Transcriptome analysis and molecular signature of human retinal pigment epithelium.

Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed 'signature' genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases.

Hourglass cell development in the soybean seed coat.

BACKGROUND AND AIMS: Hourglass cells (HGCs) are prominent cells in the soybean seed coat, and have potential use as 'phytofactories' to produce specific proteins of interest. Previous studies have shown that HGCs initiate differentiation at about 9 d post-anthesis (dpa), assuming their characteristic morphology by 18 dpa. This study aims to document the structural changes in HGCs during this critical period, and to relate these changes to the concurrent development of a specific soybean peroxidase (SBP) encoded by the Ep gene. METHODS: Pods were collected from plants at specific growth stages. Fresh material was processed for analysis of Ep peroxidase activity. Tissues were processed for scanning and transmission electron microscopy, as well as extracted for western blotting. A null variety lacking expression of Ep peroxidase was grown as a control. KEY RESULTS AND CONCLUSIONS: At 9 dpa, HGCs are typical undifferentiated plant cells, but from 12-18 dpa they undergo rapid changes in their internal and external structure. By 18 dpa, they have assumed the characteristic hourglass shape with thick cell walls, intercellular air spaces and large central vacuoles. By 45 dpa, all organelles in HGCs have been degraded. Additional observations indicate that plasmodesmata connect all cell types. SBP activity and SBP protein are detectable in the HGC before they are fully differentiated (approx. 18 dpa). In very early stages, SBP activity appears localized in a vacuole as previously predicted. These results increase our understanding of the structure and development of the HGC and will be valuable for future studies aimed at protein targeting to components of the HGC endomembrane systems.

Bone cells in culture: morphologic transformation by hormones.

Hormones and purine nucleosides and nucleotides induced cultured bone cells to transform transiently from a spherical to a stellate shape. Cytochalasin B also induced the transformation. The change was blocked by colchicine and vinblastine, but not by lumicolchicine or cycloheximide. This morphologic transformation may provide a dynamic model of hormone action and bone cell modulation in vitro.

Altered fluid transport across airway epithelium in cystic fibrosis.

In cystic fibrosis (CF), absence or dysfunction of a phosphorylation-regulated chloride channel [CF transmembrane conductance regulator (CFTR)] leads to the loss or reduction of chloride secretion into the airways. Active sodium absorption is also increased in CF, and both of these ion transport changes could alter fluid transport across the airways. Under baseline conditions, cultured human airway epithelia from normal individuals absorbed fluid, and this absorption was increased in epithelia from patients with CF. In normal and CF epithelial cultures fluid absorption was inhibited by amiloride. Adenosine 3',5'-monophosphate stimulated fluid secretion in normal epithelial cultures but not in cultures from individuals with CF. In contrast, fluid secretion induced by nucleotide triphosphates (uridine triphosphate or adenosine triphosphate) was unaltered in cultures of epithelia from patients with CF, suggesting an approach to the treatment of CF.

Treatment of potato farm wastewater with sand filtration.

This study examined sand filtration as a component of a potato farm wastewater treatment system. Two different sand filter designs, saturated flow and unsaturated flow, were evaluated at three different loading rates: 34, 68, and 136 L m(-2) d(-1). Filter design had a significant effect, with unsaturated flow sand filters having significantly (p < .05) better total suspended solids (TSS) removal (89%) than saturated flow sand filters did (79%). Loading rate also had a significant (p < .05) effect, given that the lowest loading rate had higher mass removal for TSS than the higher loading rates did. Overall, all sand filters removed TSS, 5-d biochemical oxygen demand, and total phosphorus well (62-99%). Total nitrogen removal was twice as high in unsaturated flow filters (53%) than in saturated flow filters (27%), because of the recurring cycle of aerobic and anaerobic conditions during sand saturation and drying in unsaturated flow sand filters.

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

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.

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

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.

Potassium modulation of taurine transport across the frog retinal pigment epithelium.

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport.

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.

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).

Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. Measurements in the open-circuit state.

A modified version of a capacitance probe technique has been used to measure fluid transport across the isolated retinal pigment epithelium (RPE)-choroid of the bullfrog. The accuracy of this measurement is 0.5-1.0 nl/min. Experiments carried out in the absence of external osmotic or hydrostatic gradients show that the RPE-choroid transports fluid from the retinal to the choroid side of the tissue at a rate of approximately 10 nl/min (4-6 microliters/cm2 X h). Net fluid absorption (Jv) was abolished within 10 min by the mitochondrial uncoupler 2,4-dinitrophenol. It was also inhibited (70%) by the removal of bicarbonate from the bulk solutions bathing the tissue. Ouabain caused a slow decrease in Jv (no effect at 10 min, 70% at 3 h), which indicates that RPE fluid transport is not directly coupled to the activity of the Na-K pump located at the apical membrane of this epithelium. In contrast to ouabain, cyclic AMP (cAMP) produced a quick decrease in Jv (84% within 5 min). Radioisotope experiments in the open circuit show that cAMP stimulated secretory fluxes of Na and Cl, which accounted for the observed cAMP-induced decrease in Jv. The direction of net fluid absorption, the magnitudes of the net ionic fluxes in the open circuit, and the dependence of Jv on external bicarbonate concentration strongly suggest that fluid absorption is generated primarily by the active absorption of bicarbonate.

Preparation of soybean seed samples for FT-IR microspectroscopy.

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4 degrees C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.

Experimental Bin Drenching System for Testing Biocontrol Agents to Control Postharvest Decay of Apples.

A portable drencher capable of drenching a single bin of fruit was built to simulate the commercial application of chemicals to harvested apples in small orchard operations in the central and eastern United States. The drencher required as little as 125 liters of the treatment solution and permitted various bin travel speeds. Wounded apples were placed midway between the bottom and top of the bin, in the center, and near the four corners of the bin (20 fruit per location) and covered with enough unwounded apples to fill the bin. The bins were drenched with a suspension containing Penicillium expansum at 2 × 104 conidia per ml in 2000, 5 × 103 conidia per ml in 2001, and 3 × 103 conidia per ml in 2002 and 2003. In 2000 and 2003, the additional treatments included a combination of P. expansum with the yeast Metschnikowia pulcherrima at ~;1.2 × 107 CFU/ml, and in 2003 a combination with 2% sodium bicarbonate (SB) or a mixture of the yeast and SB. After 3 months of storage at ~;2°C, at all P. expansum conidial concentrations, more than 90% of wounded fruit developed decay on 'Golden Delicious', 'Delicious', and 'Rome' apples in the 2000-02 experiments. In 2003, 66 and 33.1% of the wounded fruit developed decay on 'Delicious' and 'Golden Delicious', respectively. The application of the antagonist reduced decay to 39 and 3.3% on 'Golden Delicious' in 2000 and 2003, respectively, and to 26% on 'Delicious' in 2003. The addition of SB reduced decay on both cultivars and, in combination with the yeast, was the most effective treatment on 'Golden Delicious'. This portable drencher can be very useful for evaluating different treatments applied to apples after harvest at the commercial level.

Alendronate and estrogen effects in postmenopausal women with low bone mineral density. Alendronate/Estrogen Study Group.

The bisphosphonate alendronate and conjugated equine estrogens are both widely used for the treatment of postmenopausal osteoporosis. Acting by different mechanisms, these two agents decrease bone resorption and thereby increase or preserve bone mineral density (BMD). The comparative and combined effects of these medications have not been rigorously studied. This prospective, double blind, placebo-controlled, randomized clinical trial examined the effects of oral alendronate and conjugated estrogen, in combination and separately, on BMD, biochemical markers of bone turnover, safety, and tolerability in 425 hysterectomized postmenopausal women with low bone mass. In addition, bone biopsy with histomorphometry was performed in a subset of subjects. Treatment included placebo, alendronate (10 mg daily), conjugated equine estrogen (CEE; 0.625 mg daily), or alendronate (10 mg daily) plus CEE (0.625 mg daily) for 2 yr. All of the women received a supplement of 500 mg calcium daily. At 2 yr, placebo-treated patients showed a mean 0.6% loss in lumbar spine BMD, compared with mean increases in women receiving alendronate, CEE, and alendronate plus CEE of 6.0% (P < 0.001 vs. placebo), 6.0% (P < 0.001 vs. placebo), and 8.3% (P < 0.001 vs. placebo and CEE; P = 0.022 vs. alendronate), respectively. The corresponding changes in total proximal femur bone mineral density were +4.0%, +3.4%, +4.7%, and +0.3% for the alendronate, estrogen, alendronate plus estrogen, and placebo groups, respectively. Both alendronate and CEE significantly decreased biochemical markers of bone turnover, specifically urinary N-telopeptide of type I collagen and serum bone-specific alkaline phosphatase. The alendronate plus CEE combination produced slightly greater decreases in these markers than either treatment alone, but the mean absolute values remained within the normal premenopausal range. Alendronate, alone or in combination with CEE, was well tolerated. In the subset of patients who underwent bone biopsies, histomorphometry showed normal bone histology with the expected decrease in bone turnover, which was somewhat more pronounced in the combination group. Thus, alendronate and estrogen produced favorable effects on BMD. Combined use of alendronate and estrogen produced somewhat larger increases in BMD than either agent alone and was well tolerated.

Ion transport mechanisms in native human retinal pigment epithelium.

Electrophysiologic techniques were used to characterize the electrical properties and the ion transport mechanisms at the apical and basolateral membranes of the human retinal pigment epithelium (RPE). These experiments used fresh native tissue from adult donor and fetal eyes. In the upper range, adult donor RPE had an apical membrane resting potential (VA) of approximately equal to -60 mV and a transepithelial potential (TEP) and resistance (Rt) of 3.5 mV and 148 omega.cm2, respectively. The means were at least 50% of these values. In RPE from fetuses of gestational age 19-23 wk, VA was -56 +/- 4 mV, TEP was 2.2 +/- 1.5 mV, Rt was 206 +/- 151 omega.cm2, and the ratio of apical to basolateral resistance was 0.70 +/- 0.50 (mean +/- standard deviation; n = 15). The apical membrane of the adult donor and fetal RPE contains a large relative K+ conductance (TK > 0.3) that is barium blockable. In fetal RPE, there is evidence for separate K+ and Cl- conductive mechanisms at the basolateral membrane. However, the evidence for the Cl- conductance is indirect. The fetal RPE apical membrane, but not the basolateral membrane, contains a ouabain-sensitive mechanism that exhibits two distinct phases of apical depolarization. The first, rapid phase suggests that the pump is electrogenic. The apical membrane of fetal RPE contains a bumetanide-sensitive mechanism and a receptor activated by nanomolar amounts of epinephrine. In fetal RPE, step changes in apical [K+]o between 5 and 2 mmol/l produced a delayed basolateral membrane hyperpolarization that in situ generates the fast oscillation trough of the electroretinogram.

Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium.

The authors used a modified capacitive probe technique to simultaneously assess the effect of apical epinephrine on fluid transport rate (Jv), transepithelial potential (TEP), and transepithelial resistance (Rt) across bovine retinal pigment epithelium (RPE). In control Ringer, the RPE absorbed fluid at a rate of 1.42 +/- 0.34 microliters/cm2.hr (mean +/- SEM; 22 tissues). Tissues with the highest TEP (8-9 mV) and Rt (160-220 omega.cm2) had maximum fluid absorption rates (3-4 microliters/cm2.hr). Apical epinephrine (100 nM) stimulated Jv by a factor of 3, from 0.70 +/- 0.18 microliter/cm2.hr to 2.17 +/- 0.24 microliters/cm2.hr and TEP from 4.6 +/- 0.4 mV to 7.0 +/- 0.6 mV (n = 6). The epinephrine-induced transport changes were inhibited by apical bumetanide (0.1 mM). The alpha-1 adrenergic antagonist prazosin (1 microM) completely blocked the epinephrine-induced stimulation of Jv and TEP. In contrast, the beta adrenergic antagonist propranolol (1 microM) had no effect on epinephrine-induced transport changes. These results, coupled with previous studies on bovine RPE, suggest that the mechanisms underlying the epinephrine-induced stimulation of fluid absorption include an apical membrane alpha-1 adrenergic receptor, a bumetanide-inhibitable apical membrane Na-K-2Cl cotransporter, and a basolateral membrane Cl conductance.

Na-dependent pHi regulatory mechanisms in native human retinal pigment epithelium.

This study provides the first information about pHi regulatory mechanisms in human retinal pigment epithelium (RPE). The experiments were carried out on fresh explant tissues from adult donor and fetal eyes, and pHi was measured using fluorescence microscopy techniques and the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In adult donor RPE, the resting pHi is 7.30 +/- 0.14 (mean +/- standard deviation; n = 6) in HCO3 Ringer's solution. In HCO3 Ringer's solution, apical Na removal caused rapid cell acidification with an initial rate of 0.40 +/- 0.10 pH U/min (n = 4). This Na-dependent acidification was partially inhibited by apical amiloride (n = 1) and DIDS (n = 1). In HCO3 Ringer's solution, pHi recovery from an acid load (NH4 prepulse) also was blocked by apical Na removal. In nominally HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells. These results suggest that the apical membrane of adult human RPE contains an Na/H exchanger and possibly a Na-dependent, DIDS-inhibitable pH regulatory mechanism, perhaps a NaHCO3 cotransporter. For the fetal RPE, the resting pHi was 7.16 +/- 0.10 (n = 9) and 7.19 +/- 0.10 (n = 20) in HCO3 and HCO3-free Ringer's solution, respectively. In HCO3 and HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells and the removal of apical Na caused cell acidification with an initial rate of 0.30 +/- 0.08 (n = 32) and 0.58 +/- 0.29 (n = 6) pH U/min, respectively. The pHi recovery from an acid load also was blocked by apical amiloride and apical Na removal. These results suggest that the apical membrane Na/H exchanger is the dominant acid extrusion mechanism in human fetal RPE.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine-induced increases in [Ca2+](in) and KCl-coupled fluid absorption in bovine RPE.

PURPOSE: To define the ionic basis for the apical epinephrine-induced increase of fluid absorption (J(V)) across isolated bovine RPE-choroid. METHODS: Epinephrine-induced changes in RPE [Ca2+](in) levels were monitored with the ratioing dye fura-2. Transepithelial potential, resistance, and unidirectional fluxes of (36)Cl, (86)Rb (K substitute), and (22)Na were simultaneously determined in paired tissues from the same eye mounted in modified Ussing flux chambers. Radioisotopes (5-7 microCi) were added to the apical bath of one tissue and the basal bath of the other, and the appearance of label in the opposite bath was measured. RESULTS: Apical epinephrine (100 nM) transiently increased [Ca2+](in) by 153 +/- 78 nM. This increase was inhibited by the alpha(1)-adrenoreceptor antagonist prazosin (1 microM) and blocked by CPA(5 microM), an inhibitor of endoplasmic reticulum Ca2+-adenosine triphosphatases (ATPases). Apical epinephrine (100 nM) more than doubled the net Cl absorption rate, increased net K ((86)Rb) absorption by fivefold, and tripled net fluid absorption (J(V)), as predicted by isotonic coupling between ion and fluid transport. The epinephrine-induced increases in ion and fluid transport were completely inhibited by apical bumetanide (100 microM). CONCLUSIONS: Epinephrine increased fluid absorption across bovine RPE by activating apical membrane alpha(1)-adrenergic receptors, increasing [Ca2+](in), and stimulating bumetanide-sensitive Na,K,2Cl uptake at the apical membrane and KCl efflux at the basolateral membrane.

Lactate transport in freshly isolated human fetal retinal pigment epithelium.

PURPOSE: To study transport mechanisms for small monocarboxylic acids in the apical and basolateral membranes of freshly isolated, human fetal retinal pigment epithelium. METHODS: The epithelium was mounted in a small Ussing chamber that allowed separate perfusion of both the apical and basal compartments and simultaneous measurements of intracellular pH, transepithelial potential, and tissue resistance. Intracellular pH was measured using a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. RESULTS: When 10-100 mM lactate or pyruvate was added to the apical bath the cells acidified by 0.10-0.25 pH units. There were no differences between the initial rates of intracellular acidification produced by L-lactate and D-lactate. These rates could be described as Michaelis-Menten functions of the concentrations of lactate and pyruvate. The Km values were 42 +/- 12 mM for L-lactate and 34 +/- 8 mM for pyruvate. The rates of acidification caused by 50 mM L-lactate were reversibly reduced by 44% or 35% after apical administration of probenecid (2 mM) or alpha-cyano-4-hydroxycinnamate (2 mM), and irreversibly reduced by 78% after apical administration of the sulfhydryl-reagent mersalyl acid (2 mM). The intracellular acidifications caused by apical pyruvate (50 mM) were completely and reversibly inhibited by 50 mM apical L-lactate. Addition of 50 to 100 mM lactate to the basal bath caused intracellular alkalinizations, which could be inhibited by Na+ removal in the basal bath or by 2 mM alpha-cyano-4-hydroxycinnamate in the apical bath.

The in vitro frog pigment epithelial cell hyperpolarization in response to light.

Pigment epithelial cell membrane potentials were measured in an in vitro frog retina-pigment epithelium-choroid preparation. Light stimuli hyperpolarized the apical membrane of the pigment epithelium. This hyperpolarization was the source of the c-wave of the electroretinogram. Light stimuli also decreased retinal extracellular potassium ion concentration, [K+]O, with the same time course as the apical hyperpolarization. It is suggested that the pigment epithelial hyperpolarization, which causes the c-wave, results directly from the light-evoked decrease in retinal [K+]O.