Introduction

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Endothelin-1 (ET-1) is a member of the 21 amino acid family of potent vasoactive peptides (ET-1, ET-2, and ET-3), first isolated from vascular endothelial cells (Yanagisawa et al., 1988). In the eye, ETs are abundantly distributed and are found in the aqueous humor, iris, ciliary body, retina, and choroid (MacCumber et al., 1991; Wollensak et al., 1998). However, of the three isoforms of ET known, only ET-1 and ET-3 are present in these ocular tissues (Eichhorn and Lutjen-Drecoll, 1993; Chakravarthy et al., 1994).

Normally, ET-1 levels are found to be 2 to 3 times greater in human (16 pg/ml) and bovine (11 pg/ml) aqueous humor (AH) than that observed in the plasma (Lepple-Wienhues et al., 1992). It has been suggested that increased aqueous humor ET-1 levels in normal eyes could be indicative of a putative homeostatic function in AH outflow and intraocular pressure (IOP) regulation (Lepple-Wienhues et al., 1992; Pang and Yorio, 1997). Furthermore, ET-1 levels are significantly elevated in AH of primary open-angle glaucomatous patients (21 pg/ml; nonprimary open angle glaucoma, 16 pg/ml) and in plasma of normotensive glaucoma patients (3.5 pg/ml; non-normotensive glaucoma patients, 2.5 pg/ml), compared with nonglaucomatous patients (Sugiyama et al., 1995b; Noske et al., 1997). Recently, it has been reported that ET-1 levels in AH of dogs with acute hypertensive glaucoma are 4-fold higher than those observed in normal dogs (Kallberg et al., 2000). Although it is still unclear, elevated ET-1 levels in glaucomatous eyes could occur in response to some symptom (elevated IOP) or high ET-1 levels could themselves be the cause of glaucoma. Although elevated ET-1 in the anterior chamber could regulate IOP, excessive levels in the back of the eye could promote optic nerve damage by ischemia, probably via ET-1-mediated vasoconstriction of the retinal arteries. Also, injections of low doses of ET-1 into mammalian eyes have resulted in prolonged lowering of the IOP (Erickson-Lamy et al., 1991; MacCumber et al., 1991; Sugiyama et al., 1995a). This ET-1-induced hypotensive effect observed could be attributed to an enhanced outflow facility of AH, due to ET-1-induced contraction of the ciliary smooth muscle (Pang and Yorio, 1997), and/or to a reduction in AH formation (Taniguchi et al., 1996). The latter effect could be brought about by ET-1's actions on ion transport activity occurring at the ciliary epithelium. We have previously demonstrated that human nonpigmented ciliary epithelial (HNPE) cells secrete ET-1 following cytokine or protein kinase C stimulation (Prasanna et al., 1998b). ETs thus released could act in an autocrine manner to regulate AH formation in addition to a paracrine effect on ciliary muscle contraction.

AH, responsible for maintaining IOP, is constantly produced by pigmented and nonpigmented ciliary epithelium by passive diffusion of water coupled with active transport of ions from serosal to aqueous side (Bill, 1975; Caprioli, 1987). The Na⁺:K⁺:2Cl cotransport (electroneutral symport) and Na⁺/K⁺-ATPase pump are two major components involved in such active ion transport (Davson, 1990; Von Brauchitsch and Crook, 1993) and whose activity in other tissues is regulated by many factors, including ET-1 (Zeidel et al., 1989; Kawai et al., 1995). ET-1 has also been shown to activate a calcium-sensitive potassium channel in C6 glioma cells that is inhibited by charybdotoxin (Supattapone and Ashley, 1991). In the eye, ET-1 has been shown to inhibit the Na⁺/K⁺-ATPase activity in rabbit corneal epithelial cells and in porcine lens (Yang et al., 1998; Okafor et al., 1999). However, little is known about ET-1's effects on ion transport activity in human nonpigmented ciliary epithelium. Presently, we report on the effects of ET-1 on Na⁺/K⁺-ATPase activity and Na⁺:K⁺:2Cl cotransport in SV40-transformed HNPE cells.

	Materials and Methods

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Cell Culture. SV40-transformed HNPEs (also called ODM-2 cells; passages 12-20), a gift from Dr. Miguel Coca-Prados (Yale University, New Haven, CT), were maintained at 37°C in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) and supplemented with 44 mM NaHCO₃, 10% fetal bovine serum, and antibiotics and grown to confluency in a 24-well tissue culture plates (Corning Star, Cambridge, MA.).

⁸⁶Rb⁺ Uptake Assay. Since ⁸⁶Rb⁺ can be substituted for potassium (K⁺), its uptake by cells via the major transport mechanisms (i.e., Na⁺/K⁺-ATPase pump and Na⁺:K⁺:2Cl cotransport) was measured in this study. The method for measuring ⁸⁶Rb⁺ uptake was done according to previous reports published from our laboratory (Crider et al., 1997). Briefly, SV40-transformed HNPE cells cultured to confluency in 24-well plates (approximately 5 × 10⁵ cells/well) were washed with 0.5 ml of assay buffer (2×) (116 mM NaCl, 5 mM KCl, 1 mM CaCl₂·2H₂O, 0.8 mM MgSo₄, 1 mM Na₂HPO₄, 5.5 mM glucose, pH 7.2). The cells were incubated with 0.5 ml/well of assay buffer for 10 to 15 min at 37°C. Following this preincubation, the cells were incubated with fresh assay buffer containing 5 mM KCl and 500,000 cpm (1 µCi/well) of ⁸⁶Rb⁺ (DuPont NEN, Boston, MA). A time course experiment for ⁸⁶Rb⁺ uptake in HNPE cells was performed (0-30 min). After 30 min, the cells were washed with ice-cold assay buffer (0.5 ml, 3×) and 0.5 ml of 0.1 N NaOH was added. The cell lysate was collected and mixed with scintillation cocktail (6 ml) and counted in a beta counter.

	Results

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Time Course of ⁸⁶Rb⁺ Uptake in HNPE Cells. A linear relationship was observed between ⁸⁶Rb⁺ uptake and time in HNPE cells with a best-fit line equation of Y = 1.334X + 9.225 and squared regression coefficient value (r²) of 0.997 (Fig. 1). ⁸⁶Rb⁺ uptake was 17 ± 0.42 nmol/well/5 min (n = 3), 21 ± 0.15 nmol/well/10 min (n = 3), 30 ± 0.5 nmol/well/15 min (n = 3), and 49 ± 2 nmol/well/30 min (n = 3). The 15-min time period of ⁸⁶Rb⁺ uptake was selected for all other experiments described.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Time Course of 86Rb+ uptake in SV40-transformed HNPE cells. 86Rb+ uptake was measured in HNPE cells as described under Materials and Methods for 5, 10, 15, and 30 min. In this graph, along with the best-fit line, the line equation is also provided showing the slope, y-intercept, and r2 values. The 95% confidence interval for the regression is also given and it provides the range of variable values computed for the region containing the true relationship between the dependent (86Rb+ uptake) and independent (time) variables, for the specified level of confidence ( = 0.05). *Denotes statistical significance of mean 86Rb+ uptake (nmol/well) among different time periods as determined by one-way ANOVA and Student-Newman-Keuls test at p < 0.05 (n = 3).

Effect of Endothelin-1 on ⁸⁶Rb⁺ Uptake in HNPE Cells. ET-1 has been shown to affect the activity of both Na⁺/K⁺-ATPase and Na⁺:K⁺:2Cl either by stimulating or inhibiting ion transport in different cell and tissue types, including rat brain capillary endothelium and renal tubular epithelial cells (Zeidel et al., 1989; Kawai et al., 1995). In HNPE cells, ET-1 (100 pM and 1 nM) decreased ⁸⁶Rb⁺ uptake by 20% compared with vehicle-treated control (Fig. 2). Furthermore, ET-1 at 10 or 100 nM also significantly decreased ⁸⁶Rb⁺ uptake by 12 to 14% compared with control. However, at 1 pM dose, ET-1 failed to decrease ⁸⁶Rb⁺ uptake and was similar to control. It should be noted that although all ET-1 doses (except 1 pM) were statistically significant compared with control, no such difference was observed among the different doses.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of ET-1 (1 and 100 pM; 1, 10, and 100 nM) on 86Rb+ uptake in HNPE cells. ET-1 decreased 86Rb+ uptake by 12 to 20% compared with control. 86Rb+ uptake was measured as described under Materials and Methods. *Denotes statistical significance of control versus ET-1 doses as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05 [n = 5 for ET-1 (100 pM); n = 6 for ET-1 (100 nM); n = 3 for others]. Control 86Rb+ uptake was 30 ± 0.5 nmol/well/15 min and was taken as 100%. 86Rb+ uptake for the following ET-1 doses were as follows: 1 pM ET-1, 30 ± 0.4 nmol; 100 pM ET-1, 24 ± 1.1 nmol/well/15 min; 1 nM ET-1, 24 ± 0.1 nmol; 10 nM ET-1, 26 ± 1.3 nmol; and 100 nM ET-1, 26 ± 0.7 nmol. There was no statistical significance among various ET-1 concentrations (except 1 pM).

Effects of BQ610, an ET_A Receptor Antagonist, and BQ788, an ET_B Receptor Antagonist, on Endothelin-1-Mediated ⁸⁶Rb⁺ Uptake in HNPE Cells. It was important to identify the ET receptor subtype that mediated ET-1's effect of decreased ⁸⁶Rb⁺ uptake in HNPE cells. Therefore, the effects of BQ610, an ET_A receptor antagonist, BQ788, an ET_B receptor antagonist, and S6C, an ET_B receptor agonist, on ET-1-induced decrease in ⁸⁶Rb⁺ uptake were evaluated. BQ610 (1 µM) failed to block ET-1-induced reduction in ⁸⁶Rb⁺ uptake for both 1 and 100 nM ET-1 concentrations (Figs. 3 and 4). However, pretreatment with the ET_B antagonist BQ788 (1 µM), completely blocked ET-1's effects on ⁸⁶Rb⁺ uptake (Fig. 5). Furthermore, the ET_B agonist S6C mimicked ET-1-induced reduction in ⁸⁶Rb⁺ uptake compared with control, which could not be blocked by BQ610 pretreatment (Fig. 4). Therefore, in HNPE cells, ET-1-induced reduction in ⁸⁶Rb⁺ uptake appears to be mediated mostly by an ET_B-like receptor but not via an ET_A receptor.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of BQ610 (ETA antagonist; 1 µM) on endothelin-1-mediated 86Rb+ uptake in HNPE cells. ET-1 (1 nM) decreased 86Rb+ uptake; BQ610, however, was unable to block these effects. 86Rb+ uptake was measured as described under Materials and Methods. Control 86Rb+ uptake was 34 ± 1.2 nmol/well/15 min and was taken as 100%. *Denotes statistical significance of control versus treatments as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05 (n = 3). There was no statistical significance between ET-1 versus ET-1 + BQ610. 86Rb+ uptake for the following treatments were 1 nM ET-1, 27 ± 1.4 nmol/well/15 min; BQ610 + 1 nM ET-1, 27 ± 1.1 nmol; and BQ610, 31 ± 1.2 nmol.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of S6C (ETB receptor agonist; 100 nM), BQ610, and ET-1 (100 nM) on 86Rb+ uptake in HNPE cells. Both S6C and ET-1 caused a reduction in 86Rb+ uptake; however, BQ610 (ETA receptor antagonist; 1 µM) could not block these effects. 86Rb+ uptake for the control was 29 ± 0.6 nmol/well/15 min and was taken as 100%. 86Rb+ uptake was measured as described under Materials and Methods. 86Rb+ uptake values for the following treatments were 100 nM ET-1, 26 ± 0.2 nmol; BQ610 + ET-1, 24 ± 1.3 nmol; and BQ610, 28 ± 0.3 nmol. *Denotes statistical significance of control versus treatments as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05 (n = 3 for all treatments). Denotes statistical significance of ET-1 versus BQ610 as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05. Denotes statistical significance of S6C versus BQ610 alone and BQ610 + S6C as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05. 86Rb+ uptake for the following treatments were S6C, 23 ± 0.5 nmol/well/15 min; BQ610, 28 ± 0.5 nmol; and BQ610 + S6C, 21 ± 0.8 nmol.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Effect of BQ788 (1 µM), an ETB receptor antagonist on endothelin-1-mediated reduction of 86Rb+ uptake in HNPE cells. Although ET-1 (1 nM) alone decreased 86Rb+ uptake, its effect was blocked in the presence of BQ788. 86Rb+ uptake for the control was 24 ± 0.6 nmol/well/15 min and was taken as 100%. 86Rb+ uptake was measured as described under Materials and Methods. *Denotes statistical significance of control versus treatments as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05 (n = 3 for all treatments). Denotes statistical significance of ET-1 versus BQ788 alone and BQ788 + ET-1 as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05. 86Rb+ uptake for the following treatments were 1 nM ET-1, 19 ± 0.1 nmol/well/15 min; BQ788 + ET-1, 23 ± 1 nmol; and BQ788, 23 ± 0.6 nmol.

Effects of Ouabain, an Inhibitor of Na⁺/K⁺-ATPase, and Bumetanide, an Inhibitor of Na⁺:K⁺:2Cl, Cotransport on Endothelin-1-Mediated ⁸⁶Rb⁺ Uptake in HNPE Cells. Since basal ⁸⁶Rb⁺ uptake can be mediated by at least two different mechanisms involving the Na⁺/K⁺-ATPase and Na⁺:K⁺:2Cl cotransport, the potential involvement and relative contribution of both these transporters was investigated by using selective inhibitors of these transporters. HNPE cells were treated either singly with OUA (100 µM; an inhibitor of Na⁺/K⁺-ATPase), BUMET (100 µM; an inhibitor of Na⁺:K⁺:2Cl cotransporter), or a combination of both. As shown in Fig. 6, BUMET inhibited ⁸⁶Rb⁺ uptake by 50%, whereas the addition of BUMET + OUA nearly abolished all ⁸⁶Rb⁺ uptake.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of OUA (100 µM; Na+/K+-ATPase inhibitor) and BUMET (100 µM; Na+: K+:2Cl inhibitor) on endothelin-1-mediated 86Rb+ uptake in HNPE cells. Both OUA and BUMET sharply decreased 86Rb+ uptake, however the combination of ET-1 + BUMET had a greater inhibitory effect on 86Rb+ uptake compared with BUMET alone. No such effect was observed in the combination of ET-1 + OUA compared with OUA alone. HNPE cells were pretreated with OUA or BUMET for 30 min before the addition of 86Rb+ and ET-1 (100 nM). 86Rb+ uptake was measured as described under Materials and Methods. *Denotes statistical significance of control versus treatments as determined by one-way ANOVA and Student-Newman-Keuls multiple comparison test at p < 0.05. Denotes statistical significance of ET-1 versus OUA + ET-1 and BUMET + ET-1 as determined by one-way ANOVA and Student-Newman-Keuls test at p < 0.05. Denotes statistical significance of BUMET versus BUMET + ET-1 as determined by Student's t test at p < 0.05 [n = 6 for ET-1 (100 nM), n = 3 for others]. Control 86Rb+ uptake was 30 ± 0.5 nmol/well/15 min and was taken as 100%. 86Rb+ uptake for the following treatments were BUMET, 15 ± 0.8 nmol/well/15 min; BUMET + OUA, 3 ± 0.3 nmol; BUMET + ET-1, 11 ± 0.2 nmol; OUA, 18 ± 0.6 nmol; OUA + ET-1, 18 ± 0.5 nmol; and 100 nM ET-1, 26 ± 0.7 nmol.

Effect of Iberiotoxin, an Inhibitor of Ca²⁺-Dependent Maxi K⁺ Channels, on Endothelin-1-Mediated ⁸⁶Rb⁺ Uptake in HNPE Cells. It was also essential to demonstrate that ET-1-induced decrease in ⁸⁶Rb⁺ uptake did not involve the activation of Ca²⁺-dependent maxi K⁺ channels, since the agonist-induced activation of K⁺ efflux channels could also contribute to reduction in ⁸⁶Rb⁺ uptake. Moreover, ET-1 has been shown to activate Ca²⁺-dependent maxi K⁺ channels in other cell lines (Hill et al., 1997). HNPE cells were thus pretreated with IBTX (100 ng/ml; a specific antagonist of maxi K⁺ channel) followed by ET-1 (100 pM or 100 nM). ⁸⁶Rb⁺ uptake in the presence of IBTX alone increased by 22% from control (IBTX, 47 ± 1.5 nmol/well/15 min; control, 38 ± 1 nmol; p < 0.0001). However, in the presence of IBTX, ET-1 (both 100 pM and 100 nM) continued to decrease ⁸⁶Rb⁺ uptake compared with that of IBTX alone (value above), suggesting that ET-1 was not likely targeting the maxi K⁺ channel (IBTX + ET-1 100 pM, 30 ± 2.6 nmol/well/15 min; IBTX + ET-1 100 nM, 35 ± 1.2 nmol; p < 0.0001 versus control). Furthermore, in the same experiments ⁸⁶Rb⁺ uptake for IBTX + ET-1 (100 pM or 100 nM) was similar to that of ET-1 alone (ET-1 100 pM, 32 ± 1.4 nmol/well/15 min; ET-1 100 nM, 35 ± 1.2 nmol; not significant; one-way ANOVA/Student-Newman-Keuls test).

	Discussion

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In this study, we have demonstrated that ET-1 decreases ⁸⁶Rb⁺ uptake by inhibiting Na⁺/K⁺-ATPase activity in HNPE cells, via an ET_B-like receptor. There are several observations to support this contention. ET-1 decreased ⁸⁶Rb⁺ uptake in HNPE cells during a 15-min uptake period even in the presence of bumetanide (inhibitor of Na⁺:K⁺:Cl cotransport), whereas in the presence of ouabain (inhibitor of Na⁺/K⁺-ATPase), ET-1 did not further inhibit ⁸⁶Rb⁺ uptake, compared with that observed with ouabain alone. In other words, once the Na⁺/K⁺-ATPase was inhibited by ouabain, ET-1 had no other apparent target (bumetanide-sensitive Na⁺:K⁺:2Cl cotransport) to act upon to lower ⁸⁶Rb⁺ uptake. In addition, BQ610 (ET_A antagonist) did not prevent ET-1-induced decrease in ⁸⁶Rb⁺ uptake, whereas BQ788 (an ET_B antagonist) prevented ET-1's action and S6C (ET_B agonist) mimicked ET-1-induced decrease in ⁸⁶Rb⁺ uptake. Finally, ET-1-induced decrease in ⁸⁶Rb⁺ uptake could not be blocked by iberiotoxin, an inhibitor of Ca²⁺-activated maxi K⁺ channel. Taken together, our observations suggest that only Na⁺/K⁺-ATPase pump is inhibited following ET-1 treatment, whereas no such effects are observed on Na⁺:K⁺:Cl cotransport or Ca²⁺-activated maxi K⁺ channel.

In mammals, ETs mediate a majority of their actions via ET_A (ET-1 = ET-2 > ET-3) and ET_B (ET-1 = ET-2 = ET-3) receptors, although this signaling depends on ligand subtype, ligand affinity, ligand concentrations, activation of different phospholipases (mostly PLC- and PLA₂), and tissue type (Yanagisawa, 1994).

ET-1's inhibitory effects on ⁸⁶Rb⁺ uptake appear to be greater at lower ET-1 concentrations (100 pM and 1 nM), however, at 1 pM concentration ET-1's effects are absent. An attenuated ET-1 response at higher doses possibly reflects the recruitment of a negative feedback process involving second messenger pathways. Alternatively, the existence of different ET receptor subtypes each having a different affinity for ET-1, recruitment of different G proteins, and different second messenger systems could explain the decreased response at higher ET-1 doses. For instance, in human ciliary smooth muscle cells our laboratory has shown that both PLA₂ and PLC- are activated independently by ET-1 through ET_A receptor activation (Matsumoto et al., 1996).

These transformed HNPE cells have been widely used as a model for studying aqueous humor dynamics (Martin-Vasallo et al., 1989; Mito et al., 1995) and they express both ET_A and ET_B receptors under serum-free culture conditions (Yorio et al., 2000). Also, ET_A receptors predominate in primary and transformed HNPE cells (Tao et al., 1998; Yorio et al., 2000) and ET_A activation in other cell types has been shown to stimulate Na⁺/K⁺-ATPase (Gupta et al., 1991; Kawai et al., 1995). Since the inhibitory effect of ET-1 on Na⁺/K⁺-ATPase activity in HNPE cells is mediated only by ET_B receptor activation, the stimulation of ET_A receptors could act to counter this inhibitory action at higher doses, particularly because ET_A receptors appear to be more abundant. In transformed HNPE cells, stimulation of ET_A receptors elevate [Ca²⁺]_i (Prasanna et al., 1998a) and probably activate protein kinase C, which stimulates Na⁺/K⁺-ATPase. Thus, a cross talk could exist between both ET_A and ET_B receptors by way of distinct second messenger activation and thus possibly also explain the lack of a distinct dose response in HNPE cells.

Although ET-1-induced decrease in ⁸⁶Rb⁺ uptake in HNPE cells appears to be mediated via an ET_B-like receptor, Kawai et al. (1995) have shown that ET-1 (10 nM) increased ⁸⁶Rb⁺ uptake in cerebral capillary endothelium, via an ET_A receptor activation. Recently, both ET_A and ET_B receptors have been reported to be present in rat ciliary epithelium (Ripodas et al., 1998). However, our data show that sarafotoxin 6C (ET_B agonist) mimicked ET-1's effect in HNPE cells, whereas it had no effect in the cerebral capillary endothelium (Kawai et al., 1995). These data suggest that in HNPE cells, ET-1's action on decreasing ⁸⁶Rb⁺ uptake probably involves an ET_B-like receptor.

There are many reports that have shown endothelin-1 to inhibit the Na⁺/K⁺-ATPase activity (Zeidel et al., 1989; Garvin and Sanders, 1991). In rat proximal straight tubules, ET-1 (1 nM) was shown to decrease Na⁺/K⁺-ATPase activity by 20% (Garvin and Sanders, 1991), which is similar to what is reported here. According to Zeidel et al. (1989), ET-1 (10 nM) inhibits Na⁺/K⁺-ATPase activity by nearly 25% in intact renal tubular epithelial cells by activating specific signal transduction pathways and not by altering either the transmembrane cationic gradient or reducing intracellular ATP (i.e., by inhibiting mitochondrial metabolism). Results from Zeidel et al. (1989) also show that ⁸⁶Rb⁺ uptake in the presence of OUA and ET-1 was the same compared with that obtained with OUA alone, similar to that reported here for HNPE cells.

ET-1 has been shown to activate Ca²⁺-activated maxi K⁺ efflux channels in guinea pig mesenteric arterioles and cultured rat lactotrophs (Hill et al., 1997; Kanyicska et al., 1997). However, the sequential treatment of HNPE cells with IBTX (Ca²⁺-activated maxi K⁺ channel inhibitor) followed by ET-1 resulted in a further decrease ⁸⁶Rb⁺ uptake, confirming that the maxi K⁺ channels were not targeted by ET-1.

The Na⁺/K⁺-ATPase activity in nonpigmented ciliary epithelium has been found to be 2-fold greater than that observed in the pigmented ciliary epithelium, suggesting that the nonpigmented ciliary epithelium layer performs the bulk of AH transport into the posterior chamber of the eye (Riley and Kishida, 1986). Even though ET-1 decreases Na⁺/K⁺-ATPase activity by 12 to 20% in HNPE cells, this reduction in activity could have an impact on the normal rate of aqueous humor production in humans (2 µl/min) and consequently affect IOP. It has been shown in rabbits that intravitreal injection of ET-1 (approximately 300-400 nM) resulted in a 66% reduction in aqueous flow and IOP was lowered by 53% over a 24-h period (Taniguchi et al., 1996). Moreover, there are many previous reports showing that a single intracameral, intravitreal, or intravenous injection of ouabain (doses similar to the one used here) in rabbits or cats resulted in 30 to 40% reduction in Na⁺ transport and 40% reduction in aqueous humor formation (Becker, 1963; Bonting and Becker, 1964; Garg and Oppelt, 1970). These results further support our contention that small reductions of Na⁺/K⁺-ATPase activity, caused by ET-1 or other agents, could indeed affect aqueous humor formation.

In conclusion, we have reported for the first time that ET-1, via an ET_B-like receptor, decreases the activity of Na⁺/K⁺-ATPase in HNPE cells. The combined ability of ET-1 to decrease Na⁺/K⁺-ATPase activity in HNPE cells as well as to modulate the tone of ciliary smooth muscle is interesting from the viewpoint that ET-1 may play a role in the homeostatic regulation of AH dynamics.