Anion channels in the apical membrane of mammalian corneal epithelium primary cultures.

Chloride ion (Cl-) secretion by the rabbit corneal epithelium involves a catecholamine-stimulated Cl- conductance at the apical membrane, but the characteristics of the ion channel(s) responsible are unknown. A primary cell culture system was developed that induces stratified epithelial differentiation of rat and rabbit corneal epithelial cells, as detected by differential interference contrast light and transmission electron microscopy. In patch clamp studies, gigaOhm seal, were obtained easily during the first 1-4 days in vitro, and patches contained a voltage-dependent outwardly rectifying channel. Single-channel conductance at 24 degrees C was about 10, 29, and 72 pS in symmetric 150 mM NaCl at membrane potentials of -60, 0, and +60 mV, respectively. The current-voltage relationship measured with 75 mM NaCl and sucrose in the bath indicated anion selectivity. Increasing the temperature to 37 degrees C and replacing HEPES buffer with tricine, increased channel conductance and decreased rectification. Characteristics of this channel and a low conductance Cl- channel are compared with those of anion channels of other vertebrate epithelia.     

Divalent cation effects on lens conductance and stretch-activated cation channels.

In patch clamp studies of apical membrane from frog lens epithelium, the most frequently observed channel is 'stretch-activated', highly selective for cations over anions but showing little selectivity for Na+ vs. K+. In normal physiological saline, the open channel conductance is 25-30 pS and quite linear over +/- 100 mV. In the absence of extracellular divalent ions, the open channel conductance for inward current flow increases to about 50 pS at the normal lens resting voltage of -75 mV, whereas the conductance for outward current flow is unaffected. In the intact lens, removal of extracellular divalents causes the input conductance approximately to double and the intracellular voltage to depolarize from -74 to -58 mV. A variety of divalent ions block this change in whole lens conductance and voltage in the same order in which they block the 'stretch channels'. Single voltage-clamped epithelial cells also increase their conductance when Ca2+ is removed from their bathing medium. There are, therefore, some striking parallels between the open channel properties of the 'stretch-activated' cation channel and the response of the whole lens or single lens cells to removal of extracellular Ca2+. There are also inconsistencies. This channel is apparently not open in the normal resting lens so removal of extracellular Ca2+ must cause it to open if it is indeed responsible for the increase in lens conductance. However, we have not been able to demonstrate convincingly an increase in open probability at the single-channel level when external divalents are removed.     

Electrophysiology of rabbit Müller (glial) cells in experimental retinal detachment and PVR

PURPOSE: To determine the electrophysiological properties of Müller (glial) cells from experimentally detached rabbit retinas. METHODS: A stable local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Müller cells were acutely dissociated and studied by the whole-cell voltage-clamp technique. RESULTS: The cell membranes of Müller cells from normal retinas were dominated by a large inwardly rectifying potassium ion (K+) conductance that caused a low-input resistance (<100 M(Omega)) and a high resting membrane potential (-82 +/- 6 mV). During the first week after detachment, the Müller cells became reactive as shown by glial fibrillary acidic protein (GFAP) immunoreactivity, and their inward currents were markedly reduced, accompanied by an increased input resistance (>200 M(Omega)). After 3 weeks of detachment, the input resistance increased further (>300 M(Omega)), and some cells displayed significantly depolarized membrane potentials (mean -69 +/- 18 mV). When PVR developed (in 20% of the cases) the inward K+ currents were virtually completely eliminated. The input resistance increased dramatically (>1000 MOmega), and almost all cells displayed strongly depolarized membrane potentials (-44 +/- 16 mV). CONCLUSIONS: Reactive Müller cells are characterized by a severe reduction of their K+ inward conductance, accompanied by depolarized membrane potentials. These changes must impair physiological glial functions, such as neurotransmitter recycling and K+ ion clearance. Furthermore, the open probability of certain types of voltage-dependent ion channels (e.g., Ca2+-dependent K+ maxi channels) increases that may be a precondition for Müller cell proliferation, particularly in PVR when a dramatic downregulation of both inward current density and resting membrane potential occurs.     

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.     

Injection of RNA from carp retina induces the formation of a membrane potassium channel in Xenopus oocytes.

Total RNA was purified from freshly isolated retinas of adult carp and injected into oocytes of Xenopus laevis (stages 5-6). Two to six days after injection, depolarizing voltage-clamp steps evoked a slowly activated outward current as large as 3 microA. This current inactivated slowly with a single time constant (tau = 3.1 +/- 0.24 S.E.M., for Vm = +30 mV). The current was inhibited by tetraethylammonium (3.8 mM for half-maximal inhibition). In the presence of Co2+ (1 mM) or barium methanesulfonate (40 mM), the current-voltage relationship shifted to slightly more depolarized values (5-10 mV); the maximal value of the current that was sensitive to Co2+ or Ba2+ treatments was only a small fraction (about 10%) of the TEA-sensitive current, and its current-voltage relationship was similar to that for uninjected oocytes. The reversal potential of the membrane current was studied with [K+]0 of 1-77 mM. For [K+]0 greater than 20 mM, the reversal potential changed with a slope of 63 mV (+/- 2 mV S.E.M.) per 10-fold change in [K+]0. The conductance was induced half-maximally at 17 mV (+/- 0.9 mV S.E.M.). The depolarization required for an e-fold increase in conductance was 13 mV (+/- 0.6 mV S.E.M.). From these results, we conclude that the injection of total RNA from carp retinas induces the formation of a membrane K+ channel in Xenopus oocytes. The channel formed has many of the properties reported for the maintained outward current of goldfish horizontal and bipolar cells.     

Detection and characterization of Ca(2+)-activated K+ channels in transformed cells of human non-pigmented ciliary epithelium

Cell-attached and excised inside-out membrane patches were used to study single channel currents in a cell line derived from human non-pigmented ciliary epithelium. Most of the patches contained a Ca(2+)-dependent K+ channel with large unitary conductance (200 pS in symmetrical K+ solutions). Single channel current in cell-attached patches exposed to high K+ solution in the pipette showed a null potential of -36 mV. This value, which should yield an approximate estimation of cell membrane potential, was reversibly increased by -30 to -40 mV in the presence of Ca2+ ionophores. Tetraethylammonium up to 10 mM applied at the membrane cytoplasmic face had no effect on the channel. Addition of 1 mM BaCl2 to excised patches caused a voltage-dependent blockade of the channel. In the presence of barium the unit currents were not altered, but the channel remained closed for long periods of time and the open state probability decreased with depolarization. The possibility that this channel participates in regulation of transepithelial ciliary body secretion is discussed.     

An electrophysiologic study of rabbit ciliary epithelium

Microelectrode recordings from cells in rabbit ciliary epithelium have been made in vitro. Ionophoresis of Lucifer Yellow dye from microelectrodes during measurements of potential confirmed that the recordings were intracellular. Dye passed from the impaled cells into adjacent cells in both the nonpigmented and pigmented layers of the epithelium. Electrical coupling between epithelial cells also was observed. The mean (+/- SD) values of the potential measured across the basolateral membranes of the nonpigmented cells was -65 +/- -15 mV (n = 77); the mean value of the input resistance at this intracellular recording site was 37 +/- 28 M omega (n = 17). The membrane potential was reduced by raising the concentration of extracellular potassium but unaffected by changes in the concentrations of sodium, chloride, or bicarbonate ions. After a period of deprivation of extracellular potassium, the cells hyperpolarized without a measurable change in membrane resistance when potassium was restored to the bathing solution; this transient response to potassium was abolished by preincubation with ouabain or by bathing the epithelium in a solution lacking sodium. It was concluded that the ciliary epithelial cells are permeable to potassium but exhibit only a low permeability to sodium, chloride, or bicarbonate ions; that the cells possess an electrogenic Na/K pump; and finally, that all of the cells in the epithelium function as a syncytium.     

Single anion channels of frog rod plasma membrane

Anion channels have been found in the plasma membrane of the outer and inner segment of the isolated retinal rod by means of the patch voltage-clamp technique. The permeability of the channels for different anions follows a sequence: Cl- greater than F- greater than NO3- greater than propionate; the channel conductance in the fully open state is 200 +/- 30 pS measured in 108 mM NaCl. The non-linear character of the current-voltage relationship at membrane potentials from -40 to -20 mV suggests that these anion channels may be involved in receptor potential generation through an electrogenic mechanism.     

A non-selective cation channel in rabbit corneal endothelium activated by internal calcium and inhibited by internal ATP.

The apical membrane of the rabbit corneal endothelium contains a cation selective channel of 21.7 +/- 0.4 pS conductance which is activated by internal Ca2+ and inhibited by internal ATP. Patch clamp studies show the channel to be quite selective for cations over anions but not to select between Na+ and K+. In excised patches in the inside-out configuration, the open probability increases when the internal Ca2+ is raised above 10(-4) M. ATP decreases the open probability when its internal concentration exceeds 10 microM. At 1 mM ATP, no channel openings are ever seen. AMP and ADP also inhibit at least as well as does ATP. One microM AMP-PNP, a non-hydrolyzable ATP, also inhibits gating and so phosphorylation by a PO4(-3) cleaved from ATP is not the mechanism of ATP inhibition. The channel is apparently impermeable to divalent ions since neither Ba2+ nor Ca2+ carry any detectable current. Quinidine produces a flickery, weakly voltage dependent blockade when applied to the channel's cytoplasmic surface. Both Ca2+ and ATP work at concentrations not expected to occur inside healthy cells. It is presently uncertain if this is a physiologically important channel in these cells.     

Two Types of Stretch-Activated Channels Coexist in the Rabbit Corneal Epithelial Cell

Ion channels contribute to the regulation of cellular function through control of the membrane potential and intracellular concentration of various ions. We examined stretch-activated channels in the corneal epithelial cell. Patch clamping was applied to enzymatically dissociated corneal epithelial cells to characterize their stretch-activated ion channels. The plasma membrane was stretched by applying suction to the patch pipette in cell-attached or inside-out patch configuration. The ion selectivity, voltage-dependence, and stretch-dependence were examined. Two kinds of stretch-activated channel events were observed; the previously-reported large conductance (L) channel and a novel small conductance (S) channel. The probability of recording L vs. S channels in the cell-attached configuration was about 2:1. The L channel was potassium selective with single channel conductance (γ) of about 160 pS under the symmetrical (150 mmK⁺) solution. The S channel was permeable to Na⁺and K⁺with γof about 20 pS under the same conditions. Both L and S channels showed little activity in the absence of suction applied to the recording pipette. Channel activity was evoked by suction (negative pressure) stronger than -20 mmHg in both channels. The open probability (P_o) and the mean current increased in proportion to further applied stretch and did not saturate for applied suction as strong as -80 mmHg, the pressure at which the gigaseal started to break. Thus, two types of stretch-activated channels coexist in corneal epithelial cells; a potassium-selective L channel and non-selective S channel. The contribution of these channels to the membrane potential is discussed.     

Voltage clamp study of electrophysiologically-identified horizontal cells in carp retina.

Passive membrane properties and electromotive force of light modulated currents of L-, R/G-type and rod-driven horizontal cells were studied by voltage-clamp using double-barrelled micro-electrodes whilst perfusing with 5 microM dopamine to uncouple the gap junctions. Input impedances of horizontal cells in darkness were 31 +/- 1.4 M omega (mean +/- SE, n = 63); the resting potentials were -37 +/- 1.3 mV. Current-voltage relationships had regions of both inward and outward rectification and a region of negative resistance was commonly observed. Reversal potentials of light modulated currents were estimated on average to be -7 +/- 4 mV (n = 14), which is consistent with the involvement of K+ and Na+ and/or Ca2+ gradients. Importantly in R/G cells both depolarizing and hyperpolarizing components of the response had essentially the same reversal potential.     

Human Müller glial cells: altered potassium channel activity in proliferative vitreoretinopathy

PURPOSE: To determine differences of K+ channel activity between Müller glial cells obtained from retinas of healthy human donors and of patients with retinal detachment and proliferative vitreoretinopathy. METHODS: Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients. The whole-cell and the cell-attached configurations of the patch-clamp technique were used to characterize the current densities of different K+ channel types and the activity of single Ca2+ -activated K+ channels of big conductance (BK). RESULTS: Cells from patients displayed a less negative mean membrane potential (-52.8 mV) than cells from healthy donors (-80.6 mV). However, the membrane potentials in cells from patients scattered largely between -6 and -99 mV. The inwardly rectifying K+ permeability in cells from patients was strongly reduced (0.3 pA/pF) when compared with cells from healthy donors (6.0 pA/pF). At the resting membrane potential, single BK channels displayed a higher mean activity (open probability, Po, and channel current amplitude) in cells from patients (Po, 0.30) than in cells from healthy donors (Po: 0.03). The variations of BK current amplitudes were correlated with the variations of the membrane potential. CONCLUSIONS: The dominant expression of inwardly rectifying channels in cells from healthy donors is thought to support important glial cell functions such as the spatial buffering of extracellular K+. The downregulation of these channels and the less negative mean membrane potential in cells from patients should impair spatial buffering currents and neurotransmitter clearance. The increased activity of BK channels may support the proliferative activity of gliotic cells via feedback regulation of Ca2+ entry and membrane potential.     

Response of the intracellular potentials of cultured bovine lens cells to ions and inhibitors

Micropuncture of bovine lens epithelial cells cultured on plastic culture dishes gave values for the plasma membrane voltage (V) which remained stable for periods of up to several hours. The value of V was mainly in the range -30 to -45 mV, mean value -36.9 +/- 0.5 mV (S.E.M., n = 188). Raising extracellular [K+] from 5 to 40 mM depolarized V by 10 +/- 3 mV. The extent of this depolarization increased with increasing steady-state V. Barium (2 mM) caused a rapid, reversible depolarization of 7.9 +/- 1.2 mV. In the presence of Ba2+, the response to 40 mM K was reduced to 3.6 +/- 1.1 mV. Ouabain (10(-5) M) caused a fast depolarization by 5.3 +/- 1.2 mV. Exposure to calcium-free EGTA-Ringer's depolarized V reversibly by 19.5 +/- 5.0 mV. In Ca-free medium, the depolarization induced by 40 mM K was reduced to 3.2 +/- 2.4 mV. Whereas in control Ringer's sodium conductance (as measured by exposure to a 10 mM [Na]-Ringer's) is small as compared to potassium conductance, it increased markedly in Ca-depleted medium. Amiloride (10(-4) and 10(-3) M) had no effect on this Na conductance. An increase in the relative conductance for sodium was also elicited by Ba2+ (2 mM). Extracellular acidification led to a depolarization, alkalinization to a hyperpolarization. The extent of this effect was virtually equal in the absence or presence of HCO3-, excluding a significant pathway for bicarbonate. No evidence for a significant chloride conductance could be obtained.     

(-)-Isoproterenol modulation of maxi-K(+) channel in nonpigmented ciliary epithelial cells through a G-protein gated pathway

PURPOSE: Adrenergic agents decrease intraocular pressure by reducing aqueous humor secretion from ciliary epithelial cells. Since the ionic concentration of aqueous humor contributes to intraocular pressure, we have investigated the effect of (-)-isoproterenol, a beta-adrenergic agonist on the maxi-K( +) channel in rabbit nonpigmented ciliary epithelial (NPE) cells. METHODS: Single-channel currents were recorded from the basolateral surface of acutely isolated NPE cells using patch clamp techniques. RESULTS: A calcium dependent maxi-K(+) channel was identified in 31% of cell-attached patches. In the excised condition the channel was activated in presence of calcium. In symmetrical K(+) solution a linear current-voltage relationship and unitary conductance of 158 +/- 15 pS was observed. Replacing K(+) with Na(+) the current-voltage curve shifted to the right and approached a reversal potential for K( +) ( approximately -80 mV). Barium (2 mM) from the intracellular side or iberiotoxin (50 nM) from the extracellular side blocked the channel activity. In cell-attached patches, the beta-receptor agonist (-)-isoproterenol (2.5 microM) increased channel open probability (P(o)) only when applied directly through the patch pipette. beta(2)-adrenoceptor antagonists (ICI-118, 551, l-timolol) blocked the channel activity more efficiently than the beta(1)-adrenoceptor antagonist betaxolol. In excised patches, (-)-isoproterenol increased baseline P(o) 5-fold (0.5 +/- 0.13) when GTP (100 microM) and GTPgammaS (100 microM) were present at the cytosolic surface of the pipette (control; P(o), 0.12 +/- 0.006). GTP augmented baseline channel activity (0.1 +/- 0.004) 7-fold (0.7 +/- 0.03) when (-)-isoproterenol was included in patch pipette. CONCLUSIONS: Rabbit NPE cells expressed maxi-K(+) channels on their basolateral surface. The adrenergic agonist (-)-isoproterenol activated these channels via a beta(2)-adrenoceptor that was modulated by a direct G-protein gated pathway.     

Single potassium channels in corneal epithelium

The basal cell layers of the rabbit and human corneal epithelia contain a frequently occurring ionic channel whose unitary currents can be recorded in cell-attached or excised membrane patches by use of a patch voltage clamp. The channel is highly conductive (165 pS in 150 mM K+ salts) and is very selective for K+ over Na+ (PK/PNa greater than 40:1). Its open probability is increased by the application of suction to the recording pipette although its gating is less sensitive to suction than that of many other "stretch-activated" channels reported. The current through the channel is a saturating function of the K+ concentration in the bathing solutions with half saturation occurring at 480 mM and a single-channel current at saturation (imax) of 31 pA. In the absence of applied suction, the open probability is extremely variable from patch to patch and shows little voltage dependence over the physiologic voltage range. The channel also gates frequently to several subconductance levels. It is blocked by external Cs+ and Ba+2 in the 0.1-10 mM range but not by most other K+ channel blockers. It is also partially blocked by Ca+2 at both its internal and external surfaces. Because of its novel properties (stretch activation and large conductance), it can be used to measure the input resistance and total capacitance of single dissociated cells.     

Internal acidification modulates membrane and junctional resistance in the isolated lens of the frog Rana pipiens.

The normal internal pH (pHi) of the amphibian lens, measured using ion-sensitive microelectrodes, is 7.1 (pHo = 7.4) and the membranes appear to be relatively impermeable to hydrogen ions. Perifusing the lens with 100% CO2 appeared to be the most efficient way of decreasing pHi, which fell to 6.3 after an exposure lasting 30 min. Accompanying this acidification, there was a rapid depolarization of membrane potential (Em), a decrease in membrane resistance (Rm) and increase in internal or bulk resistance (Ri). These changes did not occur if the external pH alone was decreased. All changes were reversible, although the time course of Ri recovery was faster than the others. The decrease in membrane resistance could be prevented if the chloride concentration in the external solution was reduced, suggesting that internal acidification opens chloride channels in the amphibian lens. Since chloride ions are normally close to equilibrium across amphibian lens membranes, it is suggested that the pH-induced depolarization is due to a decrease in potassium conductance. The increase in internal resistance on perifusing with CO2 is most likely due to a closing of gap junctions between the fibre cells. The relationship between internal conductance and pHi was very similar to that obtained in other tissues and could be fitted by the Hill equation with n = 6 and pK = 6.9. Fibre junctional conductance seems sensitive to small changes in hydrogen ion concentration around the resting pH. Two agents, aspirin and cyanate, that are believed to influence cataract development, slowed the recovery of Em, Rm and Ri during recovery from an acid load.(ABSTRACT TRUNCATED AT 250 WORDS)     

Similar properties of cGMP-activated channels between cones and rods in the carp retina.

Using patch-clamp techniques, properties of cGMP-activated channel were studied at a single-channel level in order to examine (1) whether any differences are recognized between the cGMP-activated channels of rods and cones in the same animal species, and (2) whether the channel properties of the same photoreceptor class differ in different animal species. Experiments were performed on inside-out membrane patches excised from outer segments of rods and morphological subtypes of cones in the carp retina. Single-channel activities could be recorded when the patches were perfused with low concentrations of cGMP (less than 10 microM). Throughout five morphological subtypes of cones and rod, single-channel currents showed no significant rectification at membrane hyperpolarization in a low divalent cation solution, and single-channel conductances were almost the same: 13.8 +/- 0.2 pS (mean +/- S.E.M., n = 23) in cones and 12.7 +/- 0.8 pS (n = 3) in rods. These values were significantly smaller than that reported in catfish cones (about 50 pS), and that in rods of the toad and the tiger salamander (about 25 pS). In rods and all subtypes of cones of the carp, open durations of cGMP-activated channels were brief. In addition, kinetic parameters of channel openings and closings showed no differences throughout all subtypes of cones and rod.     

Effect of hypotonic media on the membrane voltage of cultured bovine corneal endothelial cells

The effects of hypotonicity on cultured bovine corneal endothelial cells were investigated using standard microelectrode and superfusion techniques. Confluent monolayers of cells were superfused with an isotonic (305 +/- 5 mosm/kg) control solution until a stable membrane voltage (V) was obtained, then with a hypotonic (240 +/- 5 mosm/kg) solution. Under control conditions, V was - 51.4 +/- 0.8 mV (means +/- SEM, n = 154). Decreasing solution osmolality resulted in an immediate depolarization: mean maximal delta V = 18.7 +/- 0.9 mV at 2.6 +/- 0.2 minutes with a gradual recovery to a new but still depolarized steady-state V (delta v = 11.1 +/- 0.9 mV at 8.2 +/- 0.3 minutes, n = 25). The depolarizing response to hypotonicity persisted in the presence of amiloride (10(-3)M), DIDS (10(-3)M), bumetanide (10(-4)M) or ouabain (10(-4)M) as well as in the absence of extracellular Cl-, Na+, HCO3- or Ca2+. Relative K+ conductance was estimated by the effect on V of increased extracellular [K+] - this was significantly reduced at 5, 10 and 20 mM K+ under hypotonic conditions. The depolarization induced by 1mM Ba2+ was also reduced from 19.6 +/- 0.5 mV (n = 8) under isotonic conditions to 15.4 +/- 0.4 mV (n = 6) under hypotonic conditions (p less than 0.001). The conductive HCO3- pathway - as judged by the hyperpolarization of V induced by increasing extracellular [HCO3-] from 28 to 60 mM, was also reduced under hypotonic conditions (delta V = 17.2 +/- 0.8 mV, n = 13 (isotonic) compared to delta V = 9.5 +/- 0.3 mV, n = 15 (hypotonic].(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Membrane potentials in retinal capillary pericytes: excitability and effect of vasoactive substances.

Retinal capillary pericytes are believed to have a contractile function and to regulate retinal blood flow at the microvascular level. Membrane potential is an important control element for contractility in smooth muscle cells. In the present study, bovine retinal capillary pericytes have been grown in tissue culture and membrane potentials have been measured using glass microelectrodes. Resting potentials averaged -31 +/- 7 mV (n = 203). Relative K+ conductance was low, with a transference number for K+ of 0.16. Readdition of K+ to K(+)-depleted cells transiently hyperpolarized the membrane potential, probably by stimulating the electrogenic Na+/K+ transport. Repetitive spike-like depolarizations (action potentials) were induced by stimulating the Na+/K(+)-ATPase, by applying norepinephrine (10(-5) mol/l), and by adding 10 mmol/l Ba2+. These action potentials depended on the presence of extracellular Ca2+ and were inhibited by the Ca2+ antagonist nifedipine (10(-6) mol/l). Norepinephrine (10(-5) mol/l) depolarized the membrane by 7.4 +/- 3.5 mV (mean +/- SD, n = 49). This response was blocked by the alpha 1-antagonist prazosin (10(-5) mol/l). Histamine also led to a membrane depolarization of 8.6 +/- 2.8 mV (n = 49), which could be inhibited by the H1-antagonist diphenhydramine. Endothelin (10(-7) mol/l), vasopressin (10(-6) mol/l), and acetylcholine (10(-4) mol/l) had no major effects on membrane potential. The conclusion is that retinal capillary pericytes are excitable cells and react to several vasoactive substances.