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.

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.

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 Potential, Current, and Ionic Fluxes across the Isolated Retinal Pigment Epithelium and Choroid

1966 ◽  
Vol 49 (5) ◽  
pp. 913-924 ◽  
Author(s):  
Arnaldo Lasansky ◽  
Felisa W. de Fisch
Keyword(s):  
Retinal Pigment Epithelium ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Short Circuit Current ◽  
Bufo Marinus ◽  
Flux Chamber ◽  
Epithelial Surface ◽  
The Mean

A flux chamber was utilized for in vitro studies of a membrane formed by the retinal pigment epithelium and choroid of the eye of the toad (Bufo arenarum and Bufo marinus). A transmembrane potential of 20 to 30 mv was found, the pigment epithelium surface positive with respect to the choroidal surface. Unidirectional fluxes of chloride, sodium, potassium, and calcium were determined in the absence of an electrochemical potential difference. A net transfer of chloride from pigment epithelium to choroid accounted for a major fraction of the mean short-circuit current. A small net flux of sodium from choroid to pigment epithelium was detected in Bufo marinus. In both species of toads, however, about one-third of the mean short-circuit current remained unaccounted for. Manometric determinations of bicarbonate suggested an uptake of this ion at the epithelial surface of the membrane but did not provide evidence of a relationship between this process and the short-circuit current.

scholarly journals Changes in apical [K+] produce delayed basal membrane responses of the retinal pigment epithelium in the gecko.

1984 ◽  
Vol 83 (2) ◽  
pp. 193-211 ◽  
Author(s):  
E R Griff ◽  
R H Steinberg
Keyword(s):  
Membrane Potential ◽  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basal Membrane ◽  
Subretinal Space ◽  
Bathing Solution ◽  
Subsequent Decrease ◽  
Opposite Sequence

We describe here a new retinal pigment epithelium (RPE) response, a delayed hyperpolarization of the RPE basal membrane, which is initiated by the light-evoked decrease of [K+]o in the subretinal space. This occurs in addition to an apical hyperpolarization previously described in cat (Steinberg et al., 1970; Schmidt and Steinberg, 1971) and in bullfrog (Oakley et al., 1977; Oakley, 1977). Intracellular and extracellular potentials and measurements of subretinal [K+]o were recorded from an in vitro preparation of neural retina-RPE-choroid from the lizard Gekko gekko in response to light. Extracellularly, the potential across the RPE, the transepithelial potential (TEP), first increased and then decreased during illumination. Whereas the light-evoked decrease in [K+]o predicted the increase in TEP, the subsequent decrease in TEP was greater than predicted by the reaccumulation of [K+]o. Intracellular RPE recordings showed that a delayed hyperpolarization generated at the RPE basal membrane produced the extra TEP decrease. At light offset, the opposite sequence of membrane potential changes occurred. RPE responses to changes in [K+]o were studied directly in the isolated gecko RPE-choroid. Decreasing [K+]o in the apical bathing solution produced first a hyperpolarization of the apical membrane, followed by a delayed hyperpolarization of the basal membrane, a sequence of membrane potential changes identical to those evoked by light. Increasing [K+]o produced the opposite sequence of membrane potential changes. In both preparations, the delayed basal membrane potentials were accompanied by changes in basal membrane conductance. The mechanism by which a change in extracellular [K+] outside the apical membrane leads to a polarization of the basal membrane remains to be determined.

Electrophysiology of the Mantle of Anodonta Cygnea

1988 ◽  
Vol 140 (1) ◽  
pp. 65-88
Author(s):  
J. COIMBRA ◽  
J. MACHADO ◽  
P. L. FERNANDES ◽  
H. G. FERREIRA ◽  
K. G. FERREIRA
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Sodium Concentration ◽  
Pure Oxygen ◽  
Bathing Solution ◽  
Anodonta Cygnea ◽  
Average Value ◽  
Apical Side ◽  
Order Of Magnitude ◽  
Net Flux

When gassed with a CO2-containing mixture, under short-circuit conditions, the isolated outer mantle epithelium (OME) of Anodonta cygnea generated a current which exhibited cyclic variations throughout the year. The intracellular potential, under short-circuit conditions, had an average value of −31±0.5mV (N = 65). The potential was sensitive to changes in concentration of potassium and chloride on the haemolymph side of the preparation, but not on the shell side, and was insensitive to changes in sodium concentration on either side. When the preparation was gassed with pure oxygen the current fell by 85±1% (N = 8). A similar fall in current (88±2%, N = 8) was observed when the solution bathing the apical side of the epithelium was prepared without bicarbonate and gassed with 95% O2 + 5% CO2. If this same bathing solution was gassed with 100% oxygen, the current fell by 67±2% (N = 8) at pH7.2 and by 92±4% (N = 8) at pH4.5. The short-circuit current was inhibited by DIDS (0.5 mmoll−1) and SITS (0.5 mmoll−1) when these drugs were applied on the haemolymph side. The current was also inhibited by DNP (1 mmoll−1), iodoacetamide (1 mmoll−1) and diamox (1 mmoll−1). Amiloride (1 mmoll−1) blocked the current but only when applied on the haemolymph side. Ouabain (0.1 mmoll−1) did not affect the current. The net fluxes of rubidium (used as a tracer for potassium), chloride and calcium, measured with 86Rb, 36Cl and 45Ca, respectively, were very small when compared with the short-circuit current. There was a small net flux of sodium (measured with 22Na) towards the haemolymph side. The net flux of bicarbonate (measured with [14C]bicarbonate) was equal to the short-circuit current and was inhibited by DIDS. The permeability of the preparation to calcium was an order of magnitude higher than the permeability to sodium, potassium or chloride. The intracellular concentrations of potassium and chloride measured with ion-sensitive microelectrodes were 26.5±1.1 (N = 16) and 7.9±0.3 (N = 30) mmoll−1, respectively. When these concentrations were measured with chemical

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)

Membrane mechanisms for electrogenic Na+-independent l-alanine transport in the lizard duodenal mucosa

2000 ◽  
Vol 279 (3) ◽  
pp. R925-R935 ◽  
Author(s):  
Mario Díaz ◽  
Virtudes Medina ◽  
Tomás Gómez ◽  
Antonio Lorenzo
Keyword(s):  
Driving Force ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Bathing Solution ◽  
Alkaline Ph ◽  
Alanine Transport ◽  
Carbonyl Cyanide ◽  
Computer Controlled

The active Na+-independent transport ofl-alanine across the duodenal mucosa of the lizard Gallotia galloti was studied in Ussing-type chambers using a computer-controlled voltage clamp. Addition of l-alanine to the Na+-free bathing solutions resulted in a significantl-alanine absorption ( J net) that was paralleled by an increase in transepithelial short-circuit current ( I sc) and potential difference (PD) without apparent changes in the tissue conductance. The concentration dependence of J net, PD, and I sc displayed Michaelis-Menten kinetics.l-alanine-induced electrical changes were completely inhibited by external alkaline pH or by the H+-ionophore carbonyl cyanide m-chlorophenyl-hydrazone in the bathing solution. The alanine-induced electrogenicity was dependent on the presence of extracellular K+ and could be blocked by serosal Ba2+ or mucosal orthovanadate. These results suggest the existence of an H+-coupledl-alanine cotransport at the apical membrane of enterocytes. The favorable H+ driving force is likely to be maintained by an apical vanadate-sensitive H+-K+-ATPase, allowing the extrusion of H+ in an exchange with K+. Potassium exit through a basolateral barium-sensitive conductance provides the key step for the electrogenicity of l-alanine absorption.

Activation of K+ conductance in basolateral membrane of toad urinary bladder by oxytocin and cAMP

1988 ◽  
Vol 254 (6) ◽  
pp. C816-C821 ◽  
Author(s):  
W. Van Driessche ◽  
D. Erlij
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Membrane Properties ◽  
Toad Urinary Bladder ◽  
Electrical Behavior ◽  
Apical Side ◽  
Camp Analogue

We incubated toad urinary bladders with Na+-free, isotonic K+ solutions on the apical side and increased the cationic conductance of the apical membrane with nystatin (150 U/ml). Under these conditions, the short-circuit current is mostly carried by K+ flowing from mucosa to serosa. Impedance measurements showed that in nystatin-treated preparations, the electrical behavior of the tissue is dominated by the basolateral membrane properties. Oxytocin (0.1 U/ml) produced an increase of the current and the conductance of the basolateral membrane. Both the resting and the oxytocin-stimulated current were rapidly and reversibly blocked by serosal Ba2+. Addition of the adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chloropheylthio)-cAMP] to the basolateral solution mimicked the effects of oxytocin. These results show that oxytocin and cAMP stimulate a potassium conductance in the basolateral membrane and that the stimulation is not related to an increase in sodium entry through the apical membrane. Addition of ouabain (10(-3) M) to the serosal solution did not modify the stimulation by oxytocin, indicating that the activated pathway is not linked to the rate of turnover of the Na+ pump.

Evidence that prostaglandin E2 stimulates chloride secretion in cultured A6 renal epithelial cells

1986 ◽  
Vol 250 (3) ◽  
pp. F511-F515 ◽  
Author(s):  
R. Keeler ◽  
N. L. Wong
Keyword(s):  
Epithelial Cells ◽  
Prostaglandin E2 ◽  
Short Circuit ◽  
Basal Medium ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Apical Surface ◽  
Renal Epithelial Cells ◽  
Apical Side ◽  
Net Flux

The effects of prostaglandin E2 (PGE2) on the transport of sodium and chloride were studied in cultured A6 renal epithelial cells. PGE2 on the basolateral but not the apical surface increased transmonolayer short-circuit current (Isc) and conductance. These changes could not be inhibited with amiloride or furosemide in the apical medium. Flux measurements showed that although Isc and net flux of sodium were equal in unstimulated cells, after addition of PGE2 the current increased with no corresponding changes in bidirectional or net flux of sodium. Immersing the cells in sodium-free or chloride-free media inhibited the effects of PGE2. Measurements of the simultaneous fluxes of sodium and chloride showed that after PGE2 was added there was a net flux of chloride from the basal to the apical side (secretion) that was equal to the change in Isc. The effects of PGE2 were inhibited by furosemide in the basal medium. We conclude that PGE2 stimulates a process of chloride secretion in A6 cells.

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