H. Fuder

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

SummaryEffects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [3H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [3H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (−log KB = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 μmol/l) and DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, 50 μmol/l) but was antagonized by the P2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 μmol/l, −log KB = 4.7)and α,β-methylene-ATP (10 μmol/l). Whereas the evoked [3H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and α,β-methylene-ATP caused a small and transient increase. Cibacron blue at 30 μmol/l failed to antagonize the inhibition of evoked [3H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [3H]-noradrenaline overflow was enhanced by cibacron blue, not changed by α,β-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A1 and P2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by α,β-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A1- but also via these P2Y-receptors.

On the opioid receptor subtype inhibiting the evoked release of 3H-noradrenaline from guinea-pig atria in vitro

Summary1.Guinea-pig isolated atria were incubated and loaded with 3H-(−)-noradrenaline. The intrinsic nerves were stimulated with trains of 5 or 35 field pulses (4 Hz), and the evoked efflux of 3H-noradrenaline and of total tritium was determined in the presence of atropine, corticosterone, desipramine, and phentolamine by liquid scintillation spectrometry.2.Ethylketocyclazocine (1.4 nmol/l, IC50), MR 2033 (9.1 nmol/l), dynorphin A (1–13) (25 nmol/l, peptidase inhibitors present), etorphine (71 nmol/l), and [d-Ala2, d-Leu5]-enkephalin (>10 μmol/l, peptidase inhibitors present) inhibited the stimulation-evoked efflux of 3H-noradrenaline in a concentration-dependent manner, but not morphine up to 10 μmol/l.3.The inhibition by ethylketocyclazocine, MR 2033, and etorphine was antagonized by naloxone 1 μmol/l. Similarly, the MR 2033 effect was antagonized by SKF 10047 1 μmol/l. All antagonists investigated failed to affect the evoked 3H-noradrenaline efflux when present in the absence of exogenous agonists.4.Arunlakshana-Schild plots were calculated for the antagonism between ethylketocyclazocine and a pair of stereoisomers, (−)-MR 2266 (20 nmol/l–5 μmol/l) and (+)-MR 2267 (0.3–10 μmol/l) at the presynaptic opioid receptor, and pA2 values were estimated. The isomeric affinity ratio was 60, with pA2 values of (−)-MR 2266, 9.06, and (+)-MR 2267, 7.28, respectively.5.The results show that the 3H-noradrenaline release can be inhibited via activation of presynaptic opioid receptors. Under the conditions presently investigated endogenous opioids do not modulate the evoked transmitter release. The results favour the idea that a single population (presumably of the K-subtype) of opioid receptors is present at guinea-pig atrial noradrenergic nerves.

The effect of dopamine on the overflow of endogenous noradrenaline from the perfused rabbit heart evoked by sympathetic nerve stimulation.

Summary1.The effects of dopamine and two dopamine receptor antagonists (pimozide, flupenthixol) on the release of endogenous noradrenaline evoked by electrical stimulation of the postganglionic sympathetic nerves and their influence on cardiac performance were measured in isolated perfused rabbit hearts.2.Dopamine 0.2μM decreased noradrenaline overflow and ventricular tension development in response to nerve stimulation.3.Dopamine 2μM increased spontaneous noradrenaline output and tension development. The noradrenaline overflow in response to nerve stimulation was greatly enhanced. This action was only partly reversed by wash out of exogenous dopamine, indicating de novo synthesis and facilitated release of noradrenaline.4.Pimozide (approximately 1μM) and flupenthixol 5μM had no effect on noradrenaline overflow and cardiac peroormance.5.In the presence of cocaine 18.3μM dopamine 2μM decreased the overflow of noradrenaline. This effect was fully antagonized by flupenthixol which on its own neither influenced transmitter overflow nor antagonized the presynaptic inhibitory effect of oxymetazoline 0.38μM.6.It is concluded that in the rabbit heart dopamine has an inhibitory effect on the release of endogenous noradrenaline which appears to be, mediated by presynaptic dopamine receptors and which differs from the prejunctional α-adrenoceptor mediated negative feedback mechanism.

The effects of several muscarinic antagonists on pre- and postsynaptic receptors in the isolated rabbit heart

SummaryIn order to reveal possible differences between pre- and postsynaptic muscarine receptors, seven antagonists were tested for their affinities on these receptor sites in the rabbit isolated perfused heart. Methacholine was used as an agonist to inhibit the noradrenaline overflow evoked by electrical stimulation (3 Hz, 3 min) of the sympathetic nerves (presynaptic parameter) and to decrease the systolic tension development of the right atrium (postsynaptic parameter). The affinity of an antagonist was expressed as pA2.A decreasing order of potency was obtained with ipratropium, scopolamine, atropine, trihexyphenidyl, amitriptyline, and gallamine, both for pre- and postsynaptic responses. The antagonists acted competitively and their effects were reversible. Furthermore, for none of the drugs did the pA2 (pre) differ from the pA2 (post).With QNB (3-quinuclidinyl benzilate) a pA2 (post) of 11.65 was obtained. However, the affinity to presynaptic receptors could not be determined as a pA2 value due to the very prolonged exposure time required for the equilibrium with QNB and for that with methacholine in the presence of QNB.It is concluded that the antagonists employed do not reveal differences between pre- and postsynaptic muscarine receptors of the rabbit heart, in spite of their greatly varying chemical structure and their individual affinities ranging over 5 orders of magnitude. The findings confirm the view of a homogeneous muscarine receptor population characterized by functional parameters.

Desensitization of inhibitory prejunctional α2-adrenoceptors and putative imidazoline receptors on rabbit heart sympathetic nerves

SummaryTo find out whether sympathetic nerves of the rabbit heart possess pharmacologically relevant prejunctional imidazoline receptors different from α-autoreceptors, the inhibition by oxymetazoline, aganodine and BDF 6143 (4-chloro-2-[2-imidazoline-2-ylamino]-isoindoline hydrochloride) of endogenous noradrenaline overflow evoked by stimulation of extrinsic postganglionic sympathetic nerves (0.66 Hz, 80 pulses) was investigated. In addition we wanted to find out whether either type of these prejunctional receptors undergoes desensitization upon pre-exposure to respective agonists.The α2-adrenoceptor agonist oxymetazoline inhibited the evoked noradrenaline overflow (2.9 nmol/l, IC50; about 90010, maximum inhibition). The inhibition was antagonized by rauwolscine (−log KB 8.20). This confirms the presence of α2-autoreceptors. Endogenous noradrenaline activated autoinhibition to a small extent as indicated by a rauwolscine-induced increase in evoked overflow by less than 2-fold.The α2- and imidazoline receptor agonist aganodine inhibited the evoked noradrenaline overflow (2.4 nmol/l, IC50; about 80%, maximum inhibition). The inhibition was antagonized by rauwolscine with a potency (−log KB 6.75), about 1/30 of that found at the α2-autoreceptor. Neither an α2-selective low concentration of rauwolscine nor the α1-adrenoceptor antagonist prazosin, nor SKF 104078, a mixed α1/2-antagonist, reduced the aganodine effect. The α2-adrenoceptor antagonist and imidazoline receptor agonist BDF 6143 inhibited the evoked noradrenaline overflow (18 nmol/l, IC50; about 70% maximum inhibition). The inhibition was insensitive to a low rauwolscine concentration.In hearts pre-exposed for 30 min (followed by washout; rauwolscine 0.1 μmol/l added later on to minimize presynaptic α2-adrenoceptor activation or blockade by drugs persisting in the biophase) to oxymetazoline 10 μmol/l, aganodine 2 μmol/l or BDF 6143 10 μmol/l, the inhibitory effects of oxymetazoline 30 nmol/l and aganodine 10 nmol/l were concomitantly reduced. No significant reduction of the agonist effect was seen after pre-exposure to BDF 6143 2 μmol/l. Pre-exposure to BDF 6143 10 μmol/l shifted the concentration for half-maximum inhibition to the right and depressed the maximum effect of both, oxymetazoline and aganodine, but did not affect the inhibitory action of the muscarinic agonist methacholine.It is concluded that inhibitory prejunctional α2-autoreceptors and putative imidazoline receptors coexist on postganglionic sympathetic nerves of the rabbit heart. They are both subject to desensitization upon exposure to a high agonist concentration. The findings are compatible with a mutual cross-desensitization under the conditions investigated.

M2 muscarinic receptors on the iris sphincter muscle differ from those on iris noradrenergic nerves

The pre- and postjunctional affinity constants of a series of muscarinic antagonists were determined in guinea pig and rabbit irises. Field stimulation-evoked [3H]noradrenaline release from superfused isolated irises was concentration dependently inhibited by (+/-)-methacholine, confirming the presence on the iris noradrenergic nerves of prejunctional inhibitory muscarinic receptors. The affinity constants of the antagonists at the pre- and postjunctional receptors are compatible with the coexistence in the iris of two different M2 receptors: the cardiac (M2 alpha) subtype on the noradrenergic nerves and the smooth muscle (M2 beta) subtype on the iris sphincter muscle. The rank order of potency of the antagonists studied at the prejunctional site was: atropine greater than himbacine greater than AF-DX 116 greater than pirenzepine greater than hexahydrosiladifenidol. The order of potency at the postjunctional receptors mediating the methacholine-induced isotonic contraction of the isolated rabbit iris sphincter was: atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than himbacine greater than AF-DX 116.

Purinoceptor-mediated modulation by endogenous and exogenous agonists of stimulation-evoked [^3H]noradrenaline release on rat iris.

SummaryTo investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [3H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [3H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated.The relatively A1-selective DPCPX 10 and 100 nmol/l increased the evoked [3H]-noradrenaline overflow by about 25%–35%a indicating a minor inhibition of evoked release by endogenous purinoceptor agonists probably via an A1 adenosine receptor. Whereas the A1/A2-antagonist 8-PT failed to increase the evoked [3H]-noradrenaline overflow in the absence of exogenous agonists (without or with dipyridamole 1 pmol/l present), the relatively A2-selective antagonist CP-66,713 (4-amino-8-chloro -1-phenyl(1,2,4)triazolo(4,3-a)quinoxaline) 100 nmol/l decreased it by 20%–30% in the absence and continuous presence of DPCPX. This may be compatible with a minor A2-mediated facilitation by an endogenous purinoceptor agonist.All exogenous agonists tested (except UTP 100 μmol/l) inhibited the evoked [3H]-noradrenaline overflow. The relative order of agonist potency (IC4o, concentration in μmol/l for inhibition of evoked release by 40%) was CPA (N6-(cyclopentyl)adenosine, 0.004) > R-PIA (R(−)N6-(2phenylisopropyl)adenosine, 0.066) = CHA (N6-(cyclohexyl)adenosine, 0.082) > NECA (N5-(ethyl-carboxamido)adenosine 0.44) > ADO (adenosine, 4.1). ATP was n early equipotent with ADO. Maximum inhibition was 70%–80% and similar for all agonists. Adenosine deaminase 1 u/ml failed to affect the ATP-induced, but abolished the adenosine-induced prejunctional inhibition. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine (NBTG) failed to enhance the potency of ADO and ATP. The A1-selective antagonist DPCPX 10 nmol/l did not reduce the ATP potency indicating an effect of ATP per se not mediated via an A1 purinoceptor.Prejunctional affinity constants of 8-PT were 6.07 when tested against adenosine (in the presence of dipyridamole), and 6.60 against CHA. The apparent -log KB of DPCPX tested against CPA was 9.71. The high DPCPX affinity is compatible with an A1 adenosine receptor mediating inhibition of sympathetic neurotransmission in rat iris. This receptor may not be the only prejunctional purinoceptor on rat iris sympathetic nerves. The receptor by which ATP acts prejunctionally in this tissue remains to be determined.

Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens

The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-prefering antagonists.Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M1 receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M1(m1)-M4(m5) receptors in functional studies and binding experiments.The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the Al receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A1 and P2 purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC50): arecaidine propargyl ester (0.062 μM), 4-Cl-McN-A-343 (0.32 μM), 4-(4-fluorophenylcarbamoyloxy)-2-butynylN-methyl-pyrrolidinium tosylate (4-F-PyMcN+; 0.48 μM) and methacholine (0.86 μM). The affinity constants of most of the muscarinic antagonists [atropine: pKB = 9.47; (R)-trihexyphenidyl: pKB = 9.18; pirenzepine: pA2 = 7.68; methoctramine: pKB = 6.90] are consistent with estimates of these antagonists at M1(m1) receptors determined in various functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F-PyMcN+ argue for the involvement of M1, and against that of M2 and M3 receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pKB of 8.30 for himbacine is not in accordance with an M1 receptor; by contrast, it would be compatible with the presence of M2 or M4 receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of noradrenaline release that are probably of the same subtype as the putative M1 receptors inhibiting neurogenic twitch contractions, and are not of the M2, M3 or m5 subtype.

A muscarinic receptor different from the M1, M2, M3 and M4 subtypes mediates the contraction of the rabbit iris sphincter

SummaryIn order to analyse the subtype of muscarinic receptors involved in the methacholine-induced contraction of the rabbit iris sphincter we have determined equilibrium dissociation constants (KB) of various antagonists in the sphincter muscle. The values were compared with those observed at M1 (rabbit vas deferens), M2 (heteroreceptors in rat iris) and M3 receptors (guinea-pig ileum), or at the muscarinic receptors in the guinea-pig uterus.The methacholine-induced contraction of the uterus from immature guinea-pigs was competitively antagonized by pirenzepine (6.64, −log KB), 4-DAMP (8.39), hexahydrodifenidol (HHD; 7.00 for the (R)- and 5.40 for the (S)-enantiomer), p-fluoro-hexahydrosiladifenidol (pF-HHSiD; 6.25) and valethamate bromide (8.04). The affinity of the antagonists is consistent with the presence of an M2 receptor.The −log KB values of the antagonists in the rabbit iris sphincter (6.43, p-F-HHSiD; 6.22, AQ-RA 741; 7.23 and 5.34, (R)- and (S)-trihexyphenidyl) were lower than, or within the lowest range of, estimates in the other experimental models, irrespective of the subtype selectivity of the antagonist. This excludes the presence of an M1, M2, M3 or M4 receptor in this smooth muscle. The affinity of UH-AH 37 in the iris was intermediate between that for M1 or M3, and M2 receptors. The low affinity of AQRA 741 and the low enantiomeric ratio of trihexyphenidyl (THP) in the iris (77.6) would be compatible with a presumed M5 receptor.Valethamate bromide and clozapine did not differentiate between M1, M2 and M3 receptors in the present functional studies; the pA2 values at the M1–M3 sites (8.46–8.57 and 7.26–7.58, respectively) were about 10 and 100 times higher, respectively, than the estimates in the iris sphincter. In absolute terms, however, our −log KB values of the THP-enantiomers, UH-AH 37 and clozapine in the iris sphincter were 0.8–2 log units lower than previous data on cloned m5 receptors suggesting receptor properties different from the presumed M5 receptor.In conclusion, the muscarinic receptors in the rabbit iris sphincter may represent a novel type differing from M1–M4 receptors.

Different muscarinic receptors mediate autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat isolated perfused heart

SummaryExperiments were carried out on rat isolated perfused hearts with both vagus nerves attached. The acetylcholine stores were labelled with [14C]-choline. The effects of muscarinic receptor antagonists on the [14C]overflow and increase in perfusion pressure evoked by vagus nerve stimulation (10 Hz, 4–10 mA) were studied in order to determine the muscarinic receptor type involved in autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat heart.Stimulation of the vagus nerves (1200 pulses) caused an increase in [14C]-overflow and in perfusion pressure which was significantly reduced by hexamethonium 500 μmol/l and abolished by tetrodotoxin 0.3 μmol/l or perfusion with Ca2+-free solution. The fractional rate of evoked [14C]-overflow per pulse upon stimulation at 10 Hz (720 pulses) was doubled in the presence of the non-selective antagonist atropine (0.01–1 μmol/l) as well as in that of the M2-selective compounds methoctramine (0.1 μmol/l) and AF-DX 116 (0.1–1 μmol/l), but remained unaffected by the M3-selective hexahydrosiladifenidol (0.1 μmol/l). The increase in perfusion pressure upon nerve stimulation was reduced by atropine (0.01 μmol/l) or hexahydrosiladifenidol (0.1 μmol/l) to approximately 50% and increased by about 50% in the presence of AF-DX 116 (0.1 μmol/l).The results show that the autoinhibition of acetylcholine release in the rat heart is mediated by M2 receptors. On the other hand, the increase in perfusion pressure upon vagus nerve stimulation is caused by a different muscarinic receptor, more sensitive to hexahydrosiladifenidol than to M2-selective antagonists.