Abstract
BackgroundA body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged. This hypothesis was further tested by studying the effects of glutamate on the tenocyte phenotype, and the impact of loading and exposure to glucocorticoids on the glutamate signaling machinery.MethodsPlantaris tendon tissue and cultured plantaris tendon derived cells were immunohisto-/cytochemically stained for glutamate, N-Methyl-D-Aspartate receptor 1 (NMDAR1) and vesicular glutamate transporter 2 (VGluT2). Primary cells were exposed to glutamate or receptor agonist NMDA. Cell death/viability was measured via LDH/MTS assays, and Western blot for cleaved caspase 3 (c-caspase 3) and cleaved poly (ADP-ribose) polymerase (c-PARP). Scleraxis mRNA (Scx)/protein(SCX) were analyzed by qPCR and Western blot, respectively. A FlexCell system was used to apply cyclic strain. The effect of glucocorticoids was studies by adding dexamethasone (Dex). The mRNA of the glutamate synthesizing enzymes Got1 and Gls, and NMDAR1 protein were measured. Levels of free glutamate were determined by a colorimetric assay.ResultsImmunoreactions for glutamate, VGluT2, and NMDAR1 were found in tenocytes and peritendinous cells in tissue sections and in cultured cells. Cell death was induced by high concentrations of glutamate but not by NMDA. Scleraxis mRNA/protein was down-regulated in response to NMDA/glutamate stimulation. Cyclic strain increased, and Dex decreased, Gls and Got1 mRNA expression. Free glutamate levels were lower after Dex exposure.ConclusionsIn conclusion, NMDA receptor stimulation leads to a reduction of scleraxis expression that may be involved in a change of phenotype in tendon cells. Glutamate synthesis is increased in tendon cells in response to strain and decreased by glucocorticoid stimulation. This implies that locally produced glutamate could be involved in the tissue changes observed in tendinopathy.
Highlights
A body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged
Expression patterns of the glutamate system Immunoreactions for glutamate were seen in tenocytes in the tendon tissue proper (Fig. 1a, b), the cultured tendon derived cells (Fig. 1c), and in the cells in the peritendinous connective tissue (Fig. 1d)
Vesicular glutamate transporter vesicular glutamate transporter 2 (VGluT2) was found to be expressed by tenocytes of the tendon tissue proper (Fig. 2a, b) and by cells in the peritendinous connective tissue (Fig. 2e) by immunohistochemistry
Summary
A body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged. This hypothesis was further tested by studying the effects of glutamate on the tenocyte phenotype, and the impact of loading and exposure to glucocorticoids on the glutamate signaling machinery. High levels of free glutamate have been detected in painful Achilles and patellar tendons and in Tennis elbow [6,7,8]. The vesicular glutamate transporter 2 (VGluT2), enabling the cells to release glutamate via synaptic-like vesicles, and various glutamate receptors, have been found to be expressed by tendon cells (tenocytes) in the Achilles and patellar tendons [10,11,12].
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