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(Investigative Ophthalmology and Visual Science. 2002;43:790-796.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Studies on Endothelin Release and Na,K Transport in Porcine Lens

Mansim C. Okafor1, Partha Mukhopadhyay2 and Nicholas A. Delamere1,3

1 From the Departments of Ophthalmology and Visual Sciences, 2 Molecular, Cellular, and Craniofacial Biology, and 3 Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. In an earlier study it was reported that thrombin significantly reduces the rate of Na,K-adenosine triphosphatase (ATPase)–mediated ion transport by porcine lens. Because thrombin stimulates the release of endogenous endothelin (ET)-1 stores from some tissues, and because ET-1 can cause Na,K-ATPase inhibition, this study was designed to determine whether thrombin causes release of ET-1 from the lens.

METHODS. Intact porcine lenses were incubated in Krebs solution. The concentration of ET-1 in the solution was determined by ELISA. The rate of Na,K-ATPase-dependent ion transport was determined by measurement of ouabain-sensitive 86Rb uptake.

RESULTS. Thrombin (1 U/mL) reduced the rate of ouabain-sensitive 86Rb uptake by approximately 40%. PD145065 (2 µM), an ET receptor antagonist, abolished the inhibitory effect of thrombin on 86Rb uptake. Added alone, PD145065 did not alter 86Rb uptake. After an incubation period of 30 minutes, thrombin increased the concentration of ET-1 in the bathing medium in a dose-dependent manner. The time course of ET-1 appearance in the bathing medium of thrombin-treated lenses showed a peak at 30 minutes followed by a gradual decline. Consistent with the idea that release of ET-1 from the lens is tightly regulated, neither the calcium ionophore A23187 (1 µM) nor depolarization by potassium-rich solution caused significant release. However, exposing the lens to insulin (150 nM) significantly increased the appearance of ET-1 in the bathing medium. In parallel studies, mRNA for prepro-ET-1 was detected in the epithelium of freshly isolated lenses.

CONCLUSIONS. The results of the study suggest that ET-1 is produced in porcine lens cells and that thrombin and insulin are capable of stimulating the release of ET-1 from the lens. Thrombin-induced inhibition of Na,K-ATPase–dependent ion transport may be mediated in part through the activation of ET-1 receptors by ET-1 released from the lens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Endothelin (ET)-1 is a 21-amino-acid peptide recognized initially for its influence on blood vessel diameter.1 It is a potent vasoconstrictive agent that exerts its effect through activation of ET(A) and ET(B) receptors on vascular smooth muscle. In addition to its effect on vascular smooth muscle, ET-1 has been shown to cause shape changes in rat intestinal villi subepithelial fibroblasts,2 to induce mitogenesis in glial cells,3 4 and to initiate a pattern of osteoblast behavior associated with bone remodeling.5 ET-1 is also known to cause changes in the activity of Na,K-ATPase. Activation of ET receptors causes a significant reduction in the rate of active sodium-potassium transport in rat proximal straight tubule6 and porcine lens.7

ET-1 has been shown to be widely distributed in a number of nonvascular tissues including glomerular epithelial cells,8 rat primary astrocytes,9 pulmonary endothelial cells,10 and rabbit tracheal epithelial cells.11 ET-1 immunoreactivity has been reported in several ocular tissues,12 13 including the lens, where it is abundant in the epithelium.14 Both aqueous humor15 16 and vitreous humor17 contain significant concentrations of ET-1.

Some tissues are known to release endothelin in response to external stimuli. One recognized ET-1–releasing factor is thrombin, which has been reported to elicit release of ET-1 from guinea pig tracheal epithelium,18 bovine vascular endothelial cells,19 and human vascular endothelial cells.20 In an earlier study, we reported that thrombin significantly reduces the rate of active sodium-potassium transport by porcine lens.21 In view of the potential for thrombin to cause release of endogenous ET-1 stores in some tissues, we tested whether stimulation of ET-release may occur in the lens. Because ET-1 can cause Na,K-ATPase inhibition,7 we also considered the possibility that the observed reduction in the rate of active sodium-potassium transport elicited by thrombin might be mediated in part through release of ET-1 stored within the lens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ET-1, thrombin, insulin, genistein, A23187, ouabain, and other general chemicals were obtained from the Sigma Chemical Co. (St. Louis, MO). To minimize autoproteolysis, thrombin-containing solutions were prepared immediately before use. 86RbCl and the ELISA kit were obtained from Amersham (Arlington Heights, IL).

Lenses
Porcine eyes were donated by the Swift Meat Packing Co. (Louisville, KY). The use of animal tissues was approved by the University of Louisville Institutional Animal Care and Use Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The lens was isolated by opening the posterior of the globe and then cutting the suspensory ligaments. Each lens was removed from the globe and transferred to a culture well containing 4 mL Krebs solution with the following composition (in millimolar): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 1 MgCl2, and 5.5 glucose at pH 7.4. Potassium-rich Krebs solution was made by increasing KCl concentration to 82 mM at the expense of an equimolar concentration of NaCl.

86Rb Uptake
Based on the assumption that Na,K-ATPase transports 86Rb similarly to potassium, the rate of ouabain-sensitive 86Rb uptake by the intact lens was used as an index of Na,K-ATPase–mediated active sodium-potassium transport. Each lens was preincubated 10 minutes in Krebs solution containing test agents before 86Rb (~0.1 µCi/mL) was added. Ouabain (1 mM) was added to half the lenses in each group simultaneously with the addition of 86Rb. Lenses were allowed to accumulate 86Rb for 30 minutes. It has been established that 86Rb uptake is linear during this time.7 At the end of the 30-minute 86Rb uptake period, each lens was removed from the radioactive Krebs solution and rinsed in nonradioactive, ice-cold Krebs solution for 2 minutes. Each lens was weighed, freeze dried, and reweighed to determine water content. Dried lenses were digested in 30% nitric acid, and radioactivity in the acid digest was quantified by scintillation counting. Taking into account the specific activity of 86Rb in the Krebs solution, uptake results were expressed as nanomoles of potassium accumulated per gram of lens water in 30 minutes.

Detection of ET-1
An ELISA technique (Amersham) was used to detect ET-1. Samples of Krebs solution were acidified with 2 N HCl before being passed through a C2 column (Amprep; Amersham). ET-1 was then eluted from the column with 80% methanol containing 0.1% trifluoroacetic acid. The methanol solution eluted from the column was dried under a stream of nitrogen. ET-1 was then measured in the reconstituted pellet according to the manufacturer’s instructions. Cross-reactivity of the assay is 0.002% for porcine Big ET-1, less than 0.001% for Big ET-1 22-38, and less than 0.001% for ET-3. Significant cross-reactivity to ET-2 could occur, but ET-2 is not known to be expressed in eye tissues.

Isolation of RNA
Total RNA was isolated according to the method described by Chomczynski and Sacchi.22 Lens epithelium was placed in guanidinium thiocyanate homogenization buffer (pH 7.0; 4 M guanidinium thiocyanate, 0.5% N-sodium lauryl sarcocinate, 25 mM sodium citrate, and 0.7% 2-mercaptoethanol) and immediately stored at -80°C. The samples were thawed and total RNA was extracted by homogenization. Sodium acetate (2 M, pH 7.0) was added and samples were mixed thoroughly by inversion. The solution was extracted with water-saturated phenol-chloroform-isoamyl alcohol and total RNA was precipitated with isopropanol. After a second extraction and isopropanol precipitation, the RNA pellet was washed with 75% ethanol, dried, and stored at -80°C until further use.

Reverse Transcription–Polyerase Chain Reaction
The primers for porcine prepro-ET-1 (ppET-1) were designed as previously reported23 and purchased from Invitrogen (Baltimore, MD). The expected size for ppET-1 PCR product is 389 bp. Yoshimura et al.23 reported that after treatment with HindIII, the ppET-1 PCR product is digested into a 286-bp fragment that stains intensely with ethidium bromide and a weaker-staining 103-bp fragment. RT-PCR was performed as described earlier.23 The reverse transcription (RT) reaction was performed in a volume of 20 µL, using 1 µg total RNA. The RT product (5 µL) was subjected to PCR amplification in a volume of 50 µL. The PCR was performed in a DNA thermal cycler, according to the following program: 94°C, 30 seconds; 55°C, 1 minute; and 72°C, 30 seconds, for 35 cycles. The PCR products were purified using a PCR purification kit (QIAquick; Qiagen, Chatsworth, CA) and were separated by electrophoresis on a 2.0% agarose gel. The gels were stained with ethidium bromide and photographed under UV light.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ouabain-sensitive potassium (86Rb) uptake was measured in intact porcine lenses exposed to thrombin at a concentration of 1 U/mL. A different group of lenses was exposed to thrombin in the presence of 2 µM PD145065, an antagonist of both ET(A) and ET(B) receptors.24 Compared with the ouabain-sensitive potassium (86Rb) uptake measured in the control (no thrombin) group, the rate determined in the thrombin-treated group of lenses was reduced by approximately 40% (Fig. 1) . PD145065 abolished the inhibitory effect of thrombin on ouabain-sensitive potassium (86Rb) uptake. Added alone, PD145065 did not significantly change the uptake rate.



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  		Figure 1. PD145065 prevents thrombin-induced inhibition of ouabain-sensitive potassium (86Rb) uptake. Lenses were preincubated for 10 minutes in the presence of either thrombin (1 U/mL) or PD145065 (2 µM) or thrombin + PD145065. Control lenses received neither thrombin nor PD145065. After the preincubation period, 86Rb was added for a further 30 minutes. Half of the lenses received ouabain (final concentration 1 mM) together with 86Rb. Data are the mean ± SE (vertical bar) of results from six lenses. *Significant difference from control (P < 0.01).

 
The ability of PD145065 to suppress the inhibitory influence of thrombin on ouabain-sensitive 86Rb uptake suggests the thrombin response involves a step that requires activation of endothelin receptors. Studies were conducted to determine whether thrombin causes release of ET-1 from the lens. Six intact lenses were preincubated for 10 minutes in Krebs buffer, and thrombin (1 U/mL) was added to the Krebs solution for an additional 30-minute incubation period. After this, ET-1 was detected at a concentration of 19.9 ± 1.9 pg/mL (mean ± SE) in the Krebs solution. Higher concentrations of ET-1 were detected in the Krebs solution surrounding different groups of lenses that were exposed to greater concentrations of thrombin (Fig. 2) . At thrombin concentrations of 0.1 U/mL or lower, the concentration of ET-1 in the bathing medium was not significantly different from the 3.6 ± 0.9 pg/mL detected in the Krebs solution surrounding six control lenses that were not treated with thrombin. No significant concentration of ET-1 was detected, either in control Krebs solution (incubated 40 minutes in the absence of a lens) or in samples of the thrombin preparation used in these studies.



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  		Figure 2. Concentration of ET-1 measured in the bathing medium surrounding lenses exposed to thrombin. Each lens was incubated 30 minutes in 4 mL of Krebs solution, and the concentration of ET-1 in the bathing medium was determined by ELISA. Data are the mean ± SE (vertical bar) of results from six lenses. The concentration of ET-1 in the bathing medium surrounding control lenses (no thrombin treatment) was 3.6 ± 0.9 pg/mL. *Significant difference from control (P < 0.01).

 
The time course of release of ET-1 by thrombin-treated lenses is shown in Figure 3 . The concentration of ET-1 in the lens incubation medium increased until it reached a maximum level 30 minutes after thrombin addition. At longer incubation times, the concentration of ET-1 diminished, suggesting that the lens may be capable of reabsorbing ET-1 from the surrounding medium. To test whether the lens is capable of accumulating ET-1 from the bathing medium, a group of lenses was incubated in Krebs solution to which ET-1 was added at a concentration of 25 pg/mL. The concentration of ET-1 in the bathing medium was observed to decline significantly (Fig. 4) . After an incubation period of 30 minutes, the concentration of ET-1 was reduced by approximately 50%. When 25 pg/mL ET-1 was added to Krebs solution alone (no lens), the concentration of ET-1 remained stable. A concentration of 22.4 pg/mL was measured after 90 minutes and this value fell within the margin of error for the ET-1 assay.



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  		Figure 3. Time course of changes in the concentration of ET-1 in the bathing medium surrounding lenses exposed to 1 U/mL thrombin. Each lens was incubated 30 minutes in 4 mL Krebs solution, and the concentration of ET-1 in the bathing medium was detected by ELISA. Data are the mean ± SE (vertical bar) of results from six lenses. *Significant difference from the concentration measured at time 0 (P < 0.01).

 


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  		Figure 4. Time course for the removal of ET-1 added exogenously to the bathing medium surrounding the lens. Each lens was incubated in 4 mL Krebs solution. At the start of the experiment, ET-1 was added to a final concentration of 25 pg/mL. After the specified incubation times, the concentration of ET-1 remaining in the Krebs solution was determined by ELISA. Data are the mean ± SE (vertical bar) of results from six lenses. *Significant difference from the initial concentration (P < 0.01).

 
In a previous study, tyrosine kinase inhibitors were found to suppress the inhibitory influence of thrombin on ouabain-sensitive 86Rb uptake by the lens.7 To determine whether tyrosine kinase inhibition alters release of ET-1, lenses were exposed to thrombin in the presence or absence of genistein (150 µM). The concentration of ET-1 detected in the bathing medium of lenses exposed to thrombin in the presence of genistein was not significantly different from the concentration of ET-1 detected in the bathing medium taken from lenses exposed to thrombin alone. Both levels were significantly higher than the concentration of ET-1 measured in the bathing medium taken from control lenses (no thrombin or genistein) or lenses that received genistein alone (Fig. 5) .



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  		Figure 5. Concentration of ET-1 measured in the bathing medium surrounding lenses exposed to thrombin and genistein. Each lens was preincubated 15 minutes in 4 mL Krebs solution in the presence or absence of genistein (150 µM) and then for a further 30 minutes, during which time some of the lenses were exposed to thrombin (1 U/mL). Control lenses received neither genistein nor thrombin. Data are the mean ± SE (vertical bar) of results from six lenses. *Significant difference from control (P < 0.01).

 
The ability of thrombin to cause release of ET-1 from the lens was compared with the influence of calcium ionophore A23187, potassium-rich solution, and insulin on release of ET-1. Exposure of the lens to either 1 µM A23187 or to 82 mM potassium for 30 minutes failed to elicit a significant increase in the concentration of ET-1 in the bathing medium (Table 1) . In contrast, the concentration of ET-1 was significantly elevated in the bathing medium of lenses exposed to 150 nM insulin for 30 minutes (Table 1) .


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  		Table 1. Concentration of ET-1 in the Lens Bathing Medium

 
Cells synthesize ppET-1 and ET-1 is a product of ppET-1 processing. Studies were conducted to examine ppET-1 mRNA in porcine lens epithelium. RT-PCR with total RNA from porcine lens epithelium led to the formation of a PCR product of the predicted size (389 bp) corresponding to the ppET-1 mRNA (Fig. 6a) . The 389-bp band was not detected when the RT-PCR was performed in the absence of reverse transcriptase, indicating that the PCR product was derived from mRNA, rather than the chromosomal DNA. To confirm the identity of the 389-bp PCR product, we used a strategy based on the work of Yoshimura et al.23 who demonstrated that digestion of ppET-1 cDNA by HindIII results in two fragments: 286 and 103 bp. HindIII digestion of cDNA with a sequence that differs from ppET-1 cDNA is highly unlikely to generate two digestion products of 286 and 103 bp. When the ppET-1 PCR product was digested with HindIII, the predicted fragments at 286 and 103 bp were observed (Fig. 6b) , confirming that the product was generated from porcine ppET-1 cDNA. As shown by Yoshimura et al.,23 it is normal for the 103-bp band to stain less intensely with ethidium bromide than does the 286-bp band. The results confirm that porcine lens epithelium expresses ppET-1 mRNA.



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  		Figure 6. Detection of ppET-1 mRNA by RT-PCR. (a) The PCR product for ppET-1 mRNA (389 bp) in porcine lens epithelium. Lane 1: 100 kb DNA ladder; lane 2: ppET-1 PCR product (389 bp); and lane 3: negative control without reverse transcriptase. (b) Digestion of the 389-bp band by HindIII. Lane 1: the ppET-1 PCR product without digestion by HindIII; lane 2: 100-kb DNA ladder; lane 3: the PCR product digested by HindIII. Bands were visible at 286 and 103 bp.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study show that thrombin and insulin are capable of stimulating the release of ET-1 from the porcine lens. The thrombin response was dose dependent, and at a thrombin concentration of 10 U/mL, the level of ET-1 detected in the 4 mL of Krebs solution bathing the lens was more than 100 pg/mL (40 pM) after a 30-minute exposure period. In comparison, when lenses were incubated under control conditions, the ET-1 concentration determined in the bathing medium was less than 7 pg/mL. Release of ET-1 by vascular endothelial cells has been extensively documented,25 but there have also been reports of release of ET-1 by other tissues including epithelial tissues, such as proximal tubule26 and nonpigmented ciliary epithelium of the eye.27 In vascular endothelium, ET-1 release is triggered by a range of stimuli, including mechanical stretching28 and oxidative stress,29 as well as by thrombin.30 In corneal epithelium, activation of ET(B) receptors has been proposed to stimulate release of ET-1 in an autocrine fashion.31 In human coronary artery, Russell et al.32 observed endothelin immunolocalization in distinct cytoplasmic compartments identified as secretory vesicles and storage granules.

In the lens, it has been reported that ET-1 is localized primarily in the epithelial cell monolayer with little immunoreactivity detectable in fibers.14 ET-1 in the lens epithelium may represent peptide accumulated from the aqueous humor as well as peptide synthesized by the lens. Detection of ppET-1 mRNA in lens epithelium suggests that lens cells are capable of synthesizing ET-1. Release of ET-1 from the lens appears to be tightly controlled, because neither depolarization by potassium-rich Krebs solution nor exposure to the calcium ionophore A23187 caused a significant increase of ET-1 in the bathing medium, even though exposure of the lens to 82 mM potassium or 1 µM A23187 is sufficient to cause marked depolarization or an increase in calcium, respectively.33 34 In contrast, 150 nM insulin significantly stimulated release of ET-1 from the lens. The ability of insulin to stimulate secretion of ET-1 in a dose-dependent manner has been reported in vascular endothelial cells.35 In rats and humans, insulin also causes an increase in the concentration of ET-1 in plasma.36 37

The time course of the appearance of ET-1 in the bathing medium after exposure of the lens to thrombin showed a peak at 30 minutes, followed by a decline in the concentration of ET-1. When exogenous ET-1 was added to the bathing medium surrounding the lens, the concentration also decreased over time. In contrast, when ET-1 was added to the bathing medium alone (no lens), the concentration did not change. One explanation for these findings is the existence of a mechanism for accumulation of ET-1 by the lens. In some cells, there is evidence that internalization of ET-1 occurs.38 It remains to be determined whether binding of ET-1 to lens ET receptors and proteolytic breakdown of internalized ET-1 contributes to the observed reduction of the concentration of ET-1 in the bathing medium.

Genistein and herbimycin both suppressed the inhibitory effect of thrombin on lens 86Rb uptake, suggesting the involvement of a tyrosine kinase step. However, genistein did not prevent the thrombin-induced stimulation of ET-1 release from the lens. This fits with an earlier proposal that stimulation of the release of ET-1 from rat lung does not involve tyrosine kinase signaling.39 It seems possible that there is a tyrosine kinase step subsequent to ET receptor activation, in that genistein is also capable of abolishing the inhibitory effect of exogenous ET-1 on lens 86Rb uptake and the increase of cytoplasmic calcium caused by exposure to ET-1.7

In several cell types, ET-1 has been shown to inhibit Na,K-ATPase–mediated ion transport.40 41 In porcine lens the mechanism of Na,K-ATPase inhibition by ET-1 appears to involve activation of ET receptors, because the inhibitory effect of ET-1 on ouabain-sensitive 86Rb uptake can be suppressed by the ET receptor antagonist PD145065.7 In the present study, we found that PD145065 abolished the inhibitory effect of thrombin on Na,K-ATPase–mediated 86Rb uptake. Based on this finding, we propose that the response of the lens to thrombin may be mediated in part by ET-1 that is released from the lens and that subsequently activates lens ET-1 receptors. The present findings raise the possibility that ET-1 acts on lens cells in an autocrine fashion. An autocrine role for ET-1 has been proposed in corneal epithelium.31 Release and reuptake of ET-1 by the lens may also influence the concentration of ET-1 in aqueous and vitreous humor.

The significance of the Na,K-ATPase inhibition response to ET-1 remains to be established. However, consideration of some possible interpretations seems appropriate. Na,K-ATPase activity must be high in some parts of the lens and low in other parts, to provide the driving force for circulating ionic currents that flow outward at the lens equator and inward at the anterior and posterior poles (for review see Mathias et al.42 ). The ionic circulation is proposed to drive the flow of water, and this may enable the lens to overcome difficulties associated with the long diffusion time required for glucose, amino acids, and other dissolved substances to reach the center of the packed cell mass. The circulating currents arise in part because of the unequal spatial distribution of Na,K-ATPase activity, potassium channels, and gap junctions in the lens cell mass. Na,K-ATPase activity is highest at the equatorial surface. High Na,K-ATPase activity in the equatorial epithelium compared with the epithelium at the anterior pole has been confirmed independently by Gao et al.43 and by Zamudio et al.44 Because Na,K-ATPase protein is abundant in all lens epithelial cells43 45 the different Na,K-ATPase activity in different regions of the epithelium may stem from factors in the aqueous humor, perhaps including ET-1, that cause inhibition or activation of Na,K-ATPase activity.

It is also the case that thrombin, endothelin, and insulin have a mitogenic influence on the lens.46 47 It is possible that the inhibitory effects of thrombin and ET-1 on lens Na,K-ATPase–mediated ion transport are associated with a mitogenic response, because an increase in cytoplasmic sodium concentration is a recognized early event in mitogenesis.48 Further study is needed in this area. However, it is clear that a significant concentration of ET-1 can be detected in aqueous humor.15 16 The same is true of insulin.49 In contrast, thrombin may enter the aqueous humor only after a defect in the blood–aqueous barrier. Although the concentrations of ET-1 and insulin present in bulk aqueous humor are lower than the concentrations used in the present study, it is possible that a high concentration of ET-1 or insulin is achieved at localized zones on the lens surface after release of these molecules from the lens, ciliary body, or iris.


   Footnotes
 
Supported by National Eye Institute Grant EY09532, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness Inc. NAD is the recipient of a Senior Scientific Investigator Award from Research to Prevent Blindness Inc.

Submitted for publication July 2, 2001; revised October 3, 2001; accepted October 24, 2001.

Commercial relationships policy: N.

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

Corresponding author: Nicholas A. Delamere, Department of Ophthalmology, University of Louisville School of Medicine, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202; delamere{at}louisville.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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