Nitric oxide promotes GABA release by activating a voltage-independent Ca2+ influx pathway in retinal amacrine cells

Retinal amacrine cells express nitric oxide (NO) synthase and produce NO, making NO available to regulate the function of amacrine cells. Here we test the hypothesis that NO can alter the GABAergic synaptic output of amacrine cells. We investigate this using whole cell voltage clamp recordings and Ca2+ imaging of cultured chick retinal amacrine cells. When recording from amacrine cells receiving synaptic input from other amacrine cells, we find that NO increases GABAergic spontaneous postsynaptic current (sPSC) frequency. This increase in sPSC frequency does not require the canonical NO receptor, soluble guanylate cyclase, or presynaptic action potentials. However, removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, we confirm that NO increases cytosolic Ca2+ in amacrine cell processes by activating a Ca2+ influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca2+ elevations are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca2+ channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca2+ influx adding to the NO-dependent, voltage-independent, Ca2+ influx. Insight into the identity of the Ca2+ influx pathway is provided by the transient receptor potential canonical (TRPC) channel inhibitor clemizole, which prevents the NO-dependent increase in sPSC frequency and cytosolic Ca2+ elevations. These data suggest that NO production in the inner retina will enhance Ca2+-dependent GABA release from amacrine cells by activating TRPC channel(s). NEW & NOTEWORTHY Our research provides evidence that nitric oxide (NO) promotes GABAergic output from retinal amacrine cells by activating a likely transient receptor potential canonical-mediated Ca2+ influx pathway. This NO-dependent mechanism promoting GABA release can be voltage independent, suggesting that, in the retina, local NO production can bypass the formal retinal circuitry and increase local inhibition.

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TRPC5 is required for the NO-dependent increase in dendritic Ca2+ and GABA release from chick retinal amacrine cells

Journal of Neurophysiology ◽

10.1152/jn.00500.2017 ◽

2018 ◽

Vol 119 (1) ◽

pp. 262-273 ◽

Cited By ~ 5

Author(s):

J. Wesley Maddox ◽

Nikka Khorsandi ◽

Evanna Gleason

Keyword(s):

Nitric Oxide ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Amacrine Cells ◽

Gaba Release ◽

Visual Signal ◽

Inner Retina ◽

Transient Receptor Potential Canonical ◽

Transient Receptor ◽

Dependent Mechanism

GABAergic signaling from amacrine cells (ACs) is a fundamental aspect of visual signal processing in the inner retina. We have previously shown that nitric oxide (NO) can elicit release of GABA independently from activation of voltage-gated Ca2+ channels in cultured retinal ACs. This voltage-independent quantal GABA release relies on a Ca2+ influx mechanism with pharmacological characteristics consistent with the involvement of the transient receptor potential canonical (TRPC) channels TRPC4 and/or TRPC5. To determine the identity of these channels, we evaluated the ability of NO to elevate dendritic Ca2+ and to stimulate GABA release from cultured ACs under conditions known to alter the function of TRPC4 and 5. We found that these effects of NO are phospholipase C dependent, have a biphasic dependence on La3+, and are unaffected by moderate concentrations of the TRPC4-selective antagonist ML204. Together, these results suggest that NO promotes GABA release by activating TRPC5 channels in AC dendrites. To confirm a role for TRPC5, we knocked down the expression of TRPC5 using CRISPR/Cas9-mediated gene knockdown and found that both the NO-dependent Ca2+ elevations and increase in GABA release are dependent on the expression of TRPC5. These results demonstrate a novel NO-dependent mechanism for regulating neurotransmitter output from retinal ACs. NEW & NOTEWORTHY Elucidating the mechanisms regulating GABAergic synaptic transmission in the inner retina is key to understanding the flexibility of retinal ganglion cell output. Here, we demonstrate that nitric oxide (NO) can activate a transient receptor potential canonical 5 (TRPC5)-mediated Ca2+ influx, which is sufficient to drive vesicular GABA release from retinal amacrine cells. This NO-dependent mechanism can bypass the need for depolarization and may have an important role in processing the visual signal by enhancing retinal amacrine cell GABAergic inhibitory output.

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TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

AJP Renal Physiology ◽

10.1152/ajprenal.00619.2013 ◽

2014 ◽

Vol 307 (6) ◽

pp. F666-F672 ◽

Cited By ~ 18

Author(s):

Pablo D. Cabral ◽

Jeffrey L. Garvin

Keyword(s):

Nitric Oxide ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Ruthenium Red ◽

Transient Receptor Potential Vanilloid ◽

No Production ◽

Thick Ascending Limb ◽

Different Types ◽

Luminal Flow ◽

Transient Receptor

Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min ( n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 μmol/l) and RN 1734 (10 μmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min ( P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min ( P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 μmol/l) enhanced NO production by 60 ± 11 AU/min ( P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min ( P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.

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Mechanotransduction of the endothelial glycocalyx mediates nitric oxide production through activation of TRP channels

AJP Cell Physiology ◽

10.1152/ajpcell.00288.2015 ◽

2016 ◽

Vol 311 (6) ◽

pp. C846-C853 ◽

Cited By ~ 22

Author(s):

Matthew A. Dragovich ◽

Daniel Chester ◽

Bingmei M. Fu ◽

Chenyu Wu ◽

Yan Xu ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Flow ◽

Transient Receptor Potential ◽

Trp Channels ◽

Receptor Potential ◽

Mechanical Stretch ◽

Endothelial Glycocalyx ◽

No Production ◽

Endothelial Surface ◽

Transient Receptor

The endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the vascular endothelium, serving critical functions in the mechanotransduction of blood flow-induced forces. One of the most important protective functions of the ESG is to mediate the production of nitric oxide (NO) in response to blood flow. However, the detailed mechanism underlying ESG's mechanotransduction of the production of NO has not been completely identified. Herein, using the cultured rat brain microvascular endothelial cells (bEnd.3) as a model system, we have implemented a combined atomic force and fluorescence microscopy approach to show that the ESG senses and transduces vertical mechanical stretch to produce NO. This rapid NO production is dependent on the presence of both heparan sulfate (HS) and hyaluronic acid (HA) in ESG, as the removal of HS and/or HA leads to a significant decrease in NO production. Moreover, the production of NO is dependent on the intake of Ca2+ via endothelial transient receptor potential (TRP) channels. Together, our results demonstrate the molecular mechanism of rapid production of NO in response to vertical mechanical stretch.

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Transient Receptor Potential Canonical Channels in Health and Disease: A 2020 Update

Cells ◽

10.3390/cells10030496 ◽

2021 ◽

Vol 10 (3) ◽

pp. 496

Author(s):

Priya R. Kirtley ◽

Gagandeep S. Sooch ◽

Fletcher A. White ◽

Alexander G. Obukhov

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Trpc Channels ◽

Special Issue ◽

25Th Anniversary ◽

Trpc Channel ◽

Transient Receptor Potential Canonical ◽

Health And Disease ◽

Transient Receptor

This 2020 Special Issue “TRPC channels” of Cells was dedicated to commemorating the 25th anniversary of discovery of the Transient Receptor Potential Canonical (TRPC) channel subfamily [...]

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