Acid incubation causes exocytic insertion of NHE3 in OKP cells

Incubation of opossum kidney clone P (OKP) cells in acid media(pH 6.8) causes activation of Na⁺/H⁺ exchanger3 (NHE3) at 6, 12, and 24 h. OKP cell NHE3 protein abundance was increased by 45% at 24 h of acid incubation but wasunaffected at 3-12 h. By contrast, apical membrane NHE3, measured by surface biotinylation, increased approximately twofold at 6, 12, and24 h, mirroring the increase in activity. Acid incubation caused a76% increase in exocytic insertion of NHE3 into the apical membranebut had no effect on endocytic internalization at 6 h. LatrunculinB, an inhibitor of microfilament organization, inhibited theacid-induced increases in apical membrane NHE3, exocytic insertion ofNHE3, and NHE3 activity at 6 h. These studies demonstrate two mechanisms for acid-induced increases in NHE3 activity. Beginning at6 h, there is an increase in apical membrane NHE3 that is due tostimulated exocytic insertion and is required for increased NHE3activity. At 24 h, there is an additional increase in total cellular NHE3.

     

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH

Chronichypokalemia increases the activity of proximal tubule apical membraneNa⁺/H⁺antiporter NHE3. The present study examined the effect ofthe incubation of OKP cells (an opossum kidney, clone P cell line) incontrol medium {K⁺ concn([K⁺]) = 5.4 mM} or low-K⁺ medium([K⁺] = 2.7 mM) onNHE3. The activity of an ethylisopropyl amiloride-resistant Na⁺/H⁺antiporter, whose characteristics were consistent with those ofNHE3, was increased inlow-K⁺ cells beginning at 8 h.NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%,respectively, at 24 h but not at 8 h. After incubation inlow-K⁺ medium, intracellular pH(pH_i) decreased by 0.27 pH units(maximum at 27 min) and then recovered to the control level.Intracellular acidosis induced by 5 mM sodium propionate increasedNa⁺/H⁺antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K⁺- andsodium propionate-induced activation of theNa⁺/H⁺antiporter at 8 and 24 h. Our results demonstrate thatlow-K⁺ medium causes an earlydecrease in pH_i, which leads to anincrease in NHE3 activity via a tyrosine kinase pathway.     

Rhinovirus increases human beta-defensin-2 and -3 mRNA expression in cultured bronchial epithelial cells

Human beta-defensins (hBDs) are antimicrobial peptides that play important roles in host defense against infection, inflammation and immunity. Previous studies showed that micro-organisms and proinflammatory mediators regulate the expression of these peptides in airway epithelial cells. The aim of the present study was to investigate the modulation of expression of hBDs in cultured primary bronchial epithelial cells (PBEC) by rhinovirus-16 (RV16), a respiratory virus responsible for the common cold and associated with asthma exacerbations. RV16 was found to induce expression of hBD-2 and -3 mRNA in PBEC, but did not affect hBD-1 mRNA. Viral replication appeared essential for rhinovirus-induced beta-defensin mRNA expression, since UV-inactivated rhinovirus did not increase expression of hBD-2 and hBD-3 mRNA. Exposure to synthetic double-stranded RNA (dsRNA) molecule polyinosinic:polycytidylic acid had a similar effect as RV16 on mRNA expression of these peptides in PBEC. In line with this, PBEC were found to express TLR3, a Toll-like receptor involved in recognition of dsRNA. This study shows that rhinovirus infection of PBEC leads to increased hBD-2 and hBD-3 mRNA expression, which may play a role in both the uncomplicated common cold and in virus-associated exacerbations of asthma.     

Testosterone increases osteoprotegerin mRNA expression in mouse osteoblast cells.

The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of Nf kappa B ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.     

Hepatitis B virus-mediated changes of apolipoprotein mRNA abundance in cultured hepatoma cells

An inverse correlation between hepatitis B virus (HBV) and steady-state levels of apolipoprotein AI and CIII mRNAs was observed in two hepatoma cell lines. Analysis of a third line containing an inducible viral genome implicated viral pregenomic RNA in apolipoprotein mRNA reduction. We conclude that HBV alters infected cells despite the absence of overt cytopathogenicity.     

Endothelin-1 chronically inhibits Na/H exchanger-3 in ET(B)-overexpressing OKP cells

Endothelin-1 (ET-1) acutely increases Na/H antiporter activity in OKPET(B)6 cells, an opossum kidney proximal tubule cell line transfected with ET(B) receptor cDNA. The purpose of the present study was to examine the chronic effect of ET-1 on Na/H antiporter activity in OKP cells and to examine whether Na/H exchanger (NHE)-3 mRNA and protein abundance are regulated by ET-1. Quiescent OKPET(B)6 cells were treated with 10 nM ET-1 for 3, 6 or 24 h and Na/H antiporter activity was assayed. The Na/H antiporter activity in 3-h ET-1-treated cells was not different from controls. However, Na/H antiporter activity was significantly decreased by 29% at 6 h and 72% at 24 h. The effect of ET-1 on Na/H antiporter activity was blocked by BQ788, an ET(B) receptor antagonist, but not BQ123, an ET(A) receptor antagonist. The NHE-3 mRNA abundance in ET-1-treated cells was not different from controls at 3 h. However, there was a significant decrease in NHE-3 mRNA abundance at 6 and 24 h. There was also a significant decrease in NHE-3 protein abundance at 6 and 24 h. In summary, ET-1 chronically inhibits NHE-3 in OKPET(B)6 cells.     

Quantifying the phenotypic information in mRNA abundance

Quantifying the dependency between mRNA abundance and downstream cellular phenotypes is a fundamental open problem in biology. Advances in multimodal single‐cell measurement technologies provide an opportunity to apply new computational frameworks to dissect the contribution of individual genes and gene combinations to a given phenotype. Using an information theory approach, we analyzed multimodal data of the expression of 83 genes in the Ca²⁺ signaling network and the dynamic Ca²⁺ response in the same cell. We found that the overall expression levels of these 83 genes explain approximately 60% of Ca²⁺ signal entropy. The average contribution of each single gene was 17%, revealing a large degree of redundancy between genes. Using different heuristics, we estimated the dependency between the size of a gene set and its information content, revealing that on average, a set of 53 genes contains 54% of the information about Ca²⁺ signaling. Our results provide the first direct quantification of information content about complex cellular phenotype that exists in mRNA abundance measurements.     

Acid increases NHE8 surface expression and activity in NRK cells

We previously demonstrated that there is a paucity of brush-border membrane NHE3 in neonates, the predominant Na(+)/H(+) exchanger in the adult proximal tubule, while NHE8 is relatively highly expressed in neonates compared with adults. We recently showed that metabolic acidosis in neonatal rodents can increase brush-border membrane NHE8 protein expression and Na(+)/H(+) exchange activity. To further examine the regulation of NHE8 by acid, we incubated NRK cells, which express NHE8 but not NHE3, with either acid or control media (6.6 vs. 7.4). There was an increase in Na(+)/H(+) exchanger activity within 6 h of incubation with acid media assessed as the rate of sodium-dependent recovery of pH from an acid load (dpH(i)/dt). The acid stimulation persisted for at least 24 h. The increase in Na(+)/H(+) exchange activity was paralleled by an increase in surface expression of NHE8, assessed by surface biotinylation and streptavidin precipitation. The increase in both apical membrane NHE8 protein expression and Na(+)/H(+) exchange activity with pH 6.6 media compared with 7.4 media was not affected by actinomycin D or cycloheximide consistent with an increase in surface expression independent of mRNA or protein synthesis. Furthermore, there was no increase in total cellular NHE8 protein abundance or mRNA abundance with acid media. Finally, we demonstrate that the increase in surface expression of NHE8 with acid media was blocked by colchicine and cytochalasin D and mediated by acid increasing the rate of exocytosis. In conclusion, NHE8 surface expression and activity are regulated by acid media by increasing the rate of trafficking to the apical membrane.     

Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by differentially increasing beta, and not alpha, subunit mRNA

In this paper we establish the response of LLC-PK1/Cl4 cells, a pig kidney cell line, to incubation in medium containing 0.25 mM K+. The amounts of the Na,K-ATPase alpha and beta subunits, determined by Western blot, increase coordinately to greater than 2-fold over control by 24 h in low K+ and remained elevated for the duration of the study period (48 h). Na,K-ATPase activity, measured enzymatically, increased 1.4-fold by 24 h and remained elevated. In order to determine if this response was initiated pretranslationally, alpha and beta subunit mRNA levels were determined by Northern blot analysis. While there was no change in alpha-mRNA levels, beta levels increased significantly, to 1.9-fold over control by 6 h of treatment and remained elevated. This selective increase in beta-mRNA was accompanied by 1.6- and 3.1-fold increases in the respective rates of accumulation of newly synthesized alpha and beta subunits, assessed by immunoprecipitating subunits from pulse-labeled cells. The degradation rates of mature Na,K-ATPase subunits did not change during 16 h of exposure to low K+, but after 16 h there was a selective decrease in the alpha degradation rate, relative to control. These results suggest that increased pretranslational regulation of the beta subunit alone is sufficient to increase accumulation of both alpha and beta subunits. These findings support the notion that in LLC-PK1 cells newly synthesized beta is rate-limiting and thus regulates, through alpha beta assembly, the number of pumps transported to the plasma membrane.     

Acid increases proliferation via ERK and p38 MAPK-mediated increases in cyclooxygenase-2 in Barrett's adenocarcinoma cells

Cyclooxygenase-2 (COX-2) has been linked to neoplastic progression in Barrett's esophagus. Acid exposure has been shown both to activate the MAPK pathways and to increase COX-2 protein expression in Barrett's metaplasia, but it is not known whether these effects are interrelated. We hypothesized that acid-induced activation of the MAPK pathways mediates an increase in COX-2 expression in Barrett's esophagus, and we tested this hypothesis in a Barrett's-associated adenocarcinoma cell line (SEG-1). We exposed SEG-1 cells to acidic or neutral media in the presence and absence of two MAPK inhibitors: U-0126 (an ERK inhibitor) or SB-203580 (a p38 inhibitor). We quantitated COX-2 protein levels using an enzyme immunometric assay and COX-2 mRNA levels using real-time PCR. We also determined how acid affects the activity of the COX-2 promoter and mRNA stability. Compared with SEG-1 cells exposed to neutral media, acid-exposed cells exhibited a 2.8-fold increase in COX-2 mRNA levels within 30 min. Both U-0126 and SB-203580 attenuated the acid-induced increase in COX-2 mRNA. Acid significantly increased COX-2 protein expression and promoter activity, and both of these effects were abolished by treatment with U-0126 and SB-203580. Acid exposure also stabilized COX-2 mRNA levels, an effect that was abolished by U-0126 but not by SB-203580. We conclude that acid increases COX-2 expression through activation of the MAPK pathways. Acid-induced activation of both ERK and p38 causes a significant increase in COX-2 promoter activity, and acid-activated ERK stabilizes COX-2 mRNA. These findings suggest potential mechanisms whereby acid reflux might promote carcinogenesis in Barrett's esophagus.