Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

ClC-2, a member of the voltage-gated Cl⁻ channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na⁺ conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl⁻ current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl⁻ current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn²⁺ sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn²⁺ (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl⁻ channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na⁺ transport in this surface epithelium under basal physiological conditions.     

Reduced Renal α-Klotho Expression in CKD Patients and Its Effect on Renal Phosphate Handling and Vitamin D Metabolism

Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)₂ vitamin D₃ (1,25VitD₃). Disruption of FGF23-α-KL signaling is thought to be an early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL expression and a reciprocal rise in serum FGF23. It remains unclear, however, whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated α-KL expression in renal biopsy samples and measured levels of several parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and urinary samples from CKD patients (n = 236). We found that although renal α-KL levels were significantly reduced and serum FGF23 levels were significantly elevated in early and intermediate CKD, serum phosphate levels remained within the normal range. Multiple regression analysis showed that the increases in FGF23 were significantly associated with reduced renal function and elevated serum phosphate, but were not associated with loss of renal α-KL. Moreover, despite falling renal α-KL levels, the increase in FGF23 enhanced urinary fractional excretion of phosphate and reduced serum 1,25VitD₃ levels in early and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell significantly over the course of CKD, and renal α-KL was a significant independent determinant of sKL. These results demonstrate that FGF23 levels rise to compensate for renal failure-related phosphate retention in early and intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate balance to be maintained despite the reduction in α-KL. In advanced CKD, however, renal α-KL declines further. This disrupts FGF23 signaling, and serum phosphate levels significantly increase, stimulating greater FGF23 secretion. Our results also suggest the serum sKL concentration may be a useful marker of renal α-KL expression levels.     

Molecular Cloning and DNA Analysis of the Orotidine-5′-Phosphate Decarboxylase Gene from the Yeast Saccharomyces exiguus Yp74L-3

The orotidine-5′-phosphate decarboxylase gene of Saccharomyces exiguus Yp74L-3 was cloned as a DNA fragment complementing a ura4 mutation of this yeast. The coding region of the gene is 807 bp in length, and represents 68.7% similarity to the corresponding gene of S. cerevisiae (URA3). The cloned URA4 gene was shown to be located on the 790-kbp Chromosome (chr) VIII of S. exiguus Yp74L-3. The neighbor-joining phylogenetic tree based on the orotidine-5′-phosphate decarboxylase coding sequences indicates that S. exiguus Yp74L-3 is closely related to Kluyveromyces yeasts, as well as to a S. cerevisiae laboratory strain. Received: 4 February 2000 / Accepted: 3 July 2000     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Uptake and transport of foreign particles in Peyer’s patches of both distal ileum and jejunum of calves

To investigate the uptake and transport patterns of variously sized particles in Peyer’s patches (PPs) of calves, intestinal loops were created in four newborn and two 2-month-old calves, and the loops were inoculated with various particles, including carbon black, fluorescein isothiocyanate (FITC)-labeled latex, FITC-labeled dextran, bovine serum, and recombinant mouse prion protein (rMPrP). The intestinal loops were recovered at 3, 6, 9, and 24 h in newborn calves and at 24 h in 2-month-old calves after inoculation, and the transport of the particles was examined by histological and immunohistochemical means. The uptake of the particles was quantified by estimation of signal intensities. A greater intensity was found in newborn calves compared with the 2-month-old calves. The peak uptake of carbon black, FITC-labeled latex, and rMPrP in the PPs of the distal ileum occurred at 6 h after inoculation in newborn calves and then progressively decreased with time. Uptake was also dependent on the site within the small intestine and the size of the particle studied. The transport of carbon black, FITC-labeled latex, and FITC-labeled dextran occurred via the bloodstream, the mesenteric lymph nodes, and the liver of newborn calves. rMPrP was found primarily in the interfollicular regions of the submucosa of the distal ileum of newborn calves. Thus, distal ileal PPs are probably more effective at particle absorption than the jejunal PPs, and the peak uptake of the PPs within the newborn calf occurs at 6 h after inoculation. This study was partly supported by a grant for BSE research from the Ministry of Health, Labour, and Welfare, Japan (17270701) and Grant-in-Aid (no. 17380180) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.