Carbenoxolone Blocks the Light-Evoked Rise in Intracellular Calcium in Isolated Melanopsin Ganglion Cell Photoreceptors

Abstract

BackgroundRetinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca2+ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca2+ signals in ipRGCs independent of gap junction blockade.Methodology/Principal FindingsTo test the possibility that carbenoxolone directly inhibits light-evoked Ca2+ responses in ipRGCs, the light-evoked rise in intracellular Ca2+ ([Ca2+]i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca2+]i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.Conclusions/SignificanceWe demonstrate that the light-evoked rise in [Ca2+]i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca2+]i in isolated ipRGCs is almost entirely due to Ca2+ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca2+]i in ipRGCs by blocking L-type voltage-gated Ca2+ channels. The ability of carbenoxolone to block evoked Ca2+ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca2+]i is the output being measured.

All Spiking, Sustained ON Displaced Amacrine Cells Receive Gap-Junction Input from Melanopsin Ganglion Cells

“…This conclusion became questionable when subsequent work showed that the percentage of light-responsive GCL neurons detected by Sekaran et al (~3%) roughly matched the percentage of ipRGCs [34], and that carbenoxolone could block the light-induced Ca 2+ dye responses of dissociated ipRGCs [35]. However, the demonstration of tracer coupling strengthened the possibility of ipRGC-amacrine coupling [10], which we validated here.…”

Carbenoxolone inhibits TRPV4 channel‐initiated oxidative urothelial injury and ameliorates cyclophosphamide‐induced bladder dysfunction

“…These channels may transmit, propagate and potentiate TRPV4 agonist‐initiated Ca 2+ signal through direct intercellular Cx channel or pannexin‐derived extracellular ATP . Furthermore, CBX has also been reported to block L‐type voltage‐gated Ca 2+ channels and prevent the light‐evoked increase in intracellular Ca 2+ in retinal ganglion cells and cultured rat cortical astroglia, respectively . Intriguingly, this channel has also reported to be implicated in diabetic bladder dysfunction .…”

Ghrelin Decreases Firing Activity of Gonadotropin-Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner

“…Ca 2+ -imaging recordings were baseline corrected, then the area-under-curve data of the records representing the net change in the intracellular free Ca 2+ -content were analyzed. Baseline correction was carried out by subtracting the background fluorescence measured from a region in the coverslip free of neurons [39]. …”