Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Neuronal Nitric Oxide Synthase Critically Regulates the Endocannabinoid Pathway in the Murine Cerebellum During Development.

The cerebellum is a major site of endocannabinoid (eCB) production and signaling. The predominant eCB within the cerebellum, 2-arachidonoylglycerol (2-AG), is produced by a metabotropic glutamate receptor type 1 (mGluR1)-initiated signaling cascade within Purkinje neurons (PNs). 2-AG retrogradely stimulates cannabinoid 1 receptors (CB1Rs) located on presynaptic terminals. The activated CB1R decreases neurotransmitter release and leads to the production of nitric oxide (NO), a gaseous molecule. Recently, our group discovered that during development in mice lacking neuronal nitric oxide synthase (nNOS-/-), PNs display an excitotoxic phenotype associated with overactivated mGluR1. Considering the importance of mGluR1 in 2-AG synthesis, the present study explored the role of nNOS-derived NO in regulating the eCB pathway within the cerebella of wildtype (WT) and nNOS-/- mice at postnatal day 7 (PD7), 2 weeks (2 W), and 7 weeks (7 W). Our analysis showed that diacylglycerol lipase α, the enzyme that catalyzes 2-AG production, was elevated at early postnatal ages, and followed by elevated levels of 2-AG in nNOS-/- cerebella compared to WT. CB1R expression in nNOS-/- cerebella was upregulated at PD7 but decreased at 2 W and 7 W when compared to age-matched WT mice cerebella. Importantly, treating organotypic nNOS-/- cerebellar slice cultures with an NO-donor-attenuated CB1R levels after 7 days in vitro. In addition, expression of the eCB hydrolases fatty acid amide hydrolase and monoacylglycerol lipase were significantly downregulated in nNOS-/- cerebella compared to WT cerebella at 7 W. Together, these results reveal a novel role for nNOS/NO in regulating eCB signaling in the cerebellum.

The expression and function of glutamate aspartate transporters in Bergmann glia are decreased in neuronal nitric oxide synthase-knockout mice during postnatal development.

Bergmann glia (BG) predominantly use glutamate/aspartate transporters (GLAST) for glutamate uptake in the cerebellum. Recently, nitric oxide (NO) treatment has been shown to upregulate GLAST function and increase glutamate uptake in vitro. We previously discovered that neuronal nitric oxide synthase knockout (nNOS-/- ) mice displayed structural and functional neuronal abnormalities in the cerebellum during development, in addition to previously reported motor deficits. Although these developmental deficits have been identified in the nNOS-/- cerebellum, it is unknown whether BG morphology and GLAST expression are also affected in the absence of nNOS in vivo. This study is the first to characterize BG morphology and GLAST expression during development in nNOS-/- mice using immunohistochemistry and western blotting across postnatal development. Results showed that BG in nNOS-/- mice exhibited abnormal morphology and decreased GLAST expression compared with wildtype (WT) mice across postnatal development. Treating ex vivo WT cerebellar slices with the NOS inhibitor L-NAME decreased GLAST expression while treating nNOS-/- slices with the slow-release NO-donor NOC-18 increased GLAST expression when compared with their respective controls. In addition, treating primary BG isolated from WT mice with the selective nNOS inhibitor 7N decreased the membrane expression of GLAST and influx of Ca2+ /Na+ , while treating nNOS-/- BG with SNAP increased the membrane expression of GLAST and Ca2+ /Na+ influx. Moreover, the effects of SNAP on GLAST expression and Ca2+ /Na+ influx in nNOS-/- BG were significantly reduced by a PKG inhibitor. Together, these results reveal a novel role for nNOS/NO signaling in BG development, regulated by a PKG-mediated mechanism.

Nitric oxide displays a biphasic effect on calcium dynamics in microglia.

Calcium is a critical secondary messenger in microglia. In response to inflammation, microglia mobilize intracellular calcium and increase the expression of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO). This study set to explore whether NO regulates intracellular calcium dynamics through transient receptor potential (TRP) channels in primary wildtype (WT) and iNOS knockout (iNOS-/-) microglia, and the BV2 microglial cell line using calcium imaging and voltage-clamp recordings. Our results demonstrated that application of the NO-donor SNAP induced a biphasic calcium response in naïve murine microglia. Specifically, phase I was characterized by a rapid decline in calcium influx that was attenuated by pretreatment of the store operated calcium channel (SOCC) inhibitor 2APB, while phase II presented as a slow calcium influx that was abolished by pretreatment with the TRP vanilloid type 2 (TRPV2) channel inhibitor tranilast. Importantly, in the presence of a protein kinase G (PKG) inhibitor, the SNAP-mediated calcium decline in phase I persisted while the calcium influx in phase II was abolished. Application of thapsigargin to activate SOCCs caused a calcium influx through a nonselective cation conductance in BV2 microglia, which was abruptly attenuated by SNAP. Importantly, iNOS-/- microglia displayed a significantly larger calcium influx though SOCCs while expressing less stromal interaction molecule 1, Orai1, and TRP canonical type 1 and 3 mRNA, when compared to WT microglia. Together, these results demonstrate that NO signaling restricts calcium influx through SOCCs independent of PKG signaling and increases calcium influx through TRPV2 channels in a PKG-dependent mechanism in microglia.

Nitric Oxide Critically Regulates Purkinje Neuron Dendritic Development Through a Metabotropic Glutamate Receptor Type 1-Mediated Mechanism.

Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous phenotypic analyses showed similar cerebellar structures between neuronal nitric oxide null (nNOS-/-) and wild-type (WT) adult male mice, despite prominent ataxic behavior within nNOS-/- mice. However, a study has yet to characterize PN molecular structure and their excitatory inputs during development in nNOS-/- mice. This study is the first to explore morphological abnormalities within the cerebellum of nNOS-/- mice, using immunohistochemistry and immunoblotting. This study sought to examine PN dendritic morphology and the expression of metabotropic glutamate receptor type 1 (mGluR1), vesicular glutamate transporter type 1 and 2 (vGluT1 and vGluT2), stromal interaction molecule 1 (STIM1), and calpain-1 within PNs of WT and nNOS-/- mice at postnatal day 7 (PD7), 2 weeks (2W), and 7 weeks (7W) of age. Results showed a decrease in PN dendritic branching at PD7 in nNOS-/- cerebella, while aberrant dendritic spine formation was noted in adult ages. Total protein expression of mGluR1 was decreased in nNOS-/- cerebella across development, while vGluT2, STIM1, and calpain-1 were significantly increased. Ex vivo treatment of WT slices with NOS inhibitor L-NAME increased calpain-1 expression, whereas treating nNOS-/- cerebellar slices with NO donor NOC-18 decreased calpain-1. Moreover, mGluR1 agonist DHPG increased calpain-1 in WT, but not in nNOS-/- slices. Together, these results indicate a novel role for nNOS/NO signaling in PN development, particularly by regulating an mGluR1-initiated calcium signaling mechanism.

Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G.

Microglia population is primarily determined by a finely-regulated proliferation process during early development of the central nervous system (CNS). Nitric oxide (NO) is known to inhibit proliferation in numerous cell types. However, how NO signaling regulates microglia proliferation remains elusive. Using wildtype (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice, this study investigated the role and underlying mechanisms of iNOS/NO signaling in microglia proliferation. Here we reported that iNOS-/- mice displayed significantly more BrdU-labeled proliferating microglia in the cortex than that in WT mice at postnatal day 10. Compared to microglia isolated from WT mouse cortex, significantly more iNOS-/- microglia displayed the specific cell-cycle markers Ki67 and phospho-histone H3 (pH3) in their nuclei. In addition, treating WT microglia with the NOS inhibitor LNAME drastically increased the percentage of cells expressing Ki67 and pH3, whereas treating iNOS-/- microglia with NOC18, a slow-release NO-donor, significantly decreased the percentage of microglia expressing the two cell-cycle markers. Moreover, inhibition of protein kinase-G (PKG) in WT microglia increased the proportion of microglia expressing Ki67 and pH3, whereas activation of PKG signaling using 8Br-cGMP in iNOS-/- microglia significantly decreased the fraction of microglia displaying Ki67 and pH3. Interestingly, in the presence of a PKG inhibitor, NOC18 increased the quantity of iNOS-/- microglia expressing Ki67 and pH3. Together, these results indicate that basal activity of iNOS/NO signaling impedes microglial cell-cycle progression and attenuates proliferation through activation of the cGMP-PKG pathway. However, NO increases microglia cell-cycle progression in the absence of cGMP-PKG signaling.

Nitric oxide upregulates microglia phagocytosis and increases transient receptor potential vanilloid type 2 channel expression on the plasma membrane.

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).

Mechanical stretch upregulates connexin43 in human trabecular meshwork cells.

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.

Nitric oxide attenuates microglia proliferation by sequentially facilitating calcium influx through TRPV2 channels, activating NFATC2, and increasing p21 transcription.

Microglia proliferation is critical for proper development and function of the central nervous system (CNS), while dysregulation of proliferation contributes to pathology. We recently reported that male inducible nitric oxide synthase knockout (iNOS-/-) mice displayed significantly more proliferating microglia in their postnatal cortex than age-matched wildtype (WT) male mice. Moreover, nitric oxide (NO) signaling in mouse microglia greatly upregulates calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. Considering that TRPV2 activity restricts astrocytic proliferation within glioma tissues, we investigated the roles of iNOS/NO signaling and TRPV2 expression in the regulation of microglial proliferation in vitro using assays of calcium imaging, immunocytochemistry, western blot, and polymerase chain reaction. Results showed that non-dividing microglia exhibited substantially higher expression of TRPV2 on the plasma membrane and significantly larger calcium influx through TRPV2 channels in comparison to dividing microglia. Additionally, non-dividing WT microglia exhibited significantly more NO production than dividing WT microglia. Furthermore, the NO-donor NOC18 increased the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 2 (NFATC2) and the mRNA of the cyclin-dependent kinase inhibitor p21 and decreased the percentage of dividing WT and iNOS-/- microglia in culture. Importantly, the presence of the TRPV2 inhibitor tranilast abolished these effects of NOC18. Together, results from this study indicated that iNOS/NO signaling inhibits microglial proliferation through TRPV2-mediated calcium influx, nuclear translocation of the transcription factor NFATC2, and p21 expression. [Figure: see text].

Administration of Nitric Oxide Through a Novel Copper-Chitosan Delivery System in Human Corneal and Limbal Epithelial Cell Injury.

Purpose: Nitric oxide (NO) has gained attention for its role in facilitating wound healing by promoting cell migration, while being cytoprotective in a variety of cell types. We determined the efficacy of NO, administered using a novel application of copper-chitosan treatments (Cu-Ch), in facilitating corneal epithelial wound healing using an in vitro model of corneal epithelial and limbal epithelial cell injury. Methods: Human corneal epithelial (HCE) and human limbal epithelial (HLE) cells were monitored under no-scratch (CON), untreated scratch (CS), scratch + plain chitosan composite (0%), scratch + 1% copper solution Cu-Ch (1%), and scratch + 2% copper solution Cu-Ch (2%) conditions. Cell migration, cytotoxicity, apoptosis, and total nitrate/nitrite concentrations were measured at 24, 48, and 72 hours after injury and treatment. iNOS expression in HLE cells also was determined using Western blot. Results: Wound closure significantly increased in HCE cells treated with Cu-Ch (1% and 2%) after 72 hours, while HLE cells showed a significant decrease in closure with Cu-Ch (1% and 2%) treatment compared to CS. Cytotoxic fragments decreased significantly with 1% and 2% Cu-Ch treatments in HCE cells. Nitrate/nitrite levels in HLE cells showed a significant increase with 2% Cu-Ch treatment compared to CS. This increase is complemented with an upregulation of iNOS. Conclusions: Overall, HCE wound healing was accelerated with administration of Cu-Ch treatment. Differences between HCE and HLE responses may be due to intrinsic differences in NO metabolism, as evidenced by differences in NO production, potentially caused by differences in iNOS expression with treatment.