μM). The co-administration of bradykinin (1 to 100 nM) with either [Leu 8]des-Arg 9-bradykinin (100 nM), AcLys[ d βNal 7,Ile 8]des-Arg 9-bradykinin (30 nM) (bradykinin B 1 receptor antagonists) or diclofenac (1 μM) (a cyclooxygenase inhibitor), shifted the facilitatory effect of bradykinin to lower concentrations. The facilitatory effect of bradykinin also was enhanced by enalaprilat (1 μM) and mergetpa (1 μM), inhibitors of angiotensin-converting enzyme (kininase II) and kininase I, respectively. In contrast, selective bradykinin B 1 receptor agonists, des-Arg 9-bradykinin (1 to 100 nM) and Sar[ d-Phe 8]des-Arg 9-bradykinin (1 to 100 nM), did not significantly affect the stimulation-induced outflow of radioactivity. Neither bradykinin (100 nM) nor des-Arg 9-bradykinin (100 nM) had any modulatory effect in B 2 knockout transgenic mice. These findings suggest that the facilitatory effect of bradykinin on noradrenaline release in the mouse atria is mediated exclusively by presynaptic bradykinin B 2 receptors which are linked to protein kinase C. The greater release of noradrenaline with bradykinin under inhibition of prostaglandins production and kininases I and II activity might be of importance in pharmacotherapies.

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.

Angiotensin converting enzyme inhibition unmasks the sympathofacilitatory effect of bradykinin in human right atrium

To investigate the role of angiotensin converting enzyme (ACE) inhibition in bradykinin-mediated modulation of noradrenaline release in human and rat atrium. Human and rat atrial slices were incubated with [3H]-noradrenaline, superfused with Krebs-Henseleit solution and stimulated electrically at 5 Hz. The stimulation-induced outflow of radioactivity was taken as an index of endogenous noradrenaline release. In the absence of ACE inhibition 0.01-1 micromol/l bradykinin failed to alter the release of noradrenaline in human atrium. In contrast, 0.001-0.1 micromol/l bradykinin enhanced the release of noradrenaline in rat atrium. In the presence of 3 micromol/l of the ACE inhibitor captopril, however, bradykinin significantly enhanced the release of noradrenaline in human atrium. The bradykinin B1-receptor agonist (Des-Arg9)-bradykinin (0.01-1 micromol/l) had no effect on the release of noradrenaline in human atrium both in the absence and in the presence of 3 micromol/l captopril. Captopril (3 micromol/l) potentiated the facilitatory effect of bradykinin in rat atrium. The selective bradykinin B2-receptor antagonist D-Arg[Hyp3,Thi5, D-Tic7,Oic8]-bradykinin (Hoe 140, 0.3 micromol/l) and the cyclo-oxygenase inhibitor indomethacin (10 micromol/l) reduced the facilitatory effect of bradykinin significantly in the presence of captopril in rat and human atrium. Prostaglandin F2alpha (0.1 micromol/l), prostaglandin E2 (0.3 micromol/l) and the thromboxane A2 receptor agonist U-46 619 (0.1 micromol/l) enhanced the release of noradrenaline in human atria, whereas 0.1 micromol/l prostaglandin I2 had no effect. These data suggest that bradykinin facilitates the release of noradrenaline in human and rat atrium by activation of bradykinin receptors of the B2-subtype and subsequent release of facilitatory prostaglandins. The facilitatory effect of bradykinin in human atrium can only be demonstrated when its enzymatic degradation is prevented by ACE inhibition.

Prejunctional neuropeptide Y receptors in human kidney and atrium.

The aim of our study was to characterize functionally prejunctional neuropeptide Y (NPY) receptors in human and rabbit renal cortex, as well as in human right atrium. Segments of human atrial appendages and of human and rabbit renal cortex were preincubated with [3H]noradrenaline, superfused with Krebs-Henseleit solution and stimulated electrically in superfusion chambers. The stimulation-induced outflow of radioactivity was taken as an index of endogenous noradrenaline release. The effects of subtype-selective NPY analogs on the stimulation-induced noradrenaline release were studied. NPY, its endogenous analog, peptide YY, and its C-terminal fragment, NPY13-36, but not its analog, [Leu31,Pro34]NPY, concentration dependently (1-100 nM) inhibited [3H]noradrenaline release in all tissues studied. NPY-induced inhibition of [3H]noradrenaline release in human and rabbit kidney was abolished by pretreatment with pertussis toxin. We conclude that prejunctional inhibition of noradrenaline release in human heart and human and rabbit kidney occurs through NPY receptors of the Y2 subtype, which appear to couple to a pertussis toxin-sensitive G protein.

Interleukin-1 beta and tumor necrosis factor-alpha inhibit the release of [3H]-noradrenaline from isolated human atrial appendages.

In the present study, we have investigated the ability of human recombinant interleukin-1 beta (hIL-1 beta) and human recombinant tumor necrosis factor-alpha (hTNF-alpha) to modulate the stimulation-induced (S-I) outflow of [3H]-noradrenaline ([3H]-NA) from isolated superfused human atria. Pieces of human right atrial appendages were excised during routine cardiac surgery. Tissues were incubated with [3H]-NA (0.2 mumol/l) for 30 min at 37 degrees C, then inserted in a Brandel suprafusion system where the radioactivity was washed for 75 min with a Krebs-Henseleit solution at a rate of 0.4 ml/min. Thereafter, the effluent was collected for the remainder of the protocol during which two trains of electrical stimulation (50 mA intensity, 5 Hz frequency, 60 s duration, 2 ms pulses) were delivered at 10 min and 45 min (short protocol) or 85 min (long protocol). The effect of drugs on the S-I outflow of [3H]-NA was determined by adding drugs 20 min (short protocol) or 60 min (long protocol) before the second stimulation. Experiments were carried out in the continuous presence of desipramine (1 mumol/l) to prevent neuronal NA reuptake. The results showed that in human atrium, hIL-1 beta (3 ng/ml) and hTNF-alpha (0.5 ng/ml) significantly inhibited the S-I release of [3H]-NA. The inhibitory effect of hIL-1 beta was blocked by human recombinant IL-1 receptor antagonist (50 ng/ml), and by the cyclooxygenase inhibitor, diclofenac (1 mumol/l), suggesting that hIL-1 beta inhibited NA release through the formation of prostaglandins. The ability of hIL-1 beta and hTNF-alpha to inhibit NA release suggest that mediators of the immune system produced locally may modulate the activity of the sympathetic nervous system in human atrial appendages.

Modulation of noradrenaline release from isolated human atrial appendages

Prejunctional modulation of noradrenaline release has been studied extensively in various experimental preparations. However, the presence and importance of prejunctional noradrenaline release modulation in human cardiac tissue is still unclear. In this study, we have used superfused human right atrial appendages excised from patients undergoing open heart surgery. The tissues were cut into six pieces and incubated with [ 3H]noradrenaline (4 μCi/ml, 0.2 μM) for 30 min at 37°C. The tissues were then inserted into a suprafusion system and washed for 75 min with a Krebs–Henseleit solution at a rate of 0.4 ml/min. The experimental protocol consisted of a 60-min perfusion period during which a field stimulation (2 ms pulses, 60 s, 50 mA, 5 Hz) was delivered at 10 and 45 min. The effect of the drugs on the stimulation-induced outflow of radioactivity was determined by adding them 20 min before the second stimulation. Each experiment was carried out with or without desipramine (1 μM) to study the influence of the reuptake blockade. Fenoterol (1–1000 nM), a β 2-adrenoceptor agonist, and angiotensin II (1–1000 nM) significantly increased noradrenaline release in a concentration-dependent manner. The administration of arecaidine propargyl ester (0.03–3 μM), a non-specific muscarinic receptor agonist, and propylnorapomorphine (0.1 nM–1 μM), a DA 2-dopaminergic agonist, produced a concentration-dependent inhibition of the stimulation-induced outflow of radioactivity. The α 2-adrenoceptor agonist, oxymetazoline (1 μM), inhibited noradrenaline release at a stimulation frequency of 2 Hz, but not at 5 and 10 Hz. The α 2-adrenoceptor antagonist, idazoxan (1 μM), significantly increased the release of noradrenaline at 2 and 5 Hz but not at 10 Hz. The results obtained in the present study demonstrated the presence of the facilitatory β 2-adrenoceptor and angiotensin II receptor as well as the presence of inhibitory α 2-adrenoceptor, muscarinic and DA 2-dopamine receptors in the human atrial appendage.

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.

Interleukin-1 beta and tumor necrosis factor-alpha inhibit the release of [3H]-noradrenaline from mice isolated atria.

In the present study, we have investigated the ability of four cytokines, interleukin-1 beta, interleukin-2, interleukin-6 and tumor necrosis factor-alpha, to modulate the stimulation-induced outflow of radioactivity from isolated superfused mouse atria which where pre-incubated with [3H]-noradrenaline. The tissues were subjected twice to field stimulation (5 Hz frequency, 50 mA intensity, 2 ms pulses for 60 s) and the drugs were added prior to the second stimulation in order to assess their modulatory effects. The results show that mouse recombinant interleukin-1 beta and tumor necrosis factor-alpha inhibited the stimulation-induced release of radioactivity from the isolated mouse atria. The effect of interleukin-1 beta was blocked by a human recombinant interleukin-1 receptor antagonist. The inhibitory effect of interleukin-1 beta was also abolished by the cyclooxygenase inhibitor, diclofenac (1 mumol/l) suggesting that the action of interleukin-1 beta might be mediated through the formation of prostaglandins. The effect of interleukin-1 beta appears to be time-dependent, since a stronger inhibition of radio-activity release was observed when the incubation time was increased from 20 to 65 minutes before the second stimulation. Interleukin-2 and interleukin-6 were ineffective in modulating release under these experimental conditions. The ability of interleukin-1 beta and tumor necrosis factor-alpha to inhibit noradrenaline release suggests that mediators of the immune system produced locally may modulate the activity of the sympathetic nervous system.