Preclinical evaluation of the effects on the gastrointestinal tract of the antineoplastic drug vincristine repeatedly administered to rats.

BACKGROUND: Vincristine is a commonly used chemotherapeutic agent. It is associated with undesirable digestive side effects. However, the impact of vincristine on gastrointestinal structure and motility or its long-term effects have not been deeply studied in animal models. This could be useful in order to develop therapeutic or preventive strategies for cancer patients. The aim of this study was to analyze such effects. METHODS: Rats received saline or vincristine (0.1 mg kg-1 , ip) daily for 10 days. Evaluations were performed during treatment and 2-6 weeks after. Somatic mechano-sensitivity was assessed using von Frey hairs. Gastrointestinal motor function was studied by means of radiographic still images and colonic propulsion of fecal pellets using fluoroscopy videos. Histological assessment of the gut morphology and immunohistochemistry for HuC/D and nNOS were performed in whole-mount myenteric plexus preparations. KEY RESULTS: Peripheral sensitivity was increased in animals treated with vincristine and did not subside 2 weeks after treatment finalization. Vincristine treatment inhibited gastrointestinal motility although this was recovered to normal values with time. Damage in the digestive wall after vincristine treatment was greater in the ileum than in the colon. Villi shortening (in ileum) and large inflammatory nodules still remained 2 weeks after treatment finalization. Finally, the proportion of nNOS-immunoreactive neurons was increased with vincristine and continued to be increased 2 weeks after treatment finalization. CONCLUSIONS AND INFERENCES: Vincristine alters gastrointestinal motility, peripheral sensitivity and mucosal architecture. Vincristine-induced neuropathy (somatic and enteric), intestinal mucosa damage and inflammatory infiltrations are relatively long-lasting.

Pepsin egg white hydrolysate ameliorates metabolic syndrome in high-fat/high-dextrose fed rats.

The aim of this study was to examine the effect of a pepsin egg white hydrolysate (EWH) on metabolic complications using a high-fat/high-dextrose diet-induced Metabolic Syndrome (MetS) experimental model. Male Wistar rats were divided into 4 groups which received: standard diet and water (C), standard diet and a solution with 1 g kg-1 day-1 of EWH (CH), high-fat/high-dextrose diet and water (MS), and high-fat/high-dextrose diet and a solution with 1 g kg-1 day-1 of EWH (MSH). EWH consumption normalized body weight gain; abdominal obesity and peripheral neuropathy developed in MetS animals, and adipose tissue and liver weight, as well as plasma glucose were reduced. Oxidative stress and inflammation biomarkers were normalized in MSH animals. In conclusion, the oral administration of EWH could be used as a functional food ingredient to improve some complications associated with MetS induced by unhealthy diets.

Blockade of sigma 1 receptors alleviates sensory signs of diabetic neuropathy in rats.

BACKGROUND: E-52862 (S1RA, 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl]-morpholine), a novel selective sigma 1 receptor (σ1R) antagonist, has demonstrated efficacy in nociceptive and neuropathic pain models. Our aim was to test if σ1R blockade with E-52862 may modify the signs of neuropathy in Zucker diabetic fatty (ZDF) rats, a type 2 diabetes model. METHODS: Mechanical and thermal response thresholds were tested on 7-, 13-, 14- and 15-week-old ZDF rats treated with saline or with E-52862 acutely administered on week 13, followed by sub-chronic administration (14 days). Axonal peripheral activity (skin-saphenous nerve preparation) and isolated aorta or mesenteric bed reactivity were analysed in 15-week-old ZDF rats treated with saline or E-52862 and in LEAN rats. RESULTS: Zucker diabetic fatty rats showed significantly decreased thermal withdrawal latency and threshold to mechanical stimulation on week 13 compared to week 7 (prediabetes) and with LEAN animals; single-dose and sub-chronic E-52862 administration restored both parameters to those recorded on week 7. Regarding axonal peripheral activity, E-52862 treatment increased the mean mechanical threshold (77.3 ± 21 mN vs. 19.6 ± 1.5 mN, saline group) and reduced the response evoked by mechanical increasing stimulation (86.4 ± 36.5 vs. 352.8 ± 41.4 spikes) or by repeated mechanical supra-threshold steps (39.4 ± 1.4 vs. 83.5 ± 0.9). E-52862 treatment also restored contractile response to phenylephrine in aorta and mesenteric bed. CONCLUSIONS: E-52862 administration reverses neuropathic (behavioural and electrophysiological) and vascular signs in the ZDF rat. SIGNIFICANCE: Blockade of σ1R avoids the development of diabetic neuropathy in rats, and may represent a potentially useful therapeutic approach to peripheral neuropathies in diabetic patients. WHAT DOES THIS STUDY ADD?: This study presents evidences for the potential usefulness of sigma receptor blockade on diabetic neuropathy in rats. The methodology includes behavioural evidences, electrophysiological data and vascular-isolated models.

Pepsin Egg White Hydrolysate Ameliorates Obesity-Related Oxidative Stress, Inflammation and Steatosis in Zucker Fatty Rats.

The aim of this work was to evaluate the effect of the administration of egg white hydrolysates on obesity-related disorders, with a focus on lipid metabolism, inflammation and oxidative stress, in Zucker fatty rats. Obese Zucker rats received water, pepsin egg white hydrolysate (750 mg/kg/day) or Rhizopus aminopeptidase egg white hydrolysate (750 mg/kg/day) for 12 weeks. Lean Zucker rats received water. Body weight, solid and liquid intakes were weekly measured. At the end of the study, urine, faeces, different organs and blood samples were collected. The consumption of egg white hydrolysed with pepsin significantly decreased the epididymal adipose tissue, improved hepatic steatosis, and lowered plasmatic concentration of free fatty acids in the obese animals. It also decreased plasma levels of tumor necrosis factor-alpha and reduced oxidative stress. Pepsin egg white hydrolysate could be used as a tool to improve obesity-related complications.

Effects of chronic dietary exposure to monosodium glutamate on feeding behavior, adiposity, gastrointestinal motility, and cardiovascular function in healthy adult rats.

BACKGROUND: Monosodium glutamate (MSG) is a flavor-enhancer widely used as a food additive. However, its safe dietary concentration and its toxicity, including its possible implication in the recent metabolic syndrome pandemia, is still a controversial issue. Therefore, a deep knowledge of its effects upon regular dietary use is needed. Our aim was to evaluate the effects of chronic exposure to MSG on feeding behavior, abdominal fat, gastrointestinal motility, and cardiovascular function in rats. METHODS: Two groups of adult male Wistar rats were used: control and treated with MSG (4 g/L in drinking water) for 6 weeks. Different functional parameters were determined and the histological structure was analyzed in tissues of interest. KEY RESULTS: Compared to control animals, chronic MSG increased water intake but did not modify food ingestion or body weight gain. Neither the abdominal fat volume nor the fat fraction, measured by magnetic resonance imaging, was modified by MSG. Monosodium glutamate did not alter general gastrointestinal motility, but significantly increased the colonic response to mechanical stimulation. It slightly reduced endothelium-dependent relaxation in aorta, without significantly modifying any other cardiovascular parameters. No significant histological alterations were detected in salivary glands, intestinal wall, aorta, heart, and kidney. CONCLUSIONS & INFERENCES: Chronic treatment with MSG in the adult rat increased water intake. This supports its potential to improve acceptance of low-fat regimens and to increase hydration in the elderly and sportspeople, often at risk of dehydration. Changes in colonic contractility and cardiovascular function could have some long-term repercussions warranting further research.

Cannabinoid-induced delayed gastric emptying is selectively increased upon intermittent administration in the rat: role of CB1 receptors.

BACKGROUND: Cannabinoids acutely administered depress central, cardiovascular and gastrointestinal functions. These effects might be modified upon repeated administration. Compared to the effects induced by daily administration, those induced by intermittent administration are less known. The effect of intermittent treatment with the CB1/CB2 cannabinoid agonist WIN55,212-2 (WIN) was studied in the rat. METHODS: Male rats received saline, vehicle or WIN at 0.5 (low-WIN) or 5 (high-WIN) mg kg(-1) week(-1) for 4 weeks. WIN effects on the central nervous system (cannabinoid tetrad tests), cardiovascular function and gastrointestinal motor function were evaluated after the first and last doses, and, where appropriate, 1 week after the last dose. To determine the involvement of CB1 receptors in the chronic effect of WIN, the CB1 receptor antagonist/inverse agonist AM251 (1 mg kg(-1)) was used. KEY RESULTS: High- (but not low-) WIN induced the four signs of the cannabinoid tetrad, and reduced gastrointestinal motility, but did not alter cardiovascular parameters. Upon chronic intermittent administration, tolerance did not clearly develop to WIN effects. Quite the opposite, depression of gastric emptying was intensified. No effect was long-lasting. Repeated administration of AM251 was more efficacious than single administration to block WIN chronic central effects, but the opposite occurred regarding lower intestinal motility. CONCLUSIONS & INFERENCES: Upon intermittent administration, hypersensitization may develop to some effects (particularly delayed gastric emptying) induced by cannabinoid agonists. CB1 antagonists/inverse agonists may show different efficacy upon repeated or single administration to block cannabinoid-induced central and gastrointestinal effects. Thus, cannabinoid effects are dependent on the pattern of drug administration.

Selective lack of tolerance to delayed gastric emptying after daily administration of WIN 55,212-2 in the rat.

The use of cannabinoids to treat gastrointestinal (GI) motor disorders has considerable potential. However, it is not clear if tolerance to their actions develops peripherally, as it does centrally. The aim of this study was to examine the chronic effects of the cannabinoid agonist WIN 55,212-2 (WIN) on GI motility, as well as those in the central nervous and cardiovascular systems. WIN was administered for 14 days, at either non-psychoactive or psychoactive doses. Cardiovascular parameters were measured in anaesthetized rats, whereas central effects and alterations in GI motor function were assessed in conscious animals using the cannabinoid tetrad and non-invasive radiographic methods, respectively. Tests were performed after first (acute effects) and last (chronic effects) administration of WIN, and 1 week after discontinuing treatment (residual effects). Food intake and body weight were also recorded throughout treatment. Blood pressure and heart rate remained unchanged after acute or chronic administration of WIN. Central activity and GI motility were acutely depressed at psychoactive doses, whereas non-psychoactive doses only slightly reduced intestinal transit. Most effects were reduced after the last administration. However, delayed gastric emptying was not and could, at least partially, account for a concomitant reduction in food intake and body weight gain. The remaining effects of WIN administration in GI motility were blocked by the CB1 antagonist AM 251, which slightly accelerated motility when administered alone. No residual effects were found 1 week after discontinuing cannabinoid treatment. The different systems show differential sensitivity to cannabinoids and tolerance developed at different rates, with delayed gastric emptying being particularly resistant to attenuation upon chronic treatment.

Characterization of the vasorelaxant mechanisms of the endocannabinoid anandamide in rat aorta.

BACKGROUND AND PURPOSE: Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta. EXPERIMENTAL APPROACH: Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide. KEY RESULTS: Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.

Vasorelaxant effect of Win 55,212-2 in rat aorta: new mechanisms involved.

R(+)-[2,3-dihydro-5-methyl-3-[(moroholinyl)methyl] pyrrolo [1,2,3-de]-1,4benzoxazinyl]-1(1-naphthalenyl) methanone mesylate (Win 55,212-2) is a synthetic cannabinoid classically classified as a potent CB(1) and CB(2) agonist with high stereoselectivity and a slight preference for CB(2) cannabinoid receptors. Its vascular actions are not always explained by its binding to these cannabinoid receptors and new targets are being proposed. The aim of this study was to further assess the vascular actions of Win 55,212-2. Isometric tension changes in response to a cumulative concentration-response curve of Win 55,212-2 (10(-9) M-10(-4) M) were recorded in aortic rings from male Wistar rats. The involvement of the endothelium, cannabinoid receptors, vanilloid receptors, and the release of calcitonin gene related peptide (CGRP) was tested. Win 55,212-2 caused a concentration-dependent vasorelaxation in rat aorta. This vascular effect was significantly inhibited by endothelial denudation, inhibition of nitric oxide synthesis, a CB(1) receptor antagonist, a transient receptor potential vanilloid-1 antagonist, capsaicin desensibilization, and a CGRP receptor antagonist (P<0.001). CB(2) and non-CB(1)/non-CB(2) receptor antagonists only caused a slight inhibitory effect in vasorelaxation to Win 55,212-2. The present findings indicate that endothelium and nitric oxide-dependent vasorelaxation induced by Win 55,212-2 mainly involves vanilloid receptors while CB(1), CB(2) and nonCB(1)/nonCB(2) cannabinoid receptors have a minor participation in its vascular effect.

Alpha-vascular responses after short-term and long-term inhibition of nitric oxide synthesis.

Apart from the direct actions of nitric oxide (NO) on vascular smooth muscle, this factor may regulate cardiovascular functions through specific actions on alpha-adrenergic constrictor mechanisms. In this study we aim to establish whether the inhibition of the synthesis of this mediator could alter the vasoconstrictor responses mediated by alpha-adrenoceptor stimulation. We have been able to demonstrate that the blockage of the NO synthase really does exist, when both short- and long-term treatments with Nomega-nitro-Larginine methyl ester (L-NAME) are carried out. We have evaluated the concentration-dependent contractions induced by the selective alpha1-adrenoceptor agonists methoxamine and phenylephrine in isolated rat aorta rings in the following groups of animals: control, short-term L-NAME-treated (100 mg/kg i.p. 20 min before subjecting the animals to the experiments) and long-term L-NAME-treated (100 mg/kg per day in the drinking water for 7, 21, or 45 days). We have also evaluated the pressor responses to methoxamine and to the selective alpha-adrenoceptor agonist B-HT 920 using the pithed rat preparation in the same groups of animals. The contractile responses to methoxamine and phenylephrine were similar in the rat aorta preparations from control and short-term L-NAME-treated animals. On the contrary, in the rat aorta preparations from long-term L-NAME-treated animals these responses were clearly reduced when compared with the corresponding responses in those from control animals, the reduction being more marked when the treatment lasted longer. The pressor responses to methoxamine were also similar in control and short-term L-NAME-treated pithed rats. Nevertheless, the responses to B-HT 920 were greater in the latter. On the other hand, the dose-response curves to both alpha-adrenoceptor agonists were shifted to the right in a non-parallel manner in rats treated long term with L-NAME, the shift being, in the case of B-HT 920, more accentuated when the treatment lasted 21 or 45 days than when it lasted only 7 days. These results indicate that the short-term decrease in NO synthesis does not modify the vascular smooth muscle responses mediated by alpha1-adrenoceptor stimulation, but it does induce a potentiation of sympathetic vasoconstriction mediated by alpha2-adrenoceptors. Nevertheless, the long-term inhibition of NO synthesis causes a compensating decrease in the alpha1- and alpha2-vascular smooth muscle contractile responses.

Mechanisms involved in SNP-induced relaxation and [Ca+]i reduction in piglet pulmonary and systemic arteries.

1. We have compared the mechanisms involved in sodium nitroprusside (SNP)-induced relaxation and [Ca2+]i reduction in isolated piglet pulmonary (PA) and mesenteric (MA) arteries. 2. SNP (10(-8) M-3x10(-5) M) evoked a concentration-dependent relaxation of PA and MA (pD2=6.66+/-0.06 and 6.74+/-0.14, respectively) stimulated by noradrenaline, which was markedly reduced by the guanylate cyclase inhibitor ODQ. In fura 2-incubated PA and MA, SNP produced a parallel reduction in contractile force and in [Ca2+]i, expressed as the ratio of emitted fluorescence at 340 and 380 nm (F340/F380). 3. The inhibition of the Na+/K+-ATPase after the incubation in a K+-free medium or the exposure to ouabain (10(-6) M) inhibited SNP-induced relaxation in MA but not in PA. SNP-induced relaxation was not attenuated by 80 mM KCl plus nifedipine (10(-6) M) but was inhibited by thapsigargin (2x10(-6) M; pD2=5.69+/-0.19 and 5.89+/-0.19 for PA and MA, respectively). 4. Pretreatment of PA with thapsigargin and MA with thapsigargin plus ouabain induced a stronger inhibition on the reduction in [Ca2+]i than on the relaxation induced by SNP, indicating the existence of Ca2+-independent mechanisms. 5. The activation of the Na+/K+-ATPase by the addition of KCl after the incubation in a K+-free medium similarly reduced [Ca2+]i in PA and MA, whereas it relaxed with much less efficacy PA than MA. 6. We conclude that SNP reduces [Ca2+]i and causes relaxation through the activation of SERCA in PA and SERCA and Na+/K+-ATPase in MA. However, Ca2+-independent mechanisms also contribute to SNP-induced effects.

Alpha-adrenoceptor-mediated pressor responses in pithed rats fed diets with different calcium contents.

Results of many clinical and experimental studies indicate an inverse relationship between dietary calcium and the prevalence of hypertension. Our study was designed to evaluate the alterations in arterial blood pressure and the changes in alpha-adrenoceptor-mediated vascular reactivity in normotensive Sprague-Dawley and spontaneously hypertensive rats (SHR) fed from weaning (3 weeks of life) three diets: normal calcium (Ca 1%), low calcium (Ca 0.1%), and high calcium (Ca 2.5%). The systolic and the diastolic arterial blood pressures were measured weekly by the tail cuff method. The plasma calcium levels in the animals were also measured regularly by colourimetric methods, and the alpha-adrenoceptor-mediated vascular reactivity was evaluated by measuring the pressor responses to alpha-adrenoceptor agonists in pithed rats. These determinations were carried out at the end of the feeding periods (9 weeks of life in Sprague-Dawley rats and 20 weeks of life in SHR) and also at the moments when maximal differences in arterial blood pressure were observed between the conscious animals fed the normal calcium diet and those fed the other two diets. Dietary calcium deficiency increased arterial blood pressure in both strains but calcium supplements were effective to lower this only in hypertensive animals. The plasma calcium levels were altered in both strains when calcium administration was not normal. The low-calcium diet did not modify the pressor responses to either the alpha(1)-adrenoceptor agonist, methoxamine, or the alpha(2)-adrenoceptor agonist, B-HT 920 (5-allyl-2-amino-5,6,7, 8-tetrahydro-4H-thiazolo-(4,5-D)-acepin-dihydrochloride, talixepole), in the normotensive and the hypertensive rats. On the contrary, the high-calcium diet caused a definite decrease in alpha(1)- and alpha(2)-adrenoceptor-mediated vascular reactivity in both strains. The changes in the alpha-adrenoceptor-mediated vasoconstrictor responses were observed in pithed 9-week old Sprague-Dawley rats and in pithed 20-week old SHR, but none were observed in pithed 15-week old SHR, although at this age maximal differences in arterial blood pressure between the animals fed the high- and the normal calcium diet were observed. The results of this study suggest that the mechanisms implicated in the effects of dietary calcium supplements on arterial blood pressure are clearly different from the mechanisms, which bring about changes in arterial blood pressure when the diet is deficient in calcium. The results of this study also show that calcium administration causes variations in alpha-adrenoceptor-mediated vascular reactivity, but this is probably not the only mechanism implicated in the calcium effect on arterial blood pressure.

Modulation of adrenergic receptors during left ventricular hypertrophy development and after regression by captopril.

The objective of this study was to analyze adrenergic receptors during cardiac hypertrophy development, after establishment of cardiac hypertrophy and after regression of cardiac hypertrophy by an angiotensin-converting enzyme inhibitor. Left ventricular hypertrophy (LVH) was induced by abdominal aortic stenosis. After surgery, plasma norepinephrine concentrations (PNE) and left ventricular adrenergic receptors from rat hearts subjected to aortic stenosis were assessed during cardiac hypertrophy development (at 3, 7, 15, and 30 days of aortic stenosis), once cardiac hypertrophy had been established (7 and 14 weeks after the stenosis) and after regression of cardiac hypertrophy by an antihypertensive dose (200 mg/kg/day) of captopril. The presence of LVH was observed from day 7 after stenosis. PNE had significantly increased after 15 days but returned to control values 30 days after surgery. The density of alpha1-adrenoceptors was found to decrease with development of hypertrophy. Once hypertrophy had been established, 7 weeks from stenosis, PNE was not different from control; however, the density of alpha1-adrenoceptors continued to diminish, whereas PNE and the density of beta-adrenoceptors were no different from control values. Fourteen weeks after stenosis, a significant decrease in PNE was recorded, and no change in alpha1- but an increase in beta-adrenoceptors was observed. LVH was reversed by treatment with captopril; PNE was similar in control and stenosed treated animals. The density of alpha1-adrenoceptors was decreased when compared with control animals, and no change in the density of beta-adrenoceptors was observed with treatment. In conclusion, a decrease of alpha1-adrenoceptors was associated with LVH development and earlier stages of established cardiac hypertrophy. Later stages of established cardiac hypertrophy were characterized by no change in alpha1- and an increase in beta-adrenoceptors. Treatment with captopril induced LVH regression and decreased the number of alpha1-adrenoceptors without any change in beta-adrenoceptors.

Parathyroid hormone and calcitriol in hypertension caused by dietary calcium deficiency in rats.

Hypertension caused by calcium deficiency in the diet has been linked with an increase in parathyroid hormone (PTH) and calcitriol levels. We evaluated arterial blood pressure (ABP), PTH, and calcitriol in normotensive Sprague-Dawley rats (SDR) and in spontaneously hypertensive rats (SHR) fed from weaning on a control diet with a normal calcium content (1%) or a low-calcium diet (0.1%). The calcemia was also measured in the rats by colorimetric methods. The low-calcium diet decreased the calcemia in both strains and brought about an increase in the ABP which was significant in adult SDR and particularly noticeable during the early hypertensive phase in SHR. The rats fed on this diet had higher hormonal plasma levels when compared with the corresponding values in rats fed on the control diet. In particular, the SDR fed on the low-calcium diet showed much higher PTH (122.6 +/- 31.0 pg/ml, p

The role of endothelium in the calcium-induced reduction of the contractile response of the rabbit aorta.

1. Increase of Ca2+ concentration in the bath solution diminishes the contractile response of isolated rabbit aorta rings to alpha 1-adrenoceptor agonists and KCl. 2. In intact preparations the contractions of methoxamine and phenylephrine were maximal when a 0.3- to 0.6-mM Ca2+ bath solution was used, and those of KCl were maximal with a 2.5-mM Ca2+ concentration. 3. The contractions of methoxamine and phenylephrine also were decreased by increasing the Ca2+ concentration above 1.25 mM in disrupted endothelium preparations and in those incubated in indomethacin (10(-5) M), N omega-nitro-L-arginine methyl ester (10(-4) M), or methylene blue (10(-6) M). 4. High organ bath Ca2+ concentrations also caused a decrease in KCl contractions using endothelium-denuded and the treated preparations, the responses being similar with 1.25 mM and 2.5-mM Ca2+ in the methylene blue-treated preparations, whereas they were greater with 1.25 mM Ca2+ in the others.