Kinetic properties of the in vivo accumulation of 3H-(−)-N-n-propylnorapomorphine in mouse brain

Summary (1) The influence of various dopamine (DA) receptor agonists and antagonists on the kinetic properties of the specific binding of ³H(–)-N-n-propylnorapomorphine (NPA) in the mouse striatum in vivo was studied. The specific binding of ³H-NPA, defined as the difference between the radioactivity in the striatum and cerebellum, was completely antagonized by the selective D-2 receptor antagonist raclopride but not by the selective D-1 antagonist SCH 23390, showing that the binding occurs exclusively to the D2 receptors. (2) The selective D-2 receptor agonists pergolide and quinpirole inhibited the ³H-NPA binding biphasically at low doses, indicating that these DA receptor agonists have high affinities for a subfraction (10 to 30%) of the NPA binding sites. (3) Increasing the synaptic DA concentration by DA release [(+)-amphetamine] or uptake blockade (amfonelic acid and methylphenidate) inhibited the ³HNPA binding in a competitive manner (unchanged B _max, increased K _D). Depletion of the DA in the synapses by -butyrolactone or reserpine decreased the apparent K _D value. (4) The possibility of estimating changes in the synaptic DA concentration from changes in the apparent K _D is discussed. According to the results obtained, the normal concentration of DA in the synaptic cleft in mouse striatum in vivo is about 40 nmol/l and this concentration is increased 2 to 3 times by (+)-amphetamine and amfonelic acid in doses which evoke hyperactivity and stereotypic behaviour.Send offprint requests to S. B. Ross at the above address     

The influence of inhibition of catechol-O-methyl transferase or of monoamine oxidase on the extraneuronal metabolism of 3H-(−)-noradrenaline in the rat heart

Summary The extraneuronal metabolism of ³H-(–)-noradrenaline (1 nmol/l) was determined in rat hearts obtained from reserpine-pretreated animals (in the presence of 30 mol/l cocaine).Inhibition of monoamine oxidase (MAO) (by pretreatment of the animals with pargyline) increased the formation of O-methylated metabolites by nearly that amount by which the formation of deaminated metabolites declined; hence, catechol-O-methyl transferase (COMT) seemed to be able to nearly fully compensate for the loss of MAO activity. However, when COMT was inhibited (by the presence of either 1 or 10 mol/l U-O521), the increase in the formation of deaminated metabolites was smaller than the decrease in the formation of O-methylated metabolites; hence, MAO seemed to be unable to fully compensate for the loss of COMT activity.These results are discussed with regard to the hypothesis that the two extraneuronal enzymes co-exist in one compartment. As inhibition of COMT causes a much greater increase in the steady-state tissue/medium ratio for ³H-(–)-noradrenaline than does inhibition of MAO, it is suggested that it is this increase in the intracellular concentration of ³H-(–)-noradrenaline which-by promoting an efflux of the unchanged amine that is proportional to the tissue/medium ratio-actually decreases the net removal of ³H-(–)-noradrenaline from the perfusion fluid.The results are compatible with (but no evidence for) the hypothesis that the two enzymes co-exist in the same extraneuronal compartment.The following abbreviations are used here NMN normetanephrine - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid - OMDA MOPEG+VMA Supported by the Deutsche Forschungsgemeinschaft     

Accumulation of 3H-(±)-noradrenaline by isolated rat liver cells

Summary Using hepatocytes isolated by collagenase perfusion, we studied the accumulation of ³-noradrenaline. Cells incubated during 15 min in the presence of 0.4 mol/l ³H-noradrenaline (without inhibition of noradrenaline metabolism) accumulated 8.32 ± 1.77 pmol/10⁶ cells (n = 3). The accumulation of ³H-noradrenaline in isolated parenchymal liver cells was sensitive to 10 mol/l cocaine (inhibition 36.6 ± 7.9%, n = 3) and 1 mol/l desipramine (inhibition 27.2 ± 6.9, n = 3). Accumulation of ³H-noradrenaline was temperature and sodium dependent (inhibition 33.2 ± 9.4%, n = 9, when Na⁺ was replaced by Tris⁺) and was influenced by the inhibition of the membrane Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-ATPase) by 150 mol/l ouabain (34.7 ± 6.9% inhibition, n = 3). Accumulation of 3H-noradrenaline in the hepatocytes was not affected by the presence of uptake₂ inhibitors, normetanephrine (30 mol/l) and corticosterone (30 mol/l), but was reduced by 30 mol/l isoprenaline (76.3 ± 5.0% inhibition, n = 6). Thus, the system that takes up and accumulates noradrenaline in the isolated rat liver cells possesses some characteristics of both, uptake₁ and uptake₂ systems and appears to be different from other extraneuronal cocaine-sensitive systems, such as the one reported for pulmonary endothelial cells. Send offprint request to M. I. Masana at the above address     

The uptake and O-methylation of 3H-(±)-isoprenaline in rat cerebral cortex slices

Summary The O-methylation and accumulation of ³H-isoprenaline in slices of the rat cerebral cortex were studied before and after inhibition of COMT. 1. Inhibition of COMT by mol/l U-0521 virtually abolished the O-methylation and increased the accumulation of ³H-isoprenaline; hence, there is evidence for the existence of a central O-methylating system (with a transport mechanism and intracellular COMT). 2. Experiments were carried out with selective uptake inhibitors for uptake, (cocaine and desipramine) or uptake2 (corticosterone and OMI), with phenoxybenzamine (known to inhibit both carriers) and with changes in the ionic composition of the incubation medium. They revealed that the central carrier differed from both, uptake, and uptake2, although exhibiting some resemblance with uptake₂ (lack of dependence on Na⁺ and Cl^–, sensitivity to K⁺ and phenoxybenzamine, ability to transport ³H-isoprenaline). 3. Although the central carrier was rather sensitive to inhibition by beta-adrenoceptor antagonists (propranolol, carteolol), the effect of propranolol was not stereoselective; hence, beta-adrenoceptors do not seem to be involved. 4. Virtually identical IC₃₀-values were obtained for inhibitors, when determined with or without inhibition of COMT. Only OMI was found to inhibit COMT as well as the central transport system; hence it was more potent in inhibiting the O-methylation than the accumulation of ³H-isoprenaline. 5. IC₅₀-values (against initial rates of accumulation of ³H-isoprenaline; COMT inhibited) were determined for various substrates and inhibitors of peripheral uptake₂. There was no correlation with the IC₅₀-values determined earlier for uptake2 in rat heart (Grohmann and Trendelenburg 1984). 6. Unlabelled catecholamines half saturated the intracellular COMT when slices were incubated with 0.22 mol/l [(±)-dobutamine] to 4.9 mol/l [(–)-noradrenaline]. As the presence of unlabelled catecholamines increased tissue levels of ³H-isoprenaline, catecholamines are substrates of the central carrier. 7. The carrier of the central O-methylating system differs from uptake₂ of peripheral organs, although it resembles the peripheral carrier in some respects.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OMI 3-Omethyl-isoprenaline Supported by the Deutsche Forschungsgemeinschaft (Tr 96 and SFB 176) and by a scholarship of the Royal Society for V. G. Wilson. Some of the results were presented to the British Pharmacological Society (Trendelenburg and Wilson 1986)     

Binding of 3H-spiperone and 3H-(−)-sulpiride to dopamine D2 receptors in rat striatal membranes: methodological considerations and demonstration of the identical nature of the binding sites for the two ligands

Summary In order to test the hypothesis emerging from the literature that ³H-sulpiride and ³H-spiperone might label different subclasses of dopamine D2 receptors in the brain, the binding properties of the two ligands were compared under optimized conditions. ³H-sulpiride was found to show a very fast rate of dissociation from the receptor. Furthermore, with this ligand, an apparently specific binding to glass fiber filters was observed. A centrifugation assay was therefore used for the characterization of ³H-sulpiride binding. The binding of ³H-spiperone was measured in a way which prevented its attachment to sites other than D2 receptors. Under these conditions, the two ligands were found to label identical dopaminergic recognition sites (D2 receptors) according to the following criteria: ³H-sulpiride and ³H-spiperone both labelled only one single site with a comparable density. Identical changes in the receptor number for both ligands were found in the tissue from animals with nigrostriatal 6-hydroxydopamine lesions. The potencies of various dopamine agonists and antagonists in displacing ³H-sulpiride or ³H-spiperone from their binding sites showed a highly significant one to one correlation. Finally, the binding of both ligands could be inactivated by pretreatment of the membranes with N-ethylmaleimide, with or without Na⁺-ions being present. This inactivation followed exactly the same kinetics for both radioligands. Thus, no indication supporting the hypothesis that ³H-sulpiride and ³H-spiperone might label different subsets of dopamine D2 receptors in the rat brain could be found.Abbreviations 6,7-ADTN 2-Amino-6,7-Dihydroxy-1,2,3,4-Tetrahydronaphthalene - NEM N-Ethyl-maleimide - 5HT Serotonin - 6-OHDA 6-hydroxydopamine Send offprint requests to S. Urwyler at the above address     

Cilostamide potentiates more the positive inotropic effects of (−)-adrenaline through β2-adrenoceptors than the effects of (−)-noradrenaline through β1-adrenoceptors in human atrial myocardium

Activation of both β₁- and β₂-adrenoceptors increases the contractility of human atrial myocardium through cyclic AMP-dependent pathways. Cyclic AMP is hydrolised by phosphodiesterases, but little is known about which isoenzymes catalyse inotropically relevant cyclic AMP accumulated upon stimulation of β-adrenoceptor subtypes. We have compared the positive inotropic effects of (−)-noradrenaline and (−)-adrenaline, mediated through β₁- and β₂-adrenoceptors, respectively, in the absence and presence of the PDE3 inhibitor cilostamide (300 nM) or PDE4 inhibitor rolipram (1 μM) on human atrial trabeculae from non-failing hearts. Cilostamide, but not rolipram, potentiated the effects of both (−)-noradrenaline and (−)-adrenaline. Cilostamide increased the −logEC₅₀M of (−)-adrenaline more than of (−)-noradrenaline (P < 0.05), regardless of whether or not the patients had been chronically treated with β-blockers. The results are consistent with a greater PDE3-catalysed hydrolysis of inotropically relevant cyclic AMP produced through β₂-adrenoceptors than β₁-adrenoceptors in human atrium.     

Investigation by NMR spectroscopy of the interaction between synthetic soluble (−)-dopa melanin and drugs

Summary In order to understand the molecular interactions of drugs with melanin, synthetic soluble (–)-dopa-melanin was prepared in deuterium buffer. The spectra of various drug moieties with the pigment at 30°C were studied employing the line width measurements obtained with a pulse NMR (AF270) instrument.As compared to drug effects in fresh melanins (48 h), the aged melanins ( 168 h) gave consistent spectral measurements, even in dilute solutions of pigment. NMR signals of aromatic and N-methyl protons of drugs were relatively easy to quantify and, in the presence of melanin, line broadening of various drug moieties occurred. The line widths of the N-methyl groups of acetylcholine (3.02 ppm), the N-methyl group of atropine (2.52 ppm), N-isopropyl of isoprenaline bitartrate (1.14 ppm) and N-ter-butyl of timolol maleate (1.22 ppm) in the presence of the pigment were increased. Line widths associated with acetate, bitartrate, maleate or tropic acid, however, were not altered by the melanin. This indicates the specificity of the interaction between drug moieties and the site(s) of melanin.Based on the line width measurements of N-methyl protons of ephedrine, two dissociation constants were obtained (K _d1 2.08 mM and K _d2 > 20 mM). The constants for atropine melanin complex were K _d1 0.79 mM and K _d2 > 6 mM. Furthermore, based on N-methyl resonances, it appears that atropine and ephedrine compete for at least one common interacting site of the melanin polymer.     

(−)-Epigallocatechin-3-gallate induces contraction of the rat aorta by a calcium influx-dependent mechanism

Although the consumption of tea has been associated with beneficial cardiovascular effects, (–)-epigallocatechin-3-gallate (EGCG), the most abundant catechin in this beverage has shown seemingly contradictory actions on vascular tissues, for example vasorelaxant activity that could contribute favourably to prevention of cardiovascular disease, and contractile activity that could act in the opposite direction. The purpose of the present work was to study the contractile effects of EGCG on isolated rat thoracic aorta rings and its effects on the cytosolic free [Ca²⁺] ([Ca²⁺]_i) measured with fura-2 in cultured rat aortic smooth muscle cell line.In partially depolarised (15 mM KCl) aortic rings EGCG (30–300 µM), (±)-BAY K 8644 (0.1 µM) and thapsigargin (1 µM) induced a Ca²⁺-dependent, endothelium-independent contraction associated with [Ca²⁺]_i elevation in RASMC. EGCG enhanced the responses elicited by (±)-BAY K 8644 and thapsigargin both in aortic rings and in RASMC. Nifedipine totally inhibited the (±)-BAY K 8644-induced contraction, but only partially blocked the contractile responses to EGCG and thapsigargin, while SKF 96365 abolished both responses. The effects of these channel blockers were associated with a decrease in [Ca²⁺]_i in RASMC. Re-introduction of Ca²⁺ in the medium after depletion of intracellular Ca²⁺ stores with thapsigargin in a Ca²⁺-free solution elicited a contraction of aortic rings and an increase in [Ca²⁺]_i in RASMC. In both cases, this response was partially sensitive to nifedipine, abolished by SKF 96365 and clearly enhanced by EGCG.These results suggest that EGCG induces a transient endothelium-independent contraction in the rat aorta, probably by increasing smooth vascular cell membrane permeability to Ca²⁺ through both non-specific and dihydropyridine-sensitive Ca²⁺ channels.     

Synergistic blockade of some dopamine-mediated behaviours by (−)-sulpiride and SCH 23390 in the rat

Several studies have indicated that the D2 dopamine receptors mediate the antidopaminergic activity of the neuroleptics; nevertheless, the selective blocker (–)-sulpiride weakly inhibits dopamine-mediated behaviour. The present study investigated whether the concomitant injection of doses of the D1 antagonist SCH 23390, which by themselves are without effect, would enable (–)-sulpiride to express fully neuroleptic activity in the rat. The benzamide YM 09151-2 that strongly inhibits dopamine-mediated behaviour was also studied. Rats receiving different doses of (–)-sulpiride, YM 09151-2 and SCH 23390 given alone or in combination were tested for exploratory activity, apomorphine-induced stereotyped behaviour and hyperactivity elicited by the D2 agonist LY 171555. When given alone, (–)-sulpiride (10, 20 and 40 mg/kg IP) had no effect on exploratory activity and stereotypy. When (–)-sulpiride was administered in combination with an ineffective dose of SCH 23390 (5 g/kg) both responses were significantly inhibited. The combined administration of subthreshold doses of (–)-sulpiride (2.5 mg/kg) and SCH 23390 (2.5 g/kg) significantly inhibited hypermotility induced by LY 171555. Moreover, the combined administration of ineffective doses of YM 09151-2 with subthreshold doses of SCH 23390 strongly inhibited all the behavioural responses. The results indicate that SCH 23390 allowed (–)-sulpiride to exhibit a wider spectrum of neuroleptic activity and potentiated the antidopaminergic activity of YM 09151-2.     

Neuronally released (−)-noradrenaline relaxes smooth muscle of calf trachea mainly through β1-adrenoceptors : comparison with (−)-adrenaline and relation to adenylate cyclase stimulation

Summary The nature of the receptors that mediate the relaxation of smooth muscle by field stimulation, (–)-noradrenaline and (–)-adrenaline was investigated in calf tracheal smooth muscle. The relation between relaxation, stimulation of the adenylate cyclase and density of -adrenoceptor subtypes was studied with the help of antagonists of ₁- and ₂-adrenoceptors. The question of the existence of catecholamine-containing nerves was also investigated. (1) Nerves with varicosities exhibiting catecholaminergic fluorescence were observed between bundles of smooth muscle cells. (2) Consistent with the existence of adrenergic nerves (–)-noradrenaline was also found. The content of (–)-noradrenaline (1 g · g^–1 w.w.) was the same in smooth muscle strips from the sublaryngeal region and from the region close to the bifurcation of the calf trachea. (–)-Adrenaline was not detected. (3) Smooth muscle relaxation by low (–)-noradrenaline concentration (0.6–2 nmol/l) was mediated through ₁-adrenoceptors. Low concentrations of (–)-adrenaline (0.06–1 nmol/l) relaxed through ₂-adrenoceptors. High concentrations of (–)-noradrenaline and (–)adrenaline also caused relaxation through ₂- and ₁-adrenoceptors respectively. (4) Field stimulation caused relaxation which was half maximal at 0.2–0.8 Hz. Blockade of ₁-adrenoceptors strongly attenuated the relaxant response to field stimulation and shifted the frequency-relaxation curves to 4 times higher frequencies. These results are consistent with a ₁-adrenoceptor-mediated relaxation caused by (–)-noradrenaline released from sympathetic nerve endings at low stimulation frequencies. (5) Blockade of ₂-adrenoceptors failed to reduce smooth muscle relaxation caused by field stimulation at low stimulation frequencies (0.1–1 Hz). However, after ₁-adrenoceptor blockade, additional blockade of ₂-adrenoceptors reduced the relaxant effects observed at high frequencies (2–400 Hz). The results suggest that high concentrations of endogenous (–)-noradrenaline cause relaxation through ₂-adrenoceptors. (6) Binding experiments with ³H-(–)-bupranolol and ³H-ICI 118,551 revealed between 10,000 and 20,000 -adrenoceptors per smooth muscle cell of which 3/4 were ₂ and 1/4 ₁. The equilibrium dissociation constant of (–)-adrenaline for both ₁- and ₂-adrenoceptors and of (–)-noradrenaline for ₁-adrenoceptors was 1 mol/l. The affinity of (–)noradrenaline for ₂-adrenoceptors was 10 to 20 times lower than for ₁-adrenoceptors. (7) The adenylate cyclase of smooth muscle cells was stimulated more through ₂-adrenoceptors by both (–)-noradrenaline (77%) and adrenaline (83%) than through ₁-adrenoceptors. Half maximum stimulation of the cyclase was observed at 2 to 4 times lower catecholamine concentrations than the concentrations that half saturate ₁- or ₂-adrenoceptors. (8) Both the low affinity and low cyclase stimulating potency of the catecholamines suggest considerable amplification of the receptor signals. Half maximum relaxation of tracheal muscle by (–)-noradrenaline is associated with the activation of less than 50 ₁-adrenoceptors per cell producing less than 250 molecules cyclic AMP per second.     

Differential inhibition of hepatic and duodenal sulfation of (−)-salbutamol and minoxidil by mefenamic acid

Objective: The aim of this investigation was to determine whether mefenamic acid and salicylic acid inhibit the sulfation of (−)-salbutamol and minoxidil in the human liver and duodenum, and if so, to ascertain whether the 50% inhibitory concentration (IC₅₀) estimates are different in the two tissues. Methods: Sulfotransferase activities were measured for 10 mM (−)-salbutamol and 5 mM minoxidil, and the concentration of 3′-phosphoadenosine-5′-phosphosulphate-[³⁵S] was 0.4 μM. Results: The IC₅₀ estimates for (−)-salbutamol and minoxidil sulfation of mefenamic acid were 72 ± 5.4 nM and 1.5 ± 0.6 μM (liver), respectively, and 161 ± 23 μM and 420 ± 18 μM (duodenum), respectively. The figures for the liver were significantly lower (P < 0.0001) than those for the duodenum. The IC₅₀ estimates for (−)-salbutamol sulfation of salicylic acid were 93 ± 11 μM (liver) and 705 ± 19 μM (duodenum, P < 0.0001). Salicylic acid was a poor inhibitor of minoxidil sulfation. Conclusion: The IC₅₀ estimates for (−)-salbutamol sulfation of mefenamic acid and salicylic acid are lower than their unbound plasma concentrations after standard dosing, suggesting that mefenamic acid and salicylic acid should inhibit the hepatic sulfation of (−)-salbutamol in vivo. Received: 11 November 1999 / Accepted in revised form: 28 April 2000     

Dietary pretreatment with green tea polyphenol, (−)-epigallocatechin-3-gallate reduces the bioavailability and hepatotoxicity of subsequent oral bolus doses of (−)-epigallocatechin-3-gallate

Human case-studies have reported an association between green tea-based dietary supplements and hepatotoxicity. Studies have demonstrated the hepatotoxicity of high-dose oral bolus dosing with the tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) in mice and dogs. We examined the effect of pretreatment with dietary EGCG on the hepatotoxicity and bioavailability of acute oral bolus dosing with EGCG in CF-1 mice. EGCG (750 mg/kg, i.g., once daily for 3 days) increased plasma alanine aminotransferase by 80-fold, decreased both reduced (by 59%) and total (by 33%) hepatic glutathione, and increased hepatic levels of phosphorylated histone 2AX. Pretreatment with dietary EGCG (3.2 mg/g diet) for 2 weeks mitigated hepatotoxicity. Acute oral EGCG also decreased mRNA expression of glutathione reductase. Dietary pretreatment prevented these decreased and increased glutathione peroxidase (Gpx)2, Gpx3, Gpx5, and Gpx7 expression. We found that dietary EGCG reduced the plasma (57% reduction) and hepatic (71% reduction) EGCG exposure following oral bolus dosing compared to mice that were not pre-treated. Overall, it appears that EGCG can modulate its own bioavailability and that dietary treatment may reduce the toxic potential of acute high oral bolus doses of EGCG. These data may partly explain the observed variation in hepatotoxic response to green tea-containing dietary supplements.     

The acceleration of the extraneuronal efflux of 3H-(±)-isoprenaline induced by high extracellular potassium

Summary After loading of the extraneuronal tissues of the perfused rat heart with ³H-isoprenaline the elevation of extracellular K⁺ concentration (from 2.7 to 15 mmol/1) in the perfusion solution about doubled the rate constant for efflux of the ³H-amine. As this increase was not seen in the presence of 100 mol/13-O-methyl-isoprenaline (OMI, a potent inhibitor of the uptake₂-carrier), it is concluded that the change in the concentration of K⁺ modulates OMI-sensitive outward transport of ³H-isoprenaline by uptake₂, not the diffusional efflux of the amine.Abbreviations MAO monoamine oxidase - COMT catechol-O-methyltransferase - OMI 3-O-methyl-isoprenaline - U-0521A 3,4-dihydroxy-2-methyl propiophenone     

Effect of endothelin 1 on the isolated human and rabbit platelet activation

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

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

Relationship between the uptake of 3H-(±) metaraminol and the density of adrenergic innervation in isolated rat tissues

Summary In this study simultaneous measurements of action potentials and force of contraction of isolated ventricular muscle from cat and calf hearts were performed under stimulated steady-state and rested-state conditions. It is shown that time-to-peak force of the rested-state contraction correlates well with electrically induced changes in the duration of the platcau phase of the corresponding action potential. Moreover, adrenaline (0.2 and 0.6 M) and theophylline (2 and 5 mM) increase the amplitude of the rested-state and stimulated steady-state contractions. The concentrations of these drugs were about equi-effective in increasing stimulated steady-state force of contraction. However, the effect of adrenaline on the rested-state contraction is small in comparison to that of theophylline. The effect of both drugs on the rested-state contraction is accompanied by an increase in the plateau amplitude of the corresponding action potential. Since the plateau phase of the cardiac action potential is maintained by the slow inward current mainly carried by Ca ions and since adrenaline and methylxanthines increase this current, it is concluded from the present study that the rested-state contraction may be regulated primarily by Ca ions flowing into the cell during the action potential, while the stimulated steady-state contraction seems to be determined by the amount of Ca ions released from the sarcoplasmic reticulum.     

Autoreceptors and α2-adrenoceptors at the serotonergic axons of rabbit brain cortex

Summary Slices of the rabbit occipito-parietal cortex were preincubated with ³H-serotonin and then superfused and stimulated electrically (2 min at 3 Hz). In the absence of drugs, the stimulation-evoked overflow of tritium was approximately 3% of the tritium content of the tissue. Unlabelled serotonin and 5-carboxamido-tryptamine, when administered in the presence of 6-nitroquipazine, reduced the evoked overflow of tritium. Their effects were antagonized by metitepin (apparent pA₂ value 8.1) and (±)-cyanopindolol (apparent pA₂ value 6.4). Metitepin, but not cyanopindolol, increased evoked tritium overflow; the effect of metitepin was greater in the presence than in the absence of nitroquipazine. The evoked overflow of tritium was also depressed by clonidine, an effect antagonized by idazoxan (apparent pA₂ value 7.0) but not by prazosin. Phenylephrine caused a decrease only at high concentrations that simultaneously accelerated basal tritium efflux. Prazosin and idazoxan did not change evoked tritium overflow, and phentolamine increased it significantly only when administered in the presence of (+)-oxaprotiline. Rauwolscine produced an inhibition that was prevented by metitepin. It is concluded that the serotonergic axons of the rabbit occipitoparietal cortex possess presynaptic, release-inhibiting serotonin autoreceptors and ₂-adrenoceptors. The receptors appear to receive an input of endogenous serotonin and, to a lesser extent, noradrenaline, under the conditions of these in vitro experiments.     

A comparative study of the modulatory effects of (−)-cubebin on the mutagenicity/recombinogenicity induced by different chemical agents

(−)-Cubebin (CUB) is a lignan isolated from dry seeds of Piper cubeba. We aimed to assess its genotoxic potential and influence on chromosomal damage (frequency of micronuclei – MN) induced by doxorubicin (DXR) in V79 cells and by urethane (URE) in somatic Drosophila melanogaster cells. Our findings indicate an absence of a CUB-mediated genotoxic effect at the concentrations tested. The results also revealed that CUB significantly reduced the frequency of MN induced by DXR, with a mean reduction of 63.88%. In a previous study, our research group demonstrated an absence of CUB-mediated mutagenic effects through the wing Somatic Mutation and Recombination Test (SMART) in Drosophila. In the present study, we used the standard and high bioactivation versions of the SMART to estimate the antigenotoxic effects of CUB associated with URE. At lower concentrations, the recombination level decreased, but at the highest concentration, the recombination level increased. Our data and previous studies suggest that CUB may act as a free radical scavenger at low concentrations, a pro-oxidant at higher concentrations when it interacts with the enzymatic system that catalyzes the metabolic detoxification of DXR or URE, and/or an inducer of recombinational DNA repair.     

Contribution of both α- and β-adrenoceptors to the inotropic effects of catecholamines in the rabbit heart

Summary The functional role of -adrenoceptors was investigated in different parts of the rabbit heart. Phenylephrine (PE) caused a marked increase in force of contraction (F_c) and a prolongation of the action potential (AP) in preparations from the left atrium and the right ventricle. The response was less pronounced in the right atrium and in the left ventricle, whereas APs of spontaneously beating sinoatrial preparations remained completely unchanged. Phentolamine as well as the diesters phorbol 12,13 dibutyrate (PDBu) or 12-O-tetradecanoylphorbol-13-acetate (TPA) eliminated the effects of PE. The contribution of a-adrenoceptors to the effects of adrenaline (Adr) and noradrenaline (NA) on F_c was determined in preparations from the right ventricle. Phentolamine and the phorbol diesters reduced the effects of Adr and NA by about 30 to 60%; the remaining response was abolished by propranolol. It can be derived from our experiments that, in some parts of the rabbit heart, a considerable amount of the effects of Adr and NA is due to the stimulation of a-adrenoceptors. The present findings therefore support the view that, in the rabbit heart, the maximally effective drive of the heart requires the stimulation of both - and -adrenoceptors. The inhibitory effects of phorbol diesters on the -adrenoceptor-mediated response indicate that the activation of protein kinase C (PKC) specifically uncouples -adrenoceptors from the effector system, whereas the response to -adrenoceptor stimulation remains unchanged.Correspondence to: H. Nawrath at the above address     

A retrospective analysis of developmental toxicity studies in rat and rabbit: What is the added value of the rabbit as an additional test species?

In contrast to most toxicological tests, developmental studies are usually required in both a rodent and a non-rodent species. This study retrospectively assessed the added value of the rabbit developmental test when a rat developmental test is available. In contrast with previous reviews, we looked at developmental toxicity instead of teratogenicity, and took into account maternal toxicity in the evaluation of developmental toxicity. We analyzed data for 54 substances classified for developmental toxicity and 73 substances considered to be teratogenic in the rabbit and not in the rat in two previous reviews. On average, the rat and the rabbit developmental toxicity studies were similarly sensitive: the average ratio of the NOAELs between the two species was about one, and for most compounds there were no differences between rat and rabbit studies in terms of classification for developmental toxicity. For certain substances the developmental study in either one of the two species appeared to be more sensitive than in the other species. However, these differences are partly due to differences between studies other than the test species used. Overall, our analysis does not clearly indicate that the evaluation of developmental toxicity, as opposed to other types of toxicity, would specifically require the rabbit as an additional test species. The discrimination between direct and indirect (i.e., as a consequence of maternal toxicity) developmental effects was often doubtful, and is one of the factors that could explain the apparent differences between the two species. A more accurate assessment of maternal toxicity might improve the reliability of the results from a single developmental toxicity study. More knowledge about the interaction between maternal and developmental effects is required before decisions on omitting the requirement for the developmental toxicity testing in a second species can be considered.