Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na⁺/H⁺ exchanger (NHE)-mediated intracellular pH (pH_i) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH₄Cl was used to induce the acid load. Results: Aldosterone (10 nM) induced a rapid (<5 min) concentration-dependent increase in pH_i recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 nM). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µM) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µM) and cariporide (1 µM). The PKC inhibitor chelerythrine chloride (1 µM) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µM), a PKC_α inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µM), a PKC_δ antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µM) and dexamethasone (100 nM) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 nM) had no significant effect. MAPK inhibition using PD98059 (25 µM) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKC_α /MAPK pathway independent of the classical MR.

Fifty years ago, Hans Ussing described the mechanism by which ions are actively transported across frog skin. Since then, an enormous amount of effort has been invested in determining the cellular and molecular specifics of the transport mechanisms and their regulatory pathways. Ion transport in high-resistance epithelia is regulated by a variety of hormonal and non-hormonal factors. In vertebrates, steroid hormones such as mineralocorticoids, glucocorticoids and estrogens are major regulators of ion and water transport and hence are central to the control of extracellular fluid volume and blood pressure. Steroid hormones act through nuclear receptors to control the transcriptional activity of specific target genes, such as ion channels, ion transporters and ion pumps. These effects are observed after a latency of several hours and can last for days leading to cellular differentiation that allows a higher transport activity. This pathway is the so-called genomic phase. However, in the past 10 years, it has become apparent that steroid hormones can regulate electrolyte and water transport in tight epithelia independently of the transcription of these ion channels and transporters by regulating ion transporter activity in a non-genomic fashion via modulation of various signal transduction pathways. The molecular mechanisms underlying the steroid hormone-induced activation of signal transduction pathways such as protein kinase C (PKC), protein kinase A (PKA), intracellular calcium, intracellular pH and mitogen-activated protein kinases (MAPKs) and how non-genomic activation of these pathways influences epithelial ion transport will be discussed in this review.

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Together, these findings demonstrate that the renal sympathetic nerves not only have a direct action to modulate tubular sodium reabsorption via stimulation of the NHE transporter, but also have an indirect effect, whereby NHE3 abundance is increased within the brush border membrane, thereby increasing the capacity for fluid reabsorption.

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.