Search

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

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

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

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

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

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

We examined male and female glucose transporter isoform-3 (GLUT3; placenta)-null [heterozygous.sup.+/-] mutation-carrying mice and compared them with age- and sex-matched [wild-type.sup.+/+] littermates. No difference in postnatal (1-2 days, 6-7 days, 12-13 days, 20-21 days), postsuckling (1-2 mo), and adult (3-6 mo) growth pattern was seen except for an increase in body weight of 9- to 11-mo-old male but not female [GLUT3.sup.+/-] mice. This change in male mutant mice was associated with increased total body fat mass, perirenal and epididymal white adipose tissue weight, and hepatic lipid infiltration. These minimally glucose-intolerant male mutant mice demonstrated no change in caloric intake but a decline in basal metabolic rate and insulin resistance. No perturbation in basal circulating glucose concentrations but an increase in insulin concentrations, triglycerides, and total cholesterol was observed in [GLUT3.sup.+/-] male mice. Tissue analysis in males and females demonstrated diminished GLU[T.sub.3] protein in [GLUT3.sup.+/-] brain and skeletal muscle with no change in brain and adipose tissue GLUT1 protein concentrations. Furthermore, the male [GLUT3.sup.+/-] mice expressed decreased insulin-responsive GLUT4 in white adipose tissue and skeletal muscle sarcolemma. We conclude that the [GLUT3.sup.+/-] male mice develop adult-onset adiposity with insulin resistance. fetal programming; hepatic steatosis; transplacental glucose transport; type 2 diabetes mellitus

We have utilized small interfering RNA (siRNA)-mediated depletion of the [beta]-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in [beta]-COP levels causes the colocalization of markers for the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network (TGN), and recycling endosomes in large, globular compartments. The lack of spatial differentiation of these compartments is not due to a general collapse of all cellular organelles since markers for the early endosomes and lysosomes do not redistribute to the common structures. Anterograde trafficking of the transmembrane cargo vesicular stomatitis virus membrane glycoprotein and of a subset of soluble cargoes is arrested within the common globular compartments. Similarly, recycling traffic of transferrin through the common compartment is perturbed. Furthermore, the trafficking of caveolin-1 (Cav1), a structural protein of caveolae, is arrested within the globular structures. Importantly, Cav1 coprecipitates with the [gamma]-subunit of COP-I, suggesting that Cavl is a COP-I cargo. Our findings suggest that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP-I plays a novel role in the biosynthetic transport of Cav1. Golgi; trans-Golgi network; endoplasmic reticulum-Golgi intermediate compartment; recycling endosome; coatomer

Transporters present in the epithelium of the small intestine determine the efficiency by which dietary and biliary cholesterol are taken up into the body and thus control whole-body cholesterol balance. Niemann-Pick C1 Like Protein 1 (Npc111) transports cholesterol into the enterocyte, whereas ATP-binding cassette transporters Abca1 and Abcg5/Abcg8 are presumed to be involved in cholesterol efflux from the enterocyte toward plasma HDL and back into the intestinal lumen, respectively. Abca1, Abcg5, and Abcg8 are well-established liver X receptor (LXR) target genes. We examined the effects of a high-fat diet on expression and function of cholesterol transporters in the small intestine in mice. Npc111, Abca1, Abcg5, and Abcg8 were all downregulated after 2, 4, and 8 wk on a cholesterol-free, high-fat diet. The high-fat diet did not affect biliary cholesterol secretion but diminished fractional cholesterol absorption from 61 to 42% (P < 0.05). In an acute experiment in which triacylglycerols of unsaturated fatty acids were given by gavage, we found that this downregulation occurs within a 6-h time frame. Studies in LXR[alpha]-null mice, confirmed by in vitro data, showed that fatty acid-induced downregulation of cholesterol transporters is LXR[alpha] independent and associated with a posttranslational increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity that reflects induction of cholesterol biosynthesis as well as with a doubling of neutral fecal sterol loss. This study highlights the induction of adaptive changes in small intestinal cholesterol metabolism during exposure to dietary fat. cholesterol absorption; ABC transporters; Npc111; fatty acids

The water-soluble vitamin [B.sub.6] (pyridoxine) is important for normal cellular functions, growth, and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source, where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the latter source of the vitamin, but nothing is known about the mechanism involved and its regulation. In this study, we addressed these issues using young adult mouse colonic epithelial (YAMC) cells and human colonic apical membrane vesicles (AMV) as models and using [[sup.3] H]pyridoxine as the uptake substrate. The results showed the initial rate of [[sup.3] H]pyridoxine uptake by YAMC cells to be 1) energy- and temperature- (but not Na-) dependent and to occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent [K.sub.m] and [V.sub.max] of 2.1 [+ or -] 0.5 [micro] M and 53.4 [+ or -]4.3 pmol*mg [protein.sup-1]* 3 [min.sup.-1], respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine, and isoniazid; 4) trans-stimulated by unlabeled pyridoxine; 5) amiloride sensitive; and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carriermediated process for pyridoxine uptake by mammalian colonocytes. colonic transport; transport mechanism; transport regulation

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how [Ca.sup.2+], despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of [Ca.sup.2+] sources and targets. capacitative calcium entry; caveolae; endothelial cells: endothelial nitric oxide synthase; mathematical model

Although colonic lumen N[H.sub.4.sup.+] levels are high, 15-44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for [H.sub.4.sup.+]. More extensive studies have elucidated the transport of N[H.sub.4.sup.+] in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to N[H.sub.4.sup.+] secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete [H.sub.4.sup.+] and maintain an apical-to-basolateral N[H.sub.4.sup.+] gradient. N[H.sub.4.sup.+] transport in the secretory direction was supported by basolateral N[H.sub.4.sup.+] loading on NKCCl, [Na.sup.+]-[K.sup.+]-ATPase, and the N[H.sub.4.sup.+] transporter, RhBG. [H.sub.4.sup.+] was transported on NKCC1 in T84 cells nearly as well as [K.sup.+] as determined by bumetanide-sensitive [sup.86]Rb-uptake. [sup.86]Rb-uptake and ouabain-sensitive current measurement indicated that N[H.sub.4.sup.+] is transported by [Na.sup.+]-[K.sup.+]-ATPase in these cells to an equal extent as [K.sup.+]. T84 cells expressed mRNA for the basolateral N[H.sub.4.sup.+] transporter RhBG and the apical N[H.sub.4.sup.+] transporter RhCG. Net N[H.sub.4.sup.+] transport in the secretory direction determined by [sup.14]C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of N[H.sub.4.sup.+] transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of N[H.sub.4.sup.+] because of surface cell N[H.sub.4.sup.+] absorption. These findings suggest that we rethink the present limited understanding of N[H.sub.4.sup.+] handling by the distal colon as being due solely to passive absorption. RhBG; RhCG; colon; hyperammonemia

A medullary thick ascending limb (mTAL) cell line, termed raTAL, has been established from freshly isolated rat mTAL tubules and cultured continuously for up to 75 passages; it retains characteristics of mTAL cells even after retrieval from storage in liquid nitrogen for several months. The cells express Tamm-Horsfall glycoprotein (THP), a TAL-specific marker, grow to confluence, exhibit a polygonal morphology characteristic of epithelial cells, and form 'domes.' Detection of THP, [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter (NKCC2), [Na.sup.+]-[K.sup.+]-ATPase, and renal outer medullary [K.sup.+] channel (ROMK) was achieved using indirect immunofluorescence and confocal microscopy. Western blot analysis of NKCC2 expression using two different antibodies revealed a band of ~160 kDa, and RT-PCR analysis demonstrated the presence of NKCC2 isoforms A and F, which was confirmed by DNA sequencing; transport of [Cl.sup.-] into raTAL cells was inhibited by furosemide. Ouabain- and bumetanide-sensitive oxygen consumption, an index of ion transport activity in the mTAL, was observed in raTAL cells, and the number of domes present was reduced significantly when cells were incubated in the presence of ouabain or bumetanide. The specific activity of [Na.sup.+]-[K.sup.+]-ATPase activity was determined in raTAL cells (0.67 [+ or -] 0.18 nmol [P.sub.i] x [micro]g [protein.sup.-1] x [min.sup.-1]), primary cultures of mTAL cells (0.39 [+ or -] 0.08 nmol [P.sub.i] x [micro]g [protein.sup.-1] x [min.sup.-1]), and freshly isolated mTAL tubules (1.10 [+ or -] 0.29 nmol [P.sub.1] x [micro]g [protein.sup.-1] x [min.sup.-1]), and ~30-50% of total cellular ATPase activity was inhibited by ouabain, in accord with other mTAL preparations. This cell line will be used in studies that address biochemical, molecular, and physiological mechanisms in the mTAL. medullary thick ascending limb; cell culture; renal epithelial cells; renal cell lines

The interaction of renal basolateral organic anion transporter 3 (Oat3) with commonly used pharmacotherapeutics (e.g., NSAIDs, [beta]-lactams, and methotrexate) has been studied extensively in vitro. However, the in vivo role of Oat3 in drug disposition, in the context of other transporters, glomerular filtration, and metabolism, has not been established. Moreover, recent investigations have identified inactive human OAT3 polymorphisms. Therefore, this investigation was designed to elucidate the in vivo role of Oat3 in the disposition of penicillin G and prototypical substrates using an Oat3 knockout mouse model. Oat3 deletion resulted in a doubling of penicillin's half-life (P < 0.05) and a reduced volume of distribution (P < 0.01), together yielding a plasma clearance that was one-half (P < 0.05, males) to one-third (P < 0.001, females) of that in wild-type mice. Inhibition of Oat3 abolished the differences in penicillin G elimination between genotypes. Hepatic accumulation of penicillin was 2.3 times higher in male knockouts (P < 0.05) and 3.7 times higher in female knockouts (P < 0.001). Female knockouts also exhibited impaired estrone-3-sulfate clearance. Oat3 deletion did not impact p-aminohippurate elimination, providing correlative evidence to studies in Oat1 knockout mice that suggest Oat1 governs tubular uptake of p-aminohippurate. Collectively, these findings are the first to indicate that functional Oat3 is necessary for proper elimination of xenobiotic and endogenous compounds in vivo. Thus Oat3 plays a distinct role in determining the efficacy and toxicity of drugs. Dysfunctional human OAT3 polymorphisms or instances of polypharmacy involving OAT3 substrates may result in altered systemic accumulation of [beta]-lactams and other clinically relevant compounds. kidney; liver; transport; Oat1; drug disposition

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD. cAMP; vasopressin; concentrating mechanism; urea transport

Kidneys can maintain acid-base homeostasis, despite reduced renal mass, through adaptive changes in net acid excretion, of which ammonia excretion is the predominant component. The present study examines whether these adaptations are associated with changes in the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). We used normal Sprague-Dawley rats and a 5/6 ablation-infarction model of reduced renal mass; control rats underwent sham operation. After 1 wk, glomerular filtration rate, assessed as creatinine clearance, was decreased, serum bicarbonate was slightly increased, and [Na.sup.+] and [K.sup.+] were unchanged. Total urinary ammonia excretion was unchanged, but urinary ammonia adjusted for creatinine clearance, an index of per nephron ammonia metabolism, increased significantly. Although reduced renal mass did not alter total Rhcg protein expression, both light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated hypertrophy of both intercalated cells and principal cells in the cortical and outer medullary collecting duct that was associated with increased apical and basolateral Rhcg polarization. Rhbg expression, analyzed using immunoblot analysis, immunohistochemistry, and measurement of cell-specific expression, was unchanged. We conclude that altered subcellular localization of Rhcg contributes to adaptive changes in single-nephron ammonia metabolism and maintenance of acid-base homeostasis in response to reduced renal mass. intercalated cell; principal cell; cortical collecting duct; outer medullary collecting duct

In contrast to other sympathetic outflow tracts, renal sympathetic nerve activity (RSNA) decreases in response to hypotensive hemorrhage. The functional significance of this 'paradox' is not known. We tested the hypothesis that RSNA modulates renal perfusion and thus erythropoietin (EPO) release after transient hypotensive hemorrhage in anesthetized rats. Plasma EPO was measured before and after 30 min of transient hypotensive hemorrhage (i.e., -40 mmHg from mean baseline blood pressure, followed by reinfusion of shed blood) and 120 min thereafter in sham-denervated rats, and after renal denervation (DNX) or bilateral cervical vagotomy (VX) to abolish/blunt the RSNA decrease mediated by a cardiopulmonary reflex. RSNA, renal Doppler flow, renal vascular resistance (RVR), resistance index, and oxygen delivery/uptake ([DO.sub.2]/[[??]O.sub.2]) were measured. RSNA decreased in intact animals (-40 [+ or -] 5% from baseline, P < 0.05). This was blunted by VX. With intact nerves, EPO level did not increase. In DNX rats, EPO was increased at minute 120 (49 [+ or -] 3 vs. 74 [+ or -] 2 mU/ml; P < 0.05), in VX rats this (47 [+ or -] 2 vs. 62 [+ or -] 4 mU/ml; P < 0.05) was less pronounced. [DO.sub.2] in DNX rats was lower compared with intact and VX rats (0.25 [+ or -] 0.04 vs. 0.51 [+ or -] 0.06 and 0.54 [+ or -] 0.05 ml [O.sub.2]/min; P < 0.05) due to lower Doppler flow and increased RVR. RVR and [DO.sub.2] were similar in intact and VX rats, but resistance index differed between all groups (0.70 [+ or -] 0.02 vs. 0.78 [+ or -] 0.02 vs. 0.85 [+ or -] 0.02; P < 0.05, intact vs. VX vs. DNX), indicating differential reactivity of renal vasculature. [[??]O.sub.2] was unaffected by VX and DNX. Renal sympathoinhibition during hypotensive hemorrhage might help to preserve sufficient oxygenation of renal tissue by modulation of hemodynamic mechanisms that act to adapt renal oxygen availability to demand. renal nerve; oxygen delivery

The aim of the present study was to investigate the effect of hydrogen sulfide ([H.sub.2]S) signaling by nitric oxide (NO) in isolated rat aortas and cultured human umbilical vein endothelial cells (HUVECs). Both administration of [H.sub.2]S and NariS, as well as endogenous [H.sub.2]S, reduced NO formation, endothelial nitric oxide synthase (eNOS) activity, eNOS transcript abundance, and L-arginine (L-Arg) transport (all P < 0.01). The kinetics analysis of eNOS activity and L-Arg transport showed that [H.sub.2]S reduced [V.sub.max] values (all P < 0.01) without modifying [K.sub.m] parameters. Use of selective NOS inhibitors verified that eNOS [vs. inducible NOS (iNOS) and neuronal NOS (nNOS)] was the specific target of [H.sub.2]S regulation. [H.sub.2]S treatment (100 [micro]mol/l) reduced Akt phosphorylation and decreased eNOS phosphorylation at Ser1177. [H.sub.2]S reduced L-Arg uptake by inhibition of a system y+ transporter and decreased the CAT-1 transcript. [H.sub.2]S treatment reduced protein expression of eNOS but not of nNOS and iNOS. Pinacidil ([K.sub.ATP] channel opener) exhibited the similar inhibitory effects on the L-Arg/NOS/NO pathway. Glibenclamide ([K.sub.ATP] channel inhibitor) partly blocked the inhibitory effect of [H.sub.2]S and pinacidil. An in vivo experiment revealed that [H.sub.2]S downregulated the vascular L-Arg/eNOS/NO pathway after intraperitoneal injection of NariS (14 [micro]mol/kg) in rats. Taken together, our findings suggest that [H.sub.2]S downregulates the vascular L-Arg/NOS/NO pathway in vitro and in vivo, and the [K.sub.ATP] channel could be involved in the regulatory mechanism of [H.sub.2]S. nitric oxide; nitric oxide synthase; L-arginine transport

Myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, and myocyte death. Intracellular [Ca.sup.2+] overload with reduced activity of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) is a critical mechanism of this injury. Although upregulation of SERCA function is well documented to improve postischemic cardiac function, there are conflicting reports where pharmacological inhibition of SERCA improved postischemic function. SERCA2a is the primary cardiac isoform regulating intracellular [Ca.sup.2+] homeostasis; however, SERCA1a has been shown to substitute SERCA2a with faster [Ca.sup.2+] transport kinetics. Therefore, to further address this issue and to evaluate whether SERCAla expression could improve postischemic cardiac function and myocardial salvage, in vitro and in vivo myocardial I/R studies were performed on SERCAla transgenic (SERCA[1a.sup.+/+]) and nontransgenic (NTG) mice. Langendorff-per-fused hearts were subjected to 30 min of global ischemia followed by reperfusion. Baseline preischemic coronary flow and left ventricular developed pressure were significantly greater in [SERCA1a.sup.+/+] mice compared with NTG mice. Independent of reperfusion-induced oxidative stress, [SERCA1a.sup.+/+] hearts demonstrated greatly improved postischemic (45 min) contractile recovery with less persistent arrhythmias compared with NTG hearts. Morphometry showed betterpreserved myocardial structure with less infarction, and electron microscopy demonstrated better-preserved myofibrillar and mitochondrial ultrastructure in [SERCA1a.sup.+/+] hearts. Importantly, intraischemic [Ca.sup.2+] levels were significantly lower in [SERCA1a.sup.+/+] hearts. The cardioprotective effect of SERCA1a was also observed during in vivo regional I/R with reduced myocardial infarct size after 24 h of reperfusion. Thus [SERCA1a.sup.+/+] hearts were markedly protected against I/R injury, suggesting that expression of SERCA 1a isoform reduces postischemic [Ca.sup.2+] overload and thus provides potent myocardial protection. myocardial ischemia-reperfusion; sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; free radicals: postischemic function