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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

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

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

Micropatterning of two different cell types based on surface modification allows spatial control over two distinct cell subpopulations. This study considers a micropatterned coculture system, which has release and absorption parts alternately arranged at the base, and each part has a single cell type. A micropattern unit was defined and within each unit, there are one release part and one absorption part. The cells in the absorption parts consume species, which are secreted by the cells in the release parts. The species concentrations at the micropatterned cell base were computed from a three-dimensional numerical flow model incorporating mass transport. Different combined parameters were developed for the release and absorption parts to make the data collapse in each part. Combination of the collapse data in the release and absorption parts can be used to predict the concentration distribution through the whole channel. The correlated results were applied to predict the critical length ratio of the release and absorption parts for an actual micropatterned system (Bhatia et al., 1999, 'Effect of Cell-Cell Interactions in Preservation of Cellular Phenotype: Co-Cultivation of Hepatocytes and Nonparenchymal Cell,' FASEB J. 13, pp. 1883-1900) to avoid species insufficiency based on basic fibroblast growth factor (bFGF). The mass transfer effectiveness was found to be higher with more numbers of micropattern units. The optimal condition for micropatterned coculture bioreactors is achieved by having the product of the length ratio and the reaction ratio equal to 1. This condition was used to optimize the mass transfer in the micropatterned system (Bhatia et al., 1999, 'Effect of Cell-Cell Interactions in Preservation of Cellular henotype: Co-Cultivation of Hepatocytes and Nonparenchymal Cell,' FASEB J. 13, pp. 1883-1900) based on bFGF. Keywords: microchannel bioreactor, micropatterned coculture, species concentration

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

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I-mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor-induced Golgi-to-ER shuttling of SAC 1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.

The stem cell niche, formed by surrounding stromal cells, provides extrinsic signals that maintain stem cell self-renewal. However, it remains unclear how these extrinsic signals are regulated. In the Drosophila female germline stem cell (GSC) niche, Decapentaplegic (DPP) is an important niche factor for GSC self-renewal. The exact source of the DPP and how its transcription is regulated in this niche remain unclear. We show that dpp is expressed in somatic cells of the niche including the cap cells, a subtype of niche cells. Furthermore, our data show that the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway positively regulates dpp expression in the cap cells, suggesting that JAK/STAT activity is required in somatic niche cells to prevent precocious GSC differentiation. Our data suggest that the JAK/STAT pathway functions downstream/independently of cap cell formation induced by Notch signaling. JAK/STAT signaling may also regulate dpp expression in the male GSC niche, suggesting a common origin of female and male GSC niches.

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptormediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify [alpha]1-antitrypsin ([alpha]1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of [alpha]1 -AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of [alpha]1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE-tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.

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

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 opossum kidney (OK) cells as well as in kidney proximal tubules, [P.sub.i] depletion increases apical (A) and basolateral (B) [Na.sup.+]-dependent [P.sub.i] cell influxes. In OK cells' monolayers in contrast to proximal tubules, there is no increase in transepithelial [P.sub.i] transport. This limitation may be due to altered cell-matrix interactions. A and B cell [sup.32][P.sub.i] uptakes and transepithelial [sup.32] [P.sub.i] and [[sup.14]C]mannitol fluxes were measured in OK cells grown on uncoated or on Matrigel-coated filter inserts. Cells were exposed overnight to solution of either low (0.25 mM) or high (2.5 mM) [P.sub.i]. When grown on Matrigel, immunofluorescence of apical NaPi4 (an isoform of the sodium-phosphate cotransporter) transporters increased and A and B [sup.32][P.sub.i] uptakes into [P.sub.i] depleted cells were five and threefold higher than in [P.sub.i] replete cells (P < 0.001). [P.sub.i] deprivation resulted in larger increase in A to B (4.6x, P < 0.001) than in B to A (3.5x, P < 0.001) [P.sub.i] flux and net [P.sub.i] transport from A to B increased 10-fold (P < 0.001). With [P.sub.i] depletion increases in B to A (3.4X) and A to B (3.3x) paracellular [[sup.14]C]mannitol fluxes were similar, and its net flux was opposite to that of [P.sub.i]. In cells grown on uncoated filters, transepithelial and paracellular unidirectional and net [P.sub.i] fluxes decreased or did not change with [P.sub.i] depletion, despite twofold increases in apical and basolateral [P.sub.i] cell influxes. In summary, Matrigel-OK cell interactions, particularly in [P.sub.i]-depleted cells, led to enhanced expression of apical NaPi4 transporters resulting in higher [P.sub.i] transport rates across cell boundaries; apical [P.sub.i] readily entered the transcellular transport pool and paracellular fluxes were smaller fractions of transepithelial [P.sub.i] fluxes. These Matrigel-induced changes led to an increase in net transepithelial apical to basolateral [P.sub.i] transport. renal; mineral; transport; paracellular; transcellular; cell compartments