David S. Miller: Scientist, Mentor, Friend-a tribute and thank you.

David S. Miller was Acting Scientific Director of the Division of Intramural Research at the National Institute of Environmental Health Sciences, National Institutes of Health, and Head of the Intracellular Regulation Group in the Laboratory of Toxicology and Pharmacology before he retired in 2016. David received his Ph.D. in biochemistry from the University of Maine in 1973. David was a Group Leader at the Michigan Cancer Foundation before joining the NIEHS in 1985. His research covered a wide range from renal excretory transport mechanisms to regulation of transporters at the blood-CSF and blood-brain barriers, from fish, amphibians and birds to mammals. David was an outstanding scientist with irresistible enthusiasm for science and an incredible ability to think outside the box while being an exceptional mentor and friend.

Gender-specific expression of ATP-binding cassette (Abc) transporters and cytoprotective genes in mouse choroid plexus.

The choroid plexus (CP) and blood-brain barrier (BBB) control the movement of several drugs and endogenous compounds between the brain and systemic circulation. The multidrug resistance associated protein (Mrp) efflux transporters form part of these barriers. Several Mrp transporters are positively regulated by the transcription factor nuclear factor erythroid-2-related factor (Nrf2) in liver. The Mrps, Nrf2 and Nrf2-dependent genes are cytoprotective and our aim was to examine basal gender differences in expression of Mrp transporters, Nrf2 and Nrf2-dependent genes (Nqo1 and Ho-1) in the brain-barriers. Previous studies have shown higher expression of Mrp1, Mrp2 and Mrp4 in female mouse liver and kidney. We hypothesized that similar renal/hepatic gender-specific patterns are present in the brain-barrier epithelia interfaces. qPCR and immunoblot analyses showed that Mrp4, Ho-1 and Nqo1 expression was higher in female CP. Mrp1, Mrp2 and Nrf2 expression in the CP had no gender pattern. Female Mrp1, Mrp2 and Mrp4 mouse brain expressions in remaining brain areas, excluding CP, were higher than male. Functional analysis of Mrp4 in CP revealed active accumulation of the Mrp4 model substrate fluo-cAMP. WT female CP had 10-fold higher accumulation in the vascular spaces than males and 60% higher than Mrp4-/- females. Probenecid blocked all transport. Methotrexate did as well except in Mrp4-/- females where it had no effect, suggesting compensatory induction of transport occurred in Mrp4-/-. Collectively, our findings indicate significant gender differences in expression of Mrp transporters and cytoprotective genes in the CP and BBB.

A plasma concentration of α-ketoglutarate influences the kinetic interaction of ligands with organic anion transporter 1.

The purpose of the present study was to determine whether a physiologic plasma concentration of α-ketoglutarate (αKG) influences the kinetic interaction of ligands with organic anion transporter 1 (OAT1). The effect of extracellular αKG on the kinetics of para-aminohippurate (PAH) and cidofovir transport was examined along with its effect on the potency of 10 drugs in five different classes (uricosuric, nonsteroidal anti-inflammatories, loop diuretics, angiotensin II receptor antagonists, and ß-lactam antibiotics) to inhibit OAT1 expressed in Chinese hamster ovary cells. Extracellular αKG competitively inhibited PAH and cidofovir transport with Ki values (∼5 µM) approximating its unbound plasma concentration (determined by equilibrium dialysis). When PAH was the substrate, extracellular αKG (5 µM) significantly increased IC50 values for some inhibitors (up to 4-fold), such as probenecid, but not for others (an inhibitor-dependent effect). For some inhibitors, a significant increase in IC50 value was observed when cidofovir was the substrate, but not PAH (a substrate-dependent effect). A significant increase in IC50 value was also observed for inhibition of PAH transport by probenecid in renal basolateral membrane vesicles (5.2-fold). The substrate- and inhibitor-dependent effect of extracellular αKG on ligand interactions with OAT1 highlights the complexity of the OAT1 ligand-binding surface. The effect of extracellular αKG on the potency of OAT1 inhibition should be considered when assessing drug-drug interaction potential at the transporter.

Active removal of inorganic phosphate from cerebrospinal fluid by the choroid plexus.

The P(i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF P(i) concentration. The large sheet-like fourth CP of the shark was mounted in Ussing chambers where unidirectional (33)P(i) fluxes revealed potent active transport from CSF to the blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 Ω·cm(2) and electrical potential difference of +0.9 ± 0.17 mV (CSF side positive). Active P(i) absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na(+)-free medium, and increasing the K(+) concentration from 5 to 100 mM. Li(+) stimulated transport twofold compared with Na(+)-free medium. Phosphonoformic acid (1 mM) had no effect on active P(i) transport. RT-PCR revealed both P(i) transporter (PiT)1 and PiT2 (SLC20 family) gene expression, but no Na(+)-P(i) cotransporter II (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot analysis and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both the shark and rat. PiT1 appeared to be localized primarily to vascular endothelial cells. Taken together, these data indicate that the CP actively removes P(i) from CSF. This process has transport properties consistent with a PiT2, Na(+)-dependent transporter that is located in the apical region of the CP epithelium.

Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase.

Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-ß-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion.

Piscine PTHrP regulation of calcium and phosphate transport in winter flounder renal proximal tubule primary cultures.

Multiple factors control calcium (Ca(2+)) and inorganic phosphate (P(i)) transport in the fish nephron, and the recently discovered members of the piscine parathyroid hormone-like protein family are likely participants in such regulatory mechanisms. The effects of an NH(2)-terminal peptide (amino acids 1-34) of Takifugu rubripes parathyroid hormone-related protein, (1-34)PTHrP, on Ca(2+) and P(i) transport were investigated in winter flounder (Pseudopleuronectes americanus) proximal tubule cells in primary culture (fPTCs). RT-PCR performed on RNA extracted from fPTCs and from intact kidney tissue indicated that expression of PTHrP and types 1 and 3 PTH/PTHrP receptors occurred both in vivo and in vitro and that circulating levels of PTHrP measured by specific radioimmunoassay averaged 2.5 +/- 0.13 ng/ml. fPTC monolayers were mounted in Ussing chambers, and under neutral electrochemical conditions, addition of 10 nM (1-34)PTHrP to the basolateral side induced a slight increase in Ca(2+) transport rate from luminal to peritubular side, significantly stimulating net Ca(2+) reabsorption. (1-34)PTHrP also significantly increased the P(i) secretory flux, and slightly reduced P(i) reabsorption, evoking a significant increase in P(i) net secretion. This stimulatory effect was partially inhibited by bisindolylmaleimide, an inhibitor of protein kinase C. Incubation of ex vivo flounder renal tubules with (1-34)PTHrP resulted in apparent reduction of Na(+)-P(i) cotransporter type II (NaP(i)-II) protein in tubule membranes. PTHrP seems therefore to participate in the modulation of Ca(2+) and P(i) homeostasis by fish kidney.

Genome comparisons reveal a dominant mechanism of chromosome number reduction in grasses and accelerated genome evolution in Triticeae.

Single-nucleotide polymorphism was used in the construction of an expressed sequence tag map of Aegilops tauschii, the diploid source of the wheat D genome. Comparisons of the map with the rice and sorghum genome sequences revealed 50 inversions and translocations; 2, 8, and 40 were assigned respectively to the rice, sorghum, and Ae. tauschii lineages, showing greatly accelerated genome evolution in the large Triticeae genomes. The reduction of the basic chromosome number from 12 to 7 in the Triticeae has taken place by a process during which an entire chromosome is inserted by its telomeres into a break in the centromeric region of another chromosome. The original centromere-telomere polarity of the chromosome arms is maintained in the new chromosome. An intrachromosomal telomere-telomere fusion resulting in a pericentric translocation of a chromosome segment or an entire arm accompanied or preceded the chromosome insertion in some instances. Insertional dysploidy has been recorded in three grass subfamilies and appears to be the dominant mechanism of basic chromosome number reduction in grasses. A total of 64% and 66% of Ae. tauschii genes were syntenic with sorghum and rice genes, respectively. Synteny was reduced in the vicinity of the termini of modern Ae. tauschii chromosomes but not in the vicinity of the ancient termini embedded in the Ae. tauschii chromosomes, suggesting that the dependence of synteny erosion on gene location along the centromere-telomere axis either evolved recently in the Triticeae phylogenetic lineage or its evolution was recently accelerated.

Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4.

Birds are uricotelic and, like humans, maintain high plasma urate concentrations (approximately 300 microM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.

Temporal patterns of foliar ozone symptoms on tall milkweed (Asclepias exaltata L.) in Great Smoky Mountains National Park.

Incidence and severity of ozone-induced foliar symptoms on tall milkweed (Asclepias exaltata L.) along selected trails in Great Smoky Mountains National Park (GRSM) were determined by two surveys/season conducted from 1992 through 1996. Overall incidence was 73%, and was 84%, 44%, 90%, 58%, and 82% for 1992-1996, respectively for the same clusters. Average incidence was 61% and 84% for the 1st and 2nd surveys, respectively. Seasonal comparisons showed two distinct injury groupings regarding incidence and severity of injury: 1992, 1994 and 1996 (high injury); 1993 and 1995 (low injury). No discernible patterns were observed between symptomatic and asymptomatic plants regarding height, herbivory or flowering. Regression analyses indicated no differentiation in foliar symptoms regarding topographic position, aspect, slope or elevation over the 5-year study period. Our findings indicate other micro-site or genetic factors may control ozone sensitivity of tall milkweed in GRSM.

Trimethylamine oxide suppresses stress-induced alteration of organic anion transport in choroid plexus.

The effect of physicochemical stress on organic anion transport across the vertebrate blood-cerebrospinal fluid (CSF) barrier in the presence and absence of an endogenous cytoprotectant, trimethylamine oxide (TMAO), was investigated in isolated IVth choroid plexus (CP) of spiny dogfish shark (Squalus acanthias), an animal with naturally high levels of TMAO ( approximately 70 mmol l(-1)). Active transepithelial absorption of the organic anion, 2,4-dichlorophenoxyacetic acid (2,4-D), by IVth CP mounted in Ussing chambers was measured after in vitro stress, and a marker for the cellular stress response, inducible heat shock protein 70 (Hsp70), was assayed by immunoblot analysis. Transient heat stress (a shift from the normal 13.5 degrees C to 23.5 degrees C for 1 h) decreased 2,4-D transport by approximately 66%; however, the same stress minus TMAO (isosmotic replacement with urea) had no effect on transport rate. In the absence of TMAO, stress-induced Hsp70 accumulation was more than double that seen in the presence of TMAO. Likewise, exposure to 50 micromol l(-1) Zn for 6 h induced a twofold greater Hsp70 accumulation in the absence of TMAO than in its presence, and the higher Hsp70 level was associated with a higher 2,4-D transport rate. Heat stress and 50 micromol l(-1) Zn also induced more pronounced increases in Hsp70 mRNA in the absence of TMAO. Thus, the cellular stress response can significantly alter CP organic anion transport capacity, and an endogenous osmolyte can suppress that response.

The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two factors that share amino acid sequence homology and act via a common receptor. In tetrapods, PTH is the main endocrine factor acting in bone and kidney to regulate calcium and phosphate. PTHrP is an essential paracrine developmental factor present in many tissues and is involved in the regulation of ossification, mammary gland development, muscle relaxation, and other functions. Fish apparently lack an equivalent of the parathyroid gland and were long thought to be devoid of PTH. Only in recent years has the existence of PTH-like peptides and their receptors in fish been firmly established. Two forms of PTH, two of PTHrP, and a protein with intermediate characteristics designated PTH-L are encoded by separate genes in teleost fish. Three receptors encoded by separate genes in fish mediate PTH/PTHrP actions, whereas only two receptors have so far been found in terrestrial vertebrates. PTHrP has been more intensively studied than PTH, from lampreys to advanced teleosts. It is expressed in many tissues and is present in high concentration in fish blood. Administration of this peptide alters calcium metabolism and has marked effects on associated gene expression and enzyme activity in vivo and in vitro. This review provides a comprehensive overview of the physiological roles, distribution, and molecular relationships of the piscine PTH-like peptides.

Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture.

Birds are uricotelic, and because they excrete urate by renal tubular secretion, they provide a convenient model for examination of this process. Primary monolayer cultures of the isolated renal proximal tubule epithelium from the domestic chicken, Gallus gallus L., were mounted in Ussing chambers where several substrates/inhibitors of renal organic anion transporters were tested for the sidedness and specificity of their effects on transepithelial urate transport. Transepithelial electrical resistance, electrical potential and sodium-dependent glucose current were monitored to detect nonspecific effects. Under control short-circuited conditions the ratio of unidirectional fluxes of [(14)C]urate was found to be 3:1. Active net secretion was specifically inhibited by 1 mmol l(-1) probenecid and 10 mmol l(-1) para-aminohippuric acid (PAH). Bromocresol Green, cimetidine, nocodozole, cytochalasin D and ouabain also inhibited secretion but were toxic. Interstitial-side lithium (5 mmol l(-1)) and glutarate (1 mmol l(-1)) specifically blocked transport, but 10-100 micromol l(-1) glutarate had no effect. Interstitial estrone sulfate (ES) stimulated urate secretion at 10 micromol l(-1) but was inhibitory at 500 micromol l(-1). Active PAH secretion (5:1 flux ratio) was inhibited 34% by 330 micromol l(-1) urate. ES (500 micromol l(-1)) blocked the remainder. From the lumen side, glucose-free, Cl(-)-free and high K(+) (30 mmol l(-1)) solutions, or an alkaline pH of 7.7 had no effect on urate transport and neither did several compounds known to be uricosuric. Lumen-side methotrexate (500 micromol l(-1)) and MK571 (20 micromol l(-1)) strongly inhibited urate secretion. MK571 had no effect from the interstitial side. RT-PCR revealed mRNA for OAT1-, OAT3-, MRP2- and MRP4-like organic anion transporters in chicken proximal epithelium.

Stimulation of renal sulfate secretion by metabolic acidosis requires Na+/H+ exchange induction and carbonic anhydrase.

The acute effect of metabolic acidosis on SO(4)(2-) secretion by the marine teleost renal proximal tubule was examined. Metabolic acidosis was mimicked in primary cultures of winter flounder renal proximal tubule epithelium (fPTCs) mounted in Ussing chambers by reducing interstitial pH to 7.1 (normally 7.7). fPTCs with metabolic acidosis secreted SO(4)(2-) at a net rate that was 40% higher than in paired isohydric controls (pH 7.7 on interstitium). The stimulation was completely blocked by the carbonic anhydrase inhibitor methazolamide (100 microM). Although Na(+)/H(+) exchange (NHE) isoforms 1, 2, and 3 were identified in fPTCs by immunoblotting, administering EIPA (20 microM) to the interstitial and luminal bath solutions had no effect on net SO(4)(2-) secretion by fPTCs with a normal interstitial pH of 7.7. However, EIPA (20 microM) blocked most of the stimulation caused by acidosis when applied to the lumen but not interstitium, demonstrating that induction of brush-border NHE activity is important. In the intact flounder, serum pH dropped 0.4 pH units (pH 7.7 to 7.3, at 2-3 h) when environmental pH was lowered from 7.8 to approximately 4.3. Whereas serum [SO(4)(2-)] was not altered by acidosis, renal tubular SO(4)(2-) secretion rate was elevated 200%. Thus metabolic acidosis strongly stimulates renal sulfate excretion most likely by a direct effect on active renal proximal tubule SO(4)(2-) secretion. This stimulation appears to be dependent on inducible brush-border NHE activity.

Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion.

The renal proximal tubule of vertebrates performs an essential role in controlling plasma SO(4)(2-) concentration ([SO(4)(2-)]). Although net tubular SO(4)(2-) reabsorption is the predominate control process in terrestrial vertebrates, a facilitated secretory flux is also present. In contrast, marine teleosts obtain excess SO(4)(2-) from drinking, and increased plasma [SO(4)(2-)] is prevented predominately through net tubular secretion. Tubular SO(4)(2-) secretion is accomplished by at least two electroneutral anion exchange processes in series. Movement of SO(4)(2-) into the cell across the basolateral membrane is pH dependent, suggesting SO(4)(2-)/OH(-) exchange. Luminal HCO(3)(-) and Cl(-) can facilitate SO(4)(2-) movement out of the cell across the brush-border membrane. The molecular identities of the anion exchangers are unknown but are probably homologues of SO(4)(2-) transporters in the mammalian SLC26 gene family. In all species tested, glucocorticoids increase renal SO(4)(2-) excretion. Whereas glucocorticoids downregulate SO(4)(2-) reabsorptive mechanisms in terrestrial vertebrates, they may also stimulate a mediated secretory flux. In the marine teleost, cortisol increases the level of SO(4)(2-)/HCO(3)(-) exchange at the brush-border membrane, tubular carbonic anhydrase (CA) activity, CAII protein, and a proportion of tubular SO(4)(2-) secretion that is CA dependent. CA activity is required for about one-half of this net SO(4)(2-) secretion but is also required for about one-half of the net reabsorption in bird proximal epithelium. A CA-SO(4)(2-)/anion exchanger metabolon arrangement is proposed that may speed both the secretory and reabsorptive processes.

Cortisol alters carbonic anhydrase-mediated renal sulfate secretion.

Active transepithelial sulfate secretion rate by winter flounder renal proximal tubule epithelium in primary culture (fPTC) is dependent on intracellular carbonic anhydrase (CA) and enhanced by cortisol. To further evaluate this relationship, a partial cDNA clone (327 bp) of carbonic anhydrase II (CAII) with high sequence similarity to CAII from numerous species including fish, chicken, and human was obtained from fPTCs. The majority of CA activity and CAII protein was present in the cytosol of fPTCs; however, significant amounts of both (in addition to SDS-resistant CA activity, i.e., CAIV-like isoform) were present in concentrated plasma membranes. CAII from concentrated membranes migrated differently than purified CAII on nondenaturing PAGE gels, suggesting that CAII associates with another membrane component. Treatment of fPTCs with the cell-soluble CA inhibitor methazolamide (100 microM) caused a 58% reduction in active transepithelial SO4(2-) secretion. fPTCs that were previously cultured under high-cortisol concentrations, when subjected to 5 days of low physiological levels of cortisol, had decreased CA activity (28%), CAII protein abundance (65%), and net active SO4(2-) secretion (28%), with no effect on epithelial differentiation. Methazolamide and low-cortisol treatment in combination inhibited net active SO4(2-) secretion 56%, which was not different than the effect of methazolamide treatment alone. These data indicate that cortisol directly increases renal CA activity, CAII protein abundance, and CA-dependent SO4(2-) secretion in the marine teleost renal proximal tubule.

Ozone injury on cutleaf coneflower (Rudbeckia laciniata) and crown-beard (Verbesina occidentalis) in Great Smoky Mountains National Park.

Incidence and severity of visible foliar ozone injury on cutleaf coneflower (Rudbeckia laciniata L.) and crown-beard (Verbesina occidentalis Walt.) were determined along selected trails at three locations in Great Smoky Mountains National Park during the summers of 2000 and 2001: Clingmans Dome, Cherokee Orchard Road and Purchase Knob. Cutleaf coneflower exhibited a greater amount of foliar injury than crown-beard each year of the 2-year study. Incidence and severity of injury was significantly greater for cutleaf coneflower growing near the edge of the Clingmans Dome trail than in the interior of the stand. Injury was greater at Clingmans Dome than Purchase Knob (70% vs. 40% ozone-injured plants, respectively), coincident with greater ozone exposures. In contrast to Clingmans Dome, there were no differences in injury between plants growing near- and off-trail at Purchase Knob. Differences in sensitivity to ozone were not observed for crown-beard growing near the edge compared with the interior of the stand adjacent to the Cherokee Orchard Road Loop. Ozone injury was greatest on the lower leaves for both species sampled with over 95% of the injured leaves occurring on the lower 50% of the plant. This is the first report of foliar ozone injury on these plant species in situ, in the Park, illustrating the great variability in symptom expression with time, and within and between populations.

Active sulfate secretion by the intestine of winter flounder is through exchange for luminal chloride.

SO4(2-) transport by winter flounder intestine in Ussing chambers was characterized. With 50 mM SO4(2-) (physiological level) bathing the lumen, net absorption (lumen to blood) dominated. Under short-circuited conditions, 1 mM SO4(2-) on both sides, net active SO4(2-) secretion occurred (8.55 +/- 0.96 nmol. cm(-2). h(-1)). NaCN (10 mM), ouabain (10(-4) M), and luminal DIDS (0.2 mM) inhibited net secretion. Removal of luminal Cl- and HCO3- together (Cl--HCO3-) or Cl- alone blocked net secretion, whereas removal of luminal HCO3- alone increased net secretion. SO4(2-) uptake into foregut brush-border membrane vesicles was stimulated by a trans-Cl- gradient (in > out) and unaffected by a trans-HCO3- gradient (in > out). Short-circuiting with K+ (in = out) and valinomycin had no effect on Cl--stimulated SO4(2-) uptake, suggesting electroneutral exchange. Satiety (i.e., full stomach) stimulated the unidirectional absorptive flux, eliminating net secretion. It was concluded that the intestine is a site of SO4(2-) absorption in marine teleosts and that active SO4(2-) secretion is in exchange for luminal Cl-.

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture.

The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2 O 3 2- (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Si reabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption approximately 45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Si reabsorption up to approximately 50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Si reabsorption approximately 45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.

Transepithelial organic anion transport by shark choroid plexus.

Spiny dogfish shark (Squalus acanthias) lateral and IV choroid plexuses (CPs) are ultrastructurally similar to the corresponding tissues of rat. However, shark IV CP is proportionally larger and easily accessible. Moreover, this epithelial sheet can be halved and studied in Ussing flux chambers. We have used confocal fluorescence microscopy and radiotracer techniques to characterize transepithelial transport of the organic anions (OAs) fluorescein (FL) and 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, by shark CP. Lateral and IV CP accumulated 1 microM FL, with highest levels in the underlying extracellular spaces, intermediate levels in epithelial cells, and lowest levels in the medium. 2,4-D and probenecid inhibited FL accumulation in cells and extracellular spaces, suggesting that these substrates compete for common carriers. Unidirectional absorptive [cerebrospinal fluid (CSF)-to-blood] and secretory (blood-to-CSF) fluxes of 10 microM [(14)C]2,4-D were measured under short-circuited conditions in IV CP mounted in Ussing chambers. 2,4-D underwent net absorption, with an average flux ratio of 7. Probenecid, 2,4,5-trichlorophenoxyacetic acid, and 5-hydroxyindolacetic acid reduced net absorption, reversibly inhibiting unidirectional absorption, with no effect on secretion. Ouabain irreversibly reduced net 2,4-D absorption and cellular and extracellular accumulation of FL, suggesting energetic coupling of OA absorption to Na(+) transport. Collectively, these data indicate that shark CP actively removes OAs from CSF by a process that is specific and active.