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MS 0442 Received 13 December 1999; accepted after revision 18 February 2000.
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ABSTRACT |
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-oestradiol, but not 17
-oestradiol.
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INTRODUCTION |
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The aqueous humour is formed by a bilayered ciliary epithelium (Cole, 1977), whose cells intercommunicate through gap junctions (Spitznas & Reale, 1975; Raviola & Raviola, 1978; Green et al. 1985; Coca-Prados et al. 1992; Edelman et al. 1994; Oh et al. 1994; Schütte & Wolosin, 1996). Secretion is generally thought to reflect a primary transfer of solute, largely NaCl, from the stroma to the aqueous humour, providing an osmotic driving force for the secondary osmotic transfer of water (Cole, 1977), although a more direct coupling between water and solute may also proceed across epithelia (Meinild et al. 1998). The activity of Cl- channels is likely to be a rate-limiting factor in aqueous humour secretion, given the low baseline level of channel activity (Coca-Prados et al. 1995). Release of Cl- by the non-pigmented ciliary epithelial (NPE) cells into the adjacent aqueous humour would enhance secretion, and Cl- release by the pigmented ciliary epithelial (PE) cells into the neighbouring stroma would reduce net secretion (Civan, 1998).
Purines affect both aqueous humour dynamics and the cellular physiology of the ciliary epithelium, although the connection between these processes is unclear. At the in vivo level, A1-adenosine receptor stimulation has been shown to decrease intraocular pressure in rabbits (Crosson, 1995) and cynomologus monkeys (Tian et al. 1997) while A2-adenosine receptor agonists lead to elevated intraocular pressure in rabbits and cats (Crosson & Gray, 1996), but the effects on aqueous humour formation are uncertain. At the isolated cell level, most of the work concerns the NPE cells. In vitro studies have suggested that the NPE cells possess A1-adenosine receptors which lower [cAMP], A2-adenosine receptors which elevate [cAMP] and P2U2 receptors which increase phosphoinositide hydrolysis when stimulated (Wax et al. 1993). Adenosine acts with acetylcholine to elevate [Ca2+]i in the NPE cells (Farahbakhsh & Cilluffo, 1997), while ATP alone elevates [Ca2+]i (Shahidullah & Wilson, 1997). NPE cells have been reported to release ATP to the extracellular surface, where ATP can be metabolized to adenosine by ecto-enzymes (Mitchell et al. 1998). Single cell studies were tied to ionic, and thus aqueous, transport when adenosine agonists were shown to reduce NPE cell volume via Cl- pathways, activate whole cell Cl- channels in these same cells, and increase the short circuit current when applied to the aqueous face of iris ciliary body under conditions which isolated Cl- movement (Carré et al. 1997). The combination of these three techniques suggested that this stimulation of Cl- transport could lead to an increase in aqueous humour production and a recent report suggests this stimulation is mediated primarily by the A3-adenosine receptor (Mitchell et al. 1999).
Considerably less information is available about the effects of purines on the PE cells. The cells have been reported to possess A1, A2 and P2Y2 receptors (Wax et al. 1993) and ATP elevates [Ca2+]i (Shahidullah & Wilson, 1997). In addition, the PE cells store and release ATP, and can degrade it extracellularly (Mitchell et al. 1998). However, the functional implications of these observations on either cellular or whole tissue physiology are unclear. In the present study we have asked how ATP affects the volume of PE cells in order to provide an initial link between purines, PE cells and net aqueous humour formation.
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METHODS |
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Cellular model
The cells studied were an immortalized PE-cell line developed by one of us (M.C.-P.) from a primary culture of bovine pigmented ciliary epithelium grown from bovine eyes obtained from a local abattoir. Cells were grown in Dulbecco's modified Eagle's medium (DMEM, 11965-027, Gibco BRL, Grand Island, NY, USA and 51-43150, JRH Biosciences, Lenexa, KS, USA) with 10 % fetal bovine serum (FBS, A-1115-L, HyClone Laboratories, Inc., Logan, UT, USA) and 50 µg ml-1 gentamicin (15750-011, Gibco BRL), at 37°C in 5 % CO2 (Yantorno et al. 1989). The medium had an osmolality of 328 mosmol kg-1. Cells were passaged every 6-7 days and, after reaching confluence, were suspended in solution for study within 6-10 days after passage.
Volumetric measurements and analysis
After harvesting cells from a single T-75 flask (Corning, NY, USA) by trypsinization (Yantorno et al. 1989), a 0·5 ml aliquot of the cell suspension in DMEM (or in Cl--free medium, where appropriate), was added to 20 ml of each test solution. The standard test solution contained (mM): 110·0 NaCl, 15·0 Hepes, 2·5 CaCl2, 1·2 MgCl2, 4·7 KCl, 1·2 KH2PO4, 30·0 NaHCO3 and 10·0 glucose, with a pH of 7·4 and osmolality of 298-305 mosmol kg-1. The Cl--free solution comprised: 110·0 sodium methanesulfonate, 15·0 Hepes, 2·5 calcium methanesulfonate, 1·2 MgSO4, 4·7 potassium methanesulfonate, 1·2 KH2PO4, 30·0 NaHCO3 and 10·0 glucose, with a pH of 7·4 and osmolality of 294-304 mosmol kg-1. Parallel aliquots of cells were studied on the same day. One aliquot usually served as a control, and the others were exposed to different experimental conditions at the time of suspension. The same amount of solvent vehicle (dimethylformamide, DMSO or ethanol) was always added to the control and experimental aliquots. The sequence of studying the suspensions was varied to preclude systematic time-dependent artifacts (Civan et al. 1994).
Cell volumes of isosmotic suspensions were measured with a Coulter Counter (model ZBI-Channelyzer II), using a 100 µm aperture (Civan et al. 1992). As previously described (Yantorno et al. 1989), the cell volume (vc) of the suspension was taken as the peak of the distribution function. Cell shrinkage was fitted as a function of time (t) to the simple exponential function:
vc = (v0 - v ) × exp(-t/ ) + v,
| (1) |
where v
is the steady-state cell volume, v0 is cell volume at t = 0, and is the time constant of the shrinkage. For purposes of data reduction, the data were normalized to the first time point, taken to be 100 % isotonic volume. The baseline isotonic value was 2488 ± 203 fl (mean ± S.E.M., N = 15). Fits were obtained by non-linear least-squares regression analysis, permitting both v and to be variables (Carré et al. 1997).
Measurements of intracellular Ca2+
Bovine PE cells grown on coverslips for 2-4 days were loaded with 5 µM fura-2 AM for 30-45 min at room temperature, and 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 lens. The emitted fluorescence (510 nm) from 10-12 confluent cells was sampled at 1 Hz following excitation at 340 nm and 380 nm, and the ratio determined with a Delta-Ram system and Felix software (Photon Technology International Inc., Princeton, NJ, USA). The ratio of light excited at 340 nm to that at 380 nm (R) was converted into Ca2+ concentration using the method of Grynkiewicz et al. (1985). An in situ Kd value for fura-2 of 350 nM was used (Negulescu & Machen, 1990). Rmin was obtained by bathing cells in a Ca2+-free isotonic solution of pH 8·0 containing 10 mM EGTA and 5 µM ionomycin. Rmax 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 were performed predominantly at room temperature, but several trials were performed at 37°C using a temperature control unit from Warner Instrument Corp. (Hamden, CT, USA). Cells were perfused with an isotonic solution containing (mM): 105 NaCl, 6 Hepes (acid), 4 Hepes (Na+), 1·3 CaCl2, 1 MgCl2, 4 KCl, 5 glucose and 90 mannitol, with a pH of 7·4 and osmolality of 317 mosmol kg-1. Tamoxifen was stored as a 10 mM stock in ethanol for 2 days. When comparing the effects of ATP and of ATP + tamoxifen, 0·1 % ethanol was also added to the solutions containing ATP alone.
Statistical analysis
Values are presented as the means ± 1 S.E.M. The number of experiments is indicated by N. The null hypothesis, that the experimental and baseline measurements shared the same mean and distribution, was tested either with the upper significance limits of the F distribution or by Student's two-tailed t test.
Chemicals
All chemicals were reagent grade. Gramicidin D, tamoxifen, adenosine, ATP, 17- and 17-oestradiol, DiC8, carbachol, atropine, histamine and trifluoperazine were purchased from Sigma Chemical Co., 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS) and fura-2 AM from Molecular Probes, Inc. and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and staurosporine from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA).
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RESULTS |
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Interactive effects of tamoxifen and ATP on cell volume
When ATP and tamoxifen were added simultaneously in isotonic solution, the volume of the PE cells was substantially reduced (Fig. 1). This shrinkage was usually evident within 2 min after introduction of tamoxifen and ATP, and cell volume continued to fall over the subsequent 30 min (Figs 1, 3, 6, 8 and 9).
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Neither ATP (10 mM) nor tamoxifen (6 µM) separately produced substantial shrinkage, whereas even 10 µM ATP added together with 6 µM tamoxifen substantially enhanced the baseline shrinkage. The values of the fits were: v | ||
The ability of combined ATP and tamoxifen to reduce cell volume is particularly striking as neither substance showed a consistent effect on its own. In nine experiments, the volume of cells in tamoxifen was not significantly different from controls (difference at t = 30 min, 1·2 ± 1·3 %). In most experiments, ATP alone had no effect. As shown in Fig. 1, even increasing the concentration of ATP to 10 mM had no effect on cell volume. In 
15 % of experiments, ATP alone produced a small reduction in cell volume, but even here the addition of tamoxifen substantially increased the degree of cell shrinkage. This occasional ability to respond to ATP is likely to reflect a change in the entire cell population rather than a heterogeneity within the population as each volume measurement represents an average of 45000 cells. We were unable to identify the specific parameter that led to this occasional change. However, the reduction in cell volumes produced by ATP and tamoxifen together was always more than would be expected from the sum of their individual responses regardless of the actions of ATP alone.
In order to better define the nature of the ATP and tamoxifen interaction, concentration-response curves were constructed (Fig. 2). At a constant tamoxifen concentration of 6 µM increasing ATP concentration led to increasingly larger reductions in cell volume (Fig. 2A), with an apparent Kd of 4·4 µM. In this series, as in most experiments, adding ATP in the absence of tamoxifen had no effect on cell volume. In a parallel set of experiments, increasing the tamoxifen concentration while the level of ATP was held constant at 100 µM produced progressively larger reductions in cell volume, while tamoxifen in the absence of ATP had no effect. The apparent Kd for tamoxifen in the presence of 100 µM ATP was 3·8 µM (Fig. 2B). Thus, the ability to reduce cell volume seems to depend upon the simultaneous presence of both ATP and tamoxifen. The effects are clearly more than additive, suggesting that the two substances act synergistically to shrink cells (Chou & Talalay, 1984).
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A, in the presence of 6 µM tamoxifen, increasing concentrations of ATP led to a reduced steady-state volume, as measured at t = 30 min ( | ||
The reduction in volume while the cells were suspended in isotonic solution suggests that together ATP and tamoxifen trigger the activation of a conductance which allows the efflux of ions, followed passively by the efflux of water. Several observations suggest that the ATP, tamoxifen-activated shrinkage involved Cl- release. First, the synergism occurred even when gramicidin was present to provide a constant pathway for K+ release (Civan et al. 1994) (Fig. 3A), suggesting that the reduction in volume prompted by ATP and tamoxifen was not due to the activation of K+ efflux, but instead involved the activation of an anionic conductance. Second, removal of Cl- abolished the synergistic response (Fig. 3A). The cells were preincubated in the Cl--free solution for 50-100 min before experiments began to provide sufficient time for intracellular Cl- to equilibrate with the extracellular solution. Third, the Cl- channel blockers NPPB (100 µM) (Wangemann et al. 1986) and DIDS (500 µM) (Cabantchik & Greger, 1992) both inhibited the volume reduction (Fig. 3B).
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Gramicidin D (5 µM) was present in all suspensions to provide an exit pathway for K+. A, the simultaneous application of 6 µM tamoxifen and 10 µM ATP produced isosmotic shrinkage in the presence of Cl- to a steady-state value (v | ||
Previous studies have found that adenosine alone caused NPE cells to shrink by activating a Cl- conductance (Carré et al. 1997). As ATP can be metabolized into adenosine, the effect of adenosine on the volume of PE cells was tested. No change in PE cell volume was produced by adenosine, either alone or in combination with tamoxifen (Fig. 4). This suggested that the PE and NPE cell types responded differently to the purines ATP and adenosine. This is supported by the observation that ATP did not affect the volume in NPE cells (Carré et al. 1997).
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In contrast to the effects observed with ATP, no synergism was noted between tamoxifen (6 µM) and adenosine (10 µM) (N = 4). | ||
The shrinkage of PE cells produced by ATP and tamoxifen also suggested that PE and NPE cells respond differently to tamoxifen. Tamoxifen can inhibit swelling-activated Cl- channels in many cells (Valverde et al. 1993; Zhang et al. 1994; Nilius et al. 1994), including bovine NPE cells (Wu et al. 1996), and had no effect on swelling-activated Cl- channels in PE cells (Mitchell et al. 1997b). Therefore, we re-examined the effect of tamoxifen on the swelling-activated Cl- channels of the immortalized human NPE cells we have previously characterized (Yantorno et al. 1989; Fig. 5). After hypotonic swelling, the cell volume spontaneously fell (the regulatory volume decrease, RVD), reflecting the release of KCl and secondarily water (Civan et al. 1994). Addition of tamoxifen 10 min later, after the conclusion of the RVD, did not affect cell volume, but addition 5 min after hypotonic suspension reduced the magnitude of the RVD. Inclusion of tamoxifen at the time of the initial hypotonic suspension completely abolished the RVD, consistent with the earlier report that tamoxifen blocks swelling-activated Cl- channels of NPE cells (Wu et al. 1996). Tamoxifen also markedly slowed the rate of hypotonic swelling (Fig. 5), raising the possibility that it also blocks the aquaporin-1 (AQP1) water channels of the NPE cells (Stamer et al. 1994; Lee et al. 1998). This interesting line of investigation was beyond the scope of the present study and was not pursued further at this time.
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Gramicidin D (5 µM) was present in all suspensions to provide an exit pathway for K+. In the absence of tamoxifen, the NPE cells displayed a regulatory volume response to hypotonic swelling with a half-time of ~5 min (N = 5). Adding tamoxifen at the conclusion of the RVD (t = 10 min) had no effect on cell volume, adding tamoxifen at t = 5 min partially inhibited the steady-state response, and adding tamoxifen at the same time as applying the hypotonic stress both slowed the rate of initial slowing and abolished the RVD. | ||
Mode of action of tamoxifen
In addition to its actions on nuclear oestrogen receptors (Klinge et al. 1992) and swelling-activated Cl- channels (Valverde et al. 1993; Zhang et al. 1994; Nilius et al. 1994; Wu et al. 1996), tamoxifen has been reported to produce multiple other effects. Experiments were conducted in order to address the possibility that the synergistic interaction between ATP and tamoxifen which led to cell shrinkage could have resulted from one of the following roles of tamoxifen: interaction with histamine and muscarinic receptors, antagonism of calcium-calmodulin, inhibition of protein kinase C, and antagonism of plasma- or nuclear membrane oestrogen receptors.
Interaction with histamine and muscarinic receptors
Tamoxifen is known to interact with histamine (Brandes & Bogdanovic, 1986) and muscarinic receptors (Ben-Baruch et al. 1981) in other preparations. The results of Fig. 6A indicated that 10 µM histamine did not enhance the volumetric response to 100 µM ATP (N = 4). The non-metabolizable muscarinic agonist carbachol did trigger a prompt shrinkage in the presence of 100 µM ATP (Fig. 6A, N = 4). However, carbachol triggered approximately the same response whether or not ATP was present, and 10 µM atropine abolished that response (Fig. 6B, N = 3). In contrast, tamoxifen had little effect in the absence of ATP (Figs 1 and 4), and 10 µM atropine did not alter the response to the combined presence of tamoxifen and ATP (Fig. 6C, N = 4). The actions of carbachol may be mediated by K+ channels, as previously reported in fresh bovine PE cells (Stelling & Jacob, 1996). The data of Fig. 6 indicate that the volumetric actions of tamoxifen cannot be mediated by either histamine or muscarinic receptors.
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A, in the presence of 100 µM ATP, either 10 µM carbachol or 10 µM tamoxifen, but not 10 µM histamine, enhanced shrinkage (N = 4). The values of the fits were: v | ||
Antagonism of calcium-calmodulin
Tamoxifen can inhibit calcium-calmodulin at the same concentration (10 µM) customarily used to block Cl- channels in NPE cells (Lam, 1984; Wu et al. 1996). In PE cells, trifluoperazine triggered a partial shrinkage of the bovine PE cells in the presence of ATP, but this effect was not synergistic as similar effects were observed in the absence of ATP (Fig. 7).
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Lack of synergism with trifluoperazine (10 µM) and ATP (100 µM) on cell volume (N = 5). The shrinkage triggered by trifluoperazine was the same whether or not ATP was present (P
> 0·05). The values of the fits were: v | ||
Protein kinase C inhibition
Tamoxifen can also inhibit protein kinase C (PKC), with a Ki of 5-100 µM depending on the assay system (O'Brien et al. 1985). However, the effects of tamoxifen in the presence of ATP were not mimicked by inhibiting PKC activity with staurosporine (Fig. 8). Activating PKC with DiC8 in the presence of 100 µM ATP also had no significant effect on cell volume. We conclude that the synergistic effect of tamoxifen cannot be mediated by its inhibition of baseline PKC activity.
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Interactions of 250 µM DiC8, 0·3 µM staurosporine and 10 µM tamoxifen with 100 µM ATP on cell volume (N = 4). Only the aliquots exposed to tamoxifen and ATP displayed shrinkage (v | ||
Antagonism of oestrogen receptors
Tamoxifen is best known as a type II antioestrogen, and shows mixed oestrogenic/antioestrogenic activity (MacGregor & Jordan, 1998). This dual action suggests that the effect of tamoxifen could be either mimicked by or antagonized by oestrogen. In the absence of ATP, 17- and 17-oestradiol (the inactive and active forms, respectively) had small effects (Fig. 9A). In the presence of ATP, the effect of 17-oestradiol was also negligible (Fig. 9B). However, 17-oestradiol seemed to block the effect of tamoxifen when added in the presence of ATP (Fig. 9C). This blockage was specific, for 17-oestradiol had little effect on cell volume.
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A, effect of oestradiols on cell volume. At the same 100 nM concentration, the active (17 | ||
Potential role of Ca2+
NPE cells show a synergistic elevation in free intracellular Ca2+ concentration ([Ca2+]i) upon simultaneous presentation of certain drug pairs, and this synergism may involve the activation of the G-protein Gi (Farahbakhsh & Cilluffo, 1997). We asked whether the signalling cascade for the synergistic shrinkage produced by tamoxifen and ATP could reflect a synergistic change in [Ca2+]i. Tamoxifen (10 µM) itself triggered no significant change (
) in [Ca2+]i ( = 7 ± 6 nM, N = 4, data not shown). Although [Ca2+]i increased in response to both ATP and ATP + tamoxifen, the comparison of the response was complicated by the attenuation of the Ca2+ spike with repeated exposure, and the variation between preparations. A 3 min application of either 100 µM ATP or 100 µM ATP + 10 µM tamoxifen usually produced an elevation in [Ca2+], but it was difficult to elicit a response of similar magnitude to a second application 5 min later. Elevating the temperature to 37°C did not eliminate the attenuation.
Nevertheless, it did prove possible to compare the magnitudes of successive Ca2+ responses when each drug application was limited to periods of 20 s (Fig. 10). Experiments were performed by alternating 20 s exposures to 100 µM ATP + 10 µM tamoxifen with 20 s exposures to 100 µM ATP alone (including 0·1 % ethanol as a vehicle control for the tamoxifen). Cells were washed in isotonic solution for 5 min between drug applications, and four or five applications were possible per trial (Fig. 10A). The order of drug application shown in Fig. 10A, beginning first with 100 µM ATP + 10 µM tamoxifen, is termed the T series. A parallel set of experiments termed the A series was performed which began with the application of ATP alone followed 5 min later by ATP + tamoxifen.
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A, ATP (100 µM) or 100 µM ATP + tamoxifen (TMX) was applied for 20 s as indicated by the horizontal bars. The drugs were washed off for 5 min between applications. Although the response attenuated, the presence of tamoxifen did not alter the magnitude of the response. Tamoxifen alone had no significant effect on [Ca2+]i. B, mean results from seven experiments in which 20 s applications of 100 µM ATP were alternated with 20 s applications of 100 µM ATP + 10 µM TMX. In order to adjust for the attenuation, two sets of experiments were performed. In the first set illustrated in A, the order of drugs was (1) ATP + TMX, (2) ATP, (3) ATP + TMX and (4) ATP. In the second type of experiment the order was inverted. The magnitude of the Ca2+ response when the first application was ATP alone (A1) was compared with experiments in which the first application was ATP + TMX (T1). Trials in which the second application was with ATP (A2) were compared with trials in which the second application was ATP + TMX (T2), and likewise for the third and fourth applications. There was no significant difference between any of the four pairs (P < 0·1, N = 3-4). | ||
To check for synergism while compensating for the attenuation, the responses to each application for those experiments where ATP was added first (A series) were compared with those where ATP + tamoxifen was added first (T series). Comparing the responses to successive applications of drugs, it is clear that there was no significant difference between the two series, whether ATP alone or ATP + tamoxifen was added at a given point in time (Fig. 10B). The presence of tamoxifen did not affect the size of the Ca2+ response to ATP regardless of whether it was included in the first, second, third or fourth application (P > 0·05 for applications 1-4, N = 3-4). We conclude that ATP and tamoxifen did not produce a synergistic elevation in the level of intracellular Ca2+. These results also suggest that the presence of tamoxifen does not affect the attenuation of the Ca2+ response to ATP. This attenuation has been reported previously in ciliary epithelial cells, and it has been suggested that the attenuation is mediated by the inhibition of IP3 production by increasingly elevated levels of PKC (Shahidullah & Wilson, 1997). The inability of tamoxifen to modify the rate of attenuation supports our interpretation of the data of Fig. 8, that tamoxifen does not act by modifying PKC in these cells.
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DISCUSSION |
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Combined actions of ATP and tamoxifen
The present results demonstrate that tamoxifen and ATP interact to produce a marked reduction in cell volume of cultured bovine pigmented ciliary epithelial cells. The effect was dependent upon the simultaneous presence of both ATP and tamoxifen, as tamoxifen alone did not have any effect, while ATP rarely affected cell volume; even when it did trigger shrinkage, the response was substantially enhanced by the inclusion of tamoxifen. The synergism was detected at ATP and tamoxifen concentrations likely to be physiologically and clinically relevant. In the presence of tamoxifen, an approximately half-maximal response was elicited by 4·4 µM ATP (Fig. 2A), a concentration probably reached physiologically by ATP release into the constrained space between the PE cells and the underlying basement membrane (Mitchell et al. 1998). Tamoxifen is used clinically as an antioestrogen (Klinge et al. 1992), and the concentrations applied here (6-10 µM) also appear to be clinically relevant (Stuart et al. 1992).
Mode of tamoxifen action
The precise signalling pathway involved in the modulating action of tamoxifen cannot as yet be identified. In addition to binding to nuclear oestrogen receptors (Klinge et al. 1992), and blocking plasma-membrane swelling-activated Cl- channels (Valverde et al. 1993; Zhang et al. 1994; Nilius et al. 1994; Wu et al. 1996), tamoxifen has been observed to affect: histamine receptors (Brandes & Boddanovic, 1986), muscarinic receptors (Ben-Baruch et al. 1981), activation by calcium-calmodulin (Lam, 1984), PKC activity (O'Brien et al. 1985), the Cl- permeability of lipid membranes (Chen et al. 1999), oestrogen receptors (Hardy & Valverde, 1994), and the fluidity of lipid membranes (Wiseman et al. 1993). The current data indicate that the first four of these seven effects play no role in the synergism between tamoxifen and ATP. (1) Histamine does not alter the response of cell volume to ATP (Fig. 6A), so histamine receptors are irrelevant in the present context. (2) Although carbachol itself shrinks cell volume, the response is not synergistic with ATP, and atropine does not affect the tamoxifen-ATP synergistic effect (Fig. 6). Thus, muscarinic receptors are not involved. (3) PKC activity cannot be playing a major role since both activation and inhibition of enzymatic activity produced similar small reductions in volume, one to two orders of magnitude smaller than that triggered by tamoxifen and ATP together (Fig. 8). At the same concentrations used here, the PKC activator DiC8 and the non-selective PKC inhibitor staurosporine exert large and opposing actions on swelling-activated Cl- channels of non-pigmented ciliary epithelial cells (Civan et al. 1994). (4) Calcium-calmodulin antagonism is unlikely to mediate the synergistic effect since the shrinkage produced by another such antagonist (trifluoperazine) was independent of the presence of ATP (Fig. 7).
Tamoxifen may have been acting in any of at least three ways. It was recently suggested that protonated tamoxifen can permeate the lipid bilayer while bringing with it a Cl- ion (Chen et al. 1999). In this model, tamoxifen acts in effect as a Cl- carrier. In the PE cells, the cell shrinkage does seem to involve Cl-, although the simplest explanation for the block by both NPPB and DIDS is that Cl- is leaving the cells via a Cl- channel. However, both NPPB and DIDS are non-specific at higher concentrations, and thus the possibility of a non-channel conduit cannot be ruled out. In addition to this specific interaction with the lipid membrane, it has previously been suggested that tamoxifen's lipophilic nature might somehow underlie its ability to modify cationic conductances (Hardy et al. 1998), perhaps acting, as some gap junction inhibitors do, to change membrane fluidity. However, the ability of 17-oestradiol to block the actions of tamoxifen, while 17-oestradiol was ineffective suggests that the action of tamoxifen is specific and involves oestrogen receptors. Tamoxifen, and its analogue toremifene activate a large conductance Cl- channel in NIH3T3 fibroblasts by stimulating plasma-membrane receptors (Hardy & Valverde, 1994). The Cl- channel in NIH3T3 cells was activated by 15 µM tamoxifen and activation was prevented by 17-oestradiol. Bovine PE cells are known to possess a maxi-Cl- channel with voltage-dependent kinetics and conductance very close to those activated by toremifene in NIH3T3 channels (Mitchell et al. 1997a). Together, these similarities suggest that tamoxifen could stimulate a plasma membrane receptor to activate the maxi-Cl- channel in PE cells. Indeed, recent immunomicroscopic and functional studies have demonstrated a membrane version of the nuclear oestrogen receptor-alpha (Watson et al. 1999; Brubaker & Gay, 1999). This is consistent with the onset of cell shrinkage within 2 min; the majority of oestrogenic effects involve transcriptional changes and require a substantially longer time to act (Ediger et al. 1999). It is worth noting that in NIH3T3 cells, tamoxifen acted alone. The requirement for both ATP and tamoxifen in the PE cell response indicates additional mechanisms may be involved here.
The major observations of the present work are that cells derived from the pigmented ciliary epithelial cell layer can respond to extracellular ATP by releasing Cl-, and that this release is strongly modulated by tamoxifen. The relevance of these observations from cultured cells to aqueous humour secretion in vivo is unclear. ATP is stored in both the intact ciliary epithelium and cultured PE and NPE cells and is released in response to cell swelling or elevations in [Ca2+]i (Mitchell et al. 1998). However, whether an endogeneous molecule comparable to tamoxifen actually regulates purinergic control of aqueous humour formation remains to be determined.
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Supported in part by research grants EY-10691, EY-12213, and EY08343, EY01583 (for core facilities) and a respiratory training grant for C.H.M. (HL-07027), all from the NIH.
Corresponding author
M. M. Civan: Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Email: civan{at}mail.med.upenn.edu
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). Data have been fitted with a single exponential to obtain a Kd of 4·4 µM. 
, cell volume 30 min after addition of 100 µM ATP in the absence of tamoxifen. B, in the presence of 100 µM ATP, increasing concentrations of tamoxifen (TMX) led to a reduced steady-state volume, as measured at t = 30 min (