(−)- -Deprenyl attenuates apoptosis in experimental brain ischaemia

(−)- -Deprenyl protects neurons from oxidative damage and helps to maintain the mitochondrial membrane potential by influencing intracellular anti-apoptotic oncoproteins, such as Bcl-2. The cellular rescue in the penumbra region by (−)- -deprenyl administration was examined after permanent middle cerebral artery occlusion in rats. (−)- -Deprenyl was given continuously following permanent middle cerebral artery occlusion. Two days later, the rats were killed and their infarct volumes were determined. Coronal brain sections were stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate deoxyribonucleic acid (DNA) nick-end labelling (TUNEL) and caspase-3, TUNEL and anti-neuronal nuclei (NeuN) double labelling. Neural plasticity was characterized by growth-associated protein-43 (GAP-43) immunohistochemistry. A 1000×1000-μm region was sampled at both cortical margins of the TUNEL-positive area at its borders. The numbers of TUNEL-labelled and TUNEL–caspase-3-labelled cells decreased significantly. (−)- -Deprenyl treatment increased the number of GAP-43-positive cells. We conclude that (−)- -deprenyl reduced the number of affected cells and induced neuronal plasticity.     

Oral Cavity Absorption of (R)−8−Hydroxy−2−(di-n-propylamino)tetralin and (S)−8−Acetyl−2−(di-n-propylamino)tetralin in the Rat

The present communication reports on the efficacy of (R)?8?OH-DPAT ((R)?8?hydroxy?2?(di-n-propylamino)tetralin) and (S)-LY?41 ((S)?8?acetyl?2?(di-n-propylamino)tetralin) in displaying the 5?HT_1A syndrome and decreasing body temperature after administration of the compound subcutaneously into the gastric ventricle or into the oral cavity in the rat. The dose range eliciting a clear-cut 5?HT_1A syndrome and hypothermia after oral cavity administration was 1/10-1/30 that of the gastric ventricle dose range, but 10?30 times higher than the dose range used for subcutaneous administration of both (R)?8?OH-DPAT and (S)-LY?41. Determination of the concentrations of (R)?8?OH-DPAT in plasma and brain tissue confirmed a higher bioavailability after oral cavity than after gastric ventricle administration; plasma and brain tissue concentrations of the drug were found to be approximately 3 times those after 10 μmol kg^?1 orally than after 100 μmol kg^?1 gastroventrically at 15?60 min after administration of (R)?8?OH-DPAT. These findings suggest that the oral cavity may be an important site for drug delivery of 8?OH-DPAT, LY?41 and other compounds with a low gastrointestinal bioavailability.     

Effect of Dopexamine Hydrochloride on Contractions of Rabbit Isolated Aorta Evoked by Various Agonists

The inhibitory affinity of dopexamine hydrochloride to postsynaptic adrenoceptors, cholinoceptors, 5-hydroxytryptamine and histamine receptors was studied in rabbit isolated aorta. Dopexamine (10^?7–10^?5M) antagonized competitively the contractions of rabbit aorta evoked by noradrenaline (pA₂: 6.60). Neither cocaine plus corticosterone nor cocaine, corticosterone plus propranolol altered the inhibition (pA₂: 6.77 and 6.63, respectively). The antagonism of dopexamine against noradrenaline-evoked contractions was the same after 1 and 4 hr of pretreatment with dopexamine. In the presence of cocaine plus corticosterone, dopexamine antagonized the contractions evoked by phenylephrine (pA₂: 6.94). Removal of endothelium did not influence this antagonism (pA₂: 7.06). Dopexamine (10^?7–10^?5M) did not antagonize the contractions of aorta evoked by histamine (3×10^?7–6 × 10^?5M) and by 5-hydroxytryptamine (3 × 10^?7 –3 × 10^?4M). Dopexamine (10^?8 and 10^?7M) did not alter the contractions of endothelium-free aorta evoked by carbachol. Dopexamine (10^?7–10^?5M) slightly enhanced the contractions of aorta evoked by potassium (10^?2–5.5 × 10^?2M). These results suggest that dopexamine is an α₁-adrenoceptor antagonist. Furthermore, dopexamine has no affinity to cholinoceptors, histamine and 5-hydroxytryptamine (5-HT₂) receptors and is apparently not a calcium antagonist.     

Investigation of Mechanisms Involved in (−)‐Borneol‐Induced Vasorelaxant Response on Rat Thoracic Aorta

Abstract: The monoterpene (?)‐borneol is present in essential oils of several medicinal plants. The aim of this study was to evaluate (?)‐borneol effects on rat thoracic aorta artery rings. The cumulative addition of (?)‐borneol (10^?9–3 × 10^?4 M) on a phenylephrine‐induced pre‐contraction (10^?6 M) promoted a vasorelaxant effect in a concentration‐dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl‐induced pre‐contractions (80 mM). (?)‐Borneol (10^?5–3 × 10^?4 M) inhibited contractions induced by cumulative addition of CaCl₂ (10^?6–3 × 10^?2 M) in depolarizing medium without Ca²⁺ in a concentration‐dependent manner. On S‐(?) Bay K 8644‐induced pre‐contractions (10^?7 M), (?)‐borneol did not induce significant changes compared with KCl‐induced pre‐contractions. In a Ca²⁺‐free medium, (?)‐borneol (10^?5, 10^?4 or 10^?3 M) interfered in calcium mobilization from phenylephrine (10^?6 M)‐ or caffeine (20 mM)‐sensitive intracellular stores. The involvement of K⁺ channels was evaluated by tetraethylammonium (3 mM), 4‐aminopyridine (1 mM) and glibenclamide (10^?5 M) pre‐treatment, and (?)‐borneol‐induced vasorelaxation was markedly attenuated. Thus, this vasorelaxant effect can probably be attributed to calcium influx blockade through voltage‐operated calcium channels (Ca_VL), calcium mobilization from intracellular stores and potassium channels activation.     

Acetylcholinesterase Inhibition and Protection by Dizocilpine (MK−801) Enantiomers

The optical isomers of the N-methyl-d-aspartate (NMDA) receptor ion-channel blocker dizocilpine (MK?801) were shown to interact with electric eel and rat brain acetylcholinesterase (AChE) in a mixed competitive-noncompetitive way. The (-) form, pharmacologically less active, was the most potent of the two isomers as an AChE inhibitor (K₁ for electric eel and rat brain AChE being 6m?2 and 17m?9μM, respectively, compared with 200 and 450 μM, respectively, of the (+) form). Both enantiomers premixed with AChE preparations, dose-dependently protected the enzyme from inactivation by diisopropylfluorophosphate (DFP). The maximal protective effects against 40 and 10 μM DFP were in the ranges 10m?7?23m?8 and 19m?5?31m?4% of control enzymic activity for the (+) and (-) forms of dizocilpine, respectively. The extent of the protective effect against DFP was increased up to 80m?1% of control enzymic activity for (-)-dizocilpine and to 38m?4% for (+)-dizocilpine by diluting the enzymic mixtures 1000 times after treatment with the organophosphate agent. The two enantiomers added to AChE 15 min after DFP, failed to reactivate the enzyme. Finally, it was shown that (+)- and (-)-dizocilpine dose-dependently and competitively decreased the DFP bimolecular reaction constant, K_i. We conclude that dizocilpine exerts a protective action towards AChE against irreversible DFP inhibition, but the molecular mechanism of such an action is at present unclear.     

Effect of ω‐Conotoxin GVIA on Noradrenaline Release from Postganglionic Sympathetic Neurones in Rabbit Aorta

Abstract: The aim of the present work was to examine the effect of the selective N‐type calcium blocking agent ω‐conotoxin GVIA on stimulation‐evoked release of noradrenaline from sympathetic nerves in rabbit isolated aorta with regard to stimulation frequency, extracellular Ca²⁺ concentration, and transmitter uptake. Rings of rabbit isolated aorta were preloaded with (‐)‐³H‐noradrenaline and the fractional ³H‐overflow evoked by electrical‐field stimulation was determined by liquid scintillation spectrometry. ω‐Conotoxin GVIA (3×10^?10– 3×10^?8 M) did not alter the spontaneous ³H‐outflow. ω‐Conotoxin GVIA (3×10^?10– 3×10^?8 M) caused a slowly developing reduction of stimulation‐evoked ³H‐overflow at 1 and 30 Hz. The Emax for the ω‐conotoxin‐induced inhibition was less (70%) at 30 Hz than that (96%) seen at 1 Hz. Short‐term incubation with ω‐conotoxin GVIA caused a subsequent steady‐state inhibition. The inhibitory action of ω‐conotoxin GVIA (3×10^?10– 3×10^?9 M) was inversely related to the extracellular Ca²⁺ concentration (6.5×10^?4– 2.7×10^?3 M). Cocaine (3×10^?5 M) plus corticosterone (4×10^?5 M), neuronal and extraneuronal uptake inhibitors, respectively, did not alter the inhibitory effect of ω‐conotoxin GVIA (3×10^?9 M) on ³H‐overflow evoked by stimulation at a frequency of either 1 or 30 Hz. It is concluded that ω‐conotoxin GVIA acts on prejunctional N‐type calcium channels to inhibit stimulation‐evoked noradrenaline release from sympathetic neurone terminals in rabbit aorta. At a high frequency, another subtype calcium channel may possibly be involved. The action of ω‐conotoxin GVIA is independent of neuronal and extraneuronal uptake mechanisms for noradrenaline, but dependent on the amount of Ca²⁺ to be transported across the neurilemma from the extracellular space into the neurone.     

A spectropolarimetric assay of (−)-adrenaline in compendial formulations

A method is described for the determination of (?)-adrenaline in certain formulations containing adrenaline hydrogen tartrate at concentrations down to 0.18 mg ml^?1 (1:10000 adrenaline). The assay is based upon a spectropolarimetric measurement at 249 nm of sample solutions, suitably treated to remove interfering substances. The rotation of the sample solutions is corrected for the rotation of the tartaric acid species which is determined by a difference rotation measurement on equimolar sample solutions at pH 1.1 and pH 5.6. The concentration of (?)-adrenaline in the sample is calculated from the net rotation at 249 nm due to the (?)-adrenaline and the total concentration of adrenaline ((+)- and (?)-isomers) determined by a published spectrofiuorimetric method. The assay is specific for (?)-adrenaline in the presence of (+)-adrenaline, (+)-tartaric acid, adrenaline sulphonic acid and low levels of adrenochrome.     

−(−)Gossypol promotes the apoptosis of bladder cancer cells in vitro

Dysregulation of Bcl2 family member proteins has been associated with poor chemotherapeutic response in bladder cancer, suggesting that agents targeting these crucial proteins may provide an interventional strategy to slow or halt bladder cancer progression and metastasis. In this study, we investigated whether the cottonseed polyphenol, -(-)gossypol, a BH3 mimetic, can reduce the expression of pro-survival, or increase the expression of pro-apoptotic, Bcl2 family proteins and thereby effectively sensitize otherwise resistant bladder cancer cells to the standard chemotherapeutic drugs gemcitabine, paclitaxel and carboplatin. These studies show that gossypol induced apoptosis in both chemosensitive UM-UC2 and chemoresistant resistant UM-UC9 bladder cancer cells in vitro in a dose and time dependent manner via a caspase mediated death signaling pathway. Moreover, in combined treatments, gossypol synergized with gemcitabine and carboplatin to induce apoptosis in chemoresistant bladder cancer cells. This effect was associated with the down-regulation the Bcl-xl and Mcl-1 pro-survival Bcl2 family proteins and up-regulation of the Bim and Puma BH3-only Bcl2 family proteins. Overall, these studies show that gossypol sensitizes bladder cancer cells to standard chemotherapeutic drugs and may provide a promising new strategy for bladder cancer treatment.     

The Inotropic Effect of 4−Aminopyridine and pH Changes in Rabbit Papillary Muscle

The effects of 4-aminopyridine (4AP), and pH changes have been examined on tension responses and cyclic nucleotide levels in rabbit isolated right ventricular papillary muscles. 4AP augmented papillary muscle contractions in a concentration-dependent manner. However, this positive inotropic action was largely due to an increase in extracellular pH produced by 4AP, rather than an intrinsic activity of the drug. Increases in extracellular pH (from 5 to 9) produced graded and reproducible increases in contractile force which were not blocked by propranolol (1 ± 10^?7M) but were inhibited by verapamil in a concentration-dependent manner. The positive inotropic effects of Ca²⁺ were enhanced and depressed by alkaline and acidic pH, respectively. Neither 4AP nor alkaline pH significantly changed cyclic AMP concentration in rabbit papillary muscles. The cyclic GMP content, however, was increased by 4AP only and this effect was blocked by atropine. The results suggest that the positive inotropic effect associated with a rise in pH from neutrality may be due to facilitation of translocation of membrane Ca²⁺ and/or to increase the release of Ca²⁺ from sources within the cell. They also illustrate that a major component of the inotropic effect of 4AP is a result of an increase in extracellular pH.     

High‐Content Analysis of Cancer‐Cell‐Specific Apoptosis and Inhibition of in Vivo Angiogenesis by Synthetic (−)‐Pironetin and Analogs

The natural product (?)‐pironetin is a structurally simple small molecule microtubule‐perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic α,β‐unsaturated lactone moiety. We used alkaloid‐catalyzed acyl halide‐aldehyde cyclocondensation reactions in asymmetric total syntheses of (?)‐pironetin and three synthetic analogs, and evaluated their biological activities by high‐content analysis in cell culture and in a zebrafish model. Synthetic (?)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi‐drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (?)‐pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide‐aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (?)‐pironetin and 2,3‐dihydro‐3‐hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule‐perturbing agents in the context of a whole organism or under conditions of multi‐drug resistance.     

Modulation of the discriminative stimulus properties of (−)-nicotine by diazepam and ethanol

The effect of different ligands for the GABA-BZD receptor and the NMDA receptor were studied in rats trained to discriminate (?)-nicotine (1.9 μmol/kg) from saline in a standard two-bar operant conditioning paradigm with food reinforcement. MK-801 (0.03–0.3 μmol/kg), flumazenil (10–30 μmol/kg), and Ro 15–4513 (3–10 μmol/kg) did not generalize to (?)-nicotine on nicotine-trained rats, and when tested as antagonists they did not block the nicotine cue. Diazepam (3–10 μmol/kg) and ethanol (11–22 mmol/kg) did not have any effect by themselves, but they significantly attenuated the nicotine cue by 53 and 65%, respectively, without affecting the response rates of the animals. Pre-treatment with flumazenil (30 μmol/kg) reversed the effect of diazepam but it did not reverse the effect of ethanol on the discriminative stimulus properties of (?)-nicotine. The effect of ethanol was not blocked by Ro 15–4513 (10 μmol/kg). These data indicate that diazepam and ethanol modulate the expression of the nicotine cue and that the effect of diazepam is mediated via a benzodiazepine receptor mechanism. © Wiley-Liss, Inc.     

Synthesis of deuterium‐labelled (−)‐galanthamine

The synthesis of deuterium‐labelled galanthamine is reported. 6‐[²H₃]methoxy‐N‐[²H₃]methyl‐(?)‐galanthamine was obtained in seven steps from galanthamine. The synthesis was carried out by selective O‐ and N‐demethylations. The [²H₃]‐N‐methyl and [²H₃]‐O‐methyl‐groups were introduced by selective aminoreduction and O‐methylation. Copyright © 2008 John Wiley & Sons, Ltd.     

β‐Cyclodextrin‐complexed (−)‐linalool produces antinociceptive effect superior to that of (−)‐linalool in experimental pain protocols

Many plants produce (?)‐linalool, a plant‐derived monoterpene alcohol, including members of the Lamiaceae (mints) and Lauraceae family (laurels, cinnamon, rosewood). The anti‐inflammatory and analgesic effects of (?)‐linalool have been widely suggested for various studies. Poor chemical stability and short half‐life restrain the clinical applications of some essential oil and monoterpenes, including (?)‐linalool. However, β‐cyclodextrin (β‐CD) has been used to increase solubility and stability of lipophilic compounds and also to improve the pharmacological effects. In this study, the antinociceptive effect of (?)‐linalool and (?)‐linalool/β‐CD was examined using the acetic acid writhing reflex, formalin and hotplate tests in rodents. (?)‐Linalool and (?)‐linalool/β‐CD demonstrated strong antinociceptive activity in all the chemical‐ and heat‐induced mice models (p < 0.01 or p < 0.001). These findings imply the involvement of both peripheral and central antinociceptive mechanisms. In peritonitis induced by carrageenan, isolated monoterpene or β‐CD complex also reduced total leucocyte migration and TNF‐α levels in peritoneal fluid. The inclusion complexes, (?)‐linalool/β‐CD, revealed that the antinociceptive effect was significantly (p < 0.01) improved when compared with (?)‐linalool alone. Such results were unlikely to be provoked by any motor abnormality. Together, our results suggest that β‐CD might represent an important tool for improvement of analgesic and anti‐inflammatory profiles of (?)‐linalool and other water‐insoluble compounds, such as lipophilic monoterpenes or essential oils.     

Effects of Terbutaline on α-Adrenergic Responses and Ca2+ Influx in Isolated Rabbit Aorta

Effects of terbutaline applied in vivo or in vitro on α-adrenergic receptors in the rabbit aorta in normal and Ca²⁺–free solution, and on basal, high potassium-, and phenylephrine-stimulated Ca²⁺ uptake into aorta were investigated. Three day terbutaline administration (25 mg/kg, subcutaneously three times daily) to rabbits increased the pK_B for phentolamine in aorta rings (control 7.3 + 0.2, n = 9; terbutaline 7.8 +0.2, n = 15). It also depressed phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free but not those in normal Krebs solution. It did not significantly depress the basal, or phenylephrine-evoked Ca²⁺ influx into aorta rings, but decreased high potassium-induced Ca²⁺–influx (control 0.58 + 0.05 umoles/g aorta; n = 3, terbutaline 0.41 +0.06 umoles/g aorta, n = 3). In vitro application of 50 μM terbutaline did not significantly alter phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free Krebs solution or significantly depress basal or phenylephrine-induced Ca²⁺ influx into aortas, but did decrease high potassium-stimulated Ca²⁺–influx. Thus, 3-day terbutaline administration increased the affinity of α-adrenergic receptors for phentolamine and had a tendency to increase contractions of aorta rings to phenylephrine. It also decreased high potassium-stimulated Ca²⁺ influx, and depressed phenylephrine-induced contractions in Ca²⁺–free Krebs solution, while in vitro terbutaline application also decreased potassium-induced Ca²⁺ influx.     

The preparation and properties of (+)- and (−)-guanoxan

(±)-2-Aminomethyl-1, 4-benzodioxan has been resolved into its optically active isomers: (+)- and (-)-guanoxan have been synthesized from these. The pharmacological effects of racemic-guanoxan and of the two optical isomers have been compared on the isolated central ear artery of the rabbit, on the pithed rat in which pressor responses were evoked by stimulation of the thoraco-lumbar sympathetic outflow, and on the pre- and post-ganglionically stimulated nictitating membrane of the cat. The two isomers were equipotent in producing adrenergic neuron blockade. Initial catecholamine release was weak in the cat, but occurred more powerfully in the rabbit ear artery and in the rat. Ability to produce this effect resided mainly with the (+)-isomer. α-Adrenoreceptor blocking activity was detectable in the rat and was produced mainly by the (+)-isomer suggesting that its stereochemical configuration corresponds to that of D-(-)-noradrenaline. Ganglion blockade was an unimportant action of the compounds, but both isomers possessed weak atropine-like activity in the rat.     

Effects of Extracellular Calcium on Potassium and Noradrenaline Induced Contractions in the Aorta of Spontaneously Hypertensive Rats – Increased Sensitivity to Nifedipine

Abstract Isolated preparations of the thoracic aorta from spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NWR) were contracted by noradrenaline (NA) 1.8 × 10^-5M, and potassium (K⁺) 127 mM, after 30 min. pretreatment in a Ca⁺⁺-free medium. In both SHR and NWR aortae, the contractions were markedly reduced; no significant differences were found between the two types of vessels in Ca⁺⁺-free medium. On addition of Ca⁺⁺, the contractions were restored to a significantly greater extent in the NWR than in the SHR aortae. In the presence of nifedipine 7.2 × 10^-9M, the response to Ca⁺⁺ was significantly more reduced in the SHR than in the NWR preparations. Relaxation of NA and K⁺ contracted preparations was induced by wash-out of the contractile agents, by addition of nifedipine 2.9 × 10^-8M, and by introduction of a Ca⁺⁺-free medium. After wash-out of NA, relaxation was slower in SHR than in NWR vessels. Relaxation induced by nifedipine and Ca⁺⁺-free medium was more complete in SHR than in NWR preparations. After wash-out of K⁺, relaxation was more rapid in NWR than in SHR aortae. Nifedipine and Ca⁺⁺-free medium induced relaxation was more complete in SHR than in NWR preparations. The results suggest that in the SHR aortae contraction induced by NA and K⁺ is more dependent on extracellular Ca⁺⁺ than is the response in the NWR preparations; the SHR vessels are also more sensitive to the relaxing effects of nifedipine.     

Enzymatic Synthesis of (S-(−)-3-(3,4-Dihydroxyphenyl)lactic Acid

The first enzymatic synthesis of (S)-(?)-3-(3,4-dihydroxyphenyl)lactic acid, a DOPA metabolite and naturally occurring compound with a broad spectrum of pharmacological activities, is described. L-Hydroxyisocaproate dehydrogenase was used as enzyme.     

Effect of flumazenil on the memory-enhancing properties of (−)-nicotine in rodents

The effect of the benzodiazepine-receptor antagonist flumazenil on the facilitatory effect of (?)-nicotine on memory in septal-lesioned rats in a spatial task and in the inhibitory avoidance test in mice was investigated. In the two-platform spatial discrimination test, septallesioned rats exhibited a significant number of errors in comparison to sham animals, an effect that can be reversed by the administration of (?)-nicotine during the training phase. Flumazenil did not affect the performance of septal-lesioned rats but it blocked the facilitatory effect of (–)-nicotine on lesioned rats. In the inhibitory avoidance test in mice,(?)-nicotine as well as flumazenil facilitated retention of the test at 0.62 and 10 μmol/kg, respectively. However, a low-noneffective dose of flumazenil blocked the memory enhancing effect of (?)-nicotine. The blockade of the facilitatory effect of (?)-nicotine by flumazenil in normal and septal-lesioned animals suggests that the cognitive effect of (?)-nicotine requires the activation of benzodiazepine receptors. © 1994 Wiley-Liss, Inc.