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Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse-chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na, K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,KATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na, K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na, K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na, K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.

Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid-specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the luminal to the abluminal compartment was time dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and ascorbate itself. Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo. paracellular transport; ascorbate transport; anion channels; endothelial permeability

Gossypol is a natural disesquiter-pene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary ceils overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a [K.sub.i] value of 20 [micro]M. Likewise, GLUT1 fluorescence was quenched ~80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose. glucose transport; GLUT1 transporter; transporter inhibition

We studied the requirement for potassium and for potassium transport activity for the biotechnologically important bacterium Corynebacterium glutamicum, which is used for large-scale production of amino acids. Different from many other bacteria, at alkaline or neutral pH, C. glutamicum is able to grow without the addition of potassium, resulting in very low cytoplasmic potassium concentrations. In contrast, at acidic pH, the ability for growth was found to depend on the presence of [K.sup.+]. For the first time, we provide experimental evidence that a potential potassium channel (CglK) acts as the major potassium uptake system in a bacterium and proved CglK's function directly in its natural membrane environment. A full-length CglK protein and a separate soluble protein harboring the RCK domain can be translated from the cglK gene, and both are essential for full CglK functionality. As a reason for potassium-dependent growth limitation at acidic pH, we identified the impaired capacity for internal pH homeostasis, which depends on the availability and internal accumulation of potassium. Potassium uptake via CglK was found to be relevant for major physiological processes, like the activity of the respiratory chain, and to be crucial for maintenance of the internal pH, as well as for the adjustment of the membrane potential in C. glutamicum.

Recent studies of three-dimensional architecture of rat renal inner medulla (IM) and expression of membrane proteins associated with fluid and solute transport in nephrons and vasculature have revealed structural and transport properties that likely impact the IM urine concentrating mechanism. These studies have shown that 1) IM descending thin limbs (DTLs) have at least two or three functionally distinct subsegments; 2) most ascending thin limbs (ATLs) and about half the ascending vasa recta (AVR) are arranged among clusters of collecting ducts (CDs), which form the organizing motif through the first 3-3.5 mm of the IM, whereas other ATLs and AVR, along with aquaporin-1-positive DTLs and urea transporter B-positive descending vasa recta (DVR), are external to the CD clusters; 3) ATLs, AVR, CDs, and interstitial cells delimit interstitial microdomains within the CD clusters; and 4) many of the longest loops of Henle form bends that include subsegments that run transversely along CDs that lie in the terminal 500 [micro]m of the papilla tip. Based on a more comprehensive understanding of three-dimensional IM architecture, we distinguish two distinct countercurrent systems in the first 3-3.5 mm of the IM (an intra-CD cluster system and an inter-CD cluster system) and a third countercurrent system in the final 1.5-2 mm. Spatial arrangements of loop of Henle subsegments and multiple countercurrent systems throughout four distinct axial IM zones, as well as our initial mathematical model, are consistent with a solute-separation, solute-mixing mechanism for concentrating urine in the IM. kidney; countercurrent system; computer-assisted reconstruction; functional anatomy; NaCl transport; urea transport; mathematical model

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 [+ or -] 0.5% of the total dose was transported after 8 h, equivalent to 38.8 [+ or -] 2.1 pmol x [cm.sup.-2] x [h.sup.-1]. Transfer of Tf across the cell layer was much more limited; 2.4 [+ or -] 0.2% of the total dose was transported after 8 h, equivalent to 5.0 [+ or -] 0.4 pmol x [cm.sup.-2] x [h.sup.-1]. Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta. placenta; fetus; mineral metabolism

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by ~80%. Finally, we found no evidence for coimmunoprecipitation of Glut 1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway. AMP-activated protein kinase; glucose transporter 1; oxidative phosphorylation; small interfering RNA directed against AS I60

Phosphoinositides are lipid second messengers that are essential for many cellular processes, including signal transduction and cell compartmentalization. Among them, phosphatidylinositol 5-phosphate (PtdIns5P) is the least characterized, although several proteins involved in its regulation are implicated in human diseases. We studied the distribution of 32 PtdIns5P-metabolizing proteins in 39 eukaryotic genomes. Phylogenetic profiles identify four groups of co-evolving proteins, confirming known protein complexes and revealing new ones. The complexes comprise a phosphatase, a kinase and a regulator; this indicates that physical interactions between the three partners are necessary for the acute spatial regulation of PtdIns5P turnover. By examining PtdIns5P metabolism in this new perspective, we propose a role for PtdIns5P in membrane trafficking from late endosomal compartments to the plasma membrane.

In cardiac muscle, intracellular [Ca.sup.2+] and [Mg.sup.2+] are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [[Ca.sup.2+]] in the SR ([[[Ca.sup.2+]].sub.L]) stimulates the [Ca.sup.2+] release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal [Mg.sup.2+], which has not been regarded as an important regulator of [Ca.sup.2+] release. The effects of luminal [Ca.sup.2+] and [Mg.sup.2+] on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal [Ca.sup.2+] produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal [Mg.sup.2+] was observed, pointing to an important physiological role tot luminal [Mg.sup.2+] in cardiac muscle. At diastolic [[[Ca.sup.2+]].sub.C], luminal [Mg.sup.2+] inhibition wits voltage independent, with [K.sub.i] = 45 [micro]M at luminal [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.1]) = 100 [micro]M. Luminal and cytoplasmic [Mg.sup.2+] inhibition was alleviated by increasing [[[Ca.sup.2+]].sub.L] or [[[Ca.sup.2+]].sub.C]. [Ca.sup.2+] and [Mg.sup.2+] on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four [Ca.sup.2+]-sensing mechanisms on each subunit: activating luminal L-site (40-[micro]M affinity for [Mg.sup.2+] and [Ca.sup.2+]), cytoplasmic A-site (1.2 [micro]M for [Ca.sup.2+] and 60 [micro]M for [Mg.sup.2+]), inactivating cytoplasmic [I.sub.i]-site (~10 mM for [Ca.sup.2+] and [Mg.sup.2+]), and [I.sub.2]-site ( 1.2 [micro]M for [Ca.sup.2+]). Activation of three or more subunits will cause channel opening. [Mg.sup.2+] inhibition occurs primarily by [Mg.sup.2+] displacing [Ca.sup.2+] from the L- and A-sites, and [Mg.sup.2+] fails to open the channel. The model predicts that under physiological conditions, SR load--dependent [Ca.sup.2+] release (1) is mainly determined by [Ca.sup.2+] displacement of [Mg.sup.2+] from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiting Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/ TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS 160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb

Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization.

The amyloid [beta]-peptide (A[beta]) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by A[beta] and accumulation of A[beta] has been detected in mitochondria. Because A[beta] is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial A[beta] uptake. Our results from rat mitochondria show that A[beta] is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed A[beta] association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of A[beta] to mitochondrial cristae. A similar distribution pattern of A[beta] in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for A[beta] in mitochondria and demonstrate both in vitro and in vivo that A[beta] is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied A[beta] can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of A[beta], a peptide considered to be of major significance in Alzheimer's disease. Alzheimer disease | protein import | human brain biopsies

The pathways implicated in the control of epithelial [Na.sup.+] channel (ENaC)-dependent [Na.sup.+] transport in renal collecting duct cells share substantial parallels with those implicated in insulin-regulated glucose metabolism. Notably, both are inhibited by wortmannin and LY294002 and signal through phosphatidylinositol-3-kinase (PI3K)-dependent kinases SGK1 and Akt. The inhibitor pattern is thought to reflect dependence on PI3K activity since wortmannin and LY294002 are both effective inhibitors of this kinase. However, these inhibitors block a variety of kinases from different families and lack specificity within the PI3K family. To begin to dissect more precisely the pathways required for signaling and for control of [Na.sup.+] transport in renal collecting duct cells, we have examined the effect of a set of PI3K inhibitors, which selectively block distinct subsets of PI3K catalytic subunit isoforms. We have found that ENaC-dependent [Na.sup.+] transport was blocked by inhibitors of the p 110-[alpha] isoform of PI3K, but not by inhibitors of p110-[beta], -[gamma] or -[delta] Inhibitors that block [Na.sup.+] current also blocked SGK1 and Akt phosphorylation. In contrast to insulin-stimulated glucose uptake in muscle cells, p110-[beta] inhibition did not enhance sensitivity to p110-[alpha] inhibition. These data support the conclusion that ENaC-dependent [Na.sup.+] current is controlled exclusively by p110-[alpha], the same isoform that is the principal mediator of insulin effects on glucose metabolism, and lacks any dependence on p110-[beta]. These findings further underscore the extent to which [Na.sup.+] and glucose regulation are intertwined and provide additional insight into the interconnections between diabetes and hypertension. phosphoinositide; sgk; phosphatidylinositol-3-phosphokinase; insulin; aldosterone

Reduced uteroplacental blood flow is hypothesized to play a key role in altitude-associated fetal growth restriction. It is unknown whether reduced blood flow is a cause or consequence of reduced fetal size. We asked whether determinants of uteroplacental blood flow were altered prior to reduced fetal growth and whether vasoactive and/or angiogenic factors were involved. Women residing at low (LA; 1600 m, n = 18) or high altitude (HA; 3100 m, n = 25) were studied during pregnancy (20, 30, and 36 wk) and 4 mo postpartum (PP) using Doppler ultrasound. In each study, endothelin (ET-1), nitric oxide metabolites ([NO.sub.x]), soluble fins-like tyrosine kinase (sFlt-1) and placental growth factor (P1GF) levels were quantified. At HA, birth weights were lower (P < 0.01) and small-for-gestational age was more common (P < 0.05) compared with LA. HA was associated with lower uterine artery (UA) diameter (P < 0.01) and blood flow (P < 0.05). Altitude did not affect ET-1, sFlt-1 or P1GF; however, ET-1/[NO.sub.x] was greater and [N[O.sub.x] lower during pregnancy and PP at HA vs. LA. ET-1/N[O.sub.x] was negatively associated with birth weight (20 wk, P < 0.01; 36 wk, P = 0.05) at LA and HA combined. At HA, UA blood flow (30 wk) was positively associated with birth weight ([dagger]). UA blood flow and ET-1/N[O.sub.x] levels accounted for 45% (20 wk) and 32% (30 wk) of birth weight variation at LA and HA combined, primarily attributed to effects at HA. We concluded that elevated ET-1/[NO.sub.x] and altered determinants of uteroplacental blood flow occur prior to altitude-associated reductions in fetal growth, and therefore, they are likely a cause rather than a consequence of smaller fetal size. fetal growth restriction; hypoxia; small-for-gestational age; uteroplacental oxygen delivery, pregnancy

Hemoglobin-based oxygen carriers (HBOC) have been primarily studied for blood loss treatment. More recently infusions of HBOC in euvolemic subjects have been proposed for a wide variety of potential therapies in which increased tissue oxygenation would be beneficial. However, compared with the exchange transfusion models to study blood loss, less is known about HBOC oxygen delivery and vasoacitvity when it is infused in euvolemic subjects. We hypothesized that HBOC [polymerized bovine hemoglobin (PBvHb)] infusion creating hypervolemia would increase oxygen delivery to tissues during acute global hypoxia. Vascular oxygen content and hemodynamics were determined after euvolemic rats were infused with 3 ml of either lactated Ringer or PBvHb solution (13 g/dl, 1.3 g/kg) during acute hypoxia (FI[O.sub.2] = 10%, 4 h) or normoxia (FI[O.sub.2] = 21%) exposure. Our data demonstrated that compared with Ringer-infused animals, in hypoxia and normoxia, PBvHb treatment improved oxygen content but raised mean arterial pressure, lowered stroke volume, heart rate, and cardiac index, which resulted in a net reduction in blood flow and oxygen delivery to the tissues. The PBvHb vasoactive effect was similar in magnitude and direction as to the Ringer-infused animals treated with a nitric oxide synthase inhibitor nitro-L-arginine, suggesting the PBvHb effect is mediated via nitric oxide scavenging. We conclude that infusion of PBvHb is not likely to be useful in treating global hypoxia under these conditions. hemoglobin-based oxygen carriers; Nitric oxide; Cardiac output

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function. [Ca.sup.2+] signaling; exocytosis; immunofluorescence; electron microscopy; multiphoton; tissue culture

Sodium tungstate reduces glycemia and reverts the diabetic phenotype in several induced and genetic animal models of diabetes. Oral administration of this compound has recently emerged as an effective treatment for diabetes. Here we examined the effects of 30 days of oral administration of tungstate on disaccharidase and [Na.sup.+]/D-glucose cotransporter (SGLT1) activity in the jejunum of control and streptozotocin-induced diabetic rats. Diabetes increased sucrase-specific activity in the jejunal mucosa but did not affect the activity of lactase, maltase, or trehalase. The abundance and the maximal rate of transport of SGLT1 in isolated brush-border membrane vesicles also increased. Tungstate decreased sucrase activity and normalized SGLT1 expression and activity in the jejunum of diabetic rats. Furthermore, tungstate did not change the affinity of SGLT1 for D-glucose and had no effect on the diffusional component. In control animals, tungstate had no effect on disaccharidases or SGLT1 expression. Northern blot analysis showed that the amount of specific SGLT1 mRNA was the same in the jejunum from all experimental groups, thereby indicating that changes in SGLT1 abundance are due to posttranscriptional mechanisms. We conclude that the antidiabetic effect of tungstate is partly due to normalization of the activity of sucrase and SGLT1 in the brush-border membrane of enterocytes. diabetes mellitus; glucose transport; SGLT1; brush-border membrane vesicles

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (CHIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by CHIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased ~2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene. myocyte enhancer factor; glucose transporter 4; chromatin immunoprecipitation assay; histone H3 acetylation; KN93; [Ca.sup.2+]/calmodulindependent kinase II phosphorylation

Epidemiological and experimental studies have demonstrated that early postnatal nutrition has been associated with long-term effects on glucose homeostasis in adulthood. Recently, our group demonstrated that undernutrition during early lactation affects the expression and activation of key proteins of the insulin signaling cascade in rat skeletal muscle during postnatal development. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity in peripheral tissues, we investigated the insulin signaling in adipose tissue. Adipocytes were isolated from epididymal fat pads of adult male rats that were the offspring of dams fed either a normal or a protein-free diet during the first 10 days of lactation. The cells were incubated with 100 nM insulin before the assays for immunoblotting analysis, 2-deoxyglucose uptake, immunocytochemistry for GLUT4, and/or actin filaments. Following insulin stimulation, adipocytes isolated from undernourished rats presented reduced tyrosine phosphorylation of IR and IRS-1 and increased basal phosphorylation of IRS-2, Akt, and mTOR compared with controls. Basal glucose uptake was increased in adipocytes from the undernourished group, and the treatment with LY294002 induced only a partial inhibition both in basal and in insulin-stimulated glucose uptake, suggesting an involvement of phosphoinositide 3-kinase activity. These alterations were accompanied by higher GLUT4 content in the plasma membrane and alterations in the actin cytoskeleton dynamics. These data suggest that early postnatal undernutrition impairs insulin sensitivity in adulthood by promoting changes in critical steps of insulin signaling in adipose tissue, which may contribute to permanent changes in glucose homeostasis. undernutrition; glucose transporter 4; insulin signaling

Accumulated evidence over the last several years indicates that insulin regulates multiple steps in the overall translocation of GLUT4 vesicles to the fat/muscle cell surface, including formation of an intracellular storage pool of GLUT4 vesicles, its movement to the proximity of the cell surface, and the subsequent docking/fusion with the plasma membrane. Insulin-stimulated formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)[P.sub.3]; and in some cases, of its catabolite PtdIns(3,4)[P.sub.2]] plays a pivotal role in this process. PtdIns(3,4,5)[P.sub.3] is synthesized by the activated wort-mannin-sensitive class IA phosphoinositide (PI) 3-kinase and controls the rate-limiting cell surface terminal stages of the GLUT4 journey. However, recent research is consistent with the conclusion that signals by each of the remaining five PIs, i.e., PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)[P.sub.2], and PtdIns(4,5)[P.sub.2], may act in concert with that of PtdIns(3,4,5)[P.sub.3] in integrating the insulin receptor-issued signals with GLUT4 surface translocation and glucose transport activation. This review summarizes the experimental evidence supporting the complementary function of these PIs in insulin responsiveness of fat and muscle cells, with particular reference to mechanistic insights and functional significance in the regulation of overall GLUT4 vesicle dynamics. glucose transporter 4; glucose transport; fat and muscle cells; phosphatidylinositol monophosphates; phosphatidylinositol bisphosphates

Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral [Na.sup.+]-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter ([Na.sup.+]-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment. myo-inositol transport; regulation; hyperosmolarity; Madin-Darby canine kidney cells

Differentiation of intestinal epithelial cells is accompanied by alterations in levels of expression of many genes, including those involved in nutrient uptake. Effects of differentiation of intestinal epithelial cells on the physiological and molecular parameters of the intestinal folate uptake process are not well characterized. To address this issue, we used two models, Caco-2 cells and native mouse intestine. Studies with Caco-2 cells showed a significant increase in the initial rate of carrier-mediated folic acid uptake during differentiation (i.e., as the cells transitioned from preconfluent to confluent and then to postconfluent stages). This increase was associated with an increase in the level of expression of the human reduced folate carrier (hRFC) and the human proton-coupled folate transporter (hPCFT) both at the protein and mRNA levels with differentiation; it was also associated with a significant increase in activity of the hRFC and hPCFT promoters. Studies with native mouse intestine showed a significantly higher folate uptake in villus compared with crypt cells, which was again associated with a significantly higher level of expression of the mouse RFC and PCFT at the protein and mRNA levels. Together, these studies demonstrate that the intestinal folate uptake process undergoes differentiation-dependent regulation and that this regulation is mediated via changes in the level of expression of both the RFC and PCFT. In addition, the studies suggest the possible involvement (at least in part) of a transcriptional mechanism(s) in this type of regulation of the intestinal folate uptake process. transport; epithelial transport; reduced folate carrier; proton-coupled folate transporter

The advent of hyperpolarized [sup.13]C magnetic resonance (MR) has provided new potential for the real-time visualization of in vivo metabolic processes. The aim of this work was to use hyperpolarized [1-[sup.13]C]pyruvate as a metabolic tracer to assess noninvasively the flux through the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) in the rat heart, by measuring the production of bicarbonate ([H.sup.13]C[O.sup.-.sub.3]), a byproduct of the PDH-catalyzed conversion of [1-[sup.13]C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in [H.sup.13]C[O.sup.-.sub.3] production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized [sup.13]C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized [sup.13]C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased [H.sup.13]C[O.sup.-.sub.3] production with the onset of diabetes that correlated with disease severity. These observations were supported by in vitro investigations of PDH activity as reported in the literature and provided evidence that flux through the PDH enzyme complex can be monitored noninvasively, in vivo, by using hyperpolarized [sup.13]C MR. cardiac metabolism | diabetes | fasting | DNP

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)--dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithe lial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference-mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.

The protective function of skin (its low permeability) presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the possibilities to temporarily breach the barrier function of skin is using electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. In addition, the application of electric pulses to biological ceils causes the electroporation of cell membrane, increasing its permeability, thus enabling cell uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA. The electropermeabilization process in skin was described theoretically, by means of numerical modeling, leaning on data derived from our in vivo experiments previously published. The numerical models took into account the layered structure of skin, macroscopical changes of its bulk electrical properties during electroporation, as well as the presence of localized sites of increased molecular transport termed local transport regions. The output of the models was compared with the in vivo experiments, and a good agreement was obtained. In addition, a comparison of our results with already published findings on skin electropermeabilization showed that permeabilizing voltage amplitudes suggested by the model are also well in the range of the voltage amplitudes reported by other authors to cause skin permeabilization. The subject of tissue conductivity changes due to electroporation is still a rather unexplored field; however, we used the available data to describe the mechanism of the nonlinear process of the tissue electropermeabilization. Index Terms--Conductivity changes, finite element method, local transport regions (LTRs), numerical modeling, skin electroporation.

Genetic variation in [alpha]-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of [alpha]-adducin in controlling Na/K pump activity, wild-type or mutated rat and human [alpha]-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, [alpha]-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension. hypertension; Na transport: clathrin; trafficking; immunoprecipitation

Nedd4-2 has been proposed to play a critical role in regulating epithelial [Na.sup.+] channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgkl) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in [Na.sup.+] homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other [Na.sup.+] transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaC1sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder. ion channels; kidney; sodium channels

Peroxisome proliferator-activated receptor (PPAR) agonists were shown to inhibit atherosclerosis through augmentation of endothelial nitric oxide synthase (eNOS) activity. In addition, rosiglitazone exerts a beneficial effect in chronic renal failure (CRF). Since L-arginine transport by CAT-1 (the specific arginine transporter for eNOS) is inhibited in uremia, we aimed to explore the effect of rosiglitazone on arginine transport in CRF. Arginine uptake by aortic rings was studied in control animals, rats, 6 wk following 5/6 nephrectomy (CRF) and rats with CRF treated with rosiglitazone. The decrease of arginine transport in CRF was prevented by rosiglitazone. Immunobloting revealed that CAT-1 protein was decreased in CRF but remained unchanged following rosiglitazone administration. Protein content of the membrane fraction of PKC[alpha] and phosphorylated CAT-I increased significantly in CRF, effects that were prevented by rosiglitazone. PKC[alpha] phosphorylation was unchanged but significantly attenuated by rosiglitazone in CRF. Ex vivo administration of phorbol-12-myristate13-acetate to rosiglitazone-treated CRF rats significantly attenuated the effect of rosiglitazone on arginine uptake. The decrease in cGMP response to carbamyl-choline (eNOS agonist) was significantly attenuated by rosiglitazone in CRF. Western blotting and immunohistochemistry analysis revealed that protein nitration was intensified in the endothelium of CRF rats and this was attenuated by rosiglitazone. In conclusion, rosiglitazone prevents the decrease in arginine uptake in CRF through both depletion and inactivation of PKC[alpha]. These findings are associated with restoration of eNO generation and attenuation of protein nitration and therefore may serve as a novel mechanism to explain the beneficial effects of rosiglitazone on endothelial function in uremia. uremia; protein nitration; endothelial function

We examined the modulation of intrinsic (i.e., spontaneous) detrusor contractions by the urothelium and the lamina propria through optical mapping approaches. Normal adult and spinal cord-transected (SCT) rat bladders were stained with [Ca.sup.2+]- and voltage-sensitive dyes, and optical activity generated from intrinsic contractions was mapped from the mucosal surface of whole bladder sheets. Both normal adult and SCT rat bladders displayed intrinsic contractions, where normal bladders showed low-amplitude, high-frequency contractions with disorganized patterns of activity. In contrast, in the SCT animals there were high-amplitude, low-frequency contractions that displayed an organized spread of membrane potential and intracellular [Ca.sup.2+]. The difference in contractile activity was mirrored in the [Ca.sup.2+] and membrane potential maps of bladder sheets. Normal bladders showed multiple initiation sites across the mucosal surface, whereas SCT bladders showed only one or two fixed initiation sites localized to the dome. The magnitude of intrinsic contractions could be enhanced by stretch or low-dose arecaidine (50 nM), a muscarinic-specific agonist. Partial removal of the mucosa decreased the amplitude of the intrinsic contractions and decreased the response to stretch or arecaidine. Optical mapping of mucosa-denuded sheets, where enhanced spontaneous activity was abolished, or application of 1 [micro]M nifedipine to remove smooth muscle signals, but not the mucosal signals, shows that intrinsic activity in pathological bladders is driven by the mucosal layer. In summary, we suggest an urotheliogenic origin for intrinsic activity, where structures within the mucosal layer organize and thereby enhance intrinsic detrusor contractions. intrinsic or spontaneous contractions; spinal cord transection; bladder overactivity; [Ca.sup.2+]- and voltage-sensitive dyes

In the 1930s, August Krogh, Homer Smith, and Ancel Keys knew that teleost fishes were hyperosmotic to fresh water and hyposmotic to seawater, and, therefore, they were potentially salt depleted and dehydrated, respectively. Their seminal studies demonstrated that freshwater teleosts extract NaCl from the environment, while marine teleosts ingest seawater, absorb intestinal water by absorbing NaCl, and excrete the excess salt via gill transport mechanisms. During the past 70 years, their research descendents have used chemical, radioisotopic, pharmacological, cellular, and molecular techniques to further characterize the gill transport mechanisms and begin to study the signaling molecules that modulate these processes. The cellular site for these transport pathways was first described by Keys and is now known as the mitochondrion-rich cell (MRC). The model for NaCl secretion by the marine MRC is well supported, but the model for NaCl uptake by freshwater MRC is more unsettled. Importantly, these ionic uptake mechanisms also appear to be expressed in the marine gill MRC, for acid-base regulation. A large suite of potential endocrine control mechanisms have been identified, and recent evidence suggests that paracrines such as endothelin, nitric oxide, and prostaglandins might also control MRC function. gill salt transport; mitochondrion-rich cell; endothelin

We tested the hypothesis that vagal withdrawal plays a role in the rapid (phase I) cardiopulmonary response to exercise. To this aim, in five men (24.6 [+ or -] 3.4 yr, 82.1 [+ or -] 13.7 kg, maximal aerobic power 330 [+ or -] 67 W), we determined beat-by-beat cardiac output (Q), oxygen delivery ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]), and breathby-breath lung oxygen uptake ([Vo.sub.2]) at light exercise (50 and 100 W) in normoxia and acute hypoxia (fraction of inspired [O.sub.2] = 0.11), because the latter reduces resting vagal activity. We computed Q from stroke volume ([Q.sub.st], by model flow) and heart rate ([f.sub.H], electrocardiography), and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]] from Q and arterial [O.sub.2] concentration. Double exponentials were fitted to the data. In hypoxia compared with normoxia, steady-state [f.sub.H] and Q were higher, and [Q.sub.st] and [Vo.sub.2] were unchanged. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.] was unchanged at rest and lower at exercise. During transients, amplitude of phase I ([A.sub.1]) for [Vo.sub.2] was unchanged. For [f.sub.H], Q and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]], [A.sub.1] was lower. Phase I time constant ([[tau].sub.1]) for Qao, and V[O.sub.2] was unchanged. The same was the case for Q at 100 w and forfH at 50 W. Qst kinetics were unaffected. In conclusion, the results do not fully support the hypothesis that vagal withdrawal determines phase I, because it was not completely suppressed. Although we can attribute the decrease in At of [f.sub.H] to a diminished degree of vagal withdrawal in hypoxia, this is not so for [Q.sub.st]. Thus the dual origin of the phase I of Q and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]], neural (vagal) and mechanical (venous return increase by muscle pump action), would rather be confirmed. cardiovascular response

Nitric oxide (NO) modulates vasodilation in cerebral cortex during sensory activation. NO is known to inhibit the synthesis of 20-HETE, which has been implicated in arteriolar constriction during astrocyte activation in brain slices. We tested the hypothesis that the attenuated cerebral blood flow (CBF) response to whisker stimulation seen after NO synthase (NOS) inhibition requires 20-HETE synthesis and that the ability of an epoxyeicosatrienoic acids (EETs) antagonist to reduce the CBF response is blunted after NOS inhibition but restored with simultaneous blockade of 20-HETE synthesis. In anesthetized rats, the increase in CBF during whisker stimulation was attenuated after the blockade of neuronal NOS with 7-nitroindazole. Subsequent administration of the 20-HETE synthesis inhibitor N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) restored the CBF response to control levels. After the administration of 7-nitroindazole, the inhibitory effect of an EETs antagonist 14, 15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on the CBF response was lost, whereas the simultaneous administration of 7-nitroindazole and HET0016 restored the inhibitory effect of 14,15-EEZE. The administration of HET0016 alone had only a small effect on the evoked CBF response in rats. Furthermore, in neuronal NO[S.sup.+/+] and NO[S.sup.-/-] mice, HET0016 administration did not increase the CBF response to whisker stimulation. In neuronal NO[S.sup.+/+] mice, HET0016 also blocked the reduction in the response seen with acute NOS inhibition. These results indicate that 20-HETE synthesis normally does not substantially restrict functional hyperemia. Increased NO production during functional activation may act dynamically to suppress 20-HETE synthesis or downstream signaling and permit EETs-dependent vasodilation. With the chronic loss of neuronal NOS in mice, other mechanisms apparently suppress 20-HETE synthesis or signaling. eicosanoids; epoxyeicosatrienoic acid; functional activation; mouse; rat; vibrissae; 20-hydroxyeicosatetraenoic acid

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein ([DELTA]F508-CFTR) that fails to fold properly, thus mutated [DELTA]F508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulumassociated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in [DELTA]F508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfidebased cross-linker and its administration to cells carrying [DELTA]F508CFTR resulted in a greater enhancement of [DELTA]F508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; correctors; transporter protein; intracellular activation

The contribution of the liver to glucose utilization is essential to maintain glucose homeostasis. Previous data from protein tyrosine phosphatase (PTP) 1B-deficient mice demonstrated that the liver is a major site for PTP1B action in the periphery. In this study, we have investigated the consequences of PTP1B deficiency in glucose uptake in hepatocytes from neonatal and adult mice. The lack of PTP1B increased basal glucose uptake in hepatocytes from neonatal (3-5 days old) but not adult (10-12 wk old) mice. This occurs without changes in hexokinase, glucokinase, and glucose 6-phosphatase enzymatic activities. By contrast, the glucose transporter GLUT2 was upregulated at the protein level in neonatal hepatocytes and livers from PTP1B-deficient neonates. These results were accompanied by a significant increase in the net free intrahepatic glucose levels in the livers of PTP1[B.sup.-/-] neonates. The association between GLUT2 and insulin receptor (IR) A isoform was increased in PTP1[B.sup.-/-] neonatal hepatocytes compared with the wild-type. Indeed, PTP1B deficiency in neonatal hepatocytes shifted the ratio of isoforms A and B of the IR by increasing the amount of IRA and decreasing IRB. Moreover, overexpression of IRA in PTP1[B.sup.-/-] neonatal hepatocytes increased the amount of IRA/GLUT2 complexes. Conversely, hepatocytes from adult mice only expressed IRB. Since IRA plays a direct role in the regulation of glucose uptake in neonatal hepatocytes through its specific association with GLUT2, we propose the increase in IRA/ GLUT2 complexes due to PTP1B deficiency as the molecular mechanism of the increased glucose uptake in the neonatal stage. liver; insulin receptor isoforms; glucose transport; insulin sensitivity

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1[alpha] and retinoid X receptor-[alpha]:retinoic acid receptor-[alpha] binding sites in the mouse Y-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4[alpha], which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse (mNtcp). A 1.1-kb 5'-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1[alpha], HNF-4[alpha] cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4[alpha] consensus site at -345/-326 and -335/-316 bp, respectively, p-475bpmNtcpLUC is not transactivated by HNF-1[alpha] but shows a 50-fold enhanced activity upon cotransfection with HNF-4[alpha]. Gel mobility shift assays demonstrated a complex of the HNF-4[alpha]-element formed with liver nuclear extracts that was blocked by an HNF-4[alpha] specific antibody. HNF-4[alpha] binding was confirmed by chromatin immunoprecipitation. Using Hepa 1-6 cells, HNF-4[alpha]-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4[alpha] via a conserved distal cis-element independently of HNF-1[alpha]. hepatocyte-enriched transcription factors; nuclear hormone receptors; transcriptional coactivator; gene regulation; bile acid transport

Glucose metabolism is vital to most mammalian cells, and the passage of glucose across cell membranes is facilitated by a family of integral membrane transporter proteins, the GLUTs. There are currently 14 members of the SLC2 family of GLUTs, several of which have been the focus of this series of reviews. The subject of the present review is GLUT3, which, as implied by its name, was the third glucose transporter to be cloned (Kayano T, Fukumoto H, Eddy RL, Fan YS, Byers MG, Shows TB, Bell GI. J Biol Chem 263: 15245-15248, 1988) and was originally designated as the neuronal GLUT. The overriding question that drove the early work on GLUT3 was why would neurons need a separate glucose transporter isoform? What is it about GLUT3 that specifically suits the needs of the highly metabolic and oxidative neuron with its high glucose demand? More recently, GLUT3 has been studied in other cell types with quite specific requirements for glucose, including sperm, preimplantation embryos, circulating white blood cells, and an array of carcinoma cell lines. The last are sufficiently varied and numerous to warrant a review of their own and will not be discussed here. However, for each of these cases, the same questions apply. Thus, the objective of this review is to discuss the properties and tissue and cellular localization of GLUT3 as well as the features of expression, function, and regulation that distinguish it from the rest of its family and make it uniquely suited as the mediator of glucose delivery to these specific cells. neurons: sperm: preimplantation embryo; white blood cells

The very last member of the SLC2A gene family of facilitated hexose transporters to be cloned was SLC2A7 (hGLUT7). It has been assigned to the class II of the GLUT family on the basis of sequence similarity, and its closest family member is GLUT5, an intestinal fructose transporter. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testis and prostate as well. The protein is expressed in the apical membrane of the small intestine and colon, and it has a high affinity ( glucose transporter proteins; SLC2A7; intestinal hexose absorption; structure-function

To meet the high-energy demands of photoreceptor cells, the outer retina metabolizes glucose through glycolytic and oxidative pathways, resulting in large-scale production of lactate and C[O.sub.2]. Mct3, a protoncoupled monocarboxylate transporter, is critically positioned to facilitate transport of lactate and [H.sup.+] out of the retina and could therefore play a role in pH and ion homeostasis of the outer retina. Mct3 is preferentially expressed in the basolateral membrane of the retinal pigment epithelium and forms a heteromeric complex with the accessory protein CD147. To examine the physiological role of Mct3 in the retina, we generated mice with a targeted deletion in Mct3 (slc16A8). The overall retinal histology of 4- to 36-wk-old [Mct3.sup.-/-] mice appeared normal. In the absence of Mct3, expression of CD147 was lost from the basolateral but not apical RPE. The saturated a-wave amplitude ([a.sub.max]) of the scotopic electroretinogram (ERG) was reduced by approximately twofold in [Mct3.sup.-/-] mice relative to wildtype mice. A fourfold increase in lactate in the retina suggested a decrease in outer-retinal pH. In single-cell recordings from superfused retinal slices, saturating amplitudes of single rod photocurrents ([J.sub.max]) were comparable indicating that [Mct3.sup.-/-] mouse photoreceptor cells were inherently healthy. Based on these data, we hypothesize that disruption of Mct3 leads to a potentially reversible decrease in subretinal space pH, thereby reducing the magnitude of the light suppressible photoreceptor current. monocarboxylate transporter 3; lactate transport; retinal pigment epithleium; pH regulation; photoreceptor; electroretinogram

[Ca.sup.2+]/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [[[Ca.sup.2+]].sub.i] with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rhokinase and/or PKC with phospfiorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation. myosin regulatory light chain; calmodulin; bladder; Rho kinase; myosin light chain phosphatase

Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1-5 and TMs 6-10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats. alternating access mechanism | neurotransmitter:sodium symporters | serotonin | structural modeling | structural repeats

The protective function of skin and hence its low permeability presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the methods to temporarily increase skin permeability is electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. Also, the application of electric pulses to biological cells causes increased permeability of cell membrane, thus enabling the uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA, into the cell. The creation of localized sites of increased molecular transport termed local transport regions (LTRs) can be observed during electroporation, as well as changes in the bulk electric properties of skin layers. We modeled these phenomena with a numerical model and compared the output of the model with our own in vivo experiments and previously published results of skin electroporation and a good agreement was obtained. With the model presented, we used the available data to describe the nonlinear process of skin electropermeabilization from the aspect of tissue conductivity changes and the presence of local transport regions in the permeabilized stratum corneum. The observations derived from various in vivo experiments by different authors were thus confirmed theoretically. Index Terms--Conductivity changes, finite-element method, local transport regions, numerical modeling, skin electroporation.

Hydrogen sulfide ([H.sub.2]S) is rapidly emerging as a biologically significant signaling molecule. Studies published before 2000 report low or undetectable [H.sub.2]S (usually as total sulfide) levels in blood or plasma, whereas recent work has reported sulfide concentrations between 10 and 300 [micro]M, suggesting it acts as a circulating signal. In the first series of experiments, we used a recently developed polarographic sensor to measure the baseline level of endogenous [H.sub.2]S gas and turnover of exogenous [H.sub.2]S gas in real time in blood from numerous animals, including lamprey, trout, mouse, rat, pig, and cow. We found that, contrary to recent reports, [H.sub.2]S gas was essentially undetectable (< 100 nM total sulfide) in all animals. Furthermore, exogenous sulfide was rapidly removed from blood, plasma, or 5% bovine serum albumin in vitro and from intact trout in vivo. To determine if blood [H.sub.2]S could transiently increase, we measured oxygen-dependent [H.sub.2]S production by trout hearts in vitro and in vivo. [H.sub.2]S has been shown to mediate ischemic preconditioning (IPC) in mammals. [PC is present in trout and, unlike mammals, the trout myocardium obtains its oxygen from relatively hypoxic systemic venous blood. In vitro, myocardial [H.sub.2]S production was inversely related to P[O.sub.2], whereas we failed to detect [H.sub.2]S in ventral aortic blood from either normoxic or hypoxic fish in vivo. These results provide an autocrine or paracrine mechanism for myocardial coupling of hypoxia to [H.sub.2]S in [PC, i.e., oxygen sensing, but they fail to provide any evidence that [H.sub.2]S signaling is mediated by the circulation. gasotransmitter; hydrogen sulfide metabolism; vascular signaling

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 [+ or -] 0.06 nM) and candesartan-treated rats (0.34 [+ or -] 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 [+ or -] 0.37 nM) that were decreased by candesartan (0.97 [+ or -] 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 [+ or -] 13%) and base (120 [+ or -] 25%). UT-A3 abundance was increased in IM tip (123 [+ or -] 11%) and base (146 [+ or -] 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 [+ or -] 14%) and base (210 [+ or -] 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 [+ or -] 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM. angiotensin II; UT-A urea transporter; aquaporin; AQP2; NKCC2

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations (~400 mmol/1). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout (Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant (Kin) = 0.58 retool/l; maximal velocity ([V.sub.max]) = 0.12 [micro]mol x [h.sup.-1] x mg [protein.sup.-1] at lower urea concentrations ( phloretin; trifluoromethoxyphenylhydrazone; nigericin; valinomycin

Aquatic organisms and, in particular, filter feeders, such as mussels, are continuously exposed to toxicants dissolved in the water and, presumably, require adaptations to avoid the detrimental effects from such chemicals. Previous work indicates that activity of ATP-binding cassette (ABC) transporters protects mussels against toxicants, but the nature of these transporters and the structural basis of protection are not known. Here we meld studies on transporter function, gene expression, and localization of transporter protein in mussel gill tissue and show activity and expression of two xenobiofic transporter types in the gills, where they provide an effective structural barrier against chemicals. Activity of ABCB/MDR/P-glycoprotein and ABCC/MRPtype transporters was indicated by sensitivity of efflux of the test substrate calcein-AM to the ABCB inhibitor PSC-833 and the ABCC inhibitor MK-571. This activity profile is supported by our cloning of the complete sequence of two ABC transporter types from RNA in mussel tissue with a high degree of identity to transporters from the ABCB and ABCC subfamilies. Overall identity of the amino acid sequences with corresponding homologs from other organisms was 38-50% (ABCB) and 27-44% (ABCC). C219 antibody staining specific for ABCB revealed that this transporter was restricted to cells in the gill filaments with direct exposure to water flow. Taken together, our data demonstrate that ABC transporters form an active, physiological barrier at the tissue-environment interface in mussel gills, providing protection against environmental xenotoxicants. ATP-binding cassette transporters; multixenobiotic resistance

High-fat feeding (HFF) is a well-accepted model for nutritionally-induced insulin resistance. The purpose of this investigation was to assess the metabolic responses of female lean Zucker rats provided regular chow (4% fat) or a high-fat chow (50% fat) for 15 wk. HFF rats spontaneously adjusted food intake so that daily caloric intake matched that of chow-fed (CF) controls. HFF animals consumed more (P < 0.05) calories from fat (31.9 [+ or -] 1.2 vs. 2.4 [+ or -] 0.2 kcal/day) and had significantly greater final body weights (280 [+ or -] 10 vs. 250 [+ or -] 5 g) and total visceral fat (24 [+ or -] 3 vs. 10 [+ or -] 1 g). Fasting plasma insulin was 2.3-fold elevated in HFF rats. Glucose tolerance (58%) and whole body insulin sensitivity (75%) were markedly impaired in HFF animals. In HFF plantaris muscle, in vivo insulin receptor [beta]-subunit (IR-[beta]) and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and phosphorylation of Akt Ser473 and glycogen synthase kinase-3[beta] (GSK-3[beta]) Ser9, relative to circulating insulin levels, were decreased by 40-59%. In vitro insulin-stimulated glucose transport in HFF soleus was decreased by 54%, as were IRS-1 tyrosine phosphorylation (26%) and phosphorylation of Akt Ser473 (38%) and GSK-3[beta] Ser9 (25%), the latter indicative of GSK-3 overactivity. GSK-3 inhibition in HFF soleus using CT98014 increased insulin-stimulated glucose transport (28%), IRS-1 tyrosine phosphorylation (28%) and phosphorylation of Akt Ser473 (38%) and GSK-3[beta] Ser9 (48%). In summary, the female lean Zucker rat fed a high-fat diet represents an isocaloric model of nutritionally-induced insulin resistance associated with moderate visceral fat gain, hyperinsulinemia, and impairments of skeletal muscle insulin-signaling functionality, including GSK-3[beta] overactivity.

The heterogeneity across the left ventricular wall is characterized by higher rates of oxygen consumption, systolic thickening fraction, myocardial perfusion, and lower energetic state in the subendocardial layers (ENDO). During dobutamine stimulation-induced demand ischemia, the transmural distribution of energy demand and metabolic markers of ischemia are not known. In this study, hemodynamics, transmural high-energy phosphate (HEP), 2-deoxyglucose-6-phosphate (2-DGP) levels, and myocardial blood flow (MBF) were determined under basal conditions, during dobutamine infusion (DOB: 20 [micro]g x [kg.sup.-1] x [min.sup.-1] iv), and during coronary stenosis + DOB + 2-deoxyglucose (2-DG) infusion. DOB increased rate pressure products (RPP) and MBF significantly without affecting the subendocardial-to-subepicardial blood flow ratio (ENDO/EPI) or HEP levels. During coronary stenosis + DOB + 2-DG infusion, RPP, ischemic zone (IZ) MBF, and ENDO/EPI decreased significantly. The IZ ratio of creatine phosphate-to-ATP decreased significantly [2.30 [+ or -] 0.14, 2.06 [+ or -] 0.13, and 2.04 [+ or -] 0.11 to 1.77 [+ or -] 0.12, 1.70 [+ or -] 0.11, and 1.72 [+ or -] 0.12 for EPI, midmyocardial (MID), and ENDO, respectively], and 2-DGP accumulated in all layers, as evidenced by the 2-DGP/PCr (0.55 [+ or -] 0.12, 0.52 [+ or -] 0.10, and 0.37 [+ or -] 0.08 for EPI, MID, and ENDO, respectively; P < 0.05, EPI > ENDO). In the IZ the wet weight-to-dry weight ratio was significantly increased compared with the normal zone (5.9 [+ or -] 0.5 vs. 4.4 [+ or -] 0.4; P < 0.05). Thus, in the stenotic perfused bed, during dobutamine-induced high cardiac work state, despite higher blood flow, the subepicardial layers showed the greater metabolic changes characterized by a shift toward higher carbohydrate metabolism, suggesting that a homeostatic response to high-cardiac work state is characterized by more glucose utilization in energy metabolism. ischemia; energetics; glycolytic metabolism; left ventricle

Previous in vivo studies suggest that constitutive nitric oxide (cNO) can regulate Na- glucose cotransport (SGLT1) and Na-H exchange (NHE3) in rabbit intestinal villus cells. Whether these two primary Na absorbing pathways are directly regulated by cNO and the mechanisms of this regulation in the enterocyte is not known. Thus nontransformed rat small intestinal epithelial cells (IEC-18) were treated with [N.sup.G]-nitro-L-arginine methyl ester (L-NAME), which directly decreased cNO in these cells, L-NAME treatment decreased SGLT1 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of inhibition was secondary to a decrease in the affinity of the cotransporter for glucose without a change in the number of cotransporters. In contrast, L-NAME treatment increased NHE3 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of stimulation was by increasing the number of the exchangers without a change in the affinity for Na. Quantitative RT-PCR (RTQ-PCR) and Western blot analysis of SGLT1 demonstrated no change in mRNA and protein, respectively. RTQ-PCR and Western blot analysis of NHE3 indicated that NHE3 was increased by L-NAME treatment by an increase in mRNA and protein, respectively. These results indicate that decreased cNO levels directly mediate the inhibition of SGLT1 and stimulation of NHE3 in intestinal epithelial cells. Thus cNO directly but uniquely regulates the two primary Na-absorptive pathways in the mammalian small intestine. glucose absorption; regulation of transport; NHE3; SGLT1; Na absorption