European Journal of Pharmacology (v.439, #1-3)

The mechanisms underlying the pathogenesis of migraine and their possible association with serotonin (5-hydroxytryptamine; 5-HT) have not yet been elucidated. One of the major obstacles in achieving this goal is the lack of information on the mechanisms by which the monoamine could possibly trigger and/or modulate the basic pathophysiological features of the condition, that is, cranial vasodilatation and neurogenic inflammation. This information should provide a useful theoretical framework to insight the nature of the postulated fundamental triggering mechanism in the brain that ultimately results in head pain. Novel avenues for research and drug development may be envisaged upon the recent observations showing that 5-HT is actually able to produce vasodilatation of intra- and extra-cranial blood vessels through a mechanism pharmacologically resembling the 5-HT7 receptor type, and that the messenger RNA (mRNA) encoding for this receptor is highly expressed in cranial vessels. Other lines of evidence have suggested that the 5-HT7 receptor may play an excitatory role in neuronal systems and that it may be involved in hyperalgesic pain and neurogenic inflammation. On the basis of these observations, it is proposed that the 5-HT7 receptor may well represent a link between the abnormal phenomena of 5-HT processing and neurotransmission that are observed in migraine patients, and the vascular and neurogenic alterations that account for migraine headache. This view is supported by the fact that most of the migraine prophylactic 5-HT receptor antagonists display relatively high affinity for the 5-HT7 receptor, which significantly correlates with their pharmaceutically active oral doses.
Keywords: 5-HT7 receptor; Migraine pathogenesis; Prophylactic drug; 5-HT (5-hydroxytryptamine, serotonin);

Evidence for changes in the tachyphylactic property of recombinant angiotensin II AT1 receptor expressed in CHO cells by Lucimar P França; Silvana A.A Corrêa; Ana M.R.B Barbosa; Alice T Ferreira; Sang W Han; Suma I Shimuta; Antonio C.M Paiva (13-19).
The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT1 receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT1A receptor gene, as well as 56 bp of its 3′- and 52 bp of its 5′-untranslated regions. These cells (CHO-AT1) responded to angiotensin II by increases in intracellular Ca2+ concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys2]angiotensin II), this analogue induced responses in CHO-AT1 cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys2]angiotensin II after transfection with the angiotensin II AT1 receptor gene. Our findings suggest that posttranscriptional control directed by the 3′- or the 5′-untranslated regions in the angiotensin II AT1 receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis.
Keywords: Angiotensin II; [Lys2]angiotensin II; Angiotensin II AT1 receptor; Tachyphylaxis; CHO cell line; Smooth muscle cell line;

Increased cardiac workload often leads to serious complications during cardiac surgery such as pericardiopulmonary bypass. Various agents have been applied to lower peripheral resistance and cardiac workload, one of which, anisodamine, is widely used in Asia. However, the direct action of anisodamine on cardiac contractile property is essentially unknown. This study was designed to examine the influence of anisodamine on ventricular contractile function at the single cardiac myocyte level. Ventricular myocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca2+ properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), maximal velocity of shortening/relengthening (±dL/dt), intracellular Ca2+ fluorescence intensity change (ΔFFI) and decay (τ). Anisodamine exhibited a concentration-dependent (10−12–10−6 M) inhibition in PS and ΔFFI, with maximal inhibitions of 44.7% and 47.2%, respectively. Anisodamine inhibited ±dL/dt, lowered resting FFI but elicited no effect on TPS/TR90 and τ. Pretreatment with the nitric oxide synthase (NOS) inhibitor N ω-nitro-l-arginine methyl ester (l-NAME, 100 μM) abolished the inhibitory effect of anisodamine in cell shortening. In addition, anisodamine prevented cholinoceptor agonist carbachol-induced positive cardiac contractile response. This study demonstrated a direct cardiac depressive action of anisodamine at the myocyte level, which may be related to, at least in part, NO production and cholinoceptor antagonism.
Keywords: Anisodamine; Myocyte shortening; Ca2+ intracellular; Nitric oxide (NO); Cholinoceptor;

Activation of nitric oxide synthase through muscarinic receptors in rat parotid gland by Florencia Rosignoli; Claudia Pérez Leirós (27-33).
Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) activity was found coupled to muscarinic receptor activation as well as to nitric oxide-mediated amylase secretion elicited by carbachol. Parotid glands presented a predominant M3 and a minor muscarinic M1 acetylcholine receptor population, though carbachol stimulated NOS activity only through muscarinic M3 receptors as revealed in the presence of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and pirenzepine. Amylase secretion induced by carbachol appeared to be partly mediated by nitric oxide and nitric oxide-induced signaling since N-nitro-l-arginine methyl ester (l-NAME) inhibited the effect as well as did methylene blue. A negative regulation of NOS by protein kinase C activation in the presence of a high concentration of carbachol was seen in parotid glands and this inhibition was paralleled by amylase secretion.
Keywords: Nitric oxide (NO) synthase; Parotid gland; Amylase secretion; Nitric oxide (NO) signaling;

The effects of the [Ti IV (C5H5)2 NCS2] metallocene (BCDT), a Titanocene Dichloride derivative, on the growth and differentiation of granulocyte-macrophage progenitor cells [colony-forming unit-granulocyte-macrophage (CFU-GM)] and bone marrow cellularity in normal and Ehrlich ascites tumour-bearing mice were studied. As expected for the Ehrlich ascites tumour-model, concomitant myelosuppression, increased number of spleen CFU-GM and changes in bone marrow cellularity were observed. The treatment of Ehrlich ascites tumour-bearing mice with BCDT (10–30 mg/kg/day) produced a dose-dependent increase in myelopoiesis, a reduction in splenic colonies and a restoration in the total and differential marrow cell counts. We also observed an increase in CFU-GM number when bone marrow cells obtained from normal mice were incubated in vitro with serum from normal mice treated with BCDT. In addition, BCDT prolonged, in a dose-dependent manner, the survival of mice inoculated with Ehrlich ascites tumour. Although it has been previously reported that substitutions in the two halides of the titanocene do not interfere with antitumoural effect, our results with BCDT demonstrated a reduction in antitumour efficacy when compared to previous results with the original titanocene produced in our laboratory.
Keywords: Ehrlich ascites tumour; Metallocene; Titanocene Dichloride; Myelopoiesis; Bis-cyclopentadienylditiocianatetitanium (IV);

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist by Yumiko Moritani; Kazuo Sato; Takeshi Shigenaga; Nami Hisamichi; Masato Ichihara; Seijiro Akamatsu; Ken-ichi Suzuki; Tomoko Nii; Seiji Kaku; Tomihisa Kawasaki; Yuzo Matsumoto; Osamu Inagaki; Kenichi Tomioka; Isao Yanagisawa (43-52).
The pharmacological properties of YM-57029 ({4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine–glycine–aspartic acid–serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 μg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.
Keywords: Platelet glycoprotein IIb/IIIa; Antagonist; Pro-aggregatory activity; Antithrombotic agent;

Ethanol (1.5–3.5 g/kg body weight) was administered intraperitoneally to mice and the phosphorylation of MAP (mitogen-activated protein) kinase in the cerebral cortex was determined using phospho-specific MAP kinase antibodies. Ethanol inhibited the phosphorylation of MAP kinase in a dose- and time-dependent manner. Developmental studies demonstrated that the levels of phospho-MAP kinase increased from fetal cortex (prenatal) to 16-day-old mice (postnatal) and remained constant up to 4 months of age. However, ethanol (3.5 g/kg) decreased the phospho-MAP kinase staining in all of the age groups studied. Subcellular fractionation studies demonstrated that ethanol inhibited the phosphorylation of MAP kinase in both the cytosol as well as nucleus, but did not alter the levels of MAP kinase. Likewise, MK-801 (0.4 mg/kg) or flurazepam (75 mg/kg) also decreased the phospho-MAP kinase content. These data indicate that ethanol may inhibit the phosphorylation of MAP kinase in vivo by either inhibiting NMDA receptors or activating GABA receptors.
Keywords: Ethanol; MAP (mitogen-activated protein) kinase; Cerebral cortex;

Lack of persistent changes in the dopaminergic system of rats withdrawn from methamphetamine self-administration by Roman Stefanski; Sun-Hee Lee; Sevil Yasar; Jean L. Cadet; Steven R. Goldberg (59-68).
A continuing challenge for studies in the neurobiology of drug abuse is to identify and characterize long-lived neuroadaptations that can trigger craving and relapse. We previously reported that rats that had actively self-administered methamphetamine for 5 weeks and were then withdrawn from methamphetamine for 24 h showed marked decreases in somatodendritic dopamine D2 autoreceptor levels in the ventral tegmental area and median and dorsal part of the substantia nigra zona compacta with a corresponding down-regulation of dopamine D1 receptors in the shell of the nucleus accumbens. The purpose of the present study was to determine whether neuroadaptive changes in dopamine receptors or transporters in the brains of rats withdrawn for 24 h from chronic methamphetamine self-administration are persistent changes that can be demonstrated long after withdrawal. A “yoked” procedure was used in which rats were tested simultaneously in groups of three, with only one rat actively self-administering methamphetamine while the other two received yoked injections of either methamphetamine or saline. In vitro quantitative autoradiography was used to determine densities of dopamine uptake sites and dopamine D1 and D2 receptors in different brain regions following 7- and 30-day periods of withdrawal from chronic methamphetamine self-administration. No changes in dopamine transporter and dopamine receptor numbers were detected in any brain region examined in rats self-administering methamphetamine compared with littermates receiving yoked infusions of either methamphetamine or saline. Thus, neuroadaptive changes in densities of dopamine receptors or transporters in certain brain areas may contribute to the reinforcing effects of methamphetamine during the acquisition and maintenance phases of self-administration, but do not appear to contribute to the long-lasting neuroadaptive effects of chronic methamphetamine self-administration, which may trigger craving and relapse.
Keywords: Methamphetamine; Drug self-administration; Neuroadaptation; Dopamine; (Rat);

Interaction between vanilloid and glutamate receptors in the central modulation of nociception by Enza Palazzo; Vito de Novellis; Ida Marabese; Dario Cuomo; Francesca Rossi; Liberato Berrino; Francesco Rossi; Sabatino Maione (69-75).
This study investigates the effect of microinjections of capsaicin in the periaqueductal grey matter of rats on nociceptive behaviour and the possible interactions with NMDA and mGlu receptors. Intra-periaqueductal grey microinjection of capsaicin (1–3–6 nmol/rat) increased the latency of the nociceptive reaction in the plantar test. This effect was prevented by pretreatment with capsazepine (6 nmol/rat), which had no effect per se on the latency of the nociceptive reaction. 7-(Hydroxyimino)cyclopropa[b]chromen-1α-carboxylate ethyl ester (CPCCOEt, 50 nmol/rat) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP, 50 nmol/rat), antagonists of mGlu1 and mGlu5 receptors, respectively, completely blocked the effect of capsaicin. Similarly, pretreatment with dl-2-Amino-5-phosphonovaleric acid (dl-AP5, 5 nmol/rat) and riluzole (4 nmol/rat), an NMDA receptor antagonist and a voltage-dependent Na+ channels blocker which inhibits glutamate release, respectively, completely antagonized the effect of capsaicin. However, pretreatment with (2S)-α-Ethylglutamic acid (30 nmol/rat) and (RS)-α-Methylserine-O-phosphate (MSOP, 30 nmol/rat), antagonists of group II and group III mGlu receptors, respectively, had no effects on capsaicin-induced analgesia. Similarly, pretreatment with N-(piperidin-1-yl)-5-(4-chlophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 5 pmol/rat), a selective cannabinoid CB1 receptor antagonist, did not affect the capsaicin-induced antinociception. In conclusion, this study shows that capsaicin might produce antinociception at the periaqueductal grey level by increasing glutamate release, which activates postsynaptic group I mGlu and NMDA receptors.
Keywords: Nociception; Capsaicin; Glutamate; Periaqueductal grey;

Comparison of nerve conduction blocks by an opioid and a local anesthetic by Tufan Mert; Yasemin Gunes; Mustafa Guven; Ismail Gunay; Dilek Ozcengiz (77-81).
The experiments were done on frog sciatic nerves, using a sucrose-gap recording technique. The aim of our study was to investigate and to compare the tonic and phasic conduction blocking potency of tramadol and lidocaine on whole nerve and their interactions with Ca2+. The concentration of a tramadol solution producing the same amount of tonic and phasic conduction blocks was three and six times higher than that needed for lidocaine, respectively. Increasing the Ca2+ concentration in the test solution enhanced the conduction blocking potency of tramadol, but decreased that of lidocaine. It is concluded that tramadol blocks nerve conduction like a local anesthetic but with a weaker effect than that of lidocaine. Interactions of Ca2+ and these drugs suggested that these drugs might have either different binding sites or different action mechanisms.
Keywords: Lidocaine; Tramadol; Sucrose-gap; Compound action potential; Sciatic nerve; (Frog);

Arvanil-induced inhibition of spasticity and persistent pain: evidence for therapeutic sites of action different from the vanilloid VR1 receptor and cannabinoid CB1/CB2 receptors by Jason W. Brooks; Gareth Pryce; Tiziana Bisogno; Siân I. Jaggar; Deborah J.R. Hankey; Peter Brown; Daniel Bridges; Catherine Ledent; Maurizio Bifulco; Andrew S.C. Rice; Vincenzo Di Marzo; David Baker (83-92).
Activation of cannabinoid receptors causes inhibition of spasticity, in a mouse model of multiple sclerosis, and of persistent pain, in the rat formalin test. The endocannabinoid anandamide inhibits spasticity and persistent pain. It not only binds to cannabinoid receptors but is also a full agonist at vanilloid receptors of type 1 (VR1). We found here that vanilloid VR1 receptor agonists (capsaicin and N-N′-(3-methoxy-4-aminoethoxy-benzyl)-(4-tert-butyl-benzyl)-urea [SDZ-249-665]) exhibit a small, albeit significant, inhibition of spasticity that can be attenuated by the vanilloid VR1 receptor antagonist, capsazepine. Arvanil, a structural “hybrid” between capsaicin and anandamide, was a potent inhibitor of spasticity at doses (e.g. 0.01 mg/kg i.v.) where capsaicin and cannabinoid CB1 receptor agonists were ineffective. The anti-spastic effect of arvanil was unchanged in cannabinoid CB1 receptor gene-deficient mice or in wildtype mice in the presence of both cannabinoid and vanilloid receptor antagonists. Likewise, arvanil (0.1–0.25 mg/kg) exhibited a potent analgesic effect in the formalin test, which was not reversed by cannabinoid and vanilloid receptor antagonists. These findings suggest that activation by arvanil of sites of action different from cannabinoid CB1/CB2 receptors and vanilloid VR1 receptors leads to anti-spastic/analgesic effects that might be exploited therapeutically.
Keywords: Cannabinoid; Vanilloid; Vanilloid VR1 receptor; Anandamide; Arvanil;

The effects of two selective serotonin reuptake inhibitors on 5-hydroxy-tryptamine (5-HT) in the hippocampus were studied in wildtype and in 5-HT1B receptor knockout mice using in vivo microdialysis. Basal 5-HT levels in the hippocampus were not different between the two genotypes. The functional absence of 5-HT1B receptors was examined in the knockout mice by local infusion of the 5-HT1B receptor agonist, 1,4-Dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one (CP93129) into the hippocampus. CP93129 (1 μM) decreased 5-HT levels in wildtype mice, but not in 5-HT1B knockout mice. Systemic administration of the selective 5-HT reuptake inhibitor paroxetine (5 mg/kg, i.p.) increased extracellular 5-HT levels. The increase of 5-HT in 5-HT1B knockout mice was almost twofold higher than in wildtype mice. Systemic administration of selective 5-HT reuptake inhibitors stimulates both terminal 5-HT1B autoreceptors and somatodendritic 5-HT1A autoreceptors. Therefore, the selective 5-HT reuptake inhibitor, fluvoxamine, was applied locally into the hippocampus to investigate the role of the terminal 5-HT1B autoreceptors. Local administration of 0.3 μM fluvoxamine resulted in comparable increases in extracellular 5-HT in both genotypes, whereas 1.0 μM fluvoxamine produced a twofold greater increase in 5-HT levels in 5-HT1B knockout as compared to wildtype mice. In conclusion, the differences in hippocampal 5-HT output between wildtype and 5-HT1B knockout mice after local or systemic administration of selective 5-HT reuptake inhibitors show that 5-HT1B autoreceptors play a significant role in the inhibition of 5-HT release at serotonergic nerve terminals. In addition, the different dose-response to fluvoxamine suggests that 5-HT1B knockout mice have possible adaptations of 5-HT transporters in order to compensate for the loss of the terminal 5-HT1B autoreceptor.
Keywords: 5-HT1B autoreceptor; Hippocampus; Microdialysis; Knockout mouse; 5-HT (5-hydroxytryptamine, serotonin) reuptake inhibitor;

Some antiepileptic drugs have been used with success to counteract withdrawal symptoms following chronic use of sedatives, hypnotics or alcohol. We evaluated the potential of levetiracetam (Keppra™), a new antiepileptic drug, to prevent benzodiazepine withdrawal in an animal model sensitive to the anxiogenic effect resulting from drug cessation. The effects of levetiracetam (17 and 54 mg/kg) given intraperitoneally (i.p.) were determined on anxiety induced in female NMRI mice by withdrawal from 21 days of chronic administration of chlordiazepoxide. Administration of chlordiazepoxide was i.p. twice daily, in increments of 2 mg/kg, from 10 up to 40 mg/kg. Anxiety was evaluated using an elevated plus-maze test 24-h after chlordiazepoxide withdrawal. Discontinuation of chronic chlordiazepoxide induced a significant anxiogenic profile in the plus-maze test mainly characterised by a decrease in open arm exploration. This effect was dose-dependently prevented by administration of levetiracetam during the withdrawal period. The highest dose tested (54 mg/kg) induced statistically significant effects on all variables recorded but had no effect upon plus-maze exploration in normal mice. This suggests that the observed effects are dependent upon the level of stress or anxiety of the animals. These results support potential efficacy of levetiracetam in the benzodiazepine withdrawal syndrome.
Keywords: Antiepileptic drug; Anxiety; Benzodiazepine; Keppra™; Levetiracetam; Withdrawal;

Although acute neurotensin receptor stimulation exerts diverse behavioural effects that resemble those seen after administration of antipsychotic drugs, data on effects after repeated exposure to neurotensin receptor agonism is relatively sparse. Here, we demonstrate that repeated administration of the novel neurotensin-(8–13) analogue NT69L [(N-methyl-Arg), Lys, Pro, l-neo-Trp, tert-Leu, Leu] induce tolerance to its suppressant effect on conditioned avoidance behaviour in rats, a predictive assay for antipsychotic activity. In contrast, the inhibitory effect of haloperidol on this behaviour was sustained despite repeated administration of this classical antipsychotic drug. These findings indicate that repeated exposure to neurotensin receptor stimulation induces tolerance to the antipsychotic-like effects of neurotensin receptor agonists.
Keywords: Neurotensin; Antipsychotic; Conditioned avoidance behaviour; Haloperidol; NT69L;

Autoinhibitory function of the sympathetic prejunctional neuropeptide Y Y2 receptor evidenced by BIIE0246 by Rickard E. Malmström; Jon O.N. Lundberg; Eddie Weitzberg (113-119).
The significance of neuropeptide Y Y2 receptors in sympathetic nonadrenergic transmission was investigated using the novel selective antagonist BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide). In anaesthetized pigs pretreated with reserpine, and after transection of sympathetic nerves (depleted of noradrenaline), electrical stimulation of renal and splanchnic sympathetic nerves evoked vasoconstriction in, and overflow of neuropeptide Y-like immunoreactivity from, kidney and spleen, respectively. In the presence of BIIE0246, the nerve-evoked overflows of neuropeptide Y-like immunoreactivity were markedly increased and the splenic vasoconstrictor response prolonged. In addition, BIIE0246 caused splenic vasodilatation per se in this model where basal levels of circulating neuropeptide Y exceed 40 pM. It is concluded that endogenous neurogenical neuropeptide Y regulates its own release via activation of sympathetic prejunctional inhibitory neuropeptide Y Y2 receptors in both spleen and kidney in the reserpinized pig. Moreover, when circulating levels of neuropeptide Y are moderately increased, activation of neuropeptide Y Y2 receptors seems to contribute to basal splenic vascular tone.
Keywords: BIIE0246; Neuropeptide Y Y2 receptor; Prejunctional; Sympathetic transmission;

Experimental diabetes induces hyperreactivity of rabbit renal artery to 5-hydroxytryptamine by Francisco J Miranda; José A Alabadı́; Silvia Lloréns; Rosa F Ruiz de Apodaca; José M Centeno; Enrique Alborch (121-127).
The influence of diabetes on the response of isolated rabbit renal arteries to 5-hydroxytryptamine (5-HT) was examined. 5-HT induced a concentration-related contraction that was higher in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal did not significantly modify 5-HT contractions in arteries from control rabbits but enhanced the response to 5-HT in arteries from diabetic rabbits. Incubation with N G-nitro-l-arginine (l-NA) enhanced contractions to 5-HT in arteries from control and diabetic rabbits. In arteries with endothelium, this l-NA enhancement was lower in diabetic rabbits than in control rabbits. In arteries without endothelium, incubation with l-NA enhanced the maximal contractions to 5-HT in control rabbits but did not in diabetic rabbits. Indomethacin inhibited 5-HT-induced contraction of arteries from control rabbits and enhanced the maximal contraction to 5-HT of arteries from diabetic rabbits. In summary, diabetes enhances contractile response of rabbit renal artery to 5-HT. In control animals, this response is regulated by both endothelial and non-endothelial (neuronal) nitric oxide (NO) and by a vasoconstrictor prostanoid. Diabetes impairs the release of non-endothelial NO and the vasoconstrictor prostanoid.
Keywords: Endothelium; Diabetes; Nitric oxide (NO); 5-HT (5-hydroxytryptamine, serotonin); Cyclooxygenase; Renal artery;

Reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril: a role of calcitonin gene-related peptide by Zhi-Hong Zhou; Jun-lin Jiang; Jun Peng; Han-Wu Deng; Yuan-Jian Li (129-134).
Previous studies have shown that the development of tolerance to nitroglycerin is related to a decrease in the release of endogenous calcitonin gene-related peptide (CGRP). In the present study, we explored whether endogenous CGRP is involved in reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril in rats in vivo and vitro. Tolerance was induced by exposure to nitroglycerin (4.4×10−6 M) for 10 min in vitro or by pretreatment with nitroglycerin (10 mg/kg, s.c.) three times a day for 8 days in vivo. Nitroglycerin (3×10−9–10−6 M) caused a concentration-dependent relaxation in the isolated rat thoracic aorta, an effect that was reduced by CGRP-(8–37) (3×10−7 M) or capsaicin (3×10−7 M). Preincubation with nitroglycerin for 10 min significantly decreased its vasodilation, which was restored in the presence of N-acetylcysteine (10−5 M) or captopril (10−5 M). Nitroglycerin (150 μg/kg, i.v.) produced a depressor effect and an increase in concentrations of nitric oxide and CGRP, and the effects of nitroglycerin disappeared after pretreatment with nitroglycerin for 8 days. However, tolerance to nitroglycerin in vivo also was partially restored in the presence of N-acetylcysteine or captopril. The present results suggest that reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril is related to the increased release of CGRP in the rat.
Keywords: Tolerance; Nitroglycerin; CGRP (calcitonin gene-related peptide); N-acetylcysteine; Captopril;

AG-041R, a cholecystokinin-B/gastrin receptor antagonist, stimulates the repair of osteochondral defect in rabbit model by Toru Nakanishi; Kenzo Kawasaki; Yuji Uchio; Hiroko Kataoka; Masaharu Terashima; Mitsuo Ochi (135-140).
A newly synthesized compound (AG-041R), 3R-1-(2,2Diethoxyethyl)-((4methylphenyl) amino-carbonyl methyl)-3-((4methylphenyl) ureido-indoline-2-one), is a cholecystokinin-B/gastrin receptor antagonist which has stimulatory effects on the matrix synthesis of chondrocytes in vitro. In this study, we examined the effect of AG-041R on the repair of osteochondral defects (cylindrical, 4 mm diameter) in the patellar groove of the rabbit knee joint. At the time of operation, 100 μl of 1 μM of AG-041R was administered, followed by 200 μl with an osmotic pump for 14 days. Histological and biochemical evaluations were performed at 12 and 24 weeks after surgery. The histological score of the AG-041R-treated group, the quantity of glycosaminoglycan and the ratio of chondroitin sulfate in the AG-041R-treated tissue were significantly higher than in the untreated group. Moreover, the degeneration of cartilage around the defect was suppressed in the AG-041R-treated group. These findings suggest that AG-041R is effective for the repair of osteochondral defects.
Keywords: Cholecystokinin-B/gastrin receptor antagonist; Chondrocyte; Articular cartilage; Repair; (Rabbit); Osteochondral defect;

Cholesterol lowering effects of a choleretic phloracetophenone in hypercholesterolemic hamsters by Pawinee Piyachaturawat; Pornpikul Srivoraphan; Aporn Chuncharunee; Prayad Komaratat; Apichart Suksamrarn (141-147).
The plasma cholesterol-lowering effect and mechanism thereof of a choleretic phloracetophenone or 2,4,6-trihydroxyacetophenone (THA) were investigated in hypercholesterolemic male hamsters. Intragastric administration of THA (300–600 μmol/kg) twice a day for 7 days to these animals caused a dose- and time-dependent decrease in both plasma cholesterol and triglyceride levels. THA at a dose of 400 μmol/kg reduced the cholesterol and triglyceride levels in plasma to 52% and 25% of the level in corresponding cholesterol-fed controls, respectively, with decreases in both plasma very low density lipoprotein and low density lipoprotein cholesterol but not in high density lipoprotein cholesterol. THA did not significantly alter total hepatic cholesterol content but significantly increased the excretion of both bile acids and cholesterol into the intestinal lumen for elimination. Corresponding to the increase in bile acid excretion, THA caused a seven-fold increase in hepatic cholesterol 7α-hydroxylase activity. These results suggest that THA exerts its cholesterol lowering effect by increasing hepatic cholesterol 7α-hydroxylase activity which increases hepatic conversion of cholesterol to bile acid for disposal via biliary secretion. This compound may have a potential for future development as a therapeutic agent for lowering lipids in hypercholesterolemic patients.
Keywords: Bile acid; Cholesterol; Cholesterol 7α-hydroxylase; (Curcuma comosa); Phloracetophenone;

Role of tachykinins in sephadex-induced airway hyperreactivity and inflammation in guinea pigs by Manuela Tramontana; Paolo Santicioli; Sandro Giuliani; Rose-Marie Catalioto; Alessandro Lecci; Francesca Carini; Carlo Alberto Maggi (149-158).
We have studied the effect of selective tachykinin NK1 and NK2 receptor antagonists on airway hyperreactivity to acetylcholine and increase of inflammatory cells on bronchoalveolar lavage fluid induced by sephadex beads (20 mg/kg, i.v.) in guinea pigs. Airway hyperreactivity was assessed by measuring the increase of bronchial insufflation pressure to acetylcholine (0.01–30 μmol/kg, i.v.) at 3 h (early phase) and 24 h (late phase) after sephadex administration. An increase in inflammatory cells in bronchoalveolar lavage fluid (eosinophils and macrophages) was detected at 24 h (from 11.6·106 to 49.3·106 cells) but not at 3 h from sephadex administration. Neurokinin A and substance P levels in bronchoalveolar lavage fluid showed a significant increase at 24 h (from 31.7±11.6 to 561±231 pg/ml and from 5.9±2.6 to 29.3±4.1 pg/ml for neurokinin A and substance P, respectively). At this time point, the tachykinin in bronchoalveolar lavage cellular content was depleted from 232±43 to 21±20 pg/sample and from 56.6±6.7 to 2±2 pg/sample for neurokinin A and substance P, respectively. Capsaicin pretreatment abolished the early but not the late phase of airway hyperreactivity induced by sephadex without modifying bronchoalveolar lavage total cells number and bronchoalveolar lavage levels of neurokinin A and substance P. Administration of the tachykinin NK2 (nepadutant) and/or the NK1 receptor antagonist (MEN 11467 or (1R,2S)-2-N[1(H)indol-3-yl-carbonyl]-1-N[N-(p-tolylacetyl)-N-(methyl)-d-3(2-naphthyl)alanyl}diaminocyclohexane)), 5 min before sephadex, prevented the early phase of airway hyperreactivity to acetylcholine but only nepadutant prevented the late phase. Nepadutant was able to abolish the early phase of airway hyperreactivity if given after sephadex administration and reduced by about 50% the increase of cell number in bronchoalveolar lavage fluid during the late phase, without affecting the levels of neurokinin A and substance P. These findings indicate an involvement of endogenous tachykinins in the genesis of airway hyperreactivity in a guinea-pig model of non-allergic asthma. Early airway hyperreactivity apparently involves release of tachykinins from capsaicin-sensitive afferent nerves acting via tachykinin NK1/NK2 receptors. Late airway hyperreactivity involves tachykinins acting via tachykinin NK2 receptors: inflammatory cells activated/recruited in response to sephadex challenge appear a likely source of tachykinins involved in the late phase of the response.
Keywords: Sephadex; Airway hyperreactivity; (Guinea-pig); Neurokinin A; Substance P; Tachykinin receptor; Nepadutant; MEN 11467; Acetylcholine; Inflammatory cell;

No involvement of interleukin-5 or eosinophils in experimental allergic rhinitis in guinea pigs by Masashi Yamasaki; Nobuaki Mizutani; Kazutoyo Sasaki; Takeshi Nabe; Shigekatsu Kohno (159-169).
The aim of this study is to evaluate whether nasal airway eosinophilia is a true pathogenetic component of allergic rhinitis. We investigated the effects of TRFK5, an anti-interleukin-5 antibody, not only on leukocyte mobilization from the bone marrow, but also on the development of nasal symptoms and hyperresponsiveness in a guinea pig model of allergic rhinitis. Intranasally sensitized animals were repetitively challenged by exposure to Japanese cedar pollen as antigen. TRFK5 (100 μg/kg, i.p.) given 12 h before the final antigen challenge selectively prevented the antigen-induced eosinophilia in blood and the nasal airway, and suppressed the corresponding decrease in the number of cells in bone marrow; however, it failed to inhibit the immediate development of sneezing, early and late nasal blockage responses, goblet cell degranulation and nasal hyperresponsiveness to histamine. Furthermore, TRFK5 did not significantly affect the production of thromboxane A2 and cysteinyl leukotrienes in the nasal airway during the late response. These results strongly suggest that while interleukin-5 is essential for eosinophil migration from the bone marrow to the nasal airway, neither interleukin-5 nor eosinophils are required for the development of the nasal symptoms and nasal hyperresponsiveness of allergic rhinitis.
Keywords: Allergic rhinitis; Interleukin-5; Eosinophil; TRFK5;

Constitutively active melatonin MT1 receptors in male rat caudal arteries by Çaǧatay Erşahin; Monica I. Masana; Margarita L. Dubocovich (171-172).
This study assessed the state of melatonin MT1 receptor coupling in sections of male rat caudal arteries by [35S]GTPγS binding autoradiography. The melatonin MT1 receptor inverse agonist 4-phenyl-2-propionamidotetraline (4P-PDOT) (0.1–1 μM) significantly decreased [35S]GTPγS binding compared to basal, strongly suggesting the presence of constitutively active receptors. Formation of constitutively active receptors during subjective day, when the levels of melatonin are low, may be a physiological mechanism by which the organism maintains vascular tone.
Keywords: Constitutive activity; Melatonin MT1 receptor; 4P-PDOT (4-phenyl-2-propionamidotetraline);

Nitric oxide inhibits RhoA/Rho-kinase signaling to cause penile erection by Thomas M. Mills; Kanchan Chitaley; Ronald W. Lewis; R.Clinton Webb (173-174).
The RhoA/Rho-kinase pathway mediates vasoconstriction in the cavernosal circulation. Inhibition of this pathway leads to penile erection in the in vivo rat model. These studies examined the hypothesis that nitric oxide (NO) inhibits RhoA/Rho-kinase signaling as part of normal erection. The results show that NO causes increased intracavernosal pressure and that this response is potentiated by prior treatment with a threshold dose of the Rho-kinase inhibitor, (+)-(R)-trans-4-(1-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632). These results support the hypothesis that NO inhibits Rho-kinase-induced cavernosal vasoconstriction during erection.
Keywords: Penile erection; Nitric oxide (NO); RhoA/Rho-kinase;

Author index (175-176).

Keyword index (177-180).