Apigenin Alleviates Autistic-like Stereotyped Repetitive Behaviors and Mitigates Brain Oxidative Stress in Mice.

Studying the involvement of nicotinic acetylcholine receptors (nAChRs), specifically α7-nAChRs, in neuropsychiatric brain disorders such as autism spectrum disorder (ASD) has gained a growing interest. The flavonoid apigenin (APG) has been confirmed in its pharmacological action as a positive allosteric modulator of α7-nAChRs. However, there is no research describing the pharmacological potential of APG in ASD. The aim of this study was to evaluate the effects of the subchronic systemic treatment of APG (10-30 mg/kg) on ASD-like repetitive and compulsive-like behaviors and oxidative stress status in the hippocampus and cerebellum in BTBR mice, utilizing the reference drug aripiprazole (ARP, 1 mg/kg, i.p.). BTBR mice pretreated with APG (20 mg/kg) or ARP (1 mg/g, i.p.) displayed significant improvements in the marble-burying test (MBT), cotton-shredding test (CST), and self-grooming test (SGT) (all p < 0.05). However, a lower dose of APG (10 mg/kg, i.p.) failed to modulate behaviors in the MBT or SGT, but significantly attenuated the increased shredding behaviors in the CST of tested mice. Moreover, APG (10-30 mg/kg, i.p.) and ARP (1 mg/kg) moderated the disturbed levels of oxidative stress by mitigating the levels of catalase (CAT) and superoxide dismutase (SOD) in the hippocampus and cerebellum of treated BTBR mice. In patch clamp studies in hippocampal slices, the potency of choline (a selective agonist of α7-nAChRs) in activating fast inward currents was significantly potentiated following incubation with APG. Moreover, APG markedly potentiated the choline-induced enhancement of spontaneous inhibitory postsynaptic currents. The observed results propose the potential therapeutic use of APG in the management of ASD. However, further preclinical investigations in additional models and different rodent species are still needed to confirm the potential relevance of the therapeutic use of APG in ASD.

The Potent and Selective Histamine H3 Receptor Antagonist E169 Counteracts Cognitive Deficits and Mitigates Disturbances in the PI3K/AKT/GSK-3ß Signaling Pathway in MK801-Induced Amnesia in Mice.

The role of histamine H3 receptors (H3Rs) in memory and the prospective of H3R antagonists in pharmacological control of neurodegenerative disorders, e.g., Alzheimer's disease (AD), is well-accepted. Therefore, the procognitive effects of acute systemic administration of H3R antagonist E169 (2.5-10 mg/kg, i.p.) on MK801-induced amnesia in C57BL/6J mice using the novel object recognition test (NORT) were evaluated. E169 (5 mg) provided a significant memory-improving effect on MK801-induced short- and long-term memory impairments in NORT. The E169 (5 mg)-provided effects were comparable to those observed with the reference phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and were abrogated with the H3R agonist (R)-α-methylhistamine (RAMH). Additionally, our results demonstrate that E169 ameliorated MK801-induced memory deficits by antagonism of H3Rs and by modulation of the level of disturbance in the expression of PI3K, Akt, and GSK-3ß proteins, signifying that E169 mitigated the Akt-mTOR signaling pathway in the hippocampus of tested mice. Moreover, the results observed revealed that E169 (2.5-10 mg/kg, i.p.) did not alter anxiety levels and locomotor activity of animals in open field tests, demonstrating that performances improved following acute systemic administration with E169 in NORT are unrelated to changes in emotional response or in spontaneous locomotor activity. In summary, these obtained results suggest the potential of H3R antagonists such as E169, with good in silico physicochemical properties and stable retained key interactions in docking studies at H3R, in simultaneously modulating disturbed brain neurotransmitters and the imbalanced Akt-mTOR signaling pathway related to neurodegenerative disorders, e.g., AD.

Canagliflozin Ameliorates Oxidative Stress and Autistic-like Features in Valproic-Acid-Induced Autism in Rats: Comparison with Aripiprazole Action.

Based on their proven anti-inflammatory and antioxidant effects, recent studies have examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitors in neurodevelopmental disorders such as autism spectrum disorder (ASD). Therefore, the aim of this study is to assess the effects of subchronic systemic treatment with intraperitoneal (i.p.) canagliflozin (20, 50, and 100 mg/kg) compared to aripiprazole (ARP) (3 mg/g, i.p.) in a valproic acid (VPA)-induced rat model of autism. The behavioral characteristics of ASD, oxidative stress, and acetylcholinesterase (AChE) activity in rats with ASD-like behaviors, which were induced by prenatal exposure to VPA, were evaluated. The behavioral assessment methods used for this study were the open field test (OFT), the marble-burying test (MBT), and the nestlet-shredding test (NST) to examine their exploratory, anxiety, and compulsiveness-like actions, while the biochemical assessment used for this study was an ELISA colorimetric assay to measure ASD biomarker activity in the hippocampus, prefrontal cortex, and cerebellum. Rats that were pretreated with 100 mg/kg of canagliflozin displayed a significantly lower percentage of shredding (1.12 ± 0.6%, p < 0.01) compared to the ARP group (3.52 ± 1.6%). Pretreatment with (20 mg/kg, 50 mg/kg, and 100 mg/kg) canagliflozin reversed anxiety levels and hyperactivity and reduced hyper-locomotor activity significantly (161 ± 34.9 s, p < 0.05; 154 ± 44.7 s, p < 0.05; 147 ± 33.6 s, p < 0.05) when compared with the VPA group (303 ± 140 s). Moreover, canagliflozin and ARP mitigated oxidative stress status by restoring levels of glutathione (GSH) and catalase (CAT) and increasing the levels of malondialdehyde (MDA) in all tested brain regions. The observed results propose repurposing of canagliflozin in the therapeutic management of ASD. However, further investigations are still required to verify the clinical relevance of canagliflozin in ASD.

Experimental Studies Indicate That ST-2223, the Antagonist of Histamine H3 and Dopamine D2/D3 Receptors, Restores Social Deficits and Neurotransmission Dysregulation in Mouse Model of Autism.

Altered regulation of neurotransmitters may lead to many pathophysiological changes in brain disorders including autism spectrum disorder (ASD). Given the fact that there are no FDA-approved effective treatments for the social deficits in ASD, the present study determined the effects of chronic systemic treatment of the novel multiple-active H3R/D2R/D3R receptor antagonist ST-2223 on ASD-related social deficits in a male Black and Tan Brachyury (BTBR) mice. ST-2223 (2.5, 5, and 10 mg/kg, i.p.) significantly and dose-dependently mitigated social deficits and disturbed anxiety levels of BTBR mice (p < 0.05) in comparison to the effects of aripiprazole (1 mg/kg, i.p.). Moreover, levels of monoaminergic neurotransmitters quantified by LC-MS/MS in four brain regions including the prefrontal cortex, cerebellum, striatum, and hippocampus unveiled significant elevation of histamine (HA) in the cerebellum and striatum; dopamine (DA) in the prefrontal cortex and striatum; as well as acetylcholine (ACh) in the prefrontal cortex, striatum, and hippocampus following ST-2223 (5 mg/kg) administration (all p < 0.05). These in vivo findings demonstrate the mitigating effects of a multiple-active H3R/D2R/D3R antagonist on social deficits of assessed BTBR mice, signifying its pharmacological potential to rescue core ASD-related behaviors and altered monoaminergic neurotransmitters. Further studies on neurochemical alterations in ASD are crucial to elucidate the early neurodevelopmental variations behind the core symptoms and heterogeneity of ASD, leading to new approaches for the future therapeutic management of ASD.

Simultaneous Antagonism at H3R/D2R/D3R Reduces Autism-like Self-Grooming and Aggressive Behaviors by Mitigating MAPK Activation in Mice.

Dysregulation in brain neurotransmitters underlies several neuropsychiatric disorders, e.g., autism spectrum disorder (ASD). Also, abnormalities in the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway pave the way for neuroinflammation, neurodegeneration, and altered learning phenotype in ASD. Therefore, the effects of chronic systemic administration of the multiple-targeting antagonist ST-713 at the histamine H3 receptor (H3R) and dopamine D2/D3 receptors (D2/D3R) on repetitive self-grooming, aggressive behaviors, and abnormalities in the MAPK pathway in BTBR T + Itpr3tf/J (BTBR) mice were assessed. The results showed that ST-713 (2.5, 5, and 10 mg/kg, i.p.) mitigated repetitive self-grooming and aggression in BTBR mice (all p < 0.05), and the ameliorative effects of the most promising dose of ST-713 (5 mg/kg, i.p.) on behaviors were completely abrogated by co-administration of the H3R agonist (R)-α-methylhistamine or the anticholinergic drug scopolamine. Moreover, the elevated levels of several MAPK pathway proteins and induced proinflammatory markers such as tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were significantly suppressed following chronic administration of ST-713 (5 mg/kg, i.p.) (all p < 0.01). Furthermore, ST-713 significantly increased the levels of histamine and dopamine in hippocampal tissue of treated BTBR mice (all p < 0.01). The current observations signify the potential role of such multiple-targeting compounds, e.g., ST-713, in multifactorial neurodevelopmental disorders such as ASD.

Curcumin Potentiates α7 Nicotinic Acetylcholine Receptors and Alleviates Autistic-Like Social Deficits and Brain Oxidative Stress Status in Mice.

Autistic spectrum disorder (ASD) refers to a group of neurodevelopmental disorders characterized by impaired social interaction and cognitive deficit, restricted repetitive behaviors, altered immune responses, and imbalanced oxidative stress status. In recent years, there has been a growing interest in studying the role of nicotinic acetylcholine receptors (nAChRs), specifically α7-nAChRs, in the CNS. Influence of agonists for α7-nAChRs on the cognitive behavior, learning, and memory formation has been demonstrated in neuro-pathological condition such as ASD and attention-deficit hyperactivity disorder (ADHD). Curcumin (CUR), the active compound of the spice turmeric, has been shown to act as a positive allosteric modulator of α7-nAChRs. Here we hypothesize that CUR, acting through α7-nAChRs, influences the neuropathology of ASD. In patch clamp studies, fast inward currents activated by choline, a selective agonist of α7-nAChRs, were significantly potentiated by CUR. Moreover, choline induced enhancement of spontaneous inhibitory postsynaptic currents was markedly increased in the presence of CUR. Furthermore, CUR (25, 50, and 100 mg/kg, i.p.) ameliorated dose-dependent social deficits without affecting locomotor activity or anxiety-like behaviors of tested male Black and Tan BRachyury (BTBR) mice. In addition, CUR (50 and 100 mg/kg, i.p.) mitigated oxidative stress status by restoring the decreased levels of superoxide dismutase (SOD) and catalase (CAT) in the hippocampus and the cerebellum of treated mice. Collectively, the observed results indicate that CUR potentiates α7-nAChRs in native central nervous system neurons, mitigates disturbed oxidative stress, and alleviates ASD-like features in BTBR mice used as an idiopathic rodent model of ASD, and may represent a promising novel pharmacological strategy for ASD treatment.

The histamine H3R and dopamine D2R/D3R antagonist ST-713 ameliorates autism-like behavioral features in BTBR T+tf/J mice by multiple actions.

Several brain neurotransmitters, including histamine (HA), acetylcholine (ACh), and dopamine (DA) are suggested to be involved in several brain disorders including cognitive deficits, depression, schizophrenia, anxiety, and narcolepsy, all of which are comorbid with Autism spectrum disorder (ASD). Therefore, the ameliorative effects of the novel multiple-active compound ST-713 with high binding affinities at histamine H3 receptor (H3R), dopamine D2sR and D3R on ASD-like behaviors in male BTBR T+tf/J mice model were assessed. ST-713 (3-(2-chloro-10H-phenothiazin-10-yl)-N-methyl-N-(4-(3-(piperidin-1-yl)propoxy)benzyl)propan-1-amine; 2.5, 5, and 10 mg/kg, i.p.) ameliorated dose-dependently social deficits, and significantly alleviated the repetitive/compulsive behaviors of BTBR mice (all P < 0.05). Moreover, ST-713 modulated disturbed anxiety levels, but failed to obliterate increased hyperactivity of tested mice. Furthermore, ST-713 (5 mg/kg) attenuated the increased levels of hippocampal and cerebellar protein expressions of NF-κB p65, COX-2, and iNOS in BTBR mice (all P < 0.05). The ameliorative effects of ST-713 on social parameters were entirely reversed by co-administration of the H3R agonist (R)-α-methylhistamine or the anticholinergic drug scopolamine. The obtained results demonstrate the potential of multiple-active compounds for the therapeutic management of neuropsychiatric disorders, e.g. ASD.

The Multi-Targeting Ligand ST-2223 with Histamine H3 Receptor and Dopamine D2/D3 Receptor Antagonist Properties Mitigates Autism-Like Repetitive Behaviors and Brain Oxidative Stress in Mice.

Autism spectrum disorder (ASD) is a complex heterogeneous neurodevelopmental disorder characterized by social and communicative impairments, as well as repetitive and restricted behaviors (RRBs). With the limited effectiveness of current pharmacotherapies in treating repetitive behaviors, the present study determined the effects of acute systemic treatment of the novel multi-targeting ligand ST-2223, with incorporated histamine H3 receptor (H3R) and dopamine D2/D3 receptor affinity properties, on ASD-related RRBs in a male Black and Tan BRachyury (BTBR) mouse model of ASD. ST-2223 (2.5, 5, and 10 mg/kg, i.p.) significantly mitigated the increase in marble burying and self-grooming, and improved reduced spontaneous alternation in BTBR mice (all p < 0.05). Similarly, reference drugs memantine (MEM, 5 mg/kg, i.p.) and aripiprazole (ARP, 1 mg/kg, i.p.), reversed abnormally high levels of several RRBs in BTBR (p < 0.05). Moreover, ST-2223 palliated the disturbed anxiety levels observed in an open field test (all p < 0.05), but did not restore the hyperactivity parameters, whereas MEM failed to restore mouse anxiety and hyperactivity. In addition, ST-2223 (5 mg/kg, i.p.) mitigated oxidative stress status by decreasing the elevated levels of malondialdehyde (MDA), and increasing the levels of decreased glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in different brain parts of treated BTBR mice (all p < 0.05). These preliminary in vivo findings demonstrate the ameliorative effects of ST-2223 on RRBs in a mouse model of ASD, suggesting its pharmacological prospective to rescue core ASD-related behaviors. Further confirmatory investigations on its effects on various brain neurotransmitters, e.g., dopamine and histamine, in different brain regions are still warranted to corroborate and expand these initial data.

Simultaneous Blockade of Histamine H3 Receptors and Inhibition of Acetylcholine Esterase Alleviate Autistic-Like Behaviors in BTBR T+ tf/J Mouse Model of Autism.

Autism spectrum disorder (ASD) is a heterogenous neurodevelopmental disorder defined by persistent deficits in social interaction and the presence of patterns of repetitive and restricted behaviors. The central neurotransmitters histamine (HA) and acetylcholine (ACh) play pleiotropic roles in physiological brain functions that include the maintenance of wakefulness, depression, schizophrenia, epilepsy, anxiety and narcolepsy, all of which are found to be comorbid with ASD. Therefore, the palliative effects of subchronic systemic treatment using the multiple-active test compound E100 with high H3R antagonist affinity and AChE inhibitory effect on ASD-like behaviors in male BTBR T+tf/J (BTBR) mice as an idiopathic ASD model were assessed. E100 (5, 10 and 15 mg/kg, i.p.) dose-dependently palliated social deficits of BTBR mice and significantly alleviated the repetitive/compulsive behaviors of tested animals. Moreover, E100 modulated disturbed anxiety levels, but failed to modulate hyperactivity parameters, whereas the reference AChE inhibitor donepezil (DOZ, one milligram per kilogram) significantly obliterated the increased hyperactivity measures of tested mice. Furthermore, E100 mitigated the increased levels of AChE activity in BTBR mice with observed effects comparable to that of DOZ and significantly reduced the number of activated microglial cells compared to the saline-treated BTBR mice. In addition, the E100-provided effects on ASD-like parameters, AChE activity, and activated microglial cells were entirely reversed by co-administration of the H3R agonist (R)-α-methylhistamine (RAM). These initial overall results observed in an idiopathic ASD mice model show that E100 (5 mg/kg) alleviated the assessed behavioral deficits and demonstrate that simultaneous targeting of brain histaminergic and cholinergic neurotransmissions is crucial for palliation of ASD-like features, albeit further in vivo assessments on its effects on brain levels of ACh as well as HA are still needed.

The Dual-Active Histamine H3 Receptor Antagonist and Acetylcholine Esterase Inhibitor E100 Alleviates Autistic-Like Behaviors and Oxidative Stress in Valproic Acid Induced Autism in Mice.

The histamine H3 receptor (H3R) functions as auto- and hetero-receptors, regulating the release of brain histamine (HA) and acetylcholine (ACh), respectively. The enzyme acetylcholine esterase (AChE) is involved in the metabolism of brain ACh. Both brain HA and ACh are implicated in several cognitive disorders like Alzheimer's disease, schizophrenia, anxiety, and narcolepsy, all of which are comorbid with autistic spectrum disorder (ASD). Therefore, the novel dual-active ligand E100 with high H3R antagonist affinity (hH3R: Ki = 203 nM) and balanced AChE inhibitory effect (EeAChE: IC50 = 2 µM and EqBuChE: IC50 = 2 µM) was investigated on autistic-like sociability, repetitive/compulsive behaviour, anxiety, and oxidative stress in male C57BL/6 mice model of ASD induced by prenatal exposure to valproic acid (VPA, 500 mg/kg, intraperitoneal (i.p.)). Subchronic systemic administration with E100 (5, 10, and 15 mg/kg, i.p.) significantly and dose-dependently attenuated sociability deficits of autistic (VPA) mice in three-chamber behaviour (TCB) test (all p < 0.05). Moreover, E100 significantly improved repetitive and compulsive behaviors by reducing the increased percentage of marbles buried in marble-burying behaviour (MBB) (all p < 0.05). Furthermore, pre-treatment with E100 (10 and 15 mg/kg, i.p.) corrected decreased anxiety levels (p < 0.05), however, failed to restore hyperactivity observed in elevated plus maze (EPM) test. In addition, E100 (10 mg/kg, i.p.) mitigated oxidative stress status by increasing the levels of decreased glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), and decreasing the elevated levels of malondialdehyde (MDA) in the cerebellar tissues (all p < 0.05). Additionally, E100 (10 mg/kg, i.p.) significantly reduced the elevated levels of AChE activity in VPA mice (p < 0.05). These results demonstrate the promising effects of E100 on in-vivo VPA-induced ASD-like features in mice, and provide evidence that a potent dual-active H3R antagonist and AChE inhibitor (AChEI) is a potential drug candidate for future therapeutic management of autistic-like behaviours.

The dual-active histamine H3 receptor antagonist and acetylcholine esterase inhibitor E100 ameliorates stereotyped repetitive behavior and neuroinflammmation in sodium valproate induced autism in mice.

Postnatal exposure to valproic acid (VPA) in rodents induces autism-like neurobehavioral defects which are comparable to the motor and cognitive deficits observed in humans with autism spectrum disorder (ASD). Histamine H3 receptor (H3R) and acetylcholine esterase (AChE) are involved in several cognitive disorders such as Alzheimer's disease, schizophrenia, anxiety, and narcolepsy, all of which are comorbid with ASD. Therefore, the present study aimed at evaluating effect of the novel dual-active ligand E100 with high H3R antagonist affinity and balanced AChE inhibition on autistic-like repetitive behavior, anxiety parameters, locomotor activity, and neuroinflammation in a mouse model of VPA-induced ASD in C57BL/6 mice. E100 (5, 10, and 15 mg/kg) dose-dependently and significantly ameliorated repetitive and compulsive behaviors by reducing the increased percentages of nestlets shredded (all P < 0.05). Moreover, pretreatment with E100 (10 and 15 mg/kg) attenuated disturbed anxiety levels (P < 0.05) but failed to restore the hyperactivity observed in the open field test. Furthermore, pretreatment with E100 (10 mg/kg) the increased microglial activation, proinflammatory cytokines and expression of NF-κB, iNOS, and COX-2 in the cerebellum as well as the hippocampus (all P < 0.05). These results demonstrate the ameliorative effects of E100 on repetitive compulsive behaviors in a mouse model of ASD. To our knowledge, this is the first in vivo demonstration of the effectiveness of a potent dual-active H3R antagonist and AChE inhibitor against autistic-like repetitive compulsive behaviors and neuroinflammation, and provides evidence for the role of such compounds in treating ASD.

The histamine H3R antagonist DL77 attenuates autistic behaviors in a prenatal valproic acid-induced mouse model of autism.

Autistic spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in social communication and restricted/repetitive behavior patterns or interests. Antagonists targeting histamine H3 receptor (H3R) are considered potential therapeutic agents for the therapeutic management of different brain disorders, e.g., cognitive impairments. Therefore, the effects of subchronic treatment with the potent and selective H3R antagonist DL77 (5, 10, or 15 mg/kg, i.p.) on sociability, social novelty, anxiety, and aggressive/repetitive behavior in male Tuck-Ordinary (TO) mice with ASD-like behaviors induced by prenatal exposure to valproic acid (VPA, 500 mg/kg, i.p.) were evaluated using the three-chamber test (TCT), marble burying test (MBT), nestlet shredding test (NST), and elevated plus maze (EPM) test. The results showed that VPA-exposed mice exhibited significantly lower sociability and social novelty preference compared to VPA-exposed mice that were pretreated with DL77 (10 or 15 mg/kg, i.p.). VPA-exposed mice presented a significantly higher percentage of buried marbles in MBT and shredded nestlet significantly more in NST compared to the control groups. However, VPA-exposed animals pretreated with DL77 (10 or 15 mg/kg, i.p.) buried a reduced percentage of marbles in MBT and presented a significantly lower percentage of shredding behavior in NST. On the other hand, pretreatment with DL77 (5, 10, or 15 mg/kg, i.p.) failed to restore the disturbed anxiety levels and hyperactivity observed in VPA-exposed animals in EPM, whereas the reference drug donepezil (DOZ, 1 mg/kg, i.p.) significantly palliated the anxiety and reduced the hyperactivity measures of VPA-exposed mice. Furthermore, pretreatment with DL77 (10 or 15 mg/kg, i.p.) modulated oxidative stress status by increasing GSH and decreasing MDA, and it attenuated the proinflammatory cytokines IL-1ß, IL-6 and TNF-α exacerbated by lipopolysaccharide (LPS) challenge, in VPA-exposed mouse brain tissue. Taken together, these results provide evidence that modulation of brain histaminergic neurotransmission, such as by subchronic administration of the H3R antagonist DL77, may serve as an effective pharmacological therapeutic target to rescue ASD-like behaviors in VPA-exposed animals, although further investigations are necessary to corroborate and expand these initial data.

Curcumin potentiates the function of human α7-nicotinic acetylcholine receptors expressed in SH-EP1 cells.

Effects of curcumin, a biologically active ingredient of turmeric, were tested on the Ca2+ transients induced by the activation of α7 subunit of the human nicotinic acetylcholine (α7 nACh) receptor expressed in SH-EP1 cells. Curcumin caused a significant potentiation of choline (1 mM)-induced Ca2+ transients with an EC50 value of 133 nM. The potentiating effect of curcumin was not observed in Ca2+ transients induced by high K+ (60 mM) containing solutions or activation of α4ß2 nACh receptors and the extent of curcumin potentiation was not altered in the presence of Ca2+ channel antagonists nifedipine (1 µM), verapamil (1 µM), ω-conotoxin (1 µM), and bepridil (10 µM). Noticeably the effect of curcumin was not observed when curcumin and choline were co-applied without curcumin pre-incubation. The effect of curcumin on choline-induced Ca2+ transients was not reversed by pre-incubation with inhibitors of protein C, A, and CaM kinases. Metabolites of curcumin such as tetrahydrocurcumin, demethylcurcumin, and didemethylcurcumin also caused potentiation of choline-induced Ca2+ transients. Notably, specific binding of [125I]-bungarotoxin was not altered in the presence of curcumin. Collectively, our results indicate that curcumin allosterically potentiate the function of the α7-nACh receptor expressed in SH-EP1 cells.

Altered profile of mRNA expression in atrioventricular node of streptozotocin­induced diabetic rats.

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)­induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ­induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription­quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization­activated cyclic­nucleotide 2 and 3, calcium channel voltage­dependent, ß2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage­gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage­gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ­induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function.

Phenylalanine derivatives with modulating effects on human α1-glycine receptors and anticonvulsant activity in strychnine-induced seizure model in male adult rats.

The critical role of α1-glycine receptor (α1-GLYRs) in pathological conditions such as epilepsy is well known. In the present study, structure-activity relations for a series of phenylalanine derivatives carrying selected hydrogen bond acceptors were investigated on the functional properties of human α1-GLYR expressed in Xenopus oocytes. The results indicate that one particular substitution position appeared to be of special importance for control of ligand activity. Among tested ligands (1-8), the biphenyl derivative (2) provided the most promising antagonistic effect on α1-GLYRs, while its phenylbenzyl analogue (5) exhibited the highest potentiation effect. Moreover, ligand 5 with most promising potentiating effect showed in-vivo moderate protection when tested in strychnine (STR)-induced seizure model in male adult rats, whereas ligand 2 with highest antagonistic effect failed to provide appreciable anti(pro)convulsant effect. Furthermore, ligands 2 and 5 with the most promising effects on human α1-GLYRs were examined for their toxicity and potential neuroprotective effect against neurotoxin 6-hydroxydopamine (6-OHDA). The results show that ligands 2 and 5 possessed neither significant antiproliferative effects, nor necrotic and mitochondrial toxicity (up to concentration of 50µM). Moreover, ligand 2 showed weak neuroprotective effect at the 50µM against 100µM toxic dose of 6-OHDA. Our results indicate that modulatory effects of ligands 2 and 5 on human α1-GLYRs as well as on STR-induced convulsion can provide further insights for the design of therapeutic agents in treatment of epilepsy and other pathological conditions requiring enhanced activity of inhibitory glycine receptors.

Different Profile of mRNA Expression in Sinoatrial Node from Streptozotocin-Induced Diabetic Rat.

BACKGROUND: Experiments in isolated perfused heart have shown that heart rate is lower and sinoatrial node (SAN) action potential duration is longer in streptozotocin (STZ)-induced diabetic rat compared to controls. In sino-atrial preparations the pacemaker cycle length and sino-atrial conduction time are prolonged in STZ heart. To further clarify the molecular basis of electrical disturbances in the diabetic heart the profile of mRNA encoding a wide variety of proteins associated with the generation and transmission of electrical activity has been evaluated in the SAN of STZ-induced diabetic rat heart. METHODOLOGY/PRINCIPAL FINDINGS: Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of intercellular proteins, intracellular Ca2+-transport and regulatory proteins, cell membrane transport proteins and calcium, sodium and potassium channel proteins were measured in SAN and right atrial (RA) biopsies using real-time reverse transcription polymerase chain reaction techniques. Heart rate was lower in STZ (203±7 bpm) compared to control (239±11 bpm) rat. Among many differences in the profile of mRNA there are some worthy of particular emphasis. Expression of genes encoding some proteins were significantly downregulated in STZ-SAN: calcium channel, Cacng4 (7-fold); potassium channel, Kcnd2 whilst genes encoding some other proteins were significantly upregulated in STZ-SAN: gap junction, Gjc1; cell membrane transport, Slc8a1, Trpc1, Trpc6 (4-fold); intracellular Ca2+-transport, Ryr3; calcium channel Cacna1g, Cacna1h, Cacnb3; potassium channels, Kcnj5, Kcnk3 and natriuretic peptides, Nppa (5-fold) and Nppb (7-fold). CONCLUSIONS/SIGNIFICANCE: Collectively, this study has demonstrated differences in the profile of mRNA encoding a variety of proteins that are associated with the generation, conduction and regulation of electrical signals in the SAN of STZ-induced diabetic rat heart. Data from this study will provide a basis for a substantial range of future studies to investigate whether these changes in mRNA translate into changes in electrophysiological function.

Enhanced Glucose Tolerance and Pancreatic Beta Cell Function by Low Dose Aspirin in Hyperglycemic Insulin-Resistant Type 2 Diabetic Goto-Kakizaki (GK) Rats.

BACKGROUND/AIM: Type 2 diabetes is the most common metabolic disorder, characterized by insulin resistance and pancreatic islet beta-cell failure. The most common complications associated with type 2 diabetes are hyperinsulinemia, hyperglycemia, hyperlipidemia, increased inflammatory and reduced insulin response. Aspirin (ASA) and other non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with the prevention of diabetes, obesity and related cardiovascular disorders. Aspirin has been used in many clinical and experimental trials for the prevention of diabetes and associated complications. METHODS: In this study, five month old Goto-Kakizaki (GK) rats, which showed signs of mild hyperglycemia (fasting blood glucose 80-95 mg/dl vs 55-60 mg/dl Wistar control rats) were used. Two subgroups of GK and Wistar control rats were injected intraperitoneally with 100 mg aspirin/kg body weight/ day for 5 weeks. Animals were sacrificed and blood and tissues were collected after performing glucose tolerance (2 h post 2g IP glucose ingestion) tests in experimental and control groups. RESULTS: Aspirin caused a moderate decrease in hyperglycemia. However, we observed a significant improvement in glucose tolerance after ASA treatment in GK rats compared to the nondiabetic Wistar rats. Also, the ASA treated GK rats exhibited a significant decrease in insulinemia. ASA treatment also caused a marked reduction in the pro-inflammatory prostaglandin, PGE2, which was significantly higher in GK rats. On the other hand, no significant organ toxicity was observed after ASA treatment at this dose and time period. However, the total cholesterol and lipoprotein levels were significantly increased in GK rats, which decreased after ASA treatment. Immunofluorescence staining for insulin/glucagon secreting pancreatic cells showed improved beta-cell structural and functional integrity in ASA-treated rats which was also confirmed by SDS-PAGE and Western blot analysis. CONCLUSION: The improved glucose tolerance in ASA-treated GK rats may be associated with increased insulin responses due to the anti-inflammatory properties of ASA and enhanced nitric oxide (NO) level which facilitated insulin signaling and energy utilization in target tissues. These results may have implications in determining the therapeutic use of ASA in insulin-resistant type 2 diabetes.

Vitamin E ameliorates the decremental effect of paraquat on cardiomyocyte contractility in rats.

BACKGROUND: Exposure to pesticides and industrial toxins are implicated in cardiovascular disease. Paraquat (PAR) is a toxic chemical widely used as an herbicide in developing countries and described as a major suicide agent. The hypothesis tested here is that PAR induced myocardial dysfunction may be attributed to altered mechanisms of Ca(2+) transport which are in turn possibly linked to oxidative stress. The mechanisms of PAR induced myocardial dysfunction and the impact of antioxidant protection was investigated in rat ventricular myocytes. METHODOLOGY: Forty adult male Wistar rats were divided into 4 groups receiving the following daily intraperitoneal injections for 3 weeks: Group 1 PAR (10 mg/kg), Control Group 2 saline, Group 3 vitamin E (100 mg/kg) and Group 4 PAR (10 mg/kg) and vitamin E (100 mg/kg). Ventricular action potentials were measured in isolated perfused heart, shortening and intracellular Ca(2+) in electrically stimulated ventricular myocytes by video edge detection and fluorescence photometry techniques, and superoxide dismutase (SOD) and catalase (CAT) levels in heart tissue. PRINCIPAL FINDINGS: Spontaneous heart rate, resting cell length, time to peak (TPK) and time to half (THALF) relaxation of myocyte shortening were unaltered. Amplitude of shortening was significantly reduced in PAR treated rats (4.99±0.26%) and was normalized by vitamin E (7.46±0.44%) compared to controls (7.87±0.52%). PAR significantly increased myocytes resting intracellular Ca(2+) whilst TPK and THALF decay and amplitude of the Ca(2+) transient were unaltered. The fura-2-cell length trajectory during the relaxation of the twitch contraction was significantly altered in myocytes from PAR treated rats compared to controls suggesting altered myofilament sensitivity to Ca(2+) as it was normalized by vitamin E treatment. A significant increase in SOD and CAT activities was observed in both PAR and vitamin E plus PAR groups. CONCLUSIONS: PAR exposure compromised rats heart function and ameliorated by vitamin E treatment.

Biocompatibility of calcined mesoporous silica particles with ventricular myocyte structure and function.

In vivo and in vitro systems were employed to investigate the biocompatibility of two forms of calcined mesoporous silica microparticles, MCM41-cal and SBA15-cal, with ventricular myocytes. These particles have potential clinical use in delivering bioactive compounds to the heart. Ventricular myocytes were isolated from 6 to 8 week male Wistar rats. The distribution of the particles in ventricular myocytes was investigated by transmission electron microscopy and scanning electron microscopy. The distribution of particles was also examined in cardiac muscle 10 min after intravenous injection of 2.0 mg/mL MCM41-cal. Myocyte shortening and the Ca(2+) transient were determined following exposure to 200 µg/mL MCM41-cal or SBA15-cal for 10 min. Within 10 min of incubation at 25 °C, both MCM41-cal and SBA15-cal were found attached to the plasma membrane, and some particles were observed inside ventricular myocytes. MCM41-cal was more abundant inside the myocytes than SBA15-cal. The particles had a notable affinity to mitochondrial membranes, where they eventually settled. Within 10 min of intravenous injection (2.0 mg/mL), MCM41-cal traversed the perivascular space, and some particles entered ventricular myocytes and localized around the mitochondrial membranes. The amplitude of shortening was slightly reduced in myocytes superperfused with MCM41-cal or SBA15-cal. The amplitude of the Ca(2+) transient was significantly reduced in myocytes superperfused with MCM41-cal but was only slightly reduced with SBA15-cal. Overall, the results show reasonable bioavailability and biocompatibility of MCM41-cal and SBA15-cal with ventricular myocytes.