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In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

Presenilin l-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog l (Mtchl). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed. presenilin l-associated protein-mitochondrial carrier homolog 1; mitochondria; apoptosis; presenilin; Alzheimer's disease

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its N[H.sub.2] end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP 17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP 17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAPI7 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa. phosphate transport; PDZ; PDZK1; protein interaction; opossum kidney cells

We previously showed an inverse correlation between membrane cholesterol content and Na-[P.sub.i] cotransport activity during the aging process and adaptation to alterations in dietary [P.sub.i] in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-[P.sub.i] cotransport activity and apical membrane Na-[P.sub.i] protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with [beta]-methyl cyclodextrin ([beta]-MCD) resulted in a significant increase in Na-[P.sub.i] cotransport activity accompanied by a moderate increase in apical membrane Na-[P.sub.i] protein abundance and no alteration of total cellular Na-[P.sub.i] protein abundance. Conversely, acute cholesterol enrichment achieved with [beta]-MCD/cholesterol resulted in a significant decrease in Na-[P.sub.i] cotransport activity with a moderate decrease in apical membrane Na-[P.sub.i] protein abundance and no change of the total cellular Na-[P.sub.i] protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-[P.sub.i] cotransport activity and apical membrane and total cellular Na-[P.sub.i] protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-[P.sub.i] cotransport activity and apical membrane and total cellular Na-[P.sub.i] protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-[P.sub.i] cotransport activity by diverse mechanisms that also include significant interactions of Na-[P.sub.i] protein with lipid microdomains. two-photon fluorescence microscopy; filipin; lipid microdomains; laurdan; opossum kidney cells

Growth hormone (GH) is an important factor in the developmental adaptation to enhance [P.sub.i] reabsorption; however, the nephron sites and mechanisms by which GH regulates renal [P.sub.i] uptake remain unclear and are the focus of the present study. Micropuncture experiments were performed after acute thyroparathyroidectomy in the presence and absence of parathyroid hormone (PTH) in adult (14- to 17-wk old), juvenile (4-wk old), and GH-suppressed juvenile male rats. While the phosphaturic effect of PTH was blunted in the juvenile rat compared with the adult, suppression of GH in the juvenile restored fractional [P.sub.i] excretion to adult levels. In the presence or absence of PTH, GH suppression in the juvenile rat caused a significant increase in the fractional [P.sub.i] delivery to the late proximal convoluted (PCT) and early distal tubule, so that delivery was not different from that in adults. These data were confirmed by [P.sub.i] uptake studies into brush-border membrane (BBM) vesicles. Immunofluorescence studies indicate increased BBM type IIa Na[P.sub.i] cotransporter (NaPi-2) expression in the juvenile compared with adult rat, and GH suppression reduced NaPi-2 expression to levels observed in the adult. GH replacement in the [N-acetyl-[Tyr.sub.1]-D-[Arg.sup.2]]-GRF-(1-29)-[NH.sub.2]treated juveniles restored high NaPi-2 expression and [P.sub.i] uptake. Together, these novel results demonstrate that the presence of GH in the juvenile animal is crucial for the early developmental upregulation of BBM NaPi-2 and, most importantly, describe the enhanced [P.sub.i] reabsorption along the PCT and proximal straight nephron segments in the juvenile rat. development; sodium-phosphate cotransporters; parathyroid hormone; antagonist to growth hormone-releasing factor; brush-border membrane vesicles

Mulroney, Susan E., Craig B. Woda, Nabil Halaihel, Brandon Louie, Kevin McDonnell, Jay Schulkin, Aviad Haramati, and Moshe Levi. Central control of renal sodium-phosphate (NaPi-2) transporters. Am J Physiol Renal Physiol 286: F647-F652, 2004. First published December 23, 2003; 10.1152/ajprenal.00354.2002.--Regulation of phosphate ([P.sub.i]) reabsorption occurs through the up- and downregulation of the renal type-II sodium [P.sub.i] cotransporters (NaPi-2). Recently, renal NaPi2-type expression has been identified in areas of the brain. The present study determined whether brain NaPi-2 is regulated by dietary [P.sub.i] and whether the behavioral and renal adaptations to low-dietary [P.sub.i] are controlled centrally. NaPi-2-like expression in the third ventricle (3V) and amygdala of juvenile Wistar rats was regulated by dietary [P.sub.i], as in the kidneys. When cerebrospinal fluid (CSF) [P.sub.i] concentration was elevated by 3V injections of Pi in rats fed low-[P.sub.i] diet (LPD), the behavioral and renal adaptations to LPD were abolished. Most importantly, NaPi-2 expression was markedly reduced not only in the brain, but also renal proximal tubules, despite the low plasma [P.sub.i] milieu. This was confirmed by the significant reduction in the transport maximum for Pi (from 8.1 [+ or -] 0.2 in LPD + veh 3V to 1.7 [+ or -] 0.1 [micro]mol [P.sub.i]/ml glomerular filtration rate in LPD + 3V [P.sub.i], P < 0.001). These findings indicate that NaPi-2-like transporters in the brain are regulated by both dietary [P.sub.i] and CSF[P.sub.i] concentrations, and most significantly, that the central [P.sub.i] milieu can regulate renal NaPi-2 expression. We hypothesize that central 3V NaPi-2 transporters may act as Pi sensors and help regulate both brain and whole body Pi homeostasis. juvenile rats; behavior; [P.sub.i] sensors

The effect of metabolic acidosis (MA) on Na-S(sub i) cotransport activity and NaS(sub i)-1 protein and mRNA expression was investigated. In rats with MA for 24 hrs, there was substantial increase in the fractional excretion of S(sub i), which was related with a 2.4-fold reduction in brush-border membrane (BBM) Na-S(sub i) cotransport activity. Results showed that MA decreases S(sub i) reabsorption by inducing reductions in BBM Na-S(sub i) cotransport activity and that reductions in the expression of NaS(sub i)-1 protein and mRNA abundance, at least partially, have a significant role in the inhibition of Na-S(sub i) cotransport activity during MA.

Abstract Background Hepatitis E virus (HEV) infection is a serious health problem in developing countries and is also increasingly reported in industrialized regions. HEV is considered a zoonotic agent and strains isolated from swine and human sources are genetically similar. Thus, HEV is of increasing importance to both public and animal health. The aim of the present study was to evaluate the distribution of HEV in a large population of pigs from herds located in different autonomous regions throughout Spain. Results The presence of anti-HEV IgG antibodies was analyzed in 1141 swine serum samples (corresponding to 381 pigs younger than 6 months and 760 pigs older than 6 months) collected from 85 herds. Herds were located in 6 provinces in 4 autonomous regions throughout Spain. At least one pig tested positive for anti-HEV IgG in over 80% of herds. Of individual pigs, 20.4% (233/1141) were positive for anti-HEV IgG, with the prevalence being higher in adult pigs than in those under 6 months (30.2% vs. 15.5%). A subset of serum samples taken at 2- to 5-week intervals showed that seroprevalence dropped between 3 and 11 weeks of age, and then rose significantly by the 15th week. Pigs were also examined for the presence of HEV-RNA by RT-PCR. Of pigs tested for the presence of HEV-RNA 18.8% (64/341) were positive, with at least one pig in almost half of the herds testing positive. HEV-RNA amplicons from several positive pigs were sequenced and all were of genotype 3. Conclusions HEV was found to be widely distributed among swine farms across Spain, with the prevalence being highest among animals older than 6 months. These results indicate that HEV infection either is or is likely to become endemic in the Spanish swine population.

Introduction: In Nicaragua, it is required to standardize molecular tests such as real-time PCR (qPCR) that improve the diagnosis of leptospirosis in humans and animals. Objective: To evaluate two qPCR for detection of pathogenic leptospires in domestic animals of Nicaragua. Materials and methods: Primers were designed for the amplification of the LipL32 gene in SYBR Green (SYBR Green-A in this study) and TaqMan, in addition, previously described primers were used (SYBR Green-B, in this study). The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to search for probes and primers. Analytical sensitivity was determined by calculating the detectable genomic equivalent (EG), while 18 pathogenic references strains and 28 negative controls were used in the diagnostic sensitivity and specificity respectively, the methods were applied in 129 urine samples of domestic animals Results: In SYBR green-A, got a detection limit of 4 EG per reaction, in TaqMan, sensitivity was 94.4% (IC95% = 81.1-100.0), in SYBR Green-A obtained sensitivity of 77.8% (IC95% = 55.8-99.8), while SYBR Green-B 61.1% (95%CI = 35.8-86.4). In the three tests, a specificity of 100% (95% CI = 98.2-100.0) was obtained. In field samples 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B. Conclusion: The SYBR green-A presented a low detection limit, while the three evaluated techniques showed high specificity, but the TaqMan presented the highest sensitivity. [ABSTRACT FROM AUTHOR]

Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat. Am J Physiol Regulatory Integrative Comp Physiol 280: R1524-R1533, 2001.--To determine the tubular sites and mechanisms involved in enhanced renal phosphate ([P.sub.i]) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult ([is greater than] 14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal [P.sub.i] diet (NPD, 0.6% [P.sub.i]) or low [P.sub.i] diet (0.07% [P.sub.i]) for 2 days, in the presence and absence of parathyroid hormone (PTH). [P.sub.i] reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater [P.sub.i] uptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary [P.sub.i] restriction in juvenile rats resulted in a significant increase in [P.sub.i] reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that [P.sub.i] reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary [P.sub.i] restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular [P.sub.i] reabsorption. kidney; low dietary phosphate; parathyroid hormone; age; brush-border membrane; inorganic phosphate transport

Rotavirus infection impairs intestinal brush-border membrane [Na.sup.+]-solute cotransport activities in young rabbits. Am J Physiol Gastrointest Liver Physiol 279: G587-G596, 2000.--The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both [Na.sup.+]-D-glucose (SGLT1) and [Na.sup.+]-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizinbinding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of [Na.sup.+]-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established. viral diarrhea; rabbit intestine; SGLT1; sodium-solute symport

Halaihel, Nabil, Daniele Gerbaud, Monique Vasseur, and Francisco Alvarado. Heterogeneity of pig intestinal D-glucose transport systems. Am. J. Physiol. 277 (Cell Physiol. 46): C1130-C1141, 1999.--Heterogeneity of intestinal D-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two D-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a D-glucose and D-mannose transporter exhibiting no preference for [Na.sup.+] over [K.sup.+]. A nonsaturable D-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker L-glucose. It is also shown that, in this kinetic work, 1) use of v-glucose-contaminated D-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range ([is greater than or equal to] 50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion. pig intestinal transport; v-mannose transport; SGLT1

We previously showed an inverse correlation between membrane cholesterol content and Na-Pi cotransport activity during the aging process and adaptation to alterations in dietary Pi in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-Pi cotransport activity and apical membrane Na-Pi protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with β-methyl cyclodextrin (β-MCD) resulted in a significant increase in Na-Pi cotransport activity accompanied by a moderate increase in apical membrane Na-Pi protein abundance and no alteration of total cellular Na-Pi protein abundance. Conversely, acute cholesterol enrichment achieved with β-MCD/cholesterol resulted in a significant decrease in Na-Pi cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-Pi protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-Pi cotransport activity by diverse mechanisms that also include significant interactions of Na-Pi protein with lipid microdomains. [ABSTRACT FROM AUTHOR]