Assessment of tissue-level kidney functions with primary cultures.

Primary cultures of renal proximal tubule have become important tools for examination of the mechanisms and control of transepithelial transport processes. The utility of the culture preparations for study of integrated tissue functions depends upon their accurate expression of in vivo transport processes. Maintenance of differentiation in culture is enhanced by contractible collagen substratum. Epithelial monolayer primary cultures of flounder and chicken proximal tubule, prepared by enzymatic and mechanical maceration with differential centrifugation, exhibit functional properties at the tissue level that generally resemble known properties of the freshly isolated or in vivo proximal tubule. Transepithelial electrical resistances and potential differences are very similar or identical to those of intact tubules. Na+-dependent glucose transport, a hallmark of proximal tubule function, has the same properties in culture as the tissue in vivo. Similarly, where appropriate comparisons are possible, amino acid, uric acid, and organic anion and cation transepithelial transport processes are qualitatively very similar in culture and in vivo. These two non-mammalian primary proximal tubule culture systems adequately reflect in vivo function, and thus provide opportunities for experimental manipulation otherwise not available.

Taurine secretion in primary monolayer cultures of flounder renal epithelium: stimulation by low osmolality.

Transepithelial taurine fluxes determined in short-circuited monolayer cultures of flounder renal proximal cells in Ussing chambers revealed net taurine secretion. Both unidirectional secretory and reabsorptive taurine fluxes exhibited saturation kinetics contributed by two distinct saturable transepithelial taurine transport systems operating at different taurine concentration ranges. The taurine secretory system operating below 0. 5 mM had lower affinity but higher capacity than the reabsorptive system, whereas the one operating at high concentrations (0.5-3.0 mM) had higher affinity but the same capacity as the corresponding reabsorptive system. Exposure (2 h) of the cultures to hyposmotic medium in the presence of taurine increased taurine secretory flux twofold with no effect on the reabsorptive flux. The hyposmolality-induced increase in taurine secretion was associated with a decreased peritubular taurine efflux and a concurrent increased luminal taurine efflux; the latter occurred via a pathway that was not affected by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid but inhibited by probenecid. The culture response in hyposmotic medium mimics the in vivo response of the intact marine fish kidney to dilution.

Tissue-level cytoprotection.

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.

Choline uptake across the ventricular membrane of neonate rat choroid plexus.

The uptake of [3H]choline from the cerebrospinal fluid (CSF) side of the rat neonatal choroid plexus was characterized in primary cultures of the choroidal epithelium grown on solid supports. Cell-to-medium concentration ratios were approximately 5 at 1 min and as high as 70 at 30 min. Apical choline uptake was facilitated; the Km was approximately 50 microM. Several organic cations (e.g., hemicholinium-3 and N1-methylnicotinamide) inhibited uptake. The reduction or removal of external Na+ or the addition of 5 mM LiCl had no effect on uptake. However, increasing external K+ concentration from 3 to 30 mM depolarized ventricular membrane potential (-70 to -15 mV) and reduced uptake to 45% of that for the control. Treatment with 1 mM ouabain or 2 mM BaCl2 reduced uptake 45%, and intracellular acidification reduced uptake to approximately 90% of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled to Na+ influx but is sensitive to plasma membrane electrical potential.

Renal sulfate secretion is carbonic anhydrase dependent in a marine teleost, Pleuronectes americanus.

Though chemical assays indicate that carbonic anhydrase (CA) activity is present in marine teleost nephrons, CA inhibitors have no effect on urine pH or bicarbonate excretion, parameters typically CA dependent in almost all vertebrate groups. Because marine teleost renal sulfate secretion is associated with bicarbonate anion exchange, we investigated the effect of CA inhibition on transepithelial sulfate transport by flounder renal tubule primary monolayer cultures (PTC) and on renal sulfate secretion (QSO4) by intact flounder. Both methazolamide and ethoxzolamide (10 microM) inhibited PTC secretory flux by approximately 50%; reabsorptive sulfate flux, Na-dependent glucose transport, and transepithelial electrical resistance were unaffected. A CA inhibitor restricted to the extracellular space (10 microM polyoxyethylene-aminobenzolamide, 3.7 kDa) had no effect on PTC sulfate transport. Intravenous administration of methazolamide reduced QSO4 almost 40% and had no effect on glomerular filtration rate (GFR), urine flow rate, or Pi excretion rate. Serum pH was significantly reduced 0.2 units, whereas urine pH was unchanged. Together, the in vitro and in vivo results indicate that CA facilitates renal sulfate secretion in the seawater teleost.

Protein kinase C regulation of p-glycoprotein-mediated xenobiotic secretion in renal proximal tubule.

Fluorescence microscopy, fluorescent substrates [daunomycin and a fluorescent cyclosporin A (CSA) derivative] and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of p-glycoprotein in killifish renal proximal tubules. PKC activators, phorbol ester (phorbol 12-myristate 13-acetate, PMA) and dioctylglycerol, reduced luminal drug accumulation, and protein kinase inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), increased luminal accumulation; a PMA analog that does not activate PKC was without effect. PMA effects were blocked by staurosporine. The increase in luminal fluorescence caused by staurosporine was blocked by the p-glycoprotein substrate, CSA, indicating that this component of transport was indeed mediated by p-glycoprotein. Neither PMA, dioctylglycerol, nor protein kinase inhibitors altered cellular drug accumulation. Finally, in primary cultures of flounder proximal tubule cells, PMA decreased transepithelial [3H]daunomycin secretion. This pharmacological approach demonstrates that in teleost renal proximal tubule, p-glycoprotein-mediated xenobiotic secretion is negatively correlated with changes in PKC activity, a finding that conflicts with results from studies using mammalian tumor cells that express p-glycoprotein.

Heat shock-induced protection and enhancement of Na+-glucose cotransport by LLC-PK1 monolayers.

Monolayers of the porcine-derived renal epithelial cell line, LLC-PK1, were used to characterize the effects of heat stress on Na+-glucose cotransport. Transepithelial current dependent on 5 mM glucose (I(Glc)), phloridzin-sensitive current (I(phz)), and total transepithelial current (I(total)) were measured as indicators of Na+-glucose cotransport. Severe heat shock (SHS; 45 degrees C for 1 h, then 37 degrees C for measurements) decreased transepithelial electrical resistance (TER), I(Glc), I(phz), and I(total) 50-70%. Mild heat shock (MHS; 42 degrees C for 3 h, then 37 degrees C for 12 h) induced accumulation of 72-kDa heat shock protein (HSP-72), decreased damage to TER from SHS, and prevented damage to I(Glc), I(phz), and I(total). Kinetic analysis showed that SHS damaged and MHS protected total Na+-glucose transport capacity (Vmax of I(Glc)). MHS alone increased TER (50%), I(Glc) (20%), I(total) (20%), and Vmax of I(Glc) (25%). On enhancement of the Na+ gradient by depletion of intracellular Na+, MHS increased I(Glc) 50% and had no effect on transepithelial Na+-dependent sulfate reabsorptive flux measured concurrently or in Na+-replete tissues. These effects of MHS were not reflected in effects on cell survival or luminal membrane surface area as indicated by lactate dehydrogenase or alkaline phosphatase release. In conclusion, HSP-72-inducing heat treatment both protected and enhanced Na+-glucose cotransport independently of the luminal membrane Na+ gradient and selectively with respect to effects on TER, reabsorptive sulfate transport, cell survival, and luminal membrane surface area.

Human stanniocalcin inhibits renal phosphate excretion in the rat.

Stanniocalcin (STC) is a glycoprotein hormone first identified in bony fishes where it counteracts hypercalcemia by inhibiting gill calcium uptake and stimulating renal inorganic phosphate (Pi) reabsorption. Human STC (hSTC) has recently been cloned and sequenced and is highly homologous to the fish hormone at the amino acid level. The objective of this study was to examine the possible effects of hSTC on electrolyte homeostasis and renal function in the rat. Recombinant hSTC was expressed in bacteria and purified by metal-ion affinity chromatography and reverse-phase high performance liquid chromatography. Anesthetized animals were given bolus infusions of 1, 5, or 10 nmol hSTC per kilogram of body weight. Control animals received solvent alone. The most effective dosage was 5 nmol/kg, which caused significant reductions in both absolute and fractional phosphate excretion in comparison with control rats. The hSTC had no effect on the renal excretion of other ions, the glomerular filtration rate, renal blood flow, blood pressure, or plasma electrolytes (Na+, K+, Ca2+, Pi, Mg/+). The maximum effect of hSTC on phosphate excretion was observed 60-80 minutes postinjection. Lesser effects were obtained with higher and lower dosages of hormone. When renal cortical brush-border membrane vesicles were isolated from control and hormone-treated animals 80 minutes postinjection, the rate of Na+/Pi cotransport was found to be 40% higher in vesicles from hormone-treated animals (p < 0.01; 5 nmol hSTC/kg). Together, the renal clearance and membrane vesicle data indicate that hSTC participates in the renal regulation of Pi homeostasis in mammals.

Renal organic anion secretion: evidence for dopaminergic and adrenergic regulation.

To examine possible regulatory control of renal proximal tubule organic anion secretion, winter flounder (Pleuronectes americanus) proximal tubule primary cultures were mounted in Ussing chambers. Unidirectional fluxes of [2,4-(14)C]dichlorophenoxyacetic acid were determined under short-circuited conditions. Phorbol 12-myristate 13-acetate (1 microM) caused a significant (P < 0.01) inhibition of net 2,4-dichlorophenoxyacetic acid secretion. Preincubation with staurosporine (1 microM) blocked the phorbol 12-myristate 13-acetate-induced decrease in secretion. Neither forskolin (10 microM) nor W-7 (20 microM) had any effect on net transport. Elevation of intracellular calcium activity with either A-23187 or thapsigargin produced a slight, transient decrease in transport. Addition of dopamine (1 microM) to the peritubular side, but not the luminal side, caused a significant (P < 0.01) decrease in net secretion. Both the alpha-adrenergic agonist oxymetazoline (10 microM) and depletion of intracellular Na+ transiently, but significantly (P < 0.05), increased net transport. The data indicate that renal organic anion excretion may be regulated through dopaminergic inhibition and alpha-adrenergic stimulation of net transepithelial secretion.

Effect of somatolactin and related hormones on phosphate transport by flounder renal tubule primary cultures.

Winter flounder renal proximal tubule primary monolayer cultures mounted in Ussing chambers were used to determine the effect of salmon somatolactin (sSL) on transepithelial Pi and Ca2+ transport. sSL stimulated Pi reabsorption in a dose-dependent manner at physiological levels of the hormone (12.5 ng/ml). Net Pi transport was significantly altered by sSL (200 ng/ml) within 2 h after the initial exposure. Ca2+ fluxes were unchanged by the addition of 200 ng/ml sSL. The sSL-induced Pi reabsorption was abolished by 10 microM H-89, a highly specific protein kinase A inhibitor. Moreover the production and release of adenosine 3',5'-cyclic monophosphate were significantly increased after 1 and 2 h of exposure to sSL. The data indicate that sSL directly stimulates net renal Pi reabsorption by an adenosine 3',5'-cyclic monophosphate-dependent pathway. In addition to sSL, flounder SL and rat prolactin greatly, and salmon growth hormone (2.3 micrograms/ml) slightly, increased net Pi reabsorptive flux, whereas salmon prolactin had no effect.

Heat-shock-stimulated transepithelial daunomycin secretion by flounder renal proximal tubule primary cultures.

Primary monolayer cultures of winter flounder renal proximal tubule epithelium mounted in Ussing chambers were used to characterize transepithelial transport of daunomycin (Dau). Control tissues performed active net secretion of Dau (0.064 +/- 0.027 nmol.cm-2.h-1). Mild heat shock (5 degrees C elevation for 6-8 h followed by return to normal temperature) almost doubled Dau secretion (0.114 +/- 0.026 nmol.cm-2.h-1). This response was inhibited approximately 40% by addition of the protein synthesis inhibitor, cycloheximide. Dau secretion was inhibited by verapamil, vinblastine, cyclosporin A, and to a lesser degree by the organic cation, tetraethylammonium. In addition, tetraethylammonium secretion was inhibited by vinblastine. Dau secretion was not inhibited by the organic anion, p-aminohippurate, and p-aminohippurate secretion was not inhibited by vinblastine. The transepithelial reabsorptive flux of Dau and the electrical characteristics of the tissues, including rheogenic glucose transport, were unaffected by any of the above treatments. Reaction of tissues with a monoclonal antibody to P-glycoprotein (C219) revealed the presence of this transporter on only apical microvilli. The data indicate that flounder possess an active mechanism for the renal excretion of Dau that is stimulated by mild heat shock. This mechanism is distinct from organic anion, but not organic cation, transport and has characteristics consistent with transport by an apical P-glycoprotein.

Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture.

The effects of several hormones on transepithelial Pi transport were determined in primary monolayer cultures of winter flounder proximal tubule epithelium in Ussing chambers. Salmon stanniocalcin (STC) had a dose-dependent stimulatory effect on net Pi reabsorption within the normal plasma hormone concentration range, 12.5-50 ng/ml (0.25-1.0 nM). Net Pi transport was significantly altered by STC (200 ng/ml) within 30 min and progressively increased from slight net secretion (0.26 +/- 0.744 nmol.cm-2.h-1) in untreated controls to net reabsorption (1.96 +/- 0.729 nmol.cm-2.h-1) after 3 h. The STC effect was mimicked by 10 microM forskolin, whereas 10 microM H-89, a highly specific protein kinase A inhibitor, significantly decreased both STC- and forskolin-induced Pi reabsorption. The release of adenosine 3',5'-cyclic monophosphate (cAMP) was increased more than twofold after a 1-h exposure to STC. This hormone had no effect on transepithelial Ca2+ transport. The results indicate that STC directly stimulates net renal Pi reabsorption by a cAMP-dependent pathway. In addition to STC, bovine parathyroid hormone (100 nM) and ovine prolactin (100 nM) significantly increased net Pi reabsorptive flux.

Renal transepithelial phosphate secretion: luminal membrane voltage and Ca2+ dependence.

The role of apical membrane electrical potential, the possibility of K+ channel involvement, and the role of extracellular Ca2+ in transepithelial P(i) secretion were examined in primary monolayer cultures of flounder renal proximal tubule cells in Ussing chambers. Exposure to 200 nM thapsigargin (TG) significantly increased net P(i) secretion. In TG-stimulated tissues, substitution of 100 mM KCl for 100 mM NaCl in the luminal medium depolarized the apical membrane potential from -64 +/- 2.8 to -26 +/- 3.9 mV and strongly inhibited net P(i) secretion. In 32P(i)-preloaded tissues, cell-to-lumen exit of 32P(i) was significantly decreased to approximately 50% of control by high luminal K+ while cell-to-peritubular bath movement was unchanged. Addition of BaCl2 (2 mM) or charybdotoxin (20 nM) to the luminal surface significantly reduced TG-stimulated net P(i) secretion. The elevation of bath Ca2+ from 2 to 5 mM significantly increased secretory flux and decreased reabsorptive flux. The effect of TG on net P(i) secretion was reduced by the Ca2+ channel blocker verapamil (VE, 100 microM) to 65% of control and by calmodulin antagonist W-7 (20 microM) to 35% of control but it was not blocked by the protein kinase inhibitor H-7 (100 microM). VE also significantly inhibited the P(i) secretion induced by acidification of the peritubular bathing medium. The data indicate that transepithelial P(i) secretion induced by TG is significantly influenced by apical membrane electrical polarity, which may be regulated in part by Ca(2+)-activated K+ channels.

PTH-sensitive K(+)- and voltage-dependent Pi transport by chick renal brush-border membranes.

Brush-border membrane vesicles (BBMV) from chick (Gallus gallus) kidneys were used to examine possible pathways of Pi transport associated with Pi secretion. Preloading with 6 mM Pi trans-stimulated 32Pi uptake in the absence of Na+, indicating facilitation. Inside-positive voltage (100 mM K+, out > in, +valinomycin) increased Pi uptake from 161 +/- 4.4 to 241 +/- 16.1 pmol.mg protein-1.5s-1 at pH 7.5 (in = out). Gradients characterized by extravesicular pH (pHo) of 5.5 vs. intravesicular pH (pHi) of 7.5, 100 mM K+ (out > in), without and with valinomycin, further increased uptake to 664 +/- 148.5 and 946 +/- 90.8 pmol.mg protein-1.5s-1, respectively. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) had no effect on the latter response, but with 100 mM K+ (in = out), valinomycin decreased the response more than one-half, implicating a H+ diffusion potential. Generation of this potential with pHo 5.5 vs. pHi 7.5 and CCCP did not drive concentrative Pi uptake in absence of K+. Parathyroid hormone (PTH) treatment significantly increased this BBMV K(+)- and voltage-dependent Pi up-take compared with the parathyroidectomized (PTX) condition. The values of maximal uptake rate (Vmax) in PTH vs. PTX BBMV were 5,330 and 1,976 pmol.mg protein-1.5s-1, respectively. K(+)-dependent transport was inhibited by arsenate, phosphonoacetic acid, and vanadate. Together, the data indicate that this PTH-sensitive, voltage- and K(+)-dependent monovalent Pi transporter could be the mechanism by which Pi exits, cell-to-lumen, during renal tubular Pi secretion.

Interaction of structurally similar pesticides with organic anion transport by primary cultures of winter flounder renal proximal tubule.

We assessed the interaction of several pesticides with renal organic anion transport based on inhibition of the active transepithelial transport of p-aminohippuric acid ([3H]PAH) by primary cultures of winter flounder proximal tubules. Four structurally similar chlorophenoxy acid herbicides were tested. 2-Methyl-4-chlorophenoxyacetic acid at 0.1 mM had no effect on PAH transport. 2-(2,4-Dichlorophenoxy)propionic acid, 2-(2-methyl,4-chlorophenoxy)propionic acid and 2,4-dichlorophenoxyacetic acid inhibited PAH secretion with IC50 values (median inhibitory concentrations) of 0.09, 0.2 and 0.2 mM, respectively. At 2-(2-methyl,4-chlorophenoxy)propionic acid concentrations of 10(-7) to 10(-6) M, PAH secretion was stimulated to 132 and 139% of controls, respectively, at 2 hr. 2,2-bis-p-Chlorophenylacetic acid was tested at concentrations of 10(-7) to 10(-3) M and inhibited with an IC50 of 0.02 mM. At 10(-7) M 2,2-bis-p-chlorophenylacetic acid, a delayed increase was again seen in which PAH secretion increased to 154% of control at 2 hr. The anticholinesterase insecticide phosphothioic acid O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl ester), its more active metabolite O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphate and its dimethyl analog, phosphothioic acid O,O-dimethyl-O-(3,5,6-trichloro-2-pyridinyl ester) were tested at 0.1 mM. Phosphothioic acid O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl ester) increased PAH secretion significantly to 115% of control whereas the O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphate inhibited by 28%.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of thermal and chemical tolerance to transepithelial transport by cultured flounder renal epithelium.

We tested the effects of mild heat shock (MHS) on tolerance of epithelial transport processes to a chemical inhibitor and, reciprocally, chemical stress effects on tolerance to severe thermal stress. Flounder renal tubules were cultured as monolayers on native collagen for 12 to 14 days at 22 degrees C and mounted in Ussing chambers in which transepithelial electrical characteristics and unidirectional [35S]sulfate fluxes were measured. 2,4-dichlorophenoxyacetic acid (0.5 mM) lowered net active sulfate secretion 25% (93.6 +/- 7.64-69.0 +/- 9.08 nmol/cm2/hr); MHS (27 degrees C for 6 hr followed by 1.5 hr at 22 degrees C) prevented this inhibition (92.4 +/- 5.72 nmol/cm2/hr) and stimulated transport 30% (125.7 +/- 11.06 nmol/cm2/hr). Cycloheximide or actinomycin D prevented the enhancing and protective effects of MHS. Preincubation in 100 microM ZnCl2 for 6 hr at 22 degrees C followed by 1.5 hr in zinc-free medium (mild zinc stress) enhanced net sulfate flux, protected transport from severe heat stress (32 degrees C for 1.5 hr followed by 1.5 hr at 22 degrees C) and prevented 2,4-dichlorophenoxyacetic acid inhibition in a manner similar to MHS. Mild zinc shock induced heat shock protein synthesis the same as MHS. Cycloheximide prevented the mild zinc shock effect on transport. The data show that thermal or chemical stressors can independently induce tolerance of transepithelial transport to both thermal and chemical insults and that stress-induced "protection" is due to enhancement of normal function rather than prevention of damage.

Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

Primary monolayer cultures of winter flounder renal proximal-tubule cells were used to determine whether transepithelial transport could be protected from the damaging effects of extreme temperature by previous mild heat shock. Renal tubule epithelial cells were enzymatically dispersed and reorganized as confluent monolayer sheets on native rat tail collagen. Transepithelial electrical properties (potential difference, resistance, short-circuit current, and Na(+)-dependent glucose current) and unidirectional [35S]sulfate fluxes were measured in Ussing chambers at 22 degrees C. Examination of transepithelial electrical properties following acute 1-hr elevation of temperature over a range of 22-37 degrees C provided the basis for the "mild" versus "severe" thermal stress protocols. Severe elevation from 22 degrees C to 32 degrees C for 1.5 hr followed by 1.5 hr at 22 degrees C significantly decreased glucose current (7 +/- 0.7 to 3 +/- 0.8 microA/cm2) as well as net sulfate secretion [131 +/- 11 to 33 +/- 11 nmol/(cm2.hr)]. Mild heat shock of 27 degrees C for 6 hr prior to this severe heat shock completely protected both glucose transport (6 +/- 0.7 microA/cm2) and sulfate flux (149 +/- 13 nmol/(cm2.hr)]. Scanning electron microscopy showed that the number of microvilli on the apical (luminal) surface of the epithelium was decreased after a 32 degrees C heat shock. Monolayers exposed to 27 degrees C for 6 hr prior to incubation at 32 degrees C showed no loss of microvilli. SDS/PAGE analysis of protein patterns from the cultures showed that three classes of heat shock proteins were maximally induced at 27 degrees C. Inhibition of protein synthesis by cycloheximide prevented the thermoprotective effect of mild heat shock. This suggests that certain renal transport functions can be protected from sublethal but debilitating thermal stress by prior mild heat shock and that heat shock proteins may play a role in this protection.

Taurine secretion in cultured winter flounder renal epithelium.

The mechanism for the secretion of taurine by the marine fish kidney was examined in flounder renal primary cultures and teased tubules. The question addressed was whether the secretion of taurine, but not beta-alanine, a related beta-amino acid, is due to differences in their basolateral uptake, intracellular concentration, or luminal efflux mechanism. Aminooxyacetate (AOA) (10 mM), an inhibitor of beta-alanine oxidation, allowed the basolateral beta-alanine accumulation (measured in teased tubules) and the beta-alanine intracellular concentration (measured in primary cultures) to be similar to that for taurine. However the addition of AOA did not promote the net secretion of beta-alanine (measured as unidirectional fluxes in the cultured epithelium mounted in an Ussing chamber). The organic anions bromcresol green (BCG) (0.1 mM) and probenecid (1 mM) inhibited the net secretion of taurine by affecting the luminal efflux step. This suggests that a BCG- and probenecid-sensitive carrier in the luminal membrane facilitates the cell-to-lumen efflux of taurine and thus achieves net secretion.

Effects of pH on phosphate transport by flounder renal tubule primary cultures.

Transepithelial phosphate (Pi) fluxes were determined in primary monolayer cultures of the winter flounder proximal tubule in Ussing chambers. Net Pi secretion, peritubular (P)-to-lumen (L) net flux (Jnet = 17.0 +/- 3.77 nmol.cm-2.h-1), was strongly stimulated by lowering peritubular pH to 6.5 (pHP 6.5 vs. pHL 7.5) compared with control tissues at pH 7.5 (pHP 7.5 vs. pHL 7.5) where net reabsorption (L-to-P Jnet = 1.10 +/- 0.36 nmol.cm-2.h-1) occurred. The stimulation of net secretion by pHP 6.5 was inhibited to 27% of control by 200 microM amiloride. The imposition of a reversed pH gradient (pHL 6.5 vs. pHP 7.5) did not stimulate Pi secretion. Preincubation with 0.5 U/ml phospholipase C caused a nearly fivefold stimulation of Pi secretion compared with the untreated controls. H-7 (100 microM), a protein kinase inhibitor, caused a 2.5-fold reduction in phorbol ester (PE)-induced stimulation of Pi secretion. H-7 also significantly inhibited Pi secretion (50% reduction) when peritubular pH was lowered to 6.5. PE-induced net Pi secretion was significantly lower when luminal pH was lowered to 5.5 compared with controls where luminal pH was kept at 7.5. Amiloride (200 microM) significantly inhibited the PE-induced net Pi secretion in the presence of luminal pH 7.5 but had no significant effect at luminal pH 5.5. Replacement of luminal NaCl with LiCl or isosmolar mannitol significantly reduced net phosphate secretion under the conditions of lowered peritubular pH. Net Pi secretion was also dependent on the maintenance of a cellular Na+ gradient, since 100 microM ouabain inhibited PE-induced net Pi secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Organic anion secretion by winter flounder renal proximal tubule primary monolayer cultures.

Recent models of para-aminohippuric acid (PAH) transport by renal proximal tubule have been deduced from isolated membrane vesicles and stopped flow microperfusion. The flounder proximal tubule primary monlayer cultures mounted in Ussing chambers have provided a means to examine the relationship of these models to transepithelial transport. Unidirectional transepithelial 3H-PAH fluxes were determined in 12-day-old monolayers on floating collagen gels under continuously short-circuited conditions. The kinetic values of PAH secretory flux were complex. Over the range of 0.6 microM to 1.5 mM the K1/2 was 0.4 mM and Vmax was 80 nmol/cm2/h. Reabsorptive flux did not saturate. 1 mM probenecid inhibited 95% of the secretory flux (0.07 +/- 0.18 compared with control of 1.38 +/- 0.34 nmol/cm2/h at 10 microM PAH) and had no significant effect on reabsorptive flux (probenecid, 0.07 +/- 0.04; control, 0.05 +/- 0.01 nmol/cm2/h). 0.1 mM 4-acetamido-4'-isothiocyanalostilbene-2,2'-diasulfonic acid and 1 mM benzoylpropionic acid also significantly inhibited secretory flux (95 and 78%, respectively). These inhibitors appeared to be specific in that no effects on transepithelial potential difference, resistance or phlorizin-sensitive current were seen. Removal of Na during flux measurement reduced net PAH secretion to zero, and 1 mM ouabain reduced PAH secretory flux to 10% of control within 90 min with no effect on reabsorptive (leak) flux. Clamping transepithelial voltage to +/- 10 mV had no significant effect on PAH fluxes. Addition of glutarate to flounder saline significantly inhibited PAH secretory flux starting at 0.1 mM and caused 90% inhibition at 1 mM; no stimulation of transport was noted at any glutarate concentration.