scholarly journals Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. Measurements in the short-circuit state.

1984 ◽  
Vol 83 (6) ◽  
pp. 853-874 ◽  
Author(s):  
S Miller ◽  
D Farber
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cyclic Amp ◽  
Ion Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Active Absorption ◽  
Apical Side

In the frog retinal pigment epithelium (RPE), the cellular levels of cyclic AMP (cAMP) were measured in control conditions and after treatment with substances that are known to inhibit phosphodiesterase (PDE) activity (isobutyl-1-methylxanthine, SQ65442) or stimulate adenylate cyclase activity (forskolin). The cAMP levels were elevated by a factor of 5-7 compared with the controls in PDE-treated tissues and by a factor of 18 in forskolin-treated tissues. The exogenous application of cAMP (1 mM), PDE inhibitors (0.5 mM), or forskolin (0.1 mM) all produced similar changes in epithelial electrical parameters, such as transepithelial potential (TEP) and resistance (Rt), as well as changes in active ion transport. Adding 1 mM cAMP to the solution bathing the apical membrane transiently increased the short-circuit current (SCC) and the TEP (apical side positive) and decreased Rt. Microelectrode experiments showed that the elevation in TEP is due mainly to a depolarization of the basal membrane followed by, and perhaps also accompanied by, a smaller hyperpolarization of the apical membrane. The ratio of the apical to the basolateral membrane resistance increased in the presence of cAMP, and this increase, coupled with the decrease in Rt and the basolateral membrane depolarization, is consistent with a conductance increase at the basolateral membrane. Radioactive tracer experiments showed that cAMP increased the active secretion of Na (choroid to retina) and the active absorption of K (retina to choroid). Cyclic AMP also abolished the active absorption of Cl across the RPE. In sum, elevated cellular levels of cAMP affect active and passive transport mechanisms at the apical and basolateral membranes of the bullfrog RPE.

Aspects of electrolyte transport across isolated dog retinal pigment epithelium

1986 ◽  
Vol 250 (5) ◽  
pp. F781-F784 ◽  
Author(s):  
S. Tsuboi ◽  
R. Manabe ◽  
S. Iizuka
Keyword(s):  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Short Circuit Current ◽  
Bathing Solution ◽  
Apical Side ◽  
Net Flux ◽  
Net Fluxes

Transport of Na and Cl across the isolated dog retinal pigment epithelium (RPE) choroid was investigated. Under the short-circuit condition, a net Na flux was observed from choroid to retina and a net Cl flux was determined in the opposite direction. The current created by the net flux of these two ions was larger than the short-circuit current (SCC). Addition of 10(-5) M ouabain to the apical side inhibited net fluxes of both Na and Cl, whereas it reduced the SCC 84%. Addition of 10(-4) M furosemide to the apical side inhibited net Cl flux but had no effect on the net Na transport. The 10(-4) M furosemide reduced the SCC 38%. These drugs had no effect when applied to the basal side. Thus the transport of both Na and Cl depends on the Na-K-ATPase in the apical membrane of the dog RPE. A furosemide-sensitive neutral carrier at the apical membrane is suggested for the transport of Cl. Replacement of HCO3 with SO4 in the bathing solution caused an increase in the SCC, indicating the choroid-to-retina movement of HCO3 across the short-circuited dog RPE choroid.

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.

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.

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.

scholarly journals Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium.

1995 ◽  
Vol 106 (6) ◽  
pp. 1089-1122 ◽  
Author(s):  
W M Peterson ◽  
S S Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Subretinal Space ◽  
Gaba Transporter ◽  
Nipecotic Acid ◽  
Beta Alanine

Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Müller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta-alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.

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.

scholarly journals CO2-induced ion and fluid transport in human retinal pigment epithelium

2009 ◽  
Vol 133 (6) ◽  
pp. 603-622 ◽  
Author(s):  
Jeffrey Adijanto ◽  
Tina Banzon ◽  
Stephen Jalickee ◽  
Nam S. Wang ◽  
Sheldon S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Subretinal Space ◽  
Photoreceptor Outer Segments ◽  
Outer Segments

In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 ± 0.05 to 7.14 ± 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (≈10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase–mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 ± 3.3 to 24.0 ± 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 ± 1.6 to 6.7 ± 2.3 µl × cm−2 × hr−1 (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H2O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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