The functional cooperation of 5−HT1A5-HT_{1A} and mGlu4R in HEK-293 cell line

Background The serotonin 5-HT1A receptor (5-HT1AR) and metabotropic glutamate receptor 4 (mGlu4) have been implicated as sites of antipsychotic drug action. 5-HT1AR belongs to the A class of G protein-coupled receptors (GPCRs); mGlu4 is a representative of class C GPCRs. Both receptors preferentially couple with Gi protein to inhibit cAMP formation. The present work aimed to examine the possibility of mGlu4 and 5-HT1A receptor cross-talk, the phenomenon that could serve as a molecular basis of the interaction of these receptor ligands observed in behavioral studies. Methods First, in vitro studies were performed to examine the pharmacological modulation of interaction of the mGlu4 and 5-HT1A receptors in the T-REx 293 cell line using SNAP- or HALO-tag and cAMP accumulation assay. Next, the colocalization of these two receptors was examined in some regions of the mouse brain by applying RNAScope dual fluorescence in situ hybridization, immunohistochemical labeling, and proximity ligation assay (PLA). Results The ex vivo and in vitro results obtained in the present work suggest the existence of interactions between mGlu4 and 5-HT1A receptors. The changes were observed in cAMP accumulation assay and were dependent on expression and activation of mGlu4R in T-REx 293cell line. Moreover, the existence of spots with proximity expression of both receptors were showed by PLA, immunofluorescence labeling and RNAscope methods. Conclusion The existence of interactions between mGlu4 and 5-HT1A receptors may represent another signaling pathway involved in the development and treatment psychiatric disorders such as schizophrenia or depression

Pioglitazone, a specific ligand of peroxisome proliferator-activated receptor-gamma, protects pancreas against acute cerulein-induced pancreatitis

AIM: To determine the effect of pioglitazone, a specific peroxisome proliferator-activated receptor-γ (PPARγ) ligand, on the development of acute pancreatitis (AP) and on the expression of heat shock protein 70 (HSP70) in the pancreas. METHODS: AP was induced in rats by subcutaneous infusion of cerulein for 5 h. Pancreatic blood flow was measured by laser Doppler flowmetry. Plasma lipase activity, interleukin-1β (IL-1β) and IL-10 were determined. Pancreatic weight and histology were evaluated and pancreatic DNA synthesis and blood flow as well as pancreatic mRNA for IL-1β and HSP70 were assessed in rats treated with pioglitazone alone or in combination with cerulein. RESULTS: Pioglitazone administered (10-100 mg/kg i.g.) 30 min before cerulein, attenuated dose-dependently the pancreatic tissue damage in cerulein-induced pancreatitis (CIP) as demonstrated by the improvement of pancreatic histology, reduction in plasma lipase activity, plasma concentration of pro-inflammatory IL-1β and its gene expression in the pancreas and attenuation of the pancreatitis-evoked fall in pancreatic blood flow. CIP increased pancreatic HSP70 mRNA and protein expression in the pancreas and this effect was enhanced by pioglitazone treatment. CONCLUSION: Pioglitazone attenuates CIP and the beneficial effect of this pioglitazone is multifactorial probably due to its anti-inflammatory activities, to the suppression of IL-1β and to the overexpression of HSP70. PPARγ ligands could represent a new therapeutic option in the treatment of AP

Design and synthesis of new quinazolin-4-one derivatives with negative mGlu7mGlu_7 receptor modulation activity and antipsychotic-like properties

Following the glutamatergic theory of schizophrenia and based on our previous study regarding the antipsychotic-like activity of mGlu7 NAMs, we synthesized a new compound library containing 103 members, which were examined for NAM mGlu7 activity in the T-REx 293 cell line expressing a recombinant human mGlu7 receptor. Out of the twenty-two scaffolds examined, active compounds were found only within the quinazolinone chemotype. 2-(2-Chlorophenyl)-6-(2,3-dimethoxyphenyl)-3-methylquinazolin-4(3H)-one (A9-7, ALX-171, mGlu7 IC50 = 6.14 µM) was selective over other group III mGlu receptors (mGlu4 and mGlu8), exhibited satisfactory drug-like properties in preliminary DMPK profiling, and was further tested in animal models of antipsychotic-like activity, assessing the positive, negative, and cognitive symptoms. ALX-171 reversed DOI-induced head twitches and MK-801-induced disruptions of social interactions or cognition in the novel object recognition test and spatial delayed alternation test. On the other hand, the efficacy of the compound was not observed in the MK-801-induced hyperactivity test or prepulse inhibition. In summary, the observed antipsychotic activity profile of ALX-171 justifies the further development of the group of quinazolin-4-one derivatives in the search for a new drug candidate for schizophrenia treatment

Der Einfluss von Gallensäuren auf die Entwicklung des Barrett Ösophagus und die Progression des Adenocarcinom. Die Rolle von der Apoptose, COX-2, NFkB, freier Radikale und CDX-2.

The present study was undertaken to investigate molecular changes in human biopsies from normal esophagus and Barrett’s esophagus (BE) on mRNA and protein level, analysis of factors that may play important role in pathogenesis of BE and its progression to BA like: NFkB,COX-2, CDX-2, ghrelin, adiponectin etc. In experimental in vitrio part fallowing ingestion were performed: 1) effects of two different bile acids deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) in neutral and acidic environment on expression of COX-2, DNA repair enzymes (MUTYH, OGG-1) and homebox protein CDX-2 gene; 2) effects on viability and apoptosis rate in esophageal adenocarcinoma cells OE-19; 3) influence of NFkB inhibition by MG-132 and siRNA technique on inflammatory related genes and CDX-2; 4) effect of potent therapeutic agents ghrelin and adiponectin on OE-19 cell line. Results of this thesis show that patients with Barrett’s esophagus have much higher rate of pathologic biliary reflux than healthy people. Thus, bile and its components like bile salts have significant effect on development of BE and progression to BA. In the biopsies from BE, compared to the normal esophagus a significant upregulation of one of the key transcription factors NFkB was observed. Furthermore, elevated expression of genes and proteins involved in cell survival, cell cycle and cell homeostasis was demonstrated. In vitro experiment showed multiple biological effects of bile salts on Barrett’s adenocarcinoma cell line including induction of NFkB and upregulation of inflammatory related genes including COX-2. Moreover, bile salts like DCA may influence cell cycle and apoptosis and generally they can play the role as clone selection factors in development of BE. This biological activity of some bile salts enable for clonal selection and finally cancer development. Additionally, the dependence between inflammation and Barrett’s metaplasia phenotype was confirmed via network of NFkB expression, activity and CDX-2 expression. Finally adiponectin and ghrelin have a beneficial effect on the Barrett’s carcinogenesis by two different mechanisms, via increase in apoptosis by adiponectin and anti-inflammatory actions by ghrelin. The decrease in level of these two peptides observed in obesity may explain why the progression of Barrett’s carcinoma is favored in obese individuals. In conclusion bile salts contribute to the Barrett’s carcinogenesis. However, UDCA has a lower carcinogenic effect on Barrett’s cells than DCA. This is due to its weaker stimulatory effect on NFkB activation, VEGF and COX-2 expressions with consecutive VEGF and PGE2 release. Bile salts inhibit DNA repair enzymes and induce COX-2 expression. This last effect is at least partly mediated by NFkB. NFkB as an important component of inflammation forces the development of BE via CDX-2 expression.Die vorliegende Studie wurde durchgeführt, um bei Barrett-Ösophagus (BÖ) molekulare Änderungen anhand humaner Biopsien zu erforschen, basierend auf Untersuchungen der mRNA und des Proteinmuster, wobei folgende Faktoren eine entscheidende Rolle in der Pathogenese von BÖ und seiner Progression zu Barrett-Adenokarzinom (BA) zugesprochen wird: NFkB, COX-2, CDX-2, Ghrelin, Adiponektin. In experimentellen in vitro Untersuchungen wurde im Anschluss an die Nahrungsaufnahme Folgendes untersucht: 1) die Effekte zweier verschiedener Gallensäuren, Desoxycholsäure (DCA) und Ursodesoxycholsäure (UDCA), auf die Expression von COX-2, CDX-2 und DNA Reparaturenzymen (MUTYH, OGG-1) sowohl im neutralen als auch im saurem Milieu; 2) die Effekte auf die Proliferations- und Apoptoserate in esophagen Adenokarzinomzellen OE-19; 3) der Einfluss der NFkB Inhibition durch einen spezifischen Inhibitor (MG-132) oder siRNA Technik auf proinflammatorische Gene und CDX-2; 4) der Effekt der möglichen Therapeutika Ghrelin und Adiponektin auf OE-19 Zelllinien. Ergebnisse dieser Arbeit zeigen, dass Patienten mit Barett-Ösophagus mit einer deutlich höheren Rate unter dem pathologischen biliären Reflux leiden, als gesunde Menschen. Folglich übernehmen die Galle und ihre Komponenten, wie z.B. Gallensalze einen signifikanten Effekt auf die Entstehung von BÖ und auf die Progression zu BA. In den BÖ Biopsien wurde, verglichen zum gesunden Esophagus, eine signifikante Hochregulierung des Transkriptionsfaktors NFkB beobachtet. Weiterhin wurde gezeigt, dass die hochregulierten Gene und Proteine sowohl den Zellzyklus beeinflussen, als auch an der Zellhomöostase beteiligt sind, und somit das Überleben der Zelle sichern. In in vitro Experimenten wurden multiple biologische Effekte der Gallensalze auf die BA-Zellen nachgewiesen, z.B. Induktion von NFkB und Hochregulierung der inflammatorischen Gene, u.a. Zyklooxygenase-2 (COX-2). Zudem beeinflussen Gallensalze, wie DCA, den Zyklus und die Apoptose der Zellen und somit könnten sie die Rolle eines Klonselektionsfaktor in der Entwicklung von BÖ übernehmen. Diese biologische Aktivität der Gallensalze, welche die Klonalselektion ermöglichen, könnte schließlich die Krebsentstehung begünstigen. Außerdem konnte ein bestehender Zusammenhang von Inflammation und der Barrett-Metaplasie mit der zusammenhängenden Expression von NFkB und CDX-2 bestätigt werden

Serotonergic–Muscarinic Interaction within the Prefrontal Cortex as a Novel Target to Reverse Schizophrenia-Related Cognitive Symptoms

Recent studies revealed that the activation of serotonergic 5-HT1A and muscarinic M1, M4, or M5 receptors prevent MK-801-induced cognitive impairments in animal models. In the present study, the effectiveness of the simultaneous activation of 5-HT1A and muscarinic receptors at preventing MK-801-induced cognitive deficits in novel object recognition (NOR) or Y-maze tests was investigated. Activators of 5-HT1A (F15599), M1 (VU0357017), M4 (VU0152100), or M5 (VU0238429) receptors administered at top doses for seven days reversed MK-801-induced deficits in the NOR test, similar to the simultaneous administration of subeffective doses of F15599 (0.05 mg/kg) with VU0357017 (0.15 mg/kg), VU0152100 (0.05 mg/kg), or VU0238429 (1 mg/kg). The compounds did not prevent the MK-801-induced impairment when administered acutely. Their activity was less evident in the Y-maze. Pharmacokinetic studies revealed high brain penetration of F15599 (brain/plasma ratio 620%), which was detected in the frontal cortex (FC) up to 2 h after administration. Decreases in the brain penetration properties of the compounds were observed after acute administration of the combinations, which might have influenced behavioral responses. This negative effect on brain penetration was not observed when the compounds were administered repeatedly. Based on our results, prolonged administration of a 5-HT1A activator with muscarinic receptor ligands may be effective at reversing cognitive decline related to schizophrenia, and the FC may play a critical role in this interaction

Nitric Oxide-Dependent Mechanisms Underlying MK-801- or Scopolamine-Induced Memory Dysfunction in Animals: Mechanistic Studies

MK-801, an NMDA receptor antagonist, and scopolamine, a cholinergic receptor blocker, are widely used as tool compounds to induce learning and memory deficits in animal models to study schizophrenia or Alzheimer-type dementia (AD), respectively. Memory impairments are observed after either acute or chronic administration of either compound. The present experiments were performed to study the nitric oxide (NO)-related mechanisms underlying memory dysfunction induced by acute or chronic (14 days) administration of MK-801 (0.3 mg/kg, i.p.) or scopolamine (1 mg/kg, i.p.). The levels of L-arginine and its derivatives, L-citrulline, L-glutamate, L-glutamine and L-ornithine, were measured. The expression of constitutive nitric oxide synthases (cNOS), dimethylaminohydrolase (DDAH1) and protein arginine N-methyltransferases (PMRTs) 1 and 5 was evaluated, and the impact of the studied tool compounds on cGMP production and NMDA receptors was measured. The studies were performed in both the cortex and hippocampus of mice. S-nitrosylation of selected proteins, such as GLT-1, APP and tau, was also investigated. Our results indicate that the availability of L-arginine decreased after chronic administration of MK-801 or scopolamine, as both the amino acid itself as well as its level in proportion to its derivatives (SDMA and NMMA) were decreased. Additionally, among all three methylamines, SDMA was the most abundant in the brain (~70%). Administration of either compound impaired eNOS-derived NO production, increasing the monomer levels, and had no significant impact on nNOS. Both compounds elevated DDAH1 expression, and slight decreases in PMRT1 and PMRT5 in the cortex after scopolamine (acute) and MK-801 (chronic) administration were observed in the PFC, respectively. Administration of MK-801 induced a decrease in the cGMP level in the hippocampus, accompanied by decreased NMDA expression, while increased cGMP production and decreased NMDA receptor expression were observed after scopolamine administration. Chronic MK-801 and scopolamine administration affected S-nitrosylation of GLT-1 transport protein. Our results indicate that the analyzed tool compounds used in pharmacological models of schizophrenia or AD induce changes in NO-related pathways in the brain structures involved in cognition. To some extent, the changes resemble those observed in human samples