Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na⁺ (Na _o ⁺ /H _i ⁺ and Na _i ⁺ /H _o ⁺ exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na _o ⁺ /H _i ⁺ exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.     

Effect of dietary phosphate intake on phosphate transport by isolated rat renal brush-border vesicles

Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.     

High yield purification of JNK1β1 and activation by in vitro reconstitution of the MEKK1 → MKK4 → JNK MAPK phosphorylation cascade

The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His₆-tagged proteins and purified using urea refolding and Ni²⁺-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.     

Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs

The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.     

Strong dependence between phytoplankton and water chemistry in a large temperate lake: spatial and temporal perspective

In lakes, spatial and temporal variability of water chemistry and phytoplankton are characteristic phenomena although often difficult to link together. This motivated us to study their interplay in Lake Vanajanselkä, a eutrophic lake in Finland. We hypothesized that in summer spatial and temporal differences in phytoplankton and water chemistry can be extended in comparison to spring and autumn. Therefore, chlorophyll a and water chemistry was examined by six sampling campaigns with 15 sampling sites over the lake in May–October 2009–2010. In summer, chlorophyll, pH, and oxygen were horizontally and vertically unevenly distributed in the lake, and in the epilimnion pH and oxygen showed a distinct diurnal variability suggesting high photosynthesis during the day. Daily >1 pH unit difference between the sites and 2.5 pH unit difference between the epi- and hypolimnion were found. In agreement with pH and oxygen, NO₃-N and NH₄-N could be unevenly distributed in the epilimnion. In autumn no spatial differences were found, however. The results emphasized that algae and cyanobacteria were responsible, at least partly, for the variability in water chemistry in the surface layer, and short- and long-term gradients in space and time need to be considered when productive lakes are studied.     

C-terminal tail phosphorylation of N-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies NFPR1 and NFPR2

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.     

Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants.     

Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice

Intake of a low-phosphate diet stimulates transepithelial transport of P_i in small intestine as well as in renal proximal tubules. In both organs, this is paralleled by a change in the abundance of the apically localized NaP_i cotransporters NaP_i type IIa (NaP_i-IIa) and NaP_i type IIb (NaP_i-IIb), respectively. Low-P_i diet, via stimulation of the activity of the renal 25-hydroxyvitamin-D₃-1-hydroxylase (1OHase), leads to an increase in the level of 1,25-dihydroxy-vitamin D₃ [1,25(OH)₂D]. Regulation of the intestinal absorption of P_i and the abundance of NaP_i-IIb by 1,25(OH)₂D has been supposed to involve the vitamin D receptor (VDR). In this study, we investigated the adaptation to a low-P_i diet of NaP_i-IIb in small intestine as well as NaP_i-IIa in kidneys of either VDR- or 1OHase-deficient mice. In both mouse models, upregulation by a low-P_i diet of the NaP_i cotransporters NaP_i-IIa and NaP_i-IIb was normal, i.e., similar to that observed in the wild types. Also, in small intestines of VDR- and 1OHase-deficient mice, the same changes in NaP_i-IIb mRNA found in wild-type mice were observed. On the basis of the results, we conclude that the regulation of NaP_i cotransport in small intestine (via NaP_i-IIb) and kidney (via NaP_i-IIa) by low dietary intake of P_i cannot be explained by the 1,25(OH)₂D-VDR axis. NaP_i type IIb; vitamin D₃