Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium

1997 ◽  
Vol 273 (2) ◽  
pp. C456-C472 ◽  
Author(s):  
E. Kenyon ◽  
A. Maminishkis ◽  
D. P. Joseph ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ph Regulation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Cytosolic Ph ◽  
Acid Recovery ◽  
Tightly Coupled ◽  
Intracellular Microelectrodes

pH regulation was studied in fresh explant bovine retinal pigment epithelium-choroid using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and intracellular microelectrodes. Acid recovery was HCO3 dependent, inhibited by apical amiloride and apical or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and required apical and basal Na. Alkali recovery was HCO3 dependent and inhibitable by apical or basal DIDS. Three apical and two basolateral transporters were identified. Four contribute to acid extrusion, i.e., apical Na/H exchange, apical H-lactate cotransport, and apical Na-HCO3 cotransport and basolateral Na-HCO3 cotransport. At least two contribute to alkali extrusion, i.e., apical Na-HCO3 cotransport and a basolateral HCO3-dependent, DIDS-inhibitable mechanism, possibly Na-HCO3 cotransport, Cl/HCO3 exchange, or both. The apical Na-HCO3 cotransporter is electrogenic, carrying net negative charge inward. Basal Cl removal or addition of basal HCO3 caused HCO3- and Cl-dependent alkalinizations, respectively. Apical DIDS increased both responses. These cytosolic pH (pHi) regulatory mechanisms are so tightly coupled that changes in pHi can only occur after two or more of them are inhibited. In addition, these mechanisms help provide pathways for transport of Na and HCO3 across the retinal pigment epithelium between the blood and the distal retina.

Acidification stimulates chloride and fluid absorption across frog retinal pigment epithelium

1994 ◽  
Vol 266 (4) ◽  
pp. C946-C956 ◽  
Author(s):  
J. L. Edelman ◽  
H. Lin ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Open Circuit ◽  
Disulfonic Acid ◽  
Fluid Absorption

Radioactive tracers and a modified capacitance-probe technique were used to characterize the mechanisms that mediate Cl and fluid absorption across the bullfrog retinal pigment epithelium (RPE)-choroid. In control (HCO3/CO2) Ringer solution, 36Cl was actively absorbed (retina to choroid) at a mean rate of 0.34 mu eq.cm-2.h-1 (n = 34) and accounted for approximately 25% of the short-circuit current. Apical bumetanide (100 microM) or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) inhibited active Cl transport by 70 and 62%, respectively. Active Cl absorption was doubled, either by removing HCO3 from the bathing media or by elevating CO2 from 5 to 13%, and the increased flux was inhibited by apical bumetanide or basal DIDS. Open-circuit measurements of fluid absorption rate (Jv) and the net fluxes of 36Cl, 22Na, and 86Rb (K substitute) indicated that CO2-induced acidification stimulated NaCl and fluid absorption across the RPE. During acidification, bumetanide produced a twofold larger inhibition of Jv compared with control. Stimulation of net Cl absorption was most likely caused by inhibition of the the basolateral membrane intracellular pH-dependent Cl-HCO3 exchanger.

scholarly journals SLC26A7 constitutes the thiocyanate-selective anion conductance of the basolateral membrane of the retinal pigment epithelium

2020 ◽  
Vol 319 (4) ◽  
pp. C641-C656
Author(s):  
Xu Cao ◽  
Manoocher Soleimani ◽  
Bret A. Hughes
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ph Regulation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Subretinal Fluid ◽  
Wild Type Mouse ◽  
Biologically Active ◽  
Rpe Cells ◽  
Anion Conductance

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl− between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN−), a property that does not correspond with any of the Cl− channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN− permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl− channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN− conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN−-selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

scholarly journals Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

1992 ◽  
Vol 99 (2) ◽  
pp. 263-290 ◽  
Author(s):  
D P Joseph ◽  
S S Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Membrane Voltage ◽  
Disulfonic Acid ◽  
Equilibrium Potential ◽  
Ionophore A23187 ◽  
Conductance Changes ◽  
Cl Conductance

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.

Potassium-induced chloride secretion across the frog retinal pigment epithelium

1994 ◽  
Vol 266 (4) ◽  
pp. C957-C966 ◽  
Author(s):  
J. L. Edelman ◽  
H. Lin ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Chloride Secretion ◽  
Ringer Solution ◽  
Open Circuit ◽  
Disulfonic Acid ◽  
Subretinal Space ◽  
K Concentration

In the intact eye, a transition from light to dark increases K concentration ([K]o) from approximately 2 to 5 mM in the extracellular (subretinal) space between the photoreceptors and the retinal pigment epithelium (RPE) apical membrane. In control (HCO3/CO2) Ringer solution, 36Cl was actively absorbed across isolated bullfrog RPE (retina to choroid) at a rate of 0.31 +/- 0.02 (SE) mu eq.cm-2.h-1 (n = 15). Elevating apical [K]o from 2 to 5 mM reversed active 36Cl transport to secretion (choroid to retina), with a rate of 0.76 +/- 0.17 mu eq.cm-2.h-1. This reversal was completely inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in either the apical or basal bath. In open circuit, elevating [K]o induced a similar reversal of net 36Cl flux and inhibited fluid absorption by approximately 25%. Apical Ba2+ (1 mM), decreased CO2 (5 to 1%), or increased apical bath HCO3 concentration ([HCO3]o) also caused a DIDS-inhibitable reversal of active 36Cl flux. A 10-fold reduction of apical bath Na or [HCO3]o significantly inhibited [K]o, Ba2+, and low CO2-induced Cl secretion. All of these results can be understood in terms of an intracellular pH-dependent stimulation of the basolateral membrane Cl-HCO3 exchanger.

Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium

1992 ◽  
Vol 262 (2) ◽  
pp. C374-C383 ◽  
Author(s):  
S. Fujii ◽  
R. P. Gallemore ◽  
B. A. Hughes ◽  
R. H. Steinberg
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Transference Number ◽  
Disulfonic Acid ◽  
Equilibrium Potential ◽  
Cl Conductance

There is now evidence that a Cl- conductance on the basal membrane of the retinal pigment epithelium (RPE) is involved in the generation of both the fast oscillation and the light peak of the direct-current electroretinogram as well as being critical for transepithelial fluid and salt movement. In the present study, we characterized the basolateral membrane Cl- conductance of an in vitro preparation of toad RPE-choroid using conventional and Cl(-)-selective microelectrodes. Under control conditions, the potential across the apical (Vap) and basal (Vba) membranes averaged -60 +/- 2 and -45 +/- 2 mV, respectively (n = 40). Intracellular Cl- activity (aiCl = 20 +/- 1 mM) was distributed above equilibrium across both membranes, consistent with active accumulation of Cl-. A sixfold decrease in Cl- in the basal bath depolarized Vba by 12 +/- 1 mV (n = 17) and increased the apparent basal membrane resistance. By sequential measurement of aiCl and subepithelial Cl- activity during a step decrease in basal Cl-, we constructed the change in Cl- equilibrium potential (ECl) across the basal membrane. Estimation of the change in basal membrane electromotive force during the change in ECl gave an average value for the Cl- transference number (TCl) of 0.45. Further evidence for a Cl- conductance was obtained by measuring changes in aiCl induced by transepithelial current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. Both the amplitude of the Cl- diffusion potential and the current-induced changes in aiCl were reduced by basal perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250-500 microM), a blocker of Cl- channels in some epithelia.

pHi-dependent Cl-HCO3 exchange at the basolateral membrane of frog retinal pigment epithelium

1994 ◽  
Vol 266 (4) ◽  
pp. C935-C945 ◽  
Author(s):  
H. Lin ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Initial Rate ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Ringer Solution ◽  
Disulfonic Acid ◽  
Subretinal Space ◽  
K Concentration

Intracellular pH (pHi) measurements in frog retinal pigment epithelium using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein demonstrate that the basolateral membrane contains a pHi-sensitive Cl-HCO3 exchanger. In control Ringer solution, the removal of Cl from the basal bath alkalinized the cells by 0.07 +/- 0.03 (SD) pH units (n = 39) with an initial rate of 0.022 +/- 0.0013 pH units/min. This effect was blocked by 0.5 mM basal 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid or the removal of HCO3 from both the apical and basal baths. The rate of the exchange is reduced by acidification and increased by alkalinization. Increasing apical bath K concentration ([K]o) from 2 to 5 mM approximates the [K]o change in the subretinal space of the intact eye following a transition from light to dark. This [K]o change alkalinized the cells by increasing the rate of the apical membrane Na-HCO3 cotransporter. In 5 mM apical [K]o, the initial rate of the 0 Cl-induced alkalinization was significantly increased to 304 +/- 13% (n = 4) of control (2 mM [K]o). These mechanisms regulate pHi and could also buffer changes in subretinal pH.

Lactate transport mechanisms at apical and basolateral membranes of bovine retinal pigment epithelium

1994 ◽  
Vol 267 (6) ◽  
pp. C1561-C1573 ◽  
Author(s):  
E. Kenyon ◽  
K. Yu ◽  
M. La Cour ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Open Circuit ◽  
Subretinal Space ◽  
Transport Mechanisms ◽  
Lactate Transport ◽  
Space Volume

The isolated bovine retinal pigment epithelium actively transports lactate from the apical to the basal bath. Net short-circuit [14C]lactate flux in 20 mM lactate was 0.46 +/- 0.09 mu eq.cm-2.h-1 (n = 8). In open circuit, with a physiological lactate gradient, net [14C]lactate flux was 0.66-1.31 mu eq.cm-2.h-1 (n = 3). Lactate in the apical bath caused intracellular acidifications that were saturable, apparently stereospecific, and reduced in magnitude by several H-lactate cotransport inhibitors. In the basal bath, lactate caused intracellular alkalinizations that were dependent on the presence of Na. In short circuit, 20 mM lactate in both baths reversed the direction of net transepithelial 22Na transport from secretion to absorption, suggesting the presence of basolateral Na-lactate cotransport moving lactate out of the cells. Outwardly directed Na-lactate cotransport requires a lactate:Na stoichiometry > 1.4:1, consistent with the coupled movement of Na, lactate, and net negative charge across the basolateral membrane. Intracellular microelectrode recordings showed that basal lactate hyperpolarized and apical lactate depolarized the basolateral membrane. For lactate absorption, this is a novel arrangement of membrane proteins:luminal H-lactate cotransport and serosal electrogenic Na:(n)lactate cotransport. Lactate transport across the retinal pigment epithelium may play an important role in regulating retinal metabolism and subretinal space volume and composition.

Chloride current activated by swelling in retinal pigment epithelium cells

1993 ◽  
Vol 265 (4) ◽  
pp. C1037-C1045 ◽  
Author(s):  
L. M. Botchkin ◽  
G. Matthews
Keyword(s):  
Retinal Pigment Epithelium ◽  
Fluid Transport ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ionic Composition ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Subretinal Space ◽  
Channel Blockers ◽  
Membrane Stretch

A membrane conductance activated by cell swelling was characterized in cells of the retinal pigment epithelium (RPE). Manipulations of internal and external Cl concentration revealed that the conductance is permeable to Cl and somewhat permeable to the gluconate anion used for Cl substitution (ratio of gluconate to Cl permeability approximately 0.1). The conductance was blocked by the Cl channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid in a manner consistent with open-channel blockade. Both the onset and recovery of the Cl current following a transient increase in cell volume were slow. This suggests that activation of the current depends on some internal signal rather than directly on membrane stretch. Experiments to examine a possible role for intracellular Ca concentration ([Ca]i) in regulation of the current demonstrated that an increase in [Ca]i was not involved in the linkage between swelling and Cl current; activation of the current was unaffected by the calcium-buffering conditions, the current could not be activated by large increases in [Ca]i elicited by ionomycin, and no changes in [Ca]i were observed to be associated with swelling. RPE cells normally experience changes in the volume and ionic composition of the extracellular subretinal space during changes in illumination conditions; therefore, the volume-sensitive Cl conductance may play a role in volume regulation in the RPE in response to these extracellular changes and/or in transepithelial fluid transport.

Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium

1995 ◽  
Vol 268 (5) ◽  
pp. C1164-C1172 ◽  
Author(s):  
B. A. Hughes ◽  
A. Shaikh ◽  
A. Ahmad
Keyword(s):  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Dissociation Constants ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
Conductance Ratio ◽  
Inwardly Rectifying

Intracellular microelectrode techniques were employed to characterize the blocker sensitivity of the K+ conductance (gK) at the apical membrane of the toad retinal pigment epithelium (RPE). Increasing the K+ concentration in the apical bath ([K+]o) from 2 to 5 mM produced a rapid depolarization of the apical membrane potential (VA). The addition of 0.5 mM Ba2+ or 5 mM Cs+ to the apical bath rapidly depolarized VA and increased the transepithelial resistance and ratio of apical-to-basolateral membrane resistance. In the presence of apical Ba2+ or Cs+, the response of VA to delta [K+]o was markedly reduced, indicating that these ions are effective blockers of apical gK. The Ba(2+)- and Cs(+)-induced decreases in the apparent apical-to-basolateral membrane conductance ratio were concentration dependent, with apparent dissociation constants of 17 microM and 0.5 mM, respectively. The apparent blocker sensitivity of apical gK is similar to that previously demonstrated for the inwardly rectifying K+ conductance in isolated toad RPE cells, suggesting that the inwardly rectifying K+ conductance comprises much of apical gK.

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