DIDS and the Janus-faced Na⁺-K⁺-activated ATPase. Focus on “DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism”

the maintenance of mammalian cell volume fundamentally rests on net cation extrusion through Na⁺-K⁺-activated ATPase, the Na⁺ pump. The publication by Shahidullah et al. (11) persuasively identifies the series of events by which a widely used inhibitor of anion transport, DIDS, can reduce Na⁺ pump activity of porcine nonpigmented ciliary epithelial (NPE) cells. DIDS sequentially acidifies the NPE cells and activates Src family kinases (SFK), thereby triggering phosphorylation and inhibition of Na⁺-K⁺-activated ATPase (Fig. 1). A pH dependence of pump activity has been long known; Na⁺-K⁺-ATPase activity of Xenopus membrane homogenates displays a pH maximum at ∼pH 7 (10). The authors' results may be clinically relevant. By inhibiting Na⁺ pump activity, DIDS can reduce porcine aqueous humor formation and thereby lower intraocular pressure. Reducing intraocular pressure remains the only documented approach for delaying the onset and slowing the progression of irreversible blindness in glaucoma. In addition to this potential clinical implication, the authors' observations are broadly instructive in two additional ways.

Fig. 1.

DIDS inhibits aqueous humor secretion by the porcine ciliary epithelium. The results of Shahidullah et al. (11) indicate that DIDS acidifies the porcine nonpigmented ciliary epithelial cell (NPE), possibly by blocking Na⁺/nHCO₃⁻ cotransport and carbonic anhydrase (CAIV), and secondarily reducing Na⁺/H⁺ antiport activity. The reduced intracellular pH (pH_c) activates Src family kinases (SFK), leading to phosphorylation and inhibition of Na⁺-K⁺-activated ATPase (NKA) activity, and thereby reduces aqueous humor secretion. Activation of SFK exerts many other actions, including activation of ERK1/2 signaling, but in parallel with inhibition of Na⁺ pump activity.

First, even when its direct action is limited to inhibition of anion transport, DIDS can initiate a wide range of unexpected downstream effects. That the stilbene can exert nonspecific effects at concentrations in excess of 100 μM is well known. However, the work of Shahidullah et al. (11) provides a further cautionary note. Their conclusions suggest that even were DIDS applied at concentrations less than 10 μM, a range over which only anion transporters and not Cl⁻ channels are thought inhibited, unanticipated downstream signaling events could result.

Second, the observations of Shahidullah et al. (11) highlight yet another action of the SFK. SFK comprises three groups of protein tyrosine kinases, some of which are alternatively spliced in different cells. These kinases play major roles in signaling an enormous spectrum of cellular activities, apart from regulation of sodium pump activity. These roles are subject to considerable complexities (14). The interaction observed by Shahidullah et al. (11) is directionally opposite to the effect of the ouabain-treated Na⁺ pump to activate SFK, which has been documented by multiple investigators (4). Thus, the Na⁺ pump both responds to intracellular regulators (backward-looking), and in turn can modify those same regulators (forward-looking), hence a Janus-faced aspect of Na⁺-K⁺-activated ATPase (Fig. 1).

The specific action of SFK on the Na⁺ pump of these NPE cells cannot be directly extrapolated to other cells. In the present work, the authors found that SFK inhibits Na⁺-K⁺-activated ATPase of porcine NPE cells. In previous work from this group, SFK was found to exert an opposite effect on Na⁺-K⁺-activated ATPase of rabbit lens epithelial cells (13). Such a discrepancy is not entirely surprising. In the present work, SFK was activated within the framework of the signaling cascade triggered by cellular acidosis, whereas in the previous study SFK was activated as part of the P2-receptor purinergic signaling cascade. Multiple downstream and cross-reacting actions could be playing roles.

The central focus of the current publication has been the ion transport function of Na⁺-K⁺-activated ATPase. The Na⁺/K⁺ exchange activity and energy requirement of a Na⁺ pump had been anticipated from results in multiple tissues, even before the Nobel Prize-winning discovery of Na⁺-K⁺-activated ATPase by Skou in 1957. The kinetics of that exchange activity have also been long understood within the framework of the Post-Albers cycle. Despite this basic understanding, many aspects of the pump continue to surprise the research community. The current paper sheds further light on regulation of the Na⁺/K⁺ exchange function of the Na⁺ pump. An additional and initially totally unexpected development has been an increasing awareness of the scaffolding and signaling function of Na⁺-K⁺-activated ATPase and of the possible regulatory role of endogenous circulating cardiotonic steroids. Cardiotonic steroids such as ouabain, digoxin, and digitoxin have been considered completely selective inhibitors of Na⁺-K⁺-activated ATPase. Cardiotonic steroids also target ryanodine receptors, but that interaction may be largely/entirely mediated by reactive oxygen species (ROS) (5), rather than by a direct stimulation.

Multiple investigators have observed that exogenous cardiotonic steroids can exert cellular effects, such as increasing the free Ca²⁺ concentration, without detectably altering the intracellular Na⁺ and K⁺ concentrations (12). In part, such effects may be mediated by changes in cationic concentration within a restricted subvolume within the cell (8). However, even when Na⁺/K⁺ exchange is fully blocked by replacing extracellular Na⁺ with N-methyl-d-glucamine, ouabain can still inhibit cell function (6). Such transport-independent effects of cardiotonic steroids have been thought to reflect scaffolding or signaling functions of the Na⁺ pump (7). For example, Na⁺-K⁺-activated ATPase is one of more than 100 interacting protein components of the tight junction (3) and regulates its barrier function (4, 9). In part, the regulatory actions of Na⁺-K⁺-activated ATPase are mediated by the second messengers Ca²⁺ and ROS. ROS can be formed by phosphorylation of mitochondrial enzymes following ouabain-triggered activation of SFK (5). Increases in cell Ca²⁺ concentration can likely result from both the transport (8) and the signaling (4) activities of the Na⁺ pump.

Given its critical role in vertebrate life, it should not be surprising that endogenous Na⁺ pump regulators, ouabain, digoxin, and possibly other cardiotonic steroids, have been identified in humans (12). Albeit circulating at subnanomolar levels, endogenous ouabain can inhibit high-affinity α2 Na⁺ pumps and may be a critical link in the pathogenesis of salt-dependent hypertension (1).

The transport and scaffolding/signaling functions are possibly complementary in considering the potential clinical relevance of the current publication (11), the reduction of intraocular pressure. That pressure directly depends on the rate of inflow and resistance to outflow of aqueous humor. Shahidullah et al. (11) point out that inhibition of the transport role of the Na⁺ pump slows aqueous humor formation of the porcine eye, lowering intraocular pressure. Dismuke et al. (2) have demonstrated that ouabain also lowers the resistance to aqueous humor exit through the trabecular meshwork outflow pathway of the porcine eye, again tending to lower intraocular pressure. The latter effect is likely mediated by the signaling/scaffolding function of Na⁺-K⁺-activated ATPase (6). Pharmacologic agents with a dual site and mechanism of action at both the inflow and the outflow levels may be particularly effective in lowering intraocular pressure.

GRANTS

This work was supported, in part, by National Eye Institute Grant EY-13624 from the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.

AUTHOR CONTRIBUTIONS

M.M.C. drafted, edited, revised, and approved the final version of the manuscript.