Cardiovascular effects of the adenosine A<Subscript>1</Subscript> receptor agonist<Emphasis Type="Italic"> N</Emphasis><Superscript>6</Superscript>-cyclopentyladenosine (CPA) decisive for its therapeutic efficacy in sarin poisoning

Mortality and occurrence of cholinergic symptoms upon sarin intoxication (144 µg/kg s.c., ~2×LD₅₀) in rats is completely prevented by treatment with the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA, 2 mg/kg i.m.). Previously, we have shown that CPA treatment altered the distribution of sarin into the brain, presumably through its cardiovascular side effects. Therefore, the objective of the present study was to evaluate the contribution of the cardiodepressant effects of CPA to its therapeutic efficacy against sarin intoxication. Intramuscular treatment of rats with 0.5 and 2.0 mg/kg CPA 1 min after sarin poisoning attenuated most cholinergic symptoms and prevented mortality, which seemed to be directly associated with an immediate strong and long-lasting bradycardia and hypotension caused by CPA. Treatment with lower doses of CPA (0.1 and 0.05 mg/kg i.m.) caused similar levels of bradycardia and hypotension, albeit a few minutes later than at the higher doses of CPA. Upon sarin intoxication, this was correlated with increased incidence of cholinergic symptoms and decreased survival rates. Pretreatment with the peripheral adenosine A₁ receptor antagonist 8-p-sulphophenyltheophylline (8-PST, 20 mg/kg i.p.) counteracted the cardiodepressant effects of 0.05 mg/kg CPA almost completely, thereby nearly abolishing its therapeutic efficacy against sarin poisoning. In conclusion, the present results strongly indicate that bradycardia and hypotension induced by the peripheral adenosine A₁ receptor play a prominent role in the therapeutic efficacy of CPA in cases of sarin poisoning.     

Attenuation of some signs of opioid withdrawal by inhibitors of nitric oxide synthase

Effects of nitric oxide synthase (NOS) inhibitors (l-N ^G-nitroarginine,l-N ^G-nitroarginine methyl ester) on precipitated opioid withdrawal were studied in morphine-dependent rats given naloxone, in order to assess the involvement of nitric oxide (NO) in opioid dependence.l-N ^G-Nitroarginine (7.5 mg/kg, IP, 1 h before naloxone or b.i.d. on days 4–7 of an 8-day morphine treatment) reduced wet dog shakes and weight loss; when given by osmotic pumps (15 mg/kg per day), the drug reduced wet dog shakes but not weight loss.l-N ^G-Nitroarginine methyl ester (60 mg/kg, 1 h before naloxone) also reduced wet dog shakes and weight loss. The results indicate that NOS inhibitors warrant further study as potential treatment of the opioid withdrawal syndrome.Abstracts were presented at meetings of the Society for Neuroscience in New Orleans, La., November 10–15, 1991 and of the American Society for Pharmacology and Experimental Therapeutics in Orlando, Fla., August 10–18, 1992     

Intravenously injected CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock: effect is mediated by central cholinergic activation

Intravenous (i.v.) administration of cytidine-5′-diphosphate choline (CDP-choline) (100, 250 and 500 mg/kg) increased blood pressure in normal rats and reversed hypotension in haemorrhagic shock. Choline (54 mg/kg; i.v.), at the dose equimolar to 250 mg/kg CDP-choline decreased blood pressure of rats in both conditions and caused the death of all hypotensive animals within 2-5 min. Equimolar dose of cytidine (124 mg/kg; i.v.) did not change cardiovascular parameters. Choline levels in plasma, lateral cerebral ventricle and hypothalamus increased after CDP-choline administration. Intracerebroventricular (i.c.v.) hemicholinium-3 pretreatment (20 μg), greatly attenuated the pressor effect of CDP-choline in both conditions. Atropine pretreatment (10 μg; i.c.v.) did not change the pressor effect of CDP-choline while mecamylamine (50 μg; i.c.v.) abolished the pressor response to drug. Besides, acetylcholine (1 μmol; i.c.v.) produced similar increases in blood pressure in normal and hypotensive conditions to that observed in CDP-choline given rats. CDP-choline (250 mg/kg; i.v.) increased plasma catecholamines and vasopressin levels but not plasma renin activity. Pretreatment of rats with either prazosin (0.5 mg/kg; i.v.) or vasopressin V₁ receptor antagonist, [β-mercapto,β,β-cyclopentamethylenepropionyl¹,O-Me-Tyr²-Arg⁸]vasopressin (10 μg/kg; i.v.), attenuated the pressor response to CDP-choline while simultaneous administration of these antagonists before CDP-choline injection completely blocked the pressor effect. Results show that i.v. CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock. Activation of central nicotinic cholinergic mechanisms by the increases in plasma and brain choline concentrations appears to be involved in the pressor effect of this drug. Moreover, the increases in plasma catecholamines and vasopressin levels mediate these effects.     

Agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ: A new compound with potent gastroprotective and ulcer healing properties

Pioglitazone, a specific ligand for peroxisome proliferator-activated receptor gamma (PPAR-γ), was recently implicated in the control of inflammatory processes and in the modulation of the expression of various cytokines such as tumor necrosis factor alpha (TNF-α), but its role in the mechanism of gastric mucosal integrity has not been studied extensively. This study was designed to determine the effect of pioglitazone on gastric mucosal lesions induced in rats by topical application of 100% ethanol and by 3.5 h of water immersion and restraint stress (WRS) with or without pretreatment with indomethacin (5 mg/kg i.p.) to inhibit cyclooxygenase-1 (COX-1) and COX-2 enzyme activities and L-NNA (20 mg/kg i.p.) to suppress nitric oxide (NO)-synthase. In addition, the effect of pioglitazone on ulcer healing in rats with chronic acetic acid ulcers (ulcer area 28 mm²) was determined. Rats were killed 1 h and 3.5 h after ethanol administration or WRS exposure or at day 9 upon ulcer induction, and the number and area of gastric lesions were measured by planimetry, the gastric blood flow (GBF) was determined by H₂-gas clearance technique and the mucosal PGE₂ generation and gene expression and plasma concentration of TNF-α and IL-1β were also evaluated. Pre-treatment with pioglitazone dose-dependently attenuated gastric lesions induced by 100% ethanol and WRS; the dose reducing these lesions by 50% (ID₅₀) being 10 mg/kg and 7 mg/kg, respectively. The protective effect of pioglitazone was accompanied by the significant rise in the GBF, an increase in PGE₂ generation and the significant fall in the plasma TNF-α and IL-1β levels. Strong signals for IL-1β-and TNF-α mRNA were recorded in gastric mucosa exposed to ethanol or WRS, and these effects were significantly decreased by pioglitazone. Indomethacin which suppressed PG generation by about 90%, while augmenting WRS damage, and L-NNA, that suppressed NO-synthase activity, significantly attenuated the protective and hyperaemic activity of this PPAR-γ ligand. In the chronic study, pioglitazone significantly reduced the area of gastric ulcers on day 9 and significantly raised the GBF at the ulcer margin. The acceleration of ulcer healing by PPAR-γ ligand was accompanied by a significant increase in the expression of PECAM-1 protein, a marker of angiogenesis. We conclude that (1) pioglitazone exerts a potent gastroprotective and hyperaemic actions on the stomach involving endogenous PG and NO and attenuation of the expression and release of proinflammatory cytokines TNF-α and IL-1β, and (2) PPAR-γ ligand accelerates ulcer healing, possibly due to the enhancement in angiogenesis at ulcer margin.     

Calcium-antagonizing activity of S-petasin, a hypotensive sesquiterpene from Petasites formosanus, on inotropic and chronotropic responses in isolated rat atria and cardiac myocytes

Petasites formosanus, an indigenous species of Petasites, has been used to treat cardiovascular diseases such as hypertension for years. We have suggested recently that S-petasin, a major sesquiterpene from P. formosanus, inhibits vascular smooth muscle contraction through inhibition of voltage-dependent Ca²⁺ channels, a phenomenon possibly responsible for the hypotensive effect of P. formosanus. This study was designed to examine the chronotropic and inotropic actions of S-petasin in the heart in vivo and in vitro. Administration of S-petasin (0.1–1.5 mg/kg i.v.) in anesthetized rats reduced heart rate dose-dependently. This response was consistent with significant suppression of both contractile amplitude and spontaneous firing rate of isolated atria, responses that were not antagonized by atropine (1 µM). Mechanical evaluation in isolated ventricular myocytes showed that S-petasin (0.1 to 100 µM) depressed peak myocyte contraction and intracellular Ca²⁺ transients concentration-dependently. The duration of myocyte contraction was not affected. Whole-cell voltage clamp analysis revealed that S-petasin inhibited the L-type Ca²⁺ current (I_Ca,L) concentration-dependently and shifted the steady-state inactivation curve of I_Ca,L to more negative potentials. However, a receptor-binding assay failed to identify any significant interaction between S-petasin (0.1–300 µM) and the dihydropyridine binding sites of L-type voltage-dependent Ca²⁺ channels. Taken together, these data show that the negative chronotropic and inotropic properties of S-petasin that can be ascribed mainly to I_Ca,L inhibition, but not to blockade of dihydropyridine binding sites of L-type Ca²⁺ channel or to muscarinic receptor activation.     

Effect of LP-805, a releaser of endothelium-derived nitric oxide,on systemic vasodilatation in vivo

Summary We have investigated relations between hypotensive responses to LP-805, a newly synthesized vasodilator, and the production of nitric oxide (NO), in anesthetized rats. LP-805 (0.1–0.5 mg/kg, i.v.) or acetylcholine (ACh) (0.3 – 3.0 g/kg, i.v.) caused a dose-dependent transient decrease in diastolic blood pressure. The decrease induced by 0.3 mg/kg LP-805 (i.v.) was partially inhibited by pretreatment with N^G-nitro-l-arginine (LNNA), a specific inhibitor of endothelial NO synthase, but the responses to lower or higher doses of LP-805 (0.1 or 0.5 mg/kg, i.v.) were not affected. The dose-dependent decrease in diastolic blood pressure, caused by LP-805, was not affected by pretreatment with l- or d-arginine. The dose-dependent decrease in diastolic blood pressure caused by ACh was not affected by pretreatment with L-NNA or with l- or d-arginine. The hypotensive response to 20-min infusions of LP-805 (100 g/kg per min) wassignificantly inhibited by pretreatment with L-NNA (10 mg/kg, i.v.). The half-recovery times (T_1/2) of LP-805 or ACh-induced depressor responses were shortened by pretreatment with L-NNA. They were prolonged by l-arginine, but not by d-arginine. This shortening, by L-NNA, of the half-recovery time after LP-805 or ACh was reversed by l-arginine, but not by d-arginine. The T_/2 of the LP-805-induced hypotensive response was not affected by pretreatment with indomethacin (1 mg/kg, i.v.). In the presence of L-NNA (10 mg/kg, i.v.), the T_/2 of the LP-805-induced hypotensive response was not affected by pretreatment with indomethacin. The results suggest that the LP-805-induced hypotensive response may be related to direct or indirect activation of NO synthase in vascular endothelial cells, and to release of endothelium-derived NO. Correspondence to M. Inazu at the above address     

Effect of N-nitro-l-arginine on shock induced by endotoxin and by platelet activating factor in dogs

When N^G-nitro-l-arginine, a nitric oxide synthase inhibitor, administration was started 5 min prior to shock induction in anesthetized dogs, a partial restoration was observed in endotoxin-induced shock and a complete recovery in platelet activating factor (PAF)-induced shock. When N^G-nitro-l-arginine infusion was started 5 min after shock induction, no significant recovery was observed in endotoxin-induced shock and a complete recovery in PAF-induced shock. These data indicate that enhanced production of nitric oxide by vascular endothelial cells may contribute to endotoxin- or PAF-induced shock and also that some mediators including inducible nitric oxide synthase and/or cellular damage might be involved in endotoxin-induced shock.     

Involvement of neuronal processes and nitric oxide in the inhibition by endotoxin of pentagastrin-stimulated sastric acid secretion

Administration of E. coli endotoxin (1 mg kg^–1, i.v.) abolished the acid response induced by the i.v. infusion of pentagastrin (8 g kg^–1, h^-1) in the continuously perfused stomach of the anaesthetized rat. Local serosal application of tetrodotoxin (36 ng per rat) completely restored acid responses to pentagastrin in endotoxin-treated rats. However, pretreatment with atropine (0.5 mg kg^–1, s.c.), capsaicin (20, 30, and 50 mg kg^–1, s.c. 2 weeks before the study) or guanethidine (16 mg kg^–1, s.c. 3 and 16h before) did not influence the inhibitory effects of endotoxin. Continuous i.v. infusion with N^G-nitro arginine methyl ester (L-NAME, 10 mg kg^–1, h^–1) restored the secretory responses to pentagastrin in endotoxin treated rats. The effects of L-NAME were reversed by L-arginine (100 mg kg^–1, h^–1, i.v.), but not by its enantiomer D-arginine (100 mg kg^–1, h^–1, i.v.). The secretory responses elicited by pentagastrin (10^–10–10^–6 M) in the isolated lumen perfused stomach of the rat were not influenced by incubation (100 min) with endotoxin (10 g ml ^–1). These observations with tetrodotoxin indicate that inhibition of acid secretion by endotoxin in vivo involves neuronal activity, while inhibition of NO synthesis had a comparable inhibitory action. Activation of a systemic non-adrenergic non-cholinergic neuronal pathway involving NO could thus mediate the acute acid inhibitory effects of endotoxin. Correspondence to: J. V. Esplugues at the above address     

A novel kainate receptor ligand [H]-(2S,4R)-4-methylglutamate: Pharmacological characterization in rabbit brain membranes

Since kainate evokes large non-desensitizing currents at α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate is of limited use in discriminating between AMPA and kainate receptors. Following recent reports that (2S,4R)-4-methylglutamate is a kainate receptor-selective agonist, we have radiolabelled and subsequently characterized the binding of [³H]-(2S,4R)-4-methylglutamate to rabbit whole-brain membranes. [³H]-(2S,4R)-4-methylglutamate binding was rapid, reversible and labelled two sites (KD1 = 3.67 ± 0.50 nM/B_max1 = 0.54 ± 0.03 pmol/mg protein and K_D2 = 281.66 ± 12.33 nM/ B_max2 = 1.77 ± 0.09 pmol/mg protein). [³H]-(2S,4R)-4-methylglutamate binding was displaced by several non-NMDA receptor ligands: domoate > kainate -quisqualate -glutamate > 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) (S)-AMPA = (S)-5-fluorowillardiine > NMDA. Neither the metabotropic glutamate receptor agonists (1S,3R)-ACPD or -AP4, together with the -glutamate uptake inhibitor -trans-2,4-PDC, influenced binding when tested at 100 μM. We conclude that [³H]-(2S,4R)-4-methylglutamate is a useful radioligand for labelling kainate receptors. It possesses high selectivity, and possesses a pharmacology similar to that for rat cloned low-affinity (Glu5 and 6) kainate receptor subunits.     

Endothelium-derived nitric oxide partially mediates salbutamol-induced vasodilatations

This study examined the ability of salbutamol (selective β₂-adrenoceptor agonist) to cause endothelium-dependent relaxation in rat aortic rings and depressor response in conscious rats. Salbutamol (0.01–100 μM) concentration dependently relaxed preconstricted aortic rings. The relaxant response was partially attenuated by either mechanical removal of the endothelium or treatment with N^G-nitro-l-arginine methyl ester (L-NAME, 100 μM). In conscious rats, either i.v. infused phenylephrine (5 μg/kg per min) or i.v. bolus injected L-NAME (12.8 mg/kg), but not the vehicle, caused similar sustained increases in mean arterial pressure (MAP). I.v. infused salbutamol (2–128 μg/kg per min, each dose for 5 min) dose dependently decreased MAP in vehicle-treated rats; the depressor responses were potentiated by hypertension induced by phenylephrine. In contrast, the magnitudes of the depressor response to salbutamol in L-NAME-treated rats were less than those in rats pretreated with phenylephrine or the vehicle. I.v. bolus injections of salbutamol (0.25–16 μg/kg) also caused dose-dependent and transient decreases in MAP in vehicle-treated rats. The magnitude but not the duration of the depressor response to salbutamol was less in rats treated with L-NAME, compared to those in rats given phenylephrine or the vehicle. These results suggest that endothelium-derived nitric oxide is partially involved in β₂-adrenoceptor-mediated vasodilatation.     

Enhancement of blood-brain barrier permeability to sodium fluorescein by stimulation of µ opioid receptors in mice

Summary The effects of opioids on the permeability of the blood-brain barrier (BBB) were examined in mice with sodium fluorescein as an indicator of the permeability. The brain was perfused with saline 30 min after injection of sodium fluorescein (40 mg/kg, i. v.) and examined by fluorometry. Morphine hydrochloride (0.3–10 mg/kg, s. c.) markedly increased the brain level of sodium fluorescein dose-dependently without influencing the plasma level, when administered 20 min before sodium fluorescein injection. Intracerebroventricularly (i. c. v.) injected morphine hydrochloride (0.5 and 1.0 Erg) increased the brain sodium fluorescein level. Buprenorphine (0.1 and 0.5 mg/kg, s. c.) was also effective. However, pentazocine, ethylketazocine, U-50488H and SKF-10047 had no significant influence. The i.c.v. administration of [D-Ala², McPhe⁴, Gly(ol)⁵]enkephalin (0.1 g) and [D-Ala², D-Leu⁵]enkephalin (0.5 g) but not of [D-Thr², Leu⁵]enkephalin-Thr increased the brain level of sodium fluorescein significantly. A small dose of naloxone (i. p.) significantly inhibited the effects of morphine, buprenorphine, [D-Ala², McPhe⁴, Gly(ol)⁵]enkephalin and [D-Ala³, D-Leu⁵]enkephalin. ICI-174864 co-administered i. c. v. with [D-Ala², D-Leu⁵]enkephalin was ineffective in antagonizing the effect of the latter. These findings suggest that the stimulation of µ opioid receptors results in an increase in BBB permeability to sodium fluorescein. Send offprint requests to K. Saeki     

Discriminative stimulus effects of a centrally administered,delta-opioid peptide (d-Pen2-d-Pen5-enkephalin) in pigeons

The present study assessed the discriminative stimulus effects of the delta-opioid agonist [d-Pen²-d-Pen⁵]enkephalin (DPDPE) in pigeons. Food-restricted pigeons were trained to discriminate between ICV injections of 100 µg [d-Pen²-d-Pen⁵]enkephalin (DPDPE) and saline in a two-key operant procedure; acquisition of discriminative control was rapid (14–28 daily sessions). [d-Ser², Leu⁵, Thr⁶]enkephalin (DSLET) and [d-Ala²]deltorphin II, peptides selective for delta-opioid receptors, produced discriminative stimulus effects similar to DPDPE, and were approximately equipotent to DPDPE. The non-peptidic, delta-opioid agonist BW373U86 (0.032–100 mg/kg, IM) partially generalized to DPDPE. The kappa-opioid agonist U69,593 (0.01–1 mg/kg, IM), and the mu-opioid agonists, DAMGO (0.1–3.2 µg, ICV) and morphine (1–10 mg/kg, IM), did not produce discriminative stimulus effects similar to DPDPE, up to doses that markedly decreased response rates. Naltrindole (0.1 mg/kg, IM), an antagonist selective for deltaopioid receptors, produced approximately a 30-fold reduction in the potency of DPDPE. DPDPE's discriminative stimulus effect in pigeons appears to be mediated through a delta-opioid receptor; this effect may provide a procedure for assessing delta-opioid receptor function in vivo.     

N-Nitro-L-arginine protects against ischaemia-induced increases in nitric oxide and hippocampal neuro-degeneration in the gerbil

To assess the effects of the nitric oxide synthase inhibitor N^G-Nitro-L-arginine on behavioural, biochemical and histological changes following global ischaemia, the Mongolian gerbil was used. Ischaemia was induced by bilateral carotid occlusion for 5 min. N^G-Nitro-L-arginine was administered i.p. at either 1 or 10 mg/kg 30 min, 6, 24, and 48 h after surgery. 5 min bilateral carotid occluded animals were hyperactive 24, 48 and 72 h after surgery. N^G-Nitro-L-arginine caused some attenuation in this hyperactivity. The activity of nitric oxide synthase was increased in the cerebellum, brain stem, striatum, cerebral cortex and hippocampus of 5 min bilateral carotoid occluded animals. N^G-Nitro-L-arginine reversed the increase in nitric oxide synthase activity in all brain regions. Extensive neuronal death was observed in the CA₁ layer of the hippocampus in 5 min bilateral carotid occluded animals 96 h after surgery. N^G-Nitro-L-arginine significantly protected against the neuronal death of cells in the CA₁ layer.     

(−)-3H-desmethoxyverapamil labelling of putative calcium channels in brain: autoradiographic distribution and allosteric coupling to 1,4-dihydropyridine and diltiazem binding sites

Summary The optically pure phenylalkylamine Ca²⁺-antagonist (–)-³H-desmethoxyverapamil has been used to directly label putative Ca²⁺ channels in thaw-mounted guinea-pig brain sections and in hippocampus homogenates. The autoradiographic distribution of (–)-³H-desmethoxyverapamil binding shows the highest density of binding sites in the molecular layer of the hippocampus, medium levels in the cerebral cortex and low levels in the cerebellum. This anatomical distribution is the same as that found for 1,4-dihydropyridine binding sites, labelled by ³H-nimodipine. (–)-³H-desmethoxyverapamil binds to hippocampus membranes with a K _D value of 1.6±0.2 nmol/l and a B _max of 870±175 fmol per mg of protein. Binding is stereospecifically inhibited by the phenylalkylamines desmethoxyverapamil, D-600 and verapamil. 1,4-Dihydropyridines regulate (–)-³H-desmethoxyverapamil binding in a negative heterotropic allosteric manner, depending on the availability of free divalent cations. The potency series of phenylalkylamine Ca²⁺ antagonists in inhibiting high affinity (–)-³H-desmethoxyverapamil binding to hippocampus membranes and the allosteric regulation by chemically different classes of Ca²⁺ antagonists suggest that the (–)-³H-desmethoxyverapamil binding sites in hippocampus are associated with putative Ca²⁺ channels.Part of theses of D. R. Ferry and A. Goll     

Isolation and identification of antioxidants from Pedilanthus tithymaloides

A bioassay-guided methodology utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH), the trolox equivalent antioxidant capacity (TEAC), and reducing power assays, as well as an assessment of scavenging properties against O₂^·−, H₂O₂, HOCl, ROO·, ·NO, and ONOO⁻ were used to find the main antioxidant principles of Pedilanthus tithymaloides (Euphorbiaceae), a shrub used in traditional Cuban medicine. The principles were identified as kaempferol 3-O-β-D-glucopyranoside-6′′-(3-hydroxy-3-methylglutarate), quercitrin, isoquercitrin, and scopoletin. The contents of total phenolics and flavonoids were found to be 76.0 ± 4.8 mg of gallic acid equivalents/g extract and 9.8 ± 0.4 mg of rutin equivalents/g extract, respectively.     

Possible role of nitric oxide in the antinociceptive action of intraventricular bradykinin in mice

The i.c.v. administration of bradykinin (4, 8 and 16 μg) induced antinociception in mice which was resistant to naloxone; furthermore, the induction of tolerance to morphine by a single s.c. injection (100 mg/kg, 24 h before test doses of the peptide) did not affect antinociception. Since bradykinin is known to increase nitric oxide (NO) in peripheral tissues, we studied the possibility that its antinociceptive action may be related to NO effects in the central nervous system. Bradykinin effects were antagonized by previous treatment with N^G-nitro- -arginine or concomitant i.c.v. administration of bradykinin and methylene blue. The immediate precursor of NO, -arginine, which by itself produces analgesia, also reduced bradykinin effects; moreover, tolerance to -arginine significantly decreased the response to the peptide. These results suggest that NO is involved in antinociception induced by i.c.v. administration of bradykinin.     

Hyperthermia induced bym-trifluoromethylphenylpiperazine (TFMPP) orm-chlorophenylpiperazine (m-CPP) in heat-adapted rats

TFMPP andm-CPP, non-selective 5-HT agonists, administered in doses of 1–20 mg/kg evoked hyperthermia in rats at a high ambient temperature (28°C). The hyperthermic effect of TFMPP (10 mg/kg) orm-CPP (10 mg/kg) was dose-dependently antagonized by the 5-HT_1c and 5-HT₂ receptor antagonists mesulergine (0.5–4 mg/kg), ketanserin (0.6–2.5 mg/kg) and ritanserin (0.5–2 mg/kg) and by the non-selective 5-HT antagonist metergoline (0.5–1 mg/kg), or was attenuated by the 5-HT_1A, 5-HT₂ and dopamine receptor antagonist spiperone (3 mg/kg, but not 0.3 or 1 mg/kg). On the other hand, the 5-HT_1A, 5-HT_1B and adrenoceptor antagonists pindolol (2 mg/kg) and cyanopindolol (2 mg/kg), the 5-HT_1A receptor agonist/antagonist ipsapirone (10 and 35 mg/kg) and haloperidol (0.25 and 0.5 mg/kg) showed a tendency towards enhancing the TFMPP- orm-CPP-induced hyperthermia. The 5-HT_1A and ₁-adrenoceptor antagonist NAN-190 (1–4 mg/kg), the 5-HT₃ antagonists tropisetron (0.01–1 mg/kg) and zacopride (0.5 and 1 mg/kg), the-blockers betaxolol (8 mg/kg) and ICI 118, 551 (8 mg/kg), which have no affinity for 5-HT receptors and prazosin (1 mg/kg), did not affect the hyperthermic effect of TFMPP orm-CPP. The hyperthermias studied were not modified, in animals with 5-HT lesion produced byp-chloroamphetamine (PCA) either. All the drugs used as putative receptor antagonists, as well as PCA, did not change or decreased (ipsapirone) the body temperature in heat-adapted rats. The obtained results suggest that the hyperthermia induced by TFMPP orm-CPP is mediated by 5-HT₂, and maybe also by 5-HT_1c receptors — those which are located postsynaptically.     

Protective effect of the ethanol extract of <Emphasis Type="Italic">Magnolia officinalis</Emphasis> and 4-<Emphasis Type="Italic">O</Emphasis>-methylhonokiol on scopolamine-induced memory impairment and the inhibition of acetylcholinesterase activity

Magnolol, honokiol, and obovatol are well-known bioactive constituents of the bark of Magnolia officinalis and have been used as traditional Chinese medicines for the treatment of neurosis, anxiety, and stroke. We recently isolated novel active compound (named 4-O-methylhonokiol) from the ethanol extract of Magnolia officinalis. The present study aimed to test two different doses of ethanol extracts of Magnolia officinalis (5 and 10 mg/kg/mouse, p.o., 1 week) and 4-O-methylhonokiol (0.75 and 1.5 mg/kg/mouse, p.o., 1 week) administered for 7 days on memory impairment induced by scopolamine (1 mg/kg body weight i.p.) in mice. Memory and learning were evaluated using the Morris water maze and the step-down avoidance test. Both the ethanol extract of Magnolia officinalis and 4-O-methylhonokiol prevented memory impairment induced by scopolamine in a dose-dependent manner. The ethanol extract of Magnolia officinalis and 4-O-methylhonokiol also dose-dependently attenuated the scopolamine-induced increase of acetylcholinesterase (AChE) activity in the cortex and hippocampus of mice, and inhibited AChE activity in vitro with IC₅₀ (12 nM). This study, therefore, suggests that the ethanol extract of Magnolia officinalis and its major ingredient, 4-O-methylhonokiol, may be useful for the prevention of the development or progression of AD.     

A novel compound N,N-(Z)-N-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC₅₀ of 0.74 ± 0.15 µM for S6 cells or 1.07 ± 0.23 µM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.     

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5–2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N^G-nitro-l-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5–20 mg/kg, i.v.), but not by an inactive enantiomer, N^G-nitro-d-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1–100 μg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE₂ (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 μg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 μg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE₂, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin. Received: 18 November 1996 / Accepted: 30 May 1997