Endothelin-1-induced nociception

Intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of morphine to mice antagonized the abdominal constriction induced by an i.p. injection of endothelin-1 (ET-1; 0.1 mg/kg). The ED50 values (95% confidence intervals) were 39.3 (16.5-80.2) ng and 1.5 (0.8-4.9) ng, respectively. The antagonism of ET-1-induced abdominal constriction by morphine was blocked by naloxone (1.0 mg/kg, s.c.) or by 24 h pretreatment with beta-funaltrexamine (beta-FNA; 8.84 micrograms, i.c.v.). These results demonstrate for the first time that the stimulus resulting from an i.p. injection of ET-1 is transmitted via ascending (pain) pathways that are subject to attenuation by opioid (mu) receptor activation. Hence, ET-1-induced abdominal constriction is a new pain model which, given the other pharmacology of ET-1, might represent a unique model with potential specific utility for anginal or other visceral pain.     

Antipsychotic profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562) in rats

The antipsychotic profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562) was investigated using the conditioned avoidance test in rats. NRA0562 is a putative "atypical" antipsychotic agent with moderate to high affinities for dopamine D(1), D(2), D(4), 5-hydroxytryptamine(2A) receptors and alpha(1) adrenoceptor. NRA0562 (1 and 3 mg/kg, p.o.) dose-dependently and significantly impaired the conditioned avoidance response. Likewise other atypical antipsychotics such as risperidone (1 and 3 mg/kg, p.o.) and clozapine (100 mg/kg, p.o.) dose-dependently and significantly impaired the conditioned avoidance response in rats. In addition, typical antipsychotics, haloperidol (1 and 3 mg/kg, p.o.) potently impaired the conditioned avoidance response.These results suggest that antipsychotic profile of NRA0562 is consistent with profiles of clozapine or risperidone and may be considered an atypical antipsychotic agent.     

Despite minimal hemodynamic alterations endotoxemia modulates NOS and p38-MAPK phosphorylation via metalloendopeptidases

In the present study, we hypothesized that endotoxemia produces metalloendopeptidase (MEPD)-dependent generation of endothelin-1 (ET-1) and alters NOS expression correlating with p38-mitogen-activated protein kinase (MAPK) phosphorylation in thoracic aorta. Male Sprague-Dawley rats (350-400 g) were subjected to two groups randomly; sham-treated (N = 10) and lipopolysaccharide (LPS)-treated (N = 10) (E. coli LPS 2 mg/kg bolus + 2 mg/kg infusion for 30 min). The animals in each group were further subdivided into vehicle and MEPD inhibitor phosphoramidon (1 mg/kg bolus, PHOS)-treated groups. LPS produces a significant decrease in mean arterial pressure (MAP) at 2 h post endotoxemia that was blocked by PHOS. PHOS attenuated LPS-induced increase in tumor necrosis factor-alpha (TNF-alpha) concentration at 2- and 24 h post-LPS administration. LPS significantly elevated plasma concentrations of ET-1 at 2- and 24 h post endotoxemia. An upregulated preproET-1 expression following both LPS and MEPD inhibition was observed in thoracic aorta at 2 h post treatment. PHOS effectively blocked conversion of preproET-1 to ET-1 in thoracic aorta locally at 24 h post treatment in endotoxic rats. PHOS inhibited LPS-induced upregulation of inducible NOS (iNOS), downregulation of endothelial NOS (eNOS) and elevation of NO byproducts (NOx) in thoracic aorta. PHOS also blocked LPS-induced upregulated p38-MAPK phosphorylation in thoracic aorta at 24 h post endotoxemia. The data revealed that LPS induces MEPD-sensitive inflammatory response syndrome (SIRS) at 2- and 24 h post endotoxemia. We concluded that inhibition of MEPD not only decreases the levels of ET-1 but also simultaneously downregulates protein expression of iNOS and phosphorylated p38-MAPK while increasing eNOS in thoracic aorta during SIRS in endotoxemia. We suggest that MEPD-dependent ET-1 and NO mechanisms may be involved in endotoxemia-induced altered p38-MAPK phosphorylation.     

Antinociceptive properties of the methanolic extract and two triterpenes isolated from Epidendrum Mosenii stems (Orchidaceae)

The antinociceptive effect of the methanolic extract (ME) and two triterpenes isolated from E. mosenii (Orchidaceae) has been investigated in chemical and thermal models of nociception in mice. The ME of E. mosenii (0.3-30 mg kg(-1), i.p. or 50-400 mg kg(-1), p.o.) produced dose-related, significant and long-lasting (4 to 6 h) inhibition of acetic acid-induced abdominal constriction, with ID50 values of 3.9 and 137.0 mg kg(-1), respectively. Pholidotin and 24-methylenecycloartenol isolated from E. mosenii (0.1-3.0 mg kg(-1), i.p.) also produced marked and dose-related inhibition of acetic acid-induced pain, with ID50 values of 0.9 and 1.1 mg kg(-1). However, these compounds and the ME were about 3- to 13-fold more potent at the level of ID50 than diclofenac when assessed in acetic acid-induced abdominal constriction. The ME of E. mosenii in the same range of doses produced dose-related inhibition of both phases of formalin-induced licking, with mean ID50 values for the first and the second phases of 0.9, 122.0 mg kg(-1) and 0.7, 258.0 mg kg(-1), respectively by i.p. or p.o. routes. In addition, the ME (0.3-30 mg kg(-1), i.p., or 50-400 mg kg(-1), p.o.) also caused dose-related inhibition of capsaicin-induced neurogenic pain with mean ID50 values of 5.2 and 130.0 mg kg(-1), respectively. Treatment of animals with naloxone (5 mg kg(-1), i.p.) completely reversed the antinociceptive effect caused by morphine (5 mg kg(-1), s.c.) and that caused by ME of E. mosenii (1 mg kg(-1), i.p.) when assessed against either phase of the formalin-induced pain. Furthermore, when assessed in the hot-plate test, ME (100 mg kg(-1), i.p.) and morphine (10 mg kg(-1), s.c.) caused significant increase in response latency. However, ME given daily for to 7 consecutive days did not develop tolerance to itself nor did it induce cross-tolerance to morphine. Taken together these data demonstrate that the ME of E. mosenii elicited pronounced antinociception, when assessed by i.p. or p.o. routes, against several models of pain. Its actions involve, at least in part, an interaction with opioid system, seeming no to be related with a non-specific peripheral or central depressant actions. Finally, the active principle(s) responsible for the antinociceptive action of E. mosenii is likely related to the presence of the triterpenes.     

Endothelin-1[1-31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells

Endothelin-1 (ET-1)[1-31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET-1[1-21]. However, at variance with ET-1[1-21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET-1[1-31] did not elicit such effect. Both ET-1[1-21] and ET-1[1-31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10(-8) M. This effect was blocked by the ETA-receptor antagonist BQ-123 and unaffected by the ETB-receptor antagonist BQ-788. Quantitative autoradiography showed that ET-1[1-21] displaced both [(125)I]PD-151242 binding to ETA receptors and [(125)I]BQ-3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET-1[1-31] displaced only [(125)I]BQ-3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of ET-1[1-31], while the protein kinase-C (PKC) inhibitor calphostin-C significantly reduced it. ET-1[1-31] (10(-8) M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ-123. The stimulatory action of ET-1[1-31] on TK activity was annulled by tyrphostin-23, while that on MAPK activity was reduced by calphostin-C and abolished by either tyrphostin-23 and PD-98059. These data suggest that ET-1[1-31] is a selective agonist of the ETA-receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC- and TK-dependent activation of p42/p44 MAPK cascade.     

A rabbit antiserum raised against the hexapeptide endothelin(16-21) shows different binding affinities for endothelin-1, -2 and -3.

A antiserum raised against the C-terminal hexapeptide ET16-21 common to ET-1, -2 and -3 was produced and characterized with respect to its binding properties for ET-1, -2, -3, ET16-21, the C-terminal octapeptide ET14-21, its derivative Phe21-ET14-21 and human big-ET-1. The antibody reacted with the peptides with decreasing binding affinities in the order: ET-1 greater than ET-2 greater than or equal to ET16-21 = ET 14-21 much greater than Phe21-ET14-21. It showed no crossreactivity with human big-ET-1. Similar results were obtained using [125I]ET-1, -2 or -3 as tracer. Substitution of Trp21 by Phe decreased the binding affinity of ET14-21 about 10 fold. Thus, the immunologically recognized sequence of the peptides is C-terminal and Trp21 seems to be important for high binding affinities. The significant differences in binding affinity observed for ET-1, -2, -3 and ET16-21 are consistent with an interaction of the C-terminal part of the endothelins with the bicyclic N-terminal part.     

Synthesis and 5-hydroxytryptamine (5-HT) activity of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines

A series of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines was shown to exhibit 5-HT2C agonist binding and functional activity. Compound 21R inhibited food intake over 2 h in fasted, male Sprague Dawley rats with ED50 values of 2 mg/kg (i.p.) and 10 mg/kg (p.o.).     

Endothelin-1[1-31] acts as a selective ETA-receptor agonist in the rat adrenal cortex

Endothelin-1 (ET-1) is a 21-amino acid residue (ET-1[1-21]) hypertensive peptide, which together with its receptor subtypes A and B (ETA and ETB) is expressed in the rat adrenal cortex, where it stimulates steroid-hormone (aldosterone and corticosterone) secretion through the ETB receptor and the growth (proliferative activity) of the zona glomerulosa (ZG) through the ETA receptor. ET-1[1-21] is generated from bigET-1 by the endothelin-converting enzyme (ECE-1). However, recent evidence indicates the existence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, which was found to reproduce the ETA receptor-mediated vascular effects of ET-1[1-21]. We found that ET-1[1-21], but not ET-1[1-31], concentration-dependently raised steroid secretion from dispersed rat adrenocortical cells, its effect being blocked by the ETB-receptor selective antagonist BQ-788. Both ET-1s concentration-dependently increased the number of "S-phase" cells (as detected by the 5-bromo-2'-deoxyuridine immunocytochemical method) in capsule-ZG strips within a 240 min incubation. The ZG proliferogenic action of both ET-1s was blocked by the ETA-receptor antagonist BQ-123, and ET-1[1-31] was found to be significantly more potent than ET-1[1-21]. Autoradiography showed that in the rat adrenal ET-1[1-21] displaced the binding of selective ligands to both ETA ([125I]PD-151242) and ETB receptors ([125I]BQ-3020), while ET-1[1-31] eliminates only the binding to ETA receptors. Collectively, our findings provide strong evidence that ET-1[1-31] acts in the rat adrenal glands as a selective ETA-receptor agonist, mainly involved in the stimulation of ZG proliferative activity.     

Adenosine preconditions against endothelin-induced constriction of coronary arterioles

Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng. kg(-1). min(-1) administered intracoronary, n = 10) induced progressive constriction over 45 min [25 +/- 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 microgram. kg(-1). min(-1) administered intracoronary) for 20 min and 10 min of washout (n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 +/- 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50-150 micrometer) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM-5 nM) was rightward shifted after preconditioning with adenosine (1 microM) for 20 min and 10 min of washout (n = 11). Blockade of A(2) receptors [8-(3-chlorostyryl)caffeine, 1 microM, n = 9] but not A(1) receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A(2) receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.     

Antipsychotic action of selective group II metabotropic glutamate receptor agonist MGS0008 and MGS0028 on conditioned avoidance responses in the rat

The present study was designed to investigate the antipsychotic-like effects of selective group II metabotropic glutamate receptor (mGluR) agonists, 5-[2-[4-(6-fluoro-1H-indole-3-yl) piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (MGS0008) and (1R, 2S, 5S, 6S)-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate (MGS0028) on conditioned avoidance responses in rats. MGS0008 (1, 3 and 10 mg/kg, p.o.) and MGS0028 (0.3, 1 and 3 mg/kg, p.o.) significantly and reduced conditioned avoidance responses in a dose-dependent fashion. Similar effects were seen with LY418426 (0.3, 1 and 3 mg/kg, p.o.), but not with LY354740 (3, 10 and 30 mg/kg, p.o.), both of which are selective agonists for group II mGluR. Since this effect is seen with a wide range of antipsychotics, such as haloperidol and clozapine [Life Sciences 71 (2002) 947], group II mGluR agonists deserve further attention for possible antipsychotic activity.     

Enalapril attenuates endothelin-1-induced hypertension via increased kinin survival

Recent studies have shown that angiotensin-converting enzyme (ACE) inhibitors attenuate endothelin-1 (ET-1)-induced hypertension, but the mechanisms for this effect have not been clarified. Initial experiments were conducted to contrast the effect of the ACE inhibitor enalapril, the combined ACE-neutral endopeptidase inhibitor omapatrilat, and the angiotensin II receptor antagonist candesartan on the hypertensive and renal response to ET-1 in anesthetized Sprague-Dawley rats. Acute intravenous infusion of ET-1 (10 pmol x kg(-1) x min(-1)) for 60 min significantly increased mean arterial pressure (MAP) from 125 +/- 8 to 145 +/- 8 mmHg (P < 0.05) and significantly decreased glomerular filtration rate (GFR) from 0.31 +/- 0.09 to 0.13 +/- 0.05 ml x min(-1) x 100 g kidney wt(-1). Pretreatment with enalapril (10 mg/kg iv) before ET-1 infusion inhibited the increase in MAP (121 +/- 4 vs. 126 +/- 4 mmHg) before and during ET-1 infusion, respectively (P < 0.05) without blocking the effect of ET-1 on GFR. In contrast, neither omapatrilat (30 mg/kg) nor candesartan (10 mg/kg) had any effect on ET-1-induced increases in MAP or decreases in GFR. To determine whether the effect of enalapril was due to the decrease in angiotensin II or increase in kinin formation, rats were given REF-000359 (1 mg/kg iv), a selective B(2) receptor antagonist, with or without enalapril before ET-1 infusion. REF-000359 completely blocked the effect of enalapril on ET-1 infusion (MAP was 117 +/- 5 vs. 135 +/- 5 mmHg before and during ET-1 infusion, respectively, P < 0.05). REF-000359 alone had no effect on the response to ET-1 infusion (MAP was 117 +/- 4 vs. 144 +/- 4 mmHg before and during ET-1 infusion, respectively, P < 0.05). REF-000359 with or without enalapril had no significant effect on the ability of ET-1 infusion to decrease GFR. These findings support the hypothesis that decreased catabolism of bradykinin and its subsequent vasodilator activity oppose the actions of ET-1 to increase MAP.     

L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2 - dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor

L-663,536 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMN) (IC50, 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 formation by rat peripheral blood and elicited PMN. At concentrations where inhibition of leukotriene biosynthesis occurred in human whole blood (1.1 microM), no effect was seen on cyclooxygenase or 12-lipoxygenase, an effect also observed in washed human platelets. The compound had no effect on rat or porcine 5-lipoxygenase indicating that L-663,536 is not a direct 5-lipoxygenase inhibitor. When administered in vivo L-663,536 was a potent inhibitor of antigen-induced dyspnea in inbred rats pretreated with methysergide (ED50, 0.036 mg/kg p.o.) and of Ascaris-induced bronchoconstriction in squirrel monkeys (1 mg/kg p.o.). The compound inhibited leukotriene biosynthesis in vivo in a rat pleurisy model (ED50, 0.2 mg/kg p.o.), an inflamed rat paw model (ED50, 0.8 mg/kg), a model of leukotriene excretion in rat bile following antigen provocation, and a model in the guinea-pig ear where leukotriene synthesis was induced by topical challenge with ionophore A23187 (ED50, 2.5 mg/kg p.o. and 0.6 micrograms topically). The results indicate that L-663,536 is a potent inhibitor of leukotriene biosynthesis both in vitro and in vivo indicating that the compound is suitable for studying the role of leukotrienes in a variety of pathological situations.     

Rubimetide (Met-Arg-Trp) derived from Rubisco exhibits anxiolytic-like activity via the DP1 receptor in male ddY mice

In this study, we found that Met-Arg-Trp (rubimetide), which had been isolated as a hypotensive peptide from a pepsin-pancreatin digest of spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), has anxiolytic-like activity in the elevated plus-maze test at a dose of 0.1mg/kg (i.p.) or 1.0mg/kg (p.o.) in mice with p<0.01 and p<0.05, respectively. The anxiolytic-like activity of rubimetide (0.1mg/kg, i.p.) was blocked by BW A868C (60microg/kg, i.p.), an antagonist for the DP1 receptor, suggesting the anxiolytic-like activity of rubimetide is mediated by prostaglandin D2 and the DP1 receptor.     

Analgesic and antiinflammatory effects of chalcones isolated from Myracrodruon urundeuva allem?o.

The present work showed analgesic and antiinflammatory activities from a fraction containing three dimeric chalcones (chalcone enriched fraction - CEF), isolated from the stem-bark ethyl acetate extract of Myracrodruon urundeuva Allemao (Anacardiaceae). M. urundeuva is a popular medicinal plant used widely in Northeast Brazil, mainly as a topical female genital tract antiinflammatory. We observed that the CEF (5 and 10 mg/kg body wt., i.p. or p.o.) inhibited acetic acid-induced abdominal contractions in mice. In the formalin test, the CEF (5 and 10 mg/kg body wt.) was more effective intraperitoneally and inhibited predominantly the second phase of response. Naloxone reversed this effect, indicating an involvement of the opioid system. The CEF (10 and 20 mg/kg body wt.) also increased the reaction time to thermal stimuli in the hot-plate test in mice, after i.p. but not after p.o. administration. In the carrageenan-induced paw edema test in mice, the CEF (20 and 40 mg/kg body wt.) decreased paw volume significantly, after i.p. administration 2-4 hours after carrageenan injection. The CEF (40 mg/kg body wt.) was also active orally during the same period of time. The present work is the first report on peripheral and central analgesic effects and antiinflammatory activity of natural dimeric chalcones.     

Role of benzodiazepine-GABAA receptor complex in attenuation of U-50,488H-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin in mice

In the present study, the role of benzodiazepine-GABAA receptor complex in the attenuation of U-50,488H (U50), a selective kappa opioid agonist-induced analgesia and inhibition of tolerance to its analgesia by ginseng total saponin (GTS) was investigated using the mice tail-flick test. The intraperitoneal (i.p.) treatment of GTS (100 and 200 mg/kg) and diazepam (0.1-1 mg/kg) dose-dependently attenuated the U50 (40 mg/kg, i.p.)-induced analgesia. GTS (0.001-10 microg/ml) did not alter binding of [3H]naloxone to mice whole brain membrane. The attenuation effect of GTS (100 mg/ kg) and diazepam (0.5 mg/kg) on U50-induced analgesia was blocked by flumazenil (0.1 mg/kg, i.p.), a benzodiazepine receptor antagonist, and picrotoxin (1 mg/kg, i.p.), a GABAA-gated chloride channel blocker. However, bicuculline (1 mg/kg, i.p.), a GABAA receptor antagonist blocked the attenuation effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on U50-induced analgesia. Chronic treatment (day 4-day 6) of GTS (50-200 mg/kg) and diazepam (0.1-1 mg/kg) dose-dependently inhibited the tolerance to U50-induced analgesia. Flumazenil (0.1 mg/kg) and picrotoxin (1 mg/kg) on chronic treatment blocked the inhibitory effect of GTS (100 mg/kg) and diazepam (0.5 mg/kg) on tolerance to U50-induced analgesia. On the other hand, chronic treatment of bicuculline (1 mg/kg) blocked the inhibitory effect of diazepam (0.5 mg/kg) but not GTS (100 mg/kg) on tolerance to U50-induced analgesia. In conclusion, the findings suggest that GTS attenuates U50-induced analgesia and inhibits tolerance to its analgesia and this action involves benzodiazepine receptors and GABAA-gated chloride channels.     

Local formation and degradation of endothelin-1 in guinea pig airway tissues

Endothelin(ET)-1 and big ET-1 caused potent and sustained constriction of isolated guinea pig bronchus. The response to ET-1 was enhanced by phosphoramidon in a simple dose-related manner (0.01-1000 microM), while the response to big ET-1 was enhanced at lower doses (0.01-0.1 microM) but was suppressed at higher doses (100-1000 microM) of phosphoramidon. Big ET-1, when given intravenously (i.v.) to anesthetized guinea pigs, increased both bronchopulmonary inflation pressure and mean arterial blood pressure (2.5, 5, 10 nmol/kg i.v.). The pressor response to big ET-1 was attenuated by phosphoramidon dose-relatedly, while the pulmonary response was modified in a complex fashion composed of delayed onset and prolonged duration of action. These results suggest that ET converting as well as degrading enzymes coexist in the airway tissue and both enzymes are sensitive to phosphoramidon, so that phosphoramidon acts bifunctionally to reduce and stimulate the airway responses to big ET-1.     

Effects of novel 5-HT1A receptor antagonists on measures of postsynaptic 5-HT1A receptor activation in vivo

The effects of two putative 5-HT_1A antagonists, 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-iodobenzamido]ethyl]piperazine (p-MPPI) and 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-flourobenzamido]ethyl]piperazine (p-MPPF), were examined in vivo in two tests of postsynaptic 5-HT_1A receptor activation, hypothermia and reciprocal forepaw treading, in the rat. Both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (10 mg/Kg, I.p.) antagonized the hypothermia induced by the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (0.5 mg/Kg, S.c.). Neither p-MPPI nor p-MPPF administered alone at a dose of 10 mg/kg (i.p.) induced hypothermia. Similarly, both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (2.5 mg/Kg, I.p.) completely antagonized 8-OH-DPAT (2 mg/Kg, S.c.)-induced forepaw treading in rats pretreated with reserpine (1 mg/Kg, S.c., 18–24 hours prior to the experiment). p-MPPI and p-MPPF, at doses of 10 mg/kg (i.p.) did not induce forepaw treading in reserpine pretreated animals. The results of the present study demonstrate that p-MPPI and p-MPPF act as 5-HT_1A receptor antagonists in these measures of postsynaptic 5-HT_1A a receptor activation.     

Micronucleus test with benzo[a]pyrene using a single peroral administration and intraperitoneal injection in males of the MS/Ae and CD-1 mouse strains

The effect of route of administration on the outcome of the micronucleus test was examined by administering benzo[a]pyrene (B[a]P) perorally (p.o.) and intraperitoneally (i.p.) to males of the MS/Ae and CD-1 mouse strains. This study consisted of 3 parts. First, an acute toxicity study lasting 3 days was done to estimate LD50s. The LD50 was larger than 1600 mg/kg for both routes in the 2 strains. Second, pilot micronucleus tests were carried out, on the basis of which an appropriate sampling time (48 h) and dose levels (62.5, 125, 250, and 500 mg/kg) were chosen for both routes and both strains. Third, full-scale micronucleus tests were done, which indicated that (1) B[a]P induced micronuclei dose-dependently by each administration route in each strain, (2) the i.p. route induced frequencies of micronuclei almost equal to or slightly higher than did the p.o. route, and (3) the MS/Ae strain was the higher responder.     

The effect of the leucopyrokinin analogue: [2-8]-leucopyrokinin on central opioid receptors in rats

The antinociceptive effect of intracerebroventricular injections of [2–8]-leucopyrokinin (LPK), a truncated leucopyrokinin analogue, was determined in rats, by means of a tail immersion test. We found a significant antinociceptive effect of three i.c.v. doses of [2–8]-LPK: 1, 5 and 10 nmol. Pre-treating animals with naloxone hydrochloride (1 mg/kg i.p.) completely blocked the effect of two high doses of [2–8]-LPK. To determine the sub-types of opioid receptors involved in [2–8]-leucopyrokinin-induced analgesia we injected specific blockers of μ-, δ- and κ-receptors namely, β-funaltrexamine hydrochloride, naltrindole hydrochloride and nor-binaltorphimine dihydrochloride, respectively, prior to [2–8]-leucopyrokinin at equimolar doses. We conclude that the antinociceptive effect of [2–8]-leucopyrokinin is mediated mainly by central μ- and δ-opioid receptors.     

Rubiscolin-6, a delta opioid peptide derived from spinach Rubisco, has anxiolytic effect via activating sigma1 and dopamine D1 receptors

Rubiscolin-6 (Tyr-Pro-Leu-Asp-Leu-Phe) is a delta opioid peptide derived from the large subunit of spinach d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We previously reported that rubiscolin-6 had an analgesic effect and stimulated memory consolidation. Here we show that intraperitoneally (i.p.) or orally administered rubiscolin-6 has an anxiolytic effect at a dose of 10 mg/kg or 100 mg/kg, respectively, in the elevated plus-maze test in mice. The anxiolytic effects of rubscolin-6 after i.p. (10 mg/kg) and oral (100 mg/kg) administration were blocked by a delta opioid receptor antagonist, naltrindole (1 mg/kg, s.c.), suggesting that the anxiolytic activity of rubiscolin-6 is mediated by delta opioid receptor. The anxiolytic effect of rubiscolin-6 (10 mg/kg, i.p.) was also blocked by a dopamine D(1) antagonist, SCH23390 (30 microg/kg, i.p.), but not by a dopamine D(2) antagonist, raclopride (15 microg/kg, i.p.). The anxiolytic effect of rubiscolin-6 (10 mg/kg, i.p.) was blocked by sigma(1) receptor antagonist, BMY14802 (0.5 mg/kg, i.p.) or BD1047 (10 mg/kg, i.p.). Taken together, the anxiolytic effect of rubiscolin-6 is mediated by sigma(1) and dopamine D(1) receptors downstream of delta opioid receptor.