Frog Retinal Pigment Epithelium Research Articles

Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.

Antibodies against a furosemide binding protein from Ehrlich ascites tumour cells inhibit Na+, K+, Cl- co-transport in frog retinal pigment epithelium.

To investigate whether antibodies against a 100 kDa protein purified by furosemide affinity chromatography from Ehrlich ascites tumour cells could inhibit Na+, K+, Cl- co-transport in the isolated frog retinal pigment epithelium. The rate of Na+, K+, Cl- co-transport across the retinal membrane in the isolated frog RPE preparation was measured as the rate of decrease in the intracellular Cl- activity observed after administration of furosemide in the apical bath. The intracellular Cl- activity was measured with double barrelled Cl- sensitive microelectrodes. Incubation of frog retinal pigment epithelium for 30 min with antibodies reduced the rate of Na+, K+, Cl- co-transport by 43%, while leaving all other measured electrophysiological parameters intact. The antibodies inhibit Na+,K+,Cl- co-transport in the frog retinal pigment epithelium. This could be due to binding of the antibodies to the co-transporter itself or to a regulatory protein.

Genesis of the frog retinal pigment epithelium

To examine cell generation in the frog retinal pigment epithelium (RPE), representative developmental stages from tail-bud to adulthood received a single injection of [3H]thymidine. Animals were killed either 24 h or several weeks later; eyes were sectioned and processed by standard autoradiographic procedures and viewed by epi-polarised illumination. The distribution of [3H]thymidine-labelled cells indicated that the RPE is formed throughout life, including in adulthood, by cell addition at the ciliary margin, matching the pattern for the neural retina. In addition, a very small number of peripapillary RPE cells underwent division but only in the adult.

Genesis of the frog retinal pigment epithelium

To examine cell generation in the frog retinal pigment epithelium (RPE), representative developmental stages from tail-bud to adulthood received a single injection of [ 3H]thymidine. Animals were killed either 24 h or several weeks later; eyes were sectioned and processed by standard autoradiographic procedures and viewed by epi-polarised illumination. The distribution of [ 3H]thymidine-labelled cells indicated that the RPE is formed throughout life, including in adulthood, by cell addition at the ciliary margin, matching the pattern for the neural retina. In addition, a very small number of peripapillary RPE cells underwent division but only in the adult.

Regulatory volume decrease in frog retinal pigment epithelium

Pages C89–C1OO: J. S. Adorante.“Regulatory volume decrease in frog retinal pigment epithelium.” The origin line should read: School of Optometry, University of California, Berkeley, California 94720. On page C99, the following sentence should be added to the acknowledgment: This work was supported by National Eye Institute Grants EY-02205 (to S. S. Miller) and Core Grant EY-03176 and National Research Service Award EY-05968 (to J. S. Adorante).

Regulatory volume decrease in frog retinal pigment epithelium

To measure changes in cell water during cell volume regulation, retinal pigment epithelial cells were loaded with tetramethylammonium (TMA). Regulatory volume decrease (RVD) in TMA-loaded retinal pigment epithelial (RPE) cells was measured using double-barreled K(+)-specific microelectrodes. Hyposmotic removal of 12.5 mM NaCl from the apical bath caused bullfrog RPE cells to rapidly swell by approximately 10% and to recover to control level within 10-15 min. Hyposmotic RVD was inhibited by 5 mM basal but not apical BaCl2. Raising K+ in the basal bath from 2 to 12 mM also inhibited RVD. Hyposmotic swelling was accompanied by an increase in the ratio of apical to basolateral membrane resistance (Ra/Rb). The swelling-induced increase in Ra/Rb was inhibited by 5 mM BaCl2. Together, the above findings suggest that hyposmotic swelling enhances basolateral K+ conductance such that K+ and presumably anion efflux mediate net solute and water loss during RVD. RPE cells can also regulate their volume when swollen in isosmotic Ringer solution under certain conditions. When urea or apical HCO3- was used to induce cell swelling, RPE cells underwent an RVD. In contrast, isosmotic elevation of apical K+ from 2 to 5 mM resulted in an increase in RPE cell volume with no subsequent RVD. Thus the method used to swell RPE cells is an important determinant of RVD. Because changes in RPE cell volume in vivo may alter the volume and composition of the extracellular (subretinal) space surrounding the photoreceptors, isosmotic volume regulation may play an important physiological role in maintaining the integrity and health of the neural retina under normal and pathophysiological conditions.

Acidification stimulates chloride and fluid absorption across frog retinal pigment epithelium.

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.

Potassium-induced chloride secretion across the frog retinal pigment epithelium

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.

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

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.

Differential incorporation of docosahexaenoic and arachidonic acids in frog retinal pigment epithelium.

Vertebrate retinas contain a high level of docosahexaenoic acid (DHA, 22:6n-3) and a relatively low level of arachidonic acid (AA, 20:4n-6). Although it is not known how DHA is selectively concentrated in the retina, the retinal pigment epithelium (RPE) is intimately involved in this process as these cells phagocytize shed rod outer segments (containing DHA) and take up DHA from the circulation. To explore the role of the RPE in the DHA enrichment in the retina, native frog RPE cells (RPE-eyecups) were incubated with [3H]DHA or [3H]AA for short times, or pulsed for up to 1 h with the two labeled fatty acids plus labeled glycerol simultaneously, followed by a chase for up to 7 h. The incorporation of label into various lipids was monitored by thin-layer or high performance liquid chromatography. In short-time incubations, a greater amount of DHA was incorporated into neutral lipids (primarily triglycerides, TG) compared with AA, while similar amounts of both fatty acids were found in phospholipids. On a relative basis, DHA was esterified mainly into TG, with AA into phospholipids. The uptake of each fatty acid correlated linearly with concentration (30-120 nM) and the incorporation pattern was distinct. Interestingly, the uptake and incorporation of labeled DHA or AA were not affected by the presence of unlabeled AA or DHA, respectively. In a pulse-chase experiment, RPE cells were active in the de novo synthesis of glycerolipids, particularly TG, which achieved a specific activity as high as diglycerides and ten times higher than the major phospholipids. The incorporation of DHA into most glycerolipids occurred to a large extent during de novo glycerolipid synthesis, whereas AA was incorporated mainly by deacylation-reacylation reactions. Our results suggest different metabolic pathways for handling DHA and AA in frog RPE. The preferential incorporation of DHA into TG suggests that TG might play an important role in the selective enrichment of DHA in the retina and in the recycling of photoreceptor DHA between the RPE and the retina.

Metabolism in Frog Retinal Pigment Epithelium of Docosahexaenoic and Arachidonic Acids Derived from Rod Outer Segment Membranes

Frog photoreceptor cells shed about 10% of their rod outer segments (ROS) every 4th day. Packets of these membranes are phagocytized and digested by retinal pigment epithelial (RPE) cells. Large amounts of lipids must be processed daily, especially docosahexaenoic acid (22:6n-3, DHA), the major fatty acid of these membranes. To study the metabolism of ROS lipids in RPE cells, RPE-eyecups were incubated with [3H]DHA-, [3H]arachidonic acid (AA)-, or [2-3H]glycerol-labeled ROS membranes for 2 hr, followed by a chase for up to 8 hr. Lipid extracts of RPE cells and incubation media were resolved into classes and quantitated for radioactivity. In RPE cells, the relative proportion of DHA and AA label in triglycerides (TG) increased dramatically with incubation time, although the substrate ROS membranes did not contain labeled TG. Other RPE lipids showed prominent reductions or relative little change. The percentage of radioactivity in free fatty acids (FFA) was low (< 3%) in RPE cells. In the chase media, the majority (60-80%) of DHA and AA label was found in FFA, with little radioactivity in TG or phospholipids. When RPE cells were incubated with [3H]glycero-labeled ROS membranes which contained 22% of the label in TG, the most rapid reduction in relative radioactivity appeared in TG. We conclude that DHA and AA are released from phagocytized ROS membranes and are rapidly incorporated into RPE cellular lipids, primarily TG. This lipid class is very active metabolically, since TG derived from ROS are rapidly hydrolysed. Free DHA and AA of ROS origin are released from RPE cells. We suggest that RPE triglycerides are the source of FFA in the interphotoreceptor matrix.

Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding

The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22:6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22:6n-3 in the retina. The recycling of 22:6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.

Cl − transport in frog retinal pigment epithelium

Cl − transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled Cl − selective microelectrodes. Three types of experiments were performed. In the first group of experiments, the ionic dependence of Cl − influx across the retinal membrane was studied. The intracellular Cl − activity was first decreased by perfusing the retinal side of the epithelium with low Cl − solutions (3·6 m m Cl −); then the perfusate was changed to high Cl − solutions (90·1 m m), and the resulting Cl − influx was studied. In these experiments, the combined presence of extracellular Na + and K + was a necessary condition for Cl − influx across the retinal membrane. This supports the hypothesis of Na +,K +,Cl − co-transport across this membrane. In a second group of experiments, the effect of furosemide was studied. Furosemide (100 μ m) inhibited Cl − influx when the retinal extracellular Cl − concentration was increased from 3·6 to 90·1 m m. When administered to cells in steady state, furosemide in concentrations between 5 and 1000 μ m decreased the intracellular Cl − activity. Michaelis-Menten analysis yielded a K i for furosemide of 7 ± 2 μ m. The effect of furosemide on the intracellular Cl − activity required the combined presence of extracellular Na + and K +. When the retinal extracellular K + concentration was increased to between 0 and 10 m m, the furosemide-sensitive Cl − influx across the retinal membrane increased. Michaelis-Menten analysis yielded a half maximal stimulation at an extracellular K + concentration of 0·5 m m. Stimulation of the epithelium with 1 m m cAMP and 0·5 m m IBMX reduced the effect of furosemide on the intracellular Cl − activity by 26%. In a third group of experiments, the effect of transepithelial currents on the intracellular Cl − activity was investigated. Currents that depolarized the choroidal membrane potential increased the intracellular Cl − activity: currents that hyperpolarized this membrane potential decreased the intracellular Cl − activity. These findings are compatible with conductive Cl − transport across the choroidal membrane. The apparent Cl − conductance of this membrane was estimated to be 0·59 mS cm −2. This represents 27% of the total conductance in the choroidal membrane. Administration of 1 m m cAMP and 0·5 m m IBMX caused a 21% increase in the apparent Cl − conductance of the choroidal membrane.

Conditional inhibition of screening-pigment aggregation by lidocaine in crayfish photoreceptors and frog retinal pigment epithelium.

Lidocaine, at concentrations equal to or lower than those that inhibit fast axoplasmic transport, was found to interfere with the dark-adapting migration of the screening pigments along crayfish photoreceptors and within the cells of the frog retinal pigment epithelium (RPE). The effects of the anesthetic on pigment movements were studied in isolated eyes incubated under light or dark conditions in media of different ionic compositions. Treatment of crayfish eyes with 25 mmol l-1 lidocaine in normal Van Harreveld's saline arrested pigment migration to the dark-adapted position or caused migration towards the light-adapted position in the dark. Similar results were obtained with frog eyecups exposed to 5 mmol l-1 lidocaine in Ringer's solution. In each case, the inhibition of dark adaptation was reversible and dependent on the levels of Na+ and Ca2+ in the incubation medium. A dark-adapted position of both pigments was compatible with lidocaine treatment provided that low-Na+, or high-Ca2+ or Co(2+)-containing solutions were used. These results indicate that light-adapted and dark-adapted pigment positions in both types of retinal cells can occur in the absence of local nervous input. Further, the data suggest a direct effect of lidocaine upon the photoreceptors or RPE cells. The inhibition of pigment aggregation is interpreted to be a consequence of an anesthetic-induced increase in the permeability of the plasma membrane, which in turn affects the intracellular ionic balance that controls pigment position.

PH homeostasis in the frog retina: The role of Na+:HCO−3 co‐transport in the retinal pigment epithelium

Transport of acid/base equivalents across the isolated frog retinal pigment epithelium (RPE) was studied by means of pH selective microelectrodes. Abrupt changes in retinal extracellular pH, from 7.40 to values between 6.66 and 7.66, were found to cause rapid changes in both intracellular pH and choroidal extracellular pH. The initial rates of these changes were reduced by more than 60%, when the cellular Na+:HCO3- co-transport system was inhibited by Na+ removal, or by administration of 1 mM SITS. It is concluded that RPE transcellular HCO3- transport changed in response to changes in retinal extracellular pH. If present in vivo these changes in RPE HCO3- transport would tend to stabilize retinal extracellular pH. It is suggested that cellular Na+:HCO3- cotransport plays a significant role for the retinal pH homeostasis.

PHi regulation in frog retinal pigment epithelium: two apical membrane mechanisms

This study demonstrates that the apical membrane of frog retinal pigment epithelium (RPE) contains two intracellular pH (pHi) regulatory mechanisms, an electrogenic Na-HCO3 cotransporter blocked by DIDS and an amiloride-inhibitable Na-H antiporter. pHi was studied using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In these cells resting pHi equals 7.26 +/- 0.09 (n = 58). After an acid load (NH4Cl prepulse), pHi recovery required apical extracellular Na concentration ([Na]o) in HCO3 or HCO3-free Ringer. In HCO3 Ringer recovery was completely blocked by 1 mM apical DIDS (n = 5) but was not affected by absence of Cl. In HCO3-free Ringer, recovery was completely blocked by 1 mM apical amiloride (n = 3). At resting pHi, the intrinsic pH-buffering capacity of the cell is approximately 7.1 mM/pH and rises monotonically as pHi decreases. In HCO3 Ringer, the initial rate of acidification caused by apical Na removal, 0.39 +/- 0.03 pH/min (n = 26), was 80-90% inhibited by apical DIDS (n = 5) and 16% inhibited by 1 mM apical amiloride (n = 7), but not affected by absence of Cl. In HCO3 Ringer, initial rates of acidification induced by apical DIDS or amiloride were 0.11 +/- 0.06 (n = 5) and 0.03 +/- 0.02 pH/min (n = 7), respectively. These results indicate that the Na-HCO3 cotransporter accounts for 80-90% of the acid extrusion from frog RPE cells. Increasing apical [K]o from 2 to 5 mM approximates the in vivo apical [K]o changes during a light-dark transition and alkalinizes the cells. [K]o-induced alkalinization had an initial rate of 0.11 +/- 0.02 pH/min (n = 16), which was approximately 75% inhibited by apical DIDS (to 0.04 +/- 0.01 pH/min, n = 7) and completely blocked by HCO3/CO2 removal from both bathing solutions. [K]o-induced pHi changes alter RPE transport mechanisms and may affect RPE-photoreceptor interactions.

Kinetic properties and Na+ dependence of rheogenic Na(+)‐HCO3‐ co‐transport in frog retinal pigment epithelium.

1. Na(+)-HCO3- co-transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled pH-selective microelectrodes. Transient changes in the intracellular pH were monitored in response to abrupt changes in the Na+ concentration on the retinal side of the epithelium. 2. The experiments were performed as follows. The Na(+)-HCO3- co-transport was inhibited by perfusing the retinal side of the epithelium with a Na(+)-free solution. The co-transport was then stimulated by changing the perfusate from the Na(+)-free solution to a solution which contained from 5 to 110 mM-Na+. The resulting inward Na(+)-HCO3- co-transport produced an intracellular alkalinization, the initial rate of which was used to calculate the initial rate of Na(+)-HCO3- co-transport, JHCO3-. 3. The Na+ dependence of the Na(+)-HCO3- co-transport was studied at two different values of extracellular pH (7.40 and 7.10), at constant extracellular HCO3- concentration (27.5 mM) and at two different extracellular HCO3- concentrations (27.5 mM and 55 mM) at constant extracellular pH (7.40). In these experiments, the calculated values of JHCO3- followed single Michaelis-Menten kinetics with respect to the extracellular Na+ concentration. 4. The data are consistent with a model in which the co-transporter has a single binding site for the Na+ ion with an apparent affinity constant (apparent Km) of 37 mM. The apparent affinity constant for Na+ was independent of the extracellular concentration of CO3(2-) in the range of 16-65 microM, and of the extracellular HCO3- concentration in the range 27.5-55 mM. 5. The NaCO3- ion-pair hypothesis, in which sodium binds to the co-transporter and is translocated across the cell membrane as the NaCO3- ion pair, was analysed. For stoichiometries 1:2 and 1:3 of the Na(+)-HCO3- co-transport, the NaCO3- ion-pair hypothesis was found incompatible with the data. 6. The intracellular buffer capacity as measured by the CO2 method was 15 mM.

Voltage‐dependent currents in isolated cells of the frog retinal pigment epithelium.

1. Retinal pigment epithelial (RPE) cells were isolated enzymatically from bullfrog retinae. The patch-clamp technique was employed to investigate whole-cell currents under voltage-clamp conditions. 2. Isolated RPE cells were columnar or cuboidal in form, often with long processes protruding from the apical surface. Distinct apical and basal membrane domains were maintained for several hours following isolation. 3. The mean membrane capacitance was 62 pF. The resting potential averaged -30 mV, but it was as high as -75 mV in some cells. 4. Three voltage-dependent currents were observed: a time-independent and inwardly rectifying current and two time-dependent outwardly rectifying currents that had distinct kinetic properties. 5. Voltage pulses from a holding potential of -70 mV to potentials ranging from -30 to -120 mV produced membrane currents that were essentially time independent. The I-V relationship in this voltage range depended on the resting potential. It was usually inwardly rectifying in cells with resting potentials negative to about -50 mV, but tended to be linear in cells with more positive potentials. Three observations strongly suggested that the inwardly rectifying current is carried by K+. First, increasing the extracellular K+ concentration [( K+]) from 2 to 112 mM shifted the zero-current potential of the I-V relationship in the positive direction from an average value of -60 mV to 0 mV. Second, the addition of the K+ channel blockers Ba2+ (2 mM) or Cs+ (5 mM) to the extracellular solution inhibited a major component of the inwardly rectifying current. Finally, the reversal potential (Vr) of the Ba2(+)-sensitive current averaged -90 mV, near the K+ equilibrium potential (EK). 6. In approximately 50% of the cells, depolarizing voltage pulses to potentials more negative than -30 mV evoked an outward current that resembled the delayed rectifier present in other non-excitable cells. It activated with sigmoidal kinetics in less than 100 ms following a brief delay and then declined exponentially with a time constant of approximately 1 s. The peak chord conductance associated with this current was half-maximal at +14 mV. Several observations indicated that this outwardly rectifying current is carried primarily by K+: its Vr closely matched EK over a wide range of extracellular [K+]; it was inhibited 80% by exposure to the K+ channel blockers 4-aminopyridine (1 mM) and tetraethylammonium (20 mM); and it was abolished by intracellular dialysis with a K(+)-free solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Rheogenic sodium-bicarbonate co-transport across the retinal membrane of the frog retinal pigment epithelium.

1. Na+ and HCO3- transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled Na(+)- and pH-selective microelectrodes. Transient changes in the intracellular pH and in the intracellular Na+ activity were monitored in response to abrupt changes in the Na+ concentration and in the HCO3- concentration on the retinal side of the epithelium, and in response to transepithelial currents. 2. Removal of Na+ from the retinal side of the epithelium caused a depolarization of the membrane potential across the retinal membrane, a decrease in the intracellular Na+ activity and a decrease in the intracellular pH. 3. An increase in the HCO3- concentration on the retinal side of the epithelium from 27.5 to 50 mM caused a hyperpolarization of the membrane potential across the retinal membrane, an increase in the intracellular Na+ activity and an increase in the intracellular pH. 4. Passage of a transepithelial current of 30 microA in the choroid-to-retina direction caused an increase in the intracellular Na+ activity and an increase in the intracellular pH. 5. The data are interpreted as evidence for rheogenic co-transport of Na+, HCO3- across the retinal membrane of the frog retinal pigment epithelium. 6. The transient changes described under 2, 3 and 4 above were blocked by 0.5 mM-4-acetamido-4'-isothiocyanostilbene-2.2'-disulphonic acid (SITS). The Na(+)- HCO3- co-transport was not inhibited by 1 mM-amiloride.

Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium.

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).