Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells

Charles W. McLaughlin1, David Peart2, Robert D. Purves3, David A. Carré4, Kim Peterson-Yantorno4, Claire H. Mitchell4, Anthony D. C. Macknight1, and Mortimer M. Civan4,5

Departments of 1 Physiology, 2 Ophthalmology, and 3 Pharmacology, University of Otago Medical School, Dunedin, New Zealand; and Departments of 4 Physiology and 5 Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The beta -adrenergic antagonist timolol reduces ciliary epithelial secretion in glaucomatous patients. Whether inhibition is mediated by reducing cAMP is unknown. Elemental composition of rabbit ciliary epithelium was studied by electron probe X-ray microanalysis. Volume of cultured bovine pigmented ciliary epithelial (PE) cells was measured by electronic cell sizing; Ca2+ activity and pH were monitored with fura 2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 µM) produced similar K and Cl losses from ciliary epithelia in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution but had no effect in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-free solution or in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution containing the carbonic anhydrase inhibitor acetazolamide. Inhibition of Na+/H+ exchange by dimethylamiloride in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution reduced Cl and K comparably to timolol. cAMP did not reverse timolol's effects. Timolol (100 nM, 10 µM) and levobunolol (10 µM) produced cAMP-independent inhibition of the regulatory volume increase (RVI) in PE cells and increased intracellular Ca2+ and pH. Increasing Ca2+ with ionomycin also blocked the RVI. The results document a previously unrecognized cAMP-independent transport effect of timolol. Inhibition of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange may mediate timolol's inhibition of aqueous humor formation.

electron probe X-ray microanalysis; cell volume; cell pH; cell calcium; chloride/bicarbonate exchanger; sodium/hydrogen exchanger


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE BILAYERED CILIARY EPITHELIUM secretes aqueous humor into the eye in three steps: stromal uptake by the pigmented ciliary epithelial (PE) cells, movement from the PE to the nonpigmented ciliary epithelial (NPE) cells, and release from the NPE cells into the posterior chamber of the eye (14, 16, 25, 37, 44, 63, 65, 66). The major solute components, Na+ and Cl-, are taken up from the stroma by the PE cell layer, diffuse through gap junctions (21, 25, 29, 50, 53, 56) to the NPE cells, and are then released into the aqueous humor. The first step, entry from the stroma, can proceed through paired NHE-1 Na+/H+ and AE2 Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiports (22) and by a Na+-K+-2Cl- symport (23-25, 60). In the final step at the aqueous surface, Na+ is extruded by Na+-K+-ATPase and Cl- is released through Cl- channels of the NPE cells (36).

The intraocular pressure (IOP) reflects a balance between rates of formation and exit of aqueous humor. The IOP is usually elevated in glaucoma, a spectrum of blinding diseases treated with a wide range of agents, including carbonic anhydrase inhibitors, alpha -adrenergic and cholinergic agonists, prostaglandin analogs, and beta -adrenergic blockers (55). The nonselective beta -adrenergic blocker timolol (73) is among the most widely used and effective drugs in lowering the secretory rate, and thereby the IOP (28). Timolol binds to beta -adrenergic receptors of the ciliary processes with high affinity (62). Agonists to all beta -adrenergic receptors (beta 1, beta 2, and beta 3) stimulate adenylyl cyclase via interaction with Gs to increase cAMP production (34). As a known beta -blocker, timolol could act by reducing the intracellular concentrations of cAMP. However, it has long been unclear whether the putative reduction in cAMP itself causes the reduction in IOP because of the following considerations (69). First, a surprisingly high concentration of timolol is considered necessary to lower IOP. The ocular hypertensive rabbit (following water load or alpha -chymotrypsin injection) has been used to mimic the glaucomatous state. In that case, 0.5% topical timolol has been required to reduce the IOP, a concentration 500 times larger than that (0.0001%) needed to inhibit the hypotensive effect of the beta -adrenergic agonist isoproterenol in the same study (62). An ocular hypotensive effect has also been reported in normotensive rabbits at very low timolol concentration [0.01% topical timolol (31)], but the effect is small. Second, beta 1-adrenergic antagonists are effective in some models of ocular hypertension (6, 61), although the high density of beta -receptors in the ciliary process are predominantly of the beta 2 subtype. Third, D-timolol may be as effective as L-timolol (42) in decreasing aqueous flow, despite stereospecificity of the beta -adrenergic receptors for the L-isomers (41, 49). Fourth, if timolol reduces aqueous humor formation by blocking beta -adrenergic-mediated increase of cAMP production, we would expect cAMP itself to increase inflow. However, cAMP certainly does not markedly increase aqueous flow, and some investigators have observed a decrease in inflow following administration of forskolin to stimulate endogenous production of cAMP (11, 40). Fifth, one might ascribe the absence of a marked increase in inflow following cAMP administration to chronic in vivo beta -adrenergic stimulation, blunting the effects of exogenous cAMP. However, such constant agonist stimulation would desensitize the beta -adrenergic receptors, uncoupling the adenylyl cyclase system (5, 48). The foregoing considerations do not preclude the possibility that timolol reduces secretion of aqueous humor exclusively through its action as a nonselective beta -adrenergic antagonist, but have raised doubts about that hypothesis.

We have addressed the issue by electron-probe X-ray microanalysis of the ciliary epithelial cells in the intact rabbit ciliary epithelium (7, 44). The great advantage of this method is the unique capability of quantifying the Na, K, and Cl contents at visualized sites within individual cells (e.g., chapter 6 of Ref. 18). Because of the complexity of the ciliary epithelium, the electron microprobe analyses have been complemented with fluorometric and volumetric measurements of cultured bovine PE cells that we have already characterized (22, 47). The results of the present study suggest an alternative mechanism by which timolol could reduce aqueous inflow and thus IOP.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Methods used in this study have been described in detail elsewhere (7, 22, 44, 47).

Cellular model: rabbit ciliary epithelium. Dutch-belted rabbits of either sex and older than 6 wk postweaning were obtained from the Department of Laboratory Animal Sciences, University of Otago Medical School, Dunedin, New Zealand, and were treated in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Research. The animals were anesthetized with 30 mg/kg pentobarbital sodium and killed by injecting air into the marginal ear vein. After enucleation, the iris-ciliary body was excised, cut into quarters, and each quarter bonded at its edge to plastic frames with cyanoacrylate. Dissected tissue was incubated for at least 2 h in either HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free medium. Pairs of quadrants (one from each eye) were then incubated separately at room temperature (18-22°C) in a beaker for at least 30 min under the different experimental conditions. We conducted incubations at room temperature (18-22°C) for reasons discussed elsewhere (44). First, conducting the experiments at a higher temperature is expected to introduce additional complexities, both in delivering sufficient oxygen to meet the enhanced metabolic demands and in making unstirred layers more important. We have indeed found that the ciliary cells are less able to maintain a high intracellular K+ concentration and low intracellular Na+ concentration at 37°C under our experimental conditions (Bowler JM, Peart D, Purves RD, Carré DA, Macknight ADC, and Civan MM, unpublished observations). Second, qualitatively similar responses in short-circuit current are evoked by adding cardiotonic steroids or forskolin to the aqueous solution bathing rabbit iris-ciliary body at 37°C (17, 38) and at room temperature (12).

In view of the prolonged periods of study, we verified that the incubated tissues retain their transport integrity during these experiments. As expected, blocking Na+-K+-ATPase with ouabain markedly reduces the intracellular K and elevates the intracellular Na contents (see Fig. 2, Table II in Ref. 7). Hypotonic swelling also produces an expected regulatory volume decrease (RVD) in NPE cells (with release of cell K and Cl), while producing little response in adjoining PE cells of the same tissue (see Fig. 5 of Ref. 43). This is consistent with reports of the robust RVD displayed by NPE cells (26, 68) and the blunted regulatory volume decreases by freshly dissected PE cells (24) and immortalized PE cells (Ref. 46 and see Fig. 8 of Ref. 22).

Cellular model: bovine PE cells. We extended our studies of an immortalized PE cell line developed by Dr. Miguel Coca-Prados from a primary culture of bovine pigmented ciliary epithelium and characterized by several investigators (22, 47, 64). Cells were grown in DMEM (#11965-084; GIBCO BRL, Grand Island, NY) with 10% fetal bovine serum (SH30071.03; HyClone Laboratories, Logan, UT) and 50 µg/ml gentamicin (#15750-060; GIBCO BRL), at 37°C in 5% CO2 (52). The medium had an osmolality of 328 mosmol/kgH2O. Cells were passaged every 6-7 days and, after reaching confluence, were suspended in solution for study within 3-10 days after passage.

Solutions and chemicals. The isotonic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution contained (in mM) 140-145 Na+, 5.9 K+, 122.1 Cl-, 15.0 HEPES, 1.2 Mg2+, 2.5 Ca2+, 1.2 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 25-30 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and 10 glucose at pH 7.30-7.45 and 305-315 mosmol/kgH2O. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution was prepared by isosmolar replacement of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with Cl-. Hypotonic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solution (150-160 mosmol/kgH2O) was prepared by reducing the NaCl concentration by 79.5 mM. Depending on whether or not HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was included, the gas bubbled through the solution throughout incubation of the iris-ciliary bodies consisted of either 95 %O2-5 %CO2 or pure O2, respectively. Cl--free solutions were prepared by replacing MgCl2 with MgSO4 and by replacing the remaining Cl- with methylsulfonate. In conducting fluorescence measurements, bovine PE cells were perfused with a solution similar to that previously described (47) (in mM): 114 Na+, 5.0 K+, 113.6 Cl-, 10.0 HEPES, 0.5 Mg2+, 1.3 Ca2+, 5.0 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 5.0 glucose, and 60.0 mannitol at pH 7.4 and 300-305 mosmol/kgH2O.

All chemicals were reagent grade. Bovine albumin (RIA grade; Immuno Chemical Products) was dialyzed for 48-60 h, freeze-dried at -70°C, and stored at 4°C. A 30% (wt/vol) solution was prepared by dissolving the albumin in the same medium in which the tissue was incubated. Timolol maleate was added to the incubation media from stock solutions in water or ethanol, and dimethylamiloride from stock solutions in water. Dibutyryl adenosine 3'-5'-cyclic monophosphate (dibutyryl cAMP) and 8-bromoadenosine 3'-5'-cyclic monophosphate were dissolved directly in incubation media. Acetazolamide was added from stock solutions in dimethylformamide. In all cases, the same concentration of solvent vehicle (0.1% vol/vol) was applied to parallel control preparations. Timolol and propranolol were obtained from Sigma, and levobunolol was purchased as Betagan (Allergan, Hormigueros, Puerto Rico).

Electron microprobe data acquisition and reduction. After incubation, the tissues were blotted and a 30% albumin solution was applied briefly to the epithelial surface of the NPE cells (i.e., to the basement membrane supporting the NPE cells). Excess albumin was removed by blotting, and the tissue segment was then plunged into liquid propane at -180°C to freeze the preparation quickly before ions and water could undergo redistribution. Sections were then cut 0.2-0.4 µm in thickness at -80°C to -90°C with a cryoultramicrotome, freeze-dried at 10-4 Pa (equivalent to 7.5 × 10-7 Torr), and transferred for analysis to a scanning electron microscope (JEOL JSM 840) equipped with an energy-dispersive spectrometer. Unless otherwise stated, five to eight pairs of NPE and PE cells were measured in each of two sections cut from each quadrant.

Electron probe X-ray microanalysis permits both quantification and localization of intracellular elements. Using an electron microscope, we target a specific visualized area within the cell. The specimen is irradiated with a beam of electrons, which ionizes a small fraction of the atoms bombarded. After an electron is knocked from an inner atomic shell, an electron from an outer shell can take its place. The relaxation of the electron from a higher to a lower energy state generates a quantum of X-ray energy. Spectroscopic measurement of the characteristic energy and number of these quanta permits identification and quantification of the elements within the sample.

The dried sections were imaged with a transmitted electron detector. Measurements were collected with a Tracor Northern X-ray 30-mm2 detector, using a probe current of 140-200 pA for 100 s at an accelerating voltage of 20 kV. The intracellular data were obtained by the electron beam scanning a rectangular area within the nucleus of each selected NPE or PE cell, which varied from ~0.9 × 1.2 µm to ~2.4 × 3.0 µm depending on the size of the nucleus analyzed.

The elemental peaks were quantified by filtered least-square fitting to a library of monoelemental peaks (8). The library spectra for Na, Mg, Si, P, S, Cl, K, and Ca were derived from microcrystals sprayed onto a Formvar film. White counts were summed over the range 4.6-6.0 keV and corrected for the nontissue contributions arising from the Al specimen holder and Ni grid.

As previously discussed (44), for purposes of data reduction the elemental peaks were routinely normalized to the phosphorus signal obtained in the same scanned area of each cell. Phosphorus was chosen for normalization because of the constancy of the intracellular signal, which almost entirely reflects the covalently linked fraction in epithelial cells. For example, inorganic phosphate (Pi) is accumulated to only 3 mmol/kg intracellular water in the epithelial cells of frog skin (20). In such cells, the total pool of ATP, ADP, phosphocreatine, and Pi corresponds to only 5% of the total P pool (~400-500 mmol/kg dry wt) measured in the ciliary epithelial cells (7). The validity of normalizing to P has been experimentally confirmed by the close linear relationship between the contents of the two largely intracellular elements, K and P (see Fig. 3 of Ref. 7).

The values we report for Na/P, Cl/P, and K/P are the measured normalized estimates of the intracellular Na, Cl, and K contents, respectively. Because the NPE and PE cells responded similarly to the experimental changes (Table 1), the data from the two cell types have generally been pooled. Although it is not possible to calculate ion concentrations in millimoles per liter from these data, the intracellular contents of (Na + K) or of (Na + K + Cl) provide indexes of intracellular water content (1). For this reason, the measured values of (Na + K)/P and of (Na + K + Cl)/P are entered in the Table and cited in the text, where appropriate. The parameter (Na + K - Cl)/P is the normalized difference between the principle mobile cations and anion and provides an index of the tissue content of unmeasured anion; this index is also presented in the Table.

                              
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Table 1.   Effects of timolol in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solutions: all available results

Values are presented as means ± 1SE. The numbers of cells analyzed are indicated by the symbol n, while N is used to refer to numbers of experiments. With more than two groups of electron microprobe data, the differences between groups have been analyzed by ANOVA using nonparametric (Kruskal-Wallis) testing, and the probabilities (P) of the null hypothesis have been calculated with the Dunn multiple comparisons posttest. With two groups, the nonparametric Mann-Whitney test was used.

In principle, intracellular Na+, K+, and Cl- concentrations might also be measured simultaneously fluorometrically. Membrane-permeant probes are available for this purpose. However, differential bleaching and leakage of the fluorophores, limited selectivity of the K+ probe, and the effects of quenching by uncontrolled factors constitute significant challenges. For example, intracellular fluorescence of the Cl--sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium has been used to measure intracellular volume, based on quenching by unidentified intracellular anions and proteins (58). It is entirely feasible to measure intracellular pH (pHi) and Ca2+ activity simultaneously using ratiometric dyes (described in Fluorescence experiments). Because the excitation or emission spectra are different for the free and bound forms of such fluorophores, the ratio of measurements at two different frequencies permits measurement of ion activity independent of photobleaching and total dye concentration. In the absence of satisfactory ratiometric dyes for measuring intracellular Na, K, and Cl simultaneously, we conclude that electron microprobe X-ray analysis provides a unique opportunity for this purpose in preparations displaying cellular heterogeneity.

Volumetric measurements and analysis. After harvesting cells from a T-75 flask by trypsinization (19), a 0.5-ml aliquot of the cell suspension in DMEM was added to 20 ml of each test solution. Parallel aliquots of cells were studied on the same day. One or two aliquots served as control, and the others were exposed to different experimental conditions at the time of suspension. The same amount of solvent vehicle was always added to the control and experimental aliquots. The sequence of studying the suspensions was varied to preclude systematic time-dependent artifacts. Cell volumes of isosmotic suspensions were measured with a Coulter counter (model ZBI-Channelyzer II), using a 100-µm aperture. As previously described, the cell volume (vc) of the suspension was taken as the peak of the distribution function. The symbol N is used to indicate the number of experimental measurements in studying the intracellular volume and Ca2+ activity and pH (see Fluorescence experiments).

As with many other cells (35), we can trigger a secondary regulatory volume increase (RVI) by first swelling these bovine PE cells in 50% hypotonic solution (see Solutions and chemicals) and then restoring isotonicity (see Figs. 8-11 of Ref. 22). After hypotonic suspension, the cells swell to a peak volume in 2-4 min, thereafter shrinking spontaneously toward their isotonic volumes with a half time of ~20 min. This regulatory response is called the RVD, reflecting the release of intracellular solute and water. Addition of NaCl to restore isotonicity 24 min after hypotonic suspension shrinks the cells to below their isotonic volumes and triggers the RVI (see Figs. 5 and 6). When tested for their effects on the RVI, timolol, levobunolol, and cAMP were added at the same time as the NaCl. The time course of the RVI was fit to a monoexponential by nonlinear least-square analysis, and the probability of the null hypothesis was obtained from the F distribution (22). These experiments were conducted at 37°C since isolated bovine cells, whether freshly isolated (63) or continuously cultured (22), do not display an RVI at room temperature.

Fluorescence experiments. For measurements of intracellular Ca2+, cells grown on coverslips for 1-5 days were loaded in the dark with 5 µM fura 2-AM and 0.005% Pluronic F-127 (Molecular Probes, Eugene, OR) for 50-240 min at 25°C or 37°C, then rinsed and maintained in fura-free solution before beginning data acquisition. The coverslips were mounted on a Nikon Diaphot microscope and visualized with a ×40 oil-immersion fluorescence objective. The emitted fluorescence (520 nm) from 15-25 confluent cells was sampled at 1 Hz following excitation at 340 and 380 nm, and the ratio was determined with a Delta-Ram system and Felix software (Photon Technology International, Princeton, NJ). In experiments in which fura 2 was the only dye used, the ratio of light excited at 340 nm to that at 380 nm was converted into Ca2+ concentration using the method of Grynkiewicz et al. (32). An in situ dissociation constant (Kd) value for fura 2 of 350 nM was used. The ratio (Rmin) of fluorescence at 340 nm vs. 380 nm in the absence of Ca2+ was obtained by bathing cells in a Ca2+-free isotonic solution of pH 8.0 containing 10 mM EGTA and 5 µM ionomycin. The ratio (Rmax) of fluorescence at 340 nm vs. 380 nm in the presence of saturating Ca2+ was obtained by bathing the cells in isotonic solution with 1.3 mM Ca2+ and 5 µM ionomycin. Calibration was performed separately for each experiment. Baseline levels from PE cells in the absence of fura 2 were subtracted from records to control for autofluorescence.

Experiments measuring pHi were performed in a similar manner, using 2-5 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes) and 0.002-0.005% Pluronic F-127. The emitted fluorescence (520 nm) from 15-25 confluent cells was sampled at 1 Hz following excitation at 484 and 440 nm, and the ratio was determined with a Delta-Ram system and Felix software. pHi calibration, based on that of Wu et al. (67), was performed by perfusing the cells with 110 mM KCl, 20 mM NaCl, 20 µM nigericin, and 20 mM buffer (pH 6.0 solution buffered with MES, pH 7.0 with PIPES, pH 7.4 with HEPES, and pH 8.0 with TES). In experiments measuring Ca2+ and pH simultaneously, both dyes were loaded together, and the excitation wavelength alternated among 340, 380, 484, and 440 nm with 1-s exposure to each wavelength. Calibration to absolute pH or Ca2+ values was usually not performed when both dyes were present; the appropriate ratios provided indexes of Ca2+ and pH.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of timolol on epithelial cell composition in tissues incubated in the presence or absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution. Timolol was applied at a concentration of 10 µM, within the concentration range likely reached clinically in the aqueous humor. Instillation of 20-50 µl timolol (0.5%) into the rabbit conjunctival sac can be calculated to produce peak concentrations of ~8 µM (62) to 17 µM (51). As considered in the DISCUSSION, Vareilles et al. (62) found that a timolol concentration of 8 µM was required to reduce the IOP of rabbits after water loading. The same concentration we have chosen has also been used in other in vitro studies of timolol's mode of action (39).

In the nominal absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, timolol produced no significant changes in epithelial cell Na, Cl, or K (Fig. 1). In contrast, in ciliary tissue from the same eyes incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2, 10 µM timolol produced significant losses of Cl and K (Fig. 1). A time course was obtained in a separate experiment conducted with HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution. Significant losses of Cl (P < 0.001) and K (P < 0.05) were detected by 10 min.


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Fig. 1.   Effects of timolol (10 µM) on ciliary epithelial Na/P, Cl/P, or K/P ratios in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solutions. In these box plots, the medians are indicated by the central horizontal lines, the lower and upper lines include all data between the 25th and 75th percentiles, and the "whiskers" display the data range between the 10th and 90th percentiles. Circles are individual data points that lie outside of this range. The open and shaded symbols present control and experimental results, respectively. Significant differences from controls: *P < 0.05, **P < 0.01. Data were obtained from experiments using eyes from 2 animals: for each condition 8 sections were analyzed, with 6 nonpigmented ciliary epithelial (NPE) and 6 pigmented ciliary epithelial (PE) cells measured in each section, giving a total of 96 cell measurements for each condition.

Altogether, in our studies we have obtained data from 32 eyes in which tissues were incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with or without timolol for 20-30 min (Table 1). Averaging the results obtained with equal numbers of NPE and PE cells (Table 1), there were highly significant (P < 0.001), comparable losses of Cl [Delta (Cl/P) = -0.059 ± 0.006] and K [Delta (K/P) = -0.064 ± 0.010] content, accompanied by significant water loss {indicated by the reductions in both [(Na + K)/P] and [(Na + K + Cl)/P]}. In contrast, there was no significant fall in unmeasured anion content, monitored by [(Na + K - Cl)/P]. Constancy of unmeasured anion content with a fall in intracellular water suggests that the intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration rose as the tissues lost Cl. The same conclusions were reached by considering the NPE and the PE cells separately (Table 1). As previously noted and discussed (44), the phosphorus-normalized contents of Na, K, and Cl are higher in NPE than in PE cells. Neverthess, the timolol-triggered reductions of Cl were similar in the NPE (~20%) and PE (~17%) cells, reflecting the syncytial nature of the ciliary epithelium under baseline conditions.

Effects of cAMP on epithelial composition in tissues incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution. As a known beta -blocker, timolol might act by reducing cell cAMP levels (Introduction). If so, this blocking action should be circumvented by directly adding a membrane-permeant form of cAMP (dibutyryl cAMP). We have previously observed that the specific choice of membrane-permeant form is not significant (13, 19, 27). Dibutyryl cAMP (1 mM) did have an effect on ion transport by the ciliary epithelium, reducing cell K significantly (Fig. 2). However, the cAMP did not alter cell Cl (Fig. 2). Furthermore, the cyclic nucleotide did not reverse the effects of 10 µM timolol in reducing both Cl and K when both agents were added simultaneously (Fig. 2). Instead, the combination of the two agents appeared to be additive, with the Cl loss slightly greater than for timolol alone, and a loss of K that was twice as great as the loss of Cl.


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Fig. 2.   Effects of timolol (10 µM) and/or cAMP (1 mM) on ciliary epithelial Na/P, Cl/P, or K/P ratios in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. Open symbols, control conditions; hatched symbols, +cAMP; shaded symbols, +timolol; solid symbols, +cAMP and timolol. Significant differences from controls: *P < 0.05, ***P < 0.001. Data were obtained from experiments using eyes from 2 animals as follows: for controls, 8 sections were analyzed, with 6-7 NPE and PE cells measured in each section, giving a total of 98 cell measurements; for timolol, 8 sections were analyzed, with 6 NPE and PE cells measured in each section, giving a total of 96 cell measurements; for cAMP, 8 sections were analyzed, with 6 NPE and PE cells measured in each section, giving a total of 96 cell measurements; for timolol +cAMP, 10 sections were analyzed, with 3-6 NPE and PE cells measured in each section, giving a total of 96 cell measurements.

Effects of acetazolamide and timolol on epithelial composition in tissues incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution. In previous studies, we showed that the carbonic anhydrase inhibitor acetazolamide decreases cell Cl and K (7, 44). We ascribed these effects to inhibition of the rate of cellular production of H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, reducing the rates of Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. In turn, the decreased rate of Na+ entry reduced the rate of Na+ extrusion and K+ uptake by the Na+-K+- ATPase. The overall effect was a decrease in cell Cl and K, with relatively little change in cell Na. Because timolol also decreased cell Cl and K, we compared the effects of acetazolamide and timolol (Fig. 3). In this set of experiments, Cl/P was decreased by 0.5 mM acetazolamide (-0.123 ± 0.012) to a greater extent than it was by 10 µM timolol (-0.045 ± 0.013). However, the two effects were not additive, since the combination of inhibitors caused no greater reduction in Cl/P (-0.104 ± 0.021) than did acetazolamide alone.


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Fig. 3.   Effects of timolol (10 µM) and/or acetazolamide (0.5 mM) on ciliary epithelial Na/P, Cl/P, or K/P ratios in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. Open symbols, control conditions; shaded symbols, +timolol; hatched symbols, +acetazolamide; and solid symbols, +acetazolamde and timolol. Significant differences from controls: *P < 0.05, **P < 0.01, ***P < 0.001. Data were obtained from experiments using eyes from 2 animals as follows: for controls, 5 sections were analyzed, with 3-6 NPE and PE cells measured in each section, giving a total of 54 cell measurements; for timolol, 7 sections were analyzed, with 6-7 NPE and PE cells measured in each section, giving a total of 86 cell measurements; for acetazolamide, 4 sections were analyzed, with 6 NPE and PE cells measured in each section, giving a total of 48 cell measurements; for timolol +acetazolamide, 6 sections were analyzed, with 6-7 NPE and PE cells measured in each section, giving a total of 74 cell measurements.

The nonadditivity of timolol and acetazolamide was surprising in view of clinical experience. However, Berson and Epstein (4) have pointed out that the inflow inhibitions of the two drugs are less than additive. Direct addition of the drugs to the well-stirred extracellular fluid may permit more complete inhibition of the transport processes in vitro, precluding additivity of the two effects.

Effects of dimethylamiloride on epithelial composition in tissues incubated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 solution. Because it was possible that timolol was affecting some aspect of Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, we studied the effects of a known inhibitor of Na+/H+ exchange, dimethylamiloride (Fig. 4). Its effects at 50 µM were similar to those of 10 µM timolol, with significant reductions in cell Cl and K. 


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Fig. 4.   Effects of dimethylamiloride (50 µM) on ciliary epithelial Na/P, Cl/P, or K/P ratios in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. Open and hatched symbols present control and experimental results, respectively. Significant differences from controls: **P < 0.01, ***P < 0.001. Data were obtained from experiments using eyes from 2 animals as follows: for each condition 8 sections were analyzed, with 6-7 NPE and PE cells measured in each section, giving a total of 98 cell measurements for each condition.

Effects of timolol, levobunolol, and propranolol on RVI of PE cells. If timolol acts at a single site both to inhibit aqueous humor secretion and reduce epithelial cell Cl content, that site must be the PE cells at the stromal surface (see DISCUSSION). To test this hypothesis more directly, we monitored the RVI as an index of solute and water uptake by the PE cells (see MATERIALS AND METHODS). Application of 50% hypotonicity followed by restoration of isotonicity produced an RVI in control preparations (Fig. 5, A and C, and Fig. 6, A-C). Timolol blocked the RVI at 10 µM concentration (Fig. 5A). Incomplete inhibition was also observed at a 100-fold lower concentration (100 nM) in the experiments of Fig. 6A. As in the case of the timolol-triggered reduction in epithelial Cl content, cAMP did not protect against the timolol-triggered inhibition (Fig. 6B). The effect of timolol was dependent on extracellular Cl- concentration. In the absence of external Cl-, the cells displayed neither an RVI nor a response to timolol (Fig. 5B).


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Fig. 5.   Effects of timolol and levobunolol on cell volume of bovine PE cells. A: timolol (10 µM) blocked the regulatory volume increase of bovine PE cells. Data points are means of 10 control and experimental aliquots and have been normalized to their volumes (vc, in %) at t = 28 min. The control trajectories from cells suspended in Cl--containing solution in Figs. 5 and 6 have been fit to the monoexponential: vc = a · exp(t/tau ). In A, a = 2.2 ± 0.4% and tau  = 13.5 ± 4.2 min. In Fig. 5, A and C, and Fig. 6, A-C, the sets of experimental and control data points are significantly different (P < 0.01 by the F distribution). B: in the absence of extracellular Cl-, the regulatory volume increase (RVI) was abolished and timolol exerted no effect on cell volume (N = 5). C: levobunolol (10 µM) mimicked the effects of 10 µM timolol, blocking the RVI. Fit of the control data generated values of a = 3.5 ± 0.3% and tau  = 7.7 ± 1.9 min (N = 6).



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Fig. 6.   Dependence of timolol effect on concentration and interactions with cAMP and Ca2+. A: timolol partially inhibited the RVI of bovine PE cells at 100 nM. The control data have been obtained from 12 aliquots and fit with a = 9.3 ± 0.4% and tau  = 19.6 ± 1.2 min. The means at timolol concentrations of 100 nM and 10 µM were obtained from 6 aliquots each. B: cAMP (500 µM) did not prevent the inhibition of RVI triggered by exposing bovine PE cells to 10 µM timolol. Duplicate controls (N = 10) were studied in parallel with the timolol (N = 5) and cAMP + timolol (N = 5) experimental aliquots. The control data were fit with a = 7.1 ± 0.7% and tau  = 17.2 ± 2.7 min. C: the calcium ionophore ionomycin (2 µM, N = 4) blocked the RVI displayed by aliquots (N = 6) of control bovine PE cells. The control data were fit with a = 8.4 ± 1.3% and tau  = 22.5 ± 5.2 min.

Levobunolol, another nonselective beta -adrenergic receptor blocker widely used in treating glaucoma, mimicked the effects of timolol in blocking the RVI at the same 10 µM concentration (Fig. 5C). Interestingly, a third nonselective beta -blocker (propranolol), which is not commonly used for lowering IOP, was found to have no effect on the RVI at either 100 nM (N = 3) or 10 µM (N = 3, data not shown).

Effects of timolol on pHi. The preceding results suggest that timolol might reduce ciliary epithelial secretion by inhibiting either the Na+/H+ or Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>antiport, thereby interfering with the paired uptake of Na+ and Cl- in exchange for H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. To determine which of the two paired exchangers might be the primary target of timolol's action, we monitored changes in the pHi of the bovine PE cells directly with the fluorescent pH indicator BCECF. The baseline value was 7.38 ± 0.02 (N = 14). Timolol clearly and repeatedly raised pHi, and the elevation was frequently reversible on removal of timolol. Calibration showed that 10 µM timolol elevated pHi by 0.027 ± 0.007 units (N = 7, P < 0.01). The probability of elevation was concentration dependent, with 10 µM timolol raising pH in 83% of trials, whereas 100 nM produced an alkalinization only 25% of the time.

Effects of timolol on intracellular Ca2+. Timolol's actions on ion concentration and cell volume appeared unrelated to the drug's inhibition of cAMP production. However, complex interations have been noted among timolol, cAMP, and Ca2+ (33, 45, 52, 59, 70-72), so we also measured Ca2+ directly with the fluorescent indicator fura 2. Timolol clearly and repeatedly raised intracellular Ca2+. Levels rose steadily, showing little or no reduction in the continued presence of timolol for as long as 5 min (Fig. 7A). Levels of intracellular Ca2+ generally returned to baseline once the timolol was washed off. The success rate was concentration dependent, with 10 µM timolol elevating intracellular Ca2+ in 83% of attempts and 100 nM timolol elevating Ca2+ only 54% of the time. In a portion of the experiments the absolute levels of intracellular Ca2+ were calibrated from the ratio of light excited at 340 nm to that at 380 nm. At 10 µM, timolol increased intracellular Ca2+ by 57 ± 20 nM from a baseline of 77 ± 7 nM, raising intracellular Ca2+ levels to between 91 and 190 nM (N = 14, P < 0.05). The magnitude of the response was dependent on concentration, as 100 nM raised intracellular Ca2+ by 13 ± 4 nM (N = 4) and 1 nM produced only a 4 ± 1 nM rise (N = 4).


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Fig. 7.   Elevation of pH and Ca2+ by timolol. A: PE cells loaded with fura 2 show that the concentration of Ca2+ was rapidly and repeatedly increased by 10 µM timolol. Timolol produced similar elevations in a total of 32 trials. B: cells loaded with both the Ca2+-sensitive dye fura 2 and the pH-sensitive dye BCECF displayed synchronous and concentration-dependent increases in Ca2+ concentration and pH following perfusion with timolol. The presence of both dyes precluded calibration to absolute values so the appropriate ratios are shown as indexes of ion concentrations. Vertical lines indicate durations of exposure to timolol. T = 10 µM timolol, t = 100 nM timolol. pHi, intracellular pH.

Simultaneous effects of timolol on pH and Ca2+. Given the ability of timolol to elevate both pH and Ca2+, experiments were designed to measure both parameters simultaneously in an attempt to determine any temporal linkage of the two effects. As indicated by Fig. 7B, cells loaded with both fura 2 and BCECF showed that Ca2+ and pH usually rose together. Positive correlation, defined as a change, or lack thereof, in both Ca2+ and pH was seen in 17/22 experiments, indicating that the effects on the two parameters were linked. It should be noted that the lack of perfect correlation supports the independence of either measurement. Although calibration to absolute pH or Ca2+ levels was not always performed when both dyes were present, the ratio of light excited at 340 nm to that at 380 nm was used as an index of changes in free Ca2+ concentration, and the ratio of light excited at 480 nm to that at 440 nm was used to monitor changes in pH. The ratio for Ca2+ was elevated by 0.012 by 1 nM timolol (N = 1), 0.021 ± 0.006 by 100 nM (N = 6), and 0.039 ± 0.009 by 10 µM (N = 14). The ratio for pH was elevated by 0.015 (N = 2) by 1 nM timolol, 0.032 ± 0.012 by 100 nM (N = 5), and 0.067 ± 0.02 by 10 µM (N = 12). These data establish that timolol raises both the intracellular Ca2+ and pH of PE cells.

Effects of levobunolol on pH and Ca2+. The beta -blocker levobunolol also elevated intracellular Ca2+ and pH levels, although the effects were smaller. At 100 nM, levobunolol had no detectable effect on either parameter, but pHi was elevated 0.012 ± 0.003 pH units by 10 µM levobunolol (N = 8, P < 0.01), while the ratio of light excited by irradiating fura 2 at 340/380 rose 0.016 ± 0.003 (N = 13, P < 0.001).

Effects of ionomycin on RVI of PE cells. Because timolol both increased intracellular Ca2+ concentration and inhibited the RVI, we wondered whether increasing Ca2+ concentration by another means would also inhibit RVI. Application of the calcium ionophore ionomycin (2 µM) indeed blocked the RVI (Fig. 6C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The central observations of the present study are that both timolol and a membrane-permeant form of cAMP exert effects on the intracellular electrolyte composition of the NPE and PE ciliary epithelial cells, but these effects are not opposite to one another. Adding cAMP not only did not reverse the effects of timolol, but produced additive actions. Likewise, cAMP did not reverse the inhibition of the RVI produced by applying timolol to isolated bovine PE cells. Thus neither the reduction in epithelial Cl content nor the inhibition of solute uptake by PE cells produced by timolol can be ascribed to a reduction in cAMP production.

Dibutyryl cAMP triggered cell K loss in a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 bath, but a reduction of cell Cl was not detected (Fig. 3). The K loss may reflect activation of basolateral K+ channels, as seen in other secretory epithelia (15, 30). Because macroscopic electroneutrality must be preserved, this cAMP-triggered K loss may have been accompanied by a net loss of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, as found in other epithelial cells (3, 10, 57). It is known that cAMP exerts multiple effects specifically on PE cells, including direct activation of Cl- channels leading to Cl- release (27) and stimulation of Na+-K+-2Cl- cotransport through activation of protein kinase A (54), which can lead to either uptake or release of Cl- depending on the ambient thermodynamic driving forces (44). Thus the net change in Cl content produced by cAMP depends on a complex interaction of multiple effects, possibly accounting for certain apparently paradoxical results in the literature. For example, beta -adrenergic agonists such as isoproterenol, which stimulate cAMP production, have been reported to increase aqueous humor inflow (9). In contrast, forskolin, which also stimulates cAMP formation, has been found to reduce inflow (11, 40), and isoproterenol itself has also been reported to reduce IOP in water-loaded rabbits (62). Whatever the nature of the complex interactions, the effects of timolol and cAMP on the intact ciliary epithelium were additive in the present study. This finding demonstrates that timolol can act independently of cAMP-mediated pathways.

Site of action. Timolol inhibits secretion of aqueous humor, the major anionic component of which is Cl-. Timolol could inhibit secretion by blocking Cl- uptake by the PE cells at the stromal surface, thereby reducing intraepithelial Cl content. Alternatively, timolol could reduce Cl- release by the NPE cells at the aqueous surface, thereby increasing Cl content. The microanalyses of intact rabbit ciliary epithelium demonstrated a fall in intracellular Cl content, strongly suggesting that the dominant action of timolol is at the stromal surface. This deduction was tested by monitoring fluid uptake during the course of the RVI by isolated cultured bovine PE cells. The RVI reflects cellular uptake of ions to replace those lost during hypotonic exposure. In these bovine cells, RVI depends on solution HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 and Na+/H+ exchange activity (22), supporting the idea that normally much of the NaCl uptake step at the stromal surface is mediated by paired Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+/H+ antiports (37, 44, 65). Timolol blocked the RVI at the same concentration (10 µM) used in the microprobe studies and even exerted a partial block at a 100-fold lower concentration (100 nM). In the absence of Cl- in the bath, the RVI was abolished and timolol had no further effect. These data support the idea that timolol acts at the stromal side to block NaCl uptake by the paired antiports, recently identified to be NHE-1 Na+/H+ and AE2 Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers (22). Further support is provided by the observations that reducing delivery of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and H+ to the paired antiports (by inhibiting carbonic anhydrase) and directly inhibiting the Na+/H+ antiport (with dimethylamiloride) mimicked the changes in intracellular composition caused by timolol.

In principle, the roles of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+/H+ antiports could be very different in the rabbit tissue that we studied by microprobe analysis and the bovine preparation that we studied volumetrically and fluorometrically. However, recent reports indicate that these antiports likely play an important role in NaCl uptake from the stroma by PE cells from both species (22, 44, 60). The agreement of the results obtained with tissues from the two species and using different experimental techniques further supports the conclusion that timolol likely inhibits stromal uptake of NaCl by PE cells.

We have tested the effects of two additional nonspecific beta -blockers (levobunolol and propranolol) on the RVI of bovine PE cells. At 10 µM concentration, levobunolol mimicked the inhibitory effect of the same concentration of timolol. In contrast, 10 µM propranolol was ineffective. These observations conform to clinical experience. Both levobunolol and timolol have been widely used in the topical treatment of glaucoma, but propranolol has not. It should be noted that propranolol and timolol have been reported to have a number of very different effects on different cell preparations. For example, 32Pi incorporation into phosphatidylinositol 4,5-bisphosphate of cat iris and ciliary process is increased by propranolol but is decreased by timolol (70). In part, the differences reported may reflect the sevenfold higher potency of timolol over propranolol for beta -adrenergic receptors in the rabbit iris-ciliary body (2). However, given the different structures of the beta -blockers, it is also possible that actions in addition to binding to beta -adrenergic receptors may well be involved.

Transport protein target. To test whether the dominant inhibitory effect of timolol is on Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or Na+/H+ antiports, we monitored pHi of the bovine PE cells. Timolol (and to a lesser extent, levobunolol) increased pH, suggesting that the primary effect is inhibition of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, thereby slowing extrusion of base out of the cell.

Second messenger. The current data argue against the possibility that timolol exerts its transport effects solely by inhibiting cAMP formation. Given the interactions reported for timolol, cAMP, and Ca2+ (33, 45, 52, 59, 71, 72), we also monitored intracellular Ca2+ activity. Timolol increased Ca2+ activity over a broad range of concentrations (1 nM to 10 µM). We do not know whether the elevated Ca2+ triggers the putative inhibition of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, but the near synchrony of changes in Ca2+ and pH and the block of the RVI by ionomycin do raise this possibility. This suggestion is consistent with the report of Tachado et al. (59), who observed that the beta -adrenergic agonist isoproterenol produced muscle relaxation at an order of magnitude lower concentration than that required to accumulate cAMP or inhibit inositol 1,4,5-trisphosphate formation in the bovine iris sphincter. The two studies raise the possibility that the agonist [isoproterenol (59)] and the antagonist (timolol at clinically relevant concentrations in the present work) act on beta -adrenergic receptors that exert effects independent of changes in intracellular cAMP concentration. Whether or not Ca2+ indeed inhibits the anion exchanger directly and the mechanism by which timolol elevates intracellular Ca2+ activity remain to be determined.


    ACKNOWLEDGEMENTS

We thank Sylvia Zellhuber-McMillan for superb technical support in the X-ray microanalytic studies, and we are grateful to Dr. Miguel Coca-Prados for providing the bovine PE cells.


    FOOTNOTES

This work was supported in part by a Project Grant from the Health Research Council of New Zealand and Lottery Health and by National Eye Institute Research Grants EY-08343 and EY-01583 (for core facilities).

Address for reprint requests and other correspondence: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, Richards Bldg., Philadelphia, PA 19104-6085.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 January 2001; accepted in final form 18 April 2001.


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