Thrombin Inhibits Active Sodium–Potassium Transport In Porcine Lens

Abstract

purpose. Although thrombin is best known for its role in blood coagulation, it has been reported to change the activity of ion motive ATPases in some tissues. In the present study, experiments were conducted to determine the influence of thrombin on active sodium–potassium transport in porcine lenses.

methods. Ouabain-sensitive potassium (⁸⁶Rb) uptake by intact porcine lenses was used as an index of Na,K-ATPase–mediated active sodium–potassium transport. Na,K-ATPase activity was measured by determining ouabain-sensitive ATP hydrolysis in isolated membrane material.

results. In the presence of thrombin (1 unit/ml) the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake was reduced by 40% to 60%, but ouabain-insensitive potassium (⁸⁶Rb) uptake was unchanged. The inhibitory effect of thrombin on ouabain-sensitive potassium (⁸⁶Rb) uptake was suppressed in the presence of hirudin (an antagonist for thrombin receptors) but persisted in the presence of amphotericin B (a pseudo ionophore that effectively clamps plasma membrane sodium permeability at a high value). Enzyme measurements showed ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was significantly inhibited in membrane material isolated from the capsule-epithelium of lenses, which had been pretreated with thrombin for 30 minutes. However, thrombin failed to exert a direct inhibitory effect on Na,K-ATPase activity when added directly to membrane fragments isolated from the epithelium of control (nonincubated) lenses. Both genistein and herbimycin (tyrosine kinase inhibitors) suppressed the effect of thrombin on the ⁸⁶Rb uptake response. Results from Western blot studies suggested that tyrosine kinases are activated in the epithelium of lenses exposed to thrombin.

conclusions. The results suggest the inhibitory effect of thrombin on lens active sodium–potassium transport could involve the activation of a receptor–second-messenger mechanism in intact lens cells. The response appears to involve a tyrosine kinase–mediated step. The functional significance of the thrombin-mediated change of lens active sodium–potassium transport is unclear since appreciable amounts of thrombin may only be presented to the lens during instances of blood-aqueous–barrier breakdown. It is possible that lens receptors are functionally activated by other proteases, possibly cathepsins, which may enter aqueous humor from the ciliary body.

Thrombin is a serine protease best known for its role in the process of blood coagulation. ¹ Generally, thrombin is present in extracellular fluid at an extremely low concentration. Thrombin levels rise when factors released in response to trauma cause cleavage of prothrombin circulating in the bloodstream. However, prothrombin can be manufactured by both vascular and nonvascular tissues. ²  

Thrombin causes a wide range of cell responses in nonvascular tissues. ^{3 4} In some tissues, it has been shown to alter ion transport mechanisms. In studies with human blood platelets, thrombin was found to inhibit plasma membrane Ca-ATPase activity. ⁵ It has also been suggested that thrombin might cause changes in the activity of Na,K-ATPase. ⁶  

Reddan and coworkers ⁷ reported mitogenic responses consistent with the presence of thrombin receptor activation in lens epithelium. Because Na,K-ATPase in the epithelium monolayer is believed to play a key role in conducting outward sodium transport and inward potassium transport for the entire lens cell mass, ⁸ we considered the possibility that thrombin might influence active sodium–potassium transport in the lens. In studies with intact porcine lenses, thrombin caused significant Na,K-ATPase inhibition. 

Materials and Methods

⁸⁶Rb Cl was purchased from Amersham (Arlington Heights, IL). Genistein and herbimycin were obtained from Calbiochem (La Jolla, CA). Thrombin, hirudin, ouabain, and other general chemicals were obtained from the Sigma Chemical Company (St. Louis, MO). To minimize autoproteolysis, solutions containing thrombin were prepared immediately before use. 

Lenses

Porcine eyes were obtained from the Swift Meat Packing Company (Louisville, KY). The tissue collection procedures were 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. To isolate the lens, the posterior of the eye was dissected open, the suspensory ligaments of the lens were cut, and the lens was gently transferred to a Petri dish containing Krebs solution. The composition of the Krebs solution was (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

The rate of ouabain-sensitive ⁸⁶Rb uptake by the intact lens was used as a measure of Na,K-ATPase–mediated active sodium–potassium transport. It was assumed that the Na,K-ATPase mechanism transports ⁸⁶Rb similarly to potassium. Lenses were preincubated for a specified period (usually 10 minutes) in Krebs solution containing test agents, and then ⁸⁶Rb (∼0.1 μCi/ml) was added. Half of the lenses in each group also received ouabain, added to a final concentration of 1 mM simultaneously with the ⁸⁶Rb. The ⁸⁶Rb uptake period was 30 minutes. During this time, ⁸⁶Rb uptake was linear. After the 30-minute ⁸⁶Rb uptake period, each lens was removed from the radioactive Krebs solution and placed in a large volume of ice-cold nonradioactive Krebs solution for 2 minutes to wash out ⁸⁶Rb from extracellular space. After this, the lenses were weighed, lyophilized, and then reweighed to determine water content. The dried lenses were digested in nitric acid (30%), and radioactivity in the acid digest was measured by scintillation counting. As the specific activity of ⁸⁶Rb in the Krebs solution is known, the uptake results were expressed as nanomoles of potassium accumulated per gram lens water per 30 minutes. 

Measurement of Na,K-ATPase Activity

The capsule epithelium was removed from each lens and homogenized in ice-cold buffer A containing (in mM) 150 sucrose, 5 HEPES, 4 EGTA, 0.8 dithiothreitol, and protease inhibitors (in μM) 2 antipain, 2 leupeptin, 1 pepstatin A, 1 PMSF, and 2 μg/ml aprotinin. The homogenate was placed in a centrifuge at 115,000g for 60 minutes, and the pellet was resuspended in buffer A containing 600 mM KCl and then centrifuged again at 115,000g for 60 minutes. The pellet was then resuspended in buffer A and centrifuged a final time at 115,000g for 60 minutes. The final pellet containing lens capsule–epithelium membrane material was resuspended in buffer A, and the protein content of the mixture was determined using a Bio-Rad assay (Bio-Rad, Richmond, CA). To measure Na,K-ATPase activity, aliquots of lens capsule–epithelium membrane material (100 μg protein) were added to a buffer containing (mM) 100 NaCl, 10 KCl, 3 MgCl₂, 1 EGTA at pH 7.4. Ouabain (1 mM) was added to half of the samples. After a preincubation period of 5 minutes at 37°C, ATP was added to a final concentration of 1 mM. The ATP hydrolysis period was 30 minutes. The reaction was stopped by the addition of ice-cold trichloroacetic acid, and ATP hydrolysis was quantified by determining the amount of inorganic phosphate in each sample using a colorimetric method reported previously. ⁹ Na,K-ATPase activity was defined as the difference in ATP hydrolysis (inorganic phosphate release) measured in the presence and absence of ouabain. The data are calculated as nanomoles phosphate release per milligram protein per 30 minutes. 

Results

Intact porcine lenses were exposed to thrombin at a concentration of 0.001 to 1 unit/ml. After 10 minutes, ⁸⁶Rb uptake was measured either in the presence or absence of ouabain. At a concentration of 0.1 unit/ml or higher, thrombin significantly reduced the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake (Table 1) . The rate of ouabain-insensitive potassium (⁸⁶Rb) uptake was not inhibited by thrombin; the ouabain-insensitive potassium (⁸⁶Rb) uptake rate was 418 ± 34 nmoles potassium accumulated/g lens water/30 min in the presence of 1 unit/ml thrombin, which was not significantly different from the control value of 431 ± 33 (mean ± SEM; n = 18). 

In the presence of hirudin, an antagonist for thrombin receptors, the inhibitory effect of thrombin on ouabain-sensitive potassium (⁸⁶Rb) uptake was markedly suppressed; in lenses exposed to 2 units/ml hirudin, there was no significant difference between uptake in the presence or absence of thrombin (Fig. 1) . Added alone, hirudin did not alter ouabain-sensitive ⁸⁶Rb uptake. 

The rate of Na,K-ATPase–mediated ion transport can be reduced subsequent to a decrease of plasma membrane sodium permeability. To examine whether a reduction of sodium permeability causes the inhibitory effect of thrombin on ouabain-sensitive potassium (⁸⁶Rb) uptake, some lenses were exposed to thrombin in the presence of amphotericin B, which effectively clamps sodium permeability at a high value. The inhibitory effect of thrombin persisted in the presence of amphotericin B; thrombin reduced the ouabain-sensitive potassium (⁸⁶Rb) uptake rate to approximately 30% of the value observed in the presence of amphotericin B alone (Fig. 2) . As expected, the addition of amphotericin B alone stimulated the rate of ouabain-sensitive potassium (⁸⁶Rb) uptake. The finding that the inhibitory effect of thrombin on ouabain-sensitive potassium (⁸⁶Rb) uptake persists in the presence of amphotericin B suggests that thrombin might have a direct inhibitory effect on Na,K-ATPase activity. However, thrombin did not significantly inhibit Na,K-ATPase activity in membrane material isolated from the capsule epithelium of fresh (nonincubated) porcine lenses. Na,K-ATPase activity in lens epithelium membrane material measured under control conditions was 230 ± 2 nmoles phosphate released/mg protein/30 min (mean ± SE, n = 6 determinations), whereas the activity measured in the presence of 1 unit/ml thrombin was 236 ± 2. In a different set of experiments designed to measure Na,K-ATPase activity, intact porcine lenses were first incubated in Krebs solution either in the presence or absence of 1 unit/ml thrombin for 30 minutes, then membrane material was isolated from the epithelium and used for Na,K-ATPase measurements. Na,K-ATPase activity was significantly reduced in membrane material isolated from lenses that had been preincubated in the presence of thrombin (Fig. 3) . 

Thrombin appears to inhibit Na,K-ATPase activity when applied to the intact lens but not when applied to isolated cell membrane material. In several tissues, cell responses to thrombin involve the activation of nonreceptor tyrosine kinases. ^{10 11} To examine whether tyrosine kinase activation is involved in the lens response to thrombin, ⁸⁶Rb uptake studies were conducted in the presence or absence of either genistein or herbimycin, two recognized inhibitors of tyrosine kinases. Both genistein (150 μM) and herbimycin (15 μM) suppressed the inhibitory effect of 1 unit/ml thrombin on ouabain-sensitive potassium (⁸⁶Rb) uptake rate (Fig. 4) . In separate experiments, intact lenses were first incubated in Krebs solution in the presence or absence of 1 unit/ml thrombin for 30 minutes, and then membrane material was isolated from the epithelium and used for a Western blot analysis of tyrosine phosphoproteins. There was a marked increase in density of several phosphotyrosine bands in epithelium membrane material isolated from lenses that had been exposed to thrombin (Fig. 5) . However, no such changes were observed in epithelium membrane material from lenses that received thrombin in the presence of genistein. 

Discussion

Thrombin caused marked inhibition of active sodium–potassium transport in the intact porcine lens. The 1 unit/ml concentration of thrombin used in the present study was similar to that used to elicit responses in blood platelets ^{5 12} and considerably lower than that required to induce mitosis in cultured rabbit lens. ⁷ The ability of hirudin to partially suppress the thrombin response is consistent with the earlier suggestion that lens cells might express thrombin receptors. ⁷  

To our knowledge, there have been no measurements of thrombin in aqueous humor. It seems likely that the normal thrombin concentration in intraocular fluids is very low although thrombin could enter the aqueous humor after trauma, which leads to breakdown of the blood–aqueous barrier. In blood, local concentrations of thrombin are difficult to measure but it has been projected to approach 300 units/ml after trauma. ¹³ The expression of thrombomodulin in corneal endothelium, iris epithelium, ciliary epithelium, and lens capsule suggests that thrombin occassionally enters the aqueous humor compartment. ^{14 15 16}  

It is well known that inhibition of the rate of active sodium–potassium transport can follow a reduction in the flow of sodium into the cell across the plasma membrane, which might occur if sodium channels, Na/K/2Cl cotransporter, Na⁺-H⁺ exchange, or Na⁺-Ca²⁺ exchange mechanisms were inhibited. However, this does not seem a likely explanation for the thrombin response measured here in porcine lens because the inhibition of active sodium–potassium transport caused by thrombin persisted in the presence of amphotericin B. Amphotericin B is a pseudo ionophore that increases effective cation permeability, creating a pathway for cation movement that would shortcut any tendency toward reduced sodium entry in the presence of thrombin. It should be noted that the amphotericin B experiments do not rule out the possibility that thrombin-induced changes of sodium entry might have contributed in part to the observed change of ouabain-sensitive potassium (⁸⁶Rb) uptake, but the overriding response to thrombin appears to be Na,K-ATPase inhibition, as suggested by the marked reduction of ouabain-sensitive potassium (⁸⁶Rb) uptake that persists in the combined presence of thrombin and amphotericin B. Indeed, Na,K-ATPase activity was significantly reduced in membrane material isolated from the epithelium of thrombin-treated lenses. However, it is important to note that Na,K-ATPase inhibition was not observed when thrombin was added directly to isolated lens epithelium membrane material, suggesting that the Na,K-ATPase response to thrombin requires lens cells to be intact. Taken together with the ability of hirudin to partially suppress the thrombin effect on active sodium–potassium transport in the intact lens, the evidence is consistent with Na,K-ATPase inhibition after activation by thrombin of a receptor–second-messenger mechanism in lens epithelial cells. It should be noted that in freshly dissected porcine lens, there is abundant expression of theα ₁ isoform of Na,K-ATPase, whereas theα ₂ and α₃ isoforms are not detectable by immunoblot. ¹⁷ Thus, it is likely that the sodium pump inhibition observed in thrombin-treated porcine lenses stemmed for inhibition of the Na,K-ATPase α₁ isoform. 

The inhibitory effect of thrombin on lens active sodium–potassium transport was suppressed by both genistein and herbimycin, both recognized inhibitors of tyrosine kinases, ¹⁸ suggesting Na,K-ATPase inhibition could be downstream of a tyrosine kinase–mediated step. Thrombin receptors are members of a family of protease activated receptors (PARs), ¹⁹ and PAR-mediated responses often involve stimulation of cytoplasmic tyrosine kinases. ^{10 11} The inhibitory response of platelet plasma membrane Ca-ATPase to thrombin is also blocked by tyrosine kinase inhibitors and the Ca-ATPase polypeptide itself is tyrosine phosphorylated. ⁵ It is noteworthy that in the kidney, modulation of Na,K-ATPase activity might also involve activation of nonreceptor tyrosine kinases. ²⁰ The detection of increased tyrosine phosphorylation in epithelium membrane material after thrombin treatment of the intact lens confirms that thrombin activates one or more tyrosine kinases in the lens. However, additional experiments will be required in order for us to identify the multiple tyrosine kinase substrates and determine their possible role in the Na,K-ATPase response to thrombin. 

The inhibitory effect of thrombin on Na,K-ATPase is not unique to the lens. Reduced ouabain-sensitive potassium (⁸⁶Rb) uptake also was observed in cultured rabbit nonpigmented ciliary epithelium cells exposed to thrombin (data not shown). As discussed above, thrombin inhibits Na,K-ATPase in human platelets. ⁶ The functional significance of the thrombin-mediated change of lens active sodium–potassium transport is unclear since appreciable amounts of thrombin may only be presented to the lens during rare instances of blood-aqueous–barrier breakdown. However, thrombin is not the only protease that can activate PARs, ²¹ and thus it is possible that PARs in the lens are functionally activated by a ligand other than thrombin. It is noteworthy that cathepsins and other proteases are produced in the ciliary body and may be delivered by exocytosis to the aqueous humor that flows over the lens. ²² Recently, there has been a report that the level of cathepsin A is elevated in the aqueous humor of human eyes with senile cataract ²³ and following ocular trauma. ²⁴ It remains to be determined whether the high concentration of sodium known to occur in cataractous lens ²⁵ might be in part the result of Na,K-ATPase inhibition caused by proteases in the aqueous humor. 

 Reprint requests: Nicholas A. Delamere, Department of Ophthalmology and Visual Science, University of Louisville School of Medicine, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202.

 Supported by National Eye Institute, National Institutes of Health Grant EY09532, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc. Nicholas A. Delamere is the recipient of a Research to Prevent Blindness Senior Investigator Award.

 Submitted for publication November 17, 1998; revised March 19, 1999; accepted April 13, 1999.

 Proprietary interest category: N.

 

Table 1.

 

The Inhibitory Influence of Thrombin on Ouabain-Sensitive Potassium (⁸⁶Rb) Uptake

Thrombin Concentration (units/ml)	Ouabain-Sensitive Potassium 86Rb Uptake
0	100.00 ± 4.0
0.001	86.98 ± 3.9
0.01	85.12 ± 2.2
0.1	55.58 ± 4.0*
1	63.04 ± 6.1*

 Lenses were preincubated for 10 minutes in the presence of different concentrations of thrombin. After the preincubation period, ⁸⁶Rb was added for a further 30 minutes. Half of the lenses received ouabain (final concentration 1 mM) together with the ⁸⁶Rb. The data are the mean ± SE of results from 10 lenses. The control rate of ouabain-sensitive potassium (⁸⁶Rb) uptake was 1803 ± 73 nmoles/g lens water/30 min.

*  Significant difference from control (P < 0.01).

Figure 1.

 

The inhibitory influence of thrombin on ouabain-sensitive potassium (⁸⁶Rb) uptake is suppressed by hirudin. Lenses were preincubated for 10 minutes in the presence of either thrombin (1 unit/ml) or hirudin (2 units/ml) or thrombin + hirudin. Control lenses received neither hirudin nor thrombin. After the preincubation period, ⁸⁶Rb was added for a further 30 minutes. Half of the lenses received ouabain (final concentration 1 mM) together with the ⁸⁶Rb. The data are the mean ± SE (vertical bar) of results from at least 17 lenses. *Significant difference from control (P < 0.01).

Figure 2.

 

The inhibitory influence of thrombin on ouabain sensitive potassium (⁸⁶Rb) uptake persists in the presence of amphotericin B. Lenses were preincubated 10 minutes in the presence of either amphotericin B (5 μM) or thrombin (1 unit/ml) or amphotericin B plus thrombin. Control lenses received neither amphotericin B nor thrombin. After the preincubation period, ⁸⁶Rb was added for a further 30 minutes. Half of the lenses received ouabain (final concentration 1 mM) together with the ⁸⁶Rb. The data are the mean ± SE (vertical bar) of results from 12 lenses. *Significant difference from control (P < 0.01).

Figure 3.

 

Na,K-ATPase activity (ouabain-sensitive ATP hydrolysis) determined in membrane material isolated from the epithelium of lenses incubated 30 minutes in the presence or absence of thrombin (1 unit/ml). The data represent the mean ± SE (vertical bar) of results from 6 lenses. *Indicates a significant difference from control (P < 0.01).

Figure 4.

 

The influence of genistein and herbimycin on the lens thrombin response. Lenses were preincubated 10 minutes in the presence of either genistein (150 μM) or herbimycin (15 μM) or thrombin (1 unit/ml) or genistein + thrombin or herbimycin + thrombin. Control lenses received neither genistein nor herbimycin nor thrombin. After the preincubation period, ⁸⁶Rb was added for a further 30 minutes. Half of the lenses received ouabain (final concentration 1 mM) together with the ⁸⁶Rb. The data are presented as a percent of the control rate of ouabain-sensitive potassium (⁸⁶Rb) uptake and are the mean ± SE (vertical bar) of data from 6 to 11 lenses. *Indicates a significant difference from control (P < 0.01).

Figure 5.

 

The detection of tyrosine phosphoproteins in epithelium membrane material isolated from lenses exposed to thrombin. Lenses were incubated 30 minutes in the presence of thrombin (1 unit/ml) (T), genistein (150 μM) (G), or thrombin + genistein (TG). Control lenses (C) received neither thrombin nor genistein. Epithelium membrane material was isolated as described in the Materials and Methods section, and aliquots containing 75 μg protein were subjected to SDS gel electrophoresis and then blotted onto nitrocellulose and probed with an antibody directed against tyrosine phosphoprotein. Molecular size standards (reading from top to bottom) 208, 116, and 84 kDa are shown in the left hand lane.