Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells

The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.     

Ion transport mechanisms in cultured bovine corneal endothelial cells

Intracellular potential measurements of confluent monolayers of cultured bovine corneal endothelial cells were used to define passive ion transport processes in these cells. Previous studies (Jentsch et al., J. Membr. Biol. 78:103 (1984); Jentsch et al., J. Membr. Biol. 81:189 (1984] have provided the experimental basis for a cellular model, in which bicarbonate entry across the basolateral membrane is indirectly driven by a Na+/H+-exchanger, which is inhibitable by amiloride (1mM). Bicarbonate and sodium should leave the cell via an electrogenic bicarbonate sodium cotransport, which is inhibitable by the disulfonic stilbene derivates SITS or DIDS. This model is also consistent with results from transendothelial studies. In this paper, we briefly review the evidence we have obtained for this model and demonstrate, that the electrical response to sodium (depolarization upon Na+-removal) is neither due to an inhibition of Na+/K+-ATPase nor explainable in terms of changes in K+-conductance. This is concluded from the observation of these responses in the presence of ouabain (10(-4)M) or barium (1mM).     

Effect of bradykinin on cultured bovine corneal endothelial cells

PURPOSE: To clarify the effect of bradykinin on cytosolic free calcium mobilization and cell proliferation in cultured bovine corneal endothelial cells (BCEC). METHODS: The cytosolic free calcium concentration (Ca2+]i) was measured with the InCa(TM) Imaging System after the treatment of bradykinin (10(-11) to 10(-7) M) alone or with the pretreatments of EGTA, bradykinin receptor (Bk1 and Bk2) antagonists and an inhibition of phospholipase C (U-73122). Also, the effect of bradykinin on cell proliferation in BCEC was evaluated using cell counts. RESULTS: In BCEC, [Ca2+]i in the resting state was 87 +/- 9 nM. Bradykinin induced an increment of [Ca2+]i in a concentration-dependent manner and its 50% effective concentration was approximately 5 x 10(-11) M. A [Ca2+]i increment at 10(-8) M bradykinin was inhibited with the pretreatment of EGTA, an extracellular calcium chelator. U-73122 (5 x 10(-6) M) attenuated the bradykinin-induced [Ca2+]i increment. The pretreatment of HOE-140 (Bk2 antagonist) almost attenuated the bradykinin (10(-8) M)-induced [Ca2+]i increase, but des-Arg9-[Leu(8)]-bradykinin (Bk1 antagonist) did not suppress it. To investigate the physiological effect of bradykinin, the effect of bradykinin on cell proliferation was studied. 10(-8) M of bradykinin produced a significant increase in cell numbers. This mitogenic effect of bradykinin was inhibited by the Bk2 antagonist. CONCLUSIONS: Bradykinin-induced stimulation of the signal transduction pathway in BCEC is coupled with the Bk2 type receptor. Furthermore, bradykinin produces the mitogenic effect in BCEC.     

Arachidonic acid metabolism by cultured bovine corneal endothelial cells

Pathways of arachidonic acid metabolism were identified in freshly prepared and in cultured bovine corneal endothelial cells. The principal pathway of arachidonic acid metabolism in the bovine corneal endothelial cells appears to be the cyclooxygenase pathway with the resultant synthesis of PGI2, PGF2 alpha and PGE2. At least two of these products, PGI2 and PGF2 alpha, are formed by the enzymatic conversion of the substrate, PGH2. Measurements of endogenous prostaglandin production by radioimmunoassay demonstrated that PGE2 was the major arachidonic acid metabolite released, with smaller amounts of PGF2 alpha and the stable hydrolysis product of PGI2, 6-keto PGF1 alpha. The release of all three prostanoids was significantly increased by the addition of the calcium ionophore (A23187), human thrombin, bradykinin and histamine. Basal and stimulated release of prostaglandins by the corneal endothelium may contribute to the regulation of intraocular pressure and also in the modulation of the corneal response to injury.     

ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells

PURPOSE: Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs). METHODS: A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (approximately 1 microm tip) for <1 second. Dynamic changes in [Ca2+]i in the mechanically stimulated (MS) cell and in the neighboring (NB) cells were visualized with a confocal microscope, using a fluorescent dye. Normalized fluorescence (NF), calculated as the ratio of the average fluorescence of a cell to the average under resting conditions, was used as a measure of [Ca2+]i. Expression of P2Y receptors and ecto-adenosine triphosphatases (ATPases) was investigated by RT-PCR. ATP release in response to PMS was measured by luciferin-luciferase (LL) bioluminescence. RESULTS: BCECs subjected to PMS showed a transient [Ca2+]i increase. Under control conditions, the maximum NF in the MS cell occurred within 600 ms, and the fluorescence returned to baseline within 170 seconds. NB cells also presented a [Ca2+]i increase with a transient characterized by decreasing maximum NF and increasing latency as a function of the distance from the MS cell. These transients propagated as an intercellular Ca2+ wave to a distance of five or six NB cells away from the MS cell, covering areas (called active areas, AAs) up to 77,000 +/- 3,200 microm2 (N=21). The percentage of responsive cells (defined as cells showing maximum NF >1.1) decreased with increasing distance from the MS cell. The Ca2+ wave crossed cell-free lanes. Pretreatment of cells with the nonselective purinergic receptor antagonist suramin (200 microM), exogenous apyrases, which break down nucleotides (10 U/mL), or the PLC inhibitor U-73122 (10 microM) reduced the wave propagation, whereas the ecto-ATPase inhibitor ARL-67156 (100 microM) significantly enhanced it. ATP-dependent LL bioluminescence increased after PMS. RT-PCR showed mRNAs for P2Y1 and P2Y2 receptors and ecto-ATPases in BCECs. CONCLUSIONS: PMS of BCECs induces release of ATP and a concomitant intercellular Ca2+ wave, even in the absence of direct cell-cell contacts. The AA of the wave is modulated by agents that affect P2Y receptor activity. Thus, PMS-induced intercellular Ca2+ wave propagation in BCECs involves ATP-dependent paracrine IC.     

Swelling activated chloride channels in cultured bovine corneal endothelial cells

Swelling induced enhancement of anion permeability was investigated using the halide-sensitive fluorescent dye SPQ in cultured bovine corneal endothelial cells (BCEC). Rates of anion influx were quantified in terms of the rate of change of SPQ fluorescence during exposure to short duration pulses of Cl-, I-or NO3-while the cells were being perfused with I-, NO3-or Cl-Ringer, respectively. Since SPQ fluorescence is quenched to different extents by these anions, their influx or efflux causes significant changes in fluorescence. The ratio of the maximum rate of change of fluorescence during the pulse period under hyposmotic conditions to that under isosmotic conditions, referred to as the enhancement ratio (ER), was calculated as a measure of the increase in anion permeability. When cells were perfused with NO3-Ringer, exposure to I-pulses yielded an ER=9.0+/-2.6 for 110+/-5 mosmhyposmotic shock. This was higher than with Cl-/I-(6.4+/-0.7) or NO3-/Cl-(3.2+/-0.8) anion-pairs for the same level of shocks. In all cases, the enhancement occurred within approximately 100 seconds after swelling but decreased with continued progress of regulatory volume decrease (RVD). ER returned to approximately 1 within 4 minutes after returning to isosmotic conditions. The membrane potential (Em) depolarized immediately after hyposmotic shock. When cells were depolarized prior to the shocks by high [K+], changes in Emwere relatively small. ER, for the NO3-/I-anion-pair, was significantly reduced by DIDS (100% at 500 microm), NPPB ( approximately 80% at 100 microm) and tamoxifen (approximately 85% at 12 microm). Tamoxifen and NPPB also inhibited swelling induced depolarization. Increasing cationic conductance with Gramicidin D at approximately 2 minutes following hyposmotic shock induced NPPB-inhibitable secondary swelling or accelerated RVD under normal or low Na+conditions, respectively. These results demonstrate that BCEC express swelling activated Cl-channels, which facilitate RVD by enhancing anionic permeability and also by providing a favorable electrical gradient for K+efflux.     

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)     

Tight junctions and paracellular permeability in cultured bovine corneal endothelial cells

Intramembrane specializations of cultured bovine corneal endothelial cells were studied with thin section and freeze-fracture electron microscopy and related to the paracellular permeability and the transendothelial resistance (R^t) of the monolayers. The following intercellular junctions were found: single and discontinuous networks of tight junctions (TJ) which girdle the apico-lateral cell perimeter incompletely, gap junctions, and membrane undulations suggesting intermediate junctions. The macromolecular tracer ruthenium red penetrated into the lateral intercellular space beyond the level of the incomplete belt of TJ. R^t of these monolayers was 20.9±1.0 º · cm² Protamine induced a reversible increase of R^t to 118±5 % of its control value. We conclude that incomplete belts of TJ may be the morphological counterpart of the high paracellular permeability of this monolayer and functionally and morphologically resemble those of their native endothelium. Cultured corneal endothelial cells are an excellent model for studying the influence of incomplete belts of TJ on paracellular permeability of cells.The results reported here were presented in part at the 91st Congress of the German Ophthalmological Society, Mannheim, 19–22 September 1993 and published in abstract form [22].     

Na,K-ATPase of cultured bovine lens epithelial cells: H2O2 effects.

Na,K-ATPase function was studied in cultured bovine lens epithelial cells under confluent and non-confluent conditions. The affinity of the Na,K-ATPase for the cardiac glycoside, ouabain, differs between the confluent and non-confluent cultures. The confluent cells have a higher affinity for ouabain than do the non-confluent cells. The ouabain affinity of the confluent cells is similar to that for the Na,K-ATPase isolated from the bovine axolemma and the bovine lens cortex. The ouabain affinity of the non-confluent cells is similar to that for the Na,K-ATPase of the renal medulla and bovine lens epithelium. Similar results are not found with confluent and non-confluent MDCK cells. H2O2 treatment of confluent and non-confluent lens epithelial cell cultures has differing effects on the Na,K-ATPase function. In the confluent cell preparations, H2O2 affects K(+)-dependent dephosphorylation of the intermediate phosphoenzyme. In the non-confluent preparations. H2O2 appears to inhibit K(+)-occlusion.     

Binding and processing of trypsin by cultured bovine corneal endothelial cells

Bovine corneal endothelial cells in culture bind internalize and degrade [125I]-trypsin. Binding involves the active site of trypsin and increases as a function of [125I]-trypsin concentration. Saturation is observed at a concentration of 0.5-1.0 micrograms ml-1. The cell surface binding of [125I]-trypsin is specific: a seven-fold excess of unlabeled trypsin abolishes about 60% of the total cell surface-associated radioactivity. In addition, thrombin competes poorly with [125I]-trypsin cell surface binding and only 20% of the specific cell surface binding of [125I]-trypsin is subjected to competition with thrombin. This fraction of the cell surface-bound [125I]-trypsin which is accessible to competition with thrombin appears in a covalent complex of [125I]-trypsin X protease-nexin with a molecular weight of 64000 daltons. The cells, when incubated at 37 degrees C, appear to internalize the cell surface-bound [125I]-trypsin at a rate of 0.15-0.25 ng (10(6) cells)-1 min-1. Both the non-covalently cell surface-bound and the protease-nexin (PN) mediated-bound [125I]-trypsin are internalized by the cells, but the [125I]-trypsin X PN complexes contribute about 75% of the total amount of [125I]-trypsin internalized by the cells. The internalized [125I]-trypsin is degraded by the cultures at a rate of about 0.05 ng (10(6) cells)-1 min-1 and the degradation products are released by the cells into the incubation medium as a trichloroacetic acid non-precipitable material. Chloroquine inhibits about 60% of the internalization of [125I]-trypsin by the cells, and inhibits more than 80% of the degradation process of [125I]-trypsin, which indicates that the degradation of the ligand is taking place in lysosomes. Bovine corneal endothelial cells in culture have demonstrated the binding and metabolism of the serine protease trypsin. This described process may indicate the ability of corneal endothelial cells to control the activity of serine proteases in their microenvironment.     

Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells

We have previously demonstrated the presence of a Na(+)-K(+)-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive (86)Rubidium ((86)Rb) uptake in (86)Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k(1/2)) of 21.1 and 1.33 mM, respectively. Extracellular Cl- is also required for cotransport activation, but shows higher order kinetics; the k(1/2) for Cl- is 28.1 mM and the Hill coefficient 2.1. HCO(3)(-) exerts a modulating effect on cotransporter activity; at 0 HCO(3)(-) the bumetanide-sensitive K(+) uptake is reduced by 30% compared to that at 26 mm HCO(3)(-). Manipulations of the intracellular [Cl-] by preincubation in Cl- -free solution or inhibition of Cl- efflux resulted in increased uptake at low [Cl-](i) and decreased uptake at high [Cl-](i). To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k(1/2)=21 nM) and okadaic acid (k(1/2)=915 nM) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Ca(i)(2+) by preincubation in Ca2+ -free medium as well as by exposure to W-7, an inhibitor of the binding of Ca(2+) to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+ -calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+ -calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl- are requirements for the function of the CBCEC Na(+)-K(+)-2Cl(-) cotransporter, while intracellular Cl- and extracellular HCO(3)(-) modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.     

压力对体外培养的牛角膜内皮细胞的影响

目的观察压力对体外培养的牛角膜内皮细胞形态结构及细胞活力的影响。方法对体外培养的牛眼角膜内皮细胞施以2.67kPa、5.33kPa及8.00kPa的压力各6、12、24、36、48h,以不加压组作为对照组。用倒置相差显微镜及电镜观察细胞形态结构,以台盼蓝染色观察比较48h时各组细胞活力的差别,用单因素方差分析比较不同压力下阳性细胞率。结果当作用于细胞的压力为2.67kPa(1kPa=7.5mmHg),作用时间为6、12、24、36、48h时,与对照组比较,细胞形态结构均无明显变化;48h时台盼蓝计数与对照组比较差异无统计学意义(P=0.776)。当压力为5.33kPa,作用时间为24h时,细胞形态结构出现轻度的损伤,在36、48h时加重;48h时台盼蓝计数与对照组比较,差异有统计学意义(P=0.00)。当压力为8.00kPa时,6h时即出现明显细胞形态结构损害,随着压力和作用时间的进一步增加,细胞的损伤程度也进一步加重;48h时台盼蓝计数与对照组比较差异有统计学意义(P=0.000)。结论牛角膜内皮细胞在加压48h时只能承受2.67kPa的压力,当压力为5.33kPa及8.00kPa,施压48h时,将造成细胞...     

氯通道阻滞剂NPPB对培养牛角膜内皮细胞增殖的抑制作用

目的观察氯离子通道阻断剂5-硝基-2-(3-苯丙胺)苯甲酸[5-nitro-2-(3-phenyl-propylamino)-benzoic acid,NPPB]对培养牛角膜内皮细胞增殖的作用。方法实验设5组,对照组,0.1%二甲基硫氧化物DMSO(di mathyl sulfoxide)组,50μmol·L-1、100μmol·L-1、200μmol·L-1NPPB组。应用MTT比色分析法观察细胞增殖情况,计算细胞存活率。采用流式细胞仪检测细胞周期时相的变化,计算细胞增殖指数。结果NPPB使培养牛角膜内皮细胞MTT光吸收值和细胞存活率较对照组均显著降低,并具有浓度和时间依赖性。经100μmol·L-1NPPB处理48h后,与对照组相比,细胞出现明显的凋亡峰,G1期细胞比例明显增高,S期及G2期细胞比例则明显降低,增殖指数明显下降。结论NPPB能浓度依赖性抑制培养牛角膜内皮细胞的增殖,并使细胞停滞于G1期。     

Inhibition of carbonic anhydrase activity in cultured bovine corneal endothelial cells by dorzolamide

PURPOSE: Fluid transport by the corneal endothelium is dependent on the presence of HCO(3)(-) and the activity of carbonic anhydrase (CA)-II and -IV, the cytoplasmic and membrane-bound CAs, respectively. This study was conducted to examine the inhibition of CA activity in cultured bovine corneal endothelial cells (BCECs) by dorzolamide, a topical CA inhibitor used in glaucoma therapy. METHODS: BCECs were grown on glass coverslips and then perfused with HCO(3)(-)-free Ringer's. The inward flux of CO(2) was induced by exposure to CO(2)-HCO(3)(-) Ringer's and the opposing outward flux by returning to HCO(3)(-)-free Ringer's. Consequent transients in intracellular pH (pH(i)) were measured using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). During the inward flux of CO(2,) the maximum rate of change of pH(i) was taken as a quantitative measure of the overall CA activity in BCECs. RESULTS: Exposure to CO(2)-HCO(3)(-) Ringer's led to a transient decrease in pH(i) (component A), followed by a rapid increase to a new steady state (component B). However, when the CO(2)-HCO(3)(-) Ringer's was removed, the pH(i) increased transiently (component C) and then rapidly returned to the original pH(i) (component D). Component A, caused by an inward flux of CO(2) and its subsequent hydration by CA-II, was blocked by dorzolamide in a dose-dependent manner with an 50% inhibitory concentration (IC)(50) of 2.4 micro M (95% confidence interval: 0.5 -10.85 microM). However, the inhibition of the outward flux of CO(2), inward flux of HCO(3)(-), and outward flux of HCO(3)(-) (associated with components C, B, and D, respectively) was not dose dependent. Cells that were exposed to 500 nM of the drug for longer than 30 minutes did not show a significantly greater inhibition of any of the components. Dorzolamide and acetazolamide (500 microM) did not show additive inhibition of any of the components (P = 0.13; n = 6). CONCLUSIONS: Dorzolamide significantly inhibits CA activity in BCECs at micromolar levels. Because these levels are encountered in the cornea and aqueous humor after topical administration, dorzolamide may compromise corneal hydration control, especially when the functional reserve of corneal endothelium is low. Dorzolamide does not appear to accumulate in the cells, because the inhibition of CA-II did not increase after prolonged exposure to the drug.     

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)     

Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells

PURPOSE: Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. METHODS: Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. CONCLUSIONS: Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.     

Growth factors and corneal endothelial cells: II. Characterization of epidermal growth factor receptor from bovine corneal endothelial cells.

Epidermal growth factor (EGF) is a potent mitogen for corneal endothelial cells and may play a role in endothelial wound healing. To further characterize the interaction of EGF with endothelial cells, we measured biochemical parameters of 125I-EGF binding to cultured bovine corneal endothelial cells (BCEC), determined the pattern of EGF-induced protein phosphorylation, and investigated the influence of retinoic acid (RA) and transforming growth factor beta (TGF-beta) on EGF-induced DNA synthesis and receptor levels. Binding of 125I-EGF to BCEC was dependent on time, reaching a plateau after approximately 2 h at 37 degrees C, was specific for EGF, and had high affinity (Kd = 100 pM) with approximately 21,000 receptors per cell. Cellular substrates for the EGF receptor kinase, which may function as initial second messengers for EGF, were detected by autoradiography of sodium dodecyl sulfate polyacrylamide gels of 32P-labeled BCEC proteins. EGF stimulated phosphorylation of 170, 37, 21 and 20-kDa proteins. Addition of 1 nM, 100 nM, and 10 microM RA to BCEC cultured in serum-free medium for 24 h progressively inhibited DNA synthesis by up to 80% compared with control cultures. However, when added in combination with 5 nM EGF, 1 nM and 100 nM RA synergistically stimulated DNA synthesis by up to 80% above the level of EGF stimulation without altering EGF receptor levels or binding affinity. Thus, short-term exposure of BCEC to RA potentiated EGF-stimulated DNA synthesis, most likely by acting at a postreceptor step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells

PURPOSE: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC). METHODS: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis and immunoblotting. Elevation of cAMP in response to agonists of A2b receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity. RESULTS: Exposure to ADO (200 microm; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 microm; 30 min), known agonists of A2b receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 microm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 microm ADO. CGS-21680, a selective A2a agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 microM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm2. CONCLUSIONS: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity.     

Urokinase binding to bovine corneal endothelial cells

Previously, we showed that the clotting factor thrombin binds to bovine corneal endothelial cells forming a covalent complex with a protein released by these cells into the culture media. We report that the plasminogen activator, urokinase, an enzyme involved in dissolution of blood clots, binds specifically to bovine corneal endothelial cells. [125I]-urokinase is rapidly bound by corneal endothelial cells. The serine active site of urokinase is required for binding since [125I]-urokinase inactivated with diisoprophylfluorophosphate does not bind to the cells. Pre-incubation of corneal cells with unlabeled urokinase for 20 hr results in increased release of binding sites for [125I]-urokinase into the culture media and at least a 3.5-fold increase in binding by the cells. [125I]-urokinase forms a 73 300 dalton sodium dodecyl sulfate complex with a corneal endothelial cell protein. Although thrombin competes with urokinase for binding to corneal endothelial cells, urokinase has at least a 10-fold higher affinity for binding to the cells. Furthermore, these cells make a plasminogen activator identical in molecular weight to urokinase. Thus, under physiological conditions, urokinase rather than thrombin binds to corneal endothelial cells. Binding may serve to regulate endogenous urokinase activity in the anterior chamber of the eye by limiting its extracellular proteolytic activity.     

Histamine H1 receptor-mediated Ca2+ signaling in cultured bovine corneal endothelial cells.

The corneal endothelium pumps ions and water from the stroma to the aqueous humor, maintaining corneal transparency. This report investigates the possibility that cultured corneal endothelial cells express neurohormonal Ca2+ signaling pathways employed by other epithelia to regulate transport or other cellular functions. Agonist-stimulated changes in intracellular calcium ([Ca2+]i) in single bovine corneal endothelial cells (BCEC) derived from confluent cultures were measured by microspectrofluorimetry using the Ca(2+)-sensitive probe, fura 2. Mean resting [Ca2+]i in BCEC was 46 +/- 2 nM (n = 124). The muscarinic cholinergic agonist, carbachol, did not mobilize Ca2+, whereas histamine induced a rapid increase in [Ca2+]i to initial peak levels of 549 +/- 22 nM (n = 46) at maximally stimulating doses. The initial rise in [Ca2+]i in response to histamine was dose dependent, with a minimum effective dose of 50 nM, EC50 = 0.84 mumol/l, and a maximum effective dose of 10 mumol/l. [Ca2+]i decreased from the initial peak, but then stabilized to form an agonist-dependent sustained elevation or abruptly fell back to baseline to begin oscillatory fluctuations. The initial peak was insensitive to removal of extracellular calcium (Ca2+o), whereas subsequent elevations in [Ca2+]i or sustained [Ca2+]i oscillations required Ca2+o. The amplitude of the oscillations in [Ca2+]i increased with an increase in [histamine]. However, frequency was independent of [histamine] (mean = 0.62 spikes min-1 +/- 0.06, n = 33). Histamine-induced Ca2+ mobilization was inhibited by the H1 receptor antagonist triprolidine, but was unaffected by ranitidine (H2 antagonist) or thioperamide (H3 antagonist).(ABSTRACT TRUNCATED AT 250 WORDS)