European Journal of Pharmacology (v.475, #1-3)

Procaine-induced enhancement of fluid-phase endocytosis and inhibition of exocytosis in human skin fibroblasts by Marta Michalik; Małgorzata Pierzchalska; Anna Pabiańczyk-Kulka; Włodzimierz Korohoda (1-10).
Local anaesthetics are often applied directly onto the skin, and for this reason the effect of some local anaesthetics upon morphology and cytoskeleton organisation in human skin fibroblasts was investigated. In this paper the authors report that procaine (p-aminobenzoic acid diethylamino-etyl ester hydrochloride) induced vacuolisation of cytoplasm and great enhancement of neutral red accumulation in human skin fibroblasts cultured in vitro. Procaine-induced vacuolisation of cell's cytoplasm was observed to be associated with the enhanced uptake and inhibited release of fluid taken by endocytosis. All these effects appeared fully reversible. The cell vacuolisation cannot be prevented by 3-methyadenine, brefeldine A, and cytochalasine D. On the other hand, nocodazole and caffeine prevent cytoplasm vacuolisation induced by procaine. These observations suggest that procaine-induced formation of great vacuoles is due to an impairment of membrane traffic between endosomes. The authors' results also demonstrate that neutral red uptake assay, if used as a cell viability test, must be interpreted with great caution.
Keywords: Local anaesthetic; Procaine; Fluid-phase endocytosis; Exocytosis; Fibroblast; Skin; Neutral red uptake assay;

SNX482 selectively blocks P/Q Ca2+ channels and delays the inactivation of Na+ channels of chromaffin cells by Gloria Arroyo; Marcos Aldea; Jorge Fuentealba; Almudena Albillos; Antonio G. Garcı́a (11-18).
The effects of the toxin SXN482 on Ca2+ channel currents (I Ca), Na+ currents (I Na), and K+ currents (I K) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at −80 mV. Currents were elicited by depolarising pulses to 0–10 mV (I Ca and I Na) or to +60 mV (I K). SNX482 blocked I Ca in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC50 of 30.2 nM. The second low-affinity phase comprised over 70% of I Ca and had an IC50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 μM) and by ω-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined ω-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 μM and higher concentrations, SNX482 delayed the inactivation of I Na. The time constant (τ) for inactivation of I Na in control conditions doubled in the presence of 0.5 μM SNX482. At 0.3 μM, SNX482 did not affect I K. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na+ channels; (iii) SNX482 delays the inactivation of Na+ channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca2+ channel.
Keywords: Ca2+ current; Na+ current; Ion channel; Toxin; Catecholamine cell;

The anti-inflammatory effect of honokiol on neutrophils: mechanisms in the inhibition of reactive oxygen species production by Kuo-Tong Liou; Yuh-Chiang Shen; Chieh-Fu Chen; Cheng-Ming Tsao; Shen-Kou Tsai (19-27).
Reactive oxygen species produced by neutrophils contribute to the pathogenesis of focal cerebral ischemia/reperfusion injury and signal the inflammatory response. We have previously shown that honokiol, an active principle extracted from Magnolia officinalis, has a protective effect against focal cerebral ischemia/reperfusion injury in rats that paralleled a reduction in reactive oxygen species production by neutrophils. To elucidate the underlying mechanism(s) of the antioxidative effect of honokiol, peripheral neutrophils isolated from rats were activated with phorbol-12-myristate-13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence or absence of honokiol. In this study, we found that honokiol inhibited PMA- or fMLP-induced reactive oxygen species production by neutrophils by three distinct mechanisms: (1) honokiol diminished the activity of assembled-NADPH oxidase, a major reactive oxygen species producing enzyme in neutrophils by 40% without interfering with its protein kinase C (PKC)-dependent assembly; (2) two other important enzymes for reactive oxygen species generation in neutrophils, i.e., myeloperoxidase and cyclooxygenase, were also inhibited by honokiol by 20% and 70%, respectively; and (3) honokiol enhanced glutathione (GSH) peroxidase activity by 30%, an enzyme that triggers the metabolism of hydrogen peroxide (H2O2). These data suggested that honokiol, acting as a potent reactive oxygen species inhibitor/scavenger, could achieve its focal cerebral ischemia/reperfusion injury protective effect by modulating enzyme systems related to reactive oxygen species production or metabolism, including NADPH oxidase, myeloperoxidase, cyclooxygenase, and GSH peroxidase in neutrophils.
Keywords: Honokiol; Reactive oxygen species; NADPH oxidase; Myeloperoxidase; Cyclooxygenase; Glutathione peroxidase; Protein kinase C;

Pramipexole inhibits MPTP toxicity in mice by dopamine D3 receptor dependent and independent mechanisms by Andres D. Ramirez; Stephen K.-F. Wong; Frank S. Menniti (29-35).
The role of dopamine D3 receptors was investigated in mediating the neuroprotective effect of the dopamine D2/D3 receptor agonist (S)-2-amino-4,5,6,7-tetrahydro-6-propylamine-benzothiazole (pramipexole) in vivo. Pramipexole retained the ability to inhibit 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion in mice in which the dopamine D3 receptor had been deleted. However, the neuroprotective efficacy was reduced in the dopamine D3 receptor-deleted mice compared to that in littermates expressing the wildtype receptor. Furthermore, the dopamine D3 receptor selective antagonist 2-{3-[4-(2-tert-butyl-6-trifluoromethyl-4-pyrimidinyl)-1-piperazinyl]propylthio}-4-pyrimidinol (A-437203) partially inhibited the neuroprotective effect of pramipexole in dopamine D3 receptor expressing mice but not in receptor-deleted mice. These results indicate that pramipexole protects dopamine neurons from MPTP-induced toxicity by mechanisms that are both dependent and independent of an interaction with dopamine D3 receptors.
Keywords: Parkinson's disease; MPTP; Pramipexole; Dopamine D3 receptor;

Chronic MCH-1 receptor modulation alters appetite, body weight and adiposity in rats by Lauren P. Shearman; Ramon E. Camacho; D. Sloan Stribling; Dan Zhou; Maria A. Bednarek; Donna L. Hreniuk; Scott D. Feighner; Carina P. Tan; Andrew D. Howard; Lex H.T. Van der Ploeg; D.Euan MacIntyre; Gerard J. Hickey; Alison M. Strack (37-47).
Central administration of the neuropeptide melanin-concentrating hormone (MCH) stimulates feeding in rodents. We studied the effects of intracerebroventricular (i.c.v.) administration of an MCH-1 receptor agonist (Compound A) and an MCH-1 receptor antagonist (Compound B) on feeding in satiated rats. Compound B (10 μg, i.c.v.) blocked the acute orexigenic effect of Compound A (5 μg, i.c.v.). In an experiment designed to either stimulate or inhibit MCH-1 receptor signaling over an extended period, rats received continuous i.c.v. infusions of vehicle (saline), Compound A (30 μg/day), Compound B (30 or 48 μg/day) or neuropeptide Y (24 μg/day, as positive control) via implantable infusion pumps. Continuous MCH-1 receptor activation recapitulated the obese phenotype of MCH-over-expressor mice, manifest as enhanced feeding (+23%, P<0.001), caloric efficiency and body weight gain (+38%, P<0.005) over the 14-day period relative to controls. Chronic MCH-1 receptor activation also elevated plasma insulin and leptin levels significantly. Conversely, continuous MCH-1 receptor antagonism led to sustained reductions in food intake (−16%, P<0.001), body weight gain (−35%, P<0.01), and body fat gain relative to controls, without an effect on lean mass. Antagonism of the MCH-1 receptor may be an effective approach for the treatment of obesity.
Keywords: MCH (melanin-concentrating hormone); Food intake; Body weight; Neuropeptide Y; Obesity;

Previously, we reported that the elevation of plasma noradrenaline and adrenaline induced by intracerebroventricularly (i.c.v.) administered corticotropin-releasing hormone (CRH) was abolished by i.c.v. administered indomethacin, an inhibitor of cyclooxygenase, in rats [Yokotani et al., Eur. J. Pharmacol. 419, 183-189, 2001]. The result suggests the involvement of active metabolites of brain arachidonic acid in the CRH-induced activation of the central sympatho-adrenomedullary outflow. Arachidonic acid is released mainly by two different pathways: phospholipase A2-dependent pathway; phospholipase C- and diacylglycerol lipase-dependent pathway. In the present study, therefore, we tried to identify which pathway is involved in the CRH-induced elevation of plasma catecholamines in urethane-anesthetized rats. CRH (1.5 nmol/animal, i.c.v.)-induced elevation of plasma noradrenaline and adrenaline was abolished by neomycin [0.55 μmol (500 μg)/animal, i.c.v.] and 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122) [5 nmol (2.3 μg)/animal, i.c.v.] (inhibitors of phospholipase C), and also by 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267) [1.3 μmol (500 μg)/animal, i.c.v.] (an inhibitor of diacylglycerol lipase). On the other hand, mepacrine [1.1 μmol (500 μg)/animal, i.c.v.] (an inhibitor of phospholipase A2) and 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidinedione (U-73343) [5 nmol (2.3 μg)/animal, i.c.v.] (an inactive analog of U-73122) had no effect. These results suggest that CRH activates the central sympatho-adrenomedullary outflow by the brain phospholipase C- and diacylglycerol lipase-dependent mechanisms in rats.
Keywords: Catecholamine; Plasma; Corticotropin-releasing hormone (CRH); Phospholipase C; Diacylglycerol lipase; Brain; Sympatho-adrenomedullary outflow;

Role of the mesotelencephalic dopamine system in learning and memory processes in the rat by Vasile Hefco; Kiyofumi Yamada; Andreea Hefco; Lucian Hritcu; Adrian Tiron; Toshitaka Nabeshima (55-60).
The effects of lesioning the ventral tegmental area or substantia nigra pars reticulata by means of bilateral microinjections of two doses of kainic acid (50 ng/250 nl and 100 ng/500 nl) or 6-hydroxydopamine (8 μg/4 μl) were investigated to clarify the role of the mesotelencephalic dopamine system in learning and memory processes. Our findings suggest that ventral tegmental area and substantia nigra dopaminergic neurons play an important role in retention of both short-term memory, tested in the Y-maze task and long-term memory evaluated with the multi-trial passive avoidance test, without affecting memory acquisition. As compared to short-term memory, long-term memory is more susceptible to the decreased dopamine level in nervous structures involved in processing and storage of information.
Keywords: Dopamine; Kainic acid; 6-OHDA (6-hydroxydopamine); Memory; Substantia nigra; Ventral tegmental area;

Comparative effects of dexamethasone and l-canavanine in experimental septic shock by Arnaud Mansart; Pierre-Edouard Bollaert; Bruno Levy; Marie-Bénédicte Nicolas; Jean-Pierre Mallié (61-67).
Glucocorticoids can reverse hemodynamic disturbances and dependence on catecholamines in septic shock. The relevant beneficial mechanisms of steroids in septic shock are unknown, although inducible nitric oxide synthase could account for them. The aim of this study was to compare the effects of dexamethasone, a glucocorticoid and l-canavanine, a selective inhibitor of inducible nitric oxide synthase, in a rodent model of sepsis. Mean arterial pressure was restored by dexamethasone and l-canavanine administration at 24 h, no longer at 30 h. Dexamethasone but not l-canavanine improved aortic blood flow at 24 and 30 h. Although both dexamethasone and l-canavanine administration significantly reduced nitrite/nitrate production, and improved survival, steroids did better for survival. In conclusion, dexamethasone and l-canavanine displayed similar vasopressor effects. In addition, steroids improved blood flow suggesting that steroid-induced hemodynamic improvement in sepsis is not solely due to inhibition of inducible nitric oxide synthase.
Keywords: Sepsis; Steroid; Vasopressor; Hemodynamic; Nitric oxide (NO);

Effects of BIBN4096BS on cardiac output distribution and on CGRP-induced carotid haemodynamic responses in the pig by Kapil Kapoor; Udayasankar Arulmani; Jan P.C Heiligers; Edwin W Willems; Henri Doods; Carlos M Villalón; Pramod R Saxena (69-77).
Calcitonin gene related peptide (CGRP) seems to be involved in the pathogenesis of migraine, since plasma CGRP levels increase during the headache phase. In the present study, we investigated the effects of a novel CGRP receptor antagonist, BIBN4096BS (1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl] amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [R-(R*,S*)]-), on the regional cardiac output distribution and on the carotid haemodynamic changes induced by α-CGRP in anaesthetised pigs. Treatment with BIBN4096BS (100, 300 and 1000 μg kg−1, i.v.) did not affect the heart rate, mean arterial blood pressure or systemic vascular conductance, but a small decrease in cardiac output was noticed; the latter was, however, not significantly different from that in vehicle-treated animals. The highest dose of BIBN4096BS moderately decreased vascular conductance in the lungs, kidneys, spleen and adrenals. Vascular conductance in other tissues including the brain, heart, gastrointestinal system, skin and skeletal muscles remained unchanged. Intracarotid artery infusions of α-CGRP (10, 30 and 100 pmol kg−1 min−1 during 3 min) increased the total carotid blood flow and conductance, but decreased the arterial blood pressure. These responses were dose-dependently blocked by BIBN4096BS. The above results show that BIBN4096BS is a CGRP receptor antagonist in the porcine carotid and systemic circulations, but the endogenous CGRP does not seem to play an important physiological role in regulating basal vascular tone. These findings suggest that BIBN4096BS may have therapeutic usefulness in migraine.
Keywords: Arteriovenous anastomosis; BIBN4096BS; Carotid vasodilatation; Cardiac output; CGRP (calcitonin gene-related peptide); α-CGRP human; Migraine; (Pig);

Antagonist pharmacology of adenosine A2B receptors from rat, guinea pig and dog by John R Fozard; Francois Baur; Cedric Wolber (79-84).
We have sought evidence for species differences between adenosine A2B receptors by comparing the potencies of eight adenosine receptor antagonists, representing four different chemical classes, at the native adenosine A2B receptors which mediate relaxation of smooth muscle from rat colon, guinea pig aorta and dog saphenous vein. In all three assays, the antagonists caused parallel rightward shifts in the concentration–response curves to NECA and there was no depression of the maximum responses. There were highly significant correlations between the pK B values on each of the three receptors. However, the pK B values of 8-SPT (8-p-(sulphophenyl)theophylline), XAC (8-[-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), CGS 15943 (9-chloro-2,2-(furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine) and CGH 2473 N-[4-(3,4-dichloro-phenyl)-5-pyridin-4-yl-thiazol-2-yl]-acetamide) for the dog receptor exceeded by at least 0.5 log units the pK B values at the rat and guinea pig sites. Our data indicate species differences between the rat and guinea pig adenosine A2B receptors on the one hand and the dog adenosine A2B receptor on the other with respect to antagonist pharmacology.
Keywords: Adenosine A2B receptor; Species difference; Adenosine receptor antagonist; Colon; Aorta; Saphenous vein;

Major role of organic anion transporters in the uptake of phenolsulfonphthalein in the kidney by Shirou Itagaki; Mitsuru Sugawara; Michiya Kobayashi; Sachiho Nishimura; Michio Fujimoto; Katsumi Miyazaki; Ken Iseki (85-92).
Phenolsulfonphthalein is used for testing renal function. However, its excretion mechanism has not been elucidated. The purpose of this study was therefore to elucidate the transporter-mediated excretion system for phenolsulfonphthalein. p-Aminohippuric acid, a substrate of rat organic anion transporter1 (rOat1), and cimetidine, a substrate of rOat3, reduced the urinary excretion of phenolsulfonphthalein. The uptake of phenolsulfonphthalein by kidney slices was found to consist of two components. The IC50 values of rOat1 substrates were higher than those of rOat3 substrates. In the presence of cimetidine, the Eadie–Hofstee plot gave a single straight line. The profile of the phenolsulfonphthalein uptake component in the presence of cimetidine was similar to that of the low-affinity component in the absence of cimetidine. We conclude that rOat1 and rOat3 are involved in the renal uptake of phenolsulfonphthalein and that phenolsulfonphthalein is a high-affinity substrate for rOat3 but is a relatively low-affinity substrate for rOat1.
Keywords: Basolateral membrane; Organic anion transporter 1; Organic anion transporter 3; Phenolsulfonphthalein; Renal uptake; Urinary excretion;

The antibiotic erythromycin has been shown to modulate a variety of electrophysiological and mechanical responses in many cell types. We investigated whether it did so in airway smooth muscle using standard patch clamp, fura-2 fluorimetric and organ bath techniques. Erythromycin (10−4 M) evoked a small transient inward current with reversal potential and time-course similar to that of the Ca2+-dependent Cl currents seen in these cells. Unlike its effects in other cell types, however, it did not alter basal [Ca2+]i, voltage-dependent Ca2+ currents, nor mechanical tone at rest, nor the corresponding responses to cholinergic stimulation (membrane currents; release of internally sequestered Ca2+, nor contractions evoked by neural stimulation or exogenously added cholinergic agonist). In conclusion, erythromycin does exert interesting electrophysiological actions in airway smooth muscle, but does not alter mechanical activity as it has been shown to do elsewhere.
Keywords: Airway smooth muscle contraction; Airway hyperresponsiveness; Erythromycin; Macrolide antibiotic; Ca2+-dependent Cl current;

Protein kinase Cβ inhibition and aorta and corpus cavernosum function in streptozotocin-diabetic mice by Matthew R. Nangle; Mary A. Cotter; Norman E. Cameron (99-106).
Increased activity of the β-isoform of protein kinase C (PKC) has been linked to the vascular and neural complications of diabetes mellitus. Treatment with the PKCβ inhibitor, (s)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione, (LY333531), improves somatic nerve function and blood flow in diabetic rats. The aim was to assess whether LY333531 treatment could prevent nitric oxide-dependent autonomic nerve and vascular dysfunction in a diabetic mouse model. Diabetes was induced by streptozotocin; duration was 4 weeks. Aorta and corpus cavernosum were isolated and mounted in organ baths and agonist or electrical stimulation-evoked nerve-mediated tension responses were examined. Maximum nitric oxide-mediated endothelium-dependent relaxation of phenylephrine-precontracted aorta and cavernosum to acetylcholine were more than 30% reduced by diabetes. LY333531 treatment (10 mg kg−1 day−1) completely prevented the diabetic deficit in cavernosum, and 75% prevented the deficit in aorta. Maximum nitric oxide-dependent non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation of phenylephrine-precontracted cavernosum was approximately 43% reduced by diabetes; LY333531 attenuated the deficit by 44%. For diabetic aorta, but not cavernosum, sensitivity (EC50) to phenylephrine-mediated contraction was increased by approximately 0.85 log10 M units; LY333531 treatment completely prevented this effect. Thus, PKCβ activation contributes to nitric oxide-dependent vascular and autonomic nerve dysfunction in diabetic mice and could prove suitable for further study in clinical trials of diabetic autonomic neuropathy and vasculopathy.
Keywords: Protein kinase C; Diabetes mellitus; Endothelium; Smooth muscle; NANC (non-adrenergic, non-cholinergic) nerve; Nitric oxide (NO); Aorta; Corpus cavernosum;

Capsaicin-like effects of N-arachidonoyl-dopamine in the isolated guinea pig bronchi and urinary bladder by Selena Harrison; Luciano De Petrocellis; Marcello Trevisani; Francesca Benvenuti; Maurizio Bifulco; Pierangelo Geppetti; Vincenzo Di Marzo (107-114).
A capsaicin-like endogenous ligand of vanilloid (VR1) receptors, N-arachidonoyl-dopamine, was recently identified in bovine and rat nervous tissue, and found to be almost as potent as capsaicin, and 5–10-fold more potent than anandamide, on these receptors, both in isolated cells and in vivo. Here we have investigated if N-arachidonoyl-dopamine also exerts other capsaicin-like effects at VR1 receptors in some isolated organ preparations. N-arachidonoyl-dopamine exerted a potent contractile response of guinea pig isolated bronchi (EC50=12.6±1.7 μM, E max=69.2±2.4% of carbachol E max), which was blocked by pre-treatment with capsaicin or with the VR1 antagonist capsazepine, as well as by a combination of tachykinin NK1 and NK2 receptor antagonists. In this assay, N-arachidonoyl-dopamine was less and more potent and/or efficacious than capsaicin (EC50=40.0 nM; E max=93.5%) and anandamide (EC50=15.2 μM, E max=38.0%), respectively. Unlike capsaicin and anandamide, forskolin or ethanol did not enhance N-arachidonoyl-dopamine effect in this preparation, whereas epithelial denudation resulted in a 2.5-fold increase in potency without affecting the efficacy. N-arachidonoyl-dopamine also contracted the isolated guinea pig urinary bladder, although in this preparation, as well as in the isolated rat urinary bladder, the potency (EC50=3.7±0.3 and 19.9±0.1 μM) and/or efficacy (E max=12.0±0.1% and 20.7±0.7% of carbachol E max) of the compound were significantly lower than those of both capsaicin and anandamide. These data suggest that the extent to which exogenous N-arachidonoyl-dopamine activates VR1 receptor in isolated organs is largely dependent on pharmacodynamics and bioavailability.
Keywords: Vanilloid VR1 receptor; Cannabinoid CB1 receptor; Anandamide; Vanilloid; Cannabinoid;

Effects of cannabinoids on non-adrenergic non-cholinergic-mediated relaxation in guinea-pig trachea by Paola Nieri; Enrica Martinotti; Lara Testai; Cinzia Martinelli; Maria Cristina Breschi (115-118).
The effects of cannabinoid receptor agonists on the non-adrenergic non-cholinergic (NANC) inhibitory responses to electrical field stimulation in guinea-pig trachea were assessed. R-(+)-[2,3-dihydro-5-methyl-3-[(morpholilinyl) methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2; 10−5 M) significantly enhanced the frequency-dependent response to electrical stimulation. The same concentration of R-(N)-(2-hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R(+)methanandamide) and 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015) did not affect significantly the electrically induced inhibitory NANC responses. The effect of WIN 55,212-2 was not modified by the cannabinoid CB1 and CB2 receptor-selective antagonists, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10−5 M) and N-(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528; 10−5 M), respectively. Moreover, the nitric oxide synthase inhibitor, l-N G-nitro-arginine methyl ester (l-NAME; 10−4 M), but not the peptidase, α-chymotrypsin (2 U/ml), blocked the effect of WIN 55,212-2. Postsynaptically, WIN 55,212-2 did not produce any change of tracheal smooth muscle tone, either basal or histamine-induced, and did not interfere with the relaxant activity of the nitric oxide donor, sodium nitroprusside (10−8–10−4 M). In conclusion, our results suggest that (a) cannabinoid CB1 and CB2 receptor stimulation does not alter the inhibitory NANC transmission in guinea-pig trachea, and (b) WIN 55,212-2 potentiates the NO-mediated component of the NANC relaxant response to electrical stimulation through a cannabinoid receptor-independent mechanism.
Keywords: NANC (Non-adrenergic non-cholinergic) inhibitory; Cannabinoid; WIN 55,212-2; Trachea; (Guinea-pig); Nitric oxide (NO);

Comprehensive evaluation of canine renal papillary necrosis induced by nefiracetam, a neurotransmission enhancer by Yoshimi Tsuchiya; Yuji Takahashi; Toshimasa Jindo; Kazuhisa Furuhama; Kazuo T. Suzuki (119-128).
The effects of nefiracetam, a neurotransmission enhancer, on renal biochemistry and morphology with toxicokinetic disposition were investigated in both in vivo and in vitro systems. In the in vivo studies with rats, dogs, and monkeys, only the dog exhibited renal papillary necrosis. Namely, when beagle dogs were orally administered with 300 mg/kg/day of nefiracetam over 11 weeks, decreased urinary osmotic pressure was noted from week 5, followed by increases in urine volume and urinary lactate dehydrogenase from week 8. The first morphological change was necrosis of ductal epithelia in the papilla in week 8. In toxicokinetics after 3 weeks of repeated oral administration to dogs, nefiracetam showed somewhat high concentrations in serum and the renal papilla as compared with rats and monkeys. As for metabolites, although metabolite-18 (M-18) concentration in the renal papilla of dogs was between that in rats and monkeys, the concentration ratios of M-18 in the papilla to cortex and papilla to medulla were remarkably high. In the in vitro studies, while nefiracetam itself showed no effects on the synthesis of prostaglandin E2 and 6-keto-prostaglandin F, a stable metabolite of prostaglandin I2, in canine renal papillary slices, only M-18 among the metabolites clearly decreased both prostaglandin syntheses. The basal prostaglandin synthesis in canine renal papillary slices was extremely low relative to those in rats and monkeys. Taken together, certain factors such as basal prostaglandin synthesis, M-18 penetration into the renal papilla leading to an intrarenal gradient, and inhibitory potential of M-18 on prostaglandin synthesis were considered to be crucial for the occurrence of renal papillary necrosis in dogs.
Keywords: Nefiracetam; Metabolite; Renal papillary necrosis; Prostaglandin; Biomarker;

Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice by Jadranka Eric; Ghassan Bkaily; Ghassan B. Bkaily; Leonid Volkov; Bichoy H. Gabra; Pierre Sirois (129-137).
The effects of the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin and the bradykinin B2 receptor agonist, bradykinin were studied on the intracellular free Ca2+ concentration ([Ca2+]i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca2+]i in response to bradykinin (1 μM) but not to des-Arg9-bradykinin (1 μM), indicating the presence of functional bradykinin B2 receptors and the absence of B1 receptors. Such elevation in [Ca2+]i induced by bradykinin was totally inhibited by the selective bradykinin B2 receptor antagonist, d-Arg0-Hyp3-Thi5-d-Tic7-Oic8-bradykinin (HOE-140; 10 μM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg9-bradykinin, indicating the presence of both functional bradykinin B1 and B2 receptors. Eosinophils exhibited higher response to des-Arg9-bradykinin (1 μM; 485% increase in [Ca2+]i) compared to bradykinin (1 μM; 163% increase in [Ca2+]i). This des-Arg9-bradykinin-induced [Ca2+]i increase was markedly inhibited by the selective bradykinin B1 receptor antagonist, Ac-Lys-[d-βNal7, Ile8]des-Arg9-bradykinin (R-715; 10 μM). Des-Arg9-bradykinin neither modified the basal [Ca2+]i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca2+]i increase in both cell types. Our results further support the implication of the inducible bradykinin B1 receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.
Keywords: Bradykinin receptor; Bradykinin; Des-Arg9-bradykinin; HOE-140; R-715; Ovalbumin sensitization; Bronchoalveolar lavage; Eosinophil; Ca2+ intracellular; Fura-2;

Dilazep and fenofibric acid inhibit MCP-1 mRNA expression in glycoxidized LDL-stimulated human endothelial cells by Kazuo Sonoki; Masanori Iwase; Kenzo Iino; Kojiro Ichikawa; Mototaka Yoshinari; Shigehiro Ohdo; Shun Higuchi; Mitsuo Iida (139-147).
We previously reported that glycoxidized low-density lipoprotein (glycoxidized LDL) enhanced monocyte chemoattractant protein-1 (MCP-1) mRNA expression through activation of nuclear factor-kappaB (NF-κB). Here we investigated the effects of dilazep, an anti-platelet agent, and fenofibric acid, an active metabolite of fenofibrate, on glycoxidized low-density lipoprotein-(LDL)-enhanced MCP-1 mRNA expression. Both 10 μg/ml dilazep and 100 μM fenofibric acid abrogated MCP-1 mRNA expression. ZM241385, an A2a adenosine receptor antagonist, partially inhibited the suppressive effect of dilazep. NF-κB activity was also suppressed by 1 μg/ml dilazep and 10 μM fenofibric acid. The antioxidative activity of these drugs on glycation to native LDL or oxidation to glycated LDL was measured using lipid peroxidation and lyso-phosphatidylcholine contents in LDL. Dilazep but not fenofibric acid exhibited antioxidative activity. Although the mechanisms of anti-atherogenic effects of the two drugs on glycoxidized LDL are different, both dilazep and fenofibric acid could potentially prevent atherosclerosis in diabetes mellitus.
Keywords: Diabetes mellitus; Atherosclerosis; LDL (low-density lipoprotein); MCP-1 (monocyte chemoattractant protein-1); NF-κB (nuclear factor-κB); Umbilical vein endothelial cell;

Immunobiological activity of N-[2-(phosphonomethoxy)alkyl] derivatives of N 6-substituted adenines, and 2,6-diaminopurines by Zdeňek Zı́dek; Petr Potměšil; Eva Kmonı́èková; Antonı́n Holý (149-159).
Acyclic nucleoside phosphonates are novel class of virostatics effective against replication of both DNA-viruses and retroviruses. We found recently, that in addition to the antimetabolic mode of action, some acyclic nucleoside phosphonates such as 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir], which is used in treatment of human immunodeficiency virus (HIV) infection, possess immunostimulatory and immunomodulatory activities known to interfere with replication of viruses. The present experiments analyzed immunobiological effects of more than 70 novel derivatives of acyclic nucleoside phosphonates. They comprise substitutions at the N 6-amino function of adenine (A) or 2,6-diaminopurine (DAP) by monoalkyl, dialkyl, cycloalkyl, alkenyl, alkynyl or substituted alkyl group, and at the N 9-side chain represented by (R)- or (S)-enantiomeric 9-[2-(phosphonomethoxy)ethyl] (PME) and 9-[2-(phosphonomethoxy)propyl] (PMP) moieties. Their biological effects were investigated in vitro using mouse resident peritoneal macrophages. A number of the compounds under scrutiny, mainly the N 6-cycloalkyl derivatives of 9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (PMEDAP) and (R)-enantiomeric 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPDAP] stimulate secretion of cytokines [tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10)] and chemokines [“regulated-upon-activation, normal T expressed and secreted” (RANTES), macrophage inflammatory protein-1α (MIP-1α)]. Moreover, they substantially augment production of nitric oxide (NO) triggered by interferon-γ. The effects are produced in a dose-dependent fashion. The most potent derivatives, i.e. N 6-isobutyl-PMEDAP, N 6-cyclopentyl-PMEDAP, N 6-cyclooctyl-PMEDAP, N 6-dimethylaminoethyl-(R)-PMPDAP, N 6-cyclopropyl-(R)-PMPDAP, and N 6-cyclopentyl-(R)-PMPDAP are more effective than (R)-PMPA (tenofovir) itself. They exhibit immunostimulatory effects at concentrations as low as 1 to 5 μM. It is suggested that these compounds might be prospective candidates for antiviral therapeutic exploitation.
Keywords: Acyclic nucleoside phosphonate; Antiviral; Chemokine; Cytokine; Nitric oxide (NO);

Vasodilating effect of norethisterone and its 5α metabolites: a novel nongenomic action by Mercedes Perusquı́a; Carlos M Villalón; Erika Navarrete; Gustavo A Garcı́a; Gregorio Pérez-Palacios; Ana E Lemus (161-169).
Estrogens are generally administered in hormone replacement therapy in combination with synthetic progestins. Studies of cardiovascular risk factors in postmenopausal women have shown a variety of responses according to the molecular structure of the progestin used in hormone replacement therapy schemes. The present study sets out to determine the vasoactive effects of norethisterone and its 5α-dihydro (5α-norethisterone) and -tetrahydro (3α,5α-norethisterone and 3β,5α-norethisterone) metabolites in isolated precontracted rat thoracic aorta. The addition of norethisterone and 3α,5α-norethisterone in rat aorta exhibited a potent, concentration–response inhibition of noradrenaline-induced contraction, while 5α- and 3β,5α-norethisterone had very little, if any, vasorelaxing effect. Relaxation to norethisterone and 3α,5α-norethisterone had very rapid time-courses and it was neither affected by the absence of endothelium nor by the inhibitor of nitric oxide synthase, N ϖ-nitro-l-arginine methyl ester (l-NAME). The addition of specific anti-androgen, anti-progestin and anti-estrogen compounds and protein synthesis inhibitors did not preclude the vasorelaxing effect of norethisterone and its 3α,5α-reduced metabolite. The results strongly suggest that these effects are not mediated by nuclear sex steroid hormone receptors. The overall data document a novel nongenomic endothelium-independent vasorelaxing action of a 19-nor synthetic progestin and one of its A-ring-reduced derivatives.
Keywords: 19-Nor-steroid; Norethisterone; Progestin; Vasorelaxation; Hormone replacement therapy;

Author index (171-172).

Keyword index (173-176).