Characterization of histamine H3 receptors inhibiting 5-HT release from porcine enterochromaffin cells: Further evidence for H3 receptor heterogeneity

The nature of the histamine receptor mediating inhibition of 5-HT release was investigated in strips of the porcine small intestine by investigating the effects of histamine ligands on the overflow of endogenous 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). The overflow was measured by HPLC, combined with electrochemical detection and represents calcium-sensitive 5-HT release from enterochromaffin cells, as reported previously. The histamine H₃ receptor selective agonists (R)--methyl-histamine and imetit inhibited the overflow of 5-HT maximally by 50–60%, with EC₅₀ values of 48 and 3.2 nmol/l, respectively. Effects on 5-HT overflow were always accompanied by similar effects on the overflow of 5-HIAA. Thioperamide (100 nmol/l) shifted the concentration response curve of (R)--methyl-histamine to the right (pK_B value 8.38). The inhibitory effect of 1 mol/l (R)--methyl-histamine was antagonized in a concentration-dependent manner by thioperamide (IC₅₀: 65 nmol/l) and dimaprit (IC₅₀: 8.6 mol/l); however, the effect of (R)--methyl-histamine was weakly antagonized by burimamide (by 38% at 100 mol/l) and not significantly affected by other H₃ receptor antagonists, such as impromidine, betahistine and phenylbutanoyl-histamine (each up to 100 mol/l). In conclusion, H₃ receptors mediating inhibition of 5-HT release from porcine enterochromaffin cells have a particular pharmacological profile indicating that heterogeneity of H₃ receptors may exist. The data suggest that histamine H₃ receptors modulating 5-HT release in pig small intestine do not belong to either H_3A or H_3B receptors as defined in rat tissue. Correspondence to: K. Racke at the above address     

The cardiovascular effects of intraventricularly administered histamine in the anaesthetised rat

Summary In urethane-anaesthetised rats intraventricular (i.c.v.) injections of histamine (0.1–10.0 g) elicited dose-related rises in both the resting blood pressure and heart rate. These cardiovascular effects of histamine were antagonised in a dose-dependent manner by i.c.v. pretreatments with the histamine H₁-receptor antagonists mepyramine (10, 50 and 100 g) and diphenylpyraline (100 and 200 g). Pretreatment with the histamine H₂-receptor antagonist metiamide (100 and 200 g i.c.v.) failed to modify either of the responses. A dose-related antagonism of the hypertensive response to histamine i.c.v. was elicited by phentolamine (100 and 200 g i.c.v.) but the positive chronotropic effect was not modified by this pretreatment. The cardiovascular responses to histamine i.c.v. were abolished by mecamylamine (5.0 mg/kg i.v.) and greatly reduced by 6-hydroxydopamine (3×250 g i.c.v.), but only the tachycardia was significantly modified by atropine (100 g i.c.v.) and propranolol (1 mg/kg i.v.). Propranolol (100 g i.c.v.), bilateral vagotomy, or acute bilateral adrenal demedullation failed to modify the cardiovascular responses to histamine i.c.v. The results suggest that histamine is able to modify the resting blood pressure and heart rate by independent central modes of action, which involve central adrenergic and cholinergic mechanisms.Preliminary findings of this study were presented at the Autumn meeting of the British Pharmacological Society (Finch and Hicks, 1975).     

Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein by presynaptic histamine H3 receptors

Summary The human saphenous vein was used to examine whether presynaptic histamine receptors can modulate noradrenaline release and, if so, to determine their pharmacological characteristics. Strips of this blood vessel were incubated with [³H]noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. Electrically (2 Hz) evoked ³H overflow was inhibited by histamine and the H₃ receptor agonist R-(–)--methylhistamine. Histamine-induced inhibition of electrically evoked tritium overflow was not affected by ₂-adrenoceptor blockade by rauwolscine. S-(+)--methylhistamine (up to 10 mol/l) as well as the histamine H₁ and H₂ receptor agonists 2-(2-thiazolyl)ethylamine (up to 3 mol/l) and dimaprit (up to 30 mol/l), respectively, were ineffective. The selective histamine H₃ receptor antagonist thioperamide abolished the inhibitory effect of histamine. The histamine H₂ and H₁ receptor antagonists ranitidine and pheniramine, respectively, did not affect the histamine-induced inhibition of evoked tritium overflow. The present results are compatible with the suggestion that the sympathetic nerves of the human saphenous vein are endowed with inhibitory presynaptic histamine receptors of the H₃ class. Send offprint requests to M. Gothert at the above address     

In vivo modulation of histamine release by autoreceptors and muscarinic acetylcholine receptors in the rat anterior hypothalamus

The modulation of histamine release by histamine and muscarinic acetylcholine receptors was investigated by using the push-pull technique. The anterior hypothalamic area of the conscious, freely moving rat was superfused through the push-pull cannula with CSF or with CSF containing drugs and the release of endogenous histamine was determined in the superfusate.Hypothalamic superfusion with tetrodotoxin (10 mol/1) led to a pronounced and sustained decrease in the histamine release rate. Superfusion with compound 48/80 (100 mg/1) was ineffective. Hypothalamic superfusion with the H₃ agonist (R)--methylhistamine inhibited, while superfusion with the H₃ antagonist thioperamide enhanced the release of histamine. The release of histamine was inhibited on hypothalamic superfusion with the muscarinic receptor agonists carbachol or oxotremorine. Histamine release was enhanced by atropine, and this release-enhancing effect was abolished by oxotremorine. The selective M₁ antagonist pirenzepine (100 mol/I) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10 ol/1), which blocks M₁ and M₃ receptors, also enhanced the release rate of histamine. On the other hand, 50 and 100 moI/I methoctramine (M₂ receptor antagonist) 10 and 100 moI/l p-fluoro-hexahydro-siladifenidol (p-F-HHSiD, a M₃ receptor antagonist) were ineffective.It is concluded that histamine released in the hypothalamus originates predominantly from neurons. The release of histamine is modulated by H₃ autoreceptors. The histamine release is also modulated by cholinergic neurons which modify histamine release from histaminergic neurons by stimulating M₁ muscarinic acetylcholine heteroreceptors probably located on histaminergic neurons.Supported by the Fonds zur Förderung der wissenschaftlichen Forschung Correspondence to: H. Prast at the above address     

Low-affinity interactions of BODIPY-FL-GTPγS and BODIPY-FL-GppNHp with G<Subscript>i</Subscript>- and G<Subscript>s</Subscript>-proteins

BODIPY-FL-guanosine 5'-[-thio]triphosphate (B-GTPS) and BODIPY-FL-guanosine 5'-[,-imido]triphosphate (B-GppNHp) induce fluorescence changes upon binding to purified G_s/G_i-proteins and were suggested to serve as probes for monitoring receptor-mediated G-protein activation. However, B-GTPS and B-GppNHp bound to receptor-G_s/G_i fusion proteins expressed in Sf9 cell membranes with 1,100- to 5,600-fold- and 17- to 55-fold lower affinity than GTPS and GppNHp, respectively. The affinity of B-GTPS/B-GppNHp for G_s/G_i-proteins was considerably lower than the affinity of N-methylanthraniloyl (MANT)-substituted GTP analogs for G_s/G_i-proteins. B-GTPS/B-GppNHp were much less potent than GTPS/GppNHp at regulating adenylyl cyclase (AC) via G_s- and G_i-proteins. B-GTPS/B-GppNHp were similarly efficient as GTPS/GppNHp at activating G_i, but less efficient at activating G_s. In contrast to MANT-GTPS/MANT-GppNHp, B-GTPS/B-GppNHp were inefficient at directly inhibiting AC. In conclusion, the bulky BODIPY group strongly reduces the affinity of GTPS/GppNHp for G-proteins, limiting the use of B-GTPS/B-GppNHp as fluorescence probes.Abbreviations AC Adenylyl cyclase - ₂AR ₂-Adrenoceptor - ₂AR-G_olf Fusion protein consisting of the ₂-adrenoceptor and G_olf - ₂AR-G_sL Fusion protein consisting of the ₂-adrenoceptor and the long splice variant of G_s - ₂AR-G_sS Fusion protein consisting of the ₂-adrenoceptor and the short splice variant of G_s - B-GppNHp BODIPY-FL-guanosine 5'-[,-imido]triphosphate - B-GTPS BODIPY-FL-guanosine 5'-[-thio]triphosphate - FPR-G_i1,2,3 Fusion protein consisting of the formyl peptide receptor and G_i1, G_i2 or G_i3 - G Unspecified G-protein -subunit - G_i Inhibitory G-protein of adenylyl cyclase - G_olf Olfactory G-protein that activates adenylyl cyclase - G_s Stimulatory G-protein of adenylyl cyclase - G_sL Long splice variant of the stimulatory G-protein of adenylyl cyclase, G_s - G_sS Short splice variant of the stimulatory G-protein of adenylyl cyclase, G_s - GppNHp Guanosine 5'-[,-imido]triphosphate - GTPS Guanosine 5'-[-thio]triphosphate - M-GppNHp 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[,-imido]triphosphate (MANT-GppNHp) - M-GTPS 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[-thio]triphosphate (MANT-GTPS)     

Investigation into the positive inotropic effect of clondine in isolated hearts

Summary Clonidine in doses 2.5–40 g injected into the aortic cannula of isolated, perfused hearts of guinea pigs produced an increase in contraction amplitude. This positive inotropic effect was not antagonised by infusions of doberol (10 g/min), phentolamine (10 g/min) or pheniramine (50 g/min), it was antagonised by burimamide (30 g/min), a histamine H₂ receptor blocking agent. Clonidine showed some similarities to histamine such as parallel dose-response curves and during infusions of burimamide the curve of clonidine was shifted in a way which indicated competitive inhibition. It is suggested therefore that the action of clonidine is due to stimulation of histamine H₂ receptors.     

Calcium channel modulation by dihydropyridines modifies sufentanil-induced antinociception in acute and tolerant conditions

Summary The study was aimed at elucidating the possible participation of l-type Ca²⁺ channel in the acute analgesic effect of an opiate and the development of tolerance to this action. Sufentanil, a selective p agonist, and two dihydropyridines, the Ca²⁺ antagonist nimodipine and the Ca²⁺ agonist Bay K 8644, were selected. The tail-flick test was used to assess the nociceptive threshold. In naive rats, nimodipine (200 g/kg) potentiated the analgesic effect of sufentanil reducing the ED₅₀ from 0.26 to 0.08 g/kg. Similar results were observed with its (–)-enantiomer Bay N 5248, while the (+) enantiomer Bay N 5247 was ineffective. Tolerance to the opiate was induced by chronic subcutaneous administration of sufentanil with minipumps (2 g/h, 7 days). In these conditions the dose-response curve to sufentanil was displaced to the right and the ED₅₀ was increased to 1.49 g/kg. In tolerant rats, nimodipine preserved its potentiating ability and prevented the displacement to the right of the sufentanil dose response-curve (ED₅₀ = 0.48 g/kg). When nimodipine was pumped (1 g/h, 7 days) concurrently with sufentanil, the development of tolerance to the opioid was not disturbed. However, the expression of tolerance was abolished and even the effect of acutely administered sufentanil was markedly potentiated (ED₅₀ = 0.03 g/kg). Similar experiments were performed with Bay K 8644. In naive rats, Bay K 8644 at a low dose (20 g/kg) that behaves as a calcium agonist, antagonized the analgesic effect of sufentanil (ED₅₀ = 0.58 g/kg), whereas at a high dose (200 g/kg) it potentiated this action (ED₅₀ = 0.15 g/kg). In tolerant rats, Bay K 8644 (20 g/kg) preserved its antagonizing ability inducing a displacement to the right of the sufentanildose-response curve (ED₅₀ = 4.2 g/kg). When Bay K 8644 was pumped (1 g/h, 7 days) concurrently with sufentanil, it enhanced the expression of tolerance to the opiate (ED₅₀ = 3.8 g/kg). These results suggest that the calcium fluxes through the l-type channel in neurones are functionally linked to the activation of the opiate receptor: the blockade of the channel increased the potency of sufentanil, whereas its activation reduced the potency of the opiate. In chronic experiments, DHPs concurrently administered with sufentanil did not affect the development of tolerance to the opiate. However, nimodipine prevented the expression of this phenomenon. Even more, the animals became hypersensitive to the opiate suggesting that the adaptative mechanisms induced by chronic opiate could be affected by chronic nimodipine.This work was supported by grants from Universidad de Cantabria-Caja Cantabria (1988) and Bayer AG, Wuppertal, FRGPredoctoral Fellow: Fondo de Investigaciones Sanitarias de la Seguridad Social.Send offprint requests to: M. A. Hurlé at the above address     

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

Summary Effects 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 K_B = 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 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/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 ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Interaction of adenine nucleotides,UTP and suramin in mouse vas deferens: suramin-sensitive and suramin-insensitive components in the contractile effect of ATP

Summary Effects of various nucleotides, nucleosides and noradrenaline on smooth muscle tension were studied in the isolated mouse vas deferens. ,-Methylene-ATP, ATPS, noradrenaline, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine or uridine (up to 100 mol/l). Prolonged incubation with ,-methylene-ATP (concentration increased stepwise from 0 to 15 mol/l) selectively reduced contractions induced by ATP and UTP but not those induced by noradrenaline, and there was cross-tachyphylaxis between ATP and UTP. Suramin (10–300 mol/l) did not alter the response to noradrenaline but shifted the concentration-response curves for ,-methylene-ATP, ATPS, UTP and lower concentrations of ATP (0.1–1 ol/l) to the right. The pA₂-values of suramin were 5.2 against ,-methylene-ATP, 4.8 against ATPyS, 5.1 against UTP and 5.4 against lower concentrations of ATP. The effects of higher concentrations of ATP were largely resistant to suramin. The results indicate that the mouse vas deferens possesses contraction-mediating smooth muscle P_2X-receptors. UTP also acts at this receptor, and there is no evidence for a separate UTP receptor. The selective inhibition of nucleotide- but not noradrenaline-induced contractions by suramin confirms the view that suramin is a selective P₂-antagonist. The resistance against suramin of part of the effect of ATP suggests that ATP activates a suramin-insensitive site in addition to the P_2X-receptor.Send offprint requests to I. von Kügelgen at the above address     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

Summary The effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 moles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 moles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 moles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 moles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 moles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 moles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 moles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 moles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 moles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 M ZK 62711 increased the level of cyclic AMP but 100 M histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 M ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 M cimetidine and enhanced the stimulatory effect of 100 M histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC₅₀=18 M) and guinea pig gastric mucosa (IC₅₀=1.5 M). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.     

Binding of aprindine and moxaprindine to human serum, α1-acid glycoprotein and serum of healthy and diseased humans

Summary A comparison was made between the binding of the anti-arrhythmic agents aprindine and moxaprindine to human serum, to human serum albumin (HSA), to ₁-acid glycoprotein (₁-AGP) and to a mixture of HSA and ₁-AGP. In serum from healthy volunteers (n=4) the binding of aprindine-HCl 5 µg/ml (13.8 µM) was 93.8% (SD±1.0), and that of moxaprindine-HCl 5 µg/ml (12.8 µM) was 94.1% (SD±1.1). Their binding to the mixture of ₁-AGP and albumin approximated their binding to serum. For ₁-AGP, the binding was similar for both compounds, whereas for HSA the binding of aprindine was more pronounced than that of moxaprindine: for both products the affinity coefficient for binding to ₁-AGP was about 100 times greater than that for binding to albumin. In serum from rheumatoid patients and from patients with renal failure a small but significant increase in binding of aprindine and moxaprindine was observed, approximately 1%. Increased and decreased binding was seen in serum from cirrhotic patients; for example, for aprindine the range in cirrhosis was 96.7%–79.8%, and the range in controls was 95.0%–92.4%. Free drug fraction and ₁-AGP concentration were inversely correlated. The results show that ₁-AGP plays an important role in the binding of aprindine and moxaprindine, and that alteration in the binding of the two compounds in disease states to a large extent can be explained by changes in serum ₁-AGP concentration.     

Is there a need for more precise definitions of bioavailability?

Summary After evaluation of the present definitions in a set of particular cases, it was agreed that there was no need for more precise definitions and that the current ones were adequate in the majority of cases. However, it was felt that the present definitions might be improved, in particular in view of the existence of non-systemically acting drugs and future targeted drugs. Thus, the FDA definition might be modified as follows: Bioavailability means the rate and extent to which the active drug ingredient or therapeutic moiety from a drug product becomes available at the site of drug action or in a biological medium believed to reflect accessibility to a site of action.     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

Chronic β1-adrenoceptor blockade sensitises the H1 and H2 receptor systems in human atrium: role of cyclic nucleotides

We have reported that chronic treatment of patients with ₁-adrenoceptor blockers sensitises isolated atrial preparations to adrenaline, noradrenaline and 5-HT. We have now examined the effect of chronic treatment with -adrenoceptor blockers on responses to histamine of human right atrial appendages. We compared the effects of histamine on contractile force, cyclic AMP and cyclic GMP levels as well as cyclic AMP-dependent protein kinase (PKA) activity and explored the arrhythmogenic effects of histamine in preparations obtained from patients chronically treated or not treated with -adrenoceptor blockers.Histamine increased contractile force in paced preparations; the effects were blocked by the H₂ receptor antagonist famotidine (0.1–30 mol/1). The maximum inotropic response to histamine was doubled and the inotropic potency of histamine 0.4 log units greater in atria from -adrenoceptor blocker-treated compared to non -adrenoceptor blocker-treated patients. Histamine elicited frequency-dependent arrhythmias that were blocked by famotidine (30 mol/1) but not by mepyramine (1 mol/1). The incidence of arrhythmias was higher in atria from -adrenoceptor blocker-treated compared to untreated patients. Histamine increased both cyclic AMP and cyclic GMP levels, as well as PKA activity, significantly more in atria from -adrenoceptor blocker-treated compared to those from untreated patients. Mepyramine 1 mol/l prevented the histamine-evoked increase in cyclic GMP levels, reduced the inotropic hyperresponsiveness and abolished the hyperresponsiveness in cyclic AMP levels and PKA activity observed in patients chronically treated with blockers. Sodium nitroprusside 10 mol/l caused smaller increases of cyclic GMP levels than histamine and restored the contracile force depressed by mepyramine to its original level in atria from -adrenoceptor blocker-treated patients.The evidence is consistent with sensitisation of both the histamine H₁ and histamine H₂ receptor systems by chronic ₁-adrenoceptor blockade. H₁ receptor-mediated increases in cyclic GMP, enhanced through an as yet unknown mechanism by chronic ₁-adrenoceptor blockade, may inhibit phosphodiesterase 3 activity, thereby causing enhanced histamine-evoked increases in cyclic AMP levels and PKA activity, and accounting partially for the increased inotropic responses to histamine through H₂ receptors.     

Some pharmacological properties of Wy 27127 a more selective alpha2: alpha1-adrenoceptor antagonist than idazoxain in vitro

Summary Wy 27127 and idazoxan were approximately equipotent as antagonists at ₂-adrenoceptors as estimated by their ability to block clonidine-induced inhibition of electrically-evoked contractions of the rat isolated vas deferens.Idazoxan was seven times as potent as Wy 27127, as an antagonist at ₁-adrenoceptors as indicated by blockade of methoxamine-induced contractions of the rat isolated anococcygeus muscle.Thus, the ₂: ₁ selectivity ratio, as calculated from these tests was 407 for Wy 27127 and 76 for idazoxan.Wy 27127 and idazoxan were equipotent in enhancing stimulation-evoked overflow of tritium from rabbit isolated pulmonary arteries preloaded with [³H]-noradrenaline as expected for ₂-adrenoceptor antagonists. At higher concentrations both compounds reduced the stimulation-evoked contraction of the pulmonary artery but idazoxan was 15 times as potent as Wy 27127 in this respect.Neither compound had marked antagonist actions at 5-hydroxytryptamine (D), muscarinic, presynaptic dopamine or histamine (H₁) receptors or at ₁-adrenoceptors.Thus, idazoxan and Wy 27127 were equipotent ₂-adrenoceptor antagonists in vitro, however, the ₂: ₁ selectivity of Wy 27127 was considerably greater than that of idazoxan by virtue of weaker ₁-adrenoceptor antagonism.     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

The role of α1-adrenoceptor subtypes in the phasic and tonic responses to phenylephrine in the longitudinal smooth muscle of the rat portal vein

Summary The purpose of this investigation was to determine whether ₁-adrenoceptor subtypes (co)exist in the rat portal vein and, if so, whether they could be functionally associated with the phasic and tonic types of contraction as a response to ₁-adrenoceptor stimulation by phenylephrine.A low Ca²⁺ concentration (0.9 mmol/l) in the Tyrode solution enabled us to quantify changes both in the phasic myogenic activity and in the basal tone of the rat portal vein preparation very precisely. We used both competitive and non-competitive -adrenoceptor antagonists which have been employed successfully by other investigators to discriminate between ₁-adrenoceptor subtypes in vascular and other tissues. Schild analysis showed that the competitive -adrenoceptor antagonists prazosin, phentolamine, yohimbine, corynanthine, idazoxan, rauwolscine and 5-methyl-urapidil could not distinguish between the phasic and tonic responses to phenylephrine and/or different ₁-adrenoceptor subtypes in the rat portal vein. However, when we compared our pA₂ values with those found to be representative indicators according to subclassifications based on the use of selective antagonists in different tissues, the ₁-adrenoceptors in the rat portal vein appeared to belong to the _1L- or _1a-subtype. This subclassification was not in accordance with the data obtained with the irreversible -cadrenoceptor antagonist chloroethylclonidine. However, the validity of this alkylating agent as a tool for receptor classification was restricted, at least in the rat portal vein, by its effects on receptor reserve. In contrast to the competitive -adrenoceptor antagonists, the irreversible -adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine could indeed discriminate between the phasic and tonic types of contraction in response to ₁-adrenoceptor stimulation by phenylephrine, indicating two different receptor reserves for phenylephrine for the two types of responses.In conclusion, both the phasic and tonic types of contraction elicited by phenylephrine in the longitudinal smooth muscle of the rat portal vein appear to be mediated by one particular ₁-adrenoceptor subtype as defined by Schild analysis with selective, competitive -adrenoceptor antagonists. However, using the method of receptor alkylation with phenoxybenzamine, two different affinity constants for the two types of responses could be calculated for phenylephrine. This may reflect the involvement of two different subtypes of ₁-adrenoceptors or more probably, the existence of only one ₁-adrenoceptor subtype, which is coupled with two different intrinsic efficacies to the effector pathways mediating the phasic and tonic responses, respectively. Send offprint requests to H. R. Schwietert at the above address     

Nordimaprit,homodimaprit, clobenpropit and imetit: affinities for H3 binding sites and potencies in a functional H3 receptor model

Summary We determined the affinities of nordimaprit, homodimaprit, clobenpropit and imetit for H₃ binding sites (labelled by ³H-N-methylhistamine) in rat brain cortex homogenates and their potencies at presynaptic H_3A receptors on noradrenergic nerve endings in mouse brain cortex slices. ³H-N-Methylhistamine bound saturably to rat brain cortex homogenates with a K_d of 0.70 nmol/l and a B_max of 98 fmol/mg protein. Binding of ³H-N-methylhistamine was displaced monophasically by dimaprit (pK_i 6.55), nordimaprit (5.94), homodimaprit (6.44), clobenpropit (9.16), imetit (9.83), R-(–)--methylhistamine (8.87) and histamine (8.20), and biphasically by burimamide (pK_{i high} 7.73, pK_{i low} 5.97). In superfused mouse brain cortex slices preincubated with ³H-noradrenaline, the electrically (0.3 Hz) evoked tritium overflow was inhibited by imetit (pIC₃₅ 8.93),R-(–)--methylhistamine (7.87) and histamine (7.03). The effect of histamine was attenuated by nordimaprit, homodimaprit, clobenpropit and N-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline (EEDQ); EEDQ (but not nordimaprit, homodimaprit and clobenpropit) attenuated the effect of histamine also in slices pre-exposed to the drug 60–30 min prior to superfusion. The concentration-response curve of histamine was shifted to the right by homodimaprit and clobenpropit; Schild plots yielded straight lines with a slope of unity for both drugs (pA₂ 5.94 and 9.55, respectively). Nordimaprit depressed the maximum effect of histamine (pD₂ 5.55) and also slightly increased the concentration of histamine producing the half-maximum effect.In conclusion, nordimaprit and homodimaprit possess similar affinities for H₃ binding sites like dimaprit; nordimaprit and homodimaprit as well as clobenpropit and imetit do not differentiate between H_3A and H_3B binding sites. Nordimaprit is a reversible noncompetitive H₃ receptor antagonist, homodimaprit and clobenpropit are reversible competitive H₃ receptor antagonists and imetit is an H₃ receptor agonist. Correspondence to: E. Schlicker at the above address     

Presynaptic α2A-adrenoceptors inhibit the release of endogenous dopamine in rabbit caudate nucleus slices

₂-Adrenoceptors modulating the release of dopamine were identified and characterized in slices of the head of the rabbit caudate nucleus. Release of endogenous dopamine was measured by fast cyclic voltammetry as the increase in the extracellular concentration of dopamine elicited by electrical stimulation. The electrochemical signal was identified as dopamine by means of the oxidation potential, the voltammogram and the fact that the signal was not changed by desipramine, which inhibits the high affinity uptake of noradrenaline, but was greatly increased by nomifensine, which in addition inhibits the high affinity uptake of dopamine.Stimulation by 6 pulses/100 Hz increased the extracellular concentration of dopamine by about 85 nM. The selective ₂-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced this release with an EC₅₀ of 173 nM and by maximally 75%. The ₂-adrenoceptor agonists clonidine and oxymetazoline only tended to cause a decrease. Six drugs, including oxymetazoline, were tested as antagonists against UK 14,304. Their order of antagonist potency (pK_D values in brackets) was rauwolscine (8.0) > oxymetazoline (7.5) > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101; 7.3) > phentolamine (7.1) > corynanthine (5.1) prazosin (< 6). Given alone, the antagonists did not change the release of dopamine elicited by 6 pulses/100 Hz, and the same was true for the dopamine receptor antagonist sulpiride. When caudate slices were stimulated by 10 pulses/1 Hz, sulpiride increased the release of dopamine. Desipramine and rauwolscine, in contrast, again caused no change.It is concluded that dopaminergic axons in the rabbit caudate nucleus possess release-inhibiting ₂-adrenoceptors. The antagonist affinities indicate that they belong to the _2A subtype. In this, they agree with all presynaptic ₂-autoreceptors studied so far in rabbits as well as with the ₂-heteroreceptors modulating the release of serotonin in rabbit brain cortex, suggesting that at least the majority of presynaptic ₂-adrenoceptors in the rabbit are _2A. The agonist sensitivity of the caudate presynaptic ₂-adrenoceptors is low in comparison with cerebrocortical presynaptic ₂-autoreceptors, possibly due to absence of a receptor reserve. Correspondence to: N. Limberger at the above address