Role of human liver microsomal CYP2C9 in the biotransformation of lornoxicam

Objective: The nature of the enzyme(s) catalysing the biotransformation of lornoxicam to one of its major metabolites, 5-hydroxy-lornoxicam, has been investigated in human liver microsomes. The reaction kinetics were characterised, the affinity of lornoxicam for three major human drug metabolising cytochrome P-450 isozymes (CYP2C9, CYP2D6 and CYP3A4) was determined, and inhibition of the reaction by known substrates (diclofenac, ibuprofen, mefenamic acid, phenytoin, tolbutamide and warfarin) and the prototype inhibitor (sulphaphenazole) of CYP2C9 was investigated. Results: Lornoxicam 5-hydroxylation displayed single enzyme Michaelis-Menten kinetics, with a K_M of 3.6 mol·l^-1 and a V_max of 2.6 nmol·h^-1·mg^-1 microsomal protein. The apparent affinity of lornoxicam was high for CYP2C9, but negligible for CYP3A4 and CYP2D6. Inhibition of lornoxicam 5-hydroxylation by CYP2C9 substrates and sulphaphenazole was comparable in all livers preparations, values predicted from their K_M or K_i for CYP2C9 determined in separate studies assuming competitive inhibition. Sulphaphenazole competitively and completely inhibited lornoxicam 5-hydroxylation (K_i=0.31 mol·l^-1) as well as lornoxicam clearance (K_i=0.33 mol·l^-1), partial metabolic clearance (f_m)=0.95). Conclusion: 5-Hydroxylation appears to be the only cytochrome P-450 catalysed metabolic reaction of lornoxicam by human liver microsomes and this major in vivo biotransformation pathway is catalysed virtually exclusively by CYP2C9.Supported in part by Hafslund Nycomed Pharma AG (Linz, Austria) and by a grant of the Forschungsförderungsfond der Gewerblichen Wirtschaft Österreichs     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

The Mechanism of Uptake of Biodegradable Microparticles in Caco-2 Cells Is Size Dependent

Purpose. To study the uptake of biodegradable microparticles in Caco-2 cells. Methods. Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.1 m, 1 m, and 10 m containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (100 g/ml) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied. Results. The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.1 m diameter microparticles had 2.5 fold greater uptake on the weight basis than the 1 m and 6 fold greater than the 10 m diameter microparticles. Similarly in terms of number the uptake of 0.1 m diameter microparticles was 2.7 × 10³ fold greater than the 1 m and 6.7 × 10⁶ greater than the 10 m diameter microparticles. The efficiency of uptake of 0.1 m diameter microparticles at 100 g/ml concentration was 41% compared to 15% and 6% for the 1 m and the 10 m diameter microparticles, respectively. The Caco-2 cell microparticle (0.1 m) uptake increased with concentration in the range of 100 g/ml to 500 g/ml which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37°C compared to at 4°C. Conclusions. The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.1 m) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.     

The influence of hyperthyroidism on β-adrenoceptor-mediated relaxation of isolated small mesenteric arteries

We investigated the influence of hyperthyroidism on relaxant responses of small mesenteric resistance arteries to -adrenoceptor agonists and to compounds stimulating the corresponding second-messenger system. Hyperthyroidism was induced by feeding rats for 28 days with 5 mg/kg L-thyroxine (T4)-containing rat chow. This treatment produced a stable hyperthyroid state, as indicated by several biochemical/metabolic and haemodynamic parameters. Preparations of small mesenteric arteries were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for isoproterenol, noradrenaline and salbutamol as well as for forskolin, dibutyryl-cAMP and theophylline. We also determined concentration-effect curves to the -adrenoceptor agonists in the presence of ICI 118,551 and CGP 20712A (i.e., in the presence of a selective ₂- and ₁-adrenoceptor antagonist, respectively). Apparent pA₂-values were calculated to determine which -adrenoceptor subtype causes vasodilation. These experiments indicate that -adrenoceptor-mediated vasodilation involves both ₁- and ₂-adrenoceptors in mesenteric resistance vessels of both hyperthyroid and control rats. In our experiments hyperthyroidism has a sensitizing influence on vascular responses induced by the -adrenoceptor agonist isoproterenol and the selective ₂-adrenoceptor agonist salbutamol. Sensitization to isoproterenol was abolished in the presence of ICI 118,551, whereas it was emphasized in the presence of CGP 20712A. Although this was not fully supported by the results obtained with noradrenaline, these results indicate that the sensitization to -adrenoceptor agonists is probably limited to the ₂-adrenoceptor/G-protein complex and not associated with alterations of the corresponding second messenger system.     

A new technique for the investigation of some analgesic drugs on a reflexive behavior in the rat

Summary The analgetic action of a series of analgesic and psychotropic agents was tested in a situation in which variable intensities of electric shock to a rat's feet were used to elicit two distinguishable reflexive responses: a flinching response at low shock values, and a jumping response at higher shock values,By using a modified method of limits, reliable threshold for the two responses were obtained.Chlorpromazine, perphenazine, morphine, codeine, nalorphine and acetylsalicylate were found to raise the threshold to jump, but had little or no effect on the threshold to flinch.PIH, JB 835, iproniazid, reserpine, tetrabenazine, and amphetamine were found to have no effect on either the jump or the flinch thresholds.A combination of amphetamine and codeine was found to produce a synergistic potentiation. A combination of morphine and nalorphine was found to produce an antagonism.The results were discussed in terms of Beecher's hypothesis that the analgetic action of drugs is due to a diminution of the emotional components of an animal's reaction to pain and in terms of the relationship of brain amine change to analgetic action.     

Differential Effects of Sulfate and Sulfobutyl Ether of β-Cyclodextrin on Erythrocyte Membranes in Vitro

The hemolytic activity of -cyclodextrin (-CyD) on rabbit erythrocytes was reduced by the introduction of negatively-charged groups onto the hydroxyls of -CyD; the membrane disrupting abilities decreased in the order of -CyD > 2-hydroxypropyl--CyD (HP--CyD) > sulfobutyl--CyD (SB--CyD) >> -CyD sulfate (S--CyD). Under pre-hemolytic concentrations, both -CyD and SB--CyD induced shape changes of membrane invagination on the erythrocytes. In sharp contrast, S--CyD showed biphasic effect on the shape of the erythrocytes; i.e. the crenation at relatively low concentrations and the invagination at higher concentrations. The S--CyD-induced membrane crenation arose from a direct action on the membranes rather than cell metabolism-mediated effects. Unlike -CyD, S--CyD was found to bind to the erythrocytes and may be confined to the outer surface of the membrane bilayer, which may expand the exterior layer relative to the cytoplasmic half, thereby inducing the cells to crenate. On the other hand, the membrane invagination mediated by the three - CyDs was initiated by extracting specific membrane lipids from the cells, depending upon their inclusion abilities, subsequently leading to the lysis of the cells. These results indicate that SB--CyD and S--CyD interact with the erythrocyte membranes in a differential manner and possess lower membrane disrupting abilities than the parent -CyD and HP--CyD.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

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)     

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     

Different effects of vinblastine on the polymerization of isotypically purified tubulins from bovine brain

Vinblastine, a highly successful antitumor drug, targets the tubulin molecule. Tubulin, the subunit protein of microtubules, consists of an - and a -subunit, both of which consist of isotypes encoded by different genes. We have purified three isotypes of bovine brain tubulin, namely, _II, _III and _IV. Microtubule associated protein-2 (MAP2) and Tau-induced assembly of these isotypes were compared in the presence and absence of vinblastine. MAP2-induced assembly of unfractionated tubulin and all the isotypes except _II tubulin was resistant to 1M vinblastine. Vinblastine at low concentrations (<10M) progressively inhibited the assembly of all of the isotypes but the vinblastine concentration required for inhibition of MAP2-induced microtubule assembly was minimal for _II. The tau-induced assembly of unfractionated tubulin and _III were equally sensitive to 1M vinblastine whereas _II and _IV were much more sensitive to vinblastine. The microtubules obtained in the presence of tau from unfractionated tubulin, _II and _IV could be easily aggregated by 20M vinblastine whereas such as aggregation of microtubules obtained from _III and tau required approximatedly 40M vinblastine. Our results suggest that among the tubulin isotypes, _II is the most sensitive to vinblastine in the presence of MAPs while _III is the most resistant and this intrinsic resistance of _III dimers persists in the polymeric form of _III tubulin as well. These results may be relevant to the therapeutic and toxic actions of vinblastine.     

In vitro and in vivo uptake of nickel sulfides by rat lymphocytes

The uptake, the biological transformation and the interaction with cellular constituents of Ni₃S₂ and NiS have been studied in vitro and in vivo on rat lymphocytes. NiS crystals are phagocytized in vitro and no structural degradation is observed within the first 3 days of exposure. Energy dispersive spectrometry (EDS) reveals a slight dissolution characterized by the loss of sulfur. Ni₃S₂ is degraded in the extracellular space to minute particles (50–100 nm) covering the cell membrane. Smaller intracellular particles (10–30 nm) are found selectively bound to mitochondria, endoplasmic reticulum, Golgi vesicles, nuclear membranes, and the euchromatinic part of nuclei. EDS analyses reveal that the particles bound to cell membranes and euchromatin no longer contain sulfur but phosphorus and nickel as inorganic compounds. This observation suggests the formation of a Ni/P complex with the phosphate groups either of membranous phospholipids or of nuclear RNA or DNA. A similar uptake and transformation process of Ni₃S₂ is observed on lymphocytes after in vivo incubation. This leads us to consider lymphocytes as target cells, as compared with other cell types where the Ni₃S₂ uptake occurs only partially. The present findings show a difference of uptake and biological transformation between Ni₃S₂ and NiS. The identical results obtained after in vitro and in vivo bioassays enhance the in vitro experiments, at least for this cell type.     

Reproductive toxicity and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effects of a single dose of TCDD on the testis were studied in rats. The animals were treated (subcutaneously) once with TCDD doses of 0, 0.5, 1.0, 3.0, 5.0 g/kg body weight. Doses of 3.0 or 5.0 g TCDD/kg reduced the number of spermatids/testis significantly (60% of the controls). Electron microscopic inspection revealed that both doses led to a dissolution on the germinal epithelium. Altered germ cells at all developmental stages occurred in all testes evaluated. Doses of 0.5 or 1.0 g TCDD/kg did not induce any effects in the testis; therefore, under these experimental conditions of single exposure to rats the dose of 1.0 g TCDD/kg can be considered as NOAEL.     

Contraction-mediating α2-adrenoceptors in the mouse vas deferens

Summary The question of the existence of postjunctional, contraction-mediating ₂-adrenoceptors, in addition to the known ₁-adrenoceptors, was studied in the mouse isolated vas deferens. Both the ₁-selective agonist phenylephrine and the ₂-selective agonist 5-bromo-6-(2imidazolin-2-ylamino)-quinoxaline (UK 14,304) caused contraction of the vas deferens. In the presence of the ₁-selective antagonist prazosin (added in order to prevent an ₁ component in the effect of high concentrations of UK 14,304), the ₂-selective antagonists yohimbine and idazoxan shifted the concentration—response curve of UK 14,304 to the right in a manner compatible with competitive antagonism and with dissociation constants K_B indicating the involvement of ₂-adrenoceptors. The maximal contraction elicited by UK 14,304 (in the presence of prazosin) was much lower than the maximal contraction elicited by phenylephrine. The effect of UK 14,304 was not changed by the P₂-purinoceptor agonist ,-methylene-ATP and was reduced by neuropeptideY, but was markedly enhanced by relatively low concentrations of phenylephrine. When the sympathetic fibres of the vas deferens were stimulated by trains of ten widely spaced (0.5 Hz) electric pulses, the tissue responded with ten separate twitches in which purinergic and adrenergic components were isolated by prazosin and suramin, respectively. Prazosin reduced the first adrenergic twitch in these trains at concentrations close to its K_B value at ₁-adrenoceptors, whereas yohimbine and idazoxan reduced the first adrenergic twitch at concentrations far lower than their K_B values at ₁-adrenoceptors. The results indicate that the smooth muscle of the mouse vas deferens possesses contraction-mediating ₂-adrenoceptors. They are activated by UK 14,304 and probably also by noradrenaline of neural origin. Responses mediated by the ₂-adrenoceptors are enhanced by simultaneous ₁-receptor activation, an interaction that may increase the contribution of the ₂-adrenoceptors to the adrenergic phase of neurogenic contractions. Send offprint requests to R. Bültmann at the above address     

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.     

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     

Benzamide action at α2-adrenoceptors modifies catecholamine-induced contraction and relaxation of circular smooth muscle from guinea-pig stomach

Summary Dopamine was shown to act on the circular smooth muscle of the stomach body to cause contraction at a yohimbine-sensitive site (₂) and a relaxation at a prazosin-sensitive site (₁). Metoclopramide and tiapride failed to modify either response, failed to antagonise a relaxation to phenylephrine at 1(₁ sites in the same tissue, and failed to modify the contractions caused by dopamine and phenylephrine at an ₂-adrenoceptor site in the pyloric sphincter. However, (+)- and (–)-sultopride and (+)-sulpiride antagonised the dopamine-induced contractions of the stomach body indicating an ₂-antagonist action. An ability to attenuate the relaxation of this tissue may reflect a displacement of the contraction curve to the right rather than an ₂-antagonist action since the response to phenylephrine was not antagonised either in this tissue or in the pyloric sphincter. Within the central nervous system the (–)-enantiomers of sultopride and sulpiride have a highly selective dopamine receptor blocking action. This cotrasts with the present findings in the stomach musculature of a non-stereospecific antagonism at ₂-type adrenoceptors.     

Adenosine modulation of non-adrenergic non-cholinergic neurotransmission in isolated guinea-pig atria

Summary Transmural stimulation of non-adrenergic, non-cholinergic sensory nerves in guinea-pig atria, isolated from reserpine-pretreated animals, in the presence of atropine and the beta-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. Adenosine (0.1–10 M) concentration-dependently reduced the cardic response to transmural nerve stimulation, without modifying the response to exogenous calcitonin gene-related peptide; the inhibitory effect of adenosine was antagonized by 1 M 8-phenyltheophylline. Moreover, the cardiac response to field stimulation was enhanced by 8-phenyltheophylline (0.1, 1 M) and by adenosine deaminase (1 g/ml), but was reduced by dipyridamole (1 M). These findings indicate the presence of inhibitory adenosine receptors on cardiac sensory nerves and suggest a modulatory effect of endogenous adenosine on cardiac non-adrenergic, non-cholinergic neurotransmission.Send offprint requests to A. Rubino at the above address     

5-Hydroxytryptamine causes rate-dependent arrhythmias through 5-HT4 receptors in human atrium: facilitation by chronic β-adrenoceptor blockade

We have investigated the ability of 5-hydroxytryptamine (5-HT) to elicit arrhythmic contractions in isolated human atrial strips as a function of pacing rate (0.1–2 Hz) using a method recently introduced by us (Kaumann and Sanders, this journal, 1993b) and examined the nature of the 5-HT receptors involved. Right atrial appendage tissue was obtained from 14 patients undergoing cardiac surgery. None of the patients had advanced heart failure. 5-HT (0.6–20 mol/l) induced arrhythmic contractions during pacing in 4/11 atrial strips from 3/4 patients who had not received blockers and in 21/27 atrial strips from 9/10 patients who had been chronically treated with blockers (primarily ₁-selective). The incidence of arrhythmic contractions evoked by 5-HT did not reach statistical significance in the atrial tissue from the non- blocked patients but was highly significant in the atrial tissue from the chronically blocked patients. The arrhythmic contractions usually occurred more frequently at low than at high pacing rates and were observed at the physiological frequency of 1 Hz in 1/4 atrial strips from 1/4 of the non- blocked patients and 6/11 strips from 5/10 of the blocked patients. The 5-HT-evoked arrhythmic contractions were observed during blockade of ₁-adrenoceptors, ₂-adrenoceptors and 5-HT₃ receptors, ruling out the participation of these receptors. The 5-HT-evoked arrhythmic contractions were totally inhibited within 30 min by the selective 5-HT₄ receptor antagonist SB 203186 ((1-piperidinyl)ethyl 1H-indole 3-carboxylate) 100 nmol/l whereas they persisted in time-matched controls. The blockade of 5-HT-evoked arrhythmic contractions by SB 203186 was surmounted by high concentrations (400–1800 mol/l) of 5-HT. Our results demonstrate that 5-HT elicits rate-dependent arrhythmic contractions in isolated human atrium through the 5-HT₄ receptor and that they are facilitated in atrial tissue from patients treated with blockers. Our results suggest that endogenous, platelet-derived 5-HT may cause atrial arrhythmias and that exogenous 5-HT₄ agonists/partial agonists may be arrhythmogenic. Correspondence to: A. J. Kaumann at the above address     

The effects of chronic carbamazepine treatment on haem biosynthesis in man and rat

Summary Recently, there has been a variety of reports of adverse drug reactions during therapy with the flavonoid Cianidanol (Ci), a cytoprotective radical scavenger, especially involving haemolytic anaemia and drug fever.To elucidate whether the fever was due to a direct, antigen-independent interaction of Ci with immune competent cells, its effect on macrophage (M) function and early biochemical events during lymphocyte activation has been examined.A direct interaction of Ci with M was demonstrated, resulting in increased secretion of interleukin-1 (IL-1). The influence of Ci on lymphocyte activation was assessed by measuring levels of cyclic AMP and GMP. At high concentrations of Ci, cAMP levels were increased, and at low Ci concentrations cGMP levels were elevated. Both findings are correlated with lymphocyte proliferation and function, which is increased at low and decreased at high concentrations of Ci. The synthesis of prostaglandin E₂ by M, an important factor in M-mediated suppression, was reduced by increasing doses of Ci, which inhibited M-cyclooxygenase. Ci did not affect phospholipase A₂ activity.These findings indicate that flavonoid-induced fever may be due to allergic as well as pseudo-allergic mechanisms, the latter probably caused by increased antigen-independent release of IL-1, the endogeneous mediator of fever.     

Effect of ethanol on the metabolism of lithocholic acid in rat liver

Summary Lithocholic acid 24-C¹⁴ is converted by 20000x g-supernatant of rat liver homogenate into several products of higher polarity. 3-6-dihydroxy-5-cholanic acid is the main metabolite, whereas 7-hydroxylation occurs only to a small extent. Besides of the hydroxylations conjugation with taurine and the formation of a 3-sulfate ester of lithocholic acid can be demonstrated. Addition of ethanol to the enzymatic system results in an inhibition of the formation of 3,6-dihydroxy-5-cholanic acid, whereas no effect can be observed with respect to the formation of the other products. This inhibition is present also in 20000x g-supernatant obtained from rats pretreated with ethanol 100 min before being preparation from ethanol-pretreated rats amounts to 68% of the controls. The chrome P-450 mediated oxidative mechanism, which has been shown to be involved in microsomal 6-hydroxylation of bile acids.This work was supported by the Deutsche Forschungsgemeinschaft.