Development and biological applications of chloride-sensitive fluorescent indicators.

Development and biological applications of chloride-sensitive fluorescent indicators A. S. VERKMAN Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143

VERKMAN, A. S. Development and biological applications of chloridesensitive fluorescent indicators. Am. J. Physiol. 259 (Cell Physiol. 28): C375C388, 1990.-Chloride movement across cell plasma and internal membranes is of central importance for regulation of cell volume and pH, vectorial salt movement in epithelia, and, probably, intracellular traffic. Quinolinium-based chloride-sensitive fluorescent indicators provide a new approach to study chloride transport mechanisms and regulation that is complementary to 36C1 tracer methods, intracellular microelectrodes, and patch clamp. Indicator fluorescence is quenched by chloride by a collisional mechanism with Stern-Volmer constants of up to 220 M-l. Fluorescence is quenched selectively by chloride in physiological systems and responds to changes in chloride concentration in under 1 ms. The indicators are nontoxic and can be loaded into living cells for continuous measurement of intracellular chloride concentration by single-cell fluorescence microscopy. In this review, the structure-activity relationships for chloridesensitive fluorescent indicators are described. Methodology for measurement of chloride transport in isolated vesicle and liposome systems and in intact cells is evaluated critically by use of examples from epithelial cell physiology. Future directions for synthesis of tailored chloride-sensitive indicators and new applications of indicators for studies of transport regulation and intracellular ion gradients are proposed. fluorescence; 6-methoxy-N-( 3 -sulfopropyl)quinolium tubules; membrane transport; liposomes; regulation

is a major ionic constituent of cells and extracellular compartments. Chloride movement across cell plasma membranes occurs by a number of distinct conductive, cotransport, and countertransport processes that are of fundamental importance for control of cell volume and pH, and in epithelia, for control of salt secretion and reabsorption. Within cells, chloride transport may be important for control of endosomal acidification and other intracellular processes. It has become clear that the activity of many chloride-transporting systems is regulated tightly by one or more signaling pathways, including protein kinase A, protein kinase C, calcium, membrane-associated G proteins, and phospholipase A2 products (30). Chloride transport defects resulting in clinical abnormalities occur in the genetic disease cystic fibrosis (29, 44) and the chloride hypersecretory state in cholera and related toxin-induced enteropathies (13). The development of chloride-sensitive fluorescent indicators provides an alternative and sometimes exclusive CHLORIDE

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approach to study chloride transport. Because of the low specific activity of 36C1and the requirement of separating internal and external radioactivity, tracer methods are applicable for slow transport processes (generally >I s) and when relatively large and homogeneous biological samples are available. Double-barreled intracellular chloride-selective microelectrodes are a valuable invasive tool for the continuous measurement of cell chloride concentration (24, 47). However, microelectrode applications have been limited because of the technical expertise required to make reliable measurements and the imperfect chloride selectivity and response characteristics of available resins. Patch clamp is the method of choice for single-channel and whole cell studies of chloride conductance. Other approaches for studying chloride for specialized applications include nuclear magnetic resonance, X-ray probe electron microanalysis, and optical measurements of cell volume and plasma membrane potential. On the basis of the experience with calcium- and pH-

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sensitive fluorescent indicators in cell systems, the potential advantages of a chloride-sensitive fluorescent indicator over other available methods are technical simplicity, noninvasiveness, high time resolution for measurement of fast transport processes, and high spatial resolution for studies of heterogeneous systems and of intracellular chloride gradients and compartmentation. The purpose of this review is to evaluate the application of chloride-sensitive fluorescent indicators to studies of membrane transport regulation in isolated membrane and living cell systems. Many of the examples are chosen from epithelial cell physiology. Applications and limitations of chloride-sensitive indicators are evaluated critically, and new approaches to address questions in chloride transport physiology are proposed. REQUIREMENTS FLUORESCENT

OF A CHLORIDE-SENSITIVE INDICATOR

Fluorescent indicators are currently available for measurement of intracellular calcium, sodium, potassium, magnesium, and pH (19, 35, 36, 40, 42, 43, 49). These indicators contain a specific cation chelation site or titrable group coupled to a fluorescent moiety. Cellpermeant acetoxymethyl ester derivatives are loaded into cells by passive diffusion. The ester function is cleaved by intracellular esterases to give a more polar molecule that leaks out from cells at a reasonably slow rate. Significant biological advances have been made over the past decade in cell calcium signaling by use of fura- and its predecessors and in pH regulation by use of fluorescein-based dyes such as 2’,7’-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). There are a number of important criteria in the design of a fluorescent indicator for biological applications. Six major categories of requirements are described, with focus on chloride-sensitive fluorescent indicators. Sensitivity and response kinetics. The fluorescence of a chloride-sensitive indicator should be sensitive to changes in intracellular chloride activity in the physiological range of O-60 mM. The response kinetics should be rapid, ideally 0.5) and molar

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absorptivity (>50,000 M-l. cm-‘). Fluoresceins have near unity quantum yield at high pH with molar absorptivity of -lo5 M-l cm-‘. The excitation and emission maxima should be at high wavelengths in the visible spectrum (>500 nm) to minimize signal contributions from cellular and instrument autofluorescence and cell photodynamic injury from ultraviolet excitation. Indicator fluorescence should be relatively insensitive to photobleaching. The Stoke’s shift (difference between excitation and emission maxima) should be large for efficient filtration of excitation light in the detection system. Cell entrapment. The indicator should be loaded into cells by a noninvasive method. Once loaded, the indicator should remain entrapped in the cytoplasmic compartment without leakage across plasma membranes, binding to intracellular components, or passage into subcellular organelles. Specific targeting to intracellular compartments would be desirable. Calibration. It should be possible to calibrate the fluorescent indicator so that absolute intracellular chloride activity can be determined from fluorescence measurements without knowledge of local indicator concentration. This can be accomplished by calculating a ratio of fluorescence intensities at two excitation or emission wavelengths that respond differently to changes in chloride activity. Alternatively, the measurement of fluorescence lifetime or steady-state anisotropy could provide information that does not depend on the local indicator concentration. Spatial gradients in intracellular chloride activity should be measurable by high-resolution confocal imaging. These ideal criteria constitute a demanding list of characteristics that have been partially met by the available intracellular fluorescent indicators for cations and pH. As described below, the chloride-sensitive fluorescent indicators synthesized to date fulfill many but not all of the ideal criteria. l

CHEMISTRY FLUORESCENT

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The best approach for the development of a chloridesensitive fluorescent indicator would be to design a fluorescent molecule with a chloride chelation site. However, because selective chloride chelators have not been described, we chose to make use of the known sensitivity of the fluorescence of quinoline-based molecules to quenching by halides (56). After an initial screening of candidate indicators, the compound 6-methoxy-N-(3sulfopropyl)quinolinium (SPQ, Fig. 1) was found to have suitable characteristics to serve as a reference molecule for evaluation of new chloride indicators (23). SPQ is easily synthesized in large quantities by reaction of equimolar quantities of the nonpolar liquids 6methoxyquinoline and propane sultone at 90°C for 45 min under Nz (27). The product SPQ is a highly polar white solid. After three recrystallizations from ethanolwater (2:1), SPQ forms large white crystals that are chemically pure as judged by thin-layer chromatography and mass spectroscopy. Twenty-five to fifty grams of SPQ (final chemical yield -70%) are generally prepared at one time. SPQ is a zwitterionic inner salt with quinolinium


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FIG. 1. Chemical structures of quinolinium-based chloride-sensitive fluorescent indicators. Top: nomenclature for ring positions. Compound I, 6-methoxy-

4

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backbone substituted at the 6-position (see Fig. 1 for ring numbering convention) with methoxy and quaternized at the heterocyclic nitrogen with a sulfopropyl chain. SPQ is water soluble (95 mM at 20°C) and stable for long periods of time in solid form or aqueous solution. SPQ fluorescence is excited at ultraviolet wavelengths with maxima at 318 and 350 nm (Fig. 2A). There is a single broad emission peak centered at 450 nm and extending beyond 500 nm. The quantum yield of SPQ is 0.55, with molar absorptivities of 5,430 and 3,470 M-l* cm-’ at 318 and 350 nm, respectively. SPQ fluorescence is quenched by chloride with a linear Stern-Volmer relation as shown in Fig. 2B F,/F = 1 + Kcl[C1] (0 where F, is fluorescence in the absence of chloride, F is fluorescence in the presence of chloride, and Kc1 is the Stern-Volmer quenching constant (in M-l). K; provides a measure of the sensitivity of SPQ to quenching by molecule i. In aqueous buffers, & is 118 M-‘, giving a 50% decrease in fluorescence (F,/F = 2) at 8 mM chloride. In the absence of chloride, SPQ is quenched strongly by nonphysiological halides (KBr = 175 M-l, Kr = 276 M-l), thiocyanate (KscN = 211 M-l), and nitrite (KNo2 = 280 M-l). SPQ fluorescence is not altered by cations, phosphate, nitrate, and sulfate and is quenched weakly by the anions citrate (15 M-l), acetate (12 M-l), gluconate (7 M-l), bicarbonate (4 M-l), and succinate (8 M-l). Because SPQ does not contain a titrable function, its fluorescence is insensitive to pH in the range 3-10. When more than one quenching ion is present at the same time, Eq. 1 becomes F,/F = 1 + &[a] + &[b] + . . l , where a and b are quenching ions, and Ka and & are the respective Stern-Volmer constants. A linear dependence of F,/F on chloride concentration is consistent with a static (complex formation) or collisional quenching mechanism. The parallel decrease in fluorescence intensity and lifetime ratios shown in Fig. 2B indicates that the quenching mechanism is collisional and does not involve complexation between SPQ and chloride. Therefore, SPQ cannot buffer chloride. In addition, a collisional quenching mechanism implies that the quenching kinetics are diffusion limited and thus on the nanosecond time scale for chloride concentration >l mM. In stopped-flow experiments, quenching of SPQ by 50 mM chloride is >99% complete in 1 ms. The chloride indicators described below function by a similar collisional quenching mechanism. To tailor chloride-sensitive fluorescent indicators for

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FIG. 2. A: excitation and emission spectra of SPQ and MQAE in water. B: Stern-Volmer plot for quenching of SPQ and MQAE by chloride. Scales on ordinate are fluorescence in the absence of chloride divided by that in the presence of chloride (F,/F, 0) or lifetime in the absence of chloride divided by that in the presence of chloride (~JT, 0). Lifetimes are 26 ns (SPQ) and 22 ns (MQAE) in the absence of chloride. Line slopes give Stern-Volmer constants.

specialized applications, it is necessary to understand structure-activity relationships. Because there are no photophysical principles to define the mechanism by which SPQ fluorescence is quenched by chloride, an empirical set of structure-activity correlations was established by synthesizing and characterizing a series of SPQ analogues (27). A positive quaternary nitrogen in a heterocyclic ring structure was found to be necessary for chloride sensitivity and water solubility. A series of variations on ring structure and ring substituents was examined systematically. The bicyclic quinoline ring structure was the best backbone for a chloride-sensitive indicator. N-substituted sulfopropyl derivatives of isoquinoline (Stern-Vol-


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mer constant Kc1 = 36 M-l) or the tricyclic compounds acridine (5 M-l), phenanthridine (25 M-l), and 5,6- or 7,8-benzoquinoline (5 M-l) had reduced chloride sensitivity compared with N-sulfopropyl quinolinium (55 M-l). The position and nature of substituents on the quinoline ring altered chloride sensitivity remarkably. Substitution of N-sulfopropyl quinolinium with electrondonating groups including methoxy, methyl, and amino increased chloride sensitivity (~60 M-l), whereas substitution with electron-withdrawing groups including chloro, bromo, and carbonyl decreased chloride sensitivity (90% quenched by dansyl by an energy transfer mechanism (Li, Ketcham, and Verkman, unpublished data). A longer or more rigid spacer chain should overcome this difficulty. In preliminary studies, a tetramethylrhodamine-SPQ conjugate with nonoverlapping excitation and emission spectra may be suitable for ratiometric applications when different filter sets are used. Other synthetic products of the quinolinium chromophore may have interesting biological applications. Wolfbeis has synthesized a lipophilic analogue of SPQ consisting of 6-methoxyquinoline that is N-substituted by decyl alcohol (unpublished data). This compound might bind to lipophilic structures via the decyl chain, while the mobile aqueous quinoline moiety would sense local chloride concentration. A quinoline-based fiberoptic chloride sensor has been described (57); however, its sensitivity to chloride is very small compared with that of iodide. SPQ-conjugated dextrans and proteins can be readily prepared by modification of the sulfonate group of SPQ to a reactive sulfonyl chloride. The targeting of SPQ to selected sites may be useful for measurement of local chloride concentration. APPLICATIONS

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Studies of chloride transport in isolated membrane vesicles and liposomes are important for the definition of transport and regulatory mechanisms in homogeneous membrane preparations under defined conditions. Because SPQ and related analogues are highly polar and membrane impermeant, they can be entrapped in a sealed membrane preparation to function as a quantitative indicator of intravesicular chloride concentration. As demonstrated by the examples cited below, biological vesicles can be loaded with SPQ by passive incubation, by transient permeabilization procedures, including freeze thaw and hypotonic shock, and by endocytosis. Liposomes can be loaded by addition of SPQ at the time of formation. Studies of chloride transport in suspensions of sealed vesicles can be performed by cuvette or stoppedflow fluorimetry. Studies in individual vesicles can be performed by high-sensitivity quantitative imaging, or if vesicle size is large, by laser fluorescence activated cell sorting. General strategies to define chloride transport mechanisms. A variety of chloride conductive, cotransport, and


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3. Examples of chloride transport mechanisms in isolated vesicles. I: chloride conductance; II: chloride-bicarbonate exchange; III: sodium-potassium-chloride cotransport; IV: countertransport of chloride with sodium and bicarbonate. See text for additional details. FIG.

countertransport mechanisms exist for movement of chloride across membranes as shown in Fig. 3. The goal of vesicle studies is to define and characterize each transport mechanism and its regulatory properties. The well-established approach is to study the influence of ion and pH gradients, membrane potential, and transport inhibitors on chloride movement. In the selection of transport inhibitors it is important to avoid compounds that are fluorescent when excited with ultraviolet light. Stilbenes, amiloride, and bumetanide are examples of highly fluorescent compounds that cannot be used when chloride concentration is measured by indicator fluorescence. However, the dihydrostilbenes [e.g., dihydro+‘diisothiocyanostilbene-2,2’-disulfonic acid (HZDIDS)], several amiloride analogues, furosemide, and diphenylamine-2-carboxylate (DPC) are nonfluorescent when pure. Compounds that absorb ultraviolet light will decrease the apparent indicator intensity by absorption of excitation and/or emission light (inner filter effect). Chloride transport by a conductive or channel mechanism is examined from the influence of membrane potential and channel blockers on the dissipation of a chloride gradient. In the presence of slowly permeating ions, conductive chloride transport is slow because the movement of a counterion required to maintain electroneutrality is rate limiting. To make conductive chloride movement rate limiting, the membrane potential is clamped by use of an ion-ionophore pair. The potassiumvalinomycin pair is most effective; however, the gramicidin-sodium and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-proton pairs are suitable if potassium cannot be used. Gradients of potassium in the presence of valinomycin are used to establish membrane potentials to drive chloride movement. The Goldman equation predicts that a 60-mV interior-positive membrane potential will increase by -1.6-fold the rate of conductive chloride influx. Coupled chloride transport mechanisms are examined from the influence of ion gradients and selective inhibitors on chloride movement. Chloride transport would be trans-stimulated by proton-bicarbonate gradients and inhibited by stilbenes in the case of countertransport of chloride and hydroxyl or chloride and bicarbonate. Chloride transport would be &-stimulated by sodium gra-

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dients and often inhibited by furosemide in the case of sodium-chloride or sodium-potassium-chloride cotransport mechanisms. A complex coupled chloride transport mechanism such as countertransport of chloride with sodium-bicarbonate would be examined from the effects of sodium and bicarbonate gradients on chloride movement. The selective use of ion gradients and inhibitors can be used to define and characterize multiple chloride transporters in a single membrane. It must be recognized that the characterization of multiple chloride channels and the determination of channel kinetics requires single-channel electrical methods. Studies in isolated biomembrane vesicles. Highly purified plasma membrane or subcellular vesicles can be prepared from most intact tissues and cultured cells. In several types of plasma membrane vesicles with 100-200 nm diameter, SPQ can be loaded to give an acceptable fluorescence signal by passive diffusion in which suspended vesicles are incubated with 10 mM SPQ for 1224 h at 4°C or 30-60 min at 37°C (6, 7, 14, 21). SPQ can be loaded rapidly by a single freeze-thaw cycle or by a sudden hypotonic shock. The latter method is particularly useful for incorporation of specific enzymes at the time of SPQ loading (22), such as the catalytic subunit of protein kinase A (15). After entrapment, external SPQ is removed by two or three washes in buffer not containing SPQ or by Sephadex exclusion chromatography. In the biological membrane vesicles that have been studied, entrapped SPQ leaks out at rates of -‘Wldt)t=o

(2)

where F, is the total intravesicular determined from the relation. F

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where [Cl]i and [Cl], are chloride concentrations at the beginning of the experiment ([Cl]i = 0 mM) and after vesicle lysis or transmembrane chloride equilibration ([Clls = 50 mM). The determination of F, can also be accomplished by other detergents, by the Cl-OH ionophore tributyltin (see below), or by prolonged incubation.


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TIME (s) FIG. 4. Time course of fluorescence quenching in vesicles loaded with SPQ by 12- to 24-h incubation in the absence of chloride. A: data in apical (brush border) membrane vesicles from rabbit proximal tubule (6). Loading buffer consisted of 100 mM N-methyl-D-glucamine gluconate, 50 mM potassium gluconate, 100 mM sucrose, 5 mM HEPESTris, and 10 mM SPQ, pH 7.0. In the external buffer, SPQ was absent, and 50 mM gluconate was replaced by chloride. Valinomycin (5 pg/mg vesicle protein) was present. Where indicated, 0.05% Triton X-100 was added to lyse vesicles. See text for additional details. B: data in basolateralmembrane vesicles from rabbit proximal tubule (7). Loading an external buffer composition were the same as in A. pH of external buffer was 7.0 or 5.5. Arrows indicate Triton X-100 addition.

The effectiveness of different detergents and tributyltin to release SPQ or to equilibrate the chloride gradient varies considerably in different biological and artificial membranes. Figure 4B shows an application of the SPQ method to characterize chloride transport mechanisms in basolatera1 membrane vesicles from renal proximal tubule (7). An outwardly directed hydroxyl gradient (pHi, = 7.0; = 5.5) caused a remarkable increase in the initial P&t chloride flux. This increase was blocked by 100 PM H2DIDS. An outwardly directed sodium gradient stimulated chloride influx in the presence but not in the absence of bicarbonate. The dissociation constant (KD) for the stimulation of chloride influx by intracellular sodium was 7 mM. These experiments demonstrate the use of SPQ for analysis of complex chloride transport mechanisms: chloride-bicarbonate exchange and countertransport of chloride with sodium and bicarbonate. SPQ has been used to study chloride transport in a variety of biological vesicles from animal organs, includ-

ing the kidney (6, 7), trachea (14), and placenta (9, Zl), and from plants (39). Effects of chloride transport regulation by protein kinases and other effecters have been examined (32). Where tested, chloride flux kinetics determined by SPQ fluorescence were not different from those determined by 36C1 uptake. Studies of 36C1 uptake in the presence and absence of SPQ showed that SPQ did not itself inhibit or activate chloride transport. An interesting application of SPQ in vesicles is the measurement of NaCl reflection coefficient by solvent drag (38, 50). Vesicles containing SPQ but not chloride were subjected to an outwardly directly osmotic gradient in the presence of external chloride. Because of the high sensitivity of SPQ fluorescence to small increases in intravesicular chloride concentration, small amounts of chloride influx by inward solvent drag are measurable. It was found by both solvent drag and induced osmosis methods that the NaCl reflection coefficient was near unity in apical and basolateral vesicles from proximal tubule. Studies in liposomes. Chloride indicators have direct application to functional studies of reconstituted chloride transporters in liposomes. SPQ can be entrapped in liposomes by inclusion in the buffer at the time of liposome formation. Sonication, extrusion, or detergent dialysis methods for liposome formation effectively entrap SPQ. External SPQ is removed rapidly (~2 min) by exclusion chromatography. SPQ is slowly permeant across liposome membranes. In 90% phosphatidylcholine-lo% cholesterol liposomes, SPQ leakage was 07 - Cl, cence objective is essential. The glycerol immersion x40 11 5 t -I 5 mtn quartz objective from Leitz (numerical aperture 0.65) has W -Clm E C + Chl/s excellent ultraviolet transmission and numerical aper-Cl, t KSCN t ture-to-magnification ratio, low autofluorescence, and I 0 1 1 relatively long working distance (0.35 mm). For longer 09 + FUROS, working distances in Ussing chamber studies, the Leitz 08 - Cl, ~25 long-working-distance objective (7-mm working dis07 i O;\._itance, numerical aperture 0.25) has low autofluorescence. 5 min 06 I t ISO, The cells should be grown on a thin (0.08-0.10 mm) glass FIG. 8. Time course of intracellular SPQ fluorescence in Swiss 3T3 or fused silica cover slip for direct contact of the cell-free fibroblasts (A) and canine airway epithelial cells in primary culture (B surface of the cover slip with the immersion objective and C). A: fibroblasts were grown on a glass cover slip and perfused (4). Most plastics are highly fluorescent. For separate continuously (4). Chloride was added to and removed from perfusion apical and basolateral perfusion in polarized epithelia, solution by replacement of 110 mM chloride with equivalent gluconate. Where indicated, 150 mM KSCN + 5 PM valinomycin was added to the Nucleopore filter provides a good porous support quench intravesicular SPQ fluorescence. B: airway cells were grown on with acceptably low autofluorescence and little SPQ a porous filter and mounted in a dual perfusion chamber for separate binding (Chao, Hartmann, and Verkman, unpublished control of mucosal and serosal perfusion solutions (Chao et al., unpubobservations). lished observations). Chloride (110 mM) was added to and removed from mucosal solution with 0 mM chloride in the serosal solution The emitted fluorescence is filtered by a cut-on filter throughout. Where indicated, 10 PM (-)-isoproterenol (ISO) was added at 420 nm or by a broad-band interference filter centered to the serosal solution to stimulate apical membrane chloride conductat 450 nm (SPQ) or 465 nm (MQAE). The cut-on filter ance. C: chloride was first added to serosal and mucosal solutions. should be of the low autofluorescence series. The fluoWhere indicated, mucosal chloride was removed, and 100 PM serosal furosemide (FUROS) and 10 PM serosal (-)-isoproterenol were added. rescence detection system can be a camera (microchannel Serosal chloride was then removed to give maximal fluorescence (see plate intensifier, silicon-intensified target camera, or text for additional details). cooled charge-coupled device) for single-cell analysis or a photomultiplier for area-integrated detection. When lines have similar appearance immediately and at 60 min single-cell information is not essential, photomultiplier after loading with SPQ. SPQ staining was also uniform detection has the important advantages of rapid data when cells were examined with a Nipkov wheel confocal analysis and higher sensitivity so that continuous illumicroscope with 0.6-pm optical sections. Similar images mination with low light intensity in possible. Imaging of cells loaded with BCECF and fura- show a slightly methods should be reserved for studies of heterogeneous nonuniform intracellular distribution when photocell populations and for specialized microinjection appligraphed with the same microscope optics. The highly cations. uniform distribution of SPQ is probably due to its high Transport studies in cells. Figure 8A shows a continupolarity and metabolic stability, resulting in little binding ous recording of SPQ fluorescence in Swiss 3T3 fibroto intracellular structures. Once loaded, SPQ remains entrapped in cells for long blasts that have been loaded with SPQ by the hypotonic periods. As will be discussed below in reference to Fig. 8, procedure (4). The excitation source was a 100-W tungthe SPQ leakage rate from Swiss 3T3 fibroblasts and sten-halogen lamp with low output at 365 nm. The light was attenuated -103-fold by an OD-2 neutral-density cultured epithelial cells is 2-10%/h at 37OC. Continuous measurement of intracellular chloride concentration can filter, black glass filter (UGl, Schott Glass) and 365 t 5-nm six-cavity interference filter (Omega Optical). No be performed for >2 h in single cells. There are several photobleaching or photodynamic cell injury was detected important cautions. In some ion-substitution experiafter 2 h of continuous illumination. With the use of an ments in which cell volume increases, SPQ has been unattenuated arc lamp, SPQ fluorescence is photofound to leak out of cells rapidly. Addition of an imperbleached by 50% in -10 s, similar to the photobleaching meant solute (e.g., 50 mM mannitol) to the perfusion solution minimized this difficulty. Precipitation of cal- kinetics of fluoresceins. However, unlike the fluoresceins, is sensitive to solucium phosphate or other poorly soluble compounds in quinoline indicator photobleaching the perfusion buffer also causes prompt SPQ leakage. tion ionic strength and not to oxygen. In the absence of

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ions, SPQ photobleaching is decreased by X0-fold. SPQ fluorescence in fibroblasts responds to changes in the external chloride concentration (Fig. 8A). The cycles of fluorescence decrease and increase are reproducible over 2 h at 37”C, with a 6%/h fluorescence decrease due to SPQ leakage. At the end of the experiment, KSCN and valinomycin are added to give a twopoint calibration. The first calibration point is the fluorescence at 0 mM chloride at the start of the experiment. The second calibration point is the fluorescence after KSCN addition. KSCN quenches >99% of SPQ fluorescence so that the difference between the initial and final intensities represents the total intracellular SPQ signal. Equations 2 and 3 are used to calculate absolute chloride flux rates (see section on calibration below). Figure 8, B and C, shows examples of hormonal effects on conductive chloride transport in canine airway epithelial cells in primary culture (Ref. 5 and Chao et al., unpublished observations). Airway epithelial cells contain an apical membrane chloride channel that is activated by isoproterenol via the adenosine 3’,5’-cyclic monophosphate (CAMP) pathway. Experiments were performed in a dual perfusion chamber with rapid and independent control of solutions bathing the mucosal (apical) and serosal (basolateral) surfaces. In Fig. 8B, serosal application of isoproterenol causes a remarkable increase in the rate of chloride influx and efflux through the apical membrane upon chloride addition to and removal from the mucosal solution. In Fig. 8C, the conditions were modified so that stimulation of the apical chloride channel would cause a prompt and dramatic change in the time course of fluorescence. To minimize baseline chloride conductance, the cells were preincubated with 10 PM indomethacin for 15 min. Chloride was first added to both mucosal and serosal solutions to increase cell chloride activity. Furosemide was added to the serosal solution to inhibit sodium-potassium-chloride cotransport partially. Under these conditions, removal of mucosal chloride causes little decrease in cell chloride (increase in SPQ fluorescence) under basal conditions. Upon stimulation of the apical chloride channel by serosal isoproterenol, the fluorescence promptly increases to near-maximal levels (corresponding to 0 mM chloride) as shown by the small effect of serosal chloride removal. These data show that the SPQ fluorescence method is a sensitive assay of plasma membrane chloride transport mechanisms and regulation. In fibroblasts, stilbene-sensitive chloride-bicarbonate exchange, furosemide-sensitive sodium-potassium-chloride cotransport, and calcium-activated chloride conductance have been characterized (4). In airway cells, CAMP-dependent DPCinhibitable apical chloride conductance and furosemidesensitive basolateral sodium-potassium-chloride cotransport have been characterized (5). Effects of calcium elevation, protein kinase C activation, and prostaglandins have been evaluated (Chao et al., unpublished observations). In sweat duct epithelial cells in primary culture, chloride transport is greatly decreased in tissues from patients with cystic fibrosis compared with normal controls (41). In cultured kidney mesangial cells (37), SPQ was used to examine effects of angiotensin II on chloride permeability. A GABA-stimulated chloride con-

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ductance was studied in cerebellar granular cells by similar methods (12). Transport studies in intact epithelia. SPQ has been used to study chloride transport in the isolated perfused proximal tubule (26) and medullary thick ascending limb (31). In proximal tubule, loading was performed by perfusion of the tubule lumen with 20 mM SPQ for 10 min at 37°C. In thick ascending limb, loading was performed by the hypotonic shock method before tubule perfusion. In contrast to the slow leakage rates in cultured cells, SPQ leaked out of proximal tubule at a rate of 5%/min at 37OC. The rapid leakage rate may be related to the highly convoluted surface of the proximal tubule apical and basolateral membranes, resulting in a high surfaceto-volume ratio. However, despite the rapid leakage rate, SPQ fluorescence was calibrated successfully and the physiological intracellular chloride activity was measured to be 21 mM. Rapid changes in cell chloride activity were measured upon chloride addition to and removal from the bath solution. Another application of SPQ in kidney tubule physiology is the measurement of transepithelial chloride transport and solvent drag in the proximal tubule (45a). In these studies SPQ is used as an extracellular indicator of chloride concentration. The tubule lumen is perfused with SPQ and a chloride-insensitive fluorophore (fluorescein sulfonate) at nanoliter per minute flow rates. The dissipation of transepithelial chloride gradients along the length of the tubule is determined by ratio imaging of SPQ-to-fluorescein sulfonate fluorescence intensities under steady-state conditions. From the effects of transepithelial osmotic gradients on chloride transport, it was concluded that the transepithelial NaCl reflection coefficient in rabbit proximal straight tubule was in the range of 0.90-l (45a). In proximal tubule ceils loaded with SPQ, the basolateral membrane NaCl reflection coefficient was in the range of 0.95-l based on measurements of osmotic gradient effects on serosal-to-cell chloride movement. Cell toxicity. Where tested, SPQ did not affect oxygen consumption and lactate production by cultured cells and kidney tubule suspensions (4, 26). Loading of tracheal epithelia with SPQ did not alter the transepithelial short-circuit current or the response to stimulation by flagonists (5). Inclusion of 5 mM SPQ in the growth medium for 48 h did not alter the growth or appearance of Swiss 3T3 fibroblasts or renal epithelial cells (4). Intravenous injection of up to 200 mg/kg SPQ in rabbits produced no hypotension or apparent ill effects (1). These observations suggest that SPQ toxicity to cell metabolism is low. It is stressed that indicator toxicity must be established in every cell type by testing whether important cell functions are disturbed. Intracellular calibration of SPQ fluorescence. The intracellular calibration of indicator fluorescence vs. chloride activity is important for determination of absolute chloride activities and chloride flux rates. Analogous to the methods established for calibration of pH and calcium, an ionophore procedure was developed to equalize intracellular and external chloride activities. Cells are incubated with solutions containing high potassium, the potassium-proton exchange ionophore nigericin, and the


INVITED

chloride-hydroxyl exchange ionophore tributyltin. The high potassium and nigericin cause membrane depolarization and collapse of pH gradients. The tributyltin causes collapse of the chloride gradient. Figure 9A shows calibration data in LLC-PK1 cells. SPQ fluorescence responds reversibly to changes in external chloride concentration. The equalization of intracellular and external chloride concentrations by this ionophore calibration procedure was validated by 36C1 and 3-O-[3H]methylglucase double-label experiments (4). The disadvantages of the nigericin-tributyltin calibration method are the considerable cell toxicity and marginal effectiveness of tributyltin in some cell systems. An alternati .ve calibration procedure that does not involve the use of ionophores is to perfuse cells with high-potassium low-sodium buffer containing different chloride concentrations. Because primary active chloride transport does not exist, intracellular and external chloride activities will eventually equilibrate. This method was used to calibrate SPQ fluorescence in Swiss 3T3 fibroblasts (4). Simultaneous measurement of intracellular chloride activity by indicator fluorescence and chloride- ,sensitive microelectrodes has not been done. The calibration data for LLC-PK1 cells and Swiss 3T3 fibroblasts are expressed in the form of a Stern-Volmer plot in Fig. 9B. The data fit a line as in Fig. 2B; however, Kc1 (13 M-‘) is -IO-fold lower than that for SPQ in aqueous solutions (118 M-l). In intact kidney proximal tubule and several cultured cell systems examined, the Stern-Volmer constant is in the range of 12-18 M-l. The possible reasons for the decreased sensitivity of intracellular SPQ to quenching by chloride are 1) deA ki $ 1.0 t3 0.9 W 5

0.8

3 IL w

0.7

I= a>

0.3 t 0.2

0.6 0II 17 1 35

I 55

I

90

I

35

0

‘\\, I

KU

I

KSCN

ii [r

Of’.

0

20

40

60

80

100

WI mw FIG. 9. Calibration of SPQ fluorescence vs. chloride in LLC-PK1 cells and Swiss 3T3 fibroblasts (4). A: solutions contained 120 mM potassium, 25 mM sodium, 5 mM HEPES-Tris, 160 mM chlorideisethionate, and the ionophores nigericin (5 PM) and tributyltin (10 PM). B: data from A (0) and a calibration in fibroblasts (M) are expressed in the form of a Stern-Volmer plot.

C385

REVIEW

creased chloride diffusion in the intracellular milieu, 2) limited geometric access of chloride to SPQ because of SPQ binding to cytoplasmic components, and 3) quenching of SPQ by nonchloride intracellular anions even in the absence of chloride. Reasons 1 and 2 do not apply. Inc reasing t ‘he viscosity of aqueous solutions fivefold (5 cP) by addition of glycerol or dextran altered Kc1 by 40%. The cytoplasmic viscosity is ~2 CP (17). Studies of SPQ anisotropy decay in intact cells showed little binding of SPQ to intracellular components (4). However, the SPQ fluorescence lifetime in cells in the absence of chloride (3.5 ns) was -lo-fold less than that in aqueous solutions (26 ns), providing direct evidence that SPQ is quenched by components other than chloride in cells. These results stress the importance of performing a calibration experiment in each cell type for calculation of intracellular activities and absolute flux rates. In addition, the quenching of SPQ by intracellular components raises the possibility that pH and cell volume could alter SPQ fluorescence. These effects should be examined in each cell type from the influence of cell pH and volume on indicator fluorescence in the absence of chloride. In Swiss 3T3 fibroblasts, SPQ fluorescence is changed by ~5% in the pH range of 6-8, suggesting that alteration of protein charge has little influence on SPQ fluorescence in this cell type. Large changes in cell volume (>50% shrinkage) cause a small (-10%) but significant decrease in SPQ fluorescence. DATA

ANALYSIS

BY MATHEMATICAL

MODELING

To obtain biologically useful information about chloride transport and regulatory mechanisms in living cells, quantitative mathematical modeling is an extremely useful and underutilized approach. The interpretation of electrical , chemical, and fluorescence data on the cell .ular response to externa .l ion substitution or addi tion of regulatory ligands is a very difficult task, especially in polar epithelia. The response of intracellular ion activities, membrane potentials, and volume is the result of a set of complex interactions among many transport systems. Mathematical modeling provides an integrated description of cell function in which to evaluate data from widely different types of experiments. Several thermodynamic (55) and kinetic (20, 28, 51; Ishibashi, Hartmann, and Verkman, unpublished observations) approaches have been used to model epithelial systems. The immediate goal of an epithelial transport model is to calculate the time course of ion activities (Na+, K+, Cl-, HCO:, etc.), pH, membrane potentials, and cell volume in response to changes in the permeabilities of transporting systems. Transporter permeabilities might be modified by agonist or antagonist action or by ion substitution. The applications of a mathematical model are to interpret experimental data, design experiments, test hypotheses, and integrate observable physiological phenomena with single-cell data. The information necessary for the model formulation is the identities of individual transporters (ion stoichiometries and saturabilities, if known) and values for intracellular pH, ion activities, membrane potentials, and transcellular and paracellular net ion fluxes under a set of standard steady-state conditions.


C386

INVITED IFLUID

REVIEW

A

COMPOSITION1

apical

Na NEUTRAL

basolateral

I

FLUXES

*

I

K 2K 3Na

K

T ,/

, MODI

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-

CALCULATE ELECTROGENIC

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-L0

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3

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A=

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CALCULATE ACTIVITIES,

I

Na 2CI K

0 8

*

GPc

NEW CELL pH AND VOLUME

I.06 ez 0.9 Id 1

0 E 0.8 LT 2 0.7 i2 0.6 f +w-n/s

RESULTS

FIG. 10. Block diagram for numerical solution of a mathematical model of epithelial ion transport. +I and \F/*, apical and basolateral membrane potentials; Ia, Ib, and It are total apical, basolateral, and paracellular ion currents, respectively. For open-circuit conditions, I, = Ib = -It; for closed-circuit conditions, Ia = Ib and \cla = #b. See text for further explanation.

Figure 10 shows the numerical method for calculation of the time course of cell pH, ion activities, and membrane potentials in an epithelial transport model (51). The fluid composition controller specifies initial solute concentrations and changes in fluid composition and transporter activities for simulated experiments. The neutral ion fluxes that do not depend on membrane potential are calculated from the chemical gradients. The electrogenic ion fluxes are calculated from the electrochemical driving force using a guess for the membrane potentials. The correct membrane potentials are calculated iteratively by the generalized Newton’s method from the requirement of charge neutrality under opencircuit or short-circuit conditions. The new cell ion activities, pH, and volume at time t + dt are calculated from the ion and volume fluxes at time t. The equations to relate transporter turnover number to the electrochemical driving force and transporter characteristics have been developed using a hybrid thermodynamickinetic approach (5 1). Figure 11A shows a schematic of a chloride-secretory epithelial cell in the airway that has been modeled mathematically (20). A chloride conductance contralateral to a chloride symporter and potassium channel is also found in the intestine, cornea, kidney thick ascending limb,

/

/T-

5 min 1 I

+ ISOS

11. A: localization of membrane transporters in a mathematical model of ion transport in chloride-secreting airway epithelia (20). Transporters 1-4 are simple ion conductances. Transporter 5 is an ATP-driven 3Na-2K pump. Transporters 6 and 7 are cation-coupled chloride symporters. G,, indicates a generalized paracellular conductance. Proposed regulatory sites of calcium and CAMP are shown. B: simulation of the experiment shown in Fig. 8C in which apical membrane chloride permeability (transporter 3) increases lo-fold upon addition of serosal isoproterenol. Parameters were appropriate for a monolayer of canine tracheal epithelial cells (20). Before addition of isoproterenol or furosemide (to inhibit basolateral symport by 90%), steady-state transcellular fluxes (mucosal to serosal) of sodium and chloride were +O. 18 and -0.04 peg. cms2 . h-l, respectively. Intracellular ion activities were (in mM) 21 (sodium), 69 (potassium), and 37 (chloride); cell membrane potential under short-circuit conditions was -60 mV. All basolateral chloride transport was assumed to be potassium dependent. FIG.

and sweat duct. The basolateral symporter drives intracellular chloride activity above electrochemical equilibrium by secondary active transport. Chloride then exits the cell through the apical membrane by passive transport. In Fig. llB, the mathematical model is applied to simulate the complex experiment given in Fig. 8C. Model parameters were chosen for the canine tracheal epithelium where isoproterenol stimulates apical membrane chloride conductance by lo-fold (20). The model correctly predicts that removal of mucosal chloride in the absence of isoproterenol causes little chloride efflux and


INVITED

that isoproterenol addition causes nearly complete efflux with a time course similar to that measured experimentally. A similar kinetic model of chloride transport in kidney proximal tubule has been developed recently for the analysis of the basolateral chloride exit step using microelectrode measurements of basolateral membrane potential and cell chloride (Ishibashi et al., unpublished observations). A series of different proposed chloride exit mechanisms were modeled and systematically compared with experimental results. It was concluded that basolatera1 chloride exit occurs primarily through countertransport of chloride with sodium and bicarbonate and that 40% of transepithelial chloride transport occurs by a transcellular route. SUMMARY

AND

FUTURE

DIRECTIONS

Chloride-sensitive fluorescent indicators have applications in the study of transport and regulatory mechanisms in isolated membrane vesicles, reconstituted liposomes, and living cells and tissues. The currently available indicators have good chloride sensitivity and selectivity, rapid response, low membrane permeability, and low toxicity. However, the loading of these indicators into living cells requires a permeabilization procedure, and the measurement of chloride concentration by a dual-wavelength ratio is not possible. An immediate goal is the synthesis of indicators that can be loaded into cells by passive diffusion and that have a spectral shift in response to chloride. Another important goal is the synthesis of a chloride-sensitive indicator with excitation wavelength >400 nm. New classes of indicators need to be developed that are not based on the quinoline chromophore. The high sensitivity and spatial resolution of fluorescence detection makes possible a number of novel applications of chloride-sensitive fluorescent indicators. Intracellular gradients of chloride have been proposed to play an important role in neuromuscular and synaptic transmission. Although an ideal ratiometric indicator has not been synthesized, imaging of anisotropy (10, 16) or of coinjected indicators with different spectra would give direct information about local chloride gradients. The use of chloride-sensitive indicators as fluid-phase markers of endocytosis should permit the measurement of endosomal chloride concentration in intact cells using confocal microscopy. The ability to microinject SPQ together with impermeant transport regulators provides a powerful approach to examine transport regulation in single cells. This approach should have direct application to single cell complementation studies of the cystic fibrosis defect. I thank Prof. Roger Ketcham for an ongoing collaboration in the organic synthesis of chloride-sensitive fluorescent indicators and Drs. Anthony C. Chao, Thomas Hartmann, Hae-Rahn Bae, Ming Li, and Lan-Bo Shi for obtaining much of the recent data and for critical review of the manuscript. This work was supported by National Institutes of Health Grants DK-39354, DK-35124, HL-42368, and DK-16095; a grant from the National Cystic Fibrosis Foundation; and a Grant-in-Aid from the American Heart Association. A. S. Verkman is an Established Investigator of the American Heart Association. The source code and compiled software (IBM BASIC) for the mathematical model of epithelial cells is available from the author

REVIEW upon request. Address for reprint requests: A. S. Verkman, East Tower, Cardiovascular Research Institute, San Francisco, CA 94143.

C387 1065 Health Sciences Univ. of California,

REFERENCES 1. BAE, H.-R., AND A. S. VERKMAN. Regulation of chloride transport in endocytic vesicles from kidney proximal tubule which contain a proton ATPase (Abstract). Kidney Int. 37: 559, 1990. 2. BRAHM, J. The red cell anion transport system: kinetics and physiological implications. Sot. Gen. Physiol. Ser. 43: 141-150, 1988. 3. CALAFUT, T., M. SOLOMON, R. GELMAN, AND J. A. DIX. Chloride/ bicarbonate exchange in human red cells characterized by a fluorescent probe (Abstract). Biophys. J. 53: 528a, 1988. 4. CHAO, A. C., J. A. DIX, M. SELLERS, AND A. S. VERKMAN. Fluorescence measurement of chloride transport in monolayer cultured cells: mechanisms of chloride transport in fibroblasts. Biophys. J. 56: 1071-1081, 1989. 5. CHAO, A. C., J. H. WIDDICOMBE, AND A. S. VERKMAN. Chloride transport mechanisms in cultured canine tracheal epithelial cells measured by an entrapped fluorescent indicator. J. Membr. Biol. 113: 193-202,199o. 6. CHEN, P.-Y., N. P. ILLSLEY, AND A. S. VERKMAN. Renal brush border chloride transport mechanisms characterized using a fluorescent indicator. Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol. 23): F114-F120, 1988. 7. CHEN, P.-Y., AND A. S. VERKMAN. Sodium-dependent chloride transport in basolateral membranes vesicles isolated from rabbit proximal tubule. Biochemistry 27: 655-660, 1988. 8. CUPPOLETTI, J. Bumetanide-sensitive chloride channel in lysosomes measured using 6-methoxy-N[ 3-sulfopropyllquinolinium (Abstract). J. Cell Biol. 107: 354a, 1988. 9. DECHECCHI, M. C., AND G. CABRINI. Chloride conductance in membrane vesicles from human placenta using a fluorescent probe. Implications for cystic fibrosis. Biochim. Biophys. Acta 945: 113120, 1988. 10. DIX, J. A., AND A. S. VERKMAN. Mapping of anisotropy in single cells by ratio imaging: application to cytoplasmic viscosity. Biophys. J. 57: 231-240, 1990. 11. DUNN, S. M. J., C. MARTIN, M. W. AGEY, AND R. MIYAZAKI. Functional reconstitution of the bovine brain GABA receptor from solubilized components. Biochemistry 28: 2545-2551, 1989. 12. ENGBLOM, A. C., I. HOLOPAINEN, AND K. E. AKERMAN. Determination of GABA receptor-linked Cl fluxes in rat cerebellar granule cells using a fluorescent probe SPQ. Neurosci. Lett. 104: 326-330, 1989. 13. FONDACARO, J. D. Intestinal ion transport and diarrhea1 disease. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): Gl-G8, 1986. 14. FONG, P., N. P. ILLSLEY, J. H. WIDDICOMBE, AND A. S. VERKMAN. Chloride transport in apical membrane vesicles from bovine trachea. J. Membr. Biol. 104: 233-239, 1988. 15. FORSYTH, G. W., AND S. F. GARIEL. Activation of chloride conductance in pig jejunal brush border vesicles. J. Membr. Biol. 107: 137-144,1989. 16. FUSHIMI, K., J. A. DIX, AND A. S. VERKMAN. Cell membrane fluidity in the intact kidney proximal tubule measured by orientation independent anisotropy imaging. Biophys. J. 57: 241-254, 1990. 17. FUSHIMI, K., AND A. S. VERKMAN. Vasopressin does not alter fluidity of collecting tubule measured by orientation-independent anisotropy imaging and picosecond microfluorimetry (Abstract). Biophys. J. 57: 482a, 1990. 18. GARCIA-CALVO, M., A. RUIZ-GOMEZ, J. VAZQUEZ, E. MORATO, F. VALDIVIESO, AND F. MAYOR. Functional reconstitution of the glycine receptor. Biochemistry 28: 6405-6409, 1989. 19. HAROOTUNIAN, A. T., J. P. KAO, B. K. ECKERT, AND R. Y. TSIEN. Fluorescence ratio imaging for cytosolic free Na in individual fibroblasts and lymphocytes. J. Biol. Chem. 264: 19458-19467, 1989. 20. HARTMANN, T., AND A. S. VERKMAN. Model of ion transport regulation in chloride-secreting airway epithelial cells: integrated description of electrical, chemical and fluorescence measurements. Biophys. J. 58: 391-401, 1990. 21. ILLSLEY, N. P., C. GLAUBENSKLEE, B. DAVIS, AND A. S. VERKMAN. Chloride transport across the placental microvillus membrane


C388

INVITED

measured by a chloride-sensitive

fluorescent probe. Am. J. Physiol.

entrapment

AND B. DAVIS. brush border vesicles.

in placental

Anal Biochem. 175: 106-113, 1989. 23. ILLSLEY, N. P., AND A. S. VERKMAN.

Membrane chloride transport measured using a chloride-sensitive fluorescent indicator. Biochem-

istry 26: 1215-1219, 1987. K., S. SASAKI, AND 24. ISHIBASHI,

N. YOSHIYAMA. Intracellular chloride activity of rabbit proximal straight tubule. Am. J. Physiol. 255

(Renal Fluid 25. JANOSHAZI,

Electrolyte

Physiol.

Membr. 26. KRAPF,

112: 25-37, 1989. C. A. BERRY, AND

24): F49-F56,

1988.

A., AND A. K. SOLOMON. Interactions among anion, cation and glucose transport proteins in the human red cell. J. Biol.

R., A. S. VERKMAN. Estimation of intracellular chloride activity in isolated perfused rabbit proximal tubules using a fluorescent probe. Biophys. J. 53: 955-962, 1988.

27. KRAPF,

R., N. P. ILLSLEY,

H. C. TSENG,

AND A. S. VERKMAN.

Structure-activity relationships of chloride-sensitive fluorescent indicators for biological application. Anal. Biochem. 169: 142-150, 1988. 28. LATTA,

R., C. CLAUSEN,

General method for of epithelial transport

AND L. C. MOORE.

the derivation and numerical solution models. J. Membr. Biol. 82: 67-82, 1984.

J. P. CLANCY, C. M. M. J. WELSH. Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia. Science Wash. DC 244: 1353-1356,

29. LI, M., J. D. MCCANN, LIEDTKE, A. C. NAIRN,

M. P. ANDERSON, P. GREENGARD, AND

1989. 30. LIEDTKE, C. M. Regulation of chloride Rev. Physiol. 51: 143-160, 1989. 31. MALONY, D. A., AND M. D. BREYER.

40. RAJU, B., E. MURPHY,

transport in epithelia. Annu.

Measurement of intracellular Cl in isolated perfused mouse medullary thick ascending limbs with the fluorescent dye 6-methoxy-N- [3-sulfopropyl] quinolinium (SPQ) (Abstract). Kidney Int. 35: 485, 1989. 32. MALONY, D. A., AND P. S. MEHTA. CAMP dependent protein kinase stimulated conductive Cl efflux from basolateral membrane vesicles of the rabbit medullary thick ascending limb (Abstract). Kid-

R. D. HALL, AND R. E. LONDON. for measuring cytosolic free magnesium.

L. A. LEVY,

A fluorescent indicator

255 (Cell Physiol. 24): C789-C798, 1988. 22. ILLSLEY, N. P., J. GOODMAN, A. S. VERKMAN,

Macromolecule

REVIEW

Am. J. Physiol. 256 (Cell Physiol. 41. RAM,. S. J., AND K. L. KIRK. Cl

25): C540-C548,

1989.

permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl permeability in cystic fibrosis. Proc. N&Z. Acad. Sci. USA 24: 10166-10170, 1989. 42. ROE, J. N., F. C. SZOKA, AND A. S. VERKMAN. Optical measurement of aqueous potassium concentration by a hydrophobic indicator in phospholipid vesicles. Biophys. Chem. 33: 295-302, 1989. 43. ROE, J. N., F. C. SZOKA, AND A. S. VERKMAN. A fiberoptic sensor for potassium detection based on fluorescence energy transfer.

Analyst 115: 353-358, 1990. 44. SCHOUMACHER, R. A., R. L. SHOEMAKER, D. R. HALM, E. A. TALLANT, R. W. WALLACE, AND R. A. FRIZZELL. Phosphorylation

fails to activate chloride channels from cystic fibrosis airway cells.

Nature Land. 330: 752-754, 1987. 45. SHI, L.-B., D. BROWN, AND A.

S. VERKMAN. Water, urea and proton transport properties of endosomes containing the vasopressin-sensitive water channel from toad bladder. J. Gen. Physiol. 95:

941-960,199O. &a.SHI, L.-B., K. FUSHIMI, urement of transcellular

AND A. S. VERKMAN. Solvent drag measand basolateral membrane NaCl reflection coefficient in mammalian proximal tubule. J. Gen. Physiol. In press. 46. SHI, L.-B., AND A. S. VERKMAN. Very high water permeability in vasopressin-dependent endocytic vesicles in toad urinary bladder. J. Gen. Physiol. 47. STANTON, B.

94: 1101-1115,

1989.

A. Electroneutral NaCl transport by distal tubule: evidence for Na/H-C1/HC03 exchange. Am. J. Physiol. 254 (Renal

Fluid Electrolyte Physiol. 23): FBO-F86, 1988. 48. SWANSON, J. Fluorescent labeling of endocytic Methods in Cell Biology, edited by Y.-L. Wang

compartments. In: and D. L. Taylor. San Diego, CA: Academic, 1989, vol. 29, p. 137-152. 49. THOMAS, J. A. Intracellularly trapped pH indicators. Sot. Gen. Physiol. Ser. 40: 311-325, 1986. 50. VERKMAN, A. S. Mechanisms and regulation in renal epithelia. Am. J. Physiol. 257 (Cell C850,1989. 51. VERKMAN, A. S., AND R. J. ALPERN. Kinetic

of water permeability Physiol.

26):

C837-

700, 1986. 35. MINTA, A., J. P. KAO, AND R. Y. TSIEN.

transport model for cellular regulation of pH and solute concentration in the renal proximal tubule. Biophys. J. 51: 533-546, 1987. 52. VERKMAN, A. S., W. LENCER, D. BROWN, AND D. A. AUSIELLO. Endosomes from kidney collecting tubule contain the vasopressinsensitive water channel. Nature Land. 333: 268-269, 1988. 53. VERKMAN, A. S., M. SELLERS, A. C. CHAO, T. LEUNG, AND R. KETCHAM. Synthesis and characterization of improved chloridesensitive fluorescent indicators for biological applications. Anal.

J. Biol. 36. MINTA,

Biochem. 178: 355-361,1989. 54. VERKMAN, A. S., R. TAKLA, WIDDICOMBE. Quantitative

ney Int. 37: 567, 1990. P. L. Incorporation of macromolecules 33. MCNEIL, In: Methods in CeLLBiology, edited by Y.-L. Wang

into living cells. and D. L. Taylor. San Diego, CA: Academic, 1989, vol. 29, p. 153-174. 34. MELLMAN, I., R. FUCHS, AND A. HELENIUS. Acidification of the endocytic and exocytic pathways. Annu. Rev. Biochem. 55: 663Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein compounds.

Chem. 264: 8171-8178,1989. A., AND R. Y. TSIEN. Fluorescent indicators sodium. J. BioZ. Chem. 264: 19449-19457, 1989. 37. OKUDA, T., M. NARUSE, E. OGATA, AND K. KUROKAWA.

for cytosolic

Regulation of intracellular Cl concentration of mesangial cells: effects of angiotensin II, fetal calf serum and Cl channel blockers (Abstract).

Kidney Int. 37: 211, 1990. 38. PEARCE, D., AND A. S. VERKMAN.

NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles: measurement by induced osmosis and solvent drag. Biophys. J. 55: 1251-

1259,1989. 39. POPE, A.

J., AND R. A. LEIGH. The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles. Planta 53: 114-120, 1988.

transport

in phospholipid

B. SEFTON, C. BASBAUM, AND J. H. fluorescence measurement of chloride vesicles. Biochemistry 28: 4240-4244,

1989. 55. WEINSTEIN,

A. M. Nonequilibrium thermodynamic model of the rat proximal tubule epithelium. Biophys. J. 44: 153-170, 1983. 56. WOLFBEIS, 0. S., AND E. URBANO. Synthesis of fluorescent dyes. XIV. Standards for fluorescent measurement in the near neutral pH-range. J. Heterocyclic Chem. 19: 841-843, 1982. 57. WOLFBEIS, 0. S., AND E. URBANO. Optical sensors for the continuous determination of halides. Anal. Chem. 56: 427-429, 1984. 58. YE, R., L.-B. SHI, W. LENCER, AND A. S. VERKMAN. Functional colocalization of water channels and proton pumps on endocytic vesicles from proximal tubule. J. Gen. Physiol. 93: 885-902, 1989.


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