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ABSTRACT |
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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) (GTP
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 GTP
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 GTP
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.
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LETTER |
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The role of Cl channels in volume regulation in
bovine pigmented epithelial 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.
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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
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
-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
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
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 |
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 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 Edelman and Adorante also draw attention to the differences in the
pharmacology of RVD and the volume-activated Cl Finally, the use of the term "predominant" (see
DISCUSSION in Ref. 5), when referring to Cl "No, I will be the pattern of all patience; I will say nothing
more" (Shakespeare, King Lear act 3, scene 2, line 37).
REPLY
Top
Abstract
Letter
References
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.
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.
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.
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.
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REFERENCES |
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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
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
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
T. J. C. Jacob C. H. Mitchell School of Molecular and Medical Biosciences University of Wales, Cardiff CF1 3US, UK |