Na + /H + Exchange and Hypoxic Pulmonary Hypertension

Role of NHE1 in physiologic conditions

Plasma membrane NHEs play a critical role in regulating several cellular processes, including cell volume ⁵⁶ and signal transduction pathways involved in mediator release.^57–59 However, the most extensively described function of NHEs in PASMCs is in regulation of pH_i. As mentioned above, NHE1 is the only plasma membrane–localized NHE isoform known to be expressed in mouse and rat PASMCs,^27,28 and while expression of the various NHE isoforms in PASMCs remains to be defined in humans, it is likely that the ubiquitous NHE1 is a major contributor to control of PASMC pH_i across species.

Homeostasis of pH_i regulates PASMC function in several ways, including control of vasomotor tone and cell proliferation and migration. With respect to the former, in isolated, perfused ferret lungs under normoxic conditions, acute intracellular alkalinization resulted in a rise in pulmonary arterial (PA) pressure, whereas acute acidification resulted in an even more pronounced elevation in PA pressure. ⁵ Both acute alkalinization and acidification were also associated with a rise in intracellular Ca²⁺, suggesting the possibility that the acute change in vasomotor tone was mediated by Ca²⁺. In vascular smooth muscle cells, multiple pathways, including voltage-gated and store-operated Ca²⁺ channels, have been implicated in alkalosis-induced elevation of intracellular Ca²⁺. ⁶⁰ Interestingly, after the initial increase in PA pressure induced by acute intracellular alkalinization of ferret PASMCs, more prolonged alkalinization resulted in a further rise in PA pressure that was not associated with a rise in intracellular Ca²⁺. ⁵ There is evidence that pH_i exerts a direct (Ca²⁺-independent) effect on the contractile apparatus, through changes in calmodulin-dependent enzyme activity and ordering of the myofilament lattice. ⁶⁰ Intracellular alkalinization also resulted in canine PA contraction during isometric-tension studies and resulted in increased PA pressures in isolated perfused rat lungs under both normoxic and hypoxic conditions.^54,61 Thus, data from multiple organisms indicate that a rise in pH_i induces PASMC contraction, likely through both Ca²⁺-dependent and Ca²⁺-independent mechanisms.

It is important to note that the response of smooth muscle to an acute acid or base load may differ from the response to sustained acidification or alkalinization. In the absence of any data on how the pulmonary vasculature responds to sustained intracellular alkalinization, findings in the systemic vasculature may offer useful insight. Knockout of either the Na⁺/HCO₃⁻ cotransporter NBCn1, which alkalinizes cells by importing HCO₃⁻, or NHE1 resulted in intracellular acidification in mouse vascular smooth muscle cells.^62,63 In both of these knockout animals, the smooth muscle in the resistance vessels of the systemic vasculature was marked by reduced norepinephrine-stimulated, ROCK-dependent Ca²⁺ sensitivity. This suggests that ROCK activity may mediate changes in Ca²⁺ sensitivity, and thereby vascular tone, in response to prolonged changes in pH_i, but the extent to which the same mechanisms apply in the pulmonary vasculature remains unclear.

There is also a link between pH_i and cell proliferation. The correlation between alkalinization and cell proliferation was initially observed in fibroblasts ⁶⁴ and was later confirmed in bovine PASMCs, where challenge with either platelet-derived growth factor (PDGF) or epidermal growth factor induced alkalinization and proliferation. ⁵⁵ Moreover, inhibiting NHE with DMA attenuated both the alkalinization and the proliferation induced by these growth factors, ⁵⁵ indicating that NHE-mediated changes in pH_i are important in controlling PASMC proliferation. Later studies using NHE1-deficient Chinese hamster ovary cells demonstrated that loss of NHE1 decreased the rate of proliferation and that proliferation could be restored with reintroduction of NHE1. ⁶⁵ Consistent with these findings, the NHE1-specific inhibitor sabiporide ⁶⁶ or knockdown of NHE1 with silencing RNA ⁶⁷ reduced proliferation rates in cultured human PASMCs, indicating a primary role for NHE1 in PASMC growth, although a direct link between intracellular alkalosis and PASMC proliferation remains to be proven, as NHE-mediated effects on cell volume or cytoskeletal rearrangement could also be contributing. Nonetheless, increased NHE activity, coinciding with increased pH_i, is now considered to be an early event in cell proliferation.

Furthermore, NHE1 has been shown to play a role in PASMC migration. Initial work in neutrophils showed that pharmacologic activation of NHE induced neutrophil alkalinization and chemotaxis and that addition of an NHE inhibitor prevented these effects. ⁶⁸ Varying neutrophil pH_i by altering Na⁺ and H⁺ gradients demonstrated that the chemotactic response correlated with the rise in pH_i, suggesting that the migratory response was pH dependent. In addition, fibroblast migration was impaired in the setting of mutations to either the NHE1 ion translocation domain or the C-terminal cytoskeletal anchoring domain. ⁶⁹ Subsequent work in cultured human PASMCs revealed that inhibition of NHE1 by silencing RNA ⁶⁷ or sabiporide ⁶⁶ decreased cell migration. As with proliferation, these data indicate that NHE1 regulates PASMC migration, but the extent to which it does so through pH_i modulation versus cytoskeletal rearrangement remains unclear.

Role of NHE1 in acute hypoxic pulmonary vasoconstriction (HPV)

While the preceding sections describe the role of NHE in modulating PASMC function under basal conditions, NHE1 also contributes to the pulmonary vascular response to pathologic stimuli, including hypoxia. The vasoconstrictor response of the pulmonary vasculature to hypoxia was first described in detail in the cat ⁷⁰ and was noted soon thereafter in humans. ⁷¹ Since these early reports, HPV has been observed in all vertebrates, and an extensive literature on HPV has developed.^72,73 HPV arises acutely upon exposure to hypoxia, with a significant increase in pulmonary vascular resistance (PVR) within 30 minutes and, if the hypoxic exposure remains brief (minutes to a few hours), largely reverses rapidly with return to normoxia. ⁷⁴

The onset of HPV occurs in phases, with an initial phase occurring within 5 minutes that reflects Ca²⁺-dependent smooth muscle contraction and a later phase of increasing PA pressure that appears to be Ca²⁺ independent. ⁷³ The initial hypoxic increase in intracellular Ca²⁺ in PASMCs is mediated by Ca²⁺ release from the sarcoplasmic reticulum and Ca²⁺ entry through voltage-gated L-type Ca²⁺ channels, Ca²⁺ permeable nonselective cation channels, and receptor-operated Ca²⁺ channels; in addition, hypoxia increases the sensitivity of the myofilament contractile apparatus to Ca²⁺. ⁷⁵ Factors involved in the later-phase Ca²⁺-independent rise in PVR include endothelial release of factors such as nitric oxide, prostacyclin, and endothelin-1 (ET-1); ⁷⁶ modulation of nitric oxide (NO) and reactive oxygen species within the pulmonary vasculature by red blood cells;^77–80 and a potential role of neural modulation.^81–83 The contribution of pH_i, and of NHE in particular, to HPV remains a field of active inquiry.

As noted above, induction of intracellular alkalosis in PASMCs increased intracellular Ca²⁺ levels, ⁵ contracted isolated pulmonary arteries, ⁵⁴ and increased PA pressure in isolated lungs,^5,54 findings consistent with the possibility that alterations in pH_i could contribute to HPV. Supporting this hypothesis, manipulating pH_i by inducing intracellular acidification inhibited, ⁶¹ while intracellular alkalinization enhanced,^61,84 HPV. However, these reports stand in contrast to other studies, in which HPV was attenuated by hypocapnic alkalosis^85–89 and enhanced by hypercapnic acidosis.^88–90 It should be noted, however, that the magnitude of the change in pH_i under these conditions was not measured and could vary considerably from that in experiments where pH_i was manipulated directly. Nonetheless, these studies provide substantial evidence that changes in pH_i during hypoxia could modulate HPV.

Unfortunately, only a few investigators have measured the effect of acute hypoxia on PASMC pH_i, with conflicting results. In large-diameter porcine pulmonary arteries mounted for isometric-tension recording, hypoxia induced an initial, transient contraction without changes in pH_i. ⁹¹ The initial contraction was followed by dilation and decreased pH_i and a later, sustained hypoxic contraction that was associated with pH_i increasing back to normal levels. In isolated PASMCs where pH_i was directly measured in real time with fluorescent indicators, exposure to acute hypoxia increased pH_i in PASMCs derived from small-diameter cat pulmonary arteries, although hypoxia induced acidification in PASMCs from large-diameter cat pulmonary arteries. ⁶ Adding to the confusion, acute hypoxia had no effect on pH_i in PASMCs from distal rat intrapulmonary arteries. ⁹² To be certain, variations in the severity of hypoxia, location of the vessels within the vascular tree, or species could account for the differences in findings. Interestingly, the alkalinization of small-diameter feline PASMCs was attributed to changes in Cl⁻/HCO₃⁻ exchange, not to NHE. Thus, whether acute hypoxia alters the activity of NHE1 in PASMCs from other species, whether this depends on the section of the vascular tree studied, and whether alterations in NHE1 activity exert effects on pH_i during hypoxia remain to be fully resolved.

Role of NHE1 in hypoxia-induced pulmonary hypertension

Acute regulation of NHE activity

Non-pH-dependent regulation of NHE1 expression and activity occurs, in large part, through modifications to its cytosolic C-terminal domain. NHE1 turnover at the plasma membrane is controlled by C-terminal ubiquitination, which targets the exchanger for endocytosis. ¹⁰³ Factors influencing activation of NHE1 include phosphorylation and interaction with regulatory molecules. For example, binding to calmodulin, a protein whose activation is dependent on intracellular Ca²⁺ levels, activates NHE1. ³⁵ In the setting of low Ca²⁺, the high-affinity calmodulin binding site in the NHE1 C-terminus interacts with a region of the transmembrane domain, inhibiting proton exchange; in high-Ca²⁺ conditions, calmodulin binding prevents this autoinhibitory interaction. ¹⁰⁴ In addition, both PIP₂ and CHP1 are essential cofactors for proper NHE1 function, as inhibition of the NHE1-CHP1 interaction^36,37 or mutating the putative PIP₂-binding domains ³⁸ markedly decreases NHE1 ion exchange activity.

NHE1 is further regulated by phosphorylation, with all known phosphorylation targets localized to serines and threonines in the C-terminal tail. ³⁹ Several protein kinases have been shown to be involved in the phosphorylation of NHE1, including PKC,^40,41 ROCK,^42,43 MAPK-dependent pathways,^44–46 and p90^rsk.^45,47 The impact of phosphorylation on activity varies, in some cases increasing exchange activity or shifting the pH-dependent activation of NHE1 to more alkaline pH_i. ¹⁰⁵ In contrast, phosphorylation of the high-affinity calmodulin binding site at Ser-648 by protein kinase B/Akt prevents the NHE1-calmodulin interaction and thus inhibits NHE1 ion transport activity. ¹⁰⁶ In addition to direct effects on the NHE1 protein, phosphorylation of NHE1 binding partners can influence NHE1 activity. For example, phosphorylation of calmodulin by Janus kinase 2 increases NHE1-calmodulin binding and thus activates NHE1. ¹⁰⁷

Interestingly, as with calmodulin, many of the kinases and regulatory proteins known to alter NHE1 activity exhibit Ca²⁺ sensitivity (such as PKC, ROCK, and CHP), suggesting the possibility that changes in intracellular Ca²⁺ concentration could act to indirectly modulate NHE1 activity. Clearly, the mechanisms that regulate NHE activity are complex and have yet to be exhaustively delineated. Moreover, while the regulation of NHE1 by phosphorylation and regulatory proteins has been described in a variety of cell types, which of these mechanisms regulates NHE1 activity in PASMCs is currently still under investigation.

Several factors known to be involved in the pathogenesis of PH have been shown to influence NHE activity. For instance, PDGF, which is released by circulating platelets, is implicated in PH, in that increased levels of PDGF are present in the blood and lungs of patients with various forms of PH. PDGF has been shown to stimulate rat ¹⁰⁸ and bovine ⁵⁵ PASMC proliferation, as well as human PASMC proliferation and migration, ¹⁰⁹ through pathways involving upregulated capacitative Ca²⁺ entry^108,110 and increased NHE activity. ⁵⁵

Similarly, the peptide ET-1, a potent endothelial-derived vasoconstricting agent released primarily by endothelial cells, is upregulated in multiple forms of PH, including hypoxia-induced PH, and ET-1 receptor inhibitors have been successful in preventing and partially reversing hypoxic PH in animal models.^111–113 Acute incubation with ET-1 increased NHE activity in a dose-dependent fashion in normoxic rat PASMCs. ¹¹⁴ Furthermore, in the same study, pretreatment of PASMCs with a ROCK inhibitor prevented ET-1-induced upregulation of NHE activity. ¹¹⁴ ROCK is activated in multiple models of PH, perhaps secondarily to increased circulating factors such as ET-1, and is implicated in signaling leading to both vasoconstriction and vascular remodeling.^115–118 ROCK inhibitors have been shown to decrease PA pressures and PVR both in experimental models of PH and in PAH patients.^119–121 The best-characterized action of ROCK is the phosphorylation of the myosin-binding subunit of myosin light chain phosphatase (MLCP), thereby inhibiting MLCP and thus increasing phosphorylation of myosin light chains, which serves to augment contraction. However, since ROCK inhibition also prevents ET-1-induced activation of NHE in normoxic settings, it is likely that the ET-1/ROCK pathway contributes to remodeling as well.

In addition, proliferation of cultured bovine PASMCs in response to hypoxic stimuli involves the activation of PKC. ¹²² Activation of PKC increased NHE expression and ion-exchange activity in vascular smooth muscle cells,^40,123,124 and, in nonvascular cells, PKC was a required intermediary in ET-1-induced upregulation of NHE activity.^125–127 However, in normoxic rat PASMCs, pretreatment with PKC inhibitors did not prevent ET-1-mediated augmentation of NHE, ¹¹⁴ suggesting that, while NHE induction by ET-1 involves PKC signaling in certain conditions, this does not appear to be a global phenomenon. Further experiments will be required to determine whether PKC plays any role in NHE activation in PASMCs during chronic hypoxia.

Regulation of NHE1 expression

Ca²⁺ signaling

In many cell types, changes in pH_i and intracellular Ca²⁺ are tightly coupled. ⁶⁰ Elevated intracellular Ca²⁺ levels are implicated not only in pulmonary vasoconstriction but also in vascular remodeling. ¹³⁶ Thus, it is worthwhile to examine in further depth the effects of alkalinization on intracellular Ca²⁺.

In porcine coronary arteries and rat aortas, intracellular alkalinization induced a rise in intracellular Ca²⁺ that was attenuated by nifedipine, an inhibitor of L-type voltage-gated Ca²⁺ channels.^137,138 Alkalinization also increased intracellular Ca²⁺ levels in ferret PASMCs and potentiated contraction in response to potassium chloride, ⁵ indicating that L-type voltage-gated Ca²⁺ channels contributed to pH-mediated Ca²⁺ entry. Furthermore, intracellular alkalinization inhibited voltage-gated K⁺ currents, resulting in depolarization of canine PASMCs, ¹³⁹ which may serve as one of the mechanisms by which an alkaline shift in pH activates L-type Ca²⁺ channels. It is likely that other Ca²⁺ entry pathways can also be modulated by pH_i. For example, in rat aortic smooth muscle cells, alkalinization potentiated Ca²⁺ entry induced by thapsigargin (which depletes stores of Ca²⁺ in the sarcoplasmic/endoplasmic reticula and thereby induces plasmalemmal Ca²⁺ entry through store-operated Ca²⁺ channels), suggesting that store-operated Ca²⁺ channels can also contribute to pH-mediated Ca²⁺ entry. ¹⁴⁰ There is also evidence of increased release of Ca²⁺ from intracellular stores in the setting of alkalinization. In smooth muscle cells from guinea pig portal veins, elevated pH was associated with increased inositol triphosphate–induced Ca²⁺ release from the endoplasmic reticulum. ¹⁴¹ Thus, it is possible that increased NHE1 activity and alkalinization during chronic hypoxia could enhance Ca²⁺ entry into PASMCs.

ROCK, p27, and cyclin D1 signaling

Experiments utilizing Nhe1^−/− mice and in vitro NHE1 silencing have yielded further insights into downstream effectors of NHE1 signaling. In Nhe1^−/− mice, there was a decrease in ROCK expression and activity, an increase in the expression of p27 (a cyclin-dependent kinase inhibitor and known downstream factor of ROCK), and a decrease in cyclin D1 expression. ¹⁰⁰ Moreover, expression of E2F1, a transcription factor important in cell cycle progression that is known to be downstream of p27, was upregulated by hypoxia in human PASMCs, and this upregulation was inhibited in the setting of in vitro NHE1 silencing. ⁶⁷ The role of E2F1 as a downstream effector of NHE1 was further confirmed by the fact that overexpression of E2F1 prevented the decrease in PASMC proliferation and migration observed when NHE1 was silenced. ⁶⁷ Thus, hypoxia-induced upregulation of NHE1 leads to downstream ROCK activation, followed by increased p27 and subsequent E2F1 expression, which in turn mediates cell cycle progression and increased PASMC proliferation.

While the studies in Nhe1^−/− mice placed ROCK as a downstream effector of NHE1 signaling, the interaction between NHE1 and ROCK is clearly more complex. As noted above, ET-1 induced NHE activity acutely in normoxic PASMCs via a mechanism involving ROCK, thus placing ROCK upstream of NHE. ¹¹⁴ Consistent with the results obtained with acute exposure to ET-1 in PASMCs, NHE has also been shown to be downstream of ROCK and RhoA (Ras homolog A, a GTPase [guanosine triphosphatase] that increases ROCK activity) in nonvascular cell types.^{42,43,142,143} Given the temporal differences in these studies, the most likely scenario is that ROCK activation acutely phosphorylates and enhances NHE1 activity, with prolonged NHE1 upregulation in turn increasing ROCK expression. However, the exact mechanism by which NHE1 increases ROCK expression remains to be determined.

NHE1 and cytoskeletal anchoring

It has been recognized for some time that the C-terminal tail of NHE1, independent of the ion-transport domain, is involved in regulation of cell shape and cell function. Fibroblasts that express an NHE1 variant with a mutation in the ezrin-binding motif have impaired organization of actin stress fibers and an irregular cell shape, while fibroblasts expressing an NHE1 mutant with disrupted ion translocation function have normal cytoskeletal organization and cell shape. ³³ Furthermore, fibroblasts containing NHE1 with mutations in the C-terminal ezrin-binding motif also show evidence of impaired cell migration. ⁶⁹

As is implied in the name, mutations in the C-terminal ezrin-binding motif impair the ability of NHE1 to bind with ezrin, ³³ a member of a family of proteins referred to as ERM (ezrin, radixin, moesin) proteins. ¹⁴⁴ ERM proteins are characterized by their shared N-terminal FERM (4.1, ezrin, radixin, moesin) domain, which binds to various membrane proteins, and a shared C-terminal filamentous-actin binding site. As their shared structure suggests, this family of proteins plays an important role in the cross-linking of membrane proteins to the actin cytoskeleton, thereby facilitating changes in cell shape, membrane protein localization, and signal transduction. The ERM family is also characterized by a shared C-ERMAD (ERM-associated domain), which is able to intramolecularly bind to the N-terminal FERM domain. As the C-ERMAD domain overlaps the C-terminal actin-binding site, this intramolecular association prevents association of ezrin with actin and thus renders ezrin functionally inactive. ROCK regulates the activity of ezrin by phosphorylating a C-terminal threonine (T567). ¹⁴⁵ T567 phosphorylation prevents ezrin intramolecular association and thus allows activated ezrin to serve as a membrane-actin cross-linker (Fig. 4). Thus, phosphorylated ezrin serves as a structural link between the C-terminal tail of transmembrane NHE1 and the actin cytoskeleton, and it is through this interaction with phosphorylated ezrin that NHE1, acting as a membrane anchor for actin filaments, is able to regulate PASMC cell shape and migration.

OPEN IN VIEWER

Figure 4.

A, Schematic illustrating proposed mechanism by which NHE1/ezrin interactions controls pulmonary arterial smooth muscle cell (PASMC) shape changes. Ezrin phosphorylation by Rho kinase (ROCK) allows dissolution of intramolecular association between the head (FERM; [4.1, ezrin, radixin, moesin]) and tail (C-ERMAD [ERM (ezrin-radixin-moesin)-associated domain]) of ezrin. Active (phosphorylated) ezrin (P) then binds to and forms a crosslink between NHE1 and actin filaments (F-actin). B, Schematic illustrating pathways whereby chronic hypoxia (CH) leads to pathologic PASMC behavior via increased NHE1-actin cross-linking. Upregulation of the transcription factor hypoxia-inducible factor 1 (HIF-1) results in increased NHE1 expression. Activation of ROCK by CH results in phosphorylation of ezrin (P-ezrin) and binding between NHE1 and F-actin, tethering actin filaments to the cell membrane and facilitating cell movement necessary for migration and proliferation.

Early studies

Amiloride is a drug that was initially found to inhibit the sodium channel in urinary epithelia but has since been recognized to inhibit numerous ion channels. Thousands of amiloride analogs have been synthesized that inhibit Na⁺-conducting channels with varying specificity. ¹⁴⁸ DMA and EIPA are two amiloride analogs exhibiting relatively more specific inhibition of NHE ion transport. Initially, DMA was found to inhibit growth factor–induced PASMC proliferation in vitro. ⁵⁵ In vivo, it was subsequently determined that both DMA and EIPA, when given during a hypoxic exposure of 10% O₂ for 14 days, inhibited the development of PH in rats. ⁹⁹ The attenuated development of PH with DMA and EIPA was achieved through decreased hypoxia-induced pulmonary vascular remodeling, as drug-treated animals showed decreased wall thickness of intraacinous vessels, compared to untreated hypoxic controls.

NHE1-specific inhibitors

Inhibitors exhibiting high specificity for the NHE1 isoform have been developed and pursued primarily for their potential cardioprotective effects. In animal studies, these agents have consistently demonstrated cardiogenic benefits in the setting of myocardial ischemia reperfusion injury, especially when the agents are given before the ischemic insult. ¹⁴⁹ To date, 2 members of a family of specific NHE1 inhibitors, cariporide and eniporide, have advanced to human clinical trials for the treatment of ischemia reperfusion injury.^150–153 Unfortunately, the results of these studies have been disappointing, with only the GUARDIAN study showing benefit of NHE1 inhibition in a single subgroup of patients, those who were reperfused via coronary artery bypass grafting (CABG). ¹⁵² Furthermore, the subsequent EXPEDITION study, while showing decreased myocardial infarction in cariporide-treated patients undergoing high-risk CABG, also revealed an overall increase in mortality in the cariporide treatment group due to an increased rate of cerebrovascular events. ¹⁵³ Because of the ubiquitous nature of NHE1 and the concerns raised by these studies, clinical trials of NHE1 inhibitors have largely been discontinued.

Exploration of the potential benefits of NHE1-specific inhibitors in PH has been limited. Sabiporide, another member of the NHE1 inhibitor family, was shown to inhibit proliferation and migration of human PASMCs in vitro. ⁶⁶ In vivo, cariporide has been shown to attenuate the development of right heart failure in monocrotaline-treated rats. ¹⁵⁴ In that study, cariporide-treated rats experienced significant blunting of the monocrotaline-induced increase in RVSP and RVH as well as blunting of monocrotaline-induced necrosis, fibrosis, and mononuclear infiltration of right ventricular myocardial cells. The authors claimed that the beneficial effects of cariporide in this model of PH were mediated through direct protection of right ventricular myocardium, because they argued that cariporide had no effect on pulmonary vascular remodeling. However, their conclusion that cariporide did not inhibit pulmonary vascular remodeling is tempered by the small number of vessels in which pulmonary artery wall thickness was measured as well as by the absence of measurement of distal vascular neomuscularization via smooth muscle–specific actin staining. Thus, further efforts at characterizing the effect of NHE1 inhibitors on the pulmonary vasculature in vivo are needed, especially in the setting of chronic hypoxia.

It should be noted that the mechanism whereby NHE1-specific inhibitors act on the exchanger remains undefined. Cariporide was characterized as an inhibitor of NHE1 ion transport activity. ¹⁵⁵ However, the effect of known NHE inhibitors on the NHE-ezrin-actin interaction or on NHE-dependent cytoskeletal rearrangement remains unexamined. Given that the C-terminal tail of NHE is required for cytoskeletal interactions and that the NHE-ezrin interaction is important for regulating cell shape and migration, it would be of interest to assess the differential effects on the development of hypoxic PH of drugs targeted to inhibition of NHE ion exchange and drugs targeted to inhibition of NHE-mediated cytoskeletal rearrangement. Importantly, cariporide was found to inhibit monocrotaline-induced increase in NHE1 mRNA expression in right ventricular myocytes. ¹⁵⁴ Thus, NHE1-specific inhibitors could impair all mechanisms of NHE1 signaling merely by depleting NHE1 expression. The effect of NHE1 inhibitors on NHE1 mRNA or protein expression in PASMCs remains undetermined, and future work on the effect of NHE1 inhibitors on the pulmonary vasculature will have to interrogate their mechanism of action in greater detail.

HIF inhibitors

An additional mechanism of NHE inhibition is via upstream inhibition of HIF-1. As noted above, HIF-1 is a transcription factor that upregulates many genes, including NHE1, in response to hypoxia. Cardiac glycosides, including digoxin, have been found to decrease HIF-1α protein expression via inhibition of HIF-1α mRNA translation. ¹⁵⁶ Acriflavine, which historically was used as a topical antiseptic, also inhibited HIF-1 function, though via a different mechanism: it inhibits dimerization of HIF-1α with HIF-1β, thus preventing formation of a functional HIF-1 unit. ¹⁵⁷

Both medications have been found to have beneficial effects on PH in animal models. Digoxin, when given to mice during a 3-week exposure to hypoxia, successfully attenuated hypoxia-induced increases in RVSP, RVH, and pulmonary vascular remodeling. ¹⁵⁸ In the same study, when mice were allowed to develop hypoxia-induced PH over 3 weeks of hypoxic exposure and were then treated with digoxin during 2 weeks of further hypoxic exposure, digoxin-treated mice developed a lower RVSP than vehicle-treated mice, indicating that digoxin therapy can slow the progression of established hypoxic PH. In these mice, digoxin therapy prevented hypoxia-induced increase in NHE1 expression in PASMCs, suggesting that NHE inhibition may be one pathway through which digoxin protects against the development of PH. It is possible that inhibition of HIF-1 targets other than NHE1 may play a role in the prevention of PH or that digoxin may have HIF-1-independent effects that protect against PH; however, the fact that acriflavine, which inhibits HIF-1 via a different mechanism, also prevented the development of hypoxia-induced PH ¹⁵⁸ suggests that the protective effect of digoxin is likely through an HIF-dependent mechanism.

ET-1 receptor antagonists

Because ET-1 acutely increases NHE activity ¹¹⁴ and is also implicated in upregulation of NHE1 expression in the setting of hypoxia, ¹³⁵ inhibition of ET-1 may serve as another upstream method of NHE inhibition. ET-1 signaling is effected through 2 G protein–coupled receptors, ET_A and ET_B. While ET_A stimulation contributes to vasoconstriction, ET_B receptor stimulation results in more ambiguous effects, because it prompts release of both vasodilatory NO and prostacyclin from endothelial cells as well as proconstrictory agents such as thromboxane A₂. ¹⁵⁹ However, both receptors promote human PASMC proliferation and thus may contribute to vascular remodeling. ¹⁶⁰ Treatment of rats with BQ-123, a selective antagonist of the ET_A receptor, during a 2-week exposure to 10% O₂ prevented pulmonary vascular remodeling and PH ¹¹³ and significantly reversed established hypoxic PH when started in the middle of a 4-week exposure to 10% O₂. ¹¹³ Nonselective inhibition of both ET_A and ET_B with bosentan has been shown to be similarly protective in the development of hypoxic PH. ¹⁶¹ While evaluation of bosentan in patients with hypoxic PH has been limited but disappointing, ¹⁶² the use of ET receptor antagonists in PAH has proven more promising.^163–167 To date, no experiments have been performed to evaluate whether NHE expression and/or activity are altered in PASMCs from chronically hypoxic animals treated with ET receptor antagonists, and thus, as with digoxin, it remains unclear to what extent the effects of ET-1 receptor antagonists are mediated through NHE inhibition versus effects on other signaling pathways.