Characterisation of the P2Y14 receptor in the pancreas: control of vascular tone and insulin secretion

The P2Y14 receptor is the most recently identified member of the P2Y family of receptors for adenine and uridine nucleotides and nucleotide sugars. It is activated by UDP, UDP-glucose and its analogues, as well as the synthetic analogue MRS2690, which exhibits greater potency and selectivity at the P2Y14 receptor. The principle aim of this study was to investigate the functional expression of the P2Y14 receptor in porcine pancreatic arteries, and the signalling pathways underlying the vasoconstriction evoked by P2Y14 receptor agonists, together with an examination of the effects of UDP-glucose and MRS2690 on insulin secretion from the rat INS-1 823/13 β-cell line. Segments of porcine pancreatic arteries were prepared for isometric tension recordings in warmed oxygenated Krebs’-Henseleit buffer. Agonists were applied after preconstriction with U46619, a thromboxane A2 mimetic. ATP induced vasoconstriction followed by a vasorelaxation in pancreatic arteries; the contraction was blocked by NF449 (a P2X1 receptor selective antagonist), while the relaxation to ATP was blocked by an adenosine receptor antagonist. Neither the contraction, nor the relaxation to ATP were affected by removal of the endothelium. ADP evoked vasorelaxation, which was inhibited in the presence of SCH58261 (a selective adenosine A2A receptor antagonist). UTP-induced vasoconstriction was attenuated significantly in endothelium-denuded arteries. UDP, UDP-glucose and MRS2690 induced concentration-dependent contractions in porcine pancreatic arteries with a rank order of potency of MRS2690 (10-fold) > UDP-glucose = UDP. The contractions evoked by UDP-glucose and MRS2690 were significantly attenuated in the presence of PPTN (a selective P2Y14 receptor antagonist), indicating actions at P2Y14 receptors. The expression of P2Y14-like receptor was shown by immunohistochemical and contractile studies to be on the endothelium of the pancreatic arteries. UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP production. UDP-glucose and MRS2690 increased the level of MLC2 phosphorylation; this effect was blocked by PPTN, indicating the involvement of P2Y14 receptors. UDP-glucose increased the level of ERK1/2 phosphorylation. UDP-glucose and MRS2690 inhibited glucose-induced insulin release from the rat INS-1 823/13 β-cell line; this effect was blocked by PPTN, indicating actions through P2Y14 receptors. PPTN itself was able to elevate significantly basal insulin secretion from INS-1 823/13 β-cells, which may suggest a constitutive release of UDP-glucose from these cells. These results suggest that, in porcine pancreatic arteries, ATP induces a vasoconstriction mediated by P2X1 receptors followed by a vasorelaxation evoked by adenosine receptors present on the vascular smooth muscle. ADP induced a relaxation mediated by adenosine A2A receptor. Moreover, my data indicate for the first time, an endothelium-dependent contraction evoked by UTP. A novel vasocontractile role of P2Y14 receptors in porcine pancreatic arteries was also documented. The contractile response was mediated largely by the endothelium. P2Y14-mediated contraction involves a cAMP-dependent mechanism, which is consistent with P2Y14 receptor coupling to Gi protein, and an elevation in phosphorylated MLC2 and ERK1/2. Activation of the P2Y14 receptor evoked a decrease in the level of insulin secreted from the rat pancreas. The current data have identified novel roles of the P2Y14 receptor as a mediator of pancreatic artery contractility and in regulation of insulin secretion. If its role within the vasculature is shown to be more widespread, the P2Y14 receptor may be a novel target for the treatment of cardiovascular disease

Characterisation of the P2Y14 receptor in the pancreas: control of vascular tone and insulin secretion

The P2Y14 receptor is the most recently identified member of the P2Y family of receptors for adenine and uridine nucleotides and nucleotide sugars. It is activated by UDP, UDP-glucose and its analogues, as well as the synthetic analogue MRS2690, which exhibits greater potency and selectivity at the P2Y14 receptor. The principle aim of this study was to investigate the functional expression of the P2Y14 receptor in porcine pancreatic arteries, and the signalling pathways underlying the vasoconstriction evoked by P2Y14 receptor agonists, together with an examination of the effects of UDP-glucose and MRS2690 on insulin secretion from the rat INS-1 823/13 ┚-cell line. Segments of porcine pancreatic arteries were prepared for isometric tension recordings in warmed oxygenated Krebs’-Henseleit buffer. Agonists were applied after preconstriction with U46619, a thromboxane A2 mimetic. ATP induced vasoconstriction followed by a vasorelaxation in pancreatic arteries; the contraction was blocked by NF449 (a P2X1 receptor selective antagonist), while the relaxation to ATP was blocked by an adenosine receptor antagonist. Neither the contraction, nor the relaxation to ATP were affected by removal of the endothelium. ADP evoked vasorelaxation, which was inhibited in the presence of SCH58261 (a selective adenosine A2A receptor antagonist). UTP-induced vasoconstriction was attenuated significantly in endothelium-denuded arteries. UDP, UDP-glucose and MRS2690 induced concentration-dependent contractions in porcine pancreatic arteries with a rank order of potency of MRS2690 (10-fold) > UDP-glucose = UDP. The contractions evoked by UDP-glucose and MRS2690 were significantly attenuated in the presence of PPTN (a selective P2Y14 receptor antagonist), indicating actions at P2Y14 receptors. The iii expression of P2Y14-like receptor was shown by immunohistochemical and contractile studies to be on the endothelium of the pancreatic arteries. UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP production. UDP-glucose and MRS2690 increased the level of MLC2 phosphorylation; this effect was blocked by PPTN, indicating the involvement of P2Y14 receptors. UDP-glucose increased the level of ERK1/2 phosphorylation. UDP-glucose and MRS2690 inhibited glucose-induced insulin release from the rat INS-1 823/13 ┚-cell line; this effect was blocked by PPTN, indicating actions through P2Y14 receptors. PPTN itself was able to elevate significantly basal insulin secretion from INS-1 823/13 ┚-cells, which may suggest a constitutive release of UDP-glucose from these cells. These results suggest that, in porcine pancreatic arteries, ATP induces a vasoconstriction mediated by P2X1 receptors followed by a vasorelaxation evoked by adenosine receptors present on the vascular smooth muscle. ADP induced a relaxation mediated by adenosine A2A receptor. Moreover, my data indicate for the first time, an endothelium-dependent contraction evoked by UTP. A novel vasocontractile role of P2Y14 receptors in porcine pancreatic arteries was also documented. The contractile response was mediated largely by the endothelium. P2Y14-mediated contraction involves a cAMP-dependent mechanism, which is consistent with P2Y14 receptor coupling to Gi protein, and an elevation in phosphorylated MLC2 and ERK1/2. Activation of the P2Y14 receptor evoked a decrease in the level of insulin secreted from the rat pancreas. The current data have identified novel roles of the P2Y14 receptor as a mediator of pancreatic artery contractility and in regulation of insulin secretion. If its role within the vasculature is shown to be more widespread, the P2Y14 receptor may be a novel target for the treatment of cardiovascular disease

Characterisation of the P2Y14 receptor in the pancreas: control of vascular tone and insulin secretion

The P2Y14 receptor is the most recently identified member of the P2Y family of receptors for adenine and uridine nucleotides and nucleotide sugars. It is activated by UDP, UDP-glucose and its analogues, as well as the synthetic analogue MRS2690, which exhibits greater potency and selectivity at the P2Y14 receptor. The principle aim of this study was to investigate the functional expression of the P2Y14 receptor in porcine pancreatic arteries, and the signalling pathways underlying the vasoconstriction evoked by P2Y14 receptor agonists, together with an examination of the effects of UDP-glucose and MRS2690 on insulin secretion from the rat INS-1 823/13 β-cell line. Segments of porcine pancreatic arteries were prepared for isometric tension recordings in warmed oxygenated Krebs’-Henseleit buffer. Agonists were applied after preconstriction with U46619, a thromboxane A2 mimetic. ATP induced vasoconstriction followed by a vasorelaxation in pancreatic arteries; the contraction was blocked by NF449 (a P2X1 receptor selective antagonist), while the relaxation to ATP was blocked by an adenosine receptor antagonist. Neither the contraction, nor the relaxation to ATP were affected by removal of the endothelium. ADP evoked vasorelaxation, which was inhibited in the presence of SCH58261 (a selective adenosine A2A receptor antagonist). UTP-induced vasoconstriction was attenuated significantly in endothelium-denuded arteries. UDP, UDP-glucose and MRS2690 induced concentration-dependent contractions in porcine pancreatic arteries with a rank order of potency of MRS2690 (10-fold) > UDP-glucose = UDP. The contractions evoked by UDP-glucose and MRS2690 were significantly attenuated in the presence of PPTN (a selective P2Y14 receptor antagonist), indicating actions at P2Y14 receptors. The expression of P2Y14-like receptor was shown by immunohistochemical and contractile studies to be on the endothelium of the pancreatic arteries. UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP production. UDP-glucose and MRS2690 increased the level of MLC2 phosphorylation; this effect was blocked by PPTN, indicating the involvement of P2Y14 receptors. UDP-glucose increased the level of ERK1/2 phosphorylation. UDP-glucose and MRS2690 inhibited glucose-induced insulin release from the rat INS-1 823/13 β-cell line; this effect was blocked by PPTN, indicating actions through P2Y14 receptors. PPTN itself was able to elevate significantly basal insulin secretion from INS-1 823/13 β-cells, which may suggest a constitutive release of UDP-glucose from these cells. These results suggest that, in porcine pancreatic arteries, ATP induces a vasoconstriction mediated by P2X1 receptors followed by a vasorelaxation evoked by adenosine receptors present on the vascular smooth muscle. ADP induced a relaxation mediated by adenosine A2A receptor. Moreover, my data indicate for the first time, an endothelium-dependent contraction evoked by UTP. A novel vasocontractile role of P2Y14 receptors in porcine pancreatic arteries was also documented. The contractile response was mediated largely by the endothelium. P2Y14-mediated contraction involves a cAMP-dependent mechanism, which is consistent with P2Y14 receptor coupling to Gi protein, and an elevation in phosphorylated MLC2 and ERK1/2. Activation of the P2Y14 receptor evoked a decrease in the level of insulin secreted from the rat pancreas. The current data have identified novel roles of the P2Y14 receptor as a mediator of pancreatic artery contractility and in regulation of insulin secretion. If its role within the vasculature is shown to be more widespread, the P2Y14 receptor may be a novel target for the treatment of cardiovascular disease

Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation

Background Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear. Methods Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network. Results We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory–excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes. Limitations Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients. Conclusions Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1

Proteins involved in endocytosis are upregulated by ageing in the normal human brain: Implications for the development of Alzheimer's Disease

© The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. The greatest risk factor for Alzheimer's disease (AD) is advanced age, but the reason for this association remains unclear. Amyloid-β (Aβ) is produced from amyloid precursor protein (APP) primarily after APP is internalized by clathrin-mediated or clathrin-independent endocytosis. Changes in endocytosis in AD have been identified. We hypothesized that endocytic protein expression is altered during ageing, thus influencing the likelihood of developing AD by increasing Aβ production. We explored how levels of endocytic proteins, APP, its metabolites, secretase enzymes, and tau varied with age in cortical brain samples from men of three age ranges (young [20-30], middle aged [45-55], and old [70-90]) with no symptoms of dementia. Aβ40 and Aβ42 were significantly increased in old brains, while APP and secretase expression was unaffected by age. Phosphorylated GSK3β increased significantly with age, a possible precursor for neurofibrillary tangle production, although phosphorylated tau was undetectable. Significant increases in clathrin, dynamin-1, AP180, Rab-5, caveolin-2, and flotillin-2 were seen in old brains. Rab-5 also increased in middle-aged brains prior to changes in Aβ levels. This age-related increase in endocytic protein expression, not described previously, suggests an age-related upregulation of endocytosis which could predispose older individuals to develop AD by increasing APP internalization and Aβ generation

Alterations in endocytic protein expression with increasing age in the transgenic APP695 V717I London mouse model of amyloid pathology: Implications for Alzheimer's disease

Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. A major risk factor for the development of Alzheimer's disease (AD) is increasing age, but the reason behind this association has not been identified. It is thought that the changes in endocytosis seen in AD patients are causal for this condition. Thus, we hypothesized that the increased risk of developing AD associated with ageing may be because of changes in endocytosis. We investigated using Western blotting whether the expression of endocytic proteins involved in clathrin-mediated and clathrin-independent endocytosis are altered by increasing age in a mouse model of amyloid pathology. We used mice transgenic for human amyloid precursor protein containing the V717I London mutation. We compared the London mutation mice with age-matched wild-type (WT) controls at three ages, 3, 9 and 18 months, representing different stages in the development of pathology in this model. Having verified that the London mutation mice overexpressed amyloid precursor protein and β-amyloid, we found that the expression of the smallest isoform of PICALM, a key protein involved in the regulation of clathrin-coated pit formation, was significantly increased in WT mice, but decreased in the London mutation mice with age. PICALM levels in WT 18-month mice and clathrin levels in WT 9-month mice were significantly higher than those in the London mutation mice of the same ages. The expression of caveolin-1, involved in clathrin-independent endocytosis, was significantly increased with age in all mice. Our results suggest that endocytic processes could be altered by the ageing process and such changes could partly explain the association between ageing and AD

A high ratio of linoleic acid (n-6 PUFA) to alpha-linolenic acid (n-3 PUFA) adversely affects early stage of human neuronal differentiation and electrophysiological activity of glutamatergic neurons in vitro

Introduction: There is a growing interest in the possibility of dietary supplementation with polyunsaturated fatty acids (PUFAs) for treatment and prevention of neurodevelopmental and neuropsychiatric disorders. Studies have suggested that of the two important classes of polyunsaturated fatty acids, omega-6 (n-6) and omega-3 (n-3), n-3 polyunsaturated fatty acids support brain development and function, and when used as a dietary supplement may have beneficial effects for maintenance of a healthy brain. However, to date epidemiological studies and clinical trials on children and adults have been inconclusive regarding treatment length, dosage and use of specific n-3 polyunsaturated fatty acids. The aim of this study is to generate a simplified in vitro cell-based model system to test how different n-6 to n-3 polyunsaturated fatty acids ratios affect human-derived neurons activity as a cellular correlate for brain function and to probe the mechanism of their action. Methods: All experiments were performed by use of human induced pluripotent stem cells (iPSCs). In this study, we examined the effect of different ratios of linoleic acid (n-6) to alpha-linolenic acid in cell growth medium on induced pluripotent stem cell proliferation, generation of neuronal precursors and electrophysiology of cortical glutamatergic neurons by multielectrode array (MEA) analysis. Results: This study shows that at a n-6:n-3 ratio of 5:1 polyunsaturated fatty acids induce stem cell proliferation, generating a large increase in number of cells after 72 h treatment; suppress generation of neuronal progenitor cells, as measured by decreased expression of FOXG1 and Nestin in neuronal precursor cells (NPC) after 20 days of development; and disrupt neuronal activity in vitro, increasing spontaneous neuronal firing, reducing synchronized bursting receptor subunits. We observed no significant differences for neuronal precursor cells treated with ratios 1:3 and 3:1, in comparison to 1:1 control ratio, but higher ratios of n-6 to n-3 polyunsaturated fatty acids adversely affect early stages of neuronal differentiation. Moreover, a 5:1 ratio in cortical glutamatergic neurons induce expression of GABA receptors which may explain the observed abnormal electrophysiological activity

Glycyrrhizin blocks the detrimental effects of HMGB1 on cortical neurogenesis after traumatic neuronal injury

Despite medical advances, neurological recovery after severe traumatic brain injury (TBI) remains poor. Elevated levels of high mobility group box protein-1 (HMGB1) are associated with poor outcomes; likely via interaction with receptors for advanced-glycation-end-products (RAGE). We examined the hypothesis that HMGB1 post-TBI is anti-neurogenic and whether this is pharmacologically reversible. Post-natal rat cortical mixed neuro-glial cell cultures were subjected to needle-scratch injury and examined for HMGB1-activation/neuroinflammation. HMGB1-related genes/networks were examined using genome-wide RNA-seq studies in cortical perilesional tissue samples from adult mice. Post-natal rat cortical neural stem/progenitor cell cultures were generated to quantify effects of injury-condition medium (ICM) on neurogenesis with/without RAGE antagonist glycyrrhizin. Needle-injury upregulated TNF-α/NOS-2 mRNA-expressions at 6 h, increased proportions of activated microglia, and caused neuronal loss at 24 h. Transcriptome analysis revealed activation of HMGB1 pathway genes/canonical pathways in vivo at 24 h. A 50% increase in HMGB1 protein expression, and nuclear-to-cytoplasmic translocation of HMGB1 in neurons and microglia at 24 h post-injury was demonstrated in vitro. ICM reduced total numbers/proportions of neuronal cells, but reversed by 0.5 μM glycyrrhizin. HMGB1 is activated following in vivo post mechanical injury, and glycyrrhizin alleviates detrimental effects of ICM on cortical neurogenesis. Our findings highlight glycyrrhizin as a potential therapeutic agent post-TBI. View Full-Text Keywords: traumatic brain injury; neurogenesis; HMGB1; neuroinflammatio

Neuron-glia interactions increase neuronal phenotypes in tuberous sclerosis complex patient iPSC-derived models

Tuberous sclerosis complex (TSC) is a rare neurodevelopmental disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes, leading to a hyperactivated mammalian target of rapamycin (mTOR) pathway, and gray and white matter defects in the brain. To study the involvement of neuron-glia interactions in TSC phenotypes, we generated TSC patient induced pluripotent stem cell (iPSC)-derived cortical neuronal and oligodendrocyte (OL) cultures. TSC neuron mono-cultures showed increased network activity, as measured by calcium transients and action potential firing, and increased dendritic branching. However, in co-cultures with OLs, neuronal defects became more apparent, showing cellular hypertrophy and increased axonal density. In addition, TSC neuron-OL co-cultures showed increased OL cell proliferation and decreased OL maturation. Pharmacological intervention with the mTOR regulator rapamycin suppressed these defects. Our patient iPSC-based model, therefore, shows a complex cellular TSC phenotype arising from the interaction of neuronal and glial cells and provides a platform for TSC disease modeling and drug development