Complementary anti-inflammatory effects of a β-blocker and a corticosteroid in an asthma model

Glucocorticosteroids are the mainstay treatment for chronic asthma; however, adverse effects can limit their usefulness. We previously determined in experimental asthma that chronic administration of β₂-adrenoceptor inverse agonists reduced airway hyperresponsiveness and indexes of inflammation. However, the effect of co-administration of glucocorticosteroids with β₂-adrenoceptor inverse agonists is unknown. Therefore, we evaluated the anti-inflammatory effect of co-administration of dexamethasone, a glucocorticosteroid, and nadolol, a β₂-inverse agonist, in a murine asthma model. We measured eosinophils and cytokines in bronchoalveolar lavage fluid and mucin content in epithelial cells after exposure to different concentrations of dexamethasone and nadolol. Dexamethasone was administered for 3 days and nadolol for 24 days prior to ovalbumin challenge. Both drugs were continued during five daily intranasal challenges with ovalbumin. Independent administration of dexamethasone (0.4 mg/kg/day) or nadolol (25 ppm) reduced bronchoalveolar lavage eosinophils by 58% and 36%, respectively (P < 0.05). Co-administration of both drugs yielded an additive reduction in eosinophils (81%, P < 0.05). Co-administration of both drugs (dexamethasone 0.4 mg/kg/day and nadolol 25 ppm) also yielded a greater reduction in mucin volume density (83%) than either drug alone (18% for dexamethasone and 62% for nadolol) and greater than high-dose dexamethasone (71%) alone (P < 0.05). Similarly, co-administration of both drugs (dexamethasone 0.4 mg/kg/day and nadolol 25 ppm) yielded an additive effect on the reduction of type 2 cytokines in bronchoalveolar lavage fluid equivalent to the administration of a 10-fold higher dose of dexamethasone. In Summary, the simultaneous administration of a glucocorticosteroid and a β₂-adrenoceptor inverse agonist was more effective at reducing indexes of airway inflammation than either drug given alone; suggesting nadolol may possess “glucocorticoid-sparing” properties.     

The PPARγ agonist,rosiglitazone, attenuates airway inflammation and remodeling via heme oxygenase-1 in murine model of asthma

Aim:

Rosiglitazone is one of the specific PPARγ agonists showing potential therapeutic effects in asthma. Though PPARγ activation was considered protective in inhibiting airway inflammation and remodeling in asthma, the specific mechanisms are still unclear. This study was aimed to investigate whether heme oxygenase-1 (HO-1) related pathways were involved in rosiglitazone-activated PPARγ signaling in asthma treatment.

Methods:

Asthma was induced in mice by multiple exposures to ovalbumin (OVA) in 8 weeks. Prior to every OVA challenge, the mice received rosiglitazone (5 mg/kg, po). After the mice were sacrificed, the bronchoalveolar lavage fluid (BALF), blood samples and lungs were collected for analyses. The activities of HO-1, MMP-2 and MMP-9 in airway tissue were assessed, and the expression of PPARγ, HO-1 and p21 proteins was also examined.

Results:

Rosiglitazone administration significantly attenuated airway inflammation and remodeling in mice with OVA-induced asthma, which were evidenced by decreased counts of total cells, eosinophils and neutrophils, and decreased levels of IL-5 and IL-13 in BALF, and by decreased airway smooth muscle layer thickness and reduced airway collagen deposition. Furthermore, rosiglitazone administration significantly increased PPARγ, HO-1 and p21 expression and HO-1 activity, decreased MMP-2 and MMP-9 activities in airway tissue. All the therapeutic effects of rosiglitazone were significantly impaired by co-administration of the HO-1 inhibitor ZnPP.

Conclusion:

Rosiglitazone effectively attenuates airway inflammation and remodeling in OVA- induced asthma of mice by activating PPARγ/HO-1 signaling pathway.     

Systemic effects of E-2078, a stabilized dynorphin A(1–8) analog, in rhesus monkeys

  Rationale: E-2078 ([N-methyl-Tyr¹, N-methyl-Arg⁷, d-Leu⁸] dynorphin A(1–8) ethylamide) is a dynorphin A(1–8) analog with a reduced tendency to be biotransformed, when compared to the unmodified opioid peptide. E-2078 has been found to produce κ-opioid agonist effects in vivo in rodents. Objective: In the present studies, we investigated whether systemically administered E-2078 could produce κ-agonist effects in rhesus monkeys, in tests of antinociception, diuresis and ethylketocyclazocine (EKC) discrimination. Methods: E-2078 (0.32–18 mg/kg, SC, IM or IV) was tested in the warm water (50°, 55°C) tail withdrawal assay of thermal antinociception. The diuretic effects of E-2078 (0.056– 1.8 mg/kg, SC) were also compared to those of the κ-agonist, U69,593 (0.01–0.32 mg/kg, SC). Lastly, the effects of E-2078 (0.1–3.2 mg/kg, SC or IV) were studied in rhesus monkeys trained to discriminate EKC (0.0056 mg/kg SC) from vehicle, in a food-reinforced operant procedure. Results: E-2078 did not produce thermal antinociception in rhesus monkeys following SC or IM administration, up to the largest doses presently studied (i.e., 18 and 10 mg/kg, respectively). E-2078 caused thermal antinociception by the IV route, but this effect was not apparently mediated by κ- or μ-opioid receptors, as shown by its insensitivity to quadazocine (1 mg/kg) pretreatment. However, SC E-2078 caused diuresis, and this effect was blocked by quadazocine pretreatment, consistent with mediation by κ-opioid receptors. E-2078 generalized in EKC-discriminating monkeys, but only after the largest dose (3.2 mg/kg), and only following IV administration. Conclusions: The present studies suggest that systemically administered E-2078 can produce some κ-receptor mediated effects in rhesus monkeys, but its profile of action is not identical to non-peptidic κ-agonists following all routes of administration, or across all experimental situations. Received: 5 August 1998 / Final version: 6 November 1998     

The effects of (±)-Praeruptorin A on airway inflammation,remodeling and transforming growth factor-β1/Smad signaling pathway in a murine model of allergic asthma

(±)-Praeruptorin A (PA) is a pair of coumarin enantiomers isolated from the root of Peucedanum praeruptorum Dunn (PPD), a common Chinese herbal medicine for the treatment of asthma. Considering its anti-inflammatory, anti-contractile and anti-hyperplasia activities, the effects of PA on airway inflammation and airway remodeling were investigated using a murine model of chronic asthma. Ovalbumin-sensitized BALB/c mice were challenged with ovalbumin to induce asthma every other day on eight successive weeks. PA was administered intragastrically before every ovalbumin challenge. Airway responsiveness was evaluated by a lung function analysis system 48 h after the last ovalbumin challenge. The total and differential leukocytes in bronchoalveolar lavage fluid (BALF) were counted using a hemocytometer and Diff-Quick-stained smears. Lung tissue samples were used for hematoxylin and eosin, periodic acid Schiff, Masson's trichrome and α-SMA immunohistochemistry staining. Levels of cytokines in BALF, immunoglobulin (Ig) E in serum as well as expression of TGF-β1 and Smad proteins in lung tissue were measured by enzyme-linked immunosorbent assay, immunohistochemistry or western blot analysis. Compared with the model group, PA suppressed airway inflammation, airway hyperresponsive and remodeling, reduced levels of IL-4 and IL-13 in BALF, and IgE in serum, inhibited expression of TGF-β1 and pSmad2/3, up-regulated the expression of Smad7 in lung tissue, and also increased the levels of INF-γ in BALF. These results suggested that PA significantly suppressed airway inflammation and airway remodeling induced by ovalbumin challenge, and is a potential candidate for the treatment of asthma.     

The peroxisome proliferator-activated receptor-α (PPAR-α) agonist,AVE8134, attenuates the progression of heart failure and increases survival in rats

Aim:

To investigate the efficacy of the peroxisome proliferator-activated receptor-α (PPARα) agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure.

Methods:

In Sprague Dawley rats with permanent ligation of the left coronary artery (post-MI), AVE8134 was compared to the PPARγ agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134 on cardiac hypertrophy and fibrosis was investigated. Finally, AVE8134 was administered to old spontaneously hypertensive rats (SHR) at a non-blood pressure lowering dose with survival as endpoint. In cellular models, we studied AVE8134 on hypertrophy in rat cardiomyocytes, nitric oxide signaling in human endothelial cells (HUVEC) and LDL-uptake in human MonoMac-6 cells.

Results:

In post-MI rats, AVE8134 dose-dependently improved cardiac output, myocardial contractility and relaxation and reduced lung and left ventricular weight and fibrosis. In contrast, rosiglitazone exacerbated cardiac dysfunction. Treatment at AVE8134 decreased plasma proBNP and arginine and increased plasma citrulline and urinary NOx/creatinine ratio. In DOCA rats, AVE8134 prevented development of high blood pressure, myocardial hypertrophy and cardiac fibrosis, and ameliorated endothelial dysfunction. Compound treatment increased cardiac protein expression and phosphorylation of eNOS. In old SHR, treatment with a low dose of AVE8134 improved cardiac and vascular function and increased life expectancy without lowering blood pressure. AVE8134 reduced phenylephrine-induced hypertrophy in adult rat cardiomyocytes. In HUVEC, Ser-1177-eNOS phosphorylation but not eNOS expression was increased. In monocytes, AVE8134 increased the expression of CD36 and the macrophage scavenger receptor 1, resulting in enhanced uptake of oxidized LDL.

Conclusion:

The PPARα agonist AVE8134 prevents post-MI myocardial hypertrophy, fibrosis and cardiac dysfunction. AVE8134 has beneficial effects against hypertension-induced organ damages, resulting in decreased mortality. The compound exerts its protective properties by a direct effect on cardiomyocyte hypertrophy, but also indirectly via monocyte signaling and increased endothelial NO production.     

SAR150640, a selective β3-adrenoceptor agonist,prevents human myometrial remodelling and activation of matrix metalloproteinase in an in vitro model of chorioamnionitis

Background and purpose:

The uterine pathophysiology underlying inflammatory conditions such as chorioamnionitis remains largely unclear. As we have shown that β₃-adrenoceptors act as regulators of myometrial inflammation, we wanted to investigate the potential role of β₃-adrenoceptors in preventing uterine remodelling induced by inflammation.

Experimental approach:

The consequences of human chorioamnionitis on myometrial remodelling were characterized by Sirius Red staining and metalloproteinase (MMP) expression, and compared with the effects of incubating human myometrial samples with Escherichia coli lipopolysaccharide (LPS) in vitro. We also assessed the effect of SAR150640, a selective β₃-adrenoceptor agonist, on the production and activity of MMPs.

Key results:

Chorioamnionitis was associated with a 46% decrease in total collagen, as well as over-expression of MMP2 (+61%) and MMP9 (+84%); both effects were reproduced by incubation with LPS (10 µg·mL⁻¹, 48 h). LPS-induced over-expression of MMP2 and MMP9 in normal human myometrium was paralleled by an overactivity of the proteins. Both over-expression and overactivity were prevented by the β₃-adrenoceptor agonist SAR150640 in a concentration-dependent manner. SAR150640, by itself, did not exhibit any effect on MMP production in control tissues.

Conclusions and implications:

This study shows that inflammation was associated with an intense remodelling of human myometrium, a process likely to be explained by MMP activation. Our study emphasizes the potential therapeutic relevance of β₃-adrenoceptor agonists to the treatment of preterm labour and other uterine inflammatory conditions.     

Dynorphin A (2–17) attenuates the unconditioned but not the conditioned effects of opiate withdrawal in the rat

OBJECTIVES: An unbiased place preference conditioning procedure was used to examine the influence of the non-opioid peptide, dynorphin A 2-17 (DYN 2-17), upon the conditioned and unconditioned effects of opiate withdrawal in the rat. METHODS: Rats were implanted SC with two pellets containing 75 mg morphine or placebo. Single-trial place conditioning sessions with saline and the opioid receptor antagonist naloxone (0.1-1.0 mg/kg; SC) commenced 4 days later. Ten minutes before SC injections, animals received an IV infusion of saline or DYN 2-17 (0.1-5.0 mg/kg). Additional groups of placebo- and morphine-pelleted animals were conditioned with saline and DYN 2-17. During each 30-min conditioning session, somatic signs of withdrawal were quantified. Tests of place conditioning were conducted in pelleted animals 24 h later. RESULTS: Naloxone produced wet-dog shakes, body weight loss, ptosis and diarrhea in morphine-pelleted animals. Morphine-pelleted animals also exhibited significant aversions for an environment previously associated with the administration of naloxone. These effects were not observed in placebo-pelleted animals. DYN 2-17 pretreatment resulted in a dose-related attenuation of somatic withdrawal signs. However, conditioned place aversions were still observed in morphine-pelleted animals that had received DYN 2-17 in combination with naloxone. Furthermore, the magnitude of this effect did not differ from control animals. CONCLUSIONS: These data demonstrate that the administration of DYN 2-17 attenuates the somatic, but not the conditioned aversive effects of antagonist-precipitated withdrawal from morphine in the rat. Differential effects of this peptide in modulating the conditioned and unconditioned effects of opiate withdrawal are suggested.     

CHF5074, a novel γ-secretase modulator,attenuates brain β-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease

Background and purpose:

We evaluated the effects of 1-(3′,4′-dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074), a new γ-secretase modulator, on brain β-amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP).

Experimental approach:

Sixty 6-month-old hAPP mice were treated for 6 months with CHF5074 or ibuprofen (375 ppm in the diet) or standard diet. Twenty-one wild-type mice received standard diet.

Key results:

Compared with transgenic controls, CHF5074 treatment significantly reduced the area occupied by plaques in cortex (P = 0.003) and hippocampus (P = 0.004). The number of plaques were also reduced by CHF5074 in both cortex (P = 0.022) and hippocampus (P = 0.005). Plaque-associated microglia in CHF5074-treated animals was lower than in transgenic controls in cortex (P = 0.008) and hippocampus (P = 0.002). Ibuprofen treatment significantly reduced microglia area in cortex and hippocampus but not β-amyloid burden. On the last day of the Morris water maze, transgenic controls performed significantly worse than the non-transgenic animals and the CHF5074-treated transgenic mice, on the swimming path to reach the hidden platform. Ibuprofen-treated animals did not perform significantly better than transgenic controls.

Conclusions and implications:

Chronic CHF5074 treatment reduced brain β-amyloid burden, associated microglia inflammation and attenuated spatial memory deficit in hAPP mice. This novel γ-secretase modulator is a promising therapeutic agent for Alzheimer''s disease.     

Anti-arthritic effects of chlorogenic acid in interleukin-1β-induced rabbit chondrocytes and a rabbit osteoarthritis model

Cartilage degradation is one of the pathological changes of osteoarthritis (OA), and accumulating evidence suggests an excess of matrix metalloproteinases (MMPs) plays a role in this cartilage breakdown. Here, we investigated the effects of chlorogenic acid (CGA) on the mRNA and protein expression of MMPs in interleukin (IL)-1β-induced rabbit chondrocytes and evaluated the in vivo effects of CGA in experimental OA induced by anterior cruciate ligament transection (ACLT) in rabbits. Using quantitative real-time PCR and ELISA to investigate the expression levels of MMP-1, MMP-3, MMP-13, and tissue inhibitors of metalloproteinase-1(TIMP-1) in IL-1β-induced rabbit chondrocytes, we showed that CGA inhibits the expression of these MMPs while increasing TIMP-1 expression, at both the mRNA and protein levels. In addition, IL-1β-induced activation of nuclear factor kappa B (NF-κB) and the degradation of inhibitor of κB (IκB)-α were suppressed by CGA. In rabbits, CGA decreased cartilage degradation as assessed by morphological and histological analyses. The down-regulation of MMP-1, MMP-3, and MMP-13 expression and up-regulation of TIMP-1 expression were also detected in CGA-treated cartilage compared with vehicle-treated cartilage, confirming these findings in an in vivo model. Taken together, these findings indicate that CGA may be considered as a possible candidate agent in the treatment of OA.     

Possible involvement of GLP-1(9�C36) in the regional haemodynamic effects of GLP-1(7�C36) in conscious rats

BACKGROUND AND PURPOSE

The incretin hormone, glucagon-like peptide (GLP)-1(7–36), is rapidly cleaved by dipeptidyl peptidase 4 (DPP-4) into GLP-1(9–36), and although it is agreed that most, if not all, of the metabolic effects are attributable to the intact peptide, the degree to which the cardiovascular effects are due to the cleavage product is unclear. The purpose of this study was to measure the regional haemodynamic effects of GLP-1(7–36), and determine the extent to which the cardiovascular effects of GLP-1(7–36) were influenced by DPP-4 inhibition and reproduced by GLP-1(9–36). Additional experiments investigated the involvement of autonomic mechanisms in the cardiovascular effects of GLP-1(7–36).

EXPERIMENTAL APPROACH

Regional haemodynamic effects of bolus doses and 4 h infusions of GLP-1(7–36) amide and GLP-1(9–36) amide were measured in conscious, chronically instrumented rats; the influence of DPP-4 inhibition and autonomic blockade on responses to GLP-1(7–36) were also assessed.

KEY RESULTS

Glucagon-like peptide-1(7–36) had clear regional haemodynamic effects comprising tachycardia, a rise in blood pressure, renal and mesenteric vasoconstriction and hindquarters vasodilatation, whereas GLP-1(9–36) was devoid of any cardiovascular actions. The effects of GLP-1(7–36) were enhanced by DPP-4 inhibition, and the tachycardia and hindquarters vasodilatation were β-adrenoceptor-mediated.

CONCLUSIONS AND IMPLICATIONS

In conscious rats, the cardiovascular effects of GLP-1(7–36) resemble those of the GLP analogue, exendin-4, and are attributable to the intact peptide rather than the cleavage product, GLP-1(9–36).     

Lack of weight gain after angiotensin AT1 receptor blockade in diet-induced obesity is partly mediated by an angiotensin-(1–7)/ Mas-dependent pathway

Background and Purpose

Angiotensin AT₁ receptor antagonists induce weight loss; however, the mechanism underlying this phenomenon is unknown. The Mas receptor agonist angiotensin-(1-7) is a metabolite of angiotensin I and of angiotensin II. As an agonist of Mas receptors, angiotensin-(1-7) has beneficial cardiovascular and metabolic effects.

Experimental Approach

We investigated the anti-obesity effects of transgenically overexpressed angiotensin-(1-7) in rats. We secondly examined whether weight loss due to telmisartan (8 mg·kg⁻¹·d⁻¹) in diet-induced obese Sprague Dawley (SD) rats can be blocked when the animals were co-treated with the Mas receptor antagonist A779 (24 or 72 μg·kg⁻¹·d⁻¹).

Key Results

In contrast to wild-type controls, transgenic rats overexpressing angiotensin-(1-7) had 1.) diminished body weight when they were regularly fed with chow; 2.) were protected from developing obesity although they were fed with cafeteria diet (CD); 3.) showed a reduced energy intake that was mainly related to a lower CD intake; 5.) remained responsive to leptin despite chronic CD feeding; 6.) had a higher, strain-dependent energy expenditure, and 7.) were protected from developing insulin resistance despite CD feeding. Telmisartan-induced weight loss in SD rats was partially antagonized after a high, but not a low dose of A779.

Conclusions and Implications

Angiotensin-(1-7) regulated food intake and body weight and contributed to the weight loss after AT₁ receptor blockade. Angiotensin-(1-7)-like agonists may be drug candidates for treating obesity.     

Evaluation of the effects of dopexamine,a novel DA1 receptor and β2-adrenoceptor agonist,on cardiac function and splanchnic circulation in a canine model of hemorrhagic shock

Summary In the present studies, the efficacy of dopexamine hydrochloride, a novel DA₁-receptor and ₂-adrenoceptor agonist in preventing deterioration of cardiovascular function in a canine model of hemorrhagic shock was investigated. Pentobarbital-anesthetized dogs were allowed to bleed into a height-regulated reservoir and the hypotensive state (about 40 mmHg) was maintained for a period of 150 min. Subsequently, blood was reinfused and recoveries in various hemodynamic variables were monitored for an additional period of 120 min. Either aqueous solvent or dopexamine HCl was randomly selected for i. v. infusion beginning 30 min before reinfusion of the blood and until the termination of the experiment.In the solvent-treated control group, various cardiovascular variables such as cardiac output, stroke volume, celiac and superior mesenteric arterial blood flows progressively declined to 50% or less of the basal values; these changes were associated with sustained increases in the regional as well as systemic vascular resistances. Dopexamine infusion lowered vascular resistances and facilitated recoveries in various hemodynamic variables to 80% to 100% of the basal values after reinfusion of the shed blood. With the exception of a transient inotropic effect during reinfusion in the dopexamine treated group, there were no essential alterations in the myocardial contractility, during the hypotensive state and/or after reinfusion of the blood.Hence, the results indicate that the efficacy of dopexamine to reduce vascular resistance by actions at DA₁-receptors and ₂-adrenoceptors would account for its ability to improve myocardial performance (secondary to reductions in afterload) and restoration of mesenteric and celiac hemodynamics. These salutary effects of dopexamine could be useful in the clinical management of hemorrhagic shock. Correspondence to B. S. Jandhyala at the above address     

Differential behavioural and neurochemical outcomes from chronic paroxetine treatment in adolescent and adult rats: a model of adverse antidepressant effects in human adolescents?

Selective serotonin reuptake inhibitor use is associated with increased risk of suicidal ideation in adolescent humans, yet the neuropharmacological basis of this phenomenon is unknown. Consequently, we examined the behavioural and neurochemical effects of chronic paroxetine (PRX) treatment in adult and adolescent rats. Rats received PRX in their drinking water (target dose 10 mg/kg) for 22 d, during which time they were assessed for depression- and anxiety-like behaviours. Subsequent ex-vivo analyses examined serum PRX concentrations, striatal neurotransmitter content, and regional serotonin and dopamine transporter (SERT, DAT) binding density. After 11-12 d treatment, PRX-treated adolescent rats showed a significant inhibition of social interaction while adults were unaffected. After 19-20 d treatment, adolescents failed to show an antidepressant-like effect of PRX treatment on the forced swim test (FST), while PRX-treated adults showed a typical decrease in immobility and increase in swimming. Two PRX-treated adolescents died unexpectedly after the FST suggesting a compromised response to physical stress. Despite their greater apparent adverse reaction to the drug, adolescents had significantly lower plasma PRX than adults at day 22 of treatment. Chronic PRX treatment had similar effects in adults and adolescents on striatal 5-HT (unchanged relative to controls) and 5-HIAA levels (decreased), while markers of dopaminergic function (DOPAC, HVA, DA turnover) were increased in adults only. SERT density was up-regulated in the amygdala in PRX-treated adolescents only while DAT density in the nucleus accumbens was down-regulated only in PRX-treated adults. These data suggest that the immature rat brain responds differently to PRX and that this might be of use in modelling the atypical response of human adolescents to antidepressants. The age-specific PRX-induced changes in dopaminergic markers and SERT and DAT binding provide clues as to the neural mechanisms underlying adverse PRX effects in adolescent humans.     

Section 1 Ibuprofen (‘Brufen’) tested with bilateral oral surgery as a model for evaluation of anti-inflammatory effects

Summary

Twenty-four healthy patients, who had two separate operations for the removal of an impacted 3rd molar from one side or the other of the lower jaw, took part in a double-blind crossover study of ibuprofen (1200?mg. daily) and placebo given for 5 days, commencing the day before surgery. A number of objective and subjective assessments were recorded for paired comparison of the post-operative course, including swelling (measured by a special device), trismus, and pain. Ibuprofen significantly reduced pain on the day of the operation, which might have accounted in part for less trismus and patient preference for ibuprofen treatment. Patients with less swelling after ibuprofen were not always those with high serum concentrations of the drug. Side-effects were infrequent and ibuprofen did not have any significant effect on wound healing, bleeding, or various haematological parameters investigated.     

Overlapping but distinct effects of genistein and ethinyl estradiol (EE2) in female Sprague–Dawley rats in multigenerational reproductive and chronic toxicity studies

Genistein and ethinyl estradiol (EE₂) were examined in multigenerational reproductive and chronic toxicity studies that had different treatment intervals among generations. Sprague–Dawley rats received genistein (0, 5, 100, or 500 ppm) or EE₂ (0, 2, 10, or 50 ppb) in a low phytoestrogen diet. Nonneoplastic effects in females are summarized here. Genistein at 500 ppm and EE₂ at 50 ppb produced similar effects in continuously exposed rats, including decreased body weights, accelerated vaginal opening, and altered estrous cycles in young animals. At the high dose, anogenital distance was subtly affected by both compounds, and a reduction in litter size was evident in genistein-treated animals. Genistein at 500 ppm induced an early onset of aberrant cycles relative to controls in the chronic studies. EE₂ significantly increased the incidence of uterine lesions (atypical focal hyperplasia and squamous metaplasia). These compound-specific effects appeared to be enhanced in the offspring of prior exposed generations.     

Steroid withdrawal in the mouse results in anxiogenic effects of 3α,5β-THP: a possible model of premenstrual dysphoric disorder

Rationale 3α-OH-5α[β]-pregnan-20-one (THP) is a positive modulator of the GABA_A receptor (GABAR), which underlies its reported anxiolytic effect. However, there are conditions such as premenstrual dysphoric disorder (PMDD) where increases in THP levels can be associated with adverse mood.Objectives In order to test for conditions where THP might be anxiogenic, we developed a mouse model of THP withdrawal. Because δ-containing GABAR are highly sensitive to THP modulation, results were compared in wild-type and δ knockout mice.Methods Finasteride, a 5α-reductase blocker, was administered for 3 days to female wild-type or δ knockout mice. Then, animals were tested in the elevated plus maze, following acute administration of THP, lorazepam, flumazenil, or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), and results compared to vehicle-injected controls. CA1 hippocampal GABAR α4 subunit levels were assessed by Western blot.Results After THP withdrawal, THP produced anxiogenic effects, decreasing open arm entries on the elevated plus maze, following a brief shock, in contrast to its expected anxiolytic effects. As we have shown in rats, THP withdrawal also resulted in increased expression of the α4 subunit in mouse CA1 hippocampus. As expected for increases in α4-containing GABAR, THP withdrawn mice were relatively insensitive to the benzodiazepine (BDZ) lorazepam and had atypical responses to the BDZ antagonist flumazenil when tested on the plus maze. In contrast, they showed a greater anxiolytic response to THIP, which has greater efficacy at α4βδ than other GABAR. Although THP withdrawal in δ knockout mice also increased the α4 GABAR subunit, the anxiogenic effects of THP and the anxiolytic effects of THIP were not observed, implicating α4βδ GABAR in these effects.Conclusions Based on these behavioral and pharmacological findings, we suggest that THP withdrawal in the mouse may serve as a rodent model of PMDD.     

Safety of dietary conjugated α-linolenic acid (CLNA) in a neonatal pig model

The aim of the present study was to perform a short-term safety evaluation of dietary mono-conjugated α-linolenic acid isomers (CLNA; c9-t11-c15-18:3 + c9-t13-c15-18:3) using a neonatal pig model. CLNA diet was compared with three other dietary fats: (1) conjugated linoleic acid (CLA; c9-t11-18:2 + t10-c12-18:2), (2) non-conjugated n-3 PUFA and (3) n-6 PUFA. Thirty-two piglets weaned at 3 weeks of age were distributed into four dietary groups. Diets were isoenergetic and food intake was controlled by a gastric tube. Mono-CLNA diet did not significantly change body or organ weight, carcass composition and most biochemical parameters including; glucose, cholesterol, triglycerides, creatinine, blood urea nitrogen, hepatic enzymes and electrolytes levels in blood (P ⩾ 0.09). Conversely, the n-3 PUFA composition of the brain, liver and heart decreased by 6–21% in the CLNA-fed group compared to animals fed nonconjugated n-3 PUFA (P < 0.01). Responses to dietary treatments were tissue-specific, with the liver and the brain being the most deprived in n-3 PUFA. Our results support that short-term intake of mono-CLNA is safe in neonatal pigs but n-3 PUFA reduction in tissues deserves to be further investigated before using long-term nutritional supplementation in pigs and other animal models and before moving to clinical trials.     

Angiotensin (1–7) protects against stress-induced gastric lesions in rats

Stress ulcers can develop with severe physiological stress, and have been proposed as being brain-driven events. New findings continue to suggest that stress ulcers can be more effectively managed through central manipulation rather than by simply altering local gastric factors. Angiotensin (1–7) (Ang (1–7)) is present as an endogenous constituent of the brain and stomach. The beneficial effects of Ang (1–7) have been confirmed in the vessels, brain, heart, kidney, liver and lungs, but not in the stomach. Given the accumulating evidence suggesting the anti-stress activities of Ang (1–7), its potential gastroprotective effect in the context of stress requires further investigation. In the present study, rat gastric mucosal lesions were induced by 2 h of cold-restraint stress. We observed that these lesions were significantly attenuated after 1 week of intracerebroventricular treatment with Ang (1–7). This gastroprotective effect was associated with attenuated oxidative stress and suppressed acid secretion. Brain Ang (1–7) administration profoundly modified responses to stress, indicated by altered levels of several stress hormones, including Ang II, glucocorticoid, norepinephrine, serotonin, and dopamine, in blood or stress-related brain regions. These findings indicate that Ang (1–7) exerts anti-stress activities by restoring the gastric microenvironment and modulating the stress pathways. Ang (1–7) may be a promising agent for stress ulcer prophylaxis and therapy, administered through brain-permeable mimics or carriers.     

The effects of the 2-amino-tetrahydronaphthalene derivative M7, a selective α2-adrenoceptor agonist in vitro

Summary M7 was originally reported to be a selective presynaptic ₂-adrenoceptor agonist in the pithed rat preparation. Subsequent work showed that M7 also stimulated postsynaptic ₂-adrenoceptors in this preparation, producing a pressor response. We have now investigated the selectivity of M7 for ₂- and ₁-adrenoceptors in vitro. Our results demonstrate that M7 is very potent in stimulating presynaptic ₂-adrenoceptors in the rat vas deferens and postsynaptic ₂-adrenoceptors in the dog saphenous vein. However, at higher doses M7 is also an ₁-adrenoceptor agonist, its ED₅₀ at ₁-adrenoceptors being approximately 60 fold greater than that at postsynaptic ₂-adrenoceptors. It is clear that the postsynaptic effects of M7 will depend upon the relative proportions of ₁- and ₂-adrenoceptors contained in the tissue under study.