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Ouabain-Sensitive Na-K Atpase Response in the Rabbit Iris-Ciliary Body After Lensectomy-Vitrectomy

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Ouabain-Sensitive Na-K Atpase Response in the Rabbit Iris-Ciliary Body After Lensectomy-Vitrectomy Investigative Ophthalmology & Visual Science, Vol. 33, No. 1, January 1992 Copyright © Association for Research in Vision and Ophthalmology Ouabain-Sensitive Na-K ATPase Response in the Rabbit Iris-Ciliary Body After Lensectomy-Vitrectomy Takezo Mito, Sei-ichi Ishiguro, and Makoto Tomai We studied ouabain-sensitive Na-K adenosine triphosphatase (ATPase) activities in the iris-ciliary body of rabbit eyes after lensectomy-vitrectomy. Changes in enzyme activities were quantitatively investigated in the plasma membrane of iris-ciliary body at 0 or 7 hours and at days 1,3,7, and 14. The specific activity of Na-K ATPase rose to significantly higher levels than the control value at 7 hours following surgery, but returned to the baseline value after 7 days. In addition, we evaluated enzyme activities after lensectomy-vitrectomy during which SF6 or silicone oil was injected. The specific activ- ity of Na-K ATPase following the injection of SF6 or silicone oil was significantly higher than the control value at 7 hours and did not return to the normal value even after 14 days. Consensual reaction, demonstrated by increased Na-K ATPase activity, also was found in the contralateral unoperated eyes of SF6- and silicone oil-injected rabbits. The increased Na-K ATPase activity in the iris-ciliary body after experimental surgery may play an important role in restoring swollen tissues. Invest Ophthalmol Vis Sci 33:172-177,1992 Ouabain-sensitive Na-K adenosine triphosphatase lateral unoperated eyes after surgery. Moreover, we (ATPase) may contribute to the regulation of cell vol- compared those findings with the results of lensec- ume and the maintenance and regulation of the intra- tomy-vitrectomy during which SF6 or silicone oil was cellular and extracellular ionic environment.1 Further- injected. more, it has been proposed that the secretion of aqueous humor results from active Na transport by Materials and Methods Na-K ATPase.2 The ciliary body contains a high con- centration of ouabain-sensitive Na-K ATPase,3 and Domestic albino rabbits, weighing about 2 kg each, the enzyme has been localized on the plasma mem- were obtained from a local breeder and were housed brane of ciliary body epithelial cells histochemi- under the same conditions. Rabbits were anesthetized cally.4"10 with an intravenous dose of xylazine and ketamine Mechanical trauma such as paracentesis causes hydrochloride. A lensectomy-vitrectomy was per- formed using the Peyman Vitrophage 9000 in the breakdown of the blood-aqueous barrier in rabbits'' 14 1213 12 13 right eye, as described by Cottingham and Forster and primates. Bartels et al and Okisaka showed 15 that the ciliary body, particularly in the anterior pars and Kaplan and coworkers. Ringer's lactate plus so- plicata region, is a source of secondary aqueous after lution (Ringer's lactate supplemented with 5.0 raM glucose and 18.3 mM bicarbonate) was used during paracentesis. 16 19 The iris-ciliary body also is affected during vitreous the operation. " After the lensectomy-vitrectomy, surgery by mechanical trauma, prolonged intraocular 30% SF6/fluid and silicone oil/air exchanges were per- irrigation, and various methods of tamponade. In this formed. Air was passed through a cellulose nitrate study, we evaluated changes in the iris-ciliary body filter (pore size = 0.02 /im; Toyo Roshi, Tokyo). Sili- through the biochemically measured activity of Na-K cone oil (1000 centistokes, specific gravity 0.98) was ATPase after experimental lensectomy-vitrectomy. purchased from Koken Co., Tokyo. Erythromycin We found increased specific activity of Na-K ATPase ointment was applied topically to the eye immedi- activity in the iris-ciliary body of operated and contra- ately afterward. Postoperative examinations were made at 1 hour and at 1, 3, and 14 days. Eyes were excluded from analysis if they had hemorrhage, reti- From the Department of Ophthalmology, Tohoku University nal detachment, or glaucoma postoperatively. The School of Medicine, Sendai 980, Japan. left eye served as the untreated control for each ani- Submitted for publication: November 20, 1990; accepted July mal. The care and treatment of animals in this inves- 25, 1991. Reprint requests: Sei-ichi Ishiguro, Department of Ophthalmol- tigation were in compliance with the ARVO Resolu- ogy, Tohoku University School of Medicine, Sendai, Miyagi 980, tion on the Use of Animals in Research. Japan. The time course of the biochemical study was mea- 172 Downloaded from iovs.arvojournals.org on 09/29/2021 No. 1 ATPASE RESPONSE IN IRIS-CILIARY DODY / Miro er ol 173 E 10.0- Fig. 1. Specific activity of Na-K ATPase in the final pellet of iris-ciliary body after sur- gery in experimental (closed squares) and con- trol (open squares) eyes. Values indicate mean 5.0- ± SD (n = 4). *P < 0.01 (paired t-test). 1 0- I 14 days sured from completion of the operation: 0 and 7 Chifflet et al.22 For the assay, the incubation time was hours, and 1, 3, 7, and 14 days. Rabbits were killed no more than 30 min, which was in a linear range. with a lethal injection of sodium pentobarbital at each The specific activity of Na-K ATPase was expressed indicated time. The eyes were rapidly enucleated and in IJM inorganic phosphate liberated/mg protein/ hemisected posterior to the ora serrata. The ciliary hour. Ouabain was used in parallel samples, so data body and iris were dissected from the anterior half. shown reflect differences between Na-K ATPase activ- Ciliary body and iris were homogenized, as previously ity measured ± ouabain in the incubation. described by Mittag et al,20 in 0.32 M sucrose-contain- For light microscopy, the enucleated eyeballs were ing histidine (30 mM) and EGTA (1 mM) with use of fixed with periodate-lysine-paraformaldehyde23 and Biotron's homogenizer, and centrifuged at 1,000 X g were embedded in paraffin. The 3-/im-thick sections for 10 min. The supernatant was decanted and recen- were sliced and stained with hematoxylin-eosin. trifuged at 12,000 X g for 60 min. The pellet was resus- pended with 0.1% deoxycholate in 0.25 M sucrose Results and the extract was centrifuged at 100,000 X g for 60 min. The final pellet was resuspended in water. Pro- Figure 1 shows the specific activities of Na-K ATP- tein determinations were done by the method of ase. Immediately (0 hour) following surgery, the Na- 21 Lowry et al. K ATPase activity corresponded to the control value. The Na-K ATPase assay was performed at 37°C at At 7 hours after surgery, the activity was 2.3 times pH 7.5, according to the methods of Mittag et al20 and higher than the control value. On days 1 and 3, the «> 10.0- Fig. 2. The effect of SF6 (open squares) and silicone oil (open circles) on specific activities of Na-K ATPase in the final pellet of iris- ciliary body was compared with that of vitrec- tomy (Ringer's lactate plus solution, closed squares) alone. Values indicate mean ± SD (n = 4). *P < 0.01; **P < 0.05 (paired t-test). 0J 0 7h 1 14 days Downloaded from iovs.arvojournals.org on 09/29/2021 174 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / January 1992 Vol. 33 Fig. 3. Na-K ATPase response in the contra- lateral, unoperated eyes after lensectomy-vi- 1 0- trectomy. Enzyme preparation is described in Materials and Methods. Iris-ciliary body in the contralateral unoperated eyes of lensec- tomy-vitrectomy alone (Ringer's lactate plus solution, open squares) shows no consensual o « reaction. Conversely, consensual reaction, 2 6- demonstrated by increased Na-K ATPase spe- cific activity in the iris-ciliary body, is ob- served in the contralateral unoperated eyes of (0 *-* 4 - silicone oil- (closed squares) and SF6-injected (open circles) rabbits compared with those that underwent lensectomy-vitrectomy alone. 2 - Prolonged increase of the specific activity in the contralateral eyes of SF6-injected rabbits is observed. Values indicate mean ± SD (n = 4). J 0 *P < 0.01; **P < 0.05 (paired t-test). DAYS 1 4 activities were 1.5 times higher than the control value. 4c). Conversely, when SF6 injection (Fig. 4d) or sili- However, after day 7, activity decreased and corre- cone oil injection (Fig. 4e) was performed, the ciliary sponded to the control value. body remained edematous even after day 14. No swell- The results of specific activities of Na-K ATPase on ing was observed in the ciliary body of the contralat- the effect of SF6 and silicone oil are summarized in eral eyes on day 14 after SF6 injection (Fig. 4f) nor in Figure 2. In the beginning, the specific activity of Na- the iris on day 1 after lensectomy-vitrectomy alone K ATPase after the injection of SF6 or silicone oil (Fig. 4g). corresponded to the specific activities of Ringer's lac- tate plus solution. However, these increased specific Discussion activities of the enzyme did not return to the control value even after 14 days. In the rabbit, the lens is disproportionately large, Consensual reaction, that is, increased Na-K ATP- and we found it technically impossible to perform a ase specific activity, was observed in the iris-ciliary pars plicata vitrectomy without simultaneously re- body of contralateral unoperated eyes (Fig. 3). The moving the lens. Therefore, a lensectomy was in- specific activity in unoperated eyes of silicone oil-in- cluded in our experiment.1424 Technically, removing jected rabbits was significantly higher than that of residual lens capsule and cortex completely near the Ringer's lactate plus solution-substituted rabbits on ciliary body using the Peyman Vitrophage 9000 also day 1 (P < 0.01). Thereafter, it returned to the basal was difficult.
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