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1 Journal of Physiology (1992), 449, pp With 4 figures Printed in Great Britain CYCLIC AMP- AND fl-agonist-activated CHLORIDE CONDUCTANCE OF A TOAD SKIN EPITHELIUM BY NIELS J. WILLUMSEN, LARS VESTERGAARD AND E. HVIID LARSEN From the Zoophysiological Laboratory A, August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen 0, Denmark (Received 1 July 1991) SUMMARY 1. The control by intracellular cyclic AMP and,-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl--sensitive microelectrodes was performed at x 500 magnification. For blocking the active sodium current 50 ftm-amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mv, the preparations generated clamping currents of ta/cm2 (mean + S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was mv with a fractional resistance of the basolateral membrane (frb) of (n = 54), and an intracellular Cl- activity of mm (n = 24). 3. At V = 0 mv, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 /im) or a,-adrenergic agonist, isoprenaline (5 /tm) resulted in a sixfold increase in transepithelial Cl- conductance identified by standard 36Cl- tracer technique. 4. The clamping current at V = 0 mv was unaffected by cyclic AMP (short-circuit current ISC = 0 ±+0-3 jta/cm2, n = 16) indicating that subepidermal Cl--secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP- or isoprenaline-induced chloride conductance (GC1) activation (V = 0 mv) was not reflected in membrane potential and intracellular Cl- activity in principal cells. Intracellular chloride activity was constant at ; 40 mm at membrane potentials between -90 and -100 mv. Therefore, it can be concluded that the principal cells are not contributing to activated Cl- currents. 6. At V = -100 mv where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GC1 was unaffected by cyclic AMP or isoprenaline. The major effect of these treatments was a rightward displacement of the MR cell-generated Gc1-V relationship along the V axis. 7. Our results indicate that the fl-adrenergically controlled cyclic AMP-mediated chloride conductance is localized to the mitochondria-rich cells. MS 9518

2 642 N. J. WILLUMSEN, L. VESTERGAARD AND E. H. LARSEN INTRODUCTION In a number of NaCl transporting epithelia the Cl- flux is controlled by /,- adrenergic receptors on the basolateral membrane. Generally, receptor occupation results in activation of a membrane-bound adenylate cyclase which in turn leads to an increased intracellular concentration of cyclic AMP and activation of a cyclic AMP-dependent protein kinase. The subsequent phosphorylation reaction has not been identified, but one of the resultant physiological effects is an increased Clconductance in the apical cell membrane. In agreement with this scheme a cyclic AMP-stimulated anion conductance has been described for some secretory epithelia like rectal gland (Greger, Schlatter, Wang & Forrest, 1984) and trachea (Welsh, 1986), where receptor activation leads to an increased serosa-to-mucosa directed Clflux energized by a Na+ concentration gradient-driven co-transport across the basolateral membrane. In absorptive epithelia, e.g. bronchial and nasal regions of airways (Boucher, Cotton, Gatzy, Knowles & Yankaskas, 1988) and sweat duct (Pedersen, 1990) receptor activation would result in an increased inward flux of Cldriven by the electrical potential gradient. Amphibian skin belongs to a class of heterocellular absorptive epithelia also comprising the vertebrate urinary bladder and kidney collecting duct. These epithelia consist of principal cells and subpopulations of mitochondria-rich (MR) cells. In principal cells the apical membrane normally is impermeable to Cl-, and in amphibian skin the passive chloride ion flow is localized to mitochondria-rich cells (review: Larsen, 1991). Chloride fluxes in heterocellular epithelia are also activated by cyclic AMP (amphibian skin: Cuthbert & Painter, 1968; kidney collecting duct: lino, Troy & Brenner, 1981; urinary bladder: Stetson, Beauwens, Palmisano, Mittchell & Steinmetz, 1985). The aim of the present study was to apply intracellular electrophysiological recording techniques to principal cells to analyse whether this cell-type is responsible for a cyclic AMP-mediated increase in the Cl- conductance. The mechanism of receptor-mediated gating of Cl- currents was investigated by application of a cyclic AMP analogue that is known to permeate the cell membrane (dibutyryl cyclic AMP), or a /3-agonist acting via,-adrenergic membrane receptors (isoprenaline). Parts of this study have been published as a brief report (Willumsen, Vestergaard & Larsen, 1992). METHODS Preparation and chamber for electrophysiological studies Isolated skin of toads (Bufo bufo) were exposed for 2 h to serosal collagenase (cat. no , Boehringer-Mannheim GmbH, 1-2 mg/ml NaCI-Ringer). The epithelium was removed from the connective tissue and placed in a flat miniature Ussing chamber made of light-absorbing polyacetal (Delrin`) which was mounted with an exposed area of 2 cm2 on the stage of an upright microscope (Zeiss, Germany). While a coverslip (diameter = 20 mm, thickness 0-08 mm) constituted the bottom of the lower chamber, the upper half-chamber was open allowing access upwards to the epithelial surface with microelectrodes at an angle of approximately 40 deg. This construction provided excellent optical conditions allowing visual inspection of principal cell impalements with DIC- Nomarski optics (Zeiss 40/0.75, water immersion) at x 500 magnification. Each half-chamber was equipped with a current electrode of AgCl-coated silver wire and an agar bridge (o.d. = 2 mm) with control Ringer solution connected to an external calomel electrode for measuring voltage. Each half-chamber could be perfused with four different Ringer solutions which were propagated by

3 EPITHELIAL CHLORIDE CONDUCTANCE gravity and manually operated by miniature valves (HVP/D/4-5, Hamilton, Switzerland). The isolated epithelium was mounted with the serosal side facing upward on a grid of 2 mm mesh width and a negative hydrostatic pressure of 5-10 cmh2o was applied to the lower half-chamber. The electrodes were connected to a voltage clamp circuit (own workshop) delivering command voltages between mv. Generally, the experiments were performed with transepithelial potential difference (V) clamped to 0 mv, and for measuring the conductance of the preparation (G,), V was pulsed to or - 10 mv with the large pulse used for estimating the small fractional resistance of the basolateral membrane, frb = AVb/AV, where AVb is the voltage change across the basolateral membrane produced by the command voltage pulse, AV. Membrane potential and intracellular chloride activity Single-barrelled microelectrodes of > 50 MQ resistance were fabricated from filament-containing glass tubes and filled with 2 or 0 5 M-KCl (Clark Electromedical Instruments, GC120F-15; Narishige PE-2 microelectrode puller, Tokyo, Japan). The intracellular chloride activity (a',) was measured by double-barrelled microelectrodes fabricated and calibrated as described in a previous paper (Willumsen & Larsen, 1986). The electrodes were connected to the head stage of an electrometer with input resistance of 1013 Q and a bias current < 150 fa (own workshop). The basolateral membrane potential was measured by the reference half-electrode (Vref). The intracellular chloride activity was calculated from the difference between the Cl- half-electrode signal (Vcl) and Ve1f, i.e. Vdiff = VC -Vref: ai = csl yexplo(avdiff/8), here cs I is the chloride concentration in the serosal solution, y is the activity coefficient of chloride (0-787, Robinson & Stokes, 1970), and s is the selectivity of the ion-sensitive barrel. AVdiff is the measured change in Vdiff upon cell impalement. For each electrode s was measured by recording its voltage response to a shift in external chloride concentration from 100 to 10 mm (gluconate substitution); s varied between 52 and 57 mv per decalog [Cl-]. Criteria for acceptable impalements were similar to those employed in a previous study (Willumsen & Larsen, 1986). Transepithelial fluxes of chloride Unidirectional chloride fluxes were measured with 36C1- on paired whole-skin preparations by standard protocols (Katz & Larsen, 1984). Sign and symbolic conventions Transepithelial (V) and basolateral membrane (Vb) potentials were measured with the serosal bath potential as reference: V = Wm- Ts, and V'b = Tce-'T, where Tc, Tm and T,P are electrical potentials of cell water (c) and mucosal (m) and serosal (s) solutions, respectively. Inward transepithelial currents (I) are defined as positive. Solutions and chemicals The following Ringer solutions were employed (composition in mm): NaCl Ringer solution (112 Na', 114 Cl-, 24 K, 24 HCO3-,1 ICa2), gluconate Ringer solution (112 Na', 113 gluconate, 24 K+, 2-4 HCO3-, 1 Ca2+), KCl Ringer solution (112 K+, 114 Cl-, 2-4 K+, 2-4 HCO3-, 1 Ca2+). Three millimolar sodium acetate was added to the Ringer solutions, and they were also bubbled with atmospheric air to maintain a ph of 8-2. Amiloride (Sigma) was added to the mucosal solution (50 /M). The cyclic AMP analogue dibutyryl cyclic AMP (Sigma) was added to the serosal solution (500 /tm). Isobutyl methylxanthin (IBMX, 100 /tm, Sigma), isoprenaline (5 am, Sigma) and forskolin (1 /tm, Sigma) were all administered to the serosal bath. Data presentation and statistical treatment Unless otherwise indicated numbers are presented as the mean+s.e.m. Statistical significance was tested by Student's paired or unpaired t test using a 5 % significance criterion. RESULTS In the mini-chamber the epithelium generated a spontaneous transepithelial potential difference. Vsp = mv, a short circuit current (ISC) of 9±1jIA/cm2 (number of observations n = 88), and a transepithelial conductance of (G) 643

4 644 N. J. WILLUMSEN, L. VESTERGAARD AND E. H. LARSEN jts/cm2 (n = 83). Mucosal exposure of the Na' channel blocker amiloride (50 JtM) virtually abolished ISC ( /ua/cm2) and reduced Gt to gs/cm2 (n = 54). Substitution of mucosal Cl- by gluconate in the presence of amiloride caused a further large reduction of Gt to jts/cm2 (n = 9) indicating a relatively small leakage conductance of the preparation. A 1200 T- (n Fig. 5 Time (min) I 10 1A. For legend see facing page. V= o mv In the presence of amiloride and with bilateral NaCl Ringer solution, principal cells in preparations clamped at V = 0 mv generated a basolateral membrane potential of mv (n = 70) with a fractional resistance of the basolateral membrane of (n = 54). The intracellular Cl- activity was mm (n = 24, see Table 1). Given the measured intracellular potential of -96 mv, the chloride activity in principal cells is far above the equilibrium value of 1-9 mm. Cyclic AMP activates a dissipative chloride conductance At V = 0 mv and with bilateral NaCl Ringer solution, the transepithelial conductance was significantly increased by serosal addition of dibutyryl cyclic AMP (see Fig. 1A). The relationship between conductance and serosal concentration of -1 15

5 EPITHELIAL CHLORIDE CONDUCTANCE 645 B V=-80 mv /clamp (pacm2) Fig. 1. A, time course of conductance change following serosal exposure to 500,UMdibutyryl cyclic AMP. Conductance activation exhibits initial delay and full effect is obtained after 10 min. B, relationship between net inward flux of Cl- measured with 36Cland clamping current in preparations held at -80 mv and exposed on the inside to 1 mm- IBMIX. Analysis with preparations of six animals. The points are mean values of three 20 min periods with standard deviation (S.D.) for fluxes and currents indicated as vertical and horizontal bars, respectively. The line shown defines the relationship I, =',amp' TABLE 1. Transepithelial conductance (G,), principal cell membrane potential (JVb), basolateral membrane fractional resistance (frb), and intracellular Cl- activity (a',) of preparations of enzvmatically isolated toad epithelium clamped at V = 0 mv and exposed to amiloride on the outside at Vb aci (,ts/cm2) (mv) frb (mm) Amiloride (control) (54) (70) (54) (24) Cyclic AMIP + amiloride * * (17) (7) (7) Isoprenaline + amiloride * * (7) (I11) (7) (I11) Amiloride (50,UM) was added to the mucosal bath, and cyclic AMP (500,UM as dibutyryl cyclic AMP) and isoprenaline (5,UM) were added to the serosal bath. Values are means+ S.E.M., number of observations in parentheses. * Significantly different from paired control values (only amiloride added), P < 1 %. Vb and ai were not significantly changed by the addition of cyclic AMP or isoprenaline, P > 5%.

6 646 N. J. WILLUMSEN, L. VESTERGAARD AND E. H. LARSEN cyclic AMP indicated full effect at a concentration of 500 JtM (not shown) which was subsequently used in all experiments. To investigate the ionic specificity of the cyclic AMP-stimulated conductance, we performed 36C1 tracer studies on whole-skin preparations treated with a phosphodiesterase inhibitor (IBMX). The potential difference V was clamped at -80 mv to provide a large transepithelial driving force for all ion flows. As illustrated in Fig. 1B, the net flux of Cl- accounted well for the simultaneously recorded clamping currents demonstrating that in the presence of mucosal amiloride the cyclic AMP-activated conductance is carrying chloride ions. Provided V was held at 0 mv, the cyclic AMP-induced effect on transepithelial conductance was not reflected in the current which remained near zero (1SC= ±3 jta/cm2, n = 16, see also Figs 2 and 3B). Thus, cyclic AMP activates a dissipative transepithelial Cl- conductance. This is an important finding because it shows that the adrenaline-activated secretory Cl- current of subepidermal glands (Koefoed-Johnsen, Ussing & Zerahn, 1952; Mills, 1985) is absent in the isolated epithelial preparation. Response of principal cells Cyclic AMP Results of a principal cell impalement with a double-barrelled microelectrode are shown in Fig. 2. During the 30 min period following serosal addition of cyclic AMP, Gt increased from 150 to 800 1tS cm2, frb of the principal cell compartment increased from 0 01 to 0 05, while the clamping current remained near zero. Similar results were obtained in all preparations tested and they are collected in Table 1. Data presented in Fig. 2 further show that the intracellular chloride activity remained far above its thermodynamic equilibrium value (the variation of calculated ac1 between 40 and 48 mm was unsystematic). During full activation by dibutyryl cyclic AMP, V was shifted to -100 mv for 5 min. This resulted in a 5 mv hyperpolarization of the basolateral membrane and hence a 95 mv depolarization of the apical membrane. Thus the driving force imposed on the Cl- flow across the apical membrane was shifted from the outward to the inward direction. However, the intracellular Cl- activity of principal cells remained constant (see Fig. 2). Accordingly, the apical membrane of principal cells which is impermeable to Clunder control conditions (Willumsen & Larsen, 1986) remains tight to Cl- after addition of cyclic AMP. Thus, the only appreciable response of the principal cell to cyclic AMP was an increase in the fractional resistance of the basolateral membrane associated with a small hyperpolarization of the membrane. Due to the large driving force acting on Cl- ion flows and the fact that the increase of frb was associated with neither a decrease of a', nor membrane depolarization it can be concluded that the affected principal cell membrane conductance is not carrying Cl-. Accordingly, the change in frb is due to an effect on another membrane conductance (see Discussion). Isoprenaline To elicit an increased concentration of intracellular cyclic AMP by a mechanism which involves a membrane-bound,-adrenergic receptor the basolateral membrane was exposed to a fl-agonist, isoprenaline (5 jtm). Like that of cyclic AMP, the major

7 EPITHELIAL CHLORIDE CONDUCTANCE 647 isoprenaline-induced response was a large increase in tissue conductance (see Table 1). Figure 3 shows continuous recordings of the intracellular Cl- activity in principal cells before and after the addition of isoprenaline to the serosal solution. Under control conditions (Fig. 3A), voltage clamping to -100 mv resulted in a large time- 500 pm-cyclic AMP V(mV) -100 a I I I I I fill -200 L o -50 _ -100 _ I (,ua/cm2) -150_ -200 _ -250 _ -300 k Vb (mv) -350 L -100 L ioo BGr (mm) _ 50 0 O 0 Fig. 2. Continuous recording with double-barrelled microelectrode in a principal cell while the electrophysiological response to cyclic AMP is building up. Vc, is the signal of the CI-- selective barrel (reference signal not subtracted); other symbols are used as defined in text. Also shown is the response to clamping the preparation to -100 mv for 5 min.

8 648 N. J. WILLUMSEN, L. VESTERGAARD AND K H. LARSEN dependent activation of the Cl- conductance as revealed by the current increase from -28 to -78 #ta/cm2. This, however, was not reflected in the Cl- activity of principal cells. This finding confirms previous results (Willumsen & Larsen, 1986) and demonstrates that the apical membrane of principal cells remains impermeable to Cl- A I (PA/cm2) 20r B 0-20k p -80L Vb (mv) OT -40 t -80t 100 s VCI (mv) -l _. L O _ Control O OC T o I O acr (mm) _ 10 J _ 90 Fig. 3. Electrophysiological recordings of an isoprenaline-treated preparation (5 4uM in serosal bath). A, recordings from the preparation prior to additions of isoprenaline and amiloride. With the tip of a double-barrelled microelectrode in a principal cell, membrane potential and intracellular Cl- activity were measured while the Cl- conductance of MR cells was reversibly activated by pulsing V to -100 mv for 150 s. Vc, is the difference signal between the Cl- selective barrel and the reference barrel. OC denotes open circuit conditions. B, same protocol as in Fig. 3A but in the presence of mucosal amiloride (Ami) and serosal isoprenaline (Iso). after voltage activation of the transepithelial chloride conductance. The addition of isoprenaline did not significantly change the intracellular Cl- activity which remained on average about 80 mv above its thermodynamic equilibrium value (see Table 1). The recordings in Fig. 3B further show that the chloride activity of principal cells could not be perturbed by clamping the preparation to mv, despite the fact that the driving force on apical Cl- transport changed direction from about 80 mv in the outward to about 15 mv in the inward direction. This result is in agreement with what was found for the cyclic AMP-activated preparation (see Fig.

9 EPITHELIAL CHLORIDE CONDUCTANCE 2). Thus, the apical membrane of principal cells remains impermeable to Cl- after transepithelial chloride conductance activation whether this is accomplished by voltage clamping, cyclic AMP, or isoprenaline treatment. Voltage dependence of the cyclic AMP response The delayed Cl- current activation by voltage clamping shown in Fig. 3A is generated by mitochondria-rich cells (Larsen, 1991 and Discussion). Serosal 649 A Slope = 6 Vt = 0 mv Gt (p S/CM2) E. Cl cn (:3 0 Slope = AMP Vt = -100 mv Gcontrol (ps/cm2) Fig. 4. A, conductance response to serosal cyclic AMP (500,UM, V(mV) * and 0), isoprenaline (5/tM, A) or forskolin (1 /tm, DI). Preparations held at 0 mv exhibited large (sixfold) conductance increase. When V was held at -100 mv the C1- conductance was fully activated by voltage clamping and it was not further increased by cyclic AMP (line of identity shown). B, the inversed S-shaped Cl- conductance-voltage curve (Cl- in mucosal bath) obtained prior to cyclic AMP (0) was shifted to the right when cyclic AMP was present in serosal bath (A). The very small leakage conductance of the preparation is indicated by the conductance measured in the absence of mucosal Cl- (@, gluconate). application of isoprenaline did not further increase the Cl- current recorded at mv (Fig. 3B). Also the presence of cyclic AMP in the serosal bath fully activated the Cl- conductance so that clamping of the preparation to mv did not result in further activation (see Fig. 2). This finding was studied in more detail by recording the cyclic AMP effect on preparations of deactivated (V = 0 mv) and fully voltage-activated (V = mv) chloride conductance, respectively. On average, at V = 0 mv cyclic AMP increased Gt sixfold. This is shown in Fig. 4A depicting pre-cyclic AMP Gt values against postcyclic AMP Gt values for individual preparations (0). When V was clamped to

10 650 N. J. WILLUMSEN, L. VESTERGAARD AND E. H. LARSEN mv, the conductance in cyclic AMP-treated tissues was not significantly different from the conductance measured prior to addition of cyclic AMP (Fig. 4A, 0). As can be seen from the data presented in Fig. 4B, the most significant effect of cyclic AMP was a displacement of the Gt-V relationship to the right along the V axis. DISCUSSION In the present study we have investigated cyclic AMP-induced Cl- conductance activation in a heterocellular epithelium. In vivo such a regulation usually involves hormone occupation of,3-adrenergic receptors on the basolateral membrane. It is an important finding of the study that the chloride conductance of principal cells, contrast to cells of other types of epithelia, is not affected by cyclic AMP or receptor stimulation by a,l-adrenergic agonist. Yet, a transepithelial dissipative Clconductance was increased severalfold by these treatments. Principal cells Membrane effects in principal cells were studied by single-cell recordings with microelectrodes. In agreement with a previous study (Willumsen & Larsen, 1986), we found that the Cl- activity in principal cells of toad skin is above its electrochemical equilibrium. The activity was about 40 mm with an associated equilibrium potential, EC1, of- 58 log1o (86/40) = -19 mv. At a membrane potential of -96 mv (Table 1), the electrochemical driving force acting on chloride ions is Vb-Ec1 = 77 mv in the outward direction. The intracellular Cl- accumulation is due to a Na+ gradientdriven Cl- transport system in the basolateral membrane (Ussing, 1982). Taken together with a virtually eliminated clamping current at V = 0 mv, it can be concluded that under control conditions the apical membrane of principal cells does not possess a Cl- channel-mediated conductance. In this respect the Na+-conductive apical membrane of principal cells of amphibian skin is similar to the luminal membrane in the collecting duct which also contains amiloride-sensitive Na+ channels (O'Neil & Sansom, 1984) with no permeability for chloride ions (Schlatter, Greger & Schafer, 1990). Activation of the transepithelial Cl- conductance by cyclic AMP or isoprenaline was not reflected in the intracellular Cl- activity which remained far above its electrochemical equilibrium value at a membrane potential of mv (Table 1). Furthermore, by voltage clamps we imposed a large shift in driving force for apical Cl- ion flows and showed that a'1 was virtually unaffected by this manoeuvre (Figs 2 and 3). It is clear from these experiments that neither cyclic AMP nor isoprenaline activates an apical Cl- conductance. Accordingly, the cyclic AMP-activated transepithelial Cl- conductance is not localized to principal cells. Cyclic AMP or o-adrenergic receptor stimulation resulted in a significant increase in fractional resistance of the basolateral membrane (Table 1). Since the measured membrane potential of mv was close to the potassium equilibrium potential, which in our preparation was mv, n = 55 (Larsen, Willumsen & Christoffersen, 1992), our results indirectly indicate that a K+ conductance becomes activated by the increased intracellular [cyclic AMP]. To account for the increase in frb such K+ conductance must be in the apical membrane. This interpretation is in in

11 EPITHELIAL CHLORIDE CONDUCTANCE agreement with previous demonstrations of cyclic AMP-induced activation of apical K+ channels in urinary bladder (Palmer, 1986) and frog skin (Van Driessche, Aelvoet & Erlij, 1987). With an equilibrium potential for chloride of -19 mv and that of potassium being -106 mv (see above) it can further be concluded that the Cl- conductance (GC1) of these cells, which has to be located in the basolateral membrane, amounts to a small value compared to the potassium conductance (GK). With amiloride in the mucosal bath and the preparation clamped at V = 0 mv the steady-state intracellular potential was -96 mv. Thus, from circuit analysis we obtain: Gcl/GK = -(Vb-EK)/(Vb-EcI) = Results obtained in the present study (Table 1) indicate that the chloride conductance of this membrane is not activated by intracellular cyclic AMP. Previous studies have indicated that the Cl- permeability of the basolateral membrane becomes activated in osmotically swelled cells (Ussing, 1982). Mitochondria-rich cells Our results discussed above provide clear evidence that neither subepidermal glands nor principal cells contribute to cyclic AMP- or isoprenaline-evoked Clconductance activation. This points toward MR cells as likely candidates for conducting the activated chloride ion flows. Because of their deep location among principal cells and their small size, it has not been possible to impale MR cells with microelectrodes. However, previous studies using other techniques have localized the major transepithelial chloride currents to this cell type. Thus, the chloride currents activated by transepithelial voltage clamping are linearly correlated with the density of MR cells (Willumsen & Larsen, 1986), and with the vibrating probe technique large Cl1-dependent currents have been recorded above single MR cells (Foskett & Ussing, 1986; Katz & Scheffey, 1986). Voltage-induced Cl- current activations are associated with the swelling of MR cells (Foskett & Ussing, 1986; Larsen, Ussing & Spring, 1987), and uptake of bromide ions from the mucosal bath (D6rge, Rick, Beck & Nagel, 1988). Most interesting results were obtained in those experiments in which cyclic AMPinduced conductance activations were studied in preparations of deactivated and fully voltage-activated Cl- conductance, respectively (Fig. 4). The addition of cyclic AMP produced a rightward displacement of the conductance-voltage curve. As a result, at V = 0 mv the normally deactivated Cl- permeability exhibited a large conductance at this potential (Fig. 4A, V = 0 mv). However, once the Clconductance of MR cells was fully activated by voltage clamping, the Clconductance of the preparation could not be further increased by cyclic AMP (Fig. 4A, V = -100 mv). This result strongly indicates that the conductive mechanism of MR cells activated by voltage clamping is identical to that activated by cyclic AMP. Furthermore, such a hypothesis is entirely consistent with our findings discussed above that no other cellular pathways contribute to the cyclic AMP-generated chloride conductance. The study was supported by the Danish Natural Science Research Council (DNSRC ), and equipment was purchased by grants from the Carlsberg and Direkt0r Ib Henriksen's 651

12 652 N. J. WILL UMSEN, L. VESTERGAARD AND E. H. LARSEN Foundations. N. J. W. was in receipt of a DNSRC Fellowship. Technical assistance in the laboratory by Lone Pedersen and Birthe Petersen and in the workshop by Arne Nielsen is much appreciated. REFERENCES BouCHER, R. C., COTTON, C. U., GATZY, J. T., KNOWLES, M. R. & YANKASKAS, J. R. (1988). Evidence for reduced Cl- and increased Na' permeability in cystic fibrosis human primary cell cultures. Journal of Physiology 405, CUTHBERT, A. W. & PAINTER, E. (1968). Independent action of antidiuretic hormone, theophylline and cyclic 3',5'-adenosine monophosphate on cell membrane permeability in frog skin. Journal of Physiology 199, D6RGE, A., RICK, R., BECK, F. X. & NAGEL, W. (1988). Uptake of Br in mitochondria-rich and principal cells of toad skin epithelium. Pfluigers Archiv 412, FOSKETT, K. J. & USSING, H. H. (1986). Localization of chloride conductance to mitochondria-rich cells in frog skin epithelium. Journal of Membrane Biology 91, GREGER, R., SCHLATTER, E., WANG, F. & FORREST, J. N. (1984). Mechanisms of NaCl secretion in rectal gland tubules of spiny dog-fish (Squalus acanthias). III. Effects of stimulation by cyclic AMP. Pfluigers Archiv 402, IINO, Y., TROY, J. L. & BRENNER, B. M. (1981). Effects of catecholamines on electrolyte transport in cortical collecting tubule. Journal of Membrane Biology 61, KATZ, U. & LARSEN, E. H. (1984). Chloride transport in toad skin (Bufo viridis). Effect of salt adaptation. Journal of Experimental Biology 109, KATZ, Y. & SCHEFFEY, C. (1986). The voltage-dependent chloride conductance of toad skin is localized to mitochondria-rich cells. Biochimica et Biophysica Acta 861, KOEFOED-JOHNSEN, V., USSING, H. H. & ZERAHN, K. (1952). The origin of the short-circuit current in the adrenaline stimulated frog skin. Acta Physiologica Scandinavica 27, LARSEN, E. H. (1991). Chloride transport by high-resistance hetero-cellular epithelia. Physiological Reviews 71, LARSEN, E. H., USSING, H. H. & SPRING, K. R. (1987). Ion transport by mitochondria-rich cells in toad skin. Journal of Membrane Biology 99, LARSEN, E. H., WILLUMSEN, N. J. & CHRISTOFFERSEN, B. C. (1992). Role of proton pump of mitochondria-rich cells for active transport of chloride ions in toad skin epithelium. Journal of Physiology 450, MILLS, J. W. (1985). Ion transport across the exocrine glands of the frog skin. Pfiuigers Archiv, suppl. 1, S O'NEIL, R. G. & SANSOM, S. G. (1984). Characterization of apical cell membrane Na+ and K' conductances of cortical collecting duct using microelectrode techniques. American Journal of Physiology 247, F PALMER, L. G. (1986). Apical membrane K conductance in the toad urinary bladder. Journal of Membrane Biology 92, PEDERSEN, P. S. (1990). Chloride permeability regulation via a cyclic AMP pathway in cultured human sweat duct cells. Journal of Physiology 421, ROBINSON, R. A. & STOKES, R. H. (1970). Electrolyte Solutions, 2nd edn, pp Butterworth, London. SCHLATTER, E., GREGER, R. & SCHAFER, J. A. (1990). Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl- transport. Pftuigers Archiv 417, STETSON, D. L., BEAUWENS, R., PALMISANO, J., MITCHELL, P. P. & STEINMETZ, P. R. (1985). A double membrane model for urinary bicarbonate secretion. American Journal of Physiology 249, F USSING, H. H. (1982). Volume regulation in frog skin epithelium. Acta Physiologica Scandinavica 114, VAN DRIESSCHE, W., AELVOET, I. & ERLIJ, D. (1987). Oxytocin and camp stimulate monovalent cation movements through a Ca2"-sensitive, amiloride-insensitive channel in the apical membrane of toad urinary bladder. Proceedings of the National Academy of Sciences of the USA 84, WELSH, M. J. (1986). Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: cellular physiology. Journal of Membrane Biology 91,

13 EPITHELIAL CHLORIDE CONDUCTANCE 653 WVILLUMSEN, N. J. & LARSEN. E. H. (1986). Membrane potentials and Cl activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl- conductance. Journal of Membrane Biology 94, WILLIUMSEN, N. J., VESTERGAARD, L. 0. & LARSEN, E. H. (1992). Evidence of /3-agonist- and camp-stimulated chloride conductance in toad skin epithelium being localized to the mitochondria-rich cells. Journal of Physiology 446, 108P.