Pharmacological characterization of the pseudopterosins: novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.     

The analgesic potency of dexmedetomidine is enhanced after nerve injury: a possible role for peripheral alpha2-adrenoceptors

This study investigated the analgesic potency and site of action of systemic dexmedetomidine, a selective alpha2-adrenoceptor (alpha2AR) agonist, in normal and neuropathic rats. Ligation of the L5-6 spinal nerves produced a chronic mechanical and thermal neuropathic hyperalgesia in rats. von Frey fibers and a thermoelectric Peltier device were used to measure mechanical and heat withdrawal thresholds over the hindpaw. Systemic dexmedetomidine dose-dependently increased the mechanical and thermal thresholds in the control animals (50% effective dose [ED50] 144 and 180 microg/kg intraperitoneally [i.p.], respectively). Neuropathic animals responded to much smaller doses of dexmedetomidine with mechanical and thermal ED50 values of 52 and 29 microg/kg i.p., respectively. There was no difference between the control and neuropathic animals with respect to dexmedetomidine-evoked sedation, as determined by decreased grid crossings in an open-field activity chamber (ED50 12 and 9 microg/kg i.p., respectively). Atipamezole, a selective alpha2AR antagonist, blocked the analgesic and sedative actions of dexmedetomidine inboth the neuropathic and control animals. However, L-659,066, a peripherally restricted alpha2AR antagonist, could only block the analgesic actions of dexmedetomidine in the neuropathic rats, with no effect in control animals. In conclusion, nerve injury enhanced the analgesic but not the sedative potency of systemic dexmedetomidine and may have shifted the site of alpha2 analgesic action to outside the blood-brain barrier. IMPLICATIONS: We tested the analgesic efficacy of the alpha2 agonist dexmedetomidine in normal and nerve-injured rats. The analgesic potency of dexmedetomidine was enhanced after nerve injury with a site of action outside the central nervous system. Peripherally restricted alpha2 agonists may be useful in the management of neuropathic pain.     

Electrophysiological comparison of 5-Hydroxytryptamine1A receptor antagonists on dorsal raphe cell firing

Single-unit recording studies were undertaken in chloral hydrate-anesthetized rats to compare the effects on dorsal raphe cell firing of several putative 5-hydroxytryptamine (HT)1A receptor antagonists, including WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide), p-MPPI (4-(2-methoxyphenyl)1-[2'-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl] pip erazine), and two newly described 5-HT1A receptor antagonists, NDL-249 [(R)-3-(N-propylamino)-8-fluoro-3, 4-dihydro-2H-1-benzopyran-5-carboxamide] and NAD-299 [(R)-3-N, N-dicyclobutylamino-8-fluoro-3, 4-dihydro-2H-1-benzopyran-5-carboxamide]. Consistent with a 5-HT1A receptor antagonist profile, pretreatment with an approximately equimolar (0.02-0.03 micromol/kg) i.v. dose of each compound caused a significant rightward shift in the dose-response curve for 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin]. Antagonist potency was clearly highest for NAD-299 and WAY 100635, which caused shifts roughly 3 times greater than those for either p-MPPI or NDL-249 (ED50 for 8-OH-DPAT, 1.3 +/- 0.3 microg/kg; after NAD-299, 18.2 +/- 1.0 microg/kg; after WAY 100635, 16.9 +/- 2.9 microg/kg; after NDL-249, 6.0 +/- 1.2 microg/kg; after p-MPPI, 4.7 +/- 1.1 microg/kg). In separate studies, each of the antagonists was administered alone in increasing cumulative doses to evaluate whether they possessed intrinsic agonist activity in this system. At doses below 0.01 micromol/kg, none of the drugs altered firing by more than +/-20% basal rates. At higher doses (>0.1 micromol/kg), WAY 100635, NDL-249, and NAD-299 caused a dose-dependent suppression of dorsal raphe cell firing (ED50 = 0.6 +/- 0.2, 0.7 +/- 0.3, and 0. 9 +/- 0.4 micromol/kg, respectively). However, the ED50 values for inhibition by these drugs were roughly 30 times higher than the doses that antagonized effects of 8-OH-DPAT. Moreover, the inhibition by all three antagonists (but not 8-OH-DPAT) was readily reversed by d-amphetamine (3.2 mg/kg i.v.), a releaser of norepinephrine, suggesting that these effects were likely due to alpha adrenergic receptor blockade rather than to 5-HT1A receptor agonism. Thus, it was concluded that WAY 100635, NAD-299, NDL-249, and p-MPPI all fulfill criteria as 5-HT1A receptor antagonists lacking intrinsic efficacy in the dorsal raphe system. The newly described compound NAD-299 exhibits antagonist potency comparable to that of WAY 100635 in this electrophysiological assay.     

Antidepressant-like effects of endogenous histamine and of two histamine H1 receptor agonists in the mouse forced swim test

1. Effects of substances which are able to alter brain histamine levels and two histamine H1 receptor agonists were investigated in mice by means of an animal model of depression, the forced swim test. 2. Imipramine (10 and 30 mg kg(-1), i.p.) and amitriptyline (5 and 15 mg kg(-1), i.p.) were used as positive controls. Their effects were not affected by pretreatment with the histamine H3 receptor agonist, (R)-alpha-methylhistamine, at a dose (10 mg kg(-1), i.p.) which did not modify the cumulative time of immobility. 3. The histamine H3 receptor antagonist, thioperamide (2-20 mg kg(-1), s.c.), showed an antidepressant-like effect, with a maximum at the dose of 5 mg kg(-1), which was completely prevented by (R)-alpha-methylhistamine. 4. The histamine-N-methyltransferase inhibitor, metoprine (2-20 mg kg(-1), s.c.), was effective with an ED50 of 4.02 (2.71-5.96) mg kg(-1); its effect was prevented by (R)-alpha-methylhistamine. 5. The histamine precursor, L-histidine (100-1000 mg kg(-1), i.p.), dose-dependently decreased the time of immobility [ED30 587 (499-712) mg kg(-1)]. The effect of 500 mg kg(-1) L-histidine was completely prevented by the selective histidine decarboxylase inhibitor, (S)-alpha-fluoromethylhistidine (50 mg kg(-1), i.p.), administered 15 h before. 6. The highly selective histamine H1 receptor agonist, 2-(3-trifluoromethylphenyl)histamine (0.3-6.5 microg per mouse, i.c.v.), and the better known H1 agonist, 2-thiazolylethylamine (0.1-1 microg per mouse, i.c.v.), were both dose-dependently effective in decreasing the time of immobility [ED50 3.6 (1.53-8.48) and 1.34 (0.084-21.5) microg per mouse, respectively]. 7. None of the substances tested affected mouse performance in the rota rod test at the doses used in the forced swim test. 8. It was concluded that endogenous histamine reduces the time of immobility in this test, suggesting an antidepressant-like effect, via activation of H1 receptors.     

Host defense within the urinary tract. I. Bacterial adhesion initiates an uroepithelial defense mechanism

The reliability and reproducibility of provocative stimuli of growth hormone (GH) secretion in the diagnosis of GH deficiency are still controversial both in childhood and in adulthood. The combined administration of GH-releasing hormone (GHRH) and arginine (ARG), which likely acts via inhibition of hypothalamic somatostatin release, is one of the most potent stimuli known so far and has been proposed recently as the best test to explore the maximal somatotrope capacity of somatotrope cells. However, it is well known that, usually, provocative stimuli of GH secretion suffer from poor reproducibility and that of the GHRH + ARG test has still to be verified. We aimed to verify the between- and within-subject variability of the GH response to the GHRH + ARG test in normal subjects during their lifespan as well as in hypopituitaric patients with GH deficiency (GHD). In 10 normal children (C: six male and four female, age 12.3 +/- 0.9 years, body mass index (BMI) = 16.6 +/- 0.7 kg/m2, pubertal stages I-III), 18 normal young adults (Y: ten male and eight female, age 31.1 +/- 1.3 years, BMI = 21.4 +/- 0.4 kg/m2), 12 normal elderly subjects (E: two male and ten female, age 74.4 +/- 1.8 years, BMI= 22.6 +/- 0.6 kg/m2) and 15 panhypopituitaric GH-deficient patients (GHD: nine male and six female, age 40.9 +/- 4.1 years, BMI= 22.7 +/- 1.0 kg/m2), we studied the inter- and intra-individual variability of the GH response to GHRH (1 microg/kg i.v.) + ARG (0.5 g/kg i.v.) in two different sessions at least 3 days apart. The GH responses to GHRH + ARG in C (1st vs 2nd session: 61.6 +/- 8.1 vs 66.5 +/- 9.4 microg/l), Y (70.4 +/- 10.1 vs 76.2 10.7 microg/l) and E (57.9 14.8 vs 52.1 +/- 8.0 microg/l) were similar and reproducible in all groups. The somatotrope responsiveness to GHRH + ARG also showed a limited within-subject variability (r = 0.71, 0.90 and 0.89 and p < 0.02, 0.0005 and 0.0005 for C, Y and E, respectively). Similarly in GHD, the GH response to the GHRH + ARG test showed a good inter- (1st vs 2nd session: 2.3 +/- 0.5 vs 2.2 +/- 0.6 microg/l) and intra-individual reproducibility (r = 0.70, p < 0.005). The GHRH + ARG-induced GH responses in GHD were markedly lower (p < 0.0005) than those in age-matched controls and no overlap was found between GH peak responses in GHD and normal subjects. In normal subjects, the GH response to GHRH + ARG is very marked, independent of age and shows limited inter- and intra-individual variability. The GH response to the GHRH + ARG test is strikingly reduced in panhypopituitaric patients with GHD, in whom the low somatotrope responsiveness is reproducible. Thus, these findings strengthen the hypothesis that GHRH + ARG should be considered the most reliable test to evaluate the maximal secretory capacity of somatotrope cells and to distinguish normal subjects from GHD patients in adulthood.     

Fructose-1,6-diphosphate enhanced oxyradicals and nitric oxide-dependent suppressions by dexamethasone of ischemic and histamine paw edema of mice

Dexamethasone (Dex, 0.1 mg/kg, s.c.) suppressions of ischemic paw edema in mice at 1, 3, 6, 8, 18 hr were; 2, 22, 12, 11, 7%. Dex suppression in fructose-1,6-diphosphate (FDP, 100 mg/kg, i.p.)-treated mice were; 5, 49, 51, 42, 33%. Suppressions by this dose of FDP alone were less than 10% during 0-18 hr. ED30 at 6 hr of Dex +/- FDP was: 80 versus 500 mg/kg in ischemic-, and 5 versus 30 mg/kg in histamine edema. Endogenous oxyradicals or NO and protein synthesis were essential for suppressions. FDP may not change glucocorticoid receptor (GR) conformation, but increase ATP-dependent GR recycling efflux from nucleus. FDP is possible to supply this ATP. Clinical trial of FDP with low dose of Dex seems advantageous.     

Autonomic contribution to the blood pressure and heart rate variability changes in early experimental hyperthyroidism

A great deal of uncertainty persists regarding the exact nature of the interaction between autonomic nervous activity and thyroid hormones in the control of heart rate (HR) and blood pressure (BP). Thyrotoxicosis was produced by a daily intraperitoneal (i.p.) injection of L-thyroxine (0.5 mg/kg body wt in 1 ml of 5 mM NaOH for 5 days). Control rats received i.p. daily injections of the thyroxine solvant. Autonomic blockers were administered intravenously: atropine (0.5 mg/kg), atenolol (1 mg/kg), atenolol + atropine or prazosin (1 mg/kg). Eight animals were studied in each group. Thyroxine treatment was sufficient to induce a significant degree of tachycardia (423 +/- 6 vs 353 +/- 4 bpm; p < 0.001, unpaired Student's tests), systolic BP elevation (142 +/- 3 vs 127 +/- 2 mmHg; p < 0.001), pulse pressure increase (51 +/- 2 vs 41 +/- 2 mmHg, p < 0.01), cardiac hypertrophy (1.165 +/- 0.017 vs 1.006 +/- 0.012 g, p < 0.001), weight loss (-21 +/- 2 g; p < 0.001) and hyperthermia (37.8 +/- 0.1 vs 37.0 +/- 0.1 degrees C, p < 0.001). The intrinsic HR observed after double blockade (atenolol + atropine) was markedly increased after treatment with thyroxine (497 +/- 16 vs 373 +/- 10 bpm, p < 0.05). Vagal tone (difference between HR obtained after atenolol and intrinsic HR) was positively linearly related to intrinsic HR (r = 0.84; p < 0.01). Atenolol neither modified HR nor BP variability in rats with hyperthyrodism. The thyrotoxicosis was associated with a reduction of the 0.4 Hz component of BP variability (analyses on 102.4 sec segments, modulus 1.10 +/- 0.07 vs 1.41 +/- 0.06 mmHg; p < 0.01). Prazosin was without effect on this 0.4 Hz component in these animals. These data show a functional diminution of the vascular and cardiac sympathetic tone in experimental hyperthyroidism. Increased intrinsic HR resulting from the direct effect of thyroid hormone on the sinoatrial node is the main determinant of a tachycardia leading to a subsequent rise in cardiac output. The resulting BP elevation could reflexly induce a vagal activation and a sympathetic (vascular and cardiac) inhibition.     

Action of prostanoids on the emetic reflex of Suncus murinus (the house musk shrew)

Several prostanoids were investigated for a potential to induce emesis in Suncus murinus. The TP receptor agonist 11alpha,9alpha-epoxymethano-15S-hydroxyprosta-5Z,13E-dienoic acid (U46619) induced emesis at doses as low as 3 microg/kg, i.p. but the DP receptor agonist 5-(6-Carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C) was approximately 1000 times less potent. The emetic action of U46619 (300 microg/kg, i.p.) was antagonized significantly by the TP receptor antagonist, vapiprost (P<0.05). EP (prostaglandin E(2), 17-phenyl-omega-trinor prostaglandin E(2), misoprostol and sulprostone), FP (prostaglandin F(2alpha) and fluprostenol) and IP (iloprost and cicaprost) receptor agonists failed to induce consistent emesis at doses up to 300-1000 microg/kg, i.p. Fluprostenol reduced nicotine (5 mg/kg, s.c.)-but not copper sulphate (120 mg/kg, intragastric)-induced emesis; the other inconsistently emetic prostanoids were inactive to modify drug-induced emesis. The results indicate an involvement of TP and possibly DP and FP receptors in the emetic reflex of S. murinus.     

Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.     

Reversal of the sedative and sympatholytic effects of dexmedetomidine with a specific alpha2-adrenoceptor antagonist atipamezole: a pharmacodynamic and kinetic study in healthy volunteers

BACKGROUND: Specific and selective alpha2-adrenergic drugs are widely exploited in veterinary anesthesiology. Because alpha2-agonists are also being introduced to human practice, the authors studied reversal of a clinically relevant dexmedetomidine dose with atipamezole, an alpha2-antagonist, in healthy persons. METHODS: The study consisted of two parts. In an open dose-finding study (part 1), the intravenous dose of atipamezole to reverse the sedative effects of 2.5 microg/kg of dexmedetomidine given intramuscularly was determined (n = 6). Part 2 was a placebo-controlled, double-blinded, randomized cross-over study in which three doses of atipamezole (15, 50, and 150 microg/kg given intravenously in 2 min) or saline were administered 1 h after dexmedetomidine at 1-week intervals (n = 8). Subjective vigilance and anxiety, psychomotor performance, hemodynamics, and saliva secretion were determined, and plasma catecholamines and serum drug concentrations were measured for 7 h. RESULTS: The mean +/- SD atipamezole dose needed in part 1 was 104+/-44 microg/kg. In part 2, dexmedetomidine induced clear impairments of vigilance and psychomotor performance that were dose dependently reversed by atipamezole (P < 0.001). Complete resolution of sedation was evident after the highest (150 microg/kg) dose, and the degree of vigilance remained high for 7 h. Atipamezole dose dependently reversed the reductions in blood pressure (P < 0.001) and heart rate (P = 0.009). Changes in saliva secretion and plasma catecholamines were similarly biphasic (i.e., they decreased after dexmedetomidine followed by dose-dependent restoration after atipamezole). Plasma norepinephrine levels were, however, increased considerably after the 150 microg/kg dose of atipamezole. The pharmacokinetics of atipamezole were linear, and elimination half-lives for both drugs were approximately 2 h. Atipamezole did not affect the disposition of dexmedetomidine. One person had symptomatic sinus arrest, and another had transient bradycardia approximately 3 h after receiving dexmedetomidine. CONCLUSIONS: The sedative and sympatholytic effects of intramuscular dexmedetomidine were dose dependently antagonized by intravenous atipamezole. The applied infusion rate (75 microg x kg(-1) x min(-1)) for the highest atipamezole dose was, however, too fast, as evident by transient sympathoactivation. Similar elimination half-lives of these two drugs are a clear advantage considering the possible clinical applications.     

Anxiogenic effect of corticotropin-releasing hormone in the dorsal periaqueductal grey

To investigate whether the dorsal periaqueductal grey (DPAG) might be involved in the anxiogenic effect of intracerebroventricular (i.c.v.) administered corticotropin-releasing hormone (CRH), rats (n = 6-13) received microinjections into the DPAG of CRH (1 or 2 microg) or saline and were tested on the elevated plus maze. The drug caused a dose-dependent decrease in plus maze exploration (percentage of entries into open arms: saline 31.9 +/- 8.6, CRH 1 microg 19.2 +/- 4.0, CRH 2 microg 6.9 +/- 3.7; p < 0.01, ANOVA). In a second experiment the anxiogenic effect of intra-DPAG CRH (2 microg) was prevented by previous microinjection of alpha-helical-CRH(9-41) (0.5 microg), a CRH antagonist (percentage of entries into open arms: saline + CRH 20.3 +/- 3.7, alpha-helical-CRH(9-41) + CRH 45.7 +/- 1.6). These results suggest that the DPAG may be a site of the anxiogenic effect of i.c.v. injected CRH.     

Transduction of cultured oligodendrocytes from normal and twitcher mice by a retroviral vector containing human galactocerebrosidase (GALC) cDNA

We examined in mice the effect of chronic diazepam treatment on the sensitivity to isoflurane, and that of repeated isoflurane exposure on the sensitivity to diazepam. Mice were divided into four groups: group 1, treated with diazepam, 10 mg/kg i.p. twice daily; group 2, vehicle-treated controls; group 3, exposed to 3% isoflurane for 25 min twice daily; and group 4, untreated controls. After 14 days the effect of the treatment was assessed. Twenty-four hours after the last 10 mg/kg diazepam treatment, groups 1 and 2 received diazepam, 5 mg/kg i.p., and were subjected to the horizontal wire test (HWT). All control mice but only 10% of the diazepam-treated mice failed the HWT. Groups 1 and 2 were then exposed to increasing concentrations of isoflurane. Diazepam-treated mice (group 1) lost the HWT at 0.7+/-0.7%, compared with 0.6+/-0.1% in controls (group 2) (P<0.001); the ED50 was 0.75% vs. 0.65%. Group 1 mice lost the righting reflex at 0.94+/-0.07% isoflurane vs. 0.87+/-0.06% in group 2 (P<0.01); the ED50 was 0.93% vs. 0.82%. Recovery time was 175+/-161 s in group 1 vs. 343+/-275 s in group 2 (P<0.02). Twenty-four hours after the last of the repeated exposures to isoflurane, we examined the responses of groups 3 and 4 to increasing concentrations of isoflurane. Mice in group 3 lost the righting reflex at 1.0+/-0.06% isoflurane vs. 0.9+/-0.04% in controls (group 4) (P<0.001); the ED50 was 0.96% vs. 0.85%. Recovery time was 113+/-124 s vs. 208+/-126 s in groups 3 and 4 (P<0.09). Diazepam, 3 mg/kg i.p. administered to groups 3 and 4, caused loss of the HWT reflex in 33% of group 3 mice and in 82% of controls (group 4) (P<0.001). It appears that prolonged exposure to both diazepam and isoflurane caused reduced sensitivity to each drug separately, as well as to the other drug. This finding may strengthen the theory that inhalational anesthetics may act via the same mechanism as the benzodiazepines.     

A biomechanical evaluation of cyclic and static stretch in human skeletal muscle

Hexarelin (HEX) is a synthetic GHRP which acts on specific receptors at both the pituitary and the hypothalamic level to stimulate GH release both in animals and in humans. Like other GHRPs, HEX possesses also acute ACTH and cortisol-releasing activity similar to that of hCRH. The mechanisms underlying the stimulatory effect of GHRPs on hypothalamo-pituitary-adrenal (HPA) axis are still unclear, although a CNS-mediated action has been demonstrated. In 6 normal healthy young women (26-34 years) we studied the effects on ACTH and cortisol secretion of HEX (2.0 microg/kg i.v. at 0 min) alone and preceded by dexamethasone (DEXA, 1 mg p.o. at 23.00 h on the previous night) or alprazolam (ALP, 0.02 mg/kg p.o. at -90 min), a benzodiazepine which binds to GABA receptors and possesses CRH-mediated inhibitory activity on HPA axis. ACTH and cortisol secretion after saline administration as well as the GH response to HEX alone and preceded by DEXA or ALP were also studied. HEX administration elicited an increase in ACTH (peak vs. baseline, mean +/- SEM: 28.0 +/- 6.7 vs. 11.7 +/- 2.2 pg/ml, p < 0.05) and cortisol secretion (162.6 +/- 15.0 vs. 137.7 +/- 12.6 microg/l, p < 0.05). DEXA pretreatment strongly inhibited basal ACTH (3.2 +/- 0.7 pg/ml, p < 0.01) and cortisol levels (11.3 +/- 2.5 microg/l, p < 0.001) and abolished the ACTH and cortisol responses to HEX (3.6 +/- 0.9 pg/ml, p < 0.01 and 10.7 +/- 2.0 microg/l, p < 0.001), respectively. On the other hand, ALP pretreatment did not significantly modify basal ACTH (7.9 +/- 2.0 pg/ml) and cortisol levels (127.6 +/- 14.5 microg/l) but abolished the HEX-induced ACTH and cortisol secretions (8.6 +/- 2.4 pg/ml, p < 0.05 and 111.0 +/- 6.0 microg/l, p < 0.05), respectively. ACTH and cortisol levels after HEX when preceded by ALP overlapped with those recorded during saline. HEX induced a clear GH response (peak at 15 min vs. baseline: 65.5 +/- 20.5 vs. 2.2 +/- 0.7 microg/l, p < 0.03) which was blunted by ALP (peak at 15 min: 21.5 +/- 5.5 microg/l, p < 0.05) while it was not modified by DEXA pretreatment (78.7 +/- 7.6 microg/l). In conclusion, our present data demonstrate that the ACTH- and cortisol-releasing effect of HEX is abolished by either dexamethasone or alprazolam, a benzodiazepine, which is even able to blunt the GH-releasing activity of the hexapeptide. These findings suggest that, in physiological conditions, the stimulatory effect of GHRPs on HPA axis is sensitive to the negative glucocorticoid feedback and could be mediated by GABAergic mechanisms; the latter seem also involved in the GH-releasing activity of GHRPs.     

Studies on the neuronal circuits involved in the discriminative stimulus effects of 5-hydroxytryptamine1A receptor agonists in the rat

Rats were trained to discriminate 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.1 mg/kg i.p.) or 5-methoxy-N,N-dimethyltryptamine (5-OMe-DMT, 1.25 mg/kg i.p.), a selective and nonselective 5-hydroxytryptamine1A (5-HT, serotonin) receptor agonist, respectively, from saline in a two-lever procedure. The selective 5-HT1A receptor agonist ipsapirone substituted completely for 8-OH-DPAT (ED50, 1.52 mg/kg) and 5-OMe-DMT substituted partially for 8-OH-DPAT, whereas 8-OH-DPAT (ED50, 0.07 mg/kg) and ipsapirone (ED50, 4.15 mg/kg) substituted completely for 5-OMe-DMT. These results suggest that the discriminative stimulus properties of both 8-OH-DPAT and 5-OMe-DMT are 5-HT1A receptor mediated, although 5-OMe-DMT may involve an additional interaction with other 5-HT receptor subtypes. 5-OMe-DMT substituted for 8-OH-DPAT after application in the lateral ventricle (ED50, 3.0 micrograms/rat) and the dorsal raphe nucleus (DRN, 1.1 micrograms/rat). After application in the DRN (ED50 range, 1.4-5.0 micrograms/rat) and the median raphe nucleus (2.3 micrograms/rat), and after bilateral application into the CA-4 region of the dorsal hippocampus (4.1 micrograms/rat), 8-OH-DPAT also produced responding on the 8-OH-DPAT lever. Ipsapirone also substituted for 8-OH-DPAT after application into the DRN and the hippocampus (ED50S, 38 and 62 micrograms/rat, respectively). The 5-HT1A mixed agonist-antagonist (1-(2-methoxyphenyl) 4-[4-(2-pthalimido)butyl]piperazine, i.p. NAN-190) attenuated the discriminative stimulus effects of 8-OH-DPAT injected i.p. (0.1 mg/kg), into the DRN (10 micrograms) or into the hippocampus (2 x 10 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)     

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 +/- 0.19 and Dex = 0.35 +/- 0.13 vs saline (S) = 2.85 +/- 0.59; fMLP: Ptx = 0.43 +/- 0.09 and Dex 0.01 +/- 0.01 vs S = 1.08 +/- 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 +/- 1.4 and Dex = 3.06 +/- 0.76 vs S = 15.94 +/- 3.97; fMLP: Ptx = 3.85 +/- 0.56 and Dex = 0.40 +/- 0.16 vs S = 7.15 +/- 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 microns), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 +/- 0.38 vs S = 4.20 +/- 1.01; fMLP: Dex = 0.25 +/- 0.11 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 +/- 2.25 vs S = 4.20 +/- 1.01; fMLP: Ptx = 4.66 +/- 1.24 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.     

alpha-Adrenoceptor and opioid receptor modulation of clonidine-induced antinociception

1. The antinociceptive action of clonidine (Clon) and the interactions with alpha 1, alpha 2 adrenoceptor and opioid receptor antagonists was evaluated in mice by use of chemical algesiometric test (acetic acid writhing test). 2. Clon produced a dose-dependent antinociceptive action and the ED50 for intracerebroventricular (i.c.v.) was lower than for intraperitoneal (i.p.) administration (1 ng kg-1 vs 300 ng kg-1). The parallelism of the dose-response curves indicates activation of a common receptor subtype. 3. Systemic administration of prazosin and terazosin displayed antinociceptive activity. Pretreatment with prazosin produced a dual action: i.c.v. Clon effect did not change, and i.p. Clon effect was enhanced. Yohimbine i.c.v. or i.p. did not induce antinonciception, but antagonized Clon-induced activity. These results suggest that alpha 1- and alpha 2-adrenoceptors, either located at the pre- and/or post-synaptic level, are involved in the control of spinal antinociception. 4. Naloxone (NX) and naltrexone (NTX) induced antinociceptive effects at low doses (microgram kg-1 range) and a lower antinociceptive effect at higher doses (mg kg-1 range). Low doses of NX or NTX antagonized Clon antinociception, possibly in relation to a preferential mu opioid receptor antagonism. In contrast, high doses of NX or NTX increased the antinociceptive activity of Clon, which could be due to an enhanced inhibition of the release of substance P. 5. The results obtained in the present work suggest the involvement of alpha 1-, alpha 2-adrenoceptor and opioid receptors in the modulation of the antinociceptive activity of clonidine, which seems to be exerted either at spinal and/or supraspinal level.     

Inhibition of nociceptin-induced allodynia in conscious mice by prostaglandin D2

1. We recently showed that intrathecal administration of nociceptin induced allodynia by innocuous tactile stimuli and hyperalgesia by noxious thermal stimuli in conscious mice. In the present study, we examined the effect of prostaglandins on nociceptin-induced allodynia and hyperalgesia. 2. Prostaglandin D2 (PGD2) blocked the allodynia induced by nociceptin in a dose-dependent manner with an IC50 of 26 ng kg(-1), but did not affect the nociceptin-induced hyperalgesia at doses up to 500 ng kg(-1). BW 245C (an agonist for PGD (DP) receptor) blocked the allodynia with an IC50 of 83 ng kg(-1). 3. The blockade of nociceptin-induced allodynia by PGD2 was reversed by the potent and selective DP-receptor antagonist BW A868C in a dose-dependent manner with an ED50 of 42.8 ng kg(-1). 4. Glycine (500 ng kg[-1]) almost completely blocked the nociceptin-induced allodynia. A synergistic effect on the inhibition of nociceptin-evoked allodynia was observed between glycine and PGD2 at below effective doses. 5. Dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, blocked the nociceptin-induced allodynia with an IC50 of 2.9 microg kg(-1). 6. PGE2, PGF2alpha, butaprost (an EP2 agonist) and cicaprost (a PGI receptor agonist) did not affect the nociceptin-induced allodynia. 7. These results demonstrate that PGD2 inhibits the nociceptin-evoked allodynia through DP receptors in the spinal cord and that glycine may be involved in this inhibition.     

Modulation of kinin B1 but not B2 receptors-mediated rat paw edema by IL-1beta and TNFalpha

The modulatory effects of IL-1beta and TNF alpha on the rat paw edema induced by B1 agonists have been analyzed. In naive rats, i.d. injection of B1 agonists, des-Arg9-bradykinin and des-Arg10-kallidin (up to 300 nmol), causes a minimal increase in paw volume, while the B2 agonist tyrosine8-bradykinin (0.3-10 nmol) induces graded paw edema. The injection of des-Arg9-bradykinin (10-100) nmol or des-Arg10-kallidin (1-100 nmol), in paws pre-treated with IL-1beta or TNF alpha (both 5 ng/paw; 60 and 30 min prior, respectively), caused a graded edema formation. The edemas induced by des-Arg9-bradykinin (100 nmol) were evident at 15 min, reaching the maximum 60 and 30 min after treatment with IL-1beta (0.64 +/- 0.06 ml) or TNF alpha (0.47 +/- 0.05 ml), respectively, being reduced at 360 min. The B1 antagonist des-Arg9-NPC 17731 (1-30 nmol), but not the B2 antagonist Hoe 140 (10 nmol), produced marked inhibition of des-Arg9-bradykinin-induced paw edema. Dexamethasone (0.5 mg/kg, s.c., 4 h) or cycloheximide (1.5 mg/kg, s.c., 6 h) significantly prevented the edema caused by des-Arg9-bradykinin (100 nmol) in rats treated with IL-1beta (81 +/- 5% and 59 +/- 3%) or TNF alpha (78 +/- 4% and 43 +/- 2%). Indomethacin (2 mg/kg, i.p.) or meloxicam (3 mg/kg, i.p.), 1 h prior, significantly reduced the edema induced by des-Arg9-bradykinin (100 nmol) in IL-1beta (40 +/- 6% and 69 +/- 8%) or TNF alpha (43 +/- 3% and 53 +/- 9%) treated rats. It is suggested that i.d. injection of the IL-1beta or TNF alpha, produced up-regulation of B1 receptor-mediated paw edema, being this effect sensitive to dexamethasone and cycloheximide and to cyclo-oxygenase pathway.     

Supplemental dietary arginine accelerates intestinal mucosal regeneration and enhances bacterial clearance following radiation enteritis in rats

Medetomidine was administered to sheep and horses at a dose rate of 5 microg kg(-1) (i.v.). Heart rate and blood pressure were recorded. Medetomidine induced bradycardia and a biphasic blood pressure response consisting of a transient hypertension followed by hypotension. Administration of prazosin (an alpha1 adrenoceptor antagonist; 100 microg kg(-1), i.v.) had no effect on the cardiovascular response to medetomidine (5 microg kg(-1), i.v.), but inhibited the cardiovascular response of methoxamine (an alpha1 adrenoceptor agonist; 75 microg kg(-1), i.v.). L-659,066 (an alpha2 adrenoceptor antagonist which does not cross the blood brain barrier; 264 microg kg(-1), i.v.) attenuated the medetomidine induced bradycardia, but had no effect on the cardiovascular response to methoxamine. L659,066 also reduced the medetomidine induced hypertension in sheep, but had less effect on the horse. It is concluded that both alpha1 and alpha2 adrenoceptors are important in the control of cardiovascular function in horses and sheep. Medetomidine appears to act on alpha2 adrenoceptors alone in the sheep. The cardiovascular effects of medetomidine in the horse are complex and may be influenced by central alpha2 adrenoceptor regulation or effects on other receptor subtypes as well as direct stimulation of peripheral alpha2 adrenoceptors.