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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
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The 
-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
/HCO
electron probe X-ray microanalysis; cell volume; cell pH; cell calcium; chloride/bicarbonate exchanger; sodium/hydrogen exchanger
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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, 
-adrenergic and
cholinergic agonists, prostaglandin analogs, and -adrenergic
blockers (55). The nonselective -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 -adrenergic receptors of the
ciliary processes with high affinity (62). Agonists to all
-adrenergic receptors (1, 2, and
3) stimulate adenylyl cyclase via interaction with Gs to increase cAMP production (34). As a
known -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
-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 -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,
1-adrenergic antagonists are effective in some models of
ocular hypertension (6, 61), although the high density of
-receptors in the ciliary process are predominantly of the
2 subtype. Third, D-timolol may be as
effective as L-timolol (42) in decreasing
aqueous flow, despite stereospecificity of the -adrenergic receptors for the L-isomers (41, 49). Fourth, if
timolol reduces aqueous humor formation by blocking
-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 -adrenergic stimulation, blunting
the effects of exogenous cAMP. However, such constant agonist
stimulation would desensitize the -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 -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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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, 15.0 HEPES, 1.2 Mg2+, 2.5 Ca2+, 1.2 H2PO
. Hypotonic
HCO-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
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 104 Pa (equivalent to
7.5 × 107 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.
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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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 |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of timolol on epithelial cell composition in tissues
incubated in the presence or absence of
HCO
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(Cl/P) = 0.059 ± 0.006] and K [(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 HCOEffects of cAMP on epithelial composition in tissues incubated in
HCO-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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Effects of acetazolamide and timolol on epithelial composition in
tissues incubated in HCO/HCO0.123 ± 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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Effects of dimethylamiloride on epithelial composition in tissues
incubated in HCO/HCO
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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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-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 -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 in
exchange for H+ and HCO
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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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 -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).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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, -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/HCO 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
/HCO-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 -adrenergic receptors in the rabbit
iris-ciliary body (2). However, given the different
structures of the -blockers, it is also possible that actions in
addition to binding to -adrenergic receptors may well be involved.
Transport protein target.
To test whether the dominant inhibitory effect of timolol is on
Cl/HCO/HCO
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-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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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A, a = 2.2 ± 0.4% and 
