Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Differences between full and partial α-adrenoceptor agonists in eliciting phasic and tonic types of responses in the longitudinal smooth muscle of the rat portal vein

Summary The aim of the present investigation was to study, taking into account both quantitative and qualitative differences, the influence of full and partial -adrenoceptor agonists on spontaneous myogenic activity in the rat portal vein.We found that the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, phenylephrine, St 587, Sgd 101/75, B-HT 920 and UK-14,304 could increase the amplitude of the phasic myogenic contractions in the rat portal vein with apparent differences in EC₅₀ and E_max values. In addition to an increase in phasic myogenic activity, the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, and phenylephrine were also able (in higher concentrations) to increase the basal tone of the rat portal vein preparation, again with apparent differences in EC₅₀ and E_max values. Changing the extracellular Ca²⁺ concentration from 0.9 mmol/l to 2.5 mmol/1 had no influence on the phasic character and the concentration range in which St 587 and UK-14,304 increased spontaneous myogenic activity, although changes in amplitude and frequency of the spontaneous myogenic contractions were less pronounced at a higher extracellular Ca²⁺ concentration (2.5 mmol/1). By the use of Schild analysis with the competitive a-adrenoceptor antagonists prazosin (pA₂ = 8.74) and 5-methyl-urapidil (pA₂ = 8.37), it was established that the contractile responses to St 587 were mediated by the same ₁-adrenoceptor subtype as the phasic and tonic type of contraction elicited by phenylephrine as described in a previous study. The concentration-response curve of UK-14,304 was significantly shifted to the right by low concentrations of prazosin (3 nmol/1–30 nmol/1), indicating stimulation of ₁-adrenoceptors by UK-14,304 in the rat portal vein. The -adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine irreversibly blocked the contractile responses to St 587. Based on the method of receptor alkylation with phenoxybenzamine an affinity constant was calculated for St 587 (pK_a = 5.91). Phenoxybenzamine was approximately 1000-fold more potent in inactivating ₁-adrenoceptors than chloroethylclonidine.In conclusion there appeared to be a divergence in the excitation-contraction coupling of ₁-adrenoceptors in the rat portal vein, which is reflected by two types of contraction (phasic versus tonic). The extent to which both the phasic and tonic types of contraction are stimulated by agonists depends on the affinity and intrinsic efficacy for each of the receptor-coupled effector pathways. Thus, partial and full agonism can only meaningfully be discussed if confined to one particular effector pathway. Send offprint requests to H. R. Schwietert at the above address     

Postjunctional α-adrenoceptors

Summary The postsynaptic -adrenoceptors in rat aorta and in pithed rat were investigated according to their sensitivity to nine -adrenergic agonists and to the selective antagonists yohimbine (₂) and prazosin (₁) and the non-selective one, phentolamine. In addition, in radioligand binding studies, the affinity and selectivity of the drugs were determined on rat cerebral cortex using [³H] yohimbine and [³H] prazosin.On rat aorta, prazosin is 1,000 times more potent than yohimbine against each -adrenoceptor agonist, whether ₁- or ₂-selective. Rat aorta probably contains only ₁-adrenoceptors.Pressor effects in pithed rats are mediated by post-junctional ₁- and ₂-adrenoceptors. The dose-response curve for -methylnorepinephrine in the presence of prazosin, using Hofstee's plots, revealed ₁- and ₂-adrenoceptors, respective proportions being 80.5 and 19.5%     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

Summary The involvement of ₁- and ₂-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with blockers (usually ₁-selective) and 9 patients not treated with blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. ₁-adrenoceptors were activated by (–)-noradrenaline during ₂-adrenoceptor blockade with 50 nmol/l ICI 118551. ₂-adrenoceptors were activated by (–)-adrenaline during ₁-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (–)noradrenaline and (–)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (–)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a blocker and 17/17 atrial strips from 15/15 patients chronically treated with blockers. ICI 118551 (50 nmol/l) blocked the (–)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with blockers and 17/20 atrial strips from 15/18 patients chronically treated with blockers. The incidence of arrhythmic contractions evoked by both (–)-noradrenaline and (–)-adrenaline was higher in chronically blocked patients than in non blocked patients. We conclude that both ₁- and ₂-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic ₁-adrenoceptor blockade. Correspondence to: A. J. Kaumann at the Clinical Pharmacology Unit, University of Cambridge, as above     

Failure of “antigastrin” (SC-15 396) to inhibit gastric acid and pepsin secretion in anaesthetized gastric fistula cats

Summary 1. The effect of antigastrin (SC-15 396) on gastric acid and pepsin secretion produced by the gastrin-analogue tetrapeptide amide Try. Met. Asp. Phe-NH₂ and by electrical stimulation of the vagus was investigated in anaesthetized gastric fistula cats.2. Antigastrin failed to inhibit both acid and pepsin response stimulated by either the tetrapeptide or vagus excitation.3. It was concluded that the ineffectiveness of antigastrin in cats is due to a species difference between rats and dogs on the one hand and cats on the other, and that antigastrin is not a specific gastrin antagonist.Supported by the Deutsche Forschungsgemeinschaft and by the Alfred Teufel-Stiftung.     

Comparative behavioral characterization of the neuroactive steroids 3α-OH,5α-pregnan-20-one and 3α-OH,5β-pregnan-20-one in rodents

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3-hydroxy-5-pregnan-20-one (3,5-P) and 3-hydroxy-5-pregnan-20-one 3,5-P), were studied for differences in their pharmacological properties using behavioral assays. 3,5-P and 3,5-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED₅₀: 3,5-P=2.8 mg/kg and 3,5-P=3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD₅₀:3,5-P=18.8 mg/kg and 3,5-P=21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3,5-P was more efficacious than 3,5-P, though both compounds had similar potencies. In the Geller-Seifter test, 3,5-P was more potent and efficacious than 3,5-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3,5-P and 3,5-P have similar anticonvulsant activity, but the 5-isomer possesses more potent and efficacious anxiolytic properties than the 5-isomer.     

The metabolism of the amyloid precursor protein and its relevance to Alzheimer's disease

Summary The pathology of Alzheimer's disease is primarily characterized by the deposition of -amyloid/A peptide as the major component of senile or neuritic plaques. The A peptide is produced as a result of proteolytic cleavage of the transmembrane protein precursor, APP, during its normal cellular metabolism. The free amino terminus of the A peptide is generated by an endopeptidic cleavage between Met⁶⁷¹-Asp⁶⁷² by a protease termed -secretase. Increased cleavage at this site takes place in a rare, inherited double mutation (Lys⁶⁷⁰-Met⁶⁷¹ to Asn⁶⁷⁰-Leu⁶⁷¹), leading to increased A production and consequent development of Alzheimer's disease on an accelerated time scale in the affected individuals, underscoring the pathological importance of -secretase activity. Cellular studies provide direct evidence that inhibition of -secretase activity would appear to be effective in inhibiting A production as a rational approach to developing therapeutics for the disease.     

The effect of isoprenaline on plasma concentrations of immunoreactive β-endorphin and β-lipotropin in the conscious rat

Summary The effect of the -adrenoceptor agonist isoprenaline on the plasma concentrations of -endorphin (-E) and -lipotropin (-LPH) was investigated in conscious rats. Isoprenaline (i.m.) elevated plasma -endorphin-like immunoreactivity (-EI) as measured by radioimmunoassay of unextracted plasma, with peak values 24 min after drug administration. This effect was dose-dependent. The lowest effective dose of isoprenaline was 15 g kg^–1; 240 g kg^–1 exerted a maximum effect, raising plasma -EI about ten-fold above control values. Plasma vasopressin concentrations also increased in response to isoprenaline following a timecourse identical to that of plasma -EI. (±)-Propranolol (1 mg kg^–1) but not phentolamine (10 mg kg^–1) rendered isoprenaline (240 g kg^–1) injections almost ineffective. Because of the cross-reactivity of -LPH in the radioimmunoassay used, plasma was extracted by means of a cation exchange resin and subjected to gel chromatography on a Sephadex G-50 column, avoiding artefactual degradation of the peptides. In isoprenaline-treated rats about 50% of the -EI behaved similar to human -LPH, whereas 45% co-migrated with human -E; immunoreactivity corresponding to -LPH or -E comprised about 70% or 30%, respectively, in the plasma extract of vehicle-treated rats. Dexamethasone pretreatment reduced the isoprenaline-induced increase in plasma -EI by 87%, but left the simultaneous elevation of plasma vasopressin concentrations unchanged.These data demonstrate that isoprenaline stimulates -LPH and -E release in vivo. The possibility of an interrelationship between vasopressin and -E release is discussed.     

Nasal Absorption Enhancement of 17β-Estradiol by Dimethyl-β-Cyclodextrin in Rabbits and Rats

A new formulation for nasal administration containing 17-estradiol (E₂) with dimethyl--cyclodextrin (DMC) as a solubilizer and absorption enhancer is described. Nasal administration of this E₂-DMC formulation gave a significantly higher E₂ absorption than an E₂ suspension in both rabbits and rats. Relative to an intravenous injection of the E₂-DMC formulation, absolute bioavailabilities of 94.6 and 67.2% were calculated for the nasal E₂-DMC formulation in rabbits and rats, respectively. Differences in bioavailability may have resulted from differences in experimental animal conditions. The effects on human nasal ciliary activity of the E₂-DMC formulation were studied with an in vitro method. The formulation was found to exert only a minor effect on ciliary beat frequency. Thus, nasal delivery of E₂, using a cyclodextrin inclusion formulation, may have potential for clinical application, e.g., in the therapy of postmenopausal disorders.     

Alpha2-adrenoceptor-mediated responses to so-called selective alpha1-adrenoceptor agonists after partial blockade of alpha1-adrenoceptors

Summary In dog saphenous vein — a tissue possessing both postsynaptic ₁- and ₂-adrenoceptors — the effects of two selective ₁-adrenoceptor agonists (phenylephrine and methoxamine) were compared with that of the selective ₂-adrenoceptor agonist, UK-14,304, before and after phenoxybenzamine. Furthermore, the influence exerted by prazosin, yohimbine and verapamil on the effects of these agonists was also studied before and after phenoxybenzamine. In the absence of phenoxybenzamine, prazosin (56 nmol/l) caused a parallel shift of the concentration-response curves of both phenylephrine and methoxamine to the right (by 0.94 and 1.1 log units, respectively) and had no effect on the concentration-response curve of UK-14,304, while 20 nmol/l yohimbine caused a marked parallel shift of the concentration-response curve of UK-14,304 to the right (by 1.18 log units) and caused only minor displacements of those of phenylephrine and methoxamine (by 0.2 and 0.33 log units, respectively). After exposure of the strips to 30 nmol/l phenoxybenzamine, prazosin (56 nmol/l) caused small shifts of the concentration-response curves of both phenylephrine (by 0.36 log units) and methoxamine (by 0.31 log units) and did not change that of UK-14,304, while yohimbine (20 nmol/l) caused pronounced parallel shifts of the concentration-response curves (to the right) of all the agonists: phenylephrine (by 1.0 log units), methoxamine (by 0.93 log units) and UK-14,304 (by 1.28 tog units). When UK-14,304 was added to the bath during a sub-maximal contraction to phenylephrine it caused a further contraction almost up to the maximum; if this procedure was repeated after phenoxybenzamine (30 nmol/1), there was no further contraction to UK-14,304.In the absence of phenoxybenzamine, verapamil (5 mol/l) caused a parallel shift of the concentration-response curve of phenylephrine (or methoxamine) to the right and a non-parallel shift (with marked depression of the maximal effect) of that of UK-14,304. However, after phenoxybenzamine (30 nmol/l), the same concentration of verapamil caused non-parallel shifts of the concentration-response curves of the three agonists to the right with about equal depression of the maximal effects. We conclude that, after removal of ₁-adrenoceptor reserve by phenoxybenzamine, the responses to selective ₁-adrenoceptor agonists are predominantly ₂-adrenoceptor-mediated. This may explain why under these conditions, the selective ₁-and ₂-adrenoceptor agonists are equally antagonized by calcium entry blockers.This work was supported by a grant from the University of Porto (Subsidio para acção de investigação no. 36/85) Send offprint requests to S. Guimarães at the above address     

Effects of metal cations on [3H]α,β-methylene ATP binding in rat vas deferens

In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [³H],-methylene ATP ([³H]meATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [³H]meATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl₃ and AlCl₃ (0.1 to 100 M), produced the greatest increases in total binding of [³H]meATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 M and 1–10 mM, increased total binding of [³H]meATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC₅₀ values for potentiating binding of the radioligand were obtained: ZnCl₂ (5.44), MnCl₂ (4.52), CaCl₂ (4.17), CoCl₂ (4.06), MgCl₂ (3.67) and BaCl₂ (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand.The effects of ZnCl₂, CaCl₂ and MgCl₂ were examined in saturation studies. In the absence of added divalent cations, [³H]meATP labelled both high (pK_d = 9.15) and low (pK_d = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl₂ (pK_d = 9.56) and by 30 M ZnCl₂ (pK_d = 9.46) but not by 3 mM MgCl₂. The B_max of the low affinity site for [³H]meATP was increased (approximately 4 fold) by both 3 mM MgCl₂ and 30 M ZnCl₂ but not by 3 mM CaCl₂. The selective effect of CaCl₂ on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl₂ using a low concentration of [³H]meATP (1 nM); the sites exhibited the binding characteristics expected of the P_2x purinoceptor. The selective effect of MgCl₂ on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl₂ and using a high concentration of [³H]meATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [³H]meATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, meATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [³H]meATP binding sites than for the low affinity binding sites. Secondly, GTP--S and pyrophosphate were selective ligands for the low affinity [³H]meATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC₅₀ values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P_2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [³H]meATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity.In conclusion the present study has demonstrated that cations have a marked effect on the binding of [³H]meATP in rat vas deferens. Of particular interest was the ability of CaCl₂ to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl₂ to increase the B_max of the low affinity binding sites enabled these sites to be selectively labelled. Correspondence to: A. D. Michel at the above address     

Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Evidence in favour of a selective α1-adrenoceptor blocking action of WB 4101 in vivo

Summary The -adrenoceptor blocking potency of WB 4101 at ₁- and ₂-adrenoceptors has been investigated in pithed rats.WB 4101 was approximately 97 times more potent at antagonizing the vasopressor responses produced by the selective ₁-adrenoceptor agonist phenylephrine, than those produced by the selective ₂-adrenoceptor agonist M-7.A dose of WB 4101 (3 mg/kg) that caused extensive blockade of vascular ₁-adrenoceptors, but little or no blockade of vascular ₂-adrenoceptors, exerted no significant blockade of the presynaptic ₂-adrenoceptors in the rat heart.The results support the view that WB 4101 is a highly selective antagonist at ₁-adrenoceptors in vivo.     

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary ³H-noradrenaline release from rat neocortical slices induced by 15 mM K⁺ was concentration-dependently inhibited by morphine, [D-Ala²-D-Leu⁵] enkephalin (DADLE) and the calcium entry blocker Cd²⁺. Blockade of presynaptic ₂-adrenoceptors with phentolamine, almost doubling K⁺-induced ³H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd²⁺ remained unaffected. In contrast, activation of presynaptic ₂-adrenoceptors with clonidine (1 M) or TL-99 (1 M), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd²⁺. When in the presence of 1 M clonidine adenylate cyclase was activated with forskolin (10 M), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, -opioid receptor efficacy appears to be dependent on the degree of activation of ₂-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.     

Exposure to the n-3 polyunsaturated fatty acid docosahexaenoic acid impairs α1-adrenoceptor-mediated contractile responses and inositol phosphate formation in rat cardiomyocytes

The beneficial effects of n-3 polyunsaturated fatty acids of fish oil in the prevention of fatal arrhythmias in myocardial ischemia were suggested to be at least in part mediated by a modulation of dihydropyridine-sensitive L-type calcium channels. As cardiac ₁-adrenoceptor stimulation has been suggested to have no significant effect on L-type calcium channels, the aim of this study using cultured neonatal rat cardiomyocytes was to investigate whether chronic n-3 polyunsaturated fatty acid exposure may have an influence on ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias. Pretreatment of the rat cardiomyocytes for 3 days in the presence of the n-3 polyunsaturated fish oil-derived fatty acid docosahexaenoic acid (60 mol/l) markedly decreased ₁-adrenoceptor-stimulated increase in contraction velocity and induction of arrhythmias. The increase in contraction velocity of the cardiomyocytes induced by the -adrenoceptor agonist isoprenaline was also markedly reduced by the n-3 fatty acid pretreatment. Basal contractile amplitude and spontaneous beating frequency of the cardiomyocytes were not significantly altered by the docosahexaenoic acid exposure. The pretreatment of the rat cardiomyocytes for 3 days in the presence of docosahexaenoic acid (60 mol/l) decreased ₁-adrenoceptor-stimulated formation of the calcium-mobilizing second messenger IP₃ and its metabolites IP₂ and IP₁ by 55%. The depression of IP₃ formation by docosahexaenoic acid treatment was not mediated by a decreased uptake of myo-inositol into the cardiomyocytes nor by a decreased synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of phospholipase C. The level of glycerol-3-phosphate, an important substrate of the phosphoinositide cycle, was unaltered by the docosahexaenoic acid pretreatment. Receptor binding studies revealed that the dissociation constant and maximal binding capacity of the ₁-adrenoceptor antagonist (³H)prazosin was unchanged by the n-3 polyunsaturated fatty acid exposure. -Adrenoceptor-and forskolin-stimulated adenylyl cyclase activities were not diminished by the docosahexaenoic acid pretreatment. Chronic exposure of the cardiomyocytes to the n-6 polyunsaturated fatty acid arachidonic acid (60 mol/l) did neither significantly alter ₁-adrenoceptor-induced inositol phosphate formation nor ₁-adrenoceptor-stimulated increase in contraction velocity. The results presented show that chronic n-3 polyunsaturated fatty acid pretreatment of rat cardiomyocytes leads to a marked impairment of ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias concomitant with a n-3 fatty acid-induced decrease in IP₃ formation. This derangement of the phosphonositide pathway by chronic n-3 fatty acid exposure may, thus, contribute to the beneficial effects of fish oil-derived fatty acids in the prevention of fatal arrhythmias in myocardial ischemia.     

Effects of suramin and α,β-methylene ATP indicate noradrenaline-ATP co-transmission in the response of the mouse vas deferens to single and low frequency pulses

Summary Vasa deferentia from mice were field-stimulated by trains of 10 pulses delivered at 0.5 Hz. The pulses elicited separate twitches, the first of which (corresponding to a single pulse) exceeded the following ones in height and width and often was clearly biphasic. , -Methylene-ATP 1 mol/1 and suramin 100 mol/1 caused almost identical changes. They reduced the height of the first twitch in the train by about one half and also reduced its width in a manner indicating that only the second, slow phase remained, but reduced much more markedly the following twitches in which now a small second, slow phase also became detectable. Idazoxan 0.1 mol/1 or yohimbine 0.1 mol/l, when added in the presence of a, -methylene-ATP or suramin, further decreased the first twitch but enhanced twitches No. 2 to 10. These responses were then almost abolished by prazosin 0.1 mol/l. Successive addition of prazosin 0.1 mol/l and idazoxan 0.1 mol/1 to previously untreated vasa deferentia depressed the response to the first pulse by about one half in a manner indicating that only the first, rapid phase remained, but had comparatively little effect on the responses to the subsequent pulses. Suramin 100 mol/l almost abolished the contractions remaining in the presence of prazosin and idazoxan. The results indicate that the first, rapid phase of the neurogenic contractions elicited by single or low frequency pulses is mediated by ATP which substantially contributes to all responses in a train. The second, slow phase is mediated by noradrenaline which substantially contributes to the response to the first pulse only. The adrenergic contribution seems to be mediated by postjunctional ₁- as well as a ₂-adrenoceptors. Prejunctional ₂-adrenergic autoinhibition depresses the release of both ATP and noradrenaline. Send offprint request to I. v. Kügelgen at the above address     

Age-related alterations in α1- and β-adrenoceptors mediated responsiveness of rat aorta

Summary We have examined the responsiveness of - and -adrenoceptors in aortae from 1.5, 3, 6 and 24 month old rats. The isometric contraction to phenylephrine was antagonised competitively by prazosin with a pA₂ value of 9.45, suggesting that the receptor is an ₁-adrenoceptor. The potency of phenylephrine was significantly reduced in 24 months old as compared with all younger rats combined. The maximum contraction to phenylephrine was unaltered in 24 month old rats. The maximum contraction to potassium chloride was significantly less than that to phenylephrine only in 1.5 months old rats. In tissues contracted by potassium chloride, isoprenaline produced a marked relaxation in 1.5 months old animals, but there was a progressive loss with increasing age of the -adrenoceptor-mediated relaxation which was markedly reduced by 6 months and abolished in 24 months old. It is concluded that, in the rat aorta, there is a decrease in ₁-adrenoceptor responsiveness in senescence, and a loss of -adrenoceptor-mediated responses in maturation. Send offprint requests to J. R. Docherty at the above address     

P2-purinoceptor antagonists discriminate three contraction-mediating receptors for ATP in rat vas deferens

The sites of action at which ATP elicits contraction of the rat vas deferens were studied by means of the P₂-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), suramin and reactive blue 2.Increasing concentrations of PPADS (up to 1 mM), suramin (up to 1 mM) and reactive blue 2 (up to 320 M) reduced and eventually abolished contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP 3 M with IC₅₀ values of 2.1, 10.1 and 27.0 M, respectively. In contrast, PPADS and suramin caused only a partial inhibition of contractions elicited by ATP 1 mM, maximal reduction by about 40%, IC₅₀ values 1.3 and 5.0 M, respectively; reactive blue 2 did not change ATP-induced contractions. In tissues exposed to PPADS 320 M throughout, increasing concentrations of reactive blue 2 or suramin decreased contractions elicited by ATP 1 MM, IC₅₀ values 2.6 and 14.5 M, respectively. In tissues exposed to suramin 320 M throughout, increasing concentrations of PPADS decreased contractions elicited by ATP 1 mM, IC₅₀ 37.9 M, whereas reactive blue 2 slightly enhanced these contractions. In tissues exposed to reactive blue 2 100 M throughout, increasing concentrations of PPADS reduced contractions elicited by ATP 1 MM, IC₅₀ 26.6 M, whereas suramin caused no change. Pre-exposure to ,-methylene ATP 1 M to desensitize P_2X-purinoceptors reduced the response to ATP 1 mM by 91% in otherwise untreated tissues, but did not reduce the response to ATP 1 mM in tissues exposed throughout to PPADS 320 M, suramin 320 M or reactive blue 2 100 M. Neither PPADS nor suramin nor reactive blue 2 altered contractions elicited by KCl 35 mM. The P₁-purinoceptor antagonist 8-(p-sulfophenyl) theophylline 100 M did not change contractions elicited by ,-methylene ATP 3 M or ATP 1 mM.It is concluded that ATP 1 mM elicits contraction of the rat vas deferens through three sites: P_2X-purinoceptors which are blocked by PPADS, suramin and reactive blue 2; P_2Y-purinoceptors blocked by reactive blue 2 and suramin but resistant to PPADS; and non-P_2X-non-P_2Y-purinoceptors blocked by PPADS but resistant to inhibition by suramin and reactive blue 2. Correspondence to: R. Bültmann at the above address     

β 1- andβ 2-adrenoceptor binding and functional response in right and left atria of rat heart

Summary The properties of ₁- and ₂-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses to-adrenoceptor agonists were examined. [¹²⁵I](-)-pindolol (¹²⁵IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek ₁'s for association in right and left atria were 6.5×10⁹ l/mol-min and 2.3×10⁹ l/mol-min respectively, while thek _–1's for dissociation were 0.20 min^–1 and 0.17 min^–1. The kinetically determinedK _D's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumK _D's determined from Scatchard analysis of saturation isotherms of specific¹²⁵IPIN binding. Inhibition of¹²⁵IPIN binding by-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition by ₁- and ₂-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement by ₁-selective (practolol, atenolol and metoprolol) and ₂-selective (ICI 118,551) antagonists gave estimates of the proportion of ₁- and ₂-adrenoceptors present in rat atria. Right atria contained 67±4.2% ₂-adrenoceptors and 33±4.2% ₂-adrenoceptor, while left atria contained 67±2.8% ₁- and 33±2.8% ₂-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused by-adrenoceptor agonists were also measured. pA₂ values for non-subtype selective-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withK _D values determined for specific¹²⁵IPIN binding. pA₂ values for ₁- and ₂-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pK _D values of these drugs in binding to ₁-adrenoceptors, but not with the pK _D values in binding to ₂-adrenoceptors. Dose-response curves for stimulation of both rate and force by the ₂-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade of ₁-adrenoceptors with metoprolol than by selective blockade of ₂-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activating ₁-adrenoceptors. These results suggest that ₁- and ₂-adrenoceptors coexist in both left and right atria of rat heart in approximately a 21 ratio, however only ₁-adrenoceptors mediate the chronotropic and inotropic effects of-adrenoceptor agonists.Supported by a grant from the American Heart Association — Georgia Affiliate