European Journal of Pharmacology (v.459, #1)

5-Hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats by Doreen Awabdy; Lesley J. Bryan-Lluka; Janet C. Wanstall (1-7).
Pulmonary hypertension is associated with various alterations in 5-hydroxytryptamine (5-HT) physiology. In this study in platelets from hypoxic pulmonary hypertensive rats (10% O2; 1 week) and normoxic rats (room air), (i) initial rates of specific [3H]5-HT uptake were measured and (ii) potentiation of collagen- and ADP-induced aggregation by 5-HT was quantified. The platelet count was almost halved in hypoxic rats. In uptake experiments, there was a decrease in 5-HT uptake in platelets from hypoxic compared with normoxic rats, due to a 36% reduction in the maximal initial rate of uptake. The aggregation experiments showed that 5-HT (1–100 μM) increased the magnitude of responses to collagen and the duration of responses to ADP, but there was no difference between hypoxic and normoxic rats. Abnormalities in platelet function may conceivably lead to increases in plasma 5-HT levels in hypoxic pulmonary hypertension, but are unlikely to aggravate pulmonary thromboembolism.
Keywords: Platelet; 5-HT (5-hydroxytryptamine, serotonin); SERT (serotonin transporter); Hypoxic pulmonary hypertension; Aggregation;

Agonist-specific down-regulation of the human δ-opioid receptor by Takashi Okura; Eva V. Varga; Yoshiaki Hosohata; Edita Navratilova; Scott M. Cowell; Kenner Rice; Hiroshi Nagase; Victor J. Hruby; William R. Roeske; Henry I. Yamamura (9-16).
Down-regulation of the δ-opioid receptor contributes to the development of tolerance to δ-opioid receptor agonists. The involvement of the carboxy terminus of the mouse δ-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human δ-opioid receptor by structurally distinct δ-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human δ-opioid receptors were incubated with various δ-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [3H]naltrindole saturation binding. Each δ-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all δ-opioid receptor agonists, except SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [d-Pen2-d-Pen5]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human δ-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of Gi/o protein activation and subsequent downstream signaling.
Keywords: δ-Opioid receptor; Down-regulation; Agonist-directed trafficking; Phosphorylation; Pertussis toxin;

In vivo comparison of two 5-HT1A receptors agonists alnespirone (S-20499) and buspirone on locus coeruleus neuronal activity by Bernadette Astier; Laura Lambás Señas; Fabienne Soulière; Patricia Schmitt; Nadia Urbain; Nicolas Rentero; Lionel Bert; Luc Denoroy; Bernard Renaud; Monique Lesourd; Carmen Muñoz; Guy Chouvet (17-26).
The aim of the present study was to compare, in chloral-hydrate anaesthetized rats, the α2-adrenergic properties of the selective 5-HT1A receptor agonist, alnespirone (S-20499), with those of buspirone, a 5-HT1A receptor agonist exhibiting potent α2-adrenoceptor antagonist properties via its principal metabolite, 1-(2-pyrimidinyl)-piperazine. Both locus coeruleus spontaneous firing activity and noradrenaline release in the medial prefrontal cortex were potently inhibited by the α2-adrenoceptor agonist clonidine, at a dose of 40 μg/kg (i.p.). Such an inhibition was neither prevented nor reversed by alnespirone (10 mg/kg, i.p.), while buspirone, at the same dose, potently antagonized the locus coeruleus inhibitory effects of clonidine. These data demonstrate that, in contrast with some aryl-piperazine compounds (such as buspirone), alnespirone, either on its own or via a possible metabolite such as buspirone, is devoid in vivo of significant α2-adrenoceptor antagonist properties.
Keywords: Locus coeruleus; Extracellular unitary recording; Noradrenaline; Microdialysis; Alnespirone; Clonidine; α2-Adrenoceptor; 5-HT1A receptor;

Actions of cysteinyl leukotrienes in the enteric nervous system of guinea-pig stomach and small intestine by Sumei Liu; Hong-Zhen Hu; Chuanyun Gao; Na Gao; Guodu Wang; Xiyu Wang; Xiang Gao; Yun Xia; Jackie D. Wood (27-39).
Conventional intracellular microelectrodes, neuronal tracer injection techniques and immunohistochemistry were used to study the actions of cysteinyl leukotrienes (CysLTs) on electrical and synaptic behavior of enteric neurons in guinea-pig stomach and small intestine. Bath application of leukotriene C4, leukotriene D4 or leukotriene E4 evoked a slowly activating depolarizing response in most of the myenteric and submucous plexus neurons in the small intestine while no effect was observed in gastric neurons. The depolarization evoked by cysteinyl leukotrienes in intestinal neurons was associated with increased input resistance and enhanced excitability. Suppression of hyperpolarizing after-potentials occurred in AH type neurons. The depolarizing action of cysteinyl leukotrienes was resistant to tetrodotoxin and cyclooxygenase inhibitors. Neither the CysLT1 receptor antagonists (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl}-ethanone (LY 171883) and α-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV 5901), nor the dual CysLT1/CysLT2 receptor antagonist 6(R)-(4′-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BAY u9773) significantly altered the depolarizing action of the cysteinyl leukotrienes. Neurotransmission was unaffected by the cysteinyl leukotrienes. The results suggested involvement of cysteinyl leukotrienes in enteric immuno-neural communication through excitatory actions on enteric neurons. The receptor mediating these effects was distinct from currently recognized cysteinyl leukotriene receptor subtypes (CysLT1 and CysLT2 receptors) and may represent a new receptor subtype.
Keywords: Intestine; Stomach; Enteric nervous system; Immune system; Inflammation; Leukotriene; Myenteric plexus; Submucosal plexus;

The dermorphin-derived tetrapeptide Tyr-d-Arg2-Phe-Sar4 (TAPS) was tested for its ability to induce tolerance, cross-tolerance, withdrawal and its substitution properties in rats subjected to chronic intracerebroventricular (i.c.v.) infusions of μ-opiate receptor agonists. Tolerance and cross-tolerance were assessed by quantification of the thermally induced tail-flick response. Chronic intracerebroventricular infusion of TAPS resulted in antinociception at almost 1000-fold lower doses compared to morphine sulphate and [d-Ala2, MePhe4Gly(ol)5]enkephalin (DAMGO). Tolerance to the antinociceptive effect of TAPS developed similar to DAMGO and morphine sulphate. Cross-tolerance to intracerebroventricular bolus injections of DAMGO, but not of TAPS, was evident in rats rendered tolerant to morphine sulphate and TAPS. Naloxone-induced withdrawal was equally pronounced in animals treated with morphine sulphate, DAMGO or TAPS. TAPS substituted for morphine sulphate and vice versa regarding the withdrawal syndrome in a cross-over experimental design. In contrast to DAMGO, TAPS retains its antinociceptive effect following bolus administration in rats rendered tolerant to μ-opioid receptor agonists.
Keywords: Dermorphin; TAPS (Tyr-d-Arg2-Phe-Sar4); Tetrapeptide; Antinociception; Tolerance; Withdrawal;

Spinal cord dorsal horn N-methyl-d-aspartate (NMDA) receptors have been implicated in central sensitization, enhanced responsiveness to peripheral stimuli following peripheral injury. Since hyperalgesia is a behavioral consequence of central sensitization, it should be attenuated at the level of the dorsal horn with NMDA receptor antagonists. However, responsiveness to thermal and mechanical hyperalgesia may be distinct, and have thus far not been directly compared in chronic inflammatory pain models. In the present study, inflammation was induced with complete Freund's adjuvant (CFA) injected into the rat hind paw and NMDA receptor antagonists dizocilpine (MK-801) or 2-amino-5-phosphonovaleric acid (AP-5) were intrathecally injected in rats to determine the effects on both mechanical and thermal hyperalgesia. Locomotor tests and reflexes were also conducted to evaluate potential motor side effects. The NMDA receptor antagonists dose-dependently ameliorated mechanical hyperalgesia, but had marginal effects on thermal hyperalgesia. In ranges near antihyperalgesic doses, significant disruption of motor coordination was observed for both antagonists. These results suggest that, depending on the stimulus, NMDA receptors may have variable significance for central sensitization-mediated hyperalgesia, and that NMDA receptor antagonists may have therapeutic potential for some, but not all components in the clinical manifestation of inflammatory pain.
Keywords: Spinal cord; Sensitization; NMDA (N-methyl-d-aspartate); Pain;

Role of superoxide anion in pancreatic islet blood flow regulation in anesthetized rats by Annika M. Svensson; Stellan Sandler; Leif Jansson (59-64).
The aim of the investigation was to study the influence of the superoxide anion on pancreatic islet blood flow in rats. For this purpose, blood flow measurements were conducted with a microsphere technique 10 min after intravenous administration of different doses of superoxide dismutase (5, 15, 50, 100 or 1000 kU/kg body weight). In separate experiments, diethyldithiodicarbamate, an inhibitor of endogenous superoxide dismutase, was given to nontreated control rats or to rats subjected to a bilateral abdominal vagotomy before the injection. Only the highest dose of superoxide dismutase increased both whole pancreatic and islet blood flow. A 50% augmentation of fractional islet blood flow was seen. Administration of diethyldithiocarbamate induced marked hyperglycemia, which was partly prevented by vagotomy. Diethyldithiocarbamate decreased the whole pancreatic blood flow, while islet blood flow was maintained in both control and vagotomized rats. Consequently, a pronounced increase in fractional islet blood flow was noted in both these groups. We conclude that administration of superoxide dismutase and its inhibitor diethyldithiocarbamate influences pancreatic blood perfusion. In particular, superoxide dismutase causes a general increase in the whole pancreatic and islet blood flow, and an augmented fractional islet blood flow, presumably by a decrease in the local concentration of O2 •−, leading to increased concentration of NO. Diethyldithiocarbamate, on the other hand, by increasing the levels of O2 •−, decreases the whole pancreatic blood flow, whereas islet blood flow remains unaffected.
Keywords: Pancreatic islet; Superoxide; Superoxide dismutase; Blood flow; Nitric oxide (NO);

The mechanisms underlying the hydrogen peroxide-induced relaxation of the norepinephrine-contraction were studied by measuring isometric force, myosin light chain (MLC20) phosphorylation and cyclic GMP in endothelium-denuded muscle from the guinea-pig aorta. Norepinephrine (5.2±1.3 μM) produced a phasic, followed by a tonic contraction. Hydrogen peroxide (10 and 100 μM), glyceryl trinitrate (30 and 300 nM) and 8-bromo cyclic GMP (30 and 100 μM) did not change the basal tone, but reduced the norepinephrine-induced contraction. Phosphorylation of MLC20 (percentage of phosphorylated to total MLC20) was increased 1 min (5.9±1.0% vs. 35.9±4.9%) and, to a lesser extent, 20 min (3.7±1.7% vs. 13.9±1.6%) after the addition of norepinephrine. Hydrogen peroxide (100 μM) did not modify basal MLC20 phosphorylation, but reduced the increase in MLC20 phosphorylation induced by 1-min exposure to norepinephrine (20.9±4.1%). Its effect was abolished by catalase. When the tissue was incubated for 20 min with norepinephrine in the presence of hydrogen peroxide, norepinephrine-induced MLC20 phosphorylation was not changed (13.6±1.5%), as compared to that in the absence of hydrogen peroxide. Hydrogen peroxide relaxed norepinephrine-stimulated aortas in a concentration-dependent fashion with EC50 values of 5.9±0.2 μM. The relaxation was inhibited by soluble guanylate cyclase inhibitors and increased by an inhibitor of cyclic GMP-selective phosphodiesterase. In aorta precontracted with norepinephrine, hydrogen peroxide (100 μM) relaxed the tissue by 89±11% and almost doubled tissue concentrations of cyclic GMP, whereas sodium nitroprusside (1 μM) relaxed the tissue by 100% and increased cyclic GMP concentrations 30-fold. It is suggested that the inhibitory effects of hydrogen peroxide on the norepinephrine-induced phasic and sustained contractions are explained by a decrease in MLC20 phosphorylation and by an alteration in MLC20 phosphorylation-independent mechanisms, respectively. The effects of hydrogen peroxide were in part mediated by cyclic GMP.
Keywords: Aorta; cGMP; Hydrogen peroxide; Myosin light chain; Norepinephrine;

R(+)-methanandamide inhibits tracheal response to endogenously released acetylcholine via capsazepine-sensitive receptors by Paola Nieri; Enrica Martinotti; Lara Testai; Barbara Adinolfi; Vincenzo Calderone; Maria Cristina Breschi (75-81).
The effects of cannabinoid drugs on the cholinergic response evoked by electrical field stimulation (0.2 ms pulse width, 20 V amplitude, 10 Hz, 7.5 s train duration) in guinea-pig tracheal preparations were investigated. The stable analogue of the endocannabinoid anandamide, R(+)-methanandamide (10−7–10−4 M), produced a dose-dependent inhibition (up to 27±5% of control) of electrical field stimulation-mediated atropine-sensitive response. This effect was not blocked by the selective cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide hydrochloride (SR 141716A; 10−6 M), and was not reproduced with the cannabinoid CB1/CB2 receptor agonist R(+)-[2,3-dihydro-5-methyl-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate) (WIN 55,212-2; 10−8–10−5 M) or the cannabinoid CB2 receptor selective agonist 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015; 10−8–10−5 M); it was, on the contrary, antagonized by the vanilloid antagonist 2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-2 benzazepine (capsazepine; 10−6 M). At the postjunctional level, neither R(+)-methanandamide nor WIN 55,212-2 nor JWH-015 did affect tracheal contractions induced by exogenous acetylcholine (10−6 M). An inhibitory vanilloid receptor-mediated effect on the cholinergic response evoked by electrical stimulation was confirmed with the vanilloid agonist capsaicin, at doses (3–6×10−8 M) which poorly influenced the basal smooth muscle tone of trachea. In conclusion, our data indicate that in guinea-pig trachea (a) neither CB1 nor CB2 cannabinoid receptor-mediated modulation of acetylcholine release occurs; (b) vanilloid VR1-like receptors appear involved in R(+)-methanandamide inhibitory activity on the cholinergic response to electrical field stimulation.
Keywords: Cannabinoid; Vanilloid; Cholinergic; (Guinea-pig); Trachea;

Antiemetic and motor-depressive actions of CP55,940: cannabinoid CB1 receptor characterization, distribution, and G-protein activation by Nissar A. Darmani; Laura J. Sim-Selley; Billy R. Martin; Jano J. Janoyan; Jennifer L. Crim; Bavita Parekh; Christopher S. Breivogel (83-95).
Dibenzopyran (Δ9-tetrahydrocannabinol) and aminoalkylindole [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) methanone mesylate; (WIN55,212-2)] cannabinoids suppress vomiting produced by cisplatin via cannabinoid CB1 receptors. This study investigates the antiemetic potential of the “nonclassical” cannabinoid CP55,940 [1α,2β-(R)-5α]-(−)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol] against cisplatin-induced vomiting and assesses the presence and functionality of cannabinoid CB1 receptors in the least shrew (Cryptotis parva) brain. CP55,940 (0.025–0.3 mg/kg) reduced both the frequency of cisplatin-induced emesis (ID50=0.025 mg/kg) and the percentage of shrews vomiting (ID50=0.09 mg/kg). CP55,940 also suppressed shrew motor behaviors (ID50=0.06– 0.21 mg/kg) at such doses. The antiemetic and motor-suppressant actions of CP55,940 were countered by SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide], indicating both effects are cannabinoid CB1 receptor-mediated. Autoradiographic studies with [3H]-SR141716A and [35S]-GTPγS binding revealed that the distribution of the cannabinoid CB1 receptor and its activation pattern are similar to rodent brain and significant levels are present in brain loci (e.g., nucleus tractus solitarius (NTS)) that control emesis. The affinity rank order of structurally diverse cannabinoid ligands for cannabinoid CB1 receptor in shrew brain is similar to rodent brain: HU-210=CP55,940=SR141716A≥WIN55,212-2≥delta-9-tetrahydrocannabinol>methanandamide=HU-211=cannabidiol=2-arachidonoylglycerol. This affinity order is also similar and is highly correlated to the cannabinoid EC50 potency rank order for GTPγS stimulation except WIN55,212-2 and delta-9-tetrahydrocannabinol potency order were reversed. The affinity and the potency rank order of tested cannabinoids were significantly correlated with their antiemetic ID50 potency order against cisplatin-induced vomiting (CP55,940>WIN55,212-2=delta-9-tetrahydrocannabinol) as well as emesis produced by 2-arachidonoylglycerol or SR141716A (CP55,940>WIN55,212-2>delta-9-tetrahydrocannabinol).
Keywords: Vomiting; Locomotor activity; CP55,940; SR141716A; Cannabinoid CB1 receptor; (Least shrew); G-protein; Potency; Efficacy;

Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract by Maria Antonietta Casu; Anna Porcella; Stefania Ruiu; Pierluigi Saba; Giorgio Marchese; Mauro Antonio Maria Carai; Roberta Reali; Gian Luigi Gessa; Luca Pani (97-105).
Recently, the gastrointestinal pharmacology of cannabinoid CB1 receptors has been extensively explored. We employed western blotting and immunohistochemistry techniques to study the distribution of the cannabinoid CB1 receptor protein in the mouse gastroenteric tract. The cannabinoid CB1 receptor peptide was detected by western blotting only in its glycosylated form (63 kDa) with a significant differential distribution. The highest levels of expression were detected in the stomach and in the colon, while the pyloric valve was devoid of any cannabinoid CB1 receptor protein. The immunohistochemical study showed intense cannabinoid CB1 receptor immunoreactivity in ganglia subadjacent to the gastric epithelium and in the smooth muscle layers of both the small and large intestine. Only the small intestine showed (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxylpropyl) cyclohexan-1-ol) ([3H]CP 55,940) specific binding (27%). These receptors mediated pharmacologically significant effects since the cannabinoid CB1 receptor agonist R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU 210) dose dependently inhibited gastrointestinal transit up to 70%, while the cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716A) increased gastrointestinal transit. Moreover, the dose of 0.3 μg/kg of HU 210, devoid per se of any activity on mouse intestinal propulsion, blocked the increased gastroenteric transit induced by the cannabinoid CB1 antagonist SR 141716A.
Keywords: Marijuana; Gene expression; Drug abuse;

Inhibition of macrophage activation and lipopolysaccaride-induced death by seco-steroids purified from Physalis angulata L. by Milena B.P. Soares; Moema C. Bellintani; Ivone M. Ribeiro; Therezinha C.B. Tomassini; Ricardo Ribeiro dos Santos (107-112).
Physalis angulata L. is an annual herb widely used in popular medicine for the treatment of a variety of pathologies. Here, we tested immunomodulatory activities of physalins, seco-steroids purified from P. angulata extracts. Addition of physalins B, F or G, but not D, caused a reduction in nitric oxide production by macrophages stimulated with lipopolysaccaride and interferon-γ. In the presence of physalin B, macrophages stimulated with lipopolysaccaride, alone or in combination with interferon-γ, produced lower levels of tumour necrosis factor (TNF)-α, interleukin-6 and interleukin-12. The inhibitory activity of physalin B, unlike that of dexamethasone, was not reversed by RU486 [(4-dimethylamino) phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one], an antiglucocorticoid. Physalin B-treated mice had lower levels of serum TNF-α than control mice after lipopolysaccaride challenge. More importantly, mice injected with physalins B, F or G survived after a lethal lipopolysaccaride challenge. These results demonstrate that seco-steroids from P. angulata are potent immunomodulatory substances and act through a mechanism distinct from that of dexamethasone.
Keywords: Steroid; Immunomodulation; (Physalis angulata L.); Endotoxic shock;