European Journal of Pharmacology (v.419, #2-3)

Characterisation of agonist binding on human 5-HT2C receptor isoforms by Kathleen Quirk; Anthony Lawrence; Julie Jones; Anil Misra; Victoria Harvey; Helen Lamb; Dean Revell; Richard H.P. Porter; Antony R. Knight (107-112).
The 5-HT2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [3H]5-HT. Displacement studies were undertaken using [3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 ((S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-(m-chlorophenyl)-piperazine), TfMPP (N-(m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di-N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT2C receptor is pharmacologically similar to the VSV form of the 5-HT2C receptor but that it couples more efficiently to G-proteins.
Keywords: 5-HT (5-hydroxytryptamine, serotonin); 5-HT2C receptor INI; 5-HT2C receptor VSV; Isoform; G-protein;

Characterization of organic anion transport inhibitors using cells stably expressing human organic anion transporters by Michio Takeda; Shinichi Narikawa; Makoto Hosoyamada; Seok Ho Cha; Takashi Sekine; Hitoshi Endou (113-120).
The organic anion transport system is involved in the tubular excretion of various clinically important drugs. The purpose of this study was to characterize the effects of various organic anion transport inhibitors on organic anion transport using proximal tubule cells stably expressing human organic anion transporter 1 (human-OAT1) and human-OAT3, which are localized to the basolateral membrane of the proximal tubule. Organic anion transport inhibitors including betamipron, cilastatin, KW-3902 (8-(noradamantan-3-yl)-1,3-dipropylxanthine) and probenecid significantly inhibited human-OAT1- and human-OAT3-mediated organic anion uptake in a dose-dependent manner. Kinetic analyses revealed that these inhibitions were competitive. The K i values of betamipron, cilastatin, KW-3902 and probencid for human-OAT1 were 23.6, 1470, 7.82 and 12.1 μM, whereas those for human-OAT3 were 48.3, 231, 3.70 and 9.0 μM. These results suggest that betamipron and probenecid could inhibit both human-OAT1- and human-OAT3-mediated organic anion transport in vivo, whereas cilastatin could inhibit only human-OAT3-mediated one. In contrast, KW-3902 did not exert the effects of significance, whereas KW-3902 was the most potent.
Keywords: Organic anion transporter; Proximal tubule; Betamipron; Cilastatin; KW-3902; Probenecid;

FK506 inhibits Cl secretion in airway epithelium via calcineurin-independent mechanism by Soichiro Kanoh; Mitsuko Kondo; Jun Tamaoki; Hideo Kobayashi; Kazuo Motoyoshi; Atsushi Nagai (121-126).
FK506 (tacrolimus)-binding protein (FKBP) is associated with intracellular Ca2+ release channel and modulates its function. To elucidate the effect of FK506 on Ca2+ dynamics and Ca2+-mediated Cl secretion in airway epithelium, we studied intracellular Ca2+ ([Ca2+]i) concentration and Cl-dependent short-circuit current (I sc), in cultured bovine tracheal epithelial cells. Addition of ATP induced an increase in [Ca2+]i, and this response was dose dependently inhibited by FK506. Rapamycin, which binds FKBP with high affinity, likewise inhibited the [Ca2+]i rise, but cyclosporin A, a specific calcineurin inhibitor, did not. In Cl secretion studies using Ussing chamber, ATP increased Ca2+-mediated I sc in amiloride-treated cells, an effect that was inhibited by FK506 and rapamycin but not by cyclosporin A. Therefore, FK506 inhibits Ca2+ mobilization in airway epithelium via FKBP but not calcineurin-dependent mechanism, which may result in the suppression of Ca2+-activated Cl secretion.
Keywords: Airway epithelium; Adenosine 5′-triphosphate; Ion transport; FK506-binding protein;

Effect of alisol B acetate, a plant triterpene, on apoptosis in vascular smooth muscle cells and lymphocytes by Huei-Wen Chen; Ming-Jen Hsu; Chiang-Ting Chien; Huei-Chen Huang (127-138).
Glucocorticoid-induced apoptosis is a well-recognized physiological regulator of T-cell number and function. Alisol B acetate, a triterpene from Alisma Plantago-aquatica, has a glucocorticoid-like structure, and may have a similar function like glucocorticoid-induced apoptosis in both vascular smooth muscle cell line (A7r5) and human acute lymphoblastic leukemia cell line (CEM cells). For exploring its mechanism, mitochondria membrane potential and apoptosis-related gene expression were discussed. Alisol B (10−6–10−4 M) inhibited serum-stimulated DNA synthesis in a concentration-dependent manner (IC50=4.0±0.8×10−6 M in A7r5 and 2.1±1.2×10−6 M in CEM cells). The cell viability was reduced at 10−4 M of alisol B. Similar results were seen in dexamethasone treatment (a synthetic glucocorticoid, 10−6 M, 48 h). Apoptosis was induced after the cells were exposed to 10−5–10−4 M alisol B or 10−6 M dexamethasone for 48 h. The mitochondrial membrane potential (ΔΨ m) was significantly reduced after the alisol B treatment, indicating that the mitochondria might play a role in the alisol B induced cell apoptosis. Alisol B (10−5–10−4 M) increased the levels of c-myc and bax mRNA and proteins, but not on the anti-apoptotic proto-oncogene, bcl-2, in A7r5 and CEM cells. In contrast, dexamethasone (10−6 M) treatment only caused significant increase in c-myc mRNA levels. These results suggest that the increased ratio of Bax/Bcl-2 and the decreased mitochondrial membrane potential might be involved in the mechanisms of alisol B-induced cell apoptosis.
Keywords: Alisol B acetate; Apoptosis; A7r5 vascular smooth muscle cell; CEM lymphocyte; Mitochondria membrane potential;

Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts by Arkadiusz Surażyński; Jerzy Pałka; Dorota Wrześniok; Ewa Buszman; Piotr Kaczmarczyk (139-145).
One of the recognized side effects of antineoplastic anthracyclines is poor wound healing, resulting from an impairment of collagen biosynthesis. The most affected tissue is skin. The mechanism underlying the tissue specificity of the side effects of anthracyclines has not been established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanins are abundant constituents of the skin, we determined whether daunorubicin interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. Results indicated that daunorubicin forms complexes with melanin. Scatchard analysis showed that the binding of daunorubicin to melanin was heterogeneous, suggesting the presence of two classes of independent binding sites with K 1=1.83×105 M−1 and K 2=5.52×103 M−1. The number of strong binding sites was calculated as n 1=0.158 μmol/mg of melanin and the number of weak binding sites as n 2=0.255 μmol/mg of melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracycline-induced inhibition of collagen synthesis. We found that daunorubicin induced inhibition of prolidase activity (IC50=10 μM), collagen biosynthesis (IC50=70 μM) and DNA biosynthesis (IC50=10 μM) in human skin fibroblasts. Melanin (100 μg/ml) by itself produced about 25% inhibition of DNA synthesis and prolidase activity but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to daunorubicin-treated cells (at IC50 concentration) augmented the inhibitory action of daunorubicin on collagen and DNA biosynthesis without having any effect on prolidase activity. The same effect was achieved when the cells were treated with daunorubicin at one-fourth of the IC50 given at 0, 6, 12 and 18 h during a 24-h incubation. The data suggest that the melanin-induced augmentation of the inhibitory effects of daunorubicin on collagen and DNA biosynthesis may result from: (i) accumulation of the drug in the extracellular matrix, (ii) gradual dissociation of the complex, and (iii) constant action of the released drug on cell metabolism. The phenomenon may explain the potential mechanism for the organ specificity of daunorubicin-induced poor wound healing in patients administered this drug.
Keywords: Melanin; Daunorubicin; Collagen; Prolidase; Fibroblast;

The neuroprotective activity of the glycine receptor antagonist GV150526: an in vivo study by magnetic resonance imaging by Angelo Reggiani; Claudio Pietra; Roberto Arban; Pasquina Marzola; Uliano Guerrini; Luigi Ziviani; Andrea Boicelli; Andrea Sbarbati; Francesco Osculati (147-153).
The neuroprotective activity of GV150526 (3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt), a selective glycine receptor antagonist of the NMDA receptor, has been evaluated by magnetic resonance imaging (MRI) in a rat model of middle cerebral artery occlusion. The aim of the work was to evaluate, using an in vivo method, whether GV150526 was able to reduce the extent of ischemic brain damage when administered both before and after (6 h) middle cerebral artery occlusion. GV150526 was administered at a dose of 3 mg/kg i.v. T2-weighted (T2W) and diffusion weighted (DW) images were acquired at 6, 24 and 144 h after the establishment of the cerebral ischemia. Substantial neuroprotection was demonstrated at all investigated time points when GV150526 was administered before the ischemic insult. The ischemic volume was reduced by 84% and 72%, compared to control values, when measured from T2W and DW images, acquired 24 h after middle cerebral artery occlusion. Administration of the same dose of GV150526, 6 h post-ischemia, also resulted in a significant (p<0.05) neuroprotection. The ischemic volume was reduced by 48% from control values when measured from T2W images and by 45% when measured from DW images. No significant difference was found between volumes of brain ischemia obtained by either MRI or triphenyltetrazolium chloride staining. These data confirm the potential neuroprotective activity of the glycine receptor antagonist GV150526 when administered either before or up to 6 h after ischemia.
Keywords: Stroke; MRI (magnetic resonance imaging); (Rat); GV150526; Focal ischemia; Neurodegeneration;

Δ9-tetrahydrocannabinol enhances cortical and hippocampal acetylcholine release in vivo: a microdialysis study by Elio Acquas; Augusta Pisanu; Paola Marrocu; Steven R Goldberg; Gaetano Di Chiara (155-161).
The intravenous administration of synthetic cannabinoid agonists was recently shown to dose dependently increase acetylcholine release from the rat prefrontal cortex and hippocampus (Eur. J. Pharmacol. 401 (2000) 179]. We report here that the active ingredient of cannabis preparations, Δ9-tetrahydrocannabinol, administered at 10, 37.5, 75 and 150 μg/kg, dose dependently stimulated acetylcholine release from rat prefrontal cortex and hippocampus estimated by means of in vivo brain microdialysis with vertical concentric probes. At these doses, Δ9-tetrahydrocannabinol induced behavioural stimulation. The administration of the CB1 receptor antagonist, ({N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3carboxamide}HCl) SR 141716A (200 μg/kg i.p.) significantly reduced the effect of Δ9-tetrahydrocannabinol (75 μg/kg i.v.) on acetylcholine release from rat prefrontal cortex and hippocampus.
Keywords: Acetylcholine; Hippocampus; Δ9-tetrahydrocannabinol; Microdialysis, in vivo; Cortex, prefrontal; SR 141716A;

Conformationally restricted analogs of BD1008 and an antisense oligodeoxynucleotide targeting σ1 receptors produce anti-cocaine effects in mice by Rae R Matsumoto; Kari A McCracken; Michele J Friedman; Buddy Pouw; Brian R De Costa; Wayne D Bowen (163-174).
Cocaine's ability to interact with σ receptors suggests that these proteins mediate some of its behavioral effects. Therefore, three novel σ receptor ligands with antagonist activity were evaluated in Swiss Webster mice: BD1018 (3S-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane), BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine), and LR132 (1R,2S-(+)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamine). Competition binding assays demonstrated that all three compounds have high affinities for σ1 receptors. The three compounds vary in their affinities for σ2 receptors and exhibit negligible affinities for dopamine, opioid, GABAA and NMDA receptors. In behavioral studies, pre-treatment of mice with BD1018, BD1063, or LR132 significantly attenuated cocaine-induced convulsions and lethality. Moreover, post-treatment with LR132 prevented cocaine-induced lethality in a significant proportion of animals. In contrast to the protection provided by the putative antagonists, the well-characterized σ receptor agonist di-o-tolylguanidine (DTG) and the novel σ receptor agonist BD1031 (3R-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane) each worsened the behavioral toxicity of cocaine. At doses where alone, they produced no significant effects on locomotion, BD1018, BD1063 and LR132 significantly attenuated the locomotor stimulatory effects of cocaine. To further validate the hypothesis that the anti-cocaine effects of the novel ligands involved antagonism of σ receptors, an antisense oligodeoxynucleotide against σ1 receptors was also shown to significantly attenuate the convulsive and locomotor stimulatory effects of cocaine. Together, the data suggests that functional antagonism of σ receptors is capable of attenuating a number of cocaine-induced behaviors.
Keywords: Antisense; Cocaine; Convulsion; Locomotor activity; Psychomotor activity; σ Receptor; Toxicity;

Antinociceptive action of amlodipine blocking N-type Ca2+ channels at the primary afferent neurons in mice by Manabu Murakami; Osamu Nakagawasai; Shigeo Fujii; Kimiko Kameyama; Shinobu Murakami; Soichi Hozumi; Akihisa Esashi; Ryoo Taniguchi; Teruyuki Yanagisawa; Koichi Tan-no; Takeshi Tadano; Kenji Kitamura; Kensuke Kisara (175-181).
We investigated the antinociceptive action of amlodipine, a dihydropyridine derivative, which acts on both L- and N-type voltage-dependent Ca2+ channels (VDCCs), in mice. Intrathecal injection of amlodipine (300 nmol/kg) significantly shortened the licking time in the late phase of a formalin test, while no effect was found with another dihydropyridine derivative, nicardipine (300 nmol/kg). Cilnidipine and ω-conotoxin GVIA also showed marked analgesic effects under the same experimental conditions. Transcripts of α1A, α1B, α1E , α1F, α1H, β3, and β4 subunits were detected by polymerase-chain reaction (PCR) in the dorsal root ganglion, suggesting the existence of a variety of voltage-dependent Ca2+ channels. Electrophysiological experiments showed that amlodipine and cilnidipine inhibit N-type currents in the dorsal root ganglion cells. These results suggest that amlodipine, cilnidipine, and ω-conotoxin GVIA exert their antinociceptive actions by blocking N-type Ca2+ channels in the primary nociceptive afferent fibers. Blocking of the Ca2+ channels results in attenuation of synaptic transmission of nociceptive neurons. Furthermore, it is suggested that some N-type Ca2+ channel blockers might have therapeutic potential as analgesics when applied directly into the subarachnoidal space.
Keywords: Ca2+ channel; Nociception; Amlodipine;

Role of brain arachidonic acid cascade on central CRF1 receptor-mediated activation of sympatho-adrenomedullary outflow in rats by Kunihiko Yokotani; Yoshinori Murakami; Shoshiro Okada; Masakazu Hirata (183-189).
The present experiments were designed to characterize the mechanisms involved in the corticotropin releasing factor (CRF)-induced activation of central sympatho-adrenomedullary outflow in rats. Intracerebroventricularly (i.c.v.) administered CRF and urocortin (0.5, 1.5 and 3.0 nmol/animal) effectively and dose-dependently elevated plasma levels of adrenaline and noradrenaline, and the effect of urocortin was almost the same as that of CRF. The elevation of catecholamines induced by CRF and urocortin (1.5 nmol/animal) was reduced by CP-154,526(butyl-ethyl-{2,5-dimethyl-7-(2,4,6trimethylphenyl)-7H-pyrrolo [2,3-d] pyrimidin-4-yl]amine), a selective CRF1 receptor antagonist, in a dose dependent manner (1.2 and/or 2.4 μmol/animal, i.c.v.), and abolished by indomethacin (1.2 μmol/animal, i.c.v.), an inhibitor of cyclooxygenase. Furegrelate (1.8 μmol/animal, i.c.v.), an inhibitor of thromboxane A2 synthase, abolished the CRF-induced elevation of adrenaline, but had no effect on the evoked release of noradrenaline. These results suggest that activation of brain CRF1 receptor facilitates the central sympathetic and adrenomedullary outflow in distinct central pathways in rats: brain thromboxane A2 is involved in the central adrenomedullary outflow; an active metabolite of arachidonic acid other than thromboxane A2 (probably prostaglandin E2) may be involved in the central sympathetic outflow.
Keywords: Catecholamine; Plasma; Corticotropin releasing factor (CRF); CRF1 receptor; Cyclooxygenase; Thromboxane A2; Brain; Sympatho-adrenomedullary outflow;

Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide by Antonio Calignano; Giovanna La Rana; Daniele Piomelli (191-198).
The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB2 receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide·HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB1 receptor antagonist SR141716A, not the cannabinoid CB2 receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.
Keywords: Cannabinoid; Anandamide; Palmitylethanolamide; Pain; Inflammation;

Effects of repeated GBR 12909 administration on brain stimulation reward by Susan M Melnick; Carmen S Maldonado-Vlaar; James R Stellar; Monika Trzcińska (199-205).
Male rats were trained at three separate currents to bar press for intracranial self-stimulation. On days 1 and 15, all subjects were given 1-(2-bis(4-fluorophenyl)-methoxy)-ethyl-4-(3-phenylpropyl) piperazine, also known as GBR 12909 (10 mg/kg, i.p.), prior to test session. Between these days, the paired Chronic-before group was injected (every other day) with GBR 12909 prior to intracranial self-stimulation, while unpaired, Chronic-after group was given the drug just after the end of the session. A third group (Control) received saline injections (i.p.) 20 min following the session. Although GBR 12909 was found to be reward enhancing, neither sensitization nor tolerance developed to the rewarding and performance/motor effects regardless of the injection regimen. In addition, the rewarding effects of intracranial self-stimulation were found to be independent of both current and environment-specific pairing. The present data obtained for GBR 12909 agree with previous observations of the effects of repeated administration of drugs of abuse on intracranial self-stimulation.
Keywords: Conditioning; GBR 12909; Reward; Self-stimulation;

Critical role of the endogenous cannabinoid system in mouse pup suckling and growth by Ester Fride; Yoav Ginzburg; Aviva Breuer; Tiziana Bisogno; Vincenzo Di Marzo; Raphael Mechoulam (207-214).
Δ9-Tetrahydrocannabinol, the active principle in marijuana, is a cannabinoid receptor agonist. Both the crude drug and Δ9-tetrahydrocannabinol have been used as appetite promoters. The endogenous cannabinoid, arachidonoyl ethanolamide (anandamide), likewise a cannabinoid receptor agonist, has been shown to have the same effect. In contrast, the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide (SR141716A) reduces food intake. Here, we report that administration of SR141716A to newly born mouse pups (either a single administration on postnatal day 1, or daily for a week as of postnatal day 2) had a devastating effect on milk ingestion and growth. The first 24 h after birth appeared the most critical for the growth stunting effect of SR141716A. Death followed within 4–8 days. Co-administration of Δ9-tetrahydrocannabinol almost fully reversed the effect of the antagonist in the week-long regimen. Co-administration of 2-arachidonoyl glycerol, an endocannabinoid, with 2-palmitoyl glycerol and 2-linoleoyl glycerol, which enhance 2-arachidonoyl glycerol potency, resulted in a significant delay in mortality rates caused by the antagonist. We conclude that the endocannabinoid system plays a vital role in milk suckling, and hence in growth and development during the early stages of mouse life.
Keywords: Tetrahydrocannabinol; 2-Arachidonoyl glycerol; SR141716A; Cannabinoid CB1 receptor; Milk; Anandamide;

Functional and biochemical evidence for capsaicin-induced neural endothelin release in isolated working rat heart by Janos Szolcsanyi; Gabor Oroszi; Jozsef Nemeth; Zoltan Szilvassy; Ingolf E Blasig; Arpad Tosaki (215-221).
In isolated working rat heart, capsaicin elicited a concentration-dependent constriction of coronary arteries accompanied by decline of all cardiac parameters recorded (heart rate, coronary and aortic flow, left ventricular developed pressure, and first derivative of left ventricular developed pressure). The following evidence suggests that capsaicin-induced changes are mediated by endothelin of neural origin: (1) the capsaicin (10 nM)-evoked decrease in coronary flow resulting in deterioration of cardiac functions was mimicked by endothelin (0.1 nM); (2) the selective endothelin ETA receptor antagonist, cyclo (d-α-aspartyl-l-propyl-d-valyl-l-leucyl-d-tryptophyl) (1 μM), abolished the cardiac effects provoked by capsaicin (10 nM); (3) reduction of extracellular Ca2+ concentration from 2.4 to 1.2 or 0.6 mM inhibited the cardiac effects of capsaicin (10 nM) but not those induced by endothelin (0.1 nM); (4) perfusion of the heart with 0.1% (v/v) Triton X-100 damaged the endothelium and reversed the enhancement of coronary flow evoked by bethanechol (1 μM), decreased the basal flow, but was without effect on capsaicin-induced coronary constriction; (5) in response to capsaicin challenge (10–100 nM), the endothelin concentration measured in coronary effluent by means of radioimmunoassay increased up to sevenfold but remained unchanged in the presence of 0.6 mM Ca2+; (6) no reduction of coronary flow was induced by capsaicin (100 nM) applied to the heart of rats which were desensitised by capsaicin (150 mg/kg). It is concluded that, in the rat heart, capsaicin acting on VR1 capsaicin receptors elicits a release of endothelin from the sensory nerve terminals.
Keywords: Capsaicin; Endothelin; BQ-123; Cardiac function; Heart; Sensory neuropeptide;

Electrically driven (1 Hz) ventricular trabeculae from explanted failing human myocardium were indirectly examined for the localization of the α1-adrenoceptor population and the β-adrenoceptor population in relation to sympathetic nerve endings. We examined the influence of neuronal uptake blockade by cocaine upon the horizontal position of the concentration–response curves for the inotropic effects exerted by noradrenaline in the presence and absence of appropriate adrenoceptor antagonists. Cocaine shifted the concentration–response curve for α1-adrenoceptor stimulation, but not that for β-adrenoceptor stimulation, to lower concentrations of noradrenaline in a parallel manner. The concentration–response curve for combined adrenoceptor stimulation was shifted by cocaine to lower concentrations of noradrenaline in a nonparallel manner. In explanted allograft heart, cocaine had no effect upon the position of the concentration–response curve to α1-adrenoceptor stimulation. The data indicate that in the explanted native hearts the α1-adrenoceptor population is located close to or within the synaptic cleft, while the β-adrenoceptor population remaining in the failing myocardium is located more distantly to the neuronal release sites.
Keywords: α1-Adrenoceptor; β-Adrenoceptor; Localization; Failing; Heart allograft (Human); Myocardium;

Adrenergic nerves mediate acetylcholine-induced endothelium-independent vasodilation in the rat mesenteric resistance artery by Hinako Shiraki; Hiromu Kawasaki; Satoko Tezuka; Akira Nakatsuma; Hideki Nawa; Hiroaki Araki; Yutaka Gomita; Yuji Kurosaki (231-242).
Mechanisms underlying acetylcholine-induced endothelium-independent vasodilation were studied in the rat mesenteric vascular bed isolated from Wistar rats. In preparations without endothelium, and contracted by perfusion with Krebs solution containing methoxamine (2–7 μM), perfusion of acetylcholine (1–100 μM) for 1 min produced a concentration-dependent vasodilation. Denervation of denuded preparations by cold storage (4°C for 72 h) abolished the acetylcholine-induced vasodilation; 10 and 100 nM atropine abolished 1 and 10 μM acetylcholine-induced vasodilation, but it inhibited only 20% of vasodilation by 100 μM acetylcholine. The acetylcholine-induced atropine-resistant vasodilation was inhibited by 10 and 100 μM hexamethonium, 5 μM guanethidine, 50 μM bretylium, in vitro 6-hydroxydopamine (2 mM for 20 min, twice), 1 μM capsaicin and 0.5 μM calcitonin gene-related peptide (CGRP)-(8-37) (CGRP receptor antagonist). These findings suggest that the acetylcholine-induced endothelium-independent nicotinic vasodilation requires the presence of intact adrenergic nerves, and is mediated by endogenous CGRP released from CGRP-containing nerves.
Keywords: Vasodilation; Vasodilation; Nicotinic acetylcholine receptor; Adrenergic nerves; CGRP-containing nerve;

Improved survival with simendan after experimental myocardial infarction in rats by Jouko Levijoki; Piero Pollesello; Petri Kaheinen; Heimo Haikala (243-248).
This study compared the effects of simendan, a calcium sensitizer, with those of milrinone and enalapril on survival of rats with healed myocardial infarction. Seven days after ligation-induced myocardial infarction, the rats were randomized to control, milrinone, enalapril, or simendan groups. All compounds were administered via the drinking water for 312 days, at which time there was 80% mortality in the control group—the study's primary endpoint. The infarct sizes were similar across all groups. At endpoint, the mortality rates were: 63% (milrinone), 56% (enalapril) and 53% (simendan); the risk reductions were 25% (P=0.04 vs. control) and 28% (P=0.02 vs. control) with enalapril and simendan, respectively. Milrinone had no statistically significant effect on the survival rate. These findings suggest that, like enalapril, simendan improved survival in rats with healed myocardial infarction.
Keywords: Ca2+ sensitizer; Simendan; Myocardial infarction; Mortality;

Pertussis toxin-sensitive and -insensitive mechanisms of α1-adrenoceptor-mediated inotropic responses in rat heart by Hitomi Otani; Akihiro Oshiro; Masahiro Yagi; Chiyoko Inagaki (249-252).
In rat left ventricular papillary muscle, phenylephrine, an α1-adrenoceptor agonist, induced a triphasic inotropic response; an initial transient, small, positive inotropic effect followed by a transient chloroethylclonidine-sensitive negative inotropic effect and a sustained 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB4101)-sensitive positive inotropic effect. Treatment with pertussis toxin for 2 days significantly inhibited only the transient negative inotropic effect without changing the sustained positive inotropic effect. This treatment also prevented the acetylcholine (1 μM)-induced negative inotropic effect. Further, phenylephrine-induced transient negative inotropic effect was attenuated in the presence of ouabain. These results suggest that pertussis toxin-sensitive or-insensitive G-protein may be responsible for α1-adrenoceptor subtype-mediated negative inotropic effect or positive inotropic effect, respectively, in which the transient negative inotropic effect was produced via the stimulation of Na+, K+ pump, presumably through pertussis toxin-sensitive G-protein-dependent pathway.
Keywords: α1-Adrenoceptor; Cardiac contractility; Pertussis toxin; Na+, K+ pump;

Effects of fenoterol on β-adrenoceptor and muscarinic M2 receptor function in bovine tracheal smooth muscle by Berber De Vries; Ad F Roffel; Jolanda M Kooistra; Herman Meurs; Johan Zaagsma (253-259).
Prolonged (18 h) incubation of isolated bovine tracheal smooth muscle with the β2-adrenoceptor agonist fenoterol (10 μM) induced desensitization of isoprenaline-induced adenylyl cyclase activity in bovine tracheal smooth muscle membranes, characterized by a 25% decrease in maximal effect (E max) (P<0.05), while the sensitivity to the agonist (pEC50) was unchanged. The E max value of isoprenaline-induced smooth muscle relaxation of submaximal methacholine-induced contractile tones was similarly reduced by about 25% (P<0.001), while the pEC50 value was diminished by 1.0 log unit (P<0.001). As determined by 30 μM gallamine-induced muscarinic M2 receptor antagonism and pertussis toxin-induced inactivation of G, muscarinic M2 receptor-mediated functional antagonism did not play a role in isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by methacholine, both in control and in 18-h fenoterol-treated tissue. In line with these observations, we found no enhanced muscarinic M2 receptor-mediated inhibition of 1 μM forskolin-stimulated adenylyl cyclase activity after 18-h fenoterol treatment. These data indicate that 18-h fenoterol treatment of bovine tracheal smooth muscle induces β2-adrenoceptor desensitization and reduced functional antagonism of methacholine-induced contraction by β-adrenoceptor agonists, without a change of muscarinic M2 receptor function.
Keywords: β2-Adrenoceptor; Muscarinic M2 receptor; Smooth muscle; Desensitization; Functional antagonism; Pertussis toxin;

Tryptase-induced airway microvascular leakage in guinea pigs: involvement of tachykinins and leukotrienes by Scott Greenfeder; Susan Sehring; Nansie McHugh; Michel Corboz; Maria Rivelli; John C Anthes; Motasim Billah; Robert W Egan; Richard W Chapman (261-267).
Tryptase, a serine protease synthesized by and stored in mast cells, is implicated as an important mediator in the pathogenesis of airway inflammation. In this study, tryptase was evaluated for its ability to induce microvascular leakage into the airways of guinea pigs. Dose- and time-dependent increases in airway microvascular leakage were produced by intratracheal tryptase (0.3–3 μg). Intratracheal tryptase (3–30 μg) had no effect on airway tone as measured by pulmonary insufflation pressure. Tryptase-induced airway microvascular leakage was partially blocked by the tachykinin NK1 receptor antagonist CP 99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] and an inhibitor of leukotriene formation SCH 37224 (1-(1,2-dihydro-4-hydroxy-2-oxo-1-phenyl-1,8-naphthyridin-2-yl)pyrrolidinium, hydroxide inner salt). Neither CP 99994 nor SCH 37224 inhibited tryptase proteolytic activity in-vitro. Pretreatment of guinea pigs with histamine H1 receptor antagonists or a tachykinin NK2 receptor antagonist had no affect on the airway microvascular leakage induced by tryptase. It is speculated that tryptase may be important in the pathogenesis of airway inflammation, particularly in disorders that involve increased airway microvascular leakage such as asthma.
Keywords: Microvascular leakage; Leukotriene; SCH 37224; Tachykinin; Tryptase;

We investigated the effects of rhein, an active metabolite of diacerein, on the interleukin-1α-stimulated production of nitric oxide (NO) in rabbit articular chondrocytes, and the effects of diacerein on NO production in rat adjuvant-induced arthritis. At doses of 10 and 30 μM, rhein significantly inhibited the interleukin-1α-stimulated NO production in chondrocytes. In the rat adjuvant-induced arthritis model, diacerein was administered for 21 days, starting at the time of adjuvant injection. Paw swelling and plasma NO level were measured in order to assess the effect of diacerein on arthritis and NO biosynthesis in the whole body. At doses of 30 and 100 mg/kg/day, diacerein significantly suppressed the development of adjuvant-induced arthritis and the increase in plasma NO. These results suggest that the inhibitory effect of diacerein on rat adjuvant-induced arthritis is partly related to its reduction of the NO production induced by adjuvant-induced arthritis.
Keywords: Diacerein; Rhein; Nitric oxide (NO); Chondrocyte; Adjuvant-induced arthritis;

Author index (275-277).

Keyword index (279-284).