Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Abstract

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K+ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (Vm), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in Vm. Cell volume and Vm changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by Vm depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K+ or Cl− channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in Vm– define the success of RVD.

TRPV4 Contributes to Resting Membrane Potential in Retinal Müller Cells: Implications in Cell Volume Regulation

“…MIO‐M1 resting V m is −63.1 ± 2.3 mV as previously reported [Fernández et al, ]. 1% changes in fluorescence correspond to a V m variation of 2.2 mV, as calculated from the mean calibration curve (0.0045 ± 0.002, X ± SD, n = 58).…”

“…How could TRPV4 blockage affect V m without evoking detectable changes in the magnitude of cytosolic Ca 2+ levels or in the %RVD during the osmotic shock? We have previously demonstrated that in MIO‐M1 cells, as in many other systems, only a fraction of RVD is mediated by KCl efflux: there is also a major component (∼70%) mediated by organic osmolyte efflux [Pasantes‐Morales et al, ; Ando et al, ; Fernández et al, ]. Therefore it is likely that hypo‐osmotic‐induced local changes in Ca 2+ conductance mediated by TRPV4 occur close to the plasma membrane and do not affect intracellular Ca 2+ levels, though they are sufficient for BK channel activation.…”

“…Our previous studies in M€ uller cells, showed that RVD response is strongly modulated by changes in the electrochemical gradient for K þ and Cl À and, thus, by V m [Fern andez et al, 2013]. Keeping in mind that the high K þ permeability of the plasma membrane is the basis of the very negative resting V m of M€ uller cells and that TRPV4 plays a key role in modulating Ca 2þ -activated K þ channels in a variety of cell types, we investigated the putative contribution of TRPV4 in modulating the resting V m in MIO-M1 cells.…”

AQP2 can modulate the pattern of Ca2+ transients induced by store‐operated Ca2+ entry under TRPV4 activation

“…To accomplish this, we exposed WT‐ and AQP2‐RCCD 1 cells to the specific TRPV4 activator 4α‐PDD (10 µM) while recording Vm with the voltage‐sensitive fluorescent probe DIBAC 4 (3). This dye has been widely used to reproducibly measure Vm under both depolarizing and hyperpolarizing conditions in a broad range of cells . WT‐ and AQP2‐RCCD 1 cells show analogous Vm values in basal conditions and a comparable change under depolarizing conditions with a high extracellular K + solution (95 mM) (see section 2).…”

Glia–neuron interactions in the mammalian retina

“…The macrophage family includes meningeal and perivascular macrophages, blood circulating macrophages, dendritic cells derived from the bone marrow, and tissue resident macrophages (Wieghofer et al, 2014). The microglia form a population of resident macrophages within the CNS that not only has immune activity but also, that participates in the development and maintenance of neural networks, as well as in tissue homeostasis (Fernandes et al, 2014;Gertig and Hanisch, 2014;Ransohoff and Brown, 2012). Although the origin of these cells is controversial, it is believed that primitive myeloid progenitors of the egg sac are the precursors of these cells and not the bone marrow (Ginhoux et al, 2010;Schulz et al, 2012), and during adulthood a population of local progenitors ensures the continuity of the microglia in the nervous system (Ajami et al, 2007).…”