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Transport of phosphate across intestinal and renal epithelia is essential for normal phosphate balance, yet we know less about the mechanisms and regulation of intestinal phosphate absorption than we do about phosphate handling by the kidney. Recent studies have provided strong evidence that the sodium-phosphate cotransporter NaPi-IIb is responsible for sodium-dependent phosphate absorption by the small intestine, and it might be that this protein can link changes in dietary phosphate to altered renal phosphate excretion to maintain phosphate balance. Evidence is also emerging that specific regions of the small intestine adapt differently to acute or chronic changes in dietary phosphate load and that phosphatonins inhibit both renal and intestinal phosphate transport. This review summarizes our current understanding of the mechanisms and control of intestinal phosphate absorption and how it may be related to renal phosphate reabsorption; it also considers the ways in which the gut could be targeted to prevent, or limit, hyperphosphatemia in chronic and end-stage renal failure. phosphate transport; small intestine; kidney; proximal tubule; phosphatonins doi: 10.1152/ajprenal.00508.2009.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate. A m J Physiol Renal Physiol 297: F1466-F1475, 2009. First published August 12, 2009: doi:10.1152/ajprenal.00279.2009.--Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal [P.sub.i] absorption is one of the pharmacological targets that prevents it. The type II Na-[P.sub.i] cotransporter NaPi-2b is the major transporter that mediates [P.sub.i] reabsorption in the intestine. The potential role and regulation of other Na-[P.sub.i] transporters remain unknown. We have identified expression of the type III Na-[P.sub.i] cotransporter PIT-1 in the apical membrane of enterocytes. Na-[P.sub.i] transport activity and NaPi-2b and PIT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-[P.sub.i] diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-[P.sub.i] transport activity and NaPi-2b, but not PiT-l, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-[P.sub.i] diet, there is an increase in BBM Na-[P.sub.i] transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum [P.sub.i] that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-[P.sub.i] transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia. SLC34A2; PiT-1; hyperphosphatemia; chronic kidney disease; dietary [P.sub.i] doi: 10.1152/ajprenal.00279.2009.

The relationship between cardiac energy metabolism and the depression of myocardial performance during oxygen deprivation has remained enigmatic. Here, we combine in vivo [sup.31]P-NMR spectroscopy and MRI to provide the first temporal profile of in vivo cardiac energetics and cardiac performance of an anoxia-tolerant vertebrate, the freshwater turtle (Trachemys scripta) during long-term anoxia exposure (~3 h at 21[degrees]C and 11 days at 5[degrees]C). During anoxia, phosphocreatine (PCr), unbound levels of inorganic phosphate (effective [P.sup.2-.sub.i]), intracellular pH ([pH.sub.i]), and free energy of ATP hydrolysis (dG/d[xi]) exhibited asymptotic patterns of change, indicating that turtle myocardial high-energy phosphate metabolism and energetic state are reset to new, reduced steady states during long-term anoxia exposure. At 21[degrees]C, anoxia caused a reduction in pHi from 7.40 to 7.01, a 69% decrease in PCr and a doubling of effective [P.sup.2-.sub.i]. ATP content remained unchanged, but the free energy of ATP hydrolysis (dG/d[xi]) decreased from -59.6 to -52.5 k J/tool. Even so, none of these cellular changes correlated with the anoxic depression of cardiac performance, suggesting that autonomic cardiac regulation may override putative cellular feedback mechanisms. In contrast, during anoxia at 5[degrees]C, when autonomic cardiac control is severely blunted, the decrease of phi from 7.66 to 7.12, 1.9-fold increase of effective [P.sup.2-.sub.i] , and 6.4 k J/tool decrease of dG/d[xi] from -53.8 to -47.4 kJ/mol were significantly correlated to the anoxic depression of cardiac performance. Our results provide the first evidence for a close, long-term coordination of functional cardiac changes with cellular energy status in a vertebrate, with a potential for autonomic control to override these immediate relationships. high-energy phosphate metabolism: anoxic turtle cardiac performance; in vivo magnetic resonance spectroscopy

Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (>2.5 mM) induced apoptosis in two endothelial cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at ~2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease. atherosclerosis; ROS; mitochondrial dysfunction; endothelial dysfunction

A new, completely in vivo method of measuring the rate of intestinal phosphate absorption has been developed. As expected from previous in vitro and ex vivo measurements, intestinal phosphate absorption is potently and rapidly stimulated by 1,25-dihydroxyvitamin [D.sub.3]. The response is saturated with as little as 11.3 ng of 1,25-dihydroxyvitamin [D.sub.3] per day, consistent with a genomic mechanism. The effect of 1,25-dihydroxyvitamin [D.sub.3] disappears when the dosing solution of phosphate is at 2 M, suggesting that 1,25-dihydroxyvitamin D3 stimulates active transport of phosphate but not diffusion of phosphate. Finally, unlike findings resulting from in vitro or ex vivo experiments, no evidence in vivo was obtained that phosphate absorption requires sodium or is inhibited by potassium. vitamin D; phosphorus; intestine doi: 10.1152/ajpendo.00654.2006

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two factors that share amino acid sequence homology and act via a common receptor. In tetrapods, PTH is the main endocrine factor acting in bone and kidney to regulate calcium and phosphate. PTHrP is an essential paracrine developmental factor present in many tissues and is involved in the regulation of ossification, mammary gland development, muscle relaxation, and other functions. Fish apparently lack an equivalent of the parathyroid gland and were long thought to be devoid of PTH. Only in recent years has the existence of PTH-like peptides and their receptors in fish been firmly established. Two forms of PTH, two of PTHrP, and a protein with intermediate characteristics designated PTH-L are encoded by separate genes in teleost fish. Three receptors encoded by separate genes in fish mediate PTH/PTHrP actions, whereas only two receptors have so far been found in terrestrial vertebrates. PTHrP has been more intensively studied than PTH, from lampreys to advanced teleosts. It is expressed in many tissues and is present in high concentration in fish blood. Administration of this peptide alters calcium metabolism and has marked effects on associated gene expression and enzyme activity in vivo and in vitro. This review provides a comprehensive overview of the physiological roles, distribution, and molecular relationships of the piscine PTH-like peptides. parathyroid hormone-related protein; calcium regulation; phosphate regulation; molecular evolution

To study the role of early energetic abnormalities in the subsequent development of heart failure, we performed serial in vivo combined magnetic resonance imaging (MRI) and [sup.31]p magnetic resonance spectroscopy (MRS) studies in mice that underwent pressure-overload following transverse aorta constriction (TAC). After 3 wk of TAC, a significant increase in left ventricular (LV) mass (74 [+ or -] 4 vs. 140 [+ or -] 26 mg, control vs. TAC, respectively; P < 0.000005), size [end-diastolic volume (EDV): 48 [+ or -] 3 vs. 61 [+ or -] 8 [micro]l; P < 0.005], and contractile dysfunction [ejection fraction (EF): 62 [+ or -] 4 vs. 38 [+ or -] 10%; P < 0.000005] was observed, as well as depressed cardiac energetics (PCr/ATP: 2.0 [+ or -] 0.1 vs. 1.3 [+ or -] 0.4, P < 0.0005) measured by combined MRI/MRS. After an additional 3 wk, LV mass (140 [+ or -] 26 vs. 167 [+ or -] 36 rag; P < 0.01) and cavity size (EDV: 61 [+ or -] 8 vs. 76 [+ or -] 8 [micro]1; P < 0.001) increased further, but there was no additional decline in PCr/ATP or EF. Cardiac PCr/ATP correlated inversely with end-systolic volume and directly with EF at 6 wk but not at 3 wk, suggesting a role of sustained energetic abnormalities in evolving chamber dysfunction and remodeling. Indeed, reduced cardiac PCr/ATP observed at 3 wk strongly correlated with changes in EDV that developed over the ensuing 3 wk. These data suggest that abnormal energetics due to pressure overload predict subsequent LV remodeling and dysfunction. high-energy phosphates; magnetic resonance imaging; magnetic resonance spectroscopy

The general phosphate need in mammalian ceils is accommodated by members of the Pi transport (PIT) family (SLC20), which use either [Na.sup.+] or [H.sup.+] to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are [Na.sup.+]-dependent [P.sub.i] (Na[P.sub.i]) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with [sup.32][P.sub.i] as a traceable [P.sub.i] source. For PiT1, the Michaelis-Menten constant for [P.sub.i] was determined as 322.5 [+ or -] 124.5 [micro]M. PiT2 was analyzed for the first time and showed positive cooperativity in [P.sub.i] uptake with a half-maximal activity constant for [P.sub.i] of 163.5 [+ or -] 39.8 [micro]M. PiT1- and PiT2-mediated [Na.sup.+]-dependent [P.sub.i] uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Nat dependency patterns. However, only PiT2 was capable of [Na.sup.t] independent [P.sub.i] transport at acidic pH. Study of the impact of divalent cations [Ca.sub.2+] and [Mg.sub.2+] revealed that [Ca.sub.2+] was important, but not critical, for NaPi transport function of PiT proteins. To gain insight into the NaPi cotransport function, we analyzed PiT2 and a PiT2 [P.sub.i] transport knockout mutant using [sub.22][Na.sup.+] as a traceable [Na.sup.+] source. [Na.sup.+] was transported by PiT2 even without [P.sub.i] in the uptake medium and also when [P.sub.i] transport function was knocked out. This is the first time decoupling of [P.sub.i] from [Na.sup.+] transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids [E.sup.55] and [E.sup.575] are responsible for linking [P.sub.i] import to [Na.sup.+] transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20

Increases in [P.sub.i] combined with decreases in myoplasmic [Ca.sup.2+] are believed to cause a significant portion of the decrease in muscular force during fatigue. To investigate this further, we determined the effect of 30 mM [P.sub.i] on the force-[Ca.sup.2+] relationship of chemically skinned single muscle fibers at near-physiological temperature (30[degrees]C). Fibers isolated from rat soleus (slow) and gastrocnemius (fast) muscle were subjected to a series of solutions with an increasing free [Ca.sup.2+] concentration in the presence and absence of 30 mM [P.sub.i] at both low (15[degrees]C) and high (30[degrees]C) temperature. In slow fibers, 30 mM [P.sub.i] significantly increased the [Ca.sup.2+] required to elicit measurable force, referred to as the activation threshold at both low and high temperatures; however, the effect was twofold greater at the higher temperature. In fast fibers, the activation threshold was unaffected by elevating [P.sub.i] at 15[degrees]C but was significantly increased at 30[degrees]C. At both low and high temperatures, 30 mM [P.sub.i] increased the [Ca.sup.2+] required to elicit half-maximal force (p[Ca.sub.50]) in both slow and fast fibers, with the effect of Pa twofold greater at the higher temperature. These data suggest that during fatigue, reductions in the myoplasmic [Ca.sup.2+] and increases in Pi act synergistically to reduce muscular force. Consequently, the combined changes in these ions likely account for a greater portion of fatigue than previously predicted based on studies at lower temperatures or high temperatures at saturating [Ca.sup.2+] levels. force-pCa relationship; phosphate; fatigue

Fibroblast growth factor-23 (FGF23) has been recently identified as playing an important pathophysiological role in phosphate homeostasis and vitamin D metabolism. To elucidate the precise physiological regulation of FGF-23, we characterized the mouse FGF-23 5'-flanking region and analyzed its promoter activity. The 5'-flanking region of the mouse FGF-23 gene contained a TFIID site (TATA box) and several putative transcription factor binding sites, including MZF1, GATA-1 and c-Ets-1 motifs, but it did not contain the typical sequences of the vitamin D response element. Plasmids encoding 554-bp (pGL/-0.6), 364-bp (pGL/-0.4) and 200-bp (pGL/-0.13) promoter regions containing the TFIID element and + 1-bp fragments drove the downstream expression of a luciferase reporter gene in transfection assays. We also found that FGF-23 mRNA was expressed in K-562 erythroleukemia cell lines but not in MC3T3-E1, Raji, or Hep G2 human carcinoma cells. Treatment with 1,25-dihydroxyvitamin [D.sub.3] in the presence of high phosphate markedly stimulated pGL/-0.6 activity, but calcium had no effect. In addition, the plasma FGF-23 levels were affected by the dietary and plasma inorganic phosphate concentrations. Finally, the levels of plasma FGF-23 in vitamin D receptor-null mice were significantly lower than in wild-type mice. The presents study demonstrated that vitamin D and the plasma phosphate level are important regulators of the transcription of the mouse FGF-23 gene. gene regulation; vitamin D receptor; phosphate homeostasis

Osteoclasts possess inorganic phosphate ([P.sub.i]) transport systems to take up external [P.sub.i] during bone resorption. In the present study, we characterized [P.sub.i] transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-[kappa]B ligand (RANKL). In undifferentiated RAW264.7 cells, [P.sub.i] transport into the cells was [Na.sup.+] dependent, but after treatment with RANKL, [Na.sup.+]-independent [P.sub.i] transport was significantly increased. In addition, compared with neutral pH, the activity of the [Na.sup.+]-independent [P.sub.i] transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The [Na.sup.+]-independent system consisted of two components with [K.sub.m] of 0.35 mM and 7.5 mM. The inhibitors of [P.sub.i] transport, phosphonoformic acid, and arsenate substantially decreased [P.sub.i] transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a [K.sup.+] ionophore, valinomycin, significantly suppressed [P.sub.i] transport activity. Analysis of BCECF fluorescence indicated that [P.sub.i] transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular [K.sup.+] ion stimulated [P.sub.i] transport, suggesting that membrane voltage is involved in the regulation of [P.sub.i] transport activity. Finally, bone particles significantly increased [Na.sup.+]-independent [P.sub.i] transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a [P.sub.i] transport system with characteristics that are different from those of other [Na.sup.+]-dependent [P.sub.i] transporters. We conclude that stimulation of [P.sub.i] transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. [Na.sup.+]-dependent phosphate cotransporter; RAW264.7; phosphate uptake

The effect of phosphorylated phloretins on [Na.sup.+]-dependent phosphate uptake into rabbit renal brush-border membrane vesicles (BBMV) was examined. [Na.sup.+]-dependent phosphate uptake into isolated rabbit cortex BBMV was sensitive to 2'-phosphophloretin (2'-PP) and 2'-phospho-4',4,6'-trimethoxy phloretin (PTMP) in a dose-dependent and pH-dependent manner. PTMP inhibition of [Na.sup.+]-dependent phosphate uptake was maximum at alkali pH, and 2'-PP inhibition of [Na.sup.+]-dependent phosphate uptake was maximum at acidic pH. Increasing [Na.sup.+] concentrations did not increase PTMP inhibition of renal cortex BBMV [Na.sup.+]-dependent phosphate uptake at pH 6. The effect of phosphophloretins on [Na.sup.+]-dependent phosphate uptake was examined in BBMV isolated from purified proximal tubules and distal tubules. 2'-PP and PTMP inhibition of [Na.sup.+]-dependent phosphate uptake into BBMV isolated from purified proximal tubules was similar to the inhibition seen with BBMV from renal cortex. 2'-PP, but not PTMP, inhibited Na+-dependent phosphate uptake into BBMV isolated from purified distal tubules. The pH dependence of inhibition, the absence of PTMP inhibition of [Na.sup.+]-dependent phosphate uptake into distal tubule BBMV, and the inhibition of [Na.sup.+]-dependent phosphate uptake into distal tubule BI3MV suggest that NaPi-Ia is 2'-PP sensitive and NaPi-IIa is PTMP sensitive. intestinal brush-border membrane vesicles; renal brush-border membrane vesicles; sodium-phosphate cotransport; phosphate reabsorption; 2'-phosphophloretin; 2'-phospho-4',4,6'-trimethoxy phloretin

The properties of the dyad cleft can in principle significantly impact excitation-contraction coupling, but these properties are not easily amenable to experimental investigation. We simultaneously measured the time course of the rise in integrated Ca current ([I.sub.ca]) and the rise in concentration of fura 2 with Ca bound ([Ca-fura 2]) with high time resolution in rat myocytes for conditions under which Ca entry is only via L-type Ca channels and sarcoplasmic reticulum (SR) Ca release is blocked, and compared these measurements with predictions from a finite-element model of cellular Ca diffusion. We found that 1) the time course of the rise of [Ca-fura 2] follows the time course of integrated [I.sub.ca], plus a brief delay (1.36 [+ or -] 0.43 ms, n = 6 cells); 2) from the model, high-affinity Ca binding sites in the dyad cleft at the level previously envisioned would result in a much greater delay ([greater than or equal to] 3 ms) and are therefore unlikely to be present at that level; 3) including ATP in the model promoted Ca efflux from the dyad cleft by a factor of 1.57 when low-affinity cleft Ca binding sites were present; 4) the data could only be fit to the model if myofibrillar troponin C (TnC) Ca binding were low affinity (4.56 [micro]M), like that of soluble troponin C, instead of the high-affinity value usually used (0.38 [micro]M). In a 'good model,' the rate constants for Ca binding and dissociation were 0.375 times the values for soluble TnC; and 5) consequently, intracellular Ca buffering at the rise of the Ca transient is inferred to be low. excitation-contraction coupling; adenosine triphosphate; fura 2; modeling; fuzzy space

Inorganic phosphate ([P.sub.i]) is absorbed by proximal tubules through a cellular pathway that is inhibited by parathyroid hormone (PTH). The calcium-sensing receptor (CaSR) is expressed on apical membranes of proximal tubules. In the present studies, we determined the effect of luminal and/or basolateral PTH on phosphate absorption and tested the hypothesis that CaSR activation blocks PTH-inhibitable phosphate absorption. Single proximal S3 tubules were dissected from the kidneys of mice and studied by the Burg technique. Tubules were bathed with DMEM culture media supplemented with 6% BSA and perfused with an ultrafiltrate prepared from the bathing solution. [sup.33]P and FITC-inulin were added to the luminal perfusate to measure phosphate absorption ([J.sub.Pi]) and fluid absorption ([J.sub.v]), respectively. [J.sub.Pi] averaged 2.9 pmol * [min.sup.-1] * [mm.sup.-1] under control conditions and decreased by 20% upon addition of serosal PTH. PTH had no effect on [J.sub.v]. Inclusion of PTH in the luminal perfusate reduced [J.sub.Pi] to 2.1 pmol * [min.sup.-1] * [mm.sup.-1]. Combined addition of PTH to perfusate and bathing solutions reduced [J.sub.Pi] to 1.5 pmol * [min.sup.-1] * [mm.sup.-1] without affecting [J.sub.v]. Indirect immunofluorescence studies revealed abundant PTH receptor (PTH1R) expression on brush-border membranes, with lower amounts on basolateral membranes. CaSRs were localized primarily, but not exclusively, to brush-border membranes. CaSR activation with luminal [Gd.sup.3+] abolished the inhibitory action of PTH on [J.sub.Pi]. Addition of [Gd.sup.3+] to the serosal bathing solution had no effect on PTH-sensitive [J.sub.Pi]. [Gd.sup.3+] did not affect basal, i.e., PTH-independent [J.sub.Pi]. [Gd.sup.3+] had no effect on [J.sub.v] when added to lumen or bath. Dopamine-inhibitable [J.sub.Pi] was not affected by [Gd.sup.3+]. Experiments with proximal-like opossum kidney cells showed that elevated extracellular [Ca.sup.2+] or NPS R467, a type II calcimimetic, inhibited PTH action on [P.sub.i] uptake. In conclusion, PTH1Rs are expressed on apical and basolateral membranes of mouse proximal tubules. Stimulating apical or basolateral PTH1R inhibits phosphate absorption. CaSR activation specifically regulates PTH-suppressible phosphate absorption. kidney; parathyroid hormone; opossum kidney cells; dopamine; parathyroid hormone receptor

A novel single-tracer approach for the study of both oxidative and nonoxidative branches of the pentose phosphate pathway (PPP) using (1,2-(super 13)C(sub 2))glucose is presented. The synthesis of pentose phosphate and lactate production from (1,2-(super 13)C(sub 2))glucose in human hepatoma cells were measured. Results indicate that the use of (1,2-(super 13)C(sub 2))glucose permits the estimation of relative rates through the TK and TA reactions in addition to allowing the determination of PC.

Sprague-Dawley rats were studied to isolate other gene products involved in diphor-1 (Pi) deprivation. The changes in the renal cortex messenger ribonucleic acid (mRNA) expression caused by the dietary Pi inhibition by differential display-polymerase chain reaction was also studied. A study of the tissue distribution was also conducted, where the presence of Pi-related mRNA was found in the kidney and the small intestine. The results further revealed that even the diphor-1 mRNA in the small intestine was controlled by Pi deprivation.

An experiment was performed to investigate the ecto-enzymatic hydrolysis of diadenosine polyphosphates (ApnA) by adrenomedullary endothelial cells using continuous fluorometric assays. Differences in hydrolytic rates of the ecto-enzymes from endothelial cells and closely related neutral chromaffin cells were observed. Discrepancies were also seen in ecto-nucleotidases of endothelial and chromaffin cells involved in epsilon-Ado production from epsilon-nucleotide moieties from epsilon (ApnA) compounds.

The study is conducted to unite localized 31P-nuclear magnetic resonance spectroscopy (NMR) and histochemical and biochemical analysis of muscle biopsies, to find out the relationship between the phosphate metabolic content and fiber-type composition in humans. It showed that human muscles with a different fiber-type composition, resembling animal muscles, vary in their basal phosphate content. The phosphate metabolite differences in human skeletal muscles are little in comparison to the metabolite differences found in animal muscles.

13C-nuclear magnetic resonance spectroscopic analysis of the cellular metabolism of (1,6-13C) fructose 1, 6-bisphosphate in hog carotiod artery segments reveals that hog artery metabolizes (1,6-13C) fructose 1,6-bisphosphate during hypoxia and normoxia, with (3-13C) lactate as the main metabolic product. (3-13C) lactate generation is higher during hypoxia, suggesting that the cellular metabolism level is more during the energetic state of the tissue. Exogenous additions of 20 mM of fructose 1,6-bisphosphate enhances isometric force preservation in the hog carotid artery.

Studies on mice with X-linked hypophosphatemic Gy mutation suggest that the increased Na+-phosphate cotransport in the proximal tubule brush-border membrane is not the sole cause of enhanced renal phosphate conservation following phosphate deprivation. Consequently, the Gy mice possess reduced renal tubular maximum for phosphate.

Astudy of cultured osteoblasts which were obtained from Hyp mice reveals that reduction of inorganic phosphate (Pi) level does not alter sodium-dependent Pi transport. The normal Hyp mice serum does not show any variation in activity with osteoblasts from normal and mutant mice. The adaptive response to or deprivation is similar in normal and mutant cells.

Whole cell patch-clamp methods are used to investigate Na+ conductance of RPMI 8226 human B lymphoblastoid cells. The cells are hyperpolarized by amiloride which also restricts inward Na+ conductance imposed through the plasma membrane. Amiloride restricts more than 50% of the ionic conductance when Na+ is the only charge transmitter, but does not restrict ionic conductance when the exclusive charge carrier is K+. Amiloride restricts conductance, and this is selective for Na+. Expression of Na+ channels which are sensitive to amiloride is found in B lymphoid cells, and cause a part of the steady-state whole cell Na+ conductance.

Ramakrishna, Ramprasad, Jeremy S. Edwards, Andrew McCulloch, and Bernhard O. Palsson. Flux-balance analysis of mitochondrial energy metabolism: consequences of systemic stoichiometric constraints. Am J Physiol Regulatory Integrative Comp Physiol 280: R695-R704, 2001.--Mitochondrial metabolism is a critical component in the functioning and maintenance of cellular organs. The stoichiometry of biochemical reaction networks imposes constraints on mitochondrial function. A modeling framework, flux-balance analysis (FBA), was used to characterize the optimal flux distributions for maximal ATP production in the mitochondrion. The model predicted the expected ATP yields for glucose, lactate, and palmitate. Genetic defects that affect mitochondrial functions have been implicated in several human diseases. FBA can characterize the metabolic behavior due to genetic deletions at the metabolic level, and the effect of mutations in the tricarboxylic acid (TCA) cycle on mitochondrial ATP production was simulated. The mitochondrial ATP production is severely affected by TCA-cycle mutations. In addition, the model predicts the secretion of TCA-cycle intermediates, which is observed in clinical studies of mitochondriopathies such as those associated with fumarase deficiency. The model provides a systemic perspective to characterize the effect of stoichiometric constraints and specific metabolic fluxes on mitochondrial function. mitochondria; flux analysis; adenosine 5'-phosphate production; mitochondrial disease

Busk, Morten, Frank B. Jensen, and Tobias Wang. Effects of feeding on metabolism, gas transport, and acid-base balance in the bullfrog Rana catesbeiana. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 278: R185-R195, 2000.--Massive feeding in ectothermic vertebrates causes changes in metabolism and acid-base and respiratory parameters. Most investigations have focused on only one aspect of these complex changes, and different species have been used, making comparison among studies difficult. The purpose of the present study was, therefore, to provide an integrative study of the multiple physiological changes taking place after feeding. Bullfrogs (Rana catesbeiana) partly submerged in water were fed meals (mice or rats) amounting to ~1/10 of their body weight. Oxygen consumption increased and peaked at a value three times the predigestive level 72-96 h after feeding. Arterial [PO.sub.2] decreased slightly during digestion, whereas hemoglobin-bound oxygen saturation was unaffected. Yet, arterial blood oxygen content was pronouncedly elevated because of a 60% increase in hematocrit, which appeared mediated via release of red blood cells from the spleen. Gastric acid secretion was associated with a 60% increase in plasma [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration ([[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]]) 48 h after feeding. Arterial pH only increased from 7.86 to 7.94, because the metabolic alkalosis was countered by an increase in [PCO.sub.2] from 10.8 to 13.7 mmHg. Feeding also induced a small intracellular alkalosis in the sartorius muscle. Arterial pH and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] returned to control values 96-120 h after feeding. There was no sign of anaerobic energy production during digestion as plasma and tissue lactate levels remained low and intracellular ATP concentration stayed high. However, phosphocreatine was reduced in the sartorius muscle and ventricle 48 h after feeding. specific dynamic action; [O.sub.2] transport; alkaline tide; metabolites; high-energy phosphates

Meszaros, J. Gary, Annette M. Gonzalez, Yuka Endo-Mochizuki, Sonia Villegas, Francisco Villarreal, and Laurence L. Brunton. Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between [G.sub.q] and [G.sub.s]. Am. J. Physiol. Cell Physiol. 278: C154-C162, 2000.--Cardiac fibroblasts (CFs) are an important cellular component of myocardial responses to injury and to hypertrophic stimuli. We studied G protein-coupled receptors to understand how CFs integrate signals that activate [G.sub.q], [G.sub.s], and [G.sub.i]. We predicted that the second messenger pathways present in CFs were distinct from those in cardiac myocytes and that unique signaling interactions existed in the CFs. ANG II, bradykinin, ATP, and UTP stimulated inositol phosphate (IP) production 2.2- to 7-fold. Each of these agonists elevated intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) via release from the intracellular [Ca.sup.2+] storage compartment. Endothelin-1 (ET-1), carbachol, and norepinephrine failed to increase either IP production or [[[Ca.sup.2+]].sub.i]. Although agonists that activated IP and [Ca.sup.2+] transients had no effect on cAMP production when administered alone, these agents potentiated the [[Beta].sub.2]-adrenergic response two- to fourfold. Hormones known to inhibit adenylyl cyclase activity in cardiac myocytes, such as ET-1 and carbachol, failed to lower the [Beta]-adrenergic response in fibroblasts. Order of potency and inhibitor data indicate that the functional receptor subtypes in these cells are [[Beta].sub.2], [P2Y.sub.2], and [AT.sub.1] for isoproterenol, ATP, and ANG II, respectively. We conclude that CFs express functional G protein-linked receptors that couple to [G.sub.q] and [G.sub.s], with little or no coupling to [G.sub.i]. The expression of receptors and their coupling to [G.sub.q]- but not to [G.sub.i]-linked responses distinguishes the signaling in CFs from that in myocytes. Furthermore, agonists that activate [G.sub.q] in CFs potentiate stimulation of [G.sub.s], an example of signaling cross talk not observed in adult myocytes. These data suggest that G protein-mediated signaling in CFs is unique and may contribute to the specificity of hormone and drug action on individual cell types within the heart. cyclic adenosine 3',5'-cyclic monophosphate; inositol phosphates; intracellular calcium; potentiation; receptor signaling

Duke, Adrian M., and Derek S. Steele. Characteristics of phosphate-induced [Ca.sup.2+] efflux from the SR in mechanically skinned rat skeletal muscle fibers. Am. J. Physiol. Cell Physiol. 278: C126-C135, 2000.--The effects of [P.sub.i] on sarcoplasmic reticulum (SR) [Ca.sup.2+] regulation were studied in mechanically skinned rat skeletal muscle fibers. Brief application of caffeine was used to assess the SR [Ca.sup.2+] content, and changes in concentration of [Ca.sup.2+] ([[Ca.sup.2+]]) within the cytosol were detected with fura 2 fluorescence. Introduction of [P.sub.i] (1-40 mM) induced a concentration-dependent [Ca.sup.2+] efflux from the SR. In solutions lacking creatine phosphate (CP), the amplitude of the [P.sub.i]-induced [Ca.sup.2+] transient approximately doubled. A similar potentiation of [P.sub.i]-induced [Ca.sup.2+] release occurred after inhibition of creatine kinase (CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced [Ca.sup.2+] release was almost abolished, whereas [P.sub.i]-induced [Ca.sup.2+] release was unaffected. However, introduction of the SR [Ca.sup.2+] ATPase inhibitor cyclopiazonic acid effectively abolished [P.sub.i]-induced [Ca.sup.2+] release. These data suggest that [P.sub.i] induces [Ca.sup.2+] release from the SR by reversal of the SR [Ca.sup.2+] pump but not via the SR [Ca.sup.2+] channel under these conditions. If this occurs in intact skeletal muscle during fatigue, activation of a [Ca.sup.2+] efflux pathway by [P.sub.i] may contribute to the reported decrease in net [Ca.sup.2+] uptake and increase in resting [[Ca.sup.2+]]. phosphate; sarcoplasmic reticulum; [Ca.sup.2+] pump; fatigue

Gupte, Sachin A., Tasneem Rupawalla, Donald Phillibert, Jr., and Michael S. Wolin. NADPH and heme redox modulate pulmonary artery relaxation and guanylate cyclase activation by NO. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1124-L1132, 1999.--The hemoprotein oxidant ferricyanide (FeCN) converts the iron of the heme on soluble guanylate cyclase (sGC) from [Fe.sup.2+] to [Fe.sup.3+], which prevents nitric oxide (NO) from binding the heme and stimulating sGC activity. This study uses FeCN to examine whether modulation of the redox status of the heme on sGC influences the relaxation of endothelium-removed bovine pulmonary arteries (BPA) to NO. Pretreatment of the homogenate of BPA with 50 [micro]M FeCN resulted in a loss of stimulation of sGC activity by the NO donor 10 [micro]M S-nitroso-N-acetylpenicillamine (SNAP). In the FeCN-treated homogenate reconcentrated to the enzyme levels in BPA, 100 [micro]M NADPH restored NO stimulation of sGC, and this effect of NADPH was prevented by an inhibitor of flavoprotein electron transport, 1 [micro]M diphenyliodonium (DPI). In BPA the relaxation to SNAP was not altered by FeCN, inhibitors of NADPH generation by the pentose phosphate pathway [250 [micro]M 6-aminonicotinamide (6-AN) and 100 [micro]M epiandrosterone (Epi)], or 1 [micro]M DPI. However, the combination of FeCN with 6-AN, Epi, or DPI inhibited (P [is less than] 0.05) relaxation to SNAP without significantly altering the relaxation of BPA to forskolin. The inhibitory effects of 1 [micro]M 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (a probe that appears to convert NO-heme of sGC to its [Fe.sup.3+]-heme form) on relaxation to SNAP were also enhanced by DPI. These observations suggest that a flavoprotein containing NADPH oxidoreductase may influence cGMP-mediated relaxation of BPA to NO by maintaining the heme of sGC in its [Fe.sup.2+] oxidation state. NADPH oxidoreductase; nitric oxide; pentose phosphate pathway

Traebert, Martin, Olaf Hattenhauer, Heini Murer, Brigitte Kaissling, and Jurg Biber. Expression of type II Na-[P.sub.i] cotransporter in alveolar type II cells. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L868-L873, 1999.--Type II Na-[P.sub.i] cotransporters (type IIa and type IIb) represent apically located Na-[P.sub.i] cotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-[P.sub.i] cotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-[P.sub.i] diet. After a chronic adaptation to a low-[P.sub.i] diet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-[P.sub.i] cotransporter in alveolar type II cells. sodium-inorganic phosphate cotransporter; immunohistochemistry

Yanagawa, Norimoto, Chi Pham, Remi N. J. Shih, Stephen Miao, and Oak Don Jo. Chloride dependency of renal brush-border membrane phosphate transport. Am. J. Physiol. 277 (Renal Physiol. 46): F506-F512, 1999.--In our present study, we examined the effect of [Cl.sup.-] on rabbit renal brush-border membrane (BBM) phosphate ([P.sub.i]) uptake. It was found that the [Na.sup.+]-dependent BBM [sup.32]P uptake was significantly inhibited by [Cl.sup.-] replacement in the uptake solution with other anions, or by [Cl.sup.-] transport inhibitors, including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid (NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Intravesicular formate or [Cl.sup.-] increased BBM [sup.36][Cl.sup.-] uptake but did not affect BBM [sup.32]P uptake. BBM [sup.22][Na.sup.+] uptake was lowered by [Cl.sup.-] replacement in the uptake solution but not by [Cl.sup.-] transport inhibitors. Changes in transmembrane electrical potential altered BBM [sup.36][Cl.sup.-] and [sup.32]P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the [Cl.sup.-] dependent BBM Pi uptake was not affected by changes in transmembrane electrical potential. Finally, a similar [Cl.sup.-] dependency of Pi uptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of [Na.sup.+] dependent [P.sub.i] uptake was also [Cl.sup.-] dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this [Cl.sup.-] dependency remains to be identified. proximal tubule; chloride channel; chloride channel inhibitor; sodium cotransport; hereditary nephrolithiasis

Wick, Debra A., Bellur Seetharam, and Nancy M. Dahms. Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G506-G514, 1999.--We have analyzed the transport of newly synthesized mannose 6-phosphate (Man-6-P)-bearing proteins (i.e., lysosomal enzymes) in the polarized human colon adenocarcinoma cell line, Caco-2, by subjecting filter-grown cells to a pulse-chase labeling protocol using [[sup.35]S]methionine, and the resulting cell lysate, apical medium, and basolateral medium were immunoprecipitated with insulin-like growth factor II/Man-6-P receptor (IGF-II/MPR)-specific antisera. The results showed that the majority of secreted lysosomal enzymes accumulated in the apical medium at [is greater than] 2 h of chase and that this polarized distribution was facilitated by the IGF-II/MPR selectively endocytosing lysosomal enzymes from the basolateral surface. Treatment with various agents known to affect vesicular transport events demonstrated that incubations at 16[degrees]C or incubations with brefeldin A inhibited the secretion of lysosomal enzymes from both the apical and basolateral surface, whereas treatment with nocodazole selectively blocked apical secretion. In contrast, incubation with [NH.sub.4]Cl or nocodazole had a stimulatory effect on basolateral secretion. Taken together, these results demonstrate that the sorting of Man-6-P-containing proteins into the apical and basolateral secretory pathways is regulated by distinct components of the intracellular trafficking machinery. lysosomal enzymes; intracellular trafficking; insulin-like growth factor II receptor