The Inhibitory Influence of Endothelin on Active Sodium-Potassium Transport in Porcine Lens

Abstract

purpose. Endothelin (ET)-1 is known to inhibit active NaK transport by as much as 50% in kidney tubule and other tissues. The presence of low levels of ET-1 in aqueous humor combined with the potential for release of ET-1 from ciliary processes suggests that the lens could be exposed to ET-1 in vivo. In this study, experiments were conducted to examine the influence of ET-1 on active NaK transport in porcine lens.

methods. The rate of Na,K-adenosine triphosphatase (Na,K-ATPase) dependent potassium transport was determined by measurements of ouabain-sensitive potassium (⁸⁶Rb) uptake by intact lenses. Lens sodium content was measured by atomic absorption spectrophotometry. Cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. Cytoplasmic calcium concentration in cultured porcine lens epithelium was measured by a fluorescence technique using fura-2.

results. In the presence of ET-1 (0.1 nM or higher concentration), the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake was diminished. The ET receptor antagonist PD145065 (2 μM) suppressed the inhibitory effect of ET-1 (100 nM) on ⁸⁶Rb uptake. Sodium content was detectably increased in lenses exposed to ET-1 for 24 hours. Forskolin (1 μM) caused an eightfold increase of cAMP in the lens epithelium, but no increase of cAMP was detected in the epithelium of lenses treated with ET-1. Genistein (150 μM), an inhibitor of tyrosine kinases, abolished the inhibitory effects of ET-1 on lens ⁸⁶Rb uptake. ET-1 caused an increase of cytoplasmic calcium concentration in cultured porcine lens epithelium. The cytoplasmic calcium response to ET-1 was inhibited by PD145065 and genistein.

conclusions. The results of the present study suggest that ET-1 causes inhibition of lens active Na-K transport by a mechanism that involves activation of ET receptors. Activation of ET receptors also causes an increase of cytoplasmic calcium concentration in cultured lens epithelial cells. Both responses to ET-1 appear to have a tyrosine kinase step, because they could be prevented by genistein. The physiological purpose of an ET-1–induced reduction in the rate of active Na-K transport by the lens is unknown at this time.

Endothelin (ET)-1 is a small peptide best known for its potency as a vasoconstrictor. ¹ ET-1 is found in vascular endothelium, but it is also detectable in a number of nonvascular tissues, such as brain, ² spinal cord, ³ and mast cells. ⁴ ET-1 release from vascular endothelium can be elicited by a range of agonists, and the mechanism of release has been extensively studied because of its relevance to vascular disease. ⁵  

Recently, there has been increasing interest in the effects of ET-1 on nonvascular tissues. For example, ET-1 alters the behavior of osteoclasts, and it has been suggested that ET-1 may play a significant role in bone remodeling. ⁶ In a number of different tissues, it has been suggested that ET-1 acts as a mitogen. ⁷ In lung airway epithelium, ET-1 stimulates chloride secretion. ^{8 9 10} ET-1 is also known to inhibit active Na-K transport in some tissues. For example, the ability of ET-1 to suppress fluid transport in kidney proximal tubule has been attributed to ET-1–induced Na,K-ATPase inhibition. ¹¹ In cells from the inner medullary collecting duct, ET-1 causes 30% to 50% inhibition of active Na-K transport activity. ¹²  

In the eye, it has been shown that ET-1 is abundant in the ciliary processes where it appears to be densely localized in the nonpigmented ciliary epithelium (NPE). ¹³ It is noteworthy that there is evidence to suggest that adrenergic ¹⁴ and cholinergic ¹⁵ receptor activation can cause cells to release ET-1. This fits with the detection of ET-1 in aqueous humor. ^{16 17} The anterior epithelial surface of the lens is therefore likely to be exposed to ET-1 in vivo. Because Na,K-ATPase in lens epithelium is believed to play a key role in conducting active sodium extrusion and potassium import for the entire lens cell mass, we examined the influence of ET-1 on the lens sodium pump mechanism. Findings from ⁸⁶Rb uptake experiments and measurements of lens sodium content suggest that ET-1 partially inhibits lens active Na-K transport. 

Materials and Methods

⁸⁶Rb Cl was purchased from Amersham (Arlington Heights, IL). ET-1, PD145065, genistein, forskolin, ouabain, ionomycin, and other general chemicals were obtained from Sigma (St. Louis, MO). Fura-2-AM was obtained from Molecular Probes (Eugene, OR). 

Lenses

Porcine eyes were provided by Swift Meat Packing (Louisville, KY). The use of porcine eyes was approved by the University of Louisville Institutional Animal Care and Use Committee and conformed to the ARVO Resolution for the Use of Animals in Ophthalmic and Vision Research. The posterior of the eye was dissected, and the zonules were cut. The lens was removed from the globe and placed in Krebs solution. The composition of the Krebs solution was (in mM) 119 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, 1 MgCl₂, and 5.5 glucose at pH 7.4. 

Measurement of ⁸⁶Rb Uptake

⁸⁶Rb uptake was measured in intact lenses. The rate of ouabain-sensitive potassium (⁸⁶Rb) uptake was used as an index of Na,K-ATPase–mediated active potassium transport, based on the assumption that Na,K-ATPase transports ⁸⁶Rb similarly to potassium. Lenses were preincubated for a specified period in Krebs solution containing test agents, and then ⁸⁶Rb Cl (∼0.1 μCi/ml) was added. ⁸⁶Rb uptake was linear over a period of 30 minutes (Fig. 1A ). To determine ouabain-sensitive ⁸⁶Rb uptake, half of the lenses in each group also received 1 mM ouabain, added 10 minutes before ⁸⁶Rb Cl. In most experiments the ⁸⁶Rb uptake period was 30 minutes. After the ⁸⁶Rb uptake period, each lens was removed from the ⁸⁶Rb-containing Krebs solution and rinsed in ice-cold nonradioactive Krebs solution for 2 minutes. The lenses were weighed, lyophilized, and reweighed to determine water content. Dried lenses were digested in 30% nitric acid, and ⁸⁶Rb in the acid digest was measured by scintillation counting. Based on the specific activity of ⁸⁶Rb in the Krebs solution, uptake results were expressed as nanomoles of potassium accumulated per gram lens water per 30 minutes. 

Lens Epithelial Cell Culture

The capsule and epithelium was removed from the lens and placed in a petri dish with the cell side facing upward. The dishes were immediately placed in a 37°C incubator with a humidified atmosphere of 95% air and 5% CO₂. For an initial 30-minute period, culture medium M199 (Life Technologies, Grand Island, NY) containing 10% fetal calf serum and 1% gentamicin was placed around the tissue without actual contact being made. Additional culture medium was then added to cover the capsule and epithelium. After approximately 1 week, measurements of cytoplasmic calcium were made using cells that had grown off the capsule and attached to the plastic dish. 

Measurement of Cytoplasmic Calcium Concentration Using Fura-2

Fura-2 was used to measure the cytoplasmic calcium concentration in cultured lens epithelial cells superfused with artificial aqueous humor (AAH) which contained (in mM) 117 NaCl, 20 NaHCO₃, 4.5 KCl, 10 HEPES, 1.5 CaCl₂, 1.0 MgCl₂, and 6 glucose at pH 7.4. The AAH was equilibrated with 95% air and 5% CO₂. To load the cells, Fura-2-AM was dissolved in dimethylsulfoxide (DMSO) and added to the AAH for 60 minutes at 37°C at a final concentration of 5 μM Fura-2-AM and less than 1% DMSO. After the Fura-2 loading period, the cells were washed with AAH. Fura-2 fluorescence was measured using a microscope (Carl Zeiss, Thornwood, NY) equipped with an digital fluorescence imaging system (Attofluor; Atto Instruments, Rockville, MD). The lens epithelial cells were continuously superfused with AAH on a heated microscope stage that maintained temperature close to 37°C. The emission wavelength was 520 nm. Alternating excitation wavelengths of 334 nm and 380 nm were used, and fluorescence was continuously recorded. For calibration at the end of each experiment, the cells were permeabilized by exposure to 10 μM ionomycin to establish the maximum signal in AAH. Then 5 μM EGTA was added to the superfusion solution to obtain the minimum signal. 

Measurement of cAMP

Cyclic adenosine monophosphate (cAMP) was measured with a competitive binding assay kit using ³H-labeled cAMP (Amersham). Intact lenses were incubated for 60 minutes in Krebs solution containing either ET-1 or forskolin. The Krebs solution also contained 3-isobutyl-1-methylxanthine (IBMX; 2.5 mM), an inhibitor of phosphodiesterase, added to prevent cAMP breakdown. After the 60-minute period, the capsule-epithelium was removed from each lens and homogenized in ice-cold Tris-EDTA buffer containing 50 mM Tris and 4 mM EDTA. The homogenate was boiled for 5 minutes and centrifuged at 3000 rpm for 5 minutes and the supernatant assayed for cAMP. 

Measurement of Lens Sodium

Lenses were washed for 5 minutes in ice-cold isotonic (100 mM) MgCl₂ solution (pH adjusted to 7.4 with Tris base) and blotted dry. The lenses were weighed, lyophilized, and reweighed to measure water content. Each lens was digested in 30% nitric acid. Deionized water was added to dilute the sample, and the sodium concentration was measured using an atomic absorption spectrophotometer (Perkin Elmer, Norwalk, CT) at a wavelength of 566.5 nm. 

Results

The time course of ⁸⁶Rb uptake was measured in lenses incubated up to 4 hours in the presence or absence of 100 nM ET-1 (Fig. 1A) . The rate of ⁸⁶Rb uptake was markedly reduced by ET-1. In parallel experiments, the rate of ouabain-sensitive ⁸⁶Rb uptake was measured in lenses exposed to ET-1 in the concentration range of 0.1 to 1000 nM. In the presence of ET-1 at a concentration of 1 nM or higher, the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake was significantly diminished (Fig. 1B) . At a concentration of 100 nM, ET-1 reduced the ouabain-sensitive potassium (⁸⁶Rb) uptake rate by approximately 40%. At the same concentration, ET-1 did not detectably alter the rate of ouabain-insensitive ⁸⁶Rb uptake, which was 333.0 ± 31.8 nanomoles/g lens H₂O/30 minutes (mean ± SD, n = 6 lenses) in control lenses and 370.0 ± 75.9 in lenses exposed to ET-1. 

To examine whether ET receptor activation is involved in the ⁸⁶Rb uptake response, some lenses were exposed to 100 nM ET-1 in the presence of 2 μM PD145065, an antagonist for both ET_A and ET_B receptors. ¹⁸ PD145065 suppressed the inhibitory effect of 100 nM ET-1 on the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake (Table 1) . Added alone, PD145065 had no detectable effect on baseline ouabain-sensitive potassium (⁸⁶Rb) uptake. 

The ⁸⁶Rb uptake experiments suggest ET-1 partially inhibits Na,K-ATPase–mediated potassium transport by the lens. If partial inhibition of Na,K-ATPase is indeed the case, a gradual increase of sodium content might be anticipated in lenses exposed to ET-1. To test this hypothesis, lenses were incubated for 24 hours in the presence or absence of 100 nM ET-1. In lenses exposed to ET-1, the sodium content increased significantly (P < 0.01) to a value of 30.3 ± 4.1 millimoles/kg lens water from a value of 23.1 ± 3.6 measured in control (no ET-1) incubated lenses (mean ± SD, n = 6 lenses). 

In some tissues, ET-1 is known to mediate responses through an increase of cytoplasmic cAMP. ^{19 20} This did not appear to be the case in porcine lens. cAMP was measured in the capsule and epithelium removed from lenses that had previously been incubated in the presence or absence of 100 nM ET-1. As a positive control, cAMP was measured in the capsule and epithelium removed from a separate group of lenses that had been preincubated in the presence of 1 μM forskolin. cAMP was not detectably altered in the capsule and epithelium of lenses exposed to ET-1 (Table 2) , whereas, in contrast, forskolin treatment increased cAMP more than eightfold. 

ET-1 has been shown to activate nonreceptor tyrosine kinases in some tissues. ²¹ To test whether tyrosine kinase activation might be involved in the mechanism by which ET-1 causes inhibition of ouabain-sensitive potassium (⁸⁶Rb) uptake, experiments were conducted in which lenses were exposed to ET-1 in the presence of genistein, a tyrosine kinase inhibitor. Genistein (150μ M) abolished the inhibition of ouabain-sensitive potassium (⁸⁶Rb) uptake caused by ET-1 (Table 1) . Ouabain-sensitive potassium (⁸⁶Rb) uptake measured in the presence of genistein alone was not significantly different from that measured in control lenses. 

In cultured human mesangial cells ²² and in bovine adrenal chromaffin cells, ²³ ET-1 causes an increase of cytoplasmic calcium. Experiments were conducted to test whether this could also be the case in porcine lens cells. Using fura-2, cytoplasmic calcium was measured in porcine lens epithelial cells in primary culture. ET-1 (100 nM) caused an immediate, transient, threefold increase of cytoplasmic calcium concentration (Fig. 2) . After this, cytoplasmic calcium established a sustained plateau that was significantly higher than the initial baseline calcium concentration. Of particular note, PD145065 (2 μM) suppressed the cytoplasmic calcium response to ET-1 (Table 3) . The findings suggest that the increase of cytoplasmic calcium occurs subsequent to ET receptor activation. 

Because genistein was found to abolish the inhibitory effect of ET-1 on ouabain-sensitive potassium (⁸⁶Rb) uptake, studies were conducted to examine the influence of genistein on the cytoplasmic calcium response to ET-1. Added alone, 150 μM genistein caused detectable reduction of baseline cytoplasmic calcium concentration. It is important to note that genistein abolished the increase of cytoplasmic calcium elicited by ET-1 (Table 3) . 

Discussion

⁸⁶Rb uptake studies indicate that ET-1 at a concentration of 1 nM and higher reduces the rate of Na,K-ATPase–mediated potassium transport in porcine lens. At a concentration of 100 nM, ET-1 caused an approximately 40% inhibition of ouabain-sensitive potassium (⁸⁶Rb) uptake. ET-1 also caused a detectable increase in the sodium content of the lens. Taken together, these findings are consistent with partial inhibition of active Na-K transport in lenses exposed to ET-1. A similar inhibitory influence on active Na-K transport has been reported for ET-1 in cardiac muscle, ²⁴ rat glomerular mesangial cells, ²⁵ and vascular smooth muscle. ²⁶  

The 100-nM concentration of ET-1 used in the present study has been shown to cause prostaglandin E₂ release from ciliary smooth muscle, ²⁷ to elevate cytoplasmic sodium concentration in cultured rat glomerular mesangial cells, ²⁸ and to cause alkalinization of cultured rat vascular smooth muscle cells. ²⁹ However, a higher concentration of ET-1 was required to inhibit Na,K-ATPase in cultured rat vascular smooth muscle cells. ²⁸ It should also be noted that the influence of ET-1 on active Na-K transport varies in different tissues. ET-1 inhibits active Na-K transport in rat renal proximal tubule ¹¹ and inner medullary collecting duct ¹² as well as in endothelium-denuded pig mesenteric arteries. ²⁹ However, in rabbit aorta ³⁰ and rat brain capillary endothelium, ³¹ ET-1 causes significant stimulation of active Na-K transport. In the rabbit descending colon epithelium, ³² ET-1 does not detectably change the rate of active Na-K transport. 

PD145065 abolished the inhibition of lens ouabain-sensitive potassium (⁸⁶Rb) uptake that occurred in the presence of ET-1. PD145065 is a nonselective antagonist for ET_A and ET_B receptors. Its ability to suppress the ⁸⁶Rb uptake response in lens suggests that ET-1 slows active Na-K transport by a mechanism involving activation by ET receptors. This leads us to propose that ET receptors are expressed in porcine lens. The expression of ET receptors in the lens has not been widely reported, but there is evidence for ET receptor expression in ciliary epithelium, corneal endothelium, and iris. ³³ The presence of ET receptors in iris, corneal endothelium, and ciliary epithelium is noteworthy, because each of these tissues is bathed by aqueous humor, as is the lens, and aqueous humor is known to contain ET-1 with concentrations reported in the range of 15 to 226 pg/ml. ^{16 34} ET-1 levels in the aqueous humor could increase in the presence of open-angle glaucoma ³⁵ and with some forms of ocular surgery. ³⁶  

In studies of cultured nonpigmented ciliary epithelium, release of stored ET-1 has been reported in response to carbachol, which activates cholinergic receptors. ¹⁶ This suggests hormones and neurotransmitters could elicit release of ET-1 from the ciliary processes in vivo. The close anatomic proximity of the lens equator and ciliary epithelium causes us to speculate that ET-1 released from ciliary processes could activate ET-1 receptors in the lens. 

In tissues such as rat vascular smooth muscle, ET receptor activation stimulates adenylate cyclase to cause an increase of cytoplasmic cAMP. ^{19 20} This does not appear to be the case in porcine lens. Although there was an eightfold increase of cAMP detected in the epithelium removed from lenses exposed to the adenylate cyclase activator, forskolin, there was no detectable change of cAMP in the epithelium removed from lenses exposed to ET-1. 

In some tissues, ET-1 responses are thought to involve activation of tyrosine kinases. ²¹ This appears to be the case in porcine lens, because the inhibitory effect of ET-1 on ouabain-sensitive potassium (⁸⁶Rb) uptake was abolished in the presence of genistein, a broad-spectrum tyrosine kinase inhibitor. Genistein alone did not alter ⁸⁶Rb uptake. It is noteworthy that in a previous study, genistein was found to suppress the inhibitory influence of thrombin on lens active Na-K transport. ³⁷ On the basis of the present study, we are not able to determine whether lens Na,K-ATPase is directly susceptible to tyrosine phosphorylation. However, this has been suggested in the kidney, where activation of nonreceptor tyrosine kinases leads to a change of Na,K-ATPase activity. ³⁸ Tyrosine phosphorylation of plasma membrane calcium ATPase is known to alter active calcium transport in human platelets. ³⁹  

Activation of ET receptors has been reported to cause an increase of cytoplasmic calcium in many tissues. ^{22 23} In the present study, fura-2 was used to measure cytoplasmic calcium in cultured porcine lens epithelium, because technical problems hinder cytoplasmic calcium measurement in the intact lens. ET-1 caused a transient threefold increase of cytoplasmic calcium followed by a sustained plateau. As expected, PD145065 inhibited the cytoplasmic calcium response to ET-1, suggesting the cytoplasmic calcium increase lies downstream of ET receptor activation. The pattern of the calcium response to ET-1 in lens epithelium is similar to that reported in other ocular tissues. ⁴⁰ Previous investigators have suggested that the initial transient calcium increase is the result of mobilization of endoplasmic reticulum stores. ⁴¹ It is noteworthy that the transient calcium increase caused by ET-1 could be abolished by genistein. This could perhaps be attributed to the inability of IP3 receptors to be activated in genistein-treated cells that received ET-1. Inositol 1,4,5-trisphosphate (IP3) receptor activation is well known to require tyrosine phosphorylation. ⁴²  

In previous studies we have shown that an increase of lens calcium can lead to a decrease in the rate of ⁸⁶Rb uptake and an increase in lens sodium. ^{43 44} This suggests the lens responds similarly to other cells in which elevation of cytoplasmic calcium initiates mechanisms that slow the rate of active Na-K transport. ⁴⁵ This fits with the observed ability of genistein to inhibit the effects of ET-1 on active Na-K transport, because genistein also inhibits the calcium response elicited by ET-1 in cultured lens epithelium. 

The results of the present study show clearly that ET-1 can cause a decrease in the rate of lens active Na-K transport. The detection of low levels of ET-1 in aqueous humor combined with the potential for release of ET-1 from ciliary processes suggests that the lens could be exposed to ET-1 in vivo. The physiological purpose of an ET-1–induced reduction in the rate of active Na-K transport is unknown at this time. 

 Supported by US Public Health Service Research Grant EY09532, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness. NAD is the recipient of a Senior Scientific Investigator Award from Research to Prevent Blindness.

 Submitted for publication May 18, 2000; revised September 28, 2000; accepted November 2, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

(A) Time course of potassium (⁸⁶Rb) uptake by lenses incubated in the presence (▵) or absence (○) of 100 nM ET-1. Each point is the mean of results from two lenses. (B) Concentration dependence of the ET-1 effect on ouabain-sensitive potassium (⁸⁶Rb) uptake. Lenses were preincubated for 10 minutes in the presence or absence of ET-1 at a concentration of 0.1 to 1000 nM. Control lenses were not treated with ET-1. Half the lenses also received ouabain (final concentration, 1 mM) added at the same time as ET-1. After the preincubation period, ⁸⁶Rb was added for a further 30 minutes. The data are the mean ± SD (n = 11 lenses). *Significant difference from control (P < 0.01).

Table 1.

 

Effects of PD145065 and Genistein on the Ability of ET-1 to Inhibit Lens Na,K Transport

Treatment	Ouabain-Sensitive;0>Potassium (86Rb) Uptake
Control	1491.4 ± 112.7
PD145065	1691.9 ± 76.6
Genistein	1334.0 ± 140.0
ET-1	900.8 ± 94.2*
ET-1+ PD145065	1530.6 ± 113.6
ET-1+ Genistein	1387.1 ± 201.3

 Lenses were preincubated for 15 minutes in the presence or absence of PD145065 (2 μM), and ET-1 (100 nM) was added for a further 10 minutes. Control lenses received neither ET-1 nor PD145065. In other experiments, lenses were preincubated for 15 minutes in the presence or absence of genistein (150 μM), and then ET-1 (100 nM) was added for a further 10 minutes. Control lenses received neither ET-1 nor genistein. Half of the lenses also received ouabain (final concentration 1 mM) added at the same time as ET-1. After the preincubation period, ⁸⁶Rb was added for a further 30 minutes. The data are presented as nanomoles per gram H₂O per 30 minutes and are given as mean ± SD (n = 6 lenses).

*  Significant difference from control (P < 0.01).

Table 2.

 

Measurements of cAMP in Lens Capsule and Epithelium

Treatment	cAMP Concentration
;l>Control	14.0 ± 2.4
ET-1	11.8 ± 5.7
Forskolin	112.1 ± 16.4*

 Intact lenses were incubated in Krebs solution in the presence or absence of ET-1 (100 nM) or forskolin (1 μM). The Krebs solution also contained IBMX (2.5 mM), a phosphodiesterase inhibitor added to prevent cAMP breakdown. After 60 minutes, the capsule and epithelium was removed from each lens and assayed for cAMP. The data are presented as picomoles per milligram protein and are given as mean ± SD (n = 6 lenses).

*  Significant difference from control (P < 0.01).

Figure 2.

 

A typical response showing the influence of ET-1 on cytoplasmic calcium concentration in cultured porcine lens epithelial cells loaded with fura-2. The cells were continuously superfused with AAH. Arrow: Time point at which superfusate was changed to contain ET-1 (100 nM).

Table 3.

 

Influence of ET-1 on Cytoplasmic Calcium Concentration in Cultured Porcine Lens Epithelial Cells

	Peak Height	Plateau Height
ET-1 (100 nM)	159.9 ± 54.4	24.5 ± 26.8 (6)
ET-1 (100 nM)+ PD145065 (2 μM)	19.4 ± 12.1*	−0.2 ± 2.4* (4)
ET-1 (100 nM)+ genistein (150 μM)	−27.6 ± 30.0*	−17.4 ± 29.5* (4)

 Results are shown as the difference between cytoplasmic calcium measured before ET-1 application and cytoplasmic calcium measured at the peak of the response (peak height) or during the sustained plateau phase of the response (plateau height). A negative value indicates a reduction of cytoplasmic calcium concentration below baseline. Data are presented as nanomoles per liter and are given as mean ± SD, with the number of experiments in parentheses.

*  Significant difference from the value determined in the presence of ET-1 alone (P < 0.01). In each experiment, a stable baseline was established before the addition of ET-1. PD145065 or genistein was added 10 minutes before the addition of ET-1. In control cells, the baseline cytoplasmic calcium concentration was 65.6 ± 31.4 nanomoles/l, whereas in cells exposed to PD145065 (2 μM) and genistein (150 μM), the baseline was 62.3 ± 38.6 and 44.3 ± 22.4 nanomoles/l, respectively.