Search

An abstract of the article "Effect of amiloride on salt-sensitive and salt-resistant adult Nigerian subjects with and without β-ENaC polymorphisms" by S. O. Elias, O. A. Sofola and S. I. Jaja is presented.

BACKGROUND: Na+ transport via the amiloride-sensitive epithelial Na+ channel (ENaC) regulates airway surface liquid (ASL) volume and mucus clearance. In cystic fibrosis (CF) airways, increased sodium absorption depletes the ASL resulting in dysfunctional mucocilliary clearance and bacteria colonisation of the lung. Although serine protease such as prostasin is an important regulator of ENaC activity, it is largely inactive at pH seen in the acidic environment of CF airways. Here, we investigate the effect of pH and Cathepsin B (CTSB) activity on the regulation of ENaC. METHODS: The activity and expression of CTSB in polarised primary human bronchial epithelial cultures (HBECs; normal and CF) and ASL lavage were examined using fluorogenic substrates that are specifically cleaved by CTSB (Z-Arg-Arg-MCA, 50 μM) and western blotting respectively. We studied the effects of CTSB on amiloride-sensitive ENaC whole-cell currents (ΔIami) using two voltage clamp and cleavage of ENaC by surface biotinylation in Xenopus oocytes. To demonstrate the physiological relevance of CTSB in polarised HBECs, we used confocal microscopy to measure the ASL height in the presence and absence of a cell-impermeable CTSB inhibitor (CA074,10 μM). RESULTS: The activity of CTSB on the apical membrane surface and ASL lavage harvested from normal and CF polarised HBECs was significantly greater at pH 6 compared to pH 7.5, 7 or 6.5. Western blotting revealed that CTSB was secreted into the ASL in both normal and CF HBECs. Basal ΔIami in oocytes expressing rat αβγENaC and CTSB exposed to pH 6 or pH 7.3 were not significantly different. However, coexpression of ENaC and CTSB stimulated ΔIami by approximately 6-fold from 681.7 ± 61.4 to4143.3 ±417.1 nA (p< 0.0001, n = 6) at pH 7.3. In the presence of hCTSB, trypsin, but not chymotrypsin, further stimulated the ΔIami. Importantly, western blotting indicated a reduction in the molecular weight of surface-expressed γENaC in oocytes expressing ENaC and CTSB. In normal and CF HBECs, exposure to pH 6 Ringer solution significantly affect ASL regulation and reduced height from 13.4 ± 1.1 to 11.4 ± 0.9 μm (p < 0.05, n = 5) and 12.1 ± 0.8 to 7.5 ± 0.5 μm (p < 0.05, n = 5), respectively. CA074 significantly increased the ASL height in normal (11.4 ± 0.9 to 15.2 ± 1.5 μm, p < 0.05, n = 7) and CF (7.5 ± 0.5 to 11.5 ± 0.6 μm, p < 0.01, n = 7) HBECs at pH 6 but not pH 7.5. CONCLUSIONS: Taken together, these results indicate that CTSB, is secreted into the ASL, is more active at acidic pH and can regulate ASL height through the cleavage of γENaC. This is the first study to implicate CTSB in the pathphysiology of CF, suggesting that inhibitors of CTSB could potentially be used for the treatment of the disease. [ABSTRACT FROM AUTHOR]

Activity of the amiloride-sensitive Na+channel (ENaC) expressed in the distal nephronand respiratory epithelium has a profound impact on Na+ homeostasis, the regulation of blood pressure and respiratory surface fluid volume. Dysfunction of ENaC underlies human diseases such as Liddle's syndrome, pseudo hypoaldosteronism type 1 and cysticfibrosis. The activity of ENaC is tightly controlled by an array of physiological factors that exert their effect on transcription, trafficking and gating of the channel via multiple cellular signalling pathways. Hydrogen peroxide (H2O2) is one of the reactive oxygen species produced in response to oxidative stress, and it modulates a variety of physiological events. H2O2 exerts its effects via redox-sensitive proteins including transcription factors, protein tyrosine kinases and small G proteins. Previous studies suggested that H2O2 plays an important role in the regulation of ENaC, implicating it in the pathophysiology of pulmonary edema and acute lung injury. Consistent with this notion, exogenous H2O2 inhibits transepithelial ion transport and suppresses baseline expression of the α-subunit of ENaC in alveolar epithelial cells. In agreement with these reports, we found that H2O2 has a profound inhibitory effect on the amiloride-sensitive current in mouse collecting duct (M1) cells and on exogenous ENaC expressed in Fisher Rat Thyroid (FRT) cells. This inhibitory effect of H2O2 is attenuated in cells over-expressing a dominant-negative mutant of apoptosis signal-regulating kinase 1 (ASK-1), a ubiquitously expressed MAP kinase kinase kinase that is activated in response to cytotoxic stress. Our data further suggest that H2O2 inhibits the activity of ENaC via the ASK-1 /p38 MAP kinase signalling cascade by a mechanism that interferes with proteolytic activation of ENaC subunits. H2O2 is also known to be an activator of the proto-oncogenetyrosine-protein kinase (Src), a non-receptor membrane-associated tyrosine kinase that plays an important role in cytoskeletal organization and cell proliferation, tyrosine phosphorylation of many cellular signalling molecules and is involved in the cellular signalling pathway by which endothelin-1 inhibits activity of ENaC. We found that Src mediates the inhibitory effect of platelet-derived growth factor (PDGF) on ENaC. Unlike ASK-1, Src alters total ENaC protein expression and the abundance of ENaC at the cell membrane, but has no effect on proteolytic cleavage of the channel subunits. The inhibitory effect of Src requires cave-olin-1, suggesting that the effector molecules that are involved in the Src-mediated signalling system that regulates ENaC may be organised in the cholesterol-rich lipid raft sub-membrane domain. [ABSTRACT FROM AUTHOR]

There are possibly multiple pH sensitive chemoreceptors in the central nervous system. Recent findings showed that a fall of pH activates a distinct class of cation channels, the acid-sensing ion channels (ASICs) in peripheral and central nervous system (Akaike and Ueno 1994). On the other hand, the two-pore domain weakly inward rectifying K+ channel (TWIK)-related acid-sensitive potassium (TASK) is pH sensitive channel (Talley et al. 2000) too. Respiratory chemosensitivity seems to arise from interactions of multiple mechanisms, rather than a single unique channel, it has been documented that above mentioned pH sensitive channels may express in a single neuron (Chernov et al. 2010). ASIC1 in lateral hypothalamus con- tributes to breathing control (Song et al, 2012). But little is known about the composed influences of ASICs and TASK on respiratory regulation. This study investigated the cooperation of ASICs and TASK-1 in central respiratory regulation; they are both acid-sensing ion channels, the former for Na+, and the latter for K+. Sprague-Dawley rats (250-350g, n=8) of either sex were performed in the experiment. Rats were anaesthetized by intraperitoneal administration of 20% urethane (7ml/kg). Artificial cerebrospinal fluid (ACSF) with different pH values (pH = 5.5, 6.5, 7.4, 8.0 respectively, 0.1ul), Amiloride (1.0mM, 0.1ul, the blockade of ASICs), and Arachidonylethanolamide (AEA, 0.92g/mL, 0.1ul, the blockade of TASK-1), were respectively or jointly administered into lateral ventricle (LV) to observe the changes of respiratory activities via phrenic nerve discharge (PND) and its integration (PNDI). The respiratory excitability was increased as pH of ACSF decreased, significantly by pH 5.5 ACSF (P<0.05, ANOVAs). A convex concentration-effect curve of AEA was achieved within the concentration range from 10% to 100% at pH7.4. The AEA (50%) excited respiration significantly at pH 7.4 (P<0.05, ANOVAs), whereas Amiloride (1mM) inhibited it, though insignificantly. In contrast, the AEA didn't significantly excite the activated respiration at pH 5.5, while Amiloride inhibited it significantly (P<0.05, ANOVAs). The joint blockings of ASICs and TASK-1 resulted in the effect in-between that of respective blocking of these channels at both pH 7.4 and pH 5.5. The combined effect of Amiloride and AEA suggest that, medications targeted on ASICs and TASK-1 have opposite effects on phrenic nerve output. These two pH sensitive channels play a concerted role in central chemosensitivity during physiological and pathophysiological changes. [ABSTRACT FROM AUTHOR]