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The following is the abstract of the article discussed in the subsequent letter:

Mitchell, Claire H., Jin Jun Zhang, Liwei Wang, and Tim J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 (Cell Physiol. 41): C212-C222, 1997.---The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob. J. Physiol. Lond. 491: 743-755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTPgamma S-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C, an inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTPgamma S but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.

    LETTER
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The role of Cl- channels in volume regulation in bovine pigmented epithelial cells

To the Editor: We would like to comment on a recently published paper by Mitchell et al. (6) on volume-sensitive Cl- currents in bovine pigmented ciliary epithelial cells (BPCE). We believe there are some serious omissions of published findings that conflict with or question a number of statements made by the above authors. In the introduction, the authors state that very little is known about the response to osmotic swelling by pigmented ciliary epithelial cells. However, our paper (4) showed that acutely isolated BPCE cells fail to volume regulate following osmotic cell swelling. These findings contradict those of the authors; however, they are not cited in their paper. Our measurements were made on freshly isolated cells, whereas those of the authors were cultured for up to 36 h. We believe that our model system more closely reflects the native transport properties of these cells. There are some striking differences between freshly isolated and cultured ciliary epithelial cells. For example, freshly isolated pigmented ciliary epithelial cells do not possess a Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchanger (3), whereas pigmented epithelial cells in culture possess a robust exchange mechanism (7). It was not established by Mitchell et al. that these cells contain a swelling-activated Cl- channel in fresh tissue, and thus the possibility is raised that this channel is not expressed in acutely isolated cells.

That a Cl- channel is activated by cell swelling does not prove that it is involved in volume regulation (1), without the support of appropriate experiments that were not performed in the study of Mitchell et al. In fact, it has been shown that, in human breast cancer cells, the swelling-activated Cl- conductance plays no role in volume regulation (2). Miley et al. reported in a previous abstract (5) that BPCE cells perform a regulatory volume decrease (RVD) in 2-4 min and that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and tamoxifen had no effect on RVD. However, Fig. 3 of their paper clearly shows that both NPPB and DIDS significantly reduce the swelling-activated Cl- currents at membrane potentials in the physiological range. These contradictory findings suggest that additional experiments are necessary before we can assign a functional role for the swelling-induced Cl- current in cultured BPCE.

Finally, in their DISCUSSION, Mitchell et al. (6) state, "Because depolarization is generated by the efflux of negative ions, this suggests that Cl-, and not K+, is the predominant ion involved in RVD in these cells." Because conductive cation efflux must accompany conductive anion efflux for a cell shrinkage to occur following osmotic swelling, the above statement requires clarification. As pointed out by Altenberg et al. (2), three conditions are required for a Cl- current to contribute to RVD: 1) a significant increase in Cl- permeability must occur on swelling, 2) a net driving force favoring Cl- efflux should accompany cell swelling, and 3) a parallel conductive pathway providing for a sizable loss of K+ need also occur. Clearly, if Cl- were the predominant conductance following cell swelling, without sizable K+ efflux, RVD would be extremely slow or absent. Moreover, an RVD that presumably occurs in as little as 2 min (which is extremely rapid when compared with most cells that undergo RVD) must have comparable K+ and Cl- effluxes.

Because it has been suggested that the pigmented and nonpigmented cell acts in concert to secrete aqueous humor, the role each cell type plays in ion and water transport is fundamentally important. However, drawing physiological conclusions from isolated cells in culture should be done with extreme caution, unless the mechanisms under study are also present in the native tissue. Recently, the hypothesis was put forth that the function of the pigmented epithelial cell was to take up ions and water and to transmit both via gap junctions into the adjacent nonpigmented epithelial cell. Once within the nonpigmented epithelial cell, ions and water are then transported by efflux pathways residing in the basolateral membrane of the nonpigmented epithelial cell into the aqueous chamber. In our paper (4), we presented data that supported the above hypothesis. What was striking was the obvious presence of a robust regulatory volume increase (RVI) in pigmented epithelial cells and the complete lack of RVD. On the other hand, the nonpigmented epithelial cells showed no RVI response, yet were very capable of undergoing a K+-dependent RVD. It will become clear with time whether the above hypothesis is correct, but only after appropriate experiments are conducted on preparations that are representative of the intact ciliary epithelium.

    REFERENCES
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1.   Adorante, J. S., and P. M. Cala. Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells. Am. J. Physiol. 268 (Cell Physiol. 37): C721-C731, 1995[Abstract/Free Full Text].

2.   Altenburg, G. A., J. W. Dietmer, D. C. Glass, and L. Reuss. P-glycoprotein-associated Cl- currents are activated by cell swelling but do not contribute to cell volume regulation. Cancer Res. 54: 618-622, 1994[Abstract].

3.   Butler, G. A. D., M. Chen, Z. Stegman, and J. M. Wolosin. Na+-Cl- and HCO<SUP>−</SUP><SUB>3</SUB>-dependent base uptake in ciliary body pigment epithelium. Exp. Eye Res. 59: 343-359, 1994[Medline].

4.   Edelman, J. L., G. Sachs, and J. S. Adorante. Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells. Am. J. Physiol. 266 (Cell Physiol. 35): C1210-C1221, 1994[Abstract/Free Full Text].

5.   Miley, H. E., V. E. Walker, C. E. Pollard, and T. J. C. Jacob. Regulatory volume decrease in ciliary epithelial cells (Abstract). Invest. Ophthalmol. Vis. Sci. 36: S586, 1995.

6.   Mitchell, C. H., J. J. Zhang, L. Wang, and T. J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 (Cell Physiol. 41): C212-C222, 1997[Abstract/Free Full Text].

7.   Weiderholt, M., H. Helbig, and C. Korbmacher. Ion transport across the ciliary epithelium: lessons from cultured cells and proposed role of the carbonic anhydrase. In: Carbonic Anhydrase, edited by F. Botre, G. Gross, and B. T. Storey. New York: Cambridge, 1991, p. 232-244.

Joseph S. Adorante
Jeffrey L. Edelman
Department of Biological Sciences
Allergan, Inc.
Irvine, CA 92715

    REPLY
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To the Editor: We thank Drs. Adorante and Edelman for creating this opportunity to address their criticisms and reconcile what they perceive to be a possible conflict between our work and theirs. "We are as men more sinned against than sinning" (to misquote Shakespeare, King Lear act 3, scene 2, line 57). In referring to our paper on the volume-sensitive Cl- current (5), Adorante and Edelman make the point that they were unable to find volume regulation in BPCE cells following osmotic cell swelling and that we do not mention this. In our hands these same cells do volume regulate, a fact that we have reported (4). Adorante and Edelman complain that we did not refer to their work. Our choice of references reflected the emphasis of our paper and its concern with the broader issues of second messenger control of volume regulation. However, since they have now drawn attention to this matter, we can pursue the possible reason(s) for the differences in our respective findings.

In their study they used acutely isolated BCPE cells and found no volume regulation following osmotic cell swelling as determined by Coulter counter. We, on the other hand, allowed our freshly dissociated cells to attach to glass for 12-36 h and, using optical image analysis, found RVD under similar osmotic conditions. Our cells are not cultured (in the sense that they have not undergone cell division while in culture), and therefore the comparison that Adorante and Edelman make with cultured cells is inappropriate. They should also be under no illusion that their system of trypsinization followed by Percoll gradient centrifugation is physiological. It has been shown that trypsinization causes damage to membrane proteins and leads to membrane leakiness (3). Thus there are two major differences between their work and ours: 1) the use of detached vs. attached cells and 2) the detection method.

To address the first difference, the cells that Adorante and Edelman studied were detached, whereas ours were attached. In this regard Han et al. (2) have made some interesting observations. They found that they could activate virtually no volume-sensitive Cl- current in detached cells (human breast cancer cells) compared with the same cells when attached to glass. Therefore, the availability of swelling-activated Cl- current depends, to some extent and through some unknown mechanism, on attachment to a substrate. Regarding the second difference, the use of image analysis allows us to select only those healthy cells and to follow the process of volume change from beginning to end in one cell. We can discard those cells that "bleb" following osmotic shock. Blebbing is an injury marker and indicates that the cells may be dying. The formation of blebs may affect the determination of cell volume by Coulter counter. Radically different time constants for RVD are determined by the two methods, and direct measurement by image analysis gives a value of 2-4 min (4) compared with the Coulter counter measurement in which the volume did not recover to original levels within 40 min of recording (Fig. 4B in Ref. 1). This difference needs to be addressed by those who chose to use the Coulter counter method.

Edelman and Adorante also draw attention to the differences in the pharmacology of RVD and the volume-activated Cl- current. NPPB and DIDS, while blocking the volume-activated Cl- current (5), did not significantly affect the RVD (4). First, both DIDS and NPPB give a voltage-dependent block (Fig. 3 in Ref. 5), and, at the potentials the cells would be resting at in the volume studies, the block would be at its weakest; and, second, the currents are measured by an "invasive" technique of whole cell patch clamping. This can, as we explain in the DISCUSSION of Ref. 5, have the effect of artificially prolonging the volume-activated Cl- current beyond its normal activation during RVD. When exposed to hypotonic solution, the cell can never equilibrate osmotically with the bathing solution because of the "infinite" (in comparison with the cell) supply of ions in the patch pipette.

Finally, the use of the term "predominant" (see DISCUSSION in Ref. 5), when referring to Cl- efflux, does not preclude the involvement of K+. One partner in a relationship may be predominant, but that does not condemn the other partner to extinction. We mention the efflux of both ions in the introduction.

"No, I will be the pattern of all patience; I will say nothing more" (Shakespeare, King Lear act 3, scene 2, line 37).

    REFERENCES
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Abstract
Letter
References

1.   Edelman, J., G. Sachs, and J. S. Adorante. Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells. Am. J. Physiol. 266 (Cell Physiol. 35): C1210-C1220, 1994[Abstract/Free Full Text].

2.   Han, E. S., C. G. Vanoye, G. A. Altenberg, and L. Reuss. P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody. Am. J. Physiol. 270 (Cell Physiol. 39): C1370-C1378, 1996[Abstract/Free Full Text].

3.   Lamb, J. F., and P. Ogden. Transient leakiness of HeLa cells to Na and K during "rounding up" with trypsin, EDTA and pronase. J. Physiol. (Lond.) 358: 70P, 1985.

4.   Miley, H. E., V. E. Walker, C. E. Pollard, and T. J. C. Jacob. Regulatory volume decrease in ciliary epithelial cells. Invest Ophthalmol. Vis. Sci. 36: S586, 1995.

5.   Mitchell, C. H., J. J. Zhang, L. Wang, and T. J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 (Cell Physiol. 41): C212-C222, 1997[Abstract/Free Full Text].

T. J. C. Jacob
C. H. Mitchell
School of Molecular and Medical Biosciences
University of Wales, Cardiff CF1 3US, UK


AJP Cell Physiol 273(4):C1435-C1436
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society