Maria M. Winnicka

Cannabinoids enhance gastric X/A - like cells activity

It has been reported that cannabinoids may cause overeating in humans and in laboratory animals. Although, endogenous cannabinoids and their receptors (CB1) have been found in the hypothalamus, and recently also in gastrointestinal tract, the precise mechanism of appetite control by cannabinoids remains unknown. Recently, ghrelin--a hormone secreted mainly from the stomach X/A-like cells was proposed to be an appetite stimulating agent. The aim of this study was the evaluation of the influence of a single ip injection of a stable analogue of endogenous cannabinoid--anandamide, R-(+)-methanandamide (2.5 mg/kg) and CP 55,940 (0.25 mg/kg), an exogenous agonist of CB1 receptors, on ghrelin plasma concentration and on ghrelin immunoreactivity in the gastric mucosa of male Wistar rats. Four hours after a single injection of both cannabinoids or vehicle, the animals were anaesthetized and blood was taken from the abdominal aorta to determinate plasma ghrelin concentration by RIA. Subsequently, the animals underwent resection of distal part of stomach. Immunohistochemical study of gastric mucosa, using the EnVision method and specific monoclonal antibodies against ghrelin was performed. The intensity of ghrelin immunoreactivity in X/A-like cells was analyzed using Olympus Cell D image analysis system. The attenuation of ghrelin-immunoreactivity of gastric mucosa, after a single injection of R-(+)-methanandamide and CP 55,940 was accompanied by a significant increase of ghrelin plasma concentration. These results indicate that stimulation of appetite exerted by cannabinoids may be connected with an increase of ghrelin secretion from gastric X/A-like cells.

CCN2 protein is an announcing marker for cardiac remodeling following STZ-induced moderate hyperglycemia in mice

Diabetes causes changes in the myocardium, which are often called diabetic cardiomyopathy. This condition has been extensively investigated in animal models with high glucose levels. Nevertheless, it has not been investigated whether moderate hyperglycemia, in the absence of other features of metabolic syndrome, may also cause similar changes in the heart. The aim of the study was to assess changes in the myocardium in an animal model of mild type 1 diabetes. Moderate hyperglycemia was induced in 8- to 10-week-old male C57BL6J mice by 5 intraperitoneal injections of streptozotocin (40 mg/kg). After 16 weeks, they were sacrificed, and left ventricle (LV) dimensions and extent of cardiac fibrosis were assessed by morphometry. The abundance of CCN proteins in LVsamples was assessed using western blotting, while activity of metalloproteinase 2 was established in zymography. Real time PCR was used to investigate the expression of transforming growth factor beta1 (TGFbeta1) and atrial natriuretic peptide. Mice with moderate hyperglycemia presented comparable cardiac dimensions with fibrosis and hypertrophy parameters as the non-diabetic controls. However, the abundance of profibrotic CCN2 protein was significantly increased in hyperglycemic animals (1.67 +/- 0.28 vs. 1 +/- 0.47, p < 0.05). Interestingly, this change was independent from the TGFbeta1 expression, as its RNA abundance was similar in both groups. Moderate hyperglycemia also caused an increase in the activity of the metalloproteinase 2 (1.21 +/- 0.17 vs. 1 +/- 0.07, p < 0.05). Despite diabetes, no profound changes in cardiac morphology were found. In our animal model, moderate hyperglycemia caused activation of a profibrotic gene expression program, which was counterbalanced by the increase of metalloproteinase activity.

Coronary sinus concentrations of interleukin 6 and its soluble receptors are affected by reperfusion and may portend complications in patients with myocardial infarction

UNLABELLEDnInterleukin 6 (IL-6) is a pleiotropic cytokine involved in both inflammatory reaction and myocardial response to stress. Its effects largely depend on the concentration of the soluble receptors (sIL-6R and sgp130). We investigated the production of IL-6, sIL-6R and sgp130 by the heart during ischemia and reperfusion.nnnMETHODSnThe levels of IL-6 were determined in blood of 34 patients with first myocardial infarction (STEMI), left anterior descending (LAD) artery occlusion, otherwise normal coronaries, without significant co-morbidities and 16 comparable subjects with stable ischemic heart disease and lesion in LAD. Blood samples from coronary sinus (CS) and aorta (Ao) were drawn before percutaneous intervention (PCI), immediately after and at the end of the procedure. Venous blood from 30 healthy volunteers served as control.nnnRESULTSnSTEMI patients presented high IL-6 concentrations that increased further after reperfusion when its levels in CS became significantly higher than in Ao. In both groups prior to the PCI there were significantly higher concentrations of sIL-6R in Ao than in CS. This difference disappeared immediately after reperfusion. STEMI patients who experienced cardiovascular complications had higher IL-6 concentration and higher transcardiac sIL-6R gradient than patients with event-free hospitalisation. This association was confirmed in multivariate logistic regression analysis. Myocardial infarction increases concentration of IL-6 that is further elevated by reperfusion. A transcardiac gradient of sIL-6R during ischemia may indicate that large amounts of soluble IL-6 receptors are bound to the infarcted heart and thus affect signal transduction. IL-6 and initial sIL-6R gradient may portend complications in STEMI patients.

AM251, cannabinoids receptors ligand, improves recognition memory in rats

High density of cannabinoid receptors type 1 (CB1) in the brain suggests that endocannabinoid system plays an important role in the functioning of the central nervous system. Natural and synthetic cannabinoids are known to attenuate learning and memory processes. The adverse effects of cannabinoids are reversed by SR141716A, at first reported to be a selective CB1 receptor antagonist, later shown to possess also inverse agonist properties. The present study was performed in an attempt to determine the influence of different doses of AM251, a member of the same cannabinoid group as SR141716A, on recognition memory evaluated in an object recognition test. Because cannabinoids may alter motor function and affect anxiety, the influence of AM251 on psychomotor activity and anxiety was assessed in an open-field test and elevated plus maze, respectively. While the lowest dose of AM251 (1.0 mg/kg) significantly improved recognition memory, higher doses (2.5 mg/kg and 5.0 mg/kg) did not have an influence on it. Moreover, AM251 did not affect anxiety but in the highest dose significantly attenuated psychomotor activity in rats. The main finding of the present study indicates that AM251, at the dose of 1.0 mg/kg, improves recognition memory in rats without alteration of their psychomotor activity and anxiety. The pro-cognitive effect exerted by compounds belonging like AM251 to diarylpyrazole group may be beneficial in therapeutic use of these compounds, especially in patients with cognitive dysfunctions.

Effect of interleukin 6 deficiency on the expression of Bcl-2 and Bax in the murine heart

Interleukin 6 (IL-6) is a pleiotropic cytokine that is highly expressed in response to ischemia and reperfusion. It has dichotomous roles in the heart, functioning both as an inflammatory mediator as well as a protective agent. The aim of this study was to evaluate the effect of IL-6 deficiency on the expression of apoptotic regulatory proteins under both baseline conditions and following induction of ischemia and reperfusion in the mouse heart. C57BL/6J IL-6-/-(TMKopf) (IL6KO) and C57BL/6J mice (WT) were subjected to 30 minutes of local reversible myocardial ischemia in vivo or a sham operation. The expression of Bcl-2, Bax and STAT3 in the heart was assessed by western blotting. Under both baseline conditions and following the sham operation, IL-6 deficiency was associated with reduced expression of Bcl-2 and Bax. The TUNEL-FITC, Evans blue and tetrazolium chloride staining of the hearts following ischemia and reperfusion revealed similar injury in operated IL6KO and WT animals. There was increased STAT3 phosphorylation in operated mice regardless of the genotype. Bcl-2 and Bax expression was also comparable between the mouse strains following ischemia and reperfusion. In summary, these results indicated that IL-6 deficiency affected the basal expression of apoptotic regulators, but this did not profoundly alter the extent of reperfusion injury or apoptosis in the mouse heart following ischemia and reperfusion.

Remodeling of the intercalated disc related to aging in the mouse heart

BACKGROUNDnAging is related to declined cardiac hemodynamic function. As pumping performance may be significantly related to slowed ventricular depolarization and non-synchronous contraction, we hypothesized that aging may cause dysfunction of intercalated disc (ID), which is the structure responsible for intercellular electrical communication between cardiomyocytes.nnnMETHODSnMale C57BL/6J mice were used for the study at two ages: 4 and 24 months. Electrocardiographic recording was made to analyze the time of ventricular depolarization. Then mice were killed, and the hearts were harvested for examination in transmission electron microscopy (TEM) and immunofluorescence imaging. The expression of connexin 43 (Cx43), N-cadherin, and β-catenin in the myocardium of the left ventricle was evaluated using Western blotting.nnnRESULTSnIn senescent mice, analysis of averaged QRS complex showed its significant prolongation. At the ultrastructural level, we found frequent disruptions of the ID (affecting 29±5% of them), mainly at the site of adherens junction, with relatively preserved desmosomal intercellular connections and diminished number of gap junctions. Western blotting revealed significantly decreased abundance of Cx43 protein in aged animals, which may cause slowed impulse propagation through the gap junctions and contribute to the observed electrocardiographic alterations. The level of RNA for Cx43 is similar between young and old animals, which suggests a post-transcriptional mechanism of Cx43 protein downregulation.nnnCONCLUSIONSnOur study shows age-related disorganization of ID, which may be responsible for slowed conduction of the depolarization wave within the heart, and supports the hypothesis of cardiac dysfunction in senescence.

Analysis of gene expression profiles in tympanic membrane following perforation using PCR Array in rats--preliminary investigation.

OBJECTIVESnThe goal of this work was to identify genes, known to be involved in the skin wound healing, that express differentially in the healthy and injured tympanic membrane (TM), and designate the molecules potentially beneficial for treatment of TM perforation. The molecular mechanisms controlling the course of TM regeneration are far from being elucidated.nnnMETHODSnTwenty rats had their tympanic membranes perforated, while four served as a control. Animals were sacrificed on either days 1, 2, 3, 5 and 10 post injury, and TMs were immediately dissected and frozen in liquid nitrogen. Total TM RNA was isolated and reversely transcribed. qPCR was performed using Rat Wound Healing RT(2) Profiler PCR Array (QIAGEN) containing primers for 84 genes.nnnRESULTSnStatistically significant changes in the expression of 42 genes were found in various stages of TM healing. The increased expression of genes taking part in the inflammatory reaction (interleukin 6, granulocyte and macrophage chemotactic proteins) was observed from day 2. The expression of several genes of extracellular matrix components and their remodeling enzymes was also changed. Among growth factor genes: Vegfa, Igf1 and Hbegf showed increased expression at the beginning of the healing process, while Hgf expression was highest on day 3.nnnCONCLUSIONSnSeveral changes in the expression of genes involved in remodeling of extracellular matrix point to important role of connective tissue in TM healing. The molecules accelerating this process, like HbEGF and HGF, seem to be good candidates for further evaluation of their possible use in clinical treatment.

Interleukin 6 modulates PPARα and PGC-1α and is involved in high-fat diet induced cardiac lipotoxicity in mouse.

BACKGROUNDnInterleukin 6 (IL-6) may be involved in regulation of cardiac lipid metabolism and mitochondrial function through its influence on peroxisome proliferator-activated receptors (PPARs). In this study we evaluated the impact of the physiological level of IL-6 on the expression of PPARα and PGC-1α in the heart and the effect of lack of this cytokine on high-fat diet (HFD) induced lipotoxicity.nnnMETHODSnMale C57BL6/J wild type (WT) and IL-6 knock-out (IL-6KO) mice were used. 20 animals of each genotype were fed with HFD for 15-18weeks. Cardiac function was assessed using echocardiography and cardiomyocyte ultrastructure was examined using electron microscopy. QT-PCR and Western blotting were applied to estimate the expression of PPARα and PGC-1α at the transcriptional and protein levels.nnnRESULTSnAt baseline WT and IL-6KO mice had similar size and function of the left ventricle. HFD induced similar left ventricular hypertrophic response in both groups without causing heart failure, but only WT animals had increased resting ejection fraction of the LV. Ultrastructure of HFD groups showed markers of lipotoxicity, that were more pronounced in IL-6KO group. In basal conditions IL-6KO animals had lower PPARα and similar PGC-1α expression as compared to WT. HFD induced downregulation of both PPARα and PGC-1α in WT animals, while in IL-6KO mice this effect was constrained.nnnCONCLUSIONnIL-6 is involved in basal regulation of PPARα and PGC-1α expression in cardiomyocytes. The lack of this cytokine promotes high-fat diet induced lipotoxicity but without overt manifestations of cardiac failure.

Estrogens modulate RANKL-RANK/osteoprotegerin mediated interleukin-6 effect on thyrotoxicosis-related bone turnover in mice.

Interleukin-6 has been shown to cause imbalance between bone resorption and formation in thyrotoxicosis. The aim of the present study was an attempt to estimate the influence of estrogens on thyrotoxicosis-related disturbances in bone turnover in relation to RANKL-RANK/osteoprotegerin system in IL-6 deficient mice. The study was performed on 56, 12-13 weeks old, female mice: C57BL/6J (wild-type; WT) and C57BL/6J (IL6-/-Kopf) (IL-6 knock-out; IL6KO). The mice were randomly divided into 8 groups with 7 mice in each one: 1. WT controls, 2. IL6KO controls, 3. WT mice with thyrotoxicosis, 4. IL6KO mice with thyrotoxicosis, 5. WT ovariectomized, 6. IL6KO ovariectomized, 7. WT ovariectomized mice with thyrotoxicosis, and 8. IL6KO ovariectomized mice with thyrotoxicosis. Experimental model of menopause was evoked by bilateral ovariectomy carried out in 8-9 weeks old mice. Thyrotoxicosis was induced by intraperitoneal injection of levothyroxine at a dose of 1 μg/g daily over 21 days. The serum levels of TRACP5b, osteocalcin, OPG, and RANKL were determined by ELISA. RANKL serum concentrations were elevated significantly in all groups of ovariectomized mice as compared to respective controls, however, in a minor degree in IL6KO thyrotoxic mice as compared to wild-type animals. Osteoprotegerin serum levels were significantly increased in all thyrotoxic groups of mice except ovariectomized IL6KO animals. To sum up, the results of the present study suggest that IL-6 plays a key role in stimulation of RANKL-RANK/OPG system and this effect is strongly enhanced in conditions of accelerated bone turnover such as thyrotoxicosis and/or estrogen depletion.

CP55,940 attenuates spatial memory retrieval in mice

BACKGROUNDnCannabinoids constitute a varied group of lipophilic substances able to infiltrate the blood-brain barrier and influence neuronal processes. Clinical observations supported by experimental data have revealed that these compounds exert a deleterious effect on cognitive processes. The present study was carried out to determine the influence of a single systemic administration of CP55,940, a potent synthetic agonist of cannabinoid receptors, on spatial memory retrieval assessed in a Morris water maze.nnnMETHODSnC57BL/6J male mice were submitted to three consecutive days of training to find a hidden platform in the water maze. CP55,940 was given intraperitoneally once, at doses of 0.025, 0.125 or 0.25mg/kg on the fourth day, 30min before testing memory retrieval, and in separate groups before testing psychomotor activity and anxiety level in a hole-board test.nnnRESULTSnCP55,940 only at the highest dose of 0.25mg/kg significantly altered all parameters used to assess spatial memory. It increased the latency in the first crossing of the former platform location (target area), decreased the number of target area crossings and shortened the time spent in the target quadrant. Moreover, CP55,940 at doses of 0.25 and 0.125mg/kg attenuated motor and exploratory activity in hole-board test.nnnCONCLUSIONnSince the attenuated psychomotor activity after a dose of 0.125mg/kg did not interfere with memory retrieval, we assume that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be excluded.