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In angiosperms, the endosperm provides nutrients for embryogenesis and seed germination and is the primary tissue where gene imprinting occurs. To identify the imprintome of early developing maize (Zea mays) endosperm, we performed high-throughput transcriptome sequencing of whole kernels at 0, 3, and 5 d after pollination (DAP) and endosperms at 7, 10, and 15 DAP, using B73 by Mo17 reciprocal crosses. We observed gradually increased expression of paternal transcripts in 3- and 5-DAP kernels. In 7-DAP endosperm, the majority of the genes tested reached a 2:1 maternal versus paternal ratio, suggesting that paternal genes are nearly fully activated by 7 DAP. A total of 116, 234, and 63 genes exhibiting parent-specific expression were identified at 7, 10, and 15 DAP, respectively. The largest proportion of paternally expressed genes was at 7 DAP, mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP and were primarily attributed to sharply increased expression levels compared with the other stages. Gene ontology enrichment analysis of the imprinted genes suggested that 10-DAP endosperm-specific MEGs are involved in nutrient uptake and allocation and the auxin signaling pathway, coincident with the onset of starch and storage protein accumulation.

Cultured Drosophila cell lines have been developed into a powerful tool for studying a wide variety of cellular processes. Their ability to be easily and cheaply cultured as well as their susceptibility to protein knockdown via double-stranded RNA-mediated interference (RNAi) has made them the model system of choice for many researchers in the fields of cell biology and functional genomics. Here we describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton.

While the chemotherapeutic effect of curcumin, one of three major curcuminoids derived from turmeric, has been reported, largely unexplored are the effects of complex turmeric extracts more analogous to traditional medicinal preparations, as well as the relative importance of the three curcuminoids and their metabolites as anti-cancer agents. These studies document the pharmacodynamic effects of chemically-complex turmeric extracts relative to curcuminoids on human breast cancer cell growth and tumor cell secretion of parathyroid hormone-related protein (PTHrP), an important driver of cancer bone metastasis. Finally, relative effects of structurallyrelated metabolites of curcuminoids were assessed on the same endpoints. We report that 3 curcuminoid-containing turmeric extracts differing with respect to the inclusion of additional naturally occurring chemicals (essential oils and/or polar compounds) were equipotent in inhibiting human breast cancer MDA-MB-231 cell growth (IC50=10-16µg/mL) and secretion of osteolytic PTHrP (IC50=2-3µg/mL) when concentrations were normalized to curcuminoid content. Moreover, these effects were curcuminoid-specific, as botanically-related gingerol containing extracts had no effect. While curcumin and bis-demethoxycurcumin were equipotent to each other and to the naturally occurring curcuminoid mixture (IC50=58µM), demethoxycurcumin did not have any effect on cell growth. However, each of the individual curcuminoids inhibited PTHrP secretion (IC50=22-31µM) to the same degree as the curcuminoid mixture (IC50=16µM). Degradative curcuminoid metabolites (vanillin and ferulic acid) did not inhibit cell growth or PTHrP, while reduced metabolites (tetrahydrocurcuminoids) had inhibitory effects on cell growth and PTHrP secretion but only at concentrations ≥10-fold higher than the curcuminoids. These studies emphasize the structural and biological importance of curcuminoids in the anti-breast cancer effects of turmeric and contradict recent assertions that certain of the curcuminoid metabolites studied here mediate these anti-cancer effects.

We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion.

Male Sprague Dawley rats subjected to MCAO/R were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema, and neurological function were assessed at 24 hours. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed.

Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC.

Turmeric-derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.

Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated. In vitro, transforming growth factor (TGF)-β-stimulated PTHrP secretion was inhibited by curcuminoids (IC50 = 24 μM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition. Effects on TGF-β signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-β signaling. In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days. Osteolytic bone lesion area was reduced up to 51% (p < 0.01). Consistent with specific effects on bone osteolysis, osteoclast number at the bone-tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered. In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment. These data suggest that curcuminoids prevent TGF-β induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.

Various age-related diseases increase in incidence during perimenopause. However, our understanding of the effects of aging compared with hormonal changes of perimenopause in mediating these disease risks is incomplete, in part due to the lack of an experimental perimenopause model. We therefore aimed to determine whether manipulation of the transition to ovarian failure in rats via the use of 4-vinylcyclohexene diepoxide (VCD) could be used to model and accelerate hormonal changes characteristic of perimenopause. We examined long-term (11 to 20 mo), dose-dependent effects of VCD on reproductive function in 1- and 3-mo-old female Sprague-Dawley rats. Twenty-five daily doses of VCD (80 or 160 mg/kg daily compared with vehicle alone) depleted ovarian follicles in a dose-dependent fashion in rats of both ages, accelerated the onset of acyclicity, and caused dose-dependent increases in follicle-stimulating hormone that exceeded those naturally occurring with age in control rats but left serum levels of 17β-estradiol unchanged, with continued ovarian production of androstenedione. High-dose VCD caused considerable nonovarian toxicities in 3-mo-old Sprague-Dawley rats, making this an unsuitable model. In contrast, 1-mo-old rats had more robust dose-dependent increases in follicle-stimulating hormone without evidence of systemic toxicity in response to either VCD dose. Because perimenopause is characterized by an increase in follicle-stimulating hormone with continued secretion of ovarian steroids, VCD acceleration of an analogous hormonal milieu in 1-mo-old Sprague-Dawley rats may be useful for probing the hormonal effects of perimenopause on age-related disease risk.

In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization. Several female signaling components essential for pollen tube reception have been identified; however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes and degenerate upon pollen tube arrival. myb triple-mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate-active enzymes, and small peptides as candidate molecular mediators of pollen tube-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.

C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, delta isoform (PP1c delta). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PP1c delta was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PP1c, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PP1c delta, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3-5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.

The relative expression of alpha(1)- and alpha(2)-Na(+)/K(+)-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the alpha(1)-isoform serves a "housekeeping" role, whereas the alpha(2)-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the alpha(1)- or the alpha(2)-Na(+)/K(+)-ATPase isoform was ablated were utilized. Both of these knockouts, alpha(1)(-/-) and alpha(2)(-/-), are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in alpha(2)(-/-)-aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; alpha(2)(+/-)-aortas had intermediate values. In contrast, neonatal alpha(1)(+/-)-aorta was similar to WT. Western blot analysis indicated a population of 70% alpha(1)- and 30% alpha(2)-isoforms in the WT. Thus in terms of the total Na(+)/K(+)-ATPase protein, the alpha(2)(-/-)-aorta (at 70%) would be similar to the alpha(1)(+/-)-aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual alpha-isoforms of the Na(+)/K(+)-ATPase differ functionally and that the alpha(2)-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the alpha(2)-isoform is distributed differently than the alpha(1)-isoform. Importantly, these isoforms do not localize to the same regions.

Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ~4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H(+)-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.

Investigation into the causes and consequences of aberrant pH regulation is entirely dependent on the ability to measure this parameter with specificity and sensitivity. Since their beginnings in the 1950s, techniques for measuring cell and tissue pH have undergone a number of significant advances. Following each of these advances, new investigators have been brought into the field, and new discoveries have been made. From microelectrode and dye distribution studies, measurement of pH underwent a revolution with the advent of pH-sensitive dyes that could be loaded into the cytosol. A further significant advance came from the measurement of cell and tissue pH in whole organisms by magnetic resonance spectroscopy (MRS). Frontiers in pH measurement exist in the development of pH-sensitive proteins and pH-sensitive MR contrast agents. These are predicted to bring even more people into this fascinating field, and generate more important discoveries.

Epidermal growth factor (EGF) is involved in acute regulation of Na(+)/H(+) exchangers (NHEs), but the effect of chronic EGF administration on NHE gene expression is unknown. The present studies showed that EGF treatment increased NHE2-mediated intestinal brush-border membrane vesicle Na(+) absorption and NHE2 mRNA abundance by nearly twofold in 19-day-old rats. However, no changes were observed in renal NHE2 mRNA or intestinal and renal NHE3 mRNA abundance. To understand the mechanism of this regulation, we developed the rat intestinal epithelial (RIE) cell as an in vitro model to study the effect of EGF on NHE2 gene expression. EGF increased functional NHE2 activity and mRNA abundance in cultured RIE cells, and this stimulation could be blocked by actinomycin D (a transcriptional inhibitor). Additionally, NHE2 promoter reporter gene assays in transiently transfected RIE cells showed an almost twofold increase in promoter activity after EGF treatment. We conclude that rat NHE2 activity can be stimulated by chronic EGF treatment and that this response is at least partially mediated by gene transcription.

The computed tomography imaging spectrometer (CTIS) is a non-scanning instrument capable of simultaneously acquiring full spectral information (450-750 nm) from every position element within its field of view (75 microm x 75 microm). The current spatial and spectral sampling intervals of the spectrometer are 1.0 microm and 10 nm, respectively. This level of resolution is adequate to resolve signal responses from multiple fluorescence probes located within individual cells or different locations within the same cell. Spectral imaging results are presented from the CTIS combined with a commercial inverted fluorescence microscope. Results demonstrate the capability of the CTIS to monitor the spatiotemporal evolution of pH in rat insulinoma cells loaded with SNARF-1. The ability to analyze full spectral information for two-dimensional (x, y) images allows precise evaluation of heterogeneous physiological responses within cell populations. Due to low signal levels, integration times up to 2 s were required. However, reasonable modifications to the instrument design will provide higher system transmission efficiency with increased temporal and spatial resolution. Specifically, a custom optical design including the use of a larger format detector array is under development for a second-generation system.

The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic beta-cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK+ neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K+ channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and beta-cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK+ neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism.

The opioid peptide dynorphin A is known to elicit a number of pathological effects that may result from neuronal excitotoxicity. An up-regulation of this peptide has also been causally related to the dysesthesia associated with inflammation and nerve injury. These effects of dynorphin A are not mediated through opioid receptor activation but can be effectively blocked by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists, thus implicating the excitatory amino acid system as a mediator of the actions of dynorphin A and/or its fragments. A direct interaction between dynorphin A and the NMDA receptors has been well established; however the physiological relevance of this interaction remains equivocal. This study examined whether dynorphin A elicits a neuronal excitatory effect that may underlie its activation of the NMDA receptors. Calcium imaging of individual cultured cortical neurons showed that the nonopioid peptide dynorphin A(2-17) induced a time- and dose-dependent increase in intracellular calcium. This excitatory effect of dynorphin A(2-17) was insensitive to (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) pretreatment in NMDA-responsive cells. Thus dynorphin A stimulates neuronal cells via a nonopioid, non-NMDA mechanism. This excitatory action of dynorphin A could modulate NMDA receptor activity in vivo by enhancing excitatory neurotransmitter release or by potentiating NMDA receptor function in a calcium-dependent manner. Further characterization of this novel site of action of dynorphin A may provide new insight into the underlying mechanisms of dynorphin excitotoxicity and its pathological role in neuropathy.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

To examine the role of protein kinase C (PKC) in organic anion (OA) secretion, we used epifluorescence microscopy to study steady-state transepithelial secretion of 1 microM fluorescein (FL) by isolated perfused S2 segments of rabbit renal proximal tubules. Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bathing medium decreased steady-state secretion of FL by approximately 30% after 25 min. This inhibition was irreversible and, indeed, increased to approximately 40% at 25 min following removal of PMA [10 microM 1,2-dioctanoyl-sn-glycerol (DOG) produced a comparable inhibition]. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath for a 20-min preexposure followed by the addition of PMA. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion rate was not under influence of PKC. Addition of 1 microM of either the peptide hormone bradykinin (BK) or the alpha(1)-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secretion by approximately 22 and approximately 27%, respectively. However, the inhibition was completely reversible after removal of BK or PE. Pretreatment of tubules with 100 nM BIM eliminated the inhibition of FL secretion produced by exposure to PE. We conclude that PKC negatively regulates the net secretion of OAs in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating OA secretion via PKC activation.

The main purpose of this study was to determine the interstitial oxygen tension at which aerobic metabolism becomes limited (critical PO(2)) in vivo in resting skeletal muscle. Using an intravital microscope system, we determined the interstitial oxygen tension at 20-micrometer-diameter tissue sites in rat spinotrapezius muscle from the phosphorescence lifetime decay of a metalloporphyrin probe during a 1-min stoppage of muscle blood flow. In paired experiments NADH fluorescence was measured at the same sites during flow stoppage. NADH fluorescence rose significantly above control when interstitial PO(2) fell to 2.9 +/- 0.5 mmHg (n = 13) and was not significantly different (2.4 +/- 0.5 mmHg) when the two variables were first averaged for all sites and then compared. Similar values were obtained using the abrupt change in rate of PO(2) decline as the criterion for critical PO(2). With a similar protocol, we determined that NADH rose significantly at a tissue site centered 30 micrometer from a collecting venule when intravascular PO(2) fell to 7.2 +/- 1.5 mmHg. The values for critical interstitial and critical intravascular PO(2) are well below those reported during free blood flow in this and in other muscle preparations, suggesting that oxygen delivery is regulated at levels well above the minimum required for oxidative metabolism. The extracellular critical PO(2) found in this study is slightly greater than previously found in vitro, possibly due to differing local conditions rather than a difference in metabolic set point for the mitochondria.

To determine the quantitative roles of the basolateral and luminal Na(+)-dicarboxylate (Na-DC) cotransporters in establishing and maintaining the alpha-ketoglutarate (alphaKG) gradient required for renal tubular secretion of organic anions, we measured net steady-state transepithelial secretion of fluorescein (FL) in real time in isolated, perfused S2 segments of rabbit renal proximal tubules. Net "basal" FL secretion in the absence of exogenous alphaKG had a K(t) of approximately 4 microM and a maximal transepithelial secretion rate (J(max)) of approximately 380 fmol. min(-1). mm(-1) (where K(t) is the FL concentration that produces one-half the J(max)). It could be almost completely inhibited by basolateral p-aminohippurate (PAH). Selective inhibition of the basolateral Na-DC cotransporter indicated that recycling via this transporter of alphaKG that had been exchanged for FL supports approximately 25% of the "basal" FL secretion. Physiological alphaKG concentrations of 10 microM in the bath or 50 microM in the perfusate stimulated net secretion of FL by approximately 30 or approximately 20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports approximately 42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of alphaKG were additive, indicating that the combined function of the luminal and basolateral Na-DC cotransporters can support approximately 50% of the net FL secretion. This apparently occurs by their establishing and maintaining approximately 50% of the outwardly directed alphaKG gradient that is responsible for driving basolateral FL/alphaKG exchange. The remaining approximately 50% would be maintained by metabolic production of alphaKG in the cells.

No abstract given.