Investigative Ophthalmology & Visual Science, Vol. 31, No. 10, October 1990 Copyright Association for Research in Vision and Ophthalmology

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1 Investigative Ophthalmology & Visual Science, Vol. 31, No. 10, October 1990 Copyright Association for Research in Vision and Ophthalmology Alteration of Sodium, Potassium-Adenosine Triphosphatase Activity in Rabbit Ciliary Processes by Cyclic Adenosine Monophosphate-Dependent Protein Kinase Nicholas A. Delamere, Robin (V Socci, and Kara L. King The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (camp)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10~ 5 M camp, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added camp. Likewise camp alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the camp-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 Mg/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, camp may alter sodium pump activity. In parallel 86 Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl camp or by forskolin. Invest Ophthalmol Vis Sci 31: , 1990 It is widely believed that the formation of aqueous humor is somehow coupled to active solute transport by the epithelium of the ciliary processes. The dense localization of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) in the ciliary epithelium 1 together with a number of physiologic studies 2 support the notion that active sodium transport contributes to the mechanism of aqueous production. However, the precise relationship between the sodium pump of the ciliary processes and fluid secretion has yet to be resolved. Aqueous flow might be linked directly to active sodium transport, but it is also possible that the From the Department of Ophthalmology and Visual Sciences, and Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky. *Present address: Department of Physiology & Endocrinology, Medical College of Georgia, Augusta, GA Supported by USPHS research grant number EY06915, the Kentucky Lions Eye Research Foundation, and an unrestricted grant from Research To Prevent Blindness Inc. Reprint requests: N. A. Delamere, PhD, Kentucky Lions Eye Research Institute, 301 East Muhammad Ali Boulevard, Louisville, KY ion gradients set up by the Na,K-ATPase drive transport mechanisms for anions such as bicarbonate or chloride, and that fluid flow is coupled to anion movement. In either case, aqueous humor formation might be altered if Na,K-ATPase activity changed. We examined one of the ways in which the sodium pumping rate of the ciliary processes might be regulated. Experiments were done to determine whether the Na,K-ATPase activity in ciliary processes is modulated by the cyclic adenosine monophosphate (camp)-dependent protein kinase, protein kinase A. There is evidence that perturbation of camp levels and adenylate cyclase activity in the ciliary processes can change the rate of aqueous humor production; 3 therefore camp might be capable of altering transport behavior in the ciliary epithelium. 4 In addition camp alters the short-circuit current across the irisciliary body. 5 Although its mode of action in the ciliary processes is not known, the role of camp as a second messenger has been studied extensively. 6 Cellular camp responses are often mediated by campdependent protein kinase which is activated by the cyclic nucleotide. 2164

2 No. 10 CILIARY PROCESS SODIUM TRANSPORT / Delamere er ol 2165 To measure Na,K-ATPase activity in membrane material from iris-ciliary body and ciliary processes we used an approach established by Riley and KJshida 7 and modified by Socci and Delamere. 8 We tested whether Na,K-ATPase activity was altered in the presence of either camp-dependent protein kinase or the catalytic subunit of this kinase. To assess whether other transport ATPases might be influenced by the protein kinase, calcium-stimulated ATPase (Ca-ATPase) activity was also examined. In addition, 86 Rb uptake experiments were used to assess the ion transport activity of the sodium pump in intact segments of rabbit iris-ciliary body. Chemicals Materials and Methods The 3',5'-cAMP-dependent protein kinase and the catalytic subunit of 3',5'-cAMP-dependent protein kinase were obtained from Sigma (St. Louis, MO) as were dibutryl camp (N 6,2'-O-dibutryladenosine 3',5'-cyclic monophosphate) and forskolin. The 86 RbCl and ATP, as a triethylammonium salt labeled with 32 P in the terminal phosphate group, were purchased from Amersham (Arlington Heights, IL). All other chemicals were obtained from Fisher (Pittsburgh, PA) or Sigma. Membrane Preparation Tissues were obtained from 2-kg albino rabbits killed with an overdose of sodium pentobarbital administered through an ear vein. The procedures used in these studies conform to the ARVO Resolution on the Use of Animals in Research. After death, the eyes were removed and dissected open posteriorly. The lens was removed, and the ciliary body with attached iris was dissected from the eye. In some experiments, ciliary processes were gently dissected free by cutting along the length of each process from the posterior margin toward the pupil. Using either iris-ciliary body or ciliary processes, a crude membrane preparation was obtained by a technique developed by Riley and Kishida 7 (shaking the tissues with glass beads dislodges epithelial cells from the ciliary processes). This technique was described in detail by Socci and Delamere. 8 In brief, iris-ciliary bodies or ciliary processes from several eyes were shaken gently with 50 ml of calcium-, magnesium-free Kreb's solution containing glass beads to disperse the cells. The material was then filtered through coarse gauze and centrifuged at 1000 X g for 8 min at a temperature of 2 C which was maintained throughout the rest of the preparative protocol. The pellet, which contained large particulates and red blood cells, was discarded. The supernatant was then given four strokes in a Teflon/ glass douncer (Kontes, Vineland, NJ) and centrifuged at 15,000 X g for 15 min. This pellet, containing mitochondrial material, was discarded, and the supernatant was centrifuged at 80,000 X g for 75 min. Using a Teflon/glass grinder, the resulting pellet was resuspended in a buffer containing 300 mm sucrose, 10 mm HEPES, and 2 mm dithiothreitol at ph 7.4; this was then centrifuged at 80,000 X g for 75 min. The final pellet was the crude membrane material which we used for ATPase assays. It was resuspended in a small volume of buffer, divided into aliquots, and stored in liquid nitrogen. The protein content of the membrane material was determined by a modified Lowry method, 9 using bovine serum albumin as a standard. Compared with its activity in a tissue homogenate, Na,K-ATPase activity in the iris-ciliary body preparation was enriched sixfold. In selected experiments, ATPase measurements were made using crude membrane preparations obtained from either rabbit kidney or rabbit lens epithelium. Lens epithelium was collected by stripping the lens capsule, together with the epithelial layer, from the lens fiber mass. Pooled lens epithelial samples or diced pieces of kidney were homogenized and the material differentially centrifuged after a protocol similar to that for the ciliary processes. Determination of Na,K-ATPase Activity The Na,K-ATPase activity was measured in tubes containing 350 [A of solution with 50 mm histidine HC1, 10 mm KC1, 3 mm MgCl 2, 100 mm NaCl, and 0.8 mm Tris-EGTA (ethylene glycol bis(aminoethyl ether)n,n,n',n'-tetraacetic acid) at ph 7.2. The solution also contained alamethicin (11 ng/m\) which was included to make any sealed membrane vesicles permeable and counter the possibility that Na,K-ATPase activity is influenced by cation gradients. In each experiment, assays were done both in the presence and absence of 1 mm ouabain to determine the ouabaininhibitable ATPase activity, which represents the Na,K-ATPase activity. In specified experiments with camp-dependent protein kinase, the kinase was dissolved in the ATPase buffer with and without 10~ 5 M camp less than 10 min before the start of the assay. Thus, ATP hydrolysis was measured under six different sets of conditions: (1) control; (2) control and ouabain; (3) control and protein kinase; (4) control, protein kinase, and ouabain; (5) control, protein kinase, and camp; and (6) control, protein kinase, camp, and ouabain. In experiments with the protein kinase catalytic subunit, this too was dissolved immediately before use, but the solutions did not contain camp. In selected experiments, the catalytic subunit was dissolved in the ATPase buffer, quickly filtered,

3 2166 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / October 1990 Vol. 01 and washed three times using a Diaflow Ultrafilter (10,000 molecular weight cutoff) (Amicon, Lexington, MA) to remove any possible contaminants. Ultrafiltration did not noticeably alter the influence of the subunit on ATPase activity. The tubes containing the assay mixture were warmed to 37 C for 5 min, and the ATPase reaction was started with the addition of 32 P-labeled ATP to a final concentration of 1 mm. After 20 min, duplicate 100-/il aliquots of the mixture were removed and added to 50-/*l ice-cold 10% trichloroacetic acid (TCA) to stop the reaction. In separate experiments, we determined that ATP hydrolysis is linear with time until at least 45 min. The amount of radiolabeled inorganic phosphate release was determined by the method used in our previous studies, 10 and ATPase activity was expressed as /L*mol phosphate released/mg protein/hr. The Na,K-ATPase activity was calculated as the difference between the ATPase activity measured in the presence and absence of 1 mm ouabain. Approximately 30% and 50% of the ATPase activity was ouabain inhibitable in the iris-ciliary body and ciliary process membrane preparations, respectively. Determination of Ca-ATPase Activity The Ca-ATPase activity was measured in 350 /A of a solution containing 3 Mg/ml A23187 calcium ionophore, 50 mm histidine, 100 mm KC1, 3 mm MgCl 2, 5 mm NaN 3, and 0.5 mm ATP (including 32 P-labeled ATP) at ph 7.2 in the presence of either 15 mm EGTA or a Ca 2+ -EGTA buffer containing a free calcium concentration of 10" 6 M. Based on the method of Kirchberger et al," the ionophore, A23187, was used to make spontaneously forming vesicles calcium permeable. Sodium azide was used to inhibit any mitochondrial activity as described earlier. 8 The Ca 2+ - EGTA buffer was prepared to contain specified free calcium concentrations using the formulae and binding constants specified earlier. 10 The assay tubes were prewarmed 5 min, and the ATPase reaction was started with the addition of an aliquot of radiolabeled ATP solution. After 15 min, duplicate 100-/*l aliquots were taken from each tube, mixed with 50-/A ice-cold 10% TCA, and phosphate release determined as described. The ATPase activity is linear with time up to at least 45 min, and the Ca-ATPase activity was calculated as the difference in ATPase activity measured in the presence and absence of 10~ 6 M calcium. Thus, to test the influence of the protein kinase catalytic subunit on the Ca-ATPase, ATPase activity was determined under four conditions: (1) control; (2) control and 10~ 6 M Ca 2+ ; (3) control and subunit; and (4) control, subunit, and 10" 6 M Ca Rb Uptake Experiments The ciliary body with attached iris was dissected from the eye and divided into four segments which were incubated for 60 min at 37 C in control Kreb's solution containing 110 mm NaCl, 6 mm KC1, 1.1 mm KH 2 PO 4, 1 mm MgCl 2, 5.5 mm glucose, and 25 mm NaHCO 3 at ph 7.4. The solution was aerated with 95% O 2 /5% CO 2. This preincubation period was included in the protocol to rid the tissue of endogenous substances released during the dissection. After the preincubation, each tissue piece was transferred to a sealed vial containing Kreb's solution equilibrated with 95% O 2 /5% CO 2 and incubated for 45 min in the presence of approximately 1 jici/ml of 86 RbCl. Parallel uptake studies were done in the presence and absence of 1 mm ouabain. In specified experiments, dibutryl camp or forskolin was added to the radioactive Kreb's solution. At the end of 45 min, each tissue piece was blotted on moistened filter paper, weighed, and digested in 30% nitric acid. The radioactivity (dpm/ml) in the tissue digests and aliquots of the loading solution was determined by scintillation counting. The 86 Rb accumulation was used to compute the rate of ouabain-inhibitable potassium uptake by the iris-ciliary body segments expressed as nmol potassium/mg tissue wet weight/hr. Under control conditions, the linearity of the ouabain-inhibitable potassium uptake process was determined by measuring the radioactivity in tissue pieces loaded for periods of 15, 30, 45, and 60 min. The uptake was linear (correlation coefficient, 0.98) with a slope of 31 nmol K + /mg tissue wet weight/hr and an intercept that was not significantly different from zero. Na,K-ATPase Studies Results The ATPase activity was measured in a crude membrane preparation obtained from rabbit iris-ciliary body. The control ouabain-sensitive, Na,K-ATPase activity was 6.3 ± 0.5 jtimol phosphate released/ mg protein/hr (mean ± standard error [SE], n = 8); the ouabain-insensitive ATPase activity was 14.6 ± 3.4. In the presence of camp-dependent protein kinase (370 units/ml) together with 10" 5 M camp, the Na,K-ATPase activity was 3.4 ± 0.6 ^mol phosphate released/mg protein/hr which was significantly less (P < 0.05) than the control Na,K-ATPase activity. In the presence of 370 units/ml camp-dependent protein kinase but no added camp, Na,K-ATPase activity was 6.1 ± 0.8 which was not significantly different from the observed control activity. Similarly, the ouabain-insensitive ATPase activity of the

4 No. 10 CILIARY PROCESS SODIUM TRANSPORT / Delomere er ol 2167 iris-ciliary body preparation was not significantly altered by the protein kinase in either the presence or absence of camp. If the protein kinase was heated to 90 C for 5 min before use, no significant alteration of Na,K-ATPase activity was observed when it was added in the presence of camp. Similarly, camp added alone to a concentration of up to lmm did not influence Na,K-ATPase activity. These observations suggest that when camp-dependent protein kinase is activated by camp, alteration of Na,K-ATPase activity takes place. This infers that the kinase catalytic subunit might alter Na,K-ATPase activity. To test this notion, we measured Na,K-ATPase activity in the iris-ciliary body membrane preparation in the presence of camp-dependent protein kinase catalytic subunit. The catalytic subunit (370 units/ml) reduced Na,K-ATPase activity to a value of 1.1 ±0.4 from a control value of 5.9 ± 0.5 (mean ± SE, n = 4) ^mol phosphate released/mg protein/hr. To pinpoint whether Na,K-ATPase from the ciliary processes is influenced by camp-dependent protein kinase, experiments were also done using a crude membrane preparation obtained from isolated rabbit ciliary processes. The ciliary process preparation had a control Na,K-ATPase activity of 15.8 ± 2.4 fxmol phosphate released/mg protein/hr (mean ± SE, n = 5 membrane preparations); the ouabain-insensitive ATPase activity was 16.4 ± 3.9. The response of the > Si TS 2 ca a to Q- "O V- 0) f CO z 5 a Catalytic subunit concentration (ug/ml) Fig. 1. The influence of camp-dependent protein kinase catalytic subunit upon Na,K-ATPase activity in a membrane preparation obtained from rabbit ciliary processes. (1 /xg/ml catalytic subunit is equivalent to 46 u/ml.) The data are the mean ± SE of measurements obtained from five separate membrane preparations. 2 control kinase control kinase control kinase o E 3" n no added ouabain ra 1 mm ouabain Fig. 2. The influence of camp-dependent protein kinase catalytic subunit (370 units/ml) upon ATPase activity in membrane material obtained from rabbit ciliary processes and kidney and lens epithelium. Total ATPase activity was measured in the absence of ouabain (open bars). Ouabain-insensitive ATPase activity (shaded bars) was determined in the presence of I mm ouabain. ciliary process Na,K-ATPase to the kinase catalytic subunit was a dose-dependent suppression of activity; lower concentrations of the subunit caused lesser reductions of enzyme activity (Fig. 1). The ouabain-insensitive component of the ATPase activity was not altered significantly by the catalytic subunit (Fig. 2). Studies With Lens Epithelium and Kidney Na,K-ATPase To examine whether the response of the Na,K- ATPase to the kinase catalytic subunit is unique to the iris-ciliary body and ciliary processes, experiments were also done using membrane material obtained from rabbit kidney and rabbit lens epithelium (Fig. 2). When Na,K-ATPase activity was measured in the presence of 370 units/ml camp-dependent protein kinase catalytic subunit, the activity in the kidney preparation was reduced significantly (P < 0.05) from a control value of 17.2 ± 2.4 to a value of 3.4 ± 1.6 ^mol phosphate released/mg protein/hr (mean ± SE, n = 4). However, in the lens epithelium, the control Na,K-ATPase activity was 3.5 ± 0.5; its activity in the presence of 370 units/ml catalytic subunit was 2.1 ± 0.4 /imol phosphate released/mg protein/hr (mean ± SE, n = 4) which was not significantly different from the control value. Ouabain-insensitive ATPase activities were not significantly altered by the subunit in either lens or kidney material. Ca-ATPase Studies To test whether the catalytic subunit suppresses the activity of other ATPases, the effect upon ciliary pro-

5 2168 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1990 Vol. 01 cess Ca-ATPase was examined. Its activity was altered by the subunit with no added camp. However, in contrast to the Na,K-ATPase response, Ca-ATPase activity was stimulated in the presence of the catalytic subunit. The control Ca-ATPase activity was 2.6 ± 0.4; its activity measured in the presence of 370 units/ml camp-dependent protein kinase catalytic subunit was 23.8 ± 0.6 pmol phosphate released/mg protein/hr (mean ± SE, n = 4 membrane preparations). The response of the Ca-ATPase to the catalytic subunit was dose-dependent (Fig. 3). In the presence of the subunit (370 units/ml) but no added calcium, the ATPase activity was 1.6 ± 3.1 fimol phosphate released/mg protein/hr; this value was not significantly different from the control activity measured in calcium-free conditions. No detectable Ca-ATPase activity was observed in blanks that contained the catalytic subunit but no membrane protein. 86 Rb Uptake Studies The Na,K-ATPase experiments suggested that sodium pump activity might be altered if camp were to c '<D +-> o a s E >, ' T3 U CD CO CO h- CD O co o co jd O E Catalytic subunit concentration (ug/ml) Fig. 3. The influence of camp-dependent protein kinase catalytic subunit upon calcium-stimulated ATPase activity in a membrane preparation obtained from rabbit ciliary processes. (1 Mg/ml catalytic subunit is equivalent to 46 u/ml.) The data are the mean ± SE of measurements obtained from four separate membrane preparations. Table 1. The influence of dibutryl cyclic AMP and forskolin upon potassium uptake by rabbit iris ciliary body Control Dibutryl camp (1(T 3 M) Forskolin (5X 1(T 5 M) Ouabaininhibitable component 31.2 (±0.9) 22.4* (±3.0) 21.9* (±3.0) Ouabaininsensilive component 9.3 (±0.1) 9.1 (±0.3) 9.4 (±0.2) Potassium uptake was measured by determining the amount of 86 Rb accumulated by segments of iris-ciliary body during an incubation period of 1 hr. The data are presented as nmoles potassium/mg tissue wet wt/hr and are given as the mean ± SE (n = 4). * Significantly different from control (P <.05). activate the camp-dependent protein kinase in the iris-ciliary body or in the ciliary processes. Since tracer uptake studies can be done with segments of iris-ciliary body, 1213 we examined whether there was a change of 86 Rb uptake in the presence of dibutryl camp or forskolin, an activator of adenylate cyclase. The rate of sodium-potassium pumping was monitored using 86 Rb as a tracer for the inward transport of potassium. From parallel uptake studies done in the presence and absence of 1 mm ouabain, the ouabain-inhibitable uptake component was determined. Over 60 min, the ouabain-inhibitable uptake was linear with time. When the iris-ciliary body was incubated in the presence of 5 X 10~ 5 M forskolin, the rate of the ouabain-inhibitable uptake component was reduced by 30% (Table 1). Ouabain-inhibitable uptake was also suppressed to a similar extent when the irisciliary body was exposed to 10~ 3 M dibutryl camp. Lesser concentrations of dibutryl camp had no significant effect. Neither compound caused a significant alteration of the ouabain-insensitive uptake component. Discussion These studies show that rabbit iris-ciliary body Na,K-ATPase activity is diminished by a commercially available preparation of camp-dependent protein kinase when camp is added to activate the kinase. The Na,K-ATPase activity was also suppressed by the kinase catalytic subunit alone. Reduction of Na,K-ATPase activity by the subunit was also seen in membranes obtained from isolated ciliary processes. These effects were all seen with a commercial preparation of the kinase. We cannot be certain that the protein kinase present in the rabbit iris-ciliary body or ciliary processes would give the same response. The Na,K-ATPase response to the kinase subunit

6 No. 10 CILIARY PROCESS SODIUM TRANSPORT / Delomere er ol 2169 does not appear to reflect general interference with all ATPases since Ca-ATPase was stimulated under the same conditions. The ciliary process Na,K-ATPase response may not be unique since Na,K-ATPase from kidney was similarly reduced, although lens epithelium Na,K-ATPase activity was not significantly altered. These Na,K-ATPase experiments suggest that activating protein kinase A in the iris-ciliary body might suppress Na,K-ATPase activity which could slow active sodium transport. Since activation of the protein kinase to release the catalytic subunit is caused by camp, we asked whether increasing tissue camp altered the sodium-potassium pump rate. In segments of iris-ciliary body, ouabain-inhibitable potassium uptake was reduced by 30% when 1 mm dibutryl camp was added to the bathing medium. Ouabaininhibitable potassium uptake was also reduced by forskolin, which activates adenylate cyclase in rabbit ciliary processes. 14 These 86 Rb uptake experiments suggest that increasing camp in the intact iris-ciliary body may slow active sodium-potassium transport in isolated pieces of tissue, but we cannot speculate on the effect of camp on blood-to-aqueous sodium transport. Furthermore, it is unlikely that an altered Na,K-ATPase activity is the only change caused by camp. It probably causes other changes in membrane function, such as alterations of permeability and perhaps long-term alterations of the amount of ATPase sites in the plasma membrane; these changes might also contribute to the observed lowering of 86 Rb uptake. Finally, segments of iris-ciliary body contain various cell types, and these may have different responses; the 86 Rb uptake studies reflect the combined response of these cell types to camp, but we cannot identify the specific response of each component. The camp-dependent modulation of epithelial transport parameters has been observed in several tissues Both adrenergic agonist-dependent and camp-dependent increases in transcorneal short circuit current have been convincingly attributed to activation of chloride transport mechanisms In addition, there are studies that suggest the characteristics of ion channels can be altered by camp. 19 ' 20 In the rabbit, adrenergic agonists, such as isoproterenol, reduce the short-circuit current measured across the isolated iris-ciliary body; camp has the opposite effect. 5 There is evidence for camp-dependent protein phosphorylation in rabbit ciliary processes 21 and in cultured human and rabbit ciliary epithelium. 22 This protein kinase has also been isolated from rabbit ciliary processes along with other protein kinases. 23 The camp-dependent modulation of Na,K-ATPase activity has been documented previously by Lingham and Sen 24 using material from rat brain. These investigators suggested that Na,K-ATPase may be inhibited by camp-dependent protein kinase. They postulated that the kinase might work by phosphorylating an intermediate membrane protein, although the direct influence of the camp-dependent protein kinase catalytic subunit upon microsomal Na,K-ATPase was not measured. However, in shark rectal gland, Marver et al 25 obtained experimental evidence from ouabain-binding studies which suggested camp stimulates Na,K-ATPase activity. Alteration of sodium-pump activity by phorbol esters, vasopressin, and angiotensin in hepatocytes has been cautiously attributed to modulation of Na 5 K-ATPase function by another protein kinase, protein kinase C, but direct ATPase studies are lacking. 26 ' 27 The studies of Na,K-ATPase modulation are complex because the catalytic subunit of the ATPase is genetically expressed in several isoforms that have different sensitivities to a number of agents, including ouabain and insulin. 28 Both the alpha+ and alpha form of the Na,K-ATPase have been observed in ciliary epithelium. 29 The isoforms could respond differently to a protein kinase. The camp-dependent modulation of enzyme activity has been documented for Ca-ATPase from several sources. On the basis of our experiments, we cannot specify how much of the ciliary process Ca- ATPase is derived from plasma membrane and how much from endoplasmic reticulum. The two ATPases might respond to the protein kinase differently. In general, however, camp-dependent protein kinase stimulates Ca-ATPase activity and active calcium transport In the ciliary processes, there is no clear physiologic significance for an increased Ca-ATPase activity when camp-dependent protein kinase is activated. Calcium is a recognized second messenger, and it might benefit a cell to reduce cytoplasmic calcium levels at the moment when camp-mediated second-messenger events are activated. In summary, our in vitro study using commercial preparations of the camp-dependent protein kinase suggests that ciliary process Na,K-ATPase activity could be suppressed when the kinase is activated. If this is true in vivo, then events that trigger an increase of camp in the ciliary epithelium might have a tendency to change the rate of sodium transport by the tissue. This could interfere with aqueous formation, but since the precise contribution of sodium transport to aqueous formation is not clear, it would be premature to predict the outcome. Furthermore, other effects of camp, on cell membrane permeability for example, might counterbalance sodium-pump

7 2170 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / October 1990 Vol. 31 changes. Despite this, it is known that forskolin and cholera toxin, which both increase camp in the ciliary epithelium, each lower the rate of aqueous inflow in laboratory animals Key words: ciliary processes, Na,K-ATPase, Ca-ATPase, cyclic AMP, rabbit, 86 Rb accumulation, iris-ciliary body References 1. Usukura J, Fain GL, and Bok D: 3 H ouabain localization of Na-ICATPase in the epithelium of the rabbit ciliary body pars plicata. Invest Ophthalmol Vis Sci 29:606, Davson H: Aqueous humor and the intraocular pressure. In Physiology of the Eye, Davson H, editor. New York, Academic Press, 1980, pp Sears ML: Regulation of aqueousflowby the adenylate cyclase receptor complex in the ciliary epithelium. Am J Ophthalmol 100:194, Sears ML: Autonomic nervous system: Adrenergic agonists. In Pharmacology of the Eye, Sears ML, editor. Berlin, Springer- Verlag, 1984, pp Chu T-C and Candia O: Effects of adrenergic agonists and cyclic AMP on the short-circuit current across the isolated rabbit iris-ciliary body. Curr Eye Res 4:523, Berridge MJ: The molecular basis of communication within the cell. Sci Am 253:142, Riley MV and Kishida K: ATPases of ciliary epithelium: Cellular and subcellular distribution and probably role in secretion of aqueous humor. Exp Eye Res 42:559, Socci RR and Delamere NA: The effect of vanadate upon calcium-stimulated ATPase of the rabbit iris-ciliary body. Invest Ophthalmol Vis Sci 29:1866, Peterson GL: A simplification of the protein assay of Lowry et al. which is more generally applicable. Ann Biochem 83:346, Borchman D, Delamere NA, and Paterson CA: Ca-ATPase activity in the rabbit and bovine lens. Invest Ophthalmol Vis Sci 29:982, KJrchberger MA, Borchman D, and Kasinathan C: Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump. Biochemistry 25:5484, Krupin T, Fritz C, and Becker B: Maturation of Rb + and PAH accumulation by the anterior uvea and choroid plexus. Invest Ophthalmol Vis Sci 26:159, Socci RR and Delamere NA: Characteristics of ascorbate transport in the rabbit iris-ciliary body. Exp Eye Res 46:853, Caprioli J, Sears M, Bausher L, Gregory D, and Mead A: Forskolin lowers intraocular pressure by reducing aqueous inflow. Invest Ophthalmol Vis Sci 25:268, Baba WI, Smith AJ, and Townshend MM: The effects of vasopressin, theophylline and cyclic 3'-5'-adenosine monophosphate (cyclic AMP) on sodium transport across the frog skin. Q J ExpPhysiol 52:416, Field M: Ion transport in rabbit ileal mucosa: II. Effects of cyclic 3',5'-AMP. Am J Physiol 221:992, Chalfie M, Neufeld AH, and Zadunaisky JA: Action of epinephrine and other cyclic AMP-mediated agents on the chloride transport of the frog cornea. Invest Ophthalmol 11:644, Klyce SD, Neufeld AH, and Zadunaisky JA: The activation of chloride transport by epinephrine and Db cyclic-amp in the cornea of the rabbit. Invest Ophthalmol 12:127, Walsh DB and Kass RS: Regulation of a heart potassium channel by protein kinase A and C. Science 242:67, Palmer LG and Sackin H: Regulation of renal ion channels. FASEBJ 2:3061, Yoshimura N, Mittag TW, and Podos SM: Cyclic nucleotidedependent phosphorylation of proteins in rabbit ciliary processes. Invest Ophthalmol Vis Sci 30:875, Coca-Prados M: Regulation of protein phosphorylation of the intermediate-sized filament vimentin in the ciliary epithelium of the mammalian eye. J Biol Chem 260:10332, Mittag TW, Yoshimura N, and Podos SM: Phorbol ester: Effect on intraocular pressure, adenylate cyclase, and protein kinase in the rabbit eye. Invest Ophthalmol Vis Sci 28:2057, Lingham RB and Sen AK: Regulation of rat brain (Na +,K + )- ATPase activity by cyclic AMP. Biochim Biophys Acta 688:475, Marver D, Lear S, Marver LT, Silva P, and Epstein FH: Cyclic AMP-dependent stimulation of Na,K-ATPase in shark rectal gland. J Membr Biol 94:205, Lynch CJ, Wilson PB, Blackmore PF, and Exton JH: The hormone-sensitive hepatic Na + -pump: Evidence for regulation by diacylglycerol and tumor promoters. J Biol Chem 261:14551, Yingst DR: Modulation of the Na,K-ATPase by Ca and intracellular proteins. Annu Rev Physiol 50:291, Lytton J, Lin JC, and Guidotti G: Identification of two molecular forms of (Na +,K + )-ATPase in rat adipocytes. J Biol Chem 260:1177, Coca-Prados M and Lopez-Briones G: Evidence that the and a(+) isoforms of the catalytic subunit of (Na +,K + ) ATPase reside in distinct ciliary epithelial cells of the mammalian eye. Biochem Biophys Res Comm 145:460, Tada M and Katz AM: Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol 44:401, Adunyah SE and Dean WL: Regulation of human platelet membrane Ca 2+ transport by camp- and calmodulin-dependent phosphorylation. Biochim Biophys Acta 930:401, Gregory D, Sears M, Bausher L, Mishima H, and Mead A: Intraocular pressure and aqueous flow are decreased by cholera toxin. Invest Ophthalmol Vis Sci 20:371, 1981.