Biomedical subjects

He-Ping Ma

Publications and source records attributed to He-Ping Ma.

8 recordsLinked to original sources

PubMed2006

Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides.

The epithelial Na(+) channel (ENaC) is an end-effector of diverse cellular signaling cascades, including those with phosphatidylinositide second messengers. Recent evidence also suggests that in some instances, phospatidylinositides can directly interact with ENaC to increase channel activity by increasing channel open probability and/or membrane localization. We review here findings relevant to regulation of ENaC by phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatidylinositol 3,4,5-triphosphate (PIP(3)). Similar to its actions on other ion channels, PIP(2) is permissive for ENaC openings having a direct effect on gating. The PIP(2) binding site in ENaC involved in this regulation is most likely localized to the NH(2) terminus of beta-ENaC. PIP(3) also affects ENaC gating but, rather than being permissive, augments open probability. The PIP(3) binding site in ENaC involved in this regulation is localized to the proximal region of the COOH terminus of gamma-ENaC just following the second transmembrane domain. In complementary pathways, PIP(3) also impacts ENaC membrane levels through both direct actions on the channel and via a signaling cascade involving phosphoinositide 3-OH kinase (PI3-K) and the aldosterone-induced gene product serum and glucocorticoid-inducible kinase. The putative PIP(3) binding site in ENaC involved in direct regulation of channel membrane levels has not yet been identified.

Binding Sites↗

PubMed2005

Reactive species mediate inhibition of alveolar type II sodium transport during mycoplasma infection.

RATIONALE: Mycoplasma pneumoniae is a significant cause of pneumonia in humans. OBJECTIVES: To determine the impact of mycoplasma infection and the host inflammatory response on alveolar type II (ATII) cell ion transport in vivo and in vitro. METHODS: Mice were infected with M. pulmonis for measurements of alveolar fluid clearance (AFC) in vivo and isolation of ATII cells. ATII cells were infected in vivo for determination of epithelial Na+ channel (ENaC) total and cell surface protein levels by biotinylation and Western blot and in vitro for whole cell patch clamp recording and measurement of nitric oxide (NO) production by chemiluminescence. RESULTS: Mycoplasma infection significantly inhibited AFC at 24 h and total and amiloride-sensitive AFC by 48 h postinfection (pi). In contrast, infected myeloperoxidase-deficient mice had similar basal and amiloride-sensitive AFC values to uninfected control mice at 48 h pi. Addition of forskolin restored total and amiloride-sensitive AFC to control values at 48 h pi. ATII cells isolated from infected mice demonstrated normal alpha, beta, and gamma ENaC total protein levels; however, infected whole-lung cell-surface levels of gamma ENaC were significantly decreased. Patch-clamp recordings demonstrated a significant decrease in total and amiloride-sensitive Na+ currents at 24 h pi. ATII cells demonstrated a significant increase in the production of NO at 24 h pi and inhibition of NO by ATII cells before infection reversed the decrease in total Na+ currents. CONCLUSIONS: These data indicate that mycoplasma infection results in decreased AFC and functional ENaC via the production of reactive oxygen nitrogen intermediates.

Animals↗

PubMed2005

Phosphatidylinositol 3,4,5-trisphosphate mediates aldosterone stimulation of epithelial sodium channel (ENaC) and interacts with gamma-ENaC.

Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). PIP(3) significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP(3)-binding protein (Grp1-PH), which sequestered intracellular PIP(3). 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(T)-ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).

Aldosterone↗

PubMed2005

Acute regulation of epithelial sodium channel by anionic phospholipids.

Anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) are normally located in the inner leaflet of the plasma membrane, where these anionic phospholipids can regulate transmembrane proteins, including ion channels and transporters. Recent work has demonstrated that (1) ATP inhibits the renal epithelial sodium channel (ENaC) via a phospholipase C-dependent pathway that reduces PIP(2), (2) aldosterone stimulates ENaC via phosphoinositide 3-kinase, and (3) PIP(2) and PIP(3) regulate ENaC. Several lines of evidence show that ATP stimulation of purinergic P2Y receptors hydrolyzes PIP(2) and that aldosterone stimulation of steroid receptors induces PIP(3) formation. These studies together suggest that one primary mechanism for regulating ENaC is by alteration of anionic phospholipids and that the receptor-mediated and hormonal regulation of ENaC works through a variety of signaling pathways, but many of these pathways finally alter ENaC activity by regulating the formation or degradation of anionic phospholipids. Therefore, changes in the concentration of PIP(2) and PIP(3) are hypothesized to participate in the regulation of ENaC by purinergic and corticoid receptors. The underlying mechanism may be associated with a physical interaction of the positively charged cytoplasmic domains of the beta- and gamma-ENaC with the negatively charged membrane phospholipids. The exact nature of this interaction will require further investigation.

Amino Acid Sequence↗

PubMed2004

Steroids and exogenous gamma-ENaC subunit modulate cation channels formed by alpha-ENaC in human B lymphocytes.

Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both alpha- and beta- but not gamma-ENaC. Western blots showed that both alpha- and beta- but not gamma-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against alpha-ENaC but not by antisense oligonucleotide against beta-ENaC, indicating that only the alpha subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 microm) reduced the frequency of observance of 21-pS alpha-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from alpha- and beta-ENaC. After expression of exogenous gamma-ENaC, aldosterone again reduced the frequency of observance of the 21-pS alpha-ENaC channel but induced the appearance of a 5-pS channel, presumably a alphabetagamma-ENaC channel. In the absence of aldosterone, the alpha subunit forms an alpha-cryptic channel that is activated by stretch, and in the presence of aldosterone, beta and alpha subunits together form an active channel that is modulated by aldosterone.

Aldosterone↗

PubMed2004

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway.

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl- for gluconate(-) shifted the reversal potential, while Cl- channel blockers, 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPgammaS (a poorly hydrolyzable analog of ATP), 2',3'-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 microM suramin (a P2 receptor antagonist), and was partially blocked by 100 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca(2+)-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.

Adenosine Triphosphate↗

PubMed2002

ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells.

The mechanosensitivity of the epithelial sodium channel (ENaC) is controversial. Using cell-attached patch-clamp techniques, we found that mechanical stretch stimulated ENaC in A6 distal nephron cells in only three of nine cell-attached patches. However, stretch consistently activated ENaC after apical ATP was scavenged with apical hexokinase plus glucose or after P(2) receptors in the patch were blocked. The mean open probability (P(o)) of ENaC was increased from 0.31 +/- 0.04 to 0.61 +/- 0.06 (P < 0.001; n = 9) when patch pipettes contained hexokinase and glucose, or from 0.24 +/- 0.05 to 0.55 +/- 0.11 (P < 0.01; n = 7) when patch pipettes contained suramin, respectively. A poorly hydrolyzable ATP analog, ATPgammaS, in the patch pipettes inhibited ENaC, reducing the P(o) from 0.41 +/- 0.06 to 0.19 +/- 0.05 (P < 0.01; n = 8). Pretreatment of A6 cells with the phospholipase C (PLC) inhibitor U-73122 abolished the effect of ATP on ENaC activity. These data together suggest that ATP, acting through a PLC-dependent purinergic pathway, masks stretch-induced ENaC activation.

Adenosine Triphosphate↗