stromal or aqueous side of the tissue, its eVects are less than when it is present simultaneously on both sides, indicating that indeed the Naþ,Kþ activated ATPase is active on the basolateral membranes of both the PE and NPE cells. As illustrated by Fig. 8, ouabain induced changes in composition are shared by contiguous NPE and PE cells. The loss of Kþ and gain of Naþ are strongly linked within a given NPE or PE cell (Fig. 9). Furthermore, the compositions of contiguous NPE and PE cells are also consistently and strongly linked to one another even when neighboring NPE–PE cell couplets are aVected to very diVerent extents by ouabain (Fig. 10). These findings indicate that, although under physiological conditions cells are linked through gap junctions, diVerent pairs of cells diVer in the activities of their membrane pathways.

E.Relationship of the EMPA Findings to the Consensus Model for Aqueous Humor Secretion

The results from the EMPA studies are consistent with the model that has emerged from a variety of studies of ciliary epithelial function. At the basolateral membrane of the PE cells, there is uptake of Naþ and Cl through electroneutral Cl /HCO3 and Naþ/Hþ exchangers. Carbonic anhydrase both catalyzes the production of HCO3 (Meldrun and Roughton, 1933) and stimulates Cl /HCO3 (Sterling et al., 2001) and Naþ/Hþ

FIGURE 8 Relationship between elemental compositions of contiguous paired NPE and PE cells in the anterior epithelium after aqueous ouabain (McLaughlin et al., 2004). The correlation coeYcient (r) is 0.95 for both least squares fits, indicating a strong correlation between ionic changes in adjoining the NPE and PE cells. Used with the permission of the American Physiological Society.

FIGURE 9 Relationship between Na/P and K/P in paired anterior NPE (open circles) and PE cells (filled circles) after aqueous ouabain (McLaughlin et al., 2004). At the highest Naþ and lowest Kþ contents, cell Cl content was beginning to increase showing that cells have begun to swell. The fall in Kþ content is tightly linked with the increase in Naþ content within the same cell. Used with the permission of the American Physiological Society.

FIGURE 10 Naþ and Kþ contents in neighboring anterior NPE cells within a single section (McLaughlin et al., 2004). (A) Under control conditions, the elemental contents are similar.

(B) In contrast, ouabain alters the composition dramatically of neighboring pairs of PE NPE cells. Some cell pairs are hardly aVected, whereas others show marked changes in composition. Used with the permission of the American Physiological Society.

exchangers (Li et al., 2002). The Naþ and Cl diVuse through the gap junctions between the contiguous PE and NPE cells and are then released to the aqueous through Naþ,Kþ activated ATPase and Cl channels, respectively.

When compared to the posterior region, the more anterior region shows greater changes in Naþ, Kþ, and Cl in response to inclusion of CO2/HCO3 in the bath (Fig. 12), inhibition of the Naþ Kþ 2Cl cotransporter with bumetanide (Fig. 13), and inhibition of the Naþ,Kþ activated ATPase by ouabain (Fig. 7).

These regional variations might indicate that the anterior region is the more active in secreting the aqueous humor. However, when we used heptanol to block the gap junctions between contiguous PE and NPE cells, we found that the largest ion changes after ouabain were in the anterior NPE cells. After heptanol, the anterior PE cells show relatively little Naþ uptake and Kþ loss after ouabain in either the stromal or aqueous solutions. Indeed, their composition is very similar to that of the posterior cells incubated under these conditions (Fig. 14). This finding indicates that the anterior NPE cells must gain Naþ from the aqueous humor. Recent studies with acylguanidines [amiloride, benzamil, and dimethylamiloride (DMA)]

FIGURE 12 The eVects of incubation in CO2/HCO3 Ringer’s (open symbols), HCO3 free solution (gray symbols), or CO2/HCO3 Ringer’sþacetazolamide (0.5 mmol/liter) (black symbols) on cell ion contents. Note that the eVects on cell ions of the CO2/HCO3 Ringer’s are greater in the anterior and middle regions than in the posterior region of the epithelium and were largely prevented by the carbonic anhydrase inhibitor, acetazolamide (0.5 mmol/liter).

FIGURE 13 The eVects of incubation in CO2/HCO3 Ringer’s on cell ion contents in the presence (gray symbols) or absence (open symbols) of bumetanide (0.5 mmol/liter). Note that in the anterior and middle regions, cells gained Cl and Kþ after bumetanide, whereas in the posterior region there was loss of Kþ and little change in Cl .

indicate that this gain in Naþ may be mediated both by Naþ channels and Naþ/Hþ exchangers (McLaughlin et al., 2007). Thus, the anterior NPE cells appear to have a greater capacity than the posterior NPE cells to reabsorb ions from the aqueous humor.

In order to explore the possibility that diVerent regions also display diVering capacities to secrete aqueous humor, we have made use of the recent observation that hypotonic swelling of the entire bovine ciliary epithelium stimulates secretion of Cl (Do et al., 2006). The time course of the swelling activated Cl secretion follows that of the regulatory volume decrease (RVD) displayed by NPE cells freshly isolated from the same preparation (Do et al., 2006). The swelling activated Cl channels of NPE cells are functionally similar to those activated by agonists of NPE cell A3 adenosine receptors, suggesting that a single population of Cl channels may provide the final conduits for much of the Cl secretion by ciliary epithelium (Carre´ et al., 2000).

We incubated tissues in a solution in which the NaCl concentration was reduced by 50%. This gave a solution whose total osmolarity was 60% of normal. As shown in Fig. 15, this leads to rapid losses of Cl and Kþ from

FIGURE 14 EVects of ouabain (0.1 mmol/liter) on epithelial cells in the presence of heptanol (3 mmol/liter) (McLaughlin et al., 2004). Note the much greater eVects of ouabain on the anterior NPE cells. Used with permission of the American Physiological Society.

the posterior cells, with little change in Naþ content. The eVects on the anterior cells are very much smaller. In the posterior epithelial cells, the loss of Kþ greatly exceeds the loss of Cl (Figs. 15 and 16). Cell electroneutrality requires that these cells must either have lost significant quantities of HCO3 ions with Kþ or gained appreciable Hþ ions that would be buVered on cellular macromolecules, thus decreasing cellular electronegativity. Although our results cannot distinguish between these two possibilities, it is known that Cl channels are also permeable to the HCO3 ion (Tabcharani et al., 1989; Nicholl et al., 2002). In addition, a Cl /HCO3 exchanger could recycle Cl across the membrane, so that the net eVect was loss of HCO3 and Cl , rather than loss only of Cl .

FIGURE 17 Pathways for unidirectional secretion (A) and possible reabsorption (B) across the ciliary epithelium (McLaughlin et al., 2007). Used with permission of the American Physiological Society.

not the case. In further experiments, we found that inhibition of Naþ/Hþ exchange by DMA in CO2/HCO3 solution reduced Cl and K contents comparably to timolol (Fig. 18).

The results documented a previously unrecognized cAMP independent transport eVect of timolol. One possibility is that inhibition of Cl /HCO3 exchange may mediate timolol’s inhibition of aqueous humor formation. Alternatively, timolol’s ocular hypotensive eVect might be mediated by reducing cAMP, but that action is exerted at a compartmentalized membrane site, so that flooding the entire cell with cAMP triggers many additional unrelated and confounding eVects. As noted earlier (Civan, 2008), another possibility is that timolol’s actions on the ciliary epithelium may be mediated by antagonism of b adrenergic receptors, but through the arachidonic acid signaling cascade by coupling to Gi proteins.

FIGURE 18 EVects of timolol (10 mM) and/or cAMP (1 mM) on ciliary epithelial Cl/P or K/ P ratios in CO2/HCO3 solution. The open symbols at the extreme left represent control conditions. Proceeding sequentially to the right, the next four columns present data obtained with timolol, cAMP, cAMP and timolol, and DMA, respectively. Stars indicate significant differences from the control data (*P<0.05, **P<0.01, ***P<0.001).