PKC-epsilon-dependent cytosol-to-membrane translocation of pendrin in rat thyroid PC Cl3 cells

We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC Cl3 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. (125)I pre-loaded cells showed an (125)I efflux accelerated in chloride-containing buffer with respect to chloride-free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists-stimulated cells, whereas it was barely detectable in un-stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8-Br-cAMP and forskolin. Stimulation with insulin (1 microg/ml; 0-40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I(-) content in (125)I pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-delta and -epsilon. Inhibition of both PKC-delta and -epsilon activities by GF109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-delta by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-epsilon translocation inhibitor peptide and also by PKC-epsilon downregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-epsilon activity. In conclusion, our study demonstrates that, in PC Cl3 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-epsilon-dependent intracellular pathway.     

Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing airway serous cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.     

Regulation of pendrin by pH: dependence on glycosylation

Mutations in the anion exchanger pendrin are responsible for Pendred syndrome, an autosomal recessive disease characterized by deafness and goitre. Pendrin is highly expressed in kidney collecting ducts, where it acts as a chloride/bicarbonate exchanger and thereby contributes to the regulation of acid-base homoeostasis and blood pressure. The present study aimed to characterize the intrinsic properties of pendrin. Mouse pendrin was transfected in HEK (human embryonic kidney) 293 and OKP (opossum kidney proximal tubule) cells and its activity was determined by monitoring changes in the intracellular pH induced by variations of transmembrane anion gradients. Combining measurements of pendrin activity with mathematical modelling we found that its affinity for Cl-, HCO3- and OH- varies with intracellular pH, with increased activity at low intracellular pH. Maximal pendrin activity was also stimulated at low extracellular pH, suggesting the presence of both intracellular and extracellular proton regulatory sites. We identified five putative pendrin glycosylation sites, only two of which are used. Mutagenesis-induced disruption of pendrin glycosylation did not alter its cell-surface expression or polarized targeting to the apical membrane and basal activity, but fully abrogated its sensitivity to extracellular pH. The hither to unknown regulation of pendrin by external pH may constitute a key mechanism in controlling ionic exchanges across the collecting duct and inner ear.     

Molecular and functional characterization of human pendrin and its allelic variants

Pendrin (SLC26A4, PDS) is an electroneutral anion exchanger transporting I(-), Cl(-), HCO(3)(-), OH(-), SCN(-) and formate. In the thyroid, pendrin is expressed at the apical membrane of the follicular epithelium and may be involved in mediating apical iodide efflux into the follicle; in the inner ear, it plays a crucial role in the conditioning of the pH and ion composition of the endolymph; in the kidney, it may exert a role in pH homeostasis and regulation of blood pressure. Mutations of the pendrin gene can lead to syndromic and non-syndromic hearing loss with EVA (enlarged vestibular aqueduct). Functional tests of mutated pendrin allelic variants found in patients with Pendred syndrome or non-syndromic EVA (ns-EVA) revealed that the pathological phenotype is due to the reduction or loss of function of the ion transport activity. The diagnosis of Pendred syndrome and ns-EVA can be difficult because of the presence of phenocopies of Pendred syndrome and benign polymorphisms occurring in the general population. As a consequence, defining whether or not an allelic variant is pathogenic is crucial. Recently, we found that the two parameters used so far to assess the pathogenic potential of a mutation, i.e. low incidence in the control population, and substitution of evolutionary conserved amino acids, are not always reliable for predicting the functionality of pendrin allelic variants; actually, we identified mutations occurring with the same frequency in the cohort of hearing impaired patients and in the control group of normal hearing individuals. Moreover, we identified functional polymorphisms affecting highly conserved amino acids. As a general rule however, we observed a complete loss of function for all truncations and amino acid substitutions involving a proline. In this view, clinical and radiological studies should be combined with genetic and molecular studies for a definitive diagnosis. In performing genetic studies, the possibility that the mutation could affect regions other than the pendrin coding region, such as its promoter region and/or the coding regions of functionally related genes (FOXI1, KCNJ10), should be taken into account. The presence of benign polymorphisms in the population suggests that genetic studies should be corroborated by functional studies; in this context, the existence of hypo-functional variants and possible differences between the I(-)/Cl(-) and Cl(-)/HCO(3)(-) exchange activities should be carefully evaluated.     

Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.     

Functional characterization of pendrin in a polarized cell system. Evidence for pendrin-mediated apical iodide efflux

Pendred's syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter, and impaired iodide organification. It is caused by mutations in the PDS/SLC26A4 gene that encodes pendrin. Functionally, pendrin is a transporter of chloride and iodide in Xenopus oocytes and heterologous mammalian cells and a chloride/base exchanger in beta-intercalated cells of the renal cortical collecting duct. The partially impaired thyroidal iodide organification in Pendred's syndrome suggests a possible role of pendrin in iodide transport at the apical membrane of thyroid follicular cells, but experimental evidence for this concept is lacking. The iodide transport properties of pendrin were determined in polarized Madin-Darby canine kidney cells expressing the sodium iodide symporter (NIS), pendrin, or NIS and pendrin using a bicameral system-permitting measurement of iodide content in the basal, intracellular, and apical compartments. Moreover, we determined the functional consequences of two naturally occurring mutations (L676Q and FS306>309X). In polarized Madin-Darby canine kidney cells, NIS mediates uptake at the basolateral membrane. Only minimal amounts of iodide reach the apical compartment in the absence of pendrin. In cells expressing NIS and pendrin, pendrin mediates transport of iodide into the apical chamber. Wild type pendrin also mediates iodide efflux in transiently transfected cells. In contrast, both pendrin mutants lose the ability to promote iodide efflux. These results provide evidence that pendrin mediates apical iodide efflux from polarized mammalian cells loaded with iodide. Consistent with the partial organification defect observed in patients with Pendred's syndrome, naturally occurring mutations of pendrin lead to impaired transport of iodide.     

Pendrin as a novel target for diuretic therapy

The Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4, PDS) and the thiazide-sensitive NaCl cotransporter NCC (SLC12A3) are expressed on the apical membranes of distal nephron segments and mediate salt absorption, with pendrin working in tandem with the epithelial Na channel (ENaC) and NCC working by itself. Pendrin is expressed on the apical membrane of intercalated cells in late distal convoluted tubule (DCT), connecting tubule (CNT) and the cortical collecting duct (CCD) whereas the thiazide-sensitive NaCl cotransporter NCC is primarily detected on the apical membrane of DCT cells. Recent studies indicate that pendrin expression is increased in kidneys of NCC knockout mice, raising the possibility that pendrin and NCC can compensate for loss of the other by increasing their expression and activity. Current investigations in our laboratories demonstrate that pendrin plays an important role in compensatory salt absorption in response to the loop diuretics and the thiazide derivatives. These studies further demonstrate that whereas single deletion of pendrin or NCC does not cause salt wasting in mutant mice under baseline conditions, double knockout of pendrin and NCC causes profound polyuria and polydipsia, along with salt wasting under basal conditions. As a result, animals develop significant dehydration. We propose that pharmacologic inhibition of pendrin and NCC can provide a novel and strong diuretic regimen for patients with fluid overload, including those with congestive heart failure, nephrotic syndrome or renal failure.     

Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.     

The Na+-driven Cl-/HCO3- exchanger. Cloning, tissue distribution, and functional characterization

The Na(+)-driven Cl(-)/HCO(3)(-) exchanger is an important regulator of intracellular pH in various cells, but its molecular basis has not been determined. We show here the primary structure, tissue distribution, and functional characterization of Na(+)-driven chloride/bicarbonate exchanger (designated NCBE) cloned from the insulin-secreting cell line MIN6 cDNA library. The NCBE protein consists of 1088 amino acids having 74, 72, and 55% amino acid identity to the human skeletal muscle, rat smooth muscle, and human kidney sodium bicarbonate cotransporter, respectively. The protein has 10 putative membrane-spanning regions. NCBE mRNA is expressed at high levels in the brain and the mouse insulinoma cell line MIN6 and at low levels in the pituitary, testis, kidney, and ileum. Functional analyses of the NCBE protein expressed in Xenopus laevis oocytes and HEK293 cells demonstrate that it transports extracellular Na(+) and HCO(3)(-) into cells in exchange for intracellular Cl(-) and H(+), thus raising the intracellular pH. Thus, we conclude that NCBE is a Na(+)-driven Cl(-)/HCO(3)(-) exchanger that regulates intracellular pH in native cells.     

Drosophila bestrophin-1 chloride current is dually regulated by calcium and cell volume

Mutations in the human bestrophin-1 (hBest1) gene are responsible for Best vitelliform macular dystrophy, however the mechanisms leading to retinal degeneration have not yet been determined because the function of the bestrophin protein is not fully understood. Bestrophins have been proposed to comprise a new family of Cl(-) channels that are activated by Ca(2+). While the regulation of bestrophin currents has focused on intracellular Ca(2+), little is known about other pathways/mechanisms that may also regulate bestrophin currents. Here we show that Cl(-) currents in Drosophila S2 cells, that we have previously shown are mediated by bestrophins, are dually regulated by Ca(2+) and cell volume. The bestrophin Cl(-) currents were activated in a dose-dependent manner by osmotic pressure differences between the internal and external solutions. The increase in the current was accompanied by cell swelling. The volume-regulated Cl(-) current was abolished by treating cells with each of four different RNAi constructs that reduced dBest1 expression. The volume-regulated current was rescued by transfecting with dBest1. Furthermore, cells not expressing dBest1 were severely depressed in their ability to regulate their cell volume. Volume regulation and Ca(2+) regulation can occur independently of one another: the volume-regulated current was activated in the complete absence of Ca(2+) and the Ca(2+)-activated current was activated independently of alterations in cell volume. These two pathways of bestrophin channel activation can interact; intracellular Ca(2+) potentiates the magnitude of the current activated by changes in cell volume. We conclude that in addition to being regulated by intracellular Ca(2+), Drosophila bestrophins are also novel members of the volume-regulated anion channel (VRAC) family that are necessary for cell volume homeostasis.     

Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine

HCO3(-) secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO3(-) secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl(-)/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl(-)/HCO3(-) exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a approximately 90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl(-)/HCO3(-) exchanger in the small intestine.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Pendrin: the thyrocyte apical membrane iodide transporter?

In the thyroid, the transport of iodide from the extracellular space to the follicular lumen requires two steps: the transport in the cell at the basal side and in the lumen at the apical side. The first step is mediated by the Na(+)/I(-) symporter (NIS). In most reviews and textbooks, the second step is presented as mediated by pendrin. In this review, we analyze this assumption. There are several arguments supporting the concept that indeed pendrin plays an important role in thyroid physiology. However, biochemical, clinical and histological data on the thyroid of a patient with Pendred syndrome do not suggest an essential role in iodide transport, which is corroborated by the lack of a thyroid phenotype in pendrin knockout mice. Experiments in vivo and in vitro on polarized and unpolarized cells show that iodide is transported transport of iodide at the apex of the thyroid cell. Moreover, ectopic expression of pendrin in transfected non-thyroid cells is capable of mediating iodide efflux. It is concluded that pendrin may participate in the iodide efflux into thyroid lumen but not as the unique transporter. Moreover, another role of pendrin in mediating Cl(-)/HCO(3)(-) exchange and controlling luminal pH is suggested.     

Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death

Cell shrinkage, or the loss of cell volume, is a ubiquitous characteristic of programmed cell death that is observed in all examples of apoptosis, independent of the death stimulus. This decrease in cell volume occurs in synchrony with other classical features of apoptosis. The molecular basis for cell shrinkage during apoptosis involves fluxes of intracellular ions including K+, Na+, and Cl-. Here we show for the first time that these ion fluxes, but not cell shrinkage, are necessary for apoptosis. Using sodium-substituted medium during anti-Fas treatment of Jurkat cells, we observed cellular swelling, a property normally associated with necrosis, in contrast to the typical cell shrinkage. Surprisingly, these swollen cells displayed all of the other classical features of apoptosis, including chromatin condensation, externalization of phosphatidylserine, caspase activity, poly(ADP)-ribose polymerase cleavage, and internucleosomal DNA degradation. These swollen cells had a marked decrease in intracellular potassium, and subsequent inhibition of this potassium loss completely blocked apoptosis. Reintroduction of sodium ions in cell cultures reversed this cellular swelling, resulting in a dramatic loss of cell volume and the characteristic apoptotic morphology. Additionally, inhibition of sodium influx using a sodium channel blocker saxitoxin completely prevented the onset of anti-Fas-induced apoptosis in Jurkat cells. These findings suggest that sodium influx can control not only changes in cell size but also the activation of apoptosis, whereas potassium ion loss controls the progression of the cell death process. Therefore cell shrinkage can be separated from other features of apoptosis.     

Regulation of pendrin by cAMP: possible involvement in β-adrenergic-dependent NaCl retention

The sodium-independent anion exchanger pendrin is expressed in several tissues including the kidney cortical collecting duct (CCD), where it acts as a chloride/bicarbonate exchanger and has been shown to participate in the regulation of acid-base homeostasis and blood pressure. The renal sympathetic nervous system is known to play a key role in the development of salt-induced hypertension. This study aimed to determine whether pendrin may partly mediate the effects of β adrenergic receptors (β-AR) on renal salt handling. We investigated the regulation of pendrin activity by the cAMP/protein kinase A (PKA) signaling pathway, both in vitro in opossum kidney proximal (OKP) cells stably transfected with pendrin cDNA and ex vivo in isolated microperfused CCDs stimulated by isoproterenol, a β-AR agonist. We found that stimulation of the cAMP/PKA pathway in OKP cells increased the amount of pendrin at the cell surface as well as its transport activity. These effects stemmed from increased exocytosis of pendrin and were associated with its phosphorylation. Furthermore, cAMP effects on the membrane expression and activity of pendrin were abolished by mutating the serine 49 located in the intracellular N-terminal domain of pendrin. Finally, we showed that isoproterenol increases pendrin trafficking to the apical membrane as well as the reabsorption of both Cl(-) and Na(+) in microperfused CCDs. All together, our results strongly suggest that pendrin activation by the cAMP/PKA pathway underlies isoproterenol-induced stimulation of NaCl reabsorption in the kidney collecting duct, a mechanism likely involved in the sodium-retaining effect of β-adrenergic agonists.     

Regulation of Cl/HCO3 exchange in gastric parietal cells.

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.     

Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.