Microglia phagocytosis is critical for central nervous system development, and dysregulation of phagocytosis may contribute to a variety of neurological disorders. During initial stages of phagocytosis, microglia display increased nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) activity and amplified calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. The present study investigated the regulatory role of iNOS/NO signaling in microglial phagocytosis and TRPV2 channel activation using phagocytosis assay, calcium imaging, patch clamp electrophysiology, immunocytochemistry, and immunoblot assays. Results showed that primary microglia from iNOS‐knockout (iNOS−/−) mice exhibited substantial deficits in phagocytic capacity and TRPV2 channel activity relative to wild‐type (WT) controls. Specifically, iNOS−/− microglia displayed a lower level of TRPV2 protein localized on the plasma membrane (PM) without any significant change in the mRNA levels of Fc‐gamma receptors and TRPV2. In addition, iNOS−/− microglia, unlike their WT controls, failed to elicit a calcium influx in response to application of the TRPV2‐agonist 2‐aminoethoxydiphenyl borate (2APB). Importantly, the phagocytic capacity and the PM expression and activity of TRPV2 in iNOS−/− microglia were largely corrected by pretreatment with NO‐donors. Accordingly, the 2APB‐evoked calcium influx and the PM expression of TRPV2 in WT microglia were significantly decreased by selective inhibition of iNOS, protein kinase‐G (PKG), or phosphoinositide‐3‐kinase (PI3K), respectively. Together, results from this study indicated that iNOS/NO signaling upregulates microglial phagocytosis and increases TRPV2 trafficking to the PM via PKG/PI3K dependent pathway(s).

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Background Primary open angle glaucoma (POAG) patients have hallmark increases in intraocular pressure (IOP) and noted dysfunction of the trabecular meshwork (TM). Connexin43 (Cx43) is a gap junction widely expressed on the TM that is important for intercellular communication. The human gene is known as gap junction alpha‐1 (GJA1). Since the role of Cx43 in the TM is not fully understood, we set out to determine the effect of excess mechanical stretch on cultured human trabecular meshwork cells (hTMCs) and to specifically investigate the effect of stretch on Cx43 expression and function. Methods Primary hTMCs were cultured and subjected to 48 hours of 15% cyclic mechanical stretch at a frequency of 1 Hz. Levels of apoptosis and necrosis secondary to stretch were investigated using colorimetric assays. The effect of stretch on gap junction Cx43 and GJA1 was investigated by RT‐PCR, immunoblotting and immunofluorescence. The migration of Lucifer Yellow dye was used to assess intercellular communication. Results Stretch significantly increased the rates of apoptosis and necrosis in hTMCs. The increased rate of injury in stretched hTMCs was further associated with significant upregulation of GJA1 mRNA and Cx43 protein. Upregulation of Cx43 protein was concomitant to increased intercellular communication. Conclusions We have shown stretch to increase GJA1 gene and Cx43 protein expression, as well as intercellular communication. We have further shown stretch to be injurious to hTMCs. Upregulation of Cx43 in the hTM subsequent to stretch is a novel finding, which may be useful in elucidating the mechanism of TM injury in POAG patients.