The accumulation of myoinositol and rubidium ions in galactose-exposed rat lens

When rat lens is incubated in 30 mM galactose overnight, the extent of accumulation of rubidium ions (Rb) and myoinositol (MI) are affected, as well as the Na-K ATPase activity. Rb accumulation and Na-K ATPase activity are only slightly affected compared to the dramatic drop in MI accumulation. These changes are completely abolished by sorbinil, which blocks polyol formation, or by rendering the galactose medium hypertonic to offset the osmotic effect of polyol formation. On the other hand, the addition of excess MI to the galactose medium had no effect on correcting these changes. The results obtained are consistent with the polyol-osmotic theory of sugar cataract formation.     

Intracellular voltage recordings in bovine non-pigmented ciliary epithelial cells in primary culture

Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na+ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3 +/- 0.5 mV (n = 36). Relative K+ conductance was increased by extracellular alkalinization. Removing extracellular K+ led to a depolarization and readdition of K+ to K+ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na+/K+ ATPase is inhibited by K+ replacement, and that there is overshoot-activation of the pump when K+ is readded. Extracellular Cl- replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stilbene-sensitive Cl(-)-conductance. Removing HCO3- led to a Na+ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na+ was not influenced by DIDS or the extracellular HCO3(-)-concentration.     

The effects of glutathione and adenosine on plasma membrane ATPases of the corneal endothelium. An hypothesis on the stimulatory mechanism of perfused glutathione upon deturgescence

Reduced glutathione (0.3 mM) stimulates the activity of sodium-potassium activated ATPase (Na+K+ATPase) by 54% in plasma membranes prepared from bovine corneal endothelial cells. Oxidized glutathione, however, has no effect on Na+K+ATPase activity in the same tissue, although it does inhibit magnesium activated ATPase (Mg++ATPase) by approximately 30%. Adenosine neither stimulates nor inhibits either Na+K+ATPase or Mg++ATPase in these plasma membranes. It is postulated that the stimulatory effect of glutathione on deturgescence stems from the direct reaction of the reduced form of the tripeptide on sulfhydryl groups located on plasma membranes of corneal endothelial cells. It is highly probable that these sulfhydryl groups are part of the Na+k+ATPase complex itself.     

Characterization of bicarbonate-dependent potassium uptake in cultured corneal endothelial cells

Bovine corneal endothelial (BCE) cells in culture demonstrated 86Rb+ uptake which was mostly ouabain-sensitive with some (15 to 50%) ouabain-insensitive uptake that was dependent on the presence of bicarbonate in the incubation medium. Bovine smooth muscle (SM) cells demonstrated ouabain-sensitive 86Rb+ uptake but the ouabain-insensitive 86Rb+ uptake was not bicarbonate-dependent. Although omission of bicarbonate from the incubation buffer resulted in some reduction in the pH, this change was not responsible for the reduction in the ouabain-insensitive 86Rb+ uptake. Furthermore, the removal of bicarbonate decreased the 86Rb+ influx but not its efflux. This ouabain-insensitive and bicarbonate-dependent 86Rb+ influx in BCE cells proceeded at a linear rate for at least 60 min and increased as a function of bicarbonate concentration such that almost maximal uptake was observed at a concentration of about 10 to 15 mM. Saturation of the bicarbonate-dependent 86Rb+ pump in BCE cells occurred at a concentration of 2 mM Rb+ in the incubation buffer, similar to the previously observed value for the Na+, K+-ATPase. Competition experiments with both unlabeled Rb+ and K+ demonstrated that likewise in the Na+, K+-ATPase the 86Rb+ influx represented physiological influx of K+. Furthermore, the energy requirements of the bicarbonate-dependent 86Rb+ uptake were similar to those of the 86Rb+ uptake via the Na+, K+-ATPase. The results described in this work demonstrated a novel bicarbonate-dependent K+ pump in addition to the Na+, K+-ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of galactosemic-induced inhibition of PGH synthase activity in cultured lens epithelial cells

Inhibition of prostaglandin synthesis as determined by prostaglandin endoperoxide synthetase (PGH synthase) activity is associated with polyol accumulation in cultured bovine lens epithelial cells (BLECs) incubated six days in minimal essential medium (MEM) containing 40 mM galactose (Gal). In order to better understand the nature of the correlation between hypergalactosemic exposure, polyol accumulation and inhibition of prostaglandin synthesis, a series of culture media reversal and sorbinil (an aldose reductase inhibitor) addition studies were carried out. BLECs were incubated in Gal for six days, then changed to galactose-free MEM +/- sorbinil for a three day recovery period. PGH synthase activity reduced to 66% of control after six days of exposure to Gal. The simultaneous administration of sorbinil during a nine day Gal incubation significantly protected the enzymatic activity, while the activity of PGH synthase further declined to 41% of control under the same conditions in the absence of sorbinil. Within 72 hours of media reversal, PGH synthase activity equaled or exceeded control values in BLECs switched to either MEM or MEM + sorbinil. Indeed, an enhanced prostaglandin biosynthetic capacity as demonstrated by radioimmunoassay was exhibited with microsomes prepared from cells switched from Gal into Gal-free MEM +/- sorbinil, corroborating the beneficial effect of media reversal. Furthermore, following 72 hours of reversal, the cellular dulcitol level was 93 nmol/micrograms PO4 for BLECs switched to MEM alone; no detectable level of polyol was observed in BLECs changed to MEM + sorbinil. In contrast, the polyol content in BLECs after six days of exposure to Gal was 185 nmol/micrograms PO4 and increased to 334 nmol/micrograms PO4 after nine days of continuous incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of immortalized human corneal endothelial cell line using HPV 16 E6/E7 on lyophilized human amniotic membrane

PURPOSE: To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). METHODS: Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. RESULTS: Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15 +/- 10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. CONCLUSIONS: IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.     

Osmoregulatory alterations in taurine uptake by cultured human and bovine lens epithelial cells

PURPOSE: Comparative assessment of cultured human lens epithelial cells (HLECs) and bovine lens epithelial cells (BLECs) established the nature of the relationship between taurine-concentrating capability and intracellular polyol accumulation or extracellular hypertonicity. METHODS: The kinetic characteristics of active taurine accumulation based on the measurement of in vitro [3H]-taurine uptake were resolved by side-to-side review of cultured HLECs and BLECs pre-exposed to either galactose-supplemented medium or extracellular hypertonicity. Competitive RT-PCR was used to appraise variation in taurine transporter (TauT) mRNA abundance from cells maintained in hyperosmotic medium over a 72-hour exposure period. RESULTS: The capacity to accumulate [3H]-taurine was significantly lowered after prolonged (20-hour) incubation of cultured BLECs in 40 mM galactose in contrast to HLECs, the latter cells' velocity curve being indistinguishable from control cells in physiological medium. Inhibition of the intracellular taurine transport site appeared to be noncompetitive, in that there was a marked reduction in the V(max) without significant alteration in the K(m) to a high-affinity transport site. Galactitol content in BLECs exceeded five times that found in HLECs. The coadministration of the aldose reductase inhibitor, sorbinil, with 40 mM galactose completely prevented the inhibitory effect of galactose on [3H]-taurine uptake. Acute exposure (3 hours) of HLECs and BLECs to a range of 10 to 40 mM galactitol or 10 to 40 mM galactose plus sorbinil-supplemented medium suggested by Dixon plot that neither galactitol nor galactose interacted with the extracellular taurine transport site. In contrast, [3H]-taurine accumulation was markedly elevated in both HLECs and BLECs after prolonged exposure to galactose-free medium made hyperosmotic by supplementation with sodium chloride. The enhanced taurine uptake capacity involved increase in peak velocity (V(max)) without significant change in Michaelis-Menten constant (K(m)). Cultured HLECs and BLECs responded to hypertonicity with an inducible but transitory upregulation of TauT mRNA. CONCLUSIONS: These results demonstrate that lens epithelial cells express a high-affinity TauT protein capable of active uptake, but predisposed to inhibition by intracellular galactitol when the sugar alcohol is present in sufficiently high concentration to interfere with cell metabolism. Furthermore, lens epithelial cells respond to hypertonic stress by raising taurine transport activity. The increase in taurine uptake is due to an increase in the number of high-affinity TauTs expressed as a result of an increase in the manifestation of taurine mRNA stemming from exposure to hypertonic medium.     

Age as a function of ATPase activity in cultured corneal endothelial cells

As cells grown in tissue culture age, they typically become altered when compared to their in vivo counterparts. Bovine corneal endothelial cells were grown in culture for periods up to 60 days (primary = 10 days; secondary = 50 days). The plasma membrane enzymes: Na+K+ ATPase and Mg2+ ATPase were assayed for specific activity at selected intervals throughout the growth periods. In primary cultures, it was found that Na+K+ ATPase was quite low at 5 days (0.05 units), but increased fourfold at 10 days (0.22 units). By contrast, Mg2+ ATPase changed little over the same period. Tissue cultures grown secondarily had Na+K+ ATPase activity fall 0.3 units (0.52 to 0.22) for 35 days after a single trypsinization. After five trypsinizations over a 50-day period, the activity fell 0.23 units (0.33 to 0.10). The alterations in Mg2+ ATPase activity were more complex in secondary cultures. In the instance in which a single trypsinization was used, the activity fell 0.15 units at day 20, rose 0.12 units (above the starting value) at day 25, then returned to the initial level by day 35. When multiple trypsinizations were used, the activity fell 0.06 units by day 30, then remained stable for the duration. The data may be indicative of mechanisms such as receptor alteration, feedback inhibition and genetic instability when these cells are grown in culture.     

体外培养的兔角膜内皮细胞生物学特性

背景如何选择高质量、生物学特性更接近在体生理状态的角膜内皮种子细胞是组织工程角膜研究的基础。角膜内皮细胞（CECs）的培养、鉴定及生物学特性检测是优选角膜内皮种子细胞的瓶颈问题。目的建立兔CECs原代培养及鉴定的方法,检测传代后细胞的生物学特性。方法从30只新西兰大白兔的角膜组织完整撕除后弹力层及CECs层,采用胰蛋白酶消化法进行原代培养,观察培养细胞的生长状态。分别从形态学、基因水平和蛋白水平鉴定细胞：茜素红染色法观察细胞形态,并与新鲜角膜组织的内皮细胞进行对比;逆转录聚合酶链反应（RT—PCR）法检测IVα2型胶原（COL4A2）、血管内皮生长因子受体2（FLK1）、Na^-K^＋ATP酶α亚单位（ATPIA1）、水通道蛋白1（AQP1）、电压依丛性阴离子通道（VDACs）等相对特异性基因;免疫细胞化学法观察神经元特异性烯醇化酶（NSE）、Na^-K^＋ATP酶及紧密连接蛋白ZO-1的表达。采用MTT比色法测定传代细胞的增生活性变化,采用超微量ATP酶试剂盒及免疫荧光法分别测定传代细胞Na^-K^＋ATP酶活性及其表达量的变化。结果原代培养的兔CECs多在24h内贴壁,2—3d达融合,形态呈六边形。随着传代代数的增加,细胞形态逐渐发生改变,第2代、第3代细胞可见空泡样变。原代培养的细胞茜素红染色可见清晰的细胞轮廓,与在体的CECs形态相似。RT—PCR法检测可见COL4A2、FLKl、ATP1Al、AQP1、VDACs等基因在培养细胞中表达。免疫荧光染色结果显示NSE、Na^-K^＋ATP酶、ZO-1在培养细胞中呈阳性表达。MTT检测结果显示,传代后细胞的增生活性逐渐下降,尤其第2代、第3代细胞下降明显。定量检测结果显示随着传代代数的增加,兔CECs的Na^-K^＋ATP酶活性逐渐降低,各代细胞的Na^-K^＋ATP酶活性总体比较的差异有统计学意义（F=77．174,P=0．000）。结论成功用酶消化法建立了体外培养兔CECs的方法,并从多个层次建立了纯化细胞的鉴定方法。研究结果证实经传代后的CECs的增生活性和功能均有所下降,因此在进行相关的研究时应选用前2代的细胞。     

Effect of glucose on corneal endothelial ATPase assays

Background : previous biochemical methods of determining corneal endothelial ATPase (adenosine triphosphatase) activity have excluded glucose from incubation media. As glucose is an important metabolite for the formation of adenosine triphosphate (ATP) it is possible that glucose may be required in incubations investigating ATPase activity. Methods : standard ATP hydrolysis assays for ATPase activity were performed on bovine corneal endothelial homogenates using incubation media glucose levels of zero, 10, 20 and 30 mM. Results : corneal endothelial Na,K ATPase activity was not significantly different at the four glucose levels tested. Corneal endothelial Mg⁺⁺ ATPase activity was significantly reduced (13.8 per cent) in the 10, 20 and 30 mM glucose conditions when compared to the zero glucose condition. Conclusions : corneal endothelial Na,K ATPase activity is unaffected by short-term exposure to hyperglycaemic media. While the data of this study are not conclusive, it is possible that short-term exposure of corneal endothelia to glucose may be associated with a transient decrease in Mg⁺⁺ ATPase activity. Interactions between cellular ATP levels and Mg⁺⁺ ATPase activity may have a role in helping regulate Na,K ATPase activity. (Clin Exp Optom 1994; 78: 1: 21–23)     

Stimulation of glucosylated lens epithelial Na,K-ATPase by an aldose reductase inhibitor

In diabetes, glucosylation of the Na,K-ATPase of the lens epithelium makes the pump inefficient. K+ transport and ATP hydrolysis (at near saturating ATP concentrations) are inhibited and the kinetics of ATP hydrolysis become substrate inhibition type. The AR inhibitor (AL1576, Alcon Laboratories) stimulates K+ transport and ATP hydrolysis by glucosylated bovine lens Na,K-ATPase. This inhibitor has a slight stimulatory effect upon the unmodified enzyme function also. The AR inhibitor is not able to prevent glucosylation of the pump in high-glucose-containing medium.     

Direct stimulation by succinate of Na+:K+ pump in rabbit ciliary epithelium

The effects of succinate on the intracellular potential difference, PDI, were measured in isolated rabbit ciliary processes. Concentration-dependent increases in the hyperpolarization of PDI occurred between 1 and 15 mM succinate in NaCl Ringers. With 5 mM succinate, there was a 6 mV hyperpolarization. Even though the hyperpolarization of PDI was comparable with 10 and 15 mM succinate, it was more sustained at the latter two concentrations. Succinate also elicited comparable hyperpolarizations of PDI in either Cl(-)-free or HCO3(-)-free Ringers. Similarly, following incubation with either 0.1 mM DIDS or 3 mM BaCl2 the effect of succinate on PDI was unchanged. Five mM succinate had no effect if it was added after 5 mM malonate. Malonate (5 mM) rapidly reversed a 5 mM succinate-induced hyperpolarization of PDI which also suggests a metabolically mediated effect on PDI. An isosmotic substitution of Na+ with NMDG Ringers depolarized PDI, whereas PDI depolarized biphasically during exposure to 0.1 mM ouabain. The addition of 5 mM succinate had no effect on either the time course or the magnitude of the depolarization of PDI during blocking of the Na+:K+ pump with either Na(+)-free Ringers or ouabain. Taken together, these results show that succinate selectively stimulates the Na+:K+ pump, but has no effect on any Cl-, HCO3- or a Ba2(+)-sensitive K+ conductance.     

3-Fluoro-3-deoxy-D-galactose: a new probe for studies on sugar cataract.

PURPOSE: Aldose reductase (AR) activity and flux through the polyol pathway can conveniently be monitored in dog lenses by measuring the metabolism of 3-fluoro-3-deoxy-D-glucose by 19F nuclear magnetic resonance (NMR) spectroscopy. Since AR has broad substrate specificity and preferentially utilizes galactose over glucose as substrate, the ability of AR to utilize 3-fluoro-3-deoxy-D-galactose (3-FDGal) as substrate as well as the metabolism of 3-FDGal in intact dog lens and cultured lens epithelial cells has been investigated. METHODS: The suitableness of 3FDGal as a substrate was examined by incubating 3FDGal with purified dog lens aldose reductase in the presence of an NADPH generating system or with galactitol dehydrogenase in the presence of NAD+. Dog lenses and dog lens epithelial cells were cultured in 3-FDGal medium with and without the AR inhibitor AL 1576. Metabolism was studied using 19F NMR. RESULTS: AR activity with 3-FDGal as substrate is higher than that with D-galactose and its Km of 4.2 mM is ca 10-fold higher than that of D-galactose. Purified dog lens AR incubated with 3-FDGal resulted in the formation of 3-fluoro-3-deoxy-D-galactitol. Galactitol formation was prevented by the addition of AL 1576. Incubation of 3-FDGal with galactitol dehydrogenase resulted in the formation of 3-fluoro-3-deoxy-D-galactonic acid. Dog lenses cultured in 3-FDGal medium formed NMR peaks corresponding to 3-fluoro-3-deoxy-D-galactitol and 3-fluoro-3-deoxy-D-galactonic acid. The presence of AL 1576 inhibited the formation of galactitol but not galactonic acid. Lens epithelial cells cultured in 3-FDGal medium formed only 3-fluoro-3-deoxy-D-galactitol. These cells developed multiple cytoplasmic vacuoles which was prevented by the aldose reductase inhibitor AL 1576. CONCLUSIONS: The high affinity of this fluorinated sugar for aldose reductase makes this an excellent probe for investigating aldose reductase activity in dog lens tissues.     

Na+-K+ and HCO-3 ATPase activity in retina: dependence on calcium and sodium.

With the use of homogenates of whole rat retina, the activities of Na+-K+-- and HCO-3-stimulated ATPases were measured in media containing different concentrations of calcium and sodium ions. In comparison to the Na+-K+ ATPase activity observed in the presence of 2 mM calcium and 130 mM sodium, the omission of calcium from the medium led to a 16-fold increase in activity. Furthermore, the omission of calcium and the reduction of the concentration of sodium in the medium to 25 mM also resulted in an increase in activity of ninefold. This study also demonstrates, for the first time, the existence of a HCO-3 stimulated. ATPase activity in the retina. These results are discussed in relation to recent observations on the effects of calcium- and bicarbonate-free media on the electrical and metabolic activities of the rat retina.     

Vanadate effects on ocular pressure, (Na+, K+)ATPase and adenylate cyclase in rabbit eyes

Topical administration of 50 microliter of 1% Na3VO4 caused a significant fall in intraocular pressure (IOP) in the rabbit eye at 90 min. Assay of ATPase of the iris and ciliary body in vitro at 90 min posttreatment showed no differences between control and treated (Na+, K+)ATPase, ouabain sensitive ATPase or vanadate sensitive ATPase. Accumulation of 48V-labelled orthovanadate in iris and ciliary body reached a plateau of 12 pmoles/mg dry tissue weight 4 hr after a single 25-microliter topical dose of 1% orthovanadate. In vitro inhibition of Na+ sensitive ATPase by sodium metavanadate had an IC50 of 1.8 mM, whereas a 1.8-fold stimulation of adenylate cyclase in iris-ciliary body (ICB) membranes in vitro occurred at 10 mM but not at 10 microM Na metavanadate. These results indicate that the vanadate content of the iris and ciliary body at the time of lowered intraocular pressure is too small to inhibit a significant fraction (greater than 10%) of (Na+, K+)ATPase, or cause a significant stimulation of adenylate cyclase, and that other cellular mechanisms are likely to be involved in the ocular hypotensive response to vanadate.     

Human corneal storage in modified McCarey-Kaufman and K-Sol media: effect on endothelial Na+/K+ ATPase pump site density and permeability.

Endothelial permeability (Pac) to carboxyfluorescein and Na+/K+ ATPase pump site density were determined in human corneas following storage for 4 or 7 days at 4 degrees C in either modified McCarey-Kaufman (mMK) or K-Sol media. Following 4 days of storage, Pac values for mMK- and K-Sol-preserved corneas were not significantly different from those of their prestorage mates. After 7 days of storage, however, corneas stored in K-Sol media showed a significant increase in Pac compared to their prestorage mates, whereas the mMK-stored corneas showed no change in Pac. Na+/K+ ATPase pump site density determined using [3H]ouabain was similar to a control group in the K-Sol-stored tissue but higher in the mMK-stored tissue following 7 days of storage. These studies suggest that mMK medium maintains endothelial barrier function and Na+/K+ ATPase pump site density at least as well as K-Sol medium through 7 days of corneal storage.     

Influence of glucose, fructose and aldose reductase inhibition on retinal sorbitol metabolism.

The sorbitol shunt has been studied in bovine retinal tissue at incubation times from 1 to 24 h. It was shown that an elevated glucose concentration (22 mM) of the medium was accompanied by a slight increase in sorbitol content already after 3 h. At longer incubation times, but lower glucose concentration (11.1 mM) there was a similar increase. Addition of an aldose reductase (AR) inhibitor prevented the sorbitol increase. Addition of fructose to the medium significantly increased the sorbitol accumulation above the effect seen with glucose alone and this effect was not influenced by the AR inhibitor. Thus the sorbitol concentration in the retina may be increased after a short incubation time and further enhanced by the presence of fructose.     

Studies on the eye lens in poikilothermal animals. II. Stimulation of anaerobic glycolysis in rainbow trout lenses incubated with Ca2+-free medium

The effects of Ca2+-free medium on cation levels and lactic acid production of rainbow trout lens incubated at various temperatures were examined in order to study passive cation transport system. Na+-K+ ratio (Na+/K+) increased when the lens was treated with Ca2+-free medium, but the effect of Ca2+-free medium was lowest between 15 and 20 degrees C which was the optimum water temperature for rainbow trout. Lactic acid production was stimulated between 15 and 20 degrees C in Ca2+-free medium. It was also stimulated at 37 degrees C in rat lenses incubated with Ca2+-free medium. However, the production was suppressed in ouabain-treated rainbow trout lenses. The suppression of Na+/K+ increase between 15 and 20 degrees C disappeared when the lens was treated with both Ca2+-free medium and ouabain. These results suggest that Na,K-ATPase activity and glycolysis were stimulated to prevent the increase of Na+/K+ in Ca2+-free medium. In addition, these results indicate that the optimum temperature was between 10 and 20 degrees C for the rainbow trout lens incubation in vitro, which is also the optimum water temperature in vivo.     

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.