The findings of the present study suggest that Na-K-Cl cotransport
activity and intracellular volume influence C. Together with previous
findings and parallel morphologic findings, our results support the
hypothesis that the cotransporter, through regulating intracellular
volume of the TM cells, modulates C.
The results of this study are consistent with those of Gual et
al.
24 who showed in an identical preparation that
hypo-osmotic and hyper-osmotic media result in decreased and increased
C, respectively, in bovine eyes. However, our results conflict with
those of Gabelt et al.
22 who found that bumetanide did not
change C in monkey eyes in vivo or in perfused human eyes in vitro.
There are a number of possible reasons for the differing results. In
the case of the monkey studies, it is possible that there are species
differences. Alternatively, the presence of anesthesia, the washout
effect,
23 or effects on other parameters of aqueous
dynamics present in the living eye and absent in our system may have
masked bumetanide’s effect on C. In the case of the human eye
perfusions, it is likely that the conflicting results were caused by
differences in experimental techniques. Gabelt et al.
22 used a constant-flow perfusion technique, rather than the
constant-pressure technique that we use. In the constant-flow
technique, pressure varies in response to changes in C or because of
technical difficulties (e.g., pulsation of the syringe pump used to
deliver the media to the eyes). Therefore, pressure spikes are not
uncommon over the short term in this method. This can result in
instability in or even damage to the outflow tissue.
Further, the postmortem handling of tissues in Gabelt et al. was
different from the methods used in our study. In that study, tissue was
accepted undissected for experimentation up to 24 hours after donor
death. Our requirements are considerably more stringent, simply because
tissue autolysis occurs within hours of death, and if the eye tissue is
not enucleated, dissected, and refrigerated soon after death, the
tissue is no longer hormone responsive, even though it may look
acceptable by light microscopy.
16 17 Tissue viability
appears to have been a major problem in the Gabelt study, because more
than half of the human eyes were deemed “unacceptable.” Finally, in
that study only five experiments were performed at one dose of
bumetanide. It is doubtful, given the inherent instability in the short
term of the constant-flow technique, that statistical power was
sufficient to uncover an effect of bumetanide.
Anisosmotic media are well known to induce rapid changes in
intracellular volume of a variety of cell
types,
9 10 11 12 15 25 followed by activation of cell volume
regulatory mechanisms. In nearly all cells, hyper-osmotic media cause
an immediate shrinkage as water exits the cell, moving down its
concentration gradient. This is true of TM cells, vascular endothelial
cells, and a variety of other cells as well.
9 10 11 12 15 25 Cell shrinkage causes activation of Na-K-Cl cotransport or Na–H
exchange, depending on the cell type, which leads to an increased net
uptake of ions and, as water follows, a reswelling of the cell. This
process, termed RVI, generally occurs over a 30- to 60-minute period
after initial exposure to the anisosmotic medium. As volume is restored
in the cells, the ion transporters mediating the RVI decrease in
activity, returning to prestimulus levels. O’Donnell et
al.
9 have shown that in cultured TM cells, it is the
Na-K-Cl cotransporter that mediates the RVI after exposure to
hyper-osmotic media. Hypo-osmotic media, in contrast, cause a rapid
swelling of most cells, as water enters down its concentration
gradient. Cell swelling immediately activates ion flux pathways that
allow net efflux of ions from the cell. Water loss follows, and the
cell is restored to its original volume. K and Cl channels and/or KCl
cotransport, depending on the cell type, mediate this regulatory volume
decrease. We do not yet know the pathways responsible for the
regulatory volume decrease response in TM cells.
Our finding that C is rapidly increased on exposure to hyper-osmotic
media is consistent with the apparent cell shrinkage shown in
Figure 1 and the rapid cell shrinkage of cultured TM cells caused by
hyper-osmotic media.
9 The rapid decrease in C observed
with hypo-osmotic media perfusion of anterior segments is similarly
consistent with the rapid cell swelling observed in the experiment
(Fig. 1) and in cultured TM
9 with hypo-osmotic media.
Further, Rohen et al.
25 reported very similar results
after perfusing monkey eyes with hyper-osmotic and hypo-osmotic media.
Interestingly, they too reported that hypertonic media increases C,
whereas hypotonic media decreases C.
25 The finding that
the increased C observed with hyper-osmotic media is transient and
reversible by 45 minutes is consistent with the time course of the RVI
observed in cultured TM cells.
9
If Na-K-Cl cotransport activity plays a role in maintaining and
regulating TM C, it would be expected that inhibiting the transport
activity would lead to a net sustained decrease in cell volume even in
iso-osmotic conditions. O’Donnell et al.
9 have shown that
the cotransport inhibitor bumetanide decreases the intracellular volume
of cultured TM cells, as it does in a number of other cell types,
including vascular endothelial cells.
9 10 11 12 15 25 Unlike
the transient effects of hyper-osmotic media, bumetanide causes a cell
shrinkage that is sustained up to at least 350 minutes. As would be
predicted if TM cell volume is a determinant of C, we found that
perfusing anterior chamber segments with bumetanide caused a sustained
increase in C in both human and calf eyes. However, it is important to
note that the cotransporter is electroneutral so that changes in its
activity do not cause changes in membrane potential. In keeping with
this, Wiederholt et al.
26 have recently reported that
bumetanide does not alter the contractility of cultured TM cells.
Perfusion of anterior chamber segments with Cl-free media, a maneuver
that both inhibits cotransport activity and promotes reduction of cell
volume through cotransport-independent pathways, was found to be
another treatment that increased C. The finding that its effects were
additive with those of bumetanide is consistent with a greater
reduction in cell volume occurring than with bumetanide alone.
A number of hormones are known to modulate TM C. The effects of some of
these on cultured TM cell Na-K-Cl cotransport activity have been
examined previously.
9 14 In those studies, norepinephrine
acting through a β-adrenergic pathway, inhibited TM cotransport
activity. In addition, elevation of cyclic adenosine monophosphate both
inhibited cotransport activity and reduced TM cell volume. This is
consistent with the well-known outflow-increasing effects ofβ
-adrenergic agents. In contrast, the hormone vasopressin, acting
through elevation of intracellular Ca and/or activation of protein
kinase C, was shown to stimulate activity of the cotransporter. The
finding in the present study that physiological doses of vasopressin
caused a sustained decrease in C of both human and bovine anterior
chamber segments suggests a role for hormone-driven TM cell volume
changes in the modulation of C under iso-osmotic conditions.
In summary, our studies provide evidence in support of the hypothesis
that TM cell volume is a determinant of C and that the Na-K-Cl
cotransporter plays a central role in this process, by regulating TM
cell volume under isosmotic and anisosmotic conditions, and by
mediating hormone-induced changes in cell volume.