High glucose enhances intracellular Ca2+ responses triggered by purinergic stimulation in retinal neurons and microglia (original) (raw)
Related papers
2006
PURPOSE. Altered glutamatergic neurotransmission and calcium homeostasis may contribute to retinal neural cell dysfunction and apoptosis in diabetic retinopathy (DR). The purpose of this study was to determine the effect of high glucose on the protein content of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptor subunits, particularly the GluR2 subunit, because it controls Ca 2ϩ permeability of AMPA receptor-associated channels. The effect of high glucose on the concentration of cytosolic free calcium ([Ca 2ϩ ] i ) was also investigated. METHODS. The protein content of GluR1, GluR2, GluR6/7, and KA2 subunits was assessed by Western blot. Cobalt staining was used to identify cells containing calcium/cobalt-permeable AMPA receptors. The [Ca 2ϩ ] i changes evoked by KCl or kainate were recorded by live-cell confocal microscopy in R28 cells and in primary cultures of rat retina, loaded with fluo-4. RESULTS. In primary cultures, high glucose significantly decreased the protein content of GluR1 and GluR6/7 subunits and increased the protein content of GluR2 and KA2 subunits. High glucose decreased the number of cobalt-positive cells, suggesting a decrease in calcium permeability through AMPA receptor-associated channels. In high-glucose-treated cells, changes in [Ca 2ϩ ] i were greater than in control cells, and the recovery to basal levels was delayed. However, in the absence of Na ϩ , to prevent the activation of voltage-sensitive calcium channels, the [Ca 2ϩ ] i changes evoked by kainate in the presence of cyclothiazide, which inhibits AMPA receptor desensitization, were significantly lower in high-glucose-treated cells than in control cultures, further indicating that AMPA receptors were less permeable to calcium. Mannitol, used as an osmotic control, did not cause significant changes compared with the control.
Calcium signalling in sensory neurones and peripheral glia in the context of diabetic neuropathies
Cell calcium, 2014
Peripheral sensory nervous system is comprised of neurones with their axons and neuroglia that includes satellite glial cells in sensory ganglia, myelinating, non-myelinating and perisynaptic Schwann cells. Pathogenesis of peripheral diabetic polyneuropathies is associated with aberrant function of both neurones and glia. Deregulated Ca(2+) homoeostasis and aberrant Ca(2+) signalling in neuronal and glial elements contributes to many forms of neuropathology and is fundamental to neurodegenerative diseases. In diabetes both neurones and glia experience metabolic stress and mitochondrial dysfunction which lead to deregulation of Ca(2+) homeostasis and Ca(2+) signalling, which in their turn lead to pathological cellular reactions contributing to development of diabetic neuropathies. Molecular cascades responsible for Ca(2+) homeostasis and signalling, therefore, can be regarded as potential therapeutic targets.
ASN neuro, 2012
Distal symmetrical sensory neuropathy in diabetes involves the dying back of axons, and the pathology equates with axonal dystrophy generated under conditions of aberrant Ca 2+ signalling. Previous work has described abnormalities in Ca 2+ homoeostasis in sensory and dorsal horn neurons acutely isolated from diabetic rodents. We extended this work by testing the hypothesis that sensory neurons exposed to long-term Type 1 diabetes in vivo would exhibit abnormal axonal Ca 2+ homoeostasis and focused on the role of SERCA (sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase). DRG (dorsal root ganglia) sensory neurons from age-matched normal and 3-5-month-old STZ (streptozotocin)-diabetic rats (an experimental model of Type 1 diabetes) were cultured. At 1-2 days in vitro an array of parameters were measured to investigate Ca 2+ homoeostasis including (i) axonal levels of intracellular Ca 2+ , (ii) Ca 2+ uptake by the ER (endoplasmic reticulum), (iii) assessment of Ca 2+ signalling following a long-term thapsigargin-induced blockade of SERCA and (iv) determination of expression of ER mass and stress markers using immunocytochemistry and Western blotting. KCl-and caffeine-induced Ca 2+ transients in axons were 2-fold lower in cultures of diabetic neurons compared with normal neurons indicative of reduced ER calcium loading. The rate of uptake of Ca 2+ into the ER was reduced by 2-fold (P,0.05) in diabetic neurons, while markers for ER mass and ER stress were unchanged. Abnormalities in Ca 2+ homoeostasis in diabetic neurons could be mimicked via long-term inhibition of SERCA in normal neurons. In summary, axons of neurons from diabetic rats exhibited aberrant Ca 2+ homoeostasis possibly triggered by suboptimal SERCA activity that could contribute to the distal axonopathy observed in diabetes.
Microglial changes occur without neural cell death in diabetic retinopathy
Vision Research - VISION RES, 2007
Very early neuroglial changes have been observed to precede major vascular changes in the retina of diabetic patients and animal models. We investigated the sequence of these neuroglial changes further, in mice with alloxan-induced diabetes. Diabetes was induced by a single injection of Alloxan into C57/Bl6 mice, which subsequently received daily insulin injections. Diabetic and control animals were weighed and their blood glucose levels were determined weekly. Electroretinographic recordings and scanner laser ophthalmoscope (SLO) examinations were carried out 15 days, one month and three months after the onset of diabetes. Diabetes induction was confirmed by the presence of glucose in the urine, a tripling of blood glucose level, weight loss and an increase in glycated haemoglobin levels. Three months after diabetes onset, the electroretinogram b/a wave amplitude ratio was decreased at the highest light intensities and oscillatory potentials were delayed. The retinal fundus and ves...
Diabetologia, 2003
Aims/Hypothesis. To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca 2+ influx pathways in retinal microvascular smooth muscle cells. Methods. Cytosolic Ca 2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca 2+ influx was tested by measuring rises in [Ca 2+ ] i with KCl (100 mmol/l) and store-operated Ca 2+ influx was assessed by depleting [Ca 2+ ] i stores with Ca 2+ free medium containing 5 µmol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca 2+ on adding 2 mmol/l or 10 mmol/l Ca 2+ solution. Results. Ca 2+ entry through voltage-dependent L-type Ca 2+ channels was unaffected by diabetes. In contrast, store-operated Ca 2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca 2+ influx. Diabetic rats injected daily with insulin had store-operated Ca 2+ influx rates similar to non-diabetic control rats. The reduced Ca 2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCβ antagonist LY379196 (100 nmol/l) also reversed the poor Ca 2+ influx although its action was less efficacious than staurosporine. Conclusion/interpretation. These results show that store-operated Ca 2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCβ seems to play a role in mediating this effect. [Diabetologia (2003) 46:1252-1259] Keywords Diabetes, retinal arterioles, store-operated Ca 2+ channels, L-type Ca 2+ channels, protein kinase C, microvessels.
Possible neuroprotective role of P2X2 in the retina of diabetic rats
Diabetology & metabolic syndrome, 2018
Purinergic receptors are expressed in different tissues including the retina. These receptors are involved in processes like cell growth, proliferation, activation and survival. ATP is the major activator of P2 receptors. In diabetes, there is a constant ATP production and this rise of ATP leads to a persistent activation of purinergic receptors. Antagonists of these receptors are used to evaluate their inhibition effects. Recently, the P2X2 has been reported to have a neuroprotective role. We carried out a study in groups of diabetic and non-diabetic rats (N = 5) treated with intraperitoneal injections of PPADS, at 9 and 24 weeks of diabetes. Control group received only the buffer. Animals were euthanized at 34 weeks of diabetes or at a matching age. Rat retinas were analyzed with immunohistochemistry and western blot using antibodies against GFAP, P2X2, P2Y2 and VEGF-A. Diabetic animals treated with PPADS disclosed a much more extended staining of VEGF-A than diabetics without tre...
Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina
Experimental Eye Research, 2008
The anti-inflammatory glucocorticoid, triamcinolone acetonide, is used clinically for the rapid resolution of diabetic macular edema. Osmotic swelling of glial cells may contribute to the development of retinal edema. Triamcinolone inhibits the swelling of retinal glial cells of diabetic rats. Here, we determined whether the effect of triamcinolone is mediated by a receptor-dependent mechanism. Hyperglycemia was induced in rats with streptozotocin injection. After 6-10 months, the swelling properties of glial cells in retinal slices upon hypotonic challenge were determined. Nucleotide-degrading ecto-enzymes were immunostained in retinal slices and glial cells. Hypotonic challenge did not change the size of glial cell bodies from control retinas but induced swelling of cells from diabetic animals. Triamcinolone inhibited glial cell swelling; this effect was prevented by a selective antagonist of adenosine A1 receptors, an inhibitor of nucleoside transporters, inhibitors of adenylyl cyclase and protein kinase A activation, and inhibitors of potassium and chloride channels. In diabetic (but not control) retinas, the effect of triamcinolone apparently involves extracellular nucleotide degradation. Glial cells from diabetic retinas displayed immunolabeling against nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) which was not observed in control retinas. The mRNA expression for NTPDase1 was significantly increased in the retina of diabetic rats. It is suggested that triamcinolone induces the release and formation of endogenous adenosine that subsequently activates A1 receptors resulting in ion efflux through potassium and chloride channels and prevention of osmotic swelling. Whereas adenosine is liberated via facilitated transport in control retinas, an extracellular formation of adenosine contributes to the effect of triamcinolone in diabetic retinas.
Serum from diabetic BB/W rats enhances calcium currents in primary sensory neurons
Journal of neurophysiology, 1998
We examined the hypothesis that exposure of nondiabetic rat dorsal root ganglion (DRG) neurons to sera from diabetic BB/W rats would produce an increase in calcium currents associated with impaired regulation of the inhibitory G protein-calcium channel complex. Acutely dissociated rat DRGs were incubated for 18-24 h in medium supplemented with sera (10% vol/vol) from either diabetic rats with neuropathy or age-matched, nondiabetic controls. Exposure of DRG neurons to sera from diabetic BB/W rats resulted in a surface membrane immunofluorescence pattern when treated with an anti-rat light-chain antibody that was not observed in neurons exposed to control sera. Calcium current density (IDCa) was assessed with the use of the whole cell variation of the patch-clamp technique. IDCa in neurons exposed to diabetic sera was significantly increased compared with neurons exposed to control sera. Guanine nucleotide-binding (G) protein regulation of calcium channel function was examined with th...
Calcium Dobesilate Prevents Neurodegeneration and Vascular Leakage in Experimental Diabetes
Current eye research, 2017
The mechanisms involved in the reported beneficial effects of Calcium dobesilate monohydrate (CaD) for the treatment of diabetic retinopathy (DR) remain to be elucidated. The main aim of the present study is to examine whether CaD prevents early events in the pathogenesis of DR such as neurodegeneration and vascular leakage. In addition, putative mediators of both neurodegeneration (glutamate/GLAST, ET-1/ETB receptor) and early microvascular impairment (ET-1/ETA receptor, oxidative stress, VEGF, and the PKC-delta-p38 MAPK pathway) have been examined. Diabetic (db/db) mice were randomly assigned to daily oral treatment with CaD (200 mg/Kg/day) (n = 12) or vehicle (n = 12) for 14 days. In addition, 12 non-diabetic (db/+) mice matched by age were used as the control group. Functional abnormalities were assessed by electroretinography. Neurodegeneration and microvascular abnormalities were evaluated by immunohistochemistry and Western blot. Glutamate was determined by HPLC. CaD signific...
Apoptosis and Inflammation Form the Mystery of Pathogenesis of Diabetic Retinopathy
Journal of Evolution of medical and Dental Sciences, 2015
Faster anaerobic glycolysis in type 2 diabetes mellitus generates increased lactate and NADH. Uncoupling of NADH produces excessive reactive oxygen species which form the main stream of apoptosis of vascular and neural cells in retina. Increased lipid peroxidation and extracellular glutamate toxicity further augment this process. Vascular endothelial growth factor is up regulated to resist the apoptotic destruction or in response to non-perfusion of acellular capillaries. Deleterious effects of excessive vascular endothelial growth factor under an adverse biochemical circumstances causes microangiopathy in diabetic subjects. Blood levels of lactate, glutamate, malondialdehyde and vascular endothelial growth factor were seen significantly higher in mild non-proliferative diabetic retinopathy in comparison to diabetic subjects with no retinopathy.