Taste transduction and channel synapses in taste buds.

The variety of taste sensations, including sweet, umami, bitter, sour, and salty, arises from diverse taste cells, each of which expresses specific taste sensor molecules and associated components for downstream signal transduction cascades. Recent years have witnessed major advances in our understanding of the molecular mechanisms underlying transduction of basic tastes in taste buds, including the identification of the bona fide sour sensor H+ channel OTOP1, and elucidation of transduction of the amiloride-sensitive component of salty taste (the taste of sodium) and the TAS1R-independent component of sweet taste (the taste of sugar). Studies have also discovered an unconventional chemical synapse termed "channel synapse" which employs an action potential-activated CALHM1/3 ion channel instead of exocytosis of synaptic vesicles as the conduit for neurotransmitter release that links taste cells to afferent neurons. New images of the channel synapse and determinations of the structures of CALHM channels have provided structural and functional insights into this unique synapse. In this review, we discuss the current view of taste transduction and neurotransmission with emphasis on recent advances in the field.

Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies.

Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated adenosine triphosphate (ATP)-release channels involved in neural functions including neurotransmission in gustation. Here, we present the cryo-electron microscopy (EM) structures of killifish CALHM1, human CALHM2, and Caenorhabditis elegans CLHM-1 at resolutions of 2.66, 3.4, and 3.6 Å, respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonamer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the intersubunit interactions at the transmembrane domain (TMD) and the TMD-intracellular domain linker define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels.

Copper-Catalyzed Electrophilic Etherification of Arylboronic Esters with Isoxazolidines.

A copper-catalyzed electrophilic etherification of arylboronic esters is reported. Isoxazolidines are utilized as easily available and stable [RO]+ surrogates to give 1,3-amino aryl ethers. The O-selective arylation of isoxazolidines takes place without causing competitive N-arylation. In contrast to previously reported anionic conditions, our copper-catalyzed conditions are mild enough to achieve high functional group tolerance. Preliminary mechanistic studies and DFT calculations support that the reaction proceeds via a transmetalation/oxidative addition pathway, followed by a Lewis acid-promoted reductive elimination to induce the crucial O-selectivity.

All-Electrical Ca2+-Independent Signal Transduction Mediates Attractive Sodium Taste in Taste Buds.

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.

[Na+] Increases in Body Fluids Sensed by Central Nax Induce Sympathetically Mediated Blood Pressure Elevations via H+-Dependent Activation of ASIC1a.

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na+] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Nax-knockout mice in contrast to wild-type mice. We identify Nax channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na+] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H+ ions exported from Nax-positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations.

The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration.

Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD+ system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt+/- mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD+ system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels.

Effect of Osteocyte-Ablation on Inorganic Phosphate Metabolism: Analysis of Bone-Kidney-Gut Axis.

In response to kidney damage, osteocytes increase the production of several hormones critically involved in mineral metabolism. Recent studies suggest that osteocyte function is altered very early in the course of chronic kidney disease. In the present study, to clarify the role of osteocytes and the canalicular network in mineral homeostasis, we performed four experiments. In Experiment 1, we investigated renal and intestinal Pi handling in osteocyte-less (OCL) model mice [transgenic mice with the dentin matrix protein-1 promoter-driven diphtheria toxin (DT)-receptor that were injected with DT]. In Experiment 2, we administered granulocyte colony-stimulating factor to mice to disrupt the osteocyte canalicular network. In Experiment 3, we investigated the role of osteocytes in dietary Pi signaling. In Experiment 4, we analyzed gene expression level fluctuations in the intestine and liver by comparing mice fed a high Pi diet and OCL mice. Together, the findings of these experiments indicate that osteocyte ablation caused rapid renal Pi excretion (P < 0.01) before the plasma fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) levels increased. At the same time, we observed a rapid suppression of renal Klotho (P < 0.01), type II sodium phosphate transporters Npt2a (P < 0.01) and Npt2c (P < 0.05), and an increase in intestinal Npt2b (P < 0.01) protein. In OCL mice, Pi excretion in feces was markedly reduced (P < 0.01). Together, these effects of osteocyte ablation are predicted to markedly increase intestinal Pi absorption (P < 0.01), thus suggesting that increased intestinal Pi absorption stimulates renal Pi excretion in OCL mice. In addition, the ablation of osteocytes and feeding of a high Pi diet affected FGF15/bile acid metabolism and controlled Npt2b expression. In conclusion, OCL mice exhibited increased renal Pi excretion due to enhanced intestinal Pi absorption. We discuss the role of FGF23-Klotho on renal and intestinal Pi metabolism in OCL mice.

Molecular mechanisms of cadmium-induced fibroblast growth factor 23 upregulation in osteoblast-like cells.

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. Renal proximal tubules are a major target of Cd toxicity. The whole mechanism of the adverse effects of Cd remains unresolved, especially how renal damage is related to the development of bone lesions. Fibroblast growth factor 23 (FGF23) is a bone-derived phosphaturic factor that regulates vitamin D and inorganic phosphate metabolism in the kidney. To clarify the role of FGF23 on Cd toxicity, we investigated the mechanisms of Cd-induced FGF23 production in the bone. Cd injection into mice significantly increased plasma FGF23 concentrations, but did not change FGF23 mRNA expression in bone. GalNAc-T3 is involved in secreting intact FGF23. To determine potential roles of GalNAc-T3 in Cd-induced FGF23 production, we examined the effect of Cd on GalNAc-T3 mRNA expression in vivo and in vitro. GalNAc-T3 gene expression was significantly increased in the bones of Cd-injected mice. Cd also enhanced the expression of GalNAc-T3 in cultured osteosarcoma UMR106 cells and primary osteocytes. Cd activated aryl hydrocarbon receptors (AhR) and AhR were required for GalNAc-T3 gene expression induced by Cd. In addition, Cd-dependent FGF23 production was completely inhibited by an AhR antagonist. AhR siRNA markedly suppressed the stimulation of transcriptional activity by Cd. Furthermore, Cd induced AhR activation via phosphorylation of Ser-68 by p38 kinase in the nuclear export signal of AhR. Thus, Cd stimulated GalNAc-T3 gene transcription via enhanced AhR binding to the GalNAc-T3 promoter. These findings suggest that the Cd-induced increase in GalNAc-T3 suppresses proteolytic processing of FGF23 and increases serum FGF23 concentrations.

Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

[Fibroblast growth factor 23 mediates the phosphaturic actions of cadmium].

Phosphaturia has been documented following cadmium (Cd) exposure in both humans and experimental animals. Fibroblast growth factor 23 (FGF23) serves as an essential phosphate homeostasis pathway in the bone-kidney axis. In the present study, we investigated the effects of Cd on phosphate (Pi) homeostasis in mice. Following Cd injection into C57BL/6J mice, plasma FGF23 concentration significantly increased. The urinary Pi excretion level was significantly higher in the Cd-injected C57BL/6J mice than in the control group. Plasma Pi concentration decreased only slightly in the Cd-injected mice compared with the control group. No changes were observed in the concentration of the plasma parathyroid hormone and 1,25-dihydroxy vitamin D(3) in both groups of mice. We observed a decrease in phosphate transport activity and also a decrease in the expression level of renal phosphate transporter Npt2c, but not that of Npt2a. Furthermore, we examined the effect of Cd on Npt2c in Npt2a-knockout (KO) mice, which expresses Npt2c as a major NaPi cotransporter. Injecting Cd to Npt2aKO mice induced a significant increase in plasma FGF23 concentration and urinary Pi excretion level. Furthermore, we observed decreases in phosphate transport activity and renal Npt2c expression level in the Cd-injected Npt2a KO mice. The present study suggests that hypophosphatemia induced by Cd may be closely associated with FGF23.

[Phosphate sensing and intestine].

Small intestine plays an important role in the sensing of various nutrients. There is information that would imply the existence of a dietary phosphate sensing mechanism within the intestine. Recent studies suggest that intestinal factors may involve in the alteration of renal phosphate transport. The elucidation of the phosphate sensing mechanism is expected to provide molecular basis for the prevention of the hyperphosphatemia in chronic kidney disease patients.

[Information about phosphorus additives and nutritional counseling].

Hyperphosphatemia is a common disorder in patients with chronic kidney disease (CKD) , and may result in hyperparathyroidism and renal osteodystrophy. Hyperphosphatemia also may contribute to deterioration vascular calcification and increase mortality. Hence, correction and prevention of hyperphosphatemia is a main component of the management of CKD. This goal is usually approached both by administering phosphorus binders and by restricting dietary phosphorus (P) intake. Dietary intake of phosphorus (P) is derived largely from foods with high protein content or food additives and is an important determinant of P balance in patient with CKD. Food additives (PO4) can dramatically increase the amount of P consumed in the daily diet, especially because P is more readily absorbed in its inorganic form. In addition, information about the P content and type in prepared foods is often unavailable or misleading. Therefore, during dietary counseling of patients with CKD, we recommended that they consider both the absolute dietary P content and the P-to-protein ratio of foods and meals including food additives.

Identification and functional analysis of a splice variant of mouse sodium-dependent phosphate transporter Npt2c.

Mutations in the SLC34A3 gene, a sodium-dependent inorganic phosphate (Pi) cotransporter, also referred to as NaPi IIc, causes hereditary hypophosphatemic rickets with hypercalciuria (HHRH), an autosomal recessive disorder. In human and rodent, NaPi IIc is mainly localized in the apical membrane of renal proximal tubular cells. In this study, we identified mouse NaPi IIc variant (Npt2c-v1) that lacks the part of the exon 3 sequence that includes the assumed translation initiation site of Npt2c. Microinjection of mouse Npt2c-v1 cRNA into Xenopus oocytes demonstrated that Npt2c-v1 showed sodium-dependent Pi cotransport activity. The characterization of pH dependency showed activation at extracellular alkaline-pH. Furthermore, Npt2c-v1 mediated Pi transport activity was significantly higher at any pH value than those of Npt2c. In an in vitro study, the localization of the Npt2c-v1 protein was detected in the apical membrane in opossum kidney cells. The expression of Npt2c-v1 mRNA was detected in the heart, spleen, testis, uterus, placenta, femur, cerebellum, hippocampus, diencephalon and brain stem of mouse. Using mouse bone primary cultured cells, we showed the expression of Npt2c-v1 mRNA. In addition, the Npt2c protein was detected in the spermatozoa head. Thus, Npt2c-v1 was expressed in extra-renal tissues such as epididymal spermatozoa and may function as a sodium-dependent phosphate transporter.

[Calcium pros and cons significance and risk of phosphorus supplementation. The risk of dietary phosphorus intake].

Dietary intake of phosphorus (Pi) is an important determinant of Pi balance in patients who have chronic kidney disease (CKD) and a reduced GFR. High dietary Pi burden may promote vascular calcification and cardiovascular events. Recently, Ohnishi and Razzaque suggest that phosphate toxicity accelerates the mammalian aging process and that reducing the phosphate burden can delay the aging (FASEB J 24, 3562, 2010) . Dietary Pi is derived largely from foods with high protein content or food additives. Accurate information on the Pi content of foods is needed to achieve a low Pi intake and effectively manage CKD and the aging. In this review, we discuss the risk of dietary Pi intake in CKD and the aging.

Sodium-dependent phosphate cotransporters: lessons from gene knockout and mutation studies.

Inorganic phosphate (Pi) is an essential physiological compound, highlighted by the syndromes caused by hypo or hyperphosphatemic states. Hyperphosphatemia is associated with ectopic calcification, cardiovascular disease, and increased mortality in patients with chronic kidney disease (CKD). As phosphate control is not efficient with diet or dialysis, oral Pi binders are used in over 90% of patients with renal failure. However, achieving tight control of serum Pi is difficult, and lower levels of serum Pi (severe hypophosphatemia) do not lead to better outcomes. The inhibition of sodium-dependent Pi (NaPi) transporter would be a preferable method to control serum Pi levels in patients with CKD or patients undergoing dialysis. Three types of NaPi transporters (types I-III) have been identified: solute carrier series SLC17A1 (NPT1/NaPi-I/OATv1), SLC34 (NaPi-IIa, NaPi-IIb, NaPi-IIc), and SLC20 (PiT1, PiT2), respectively. Knockout mice have been created for types I-III NaPi transporters. In this review, we discuss the roles of the NaPi transporters in Pi homeostasis.