Role of intracellular by rabbit medullary
calcium in volume regulation thick ascending limb cells
CHAHRZAD MONTROSE-RAFIZADEH AND WILLIAM B. GUGGINO Physiology Department, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205
CHAHRZAD, AND WILLIAM B. GUGcalcium in volume regulation by rabbit medullary thick ascending limb cells. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F402-F409, 1991.-Previous studies demonstrated that Ca2+-activated K’ channels in luminal membrane of rabbit medullary thick ascending limb cells (MTAL) are activated on exposure of the cells to hyposmotic solutions [J. Taniguchi and W. B. Guggino. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F347-F352, 19891. In this study, we investigated the mechanism of activation of Ca2+activated K’ channels in MTAL cells exposed to hyposmotic solutions. MTAL cells swell in hyposmotic medium and regulate volume back toward the starting volume. This regulatory volume decrease (RVD) is inhibited at high medium K’ concentrations or by presence of quinine or Ba2’ in extracellular medium, suggesting involvement of K’ channels. Measurements of intracellular Ca2+ concentrations with furashow that intracellular Ca2’ rises in hyposmotic solutions and that this rise does not occur in absence of extracellular Ca2+. Nifedipine and verapamil also inhibit rise in intracellular Ca2+. Decreasing intracellular Ca2’ by removal of external Ca2’ in presence of EDTA or by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) inhibits RVD. We conclude that hypotonic solutions activate K+ efflux probably via K’ channels and Ca2+ influx via a nifedipineand verapamil-sensitive pathway. Lowering intracellular Ca2+ removes the ability of MTAL cells to regulate volume in hyposmotic solutions. MONTROSE-RAFIZADEH, GINO. Role of intracellular
intracellular stores. In either case, an increase in cellular Ca2+ results in an increase in Ca2+-activated K+ channel open probability. Taken together, these observations suggest an involvement of maxi-K+ channels in regulatory volume decrease (RVD). This study evaluates the mechanisms causing hyposmotic activation of Ca2+-activated K+ channels in MTAL cells. We have examined whether intracellular Ca2’ increases during cell swelling and have determined whether increases in intracellular Ca2+ are caused by an increase in Ca2+ entry or by a release from intracellular stores. MATERIALS
AND
METHODS
Cell culture and cell suspension. The clone, A3, of MTAL cells (7), originally derived from rabbit MTAL cells (GRB-Mali), was cultured as described previously (9). Cells were grown to confluency, usually 3-5 days before use. Cell suspensions were created by lo- to 15 min incubation of the cell monolayer with 2 ml of a Ca2+free, Mg2+-free medium containing (in mM) 145 K-gluconate, 4.5 NaCl, 17 glucose, 0.5 Na2HP04, 0.5 NaH2P04, and 20 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-tris(hydroxymethyl)aminomethane (Tris), pH 7.4. Cells were then diluted into 10 ml of an isosmotic solution (see below for composition) and cenkidney; regulatory volume decrease; furatrifuged at 200 g for 12 s, and then the cell pellet was resuspended in 6 ml of isosmotic solution. Suspended cells were gently shaken for 2 h at 25°C to allow the cells CALCIUM-ACTIVATED potassium channels are present in to recover. After the recovery period, the cells had a K+the luminal membrane of many renal cells including the to-Na+ ratio of 7.8 t 0.3 (mean t SE, n = 15). K+ and following: a medullary thick ascending limb (MTAL) cell Na+ content were measured by flame photometry as described previously (19). After the 2-h recovery period line derived from rabbit kidney (9), the cortical collecting ratios were stable for 4 h after suspension tubule of rat kidney (6), a canine kidney cell line (l), an K’-to-Na’ opossum kidney cell line (24), and the distal tubule of (data not shown). Solutions. Cells were suspended in a control solution Amphiuma kidney (13). These Ca’+-activated K+ channels probably do not contribute to the resting apical cell containing (in mM) 145 NaCl, 4.5 KCl, 1 CaC12,1 MgC12, membrane conductance in these tissues, because the 0.5 NaH2P04, 0.5 Na2HP04, 17 glucose, and 20 HEPESopen probability is very low in unstimulated cells. Re- Tris (pH 7.4), as well as 1 mg/ml of bovine serum cently, studies by Taniguchi and Guggino (23) have albumin (BSA). The osmolality of this solution was 315 shown that the open probability of maxi-K+ channels in mosmol/kgH20, as measured by vapor-pressure osmothe luminal membrane of cultured MTAL cells increases meter (model 5lOOC; Wescor, Logan, UT), and is referred when cells are exposed to hyposmotic solutions or mem- to as the control osmolality. Except where noted, hyposwere identical to the control solution, brane stretch. Activation of Ca”+-activated K+ channels motic solutions except, that the NaCl concentration was reduced to 60 by stretch has also been demonstrated in opossum kidney mM to obtain an osmolality of 180 mosmol/kgH20. For cells (24), in choroid plexus (3), and in Necturus proximal high-K+ solutions at control osmolality, 60 mM NaCl tubule cells (5). In choroid plexus and Necturus proximal tubule, stretch is thought to act directly to increase Ca2+ was omitted and KC1 concentration was increased to 60 influx, whereas in opossum kidney, Ca”+ is released from mM. To obtain high K+ at low osmolality, the NaCl F402
0363-6127/91
$1.50 Copyright
0 1991 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 10, 2019.
CA’+-ACTIVATED
K’
concentration was reduced to 4.5 mM and KC1 concentration was maintained at 60 mM. Ca”+-free media were prepared by omitting CaC12 from the media and by addition of either 0.1-l mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) or 10 mM EDTA. Electronic cell sizing. Measurements of cell volume were made using a model ZM Coulter Counter (Hialeah, FL) coupled to a pulse-height analyzer (The Nucleus, Oak Ridge, TN) and interfaced to an IBM-AT computer. At time 0, 2 ml of cells were diluted in 20 ml of experimental medium. Cells in the diluted suspension were gently mixed and maintained at 37OC. Cells were sized in the instrument by flow through a loo-pm orifice. Each volume measurement included 15-30 s of data collection. The average size of single cells was determined by cornputer. The centroid (weighted mean) of the cell size distribution of 20,000-30,000 cells was calculated and is reported in femtoliters. The instrument was calibrated with spherical latex beads of defined size (Coulter). The calibration was independent of the composition of the medium or of the different osmolarities used in this study (190-315 mosmol/kgH20). The average size of the single cells in suspension in the isosmotic solutions was 1,961 t 40 (SE) fl (n = 47). The initial rate of change in volume was measured by linear regression fit of 3-5 measurements and is given in femtoliters per second. Because of the dilution factors in the experiments (e.g., 2 ml of cells at 315 mosmol/kgH20 were added to 20 ml of media at 180 mosmol/kgH20), the final osmolarity of each solution was measured at the end of the experiment. Intracellular Ca2’ measurements. To measure intracellular Ca”+, cells were exposed to 2 PM acetoxy methylester of fura- (fura-2/AM; Molecular Probes, Eugene, OR) for 1 h at 25°C. Cells were pelleted by gentle centrifugation for 12 s using a microcentrifuge (2,000 g), and the supernatant was aspirated. The cell pellet (l-3 x 10" cells) was resuspended in 50 ,ul of isosmotic media without BSA and injected into a prewarmed (37°C) cuvette containing 3 ml of experimental media. Using a fluorescence microscope, we observed that cellular dye fluorescence was homogenous at 360 nm (Ca”+-insensitive excitation wavelength). For experiments, fluorescence was measured by a model SPF-500C SLM Aminco Spectrofluorometer (Urbana, IL) using alternating excitation wavelengths of 340 and 380 nm (1-nm bandpass) and an emission wavelength of 505 nm (20-nm bandpass). Ratios of fluorescence (R = 340/380) were measured every 2 s, automatically corrected for autofluorescence, and plotted graphically. Cellular autofluorescence was measured in an identical aliquot of cells not exposed to fura-2. Values of autofluorescence were ~5% of the fluorescence from dye-loaded cells and were measured for every experiment. To calculate free Ca2+ concentrations, the dye was calibrated using the Tsien equation (8) [Ca2+] = & x B - R), where B is the ratio of 380-nm (R ~rnin)I(~rnax fluorescence in the absence of Ca2+ to 380-nm fluorescence in the presence of saturating concentrations of Ca2’ and & is the dissociation constant. We assumed & of 224 nM (8) and estimated R,;,, R,,,, and B from two separate protocols. In the protocol used routinely, R,,;, was determined by using 0.02% digitonin (Fluka,
CHANNELS
IN
F403
RVD
Ronkonkoma, NY) to release dye from furaloaded cells into a Ca2+ -free medium plus EGTA. Subsequent addition of saturating concentrations of CaC12 (l-2 mM) from 1 M stock solution allowed the R,,, to be determined. In a second protocol, cells loaded with fura- were exposed to saturating concentrations of ionomycin (6 PM) in a Ca2+-containing medium to obtain the R,,,. After release of the dye using digitonin (0.02%), the Rmin was determined by addition of EGTA (10 mM). The R,,, and Rmin obtained with these two different protocols were similar, and for convenience the first protocol was used routinely. In the protocol used routinely, cells loaded with fura- were resuspended in 50 ~1 of Ca2+-free isosmotic media (plus 1 mM EGTA) and injected in a cuvette containing 3 ml of Ca”+ -free isosmotic media (plus 1 mM EGTA). In this protocol Rmin was 0.963 t 0.034 (SE), R,,, was 12.323 t 1.375, and B was 4.524 t 0.523 (n = 9). The initial rate of increase of intracellular Ca”+ concentration was measured by linear regression fit between the first 20-30 measurements of intracellular C a 2+ after exposure of the cells to the experimental solutions. Statistical analysis. Data are presented as means t SE. Statistical significance was evaluated in paired t tests. RESULTS
Volume regulation in hyposmotic solutions. A typical response of MTAL cells exposed to hyposmotic medium is shown in Fig. 1. In hyposmotic medium at 37°C MTAL cells swell, initially reaching a maximum cell volume within 2-4 min. The maximal increase of volume, 25 t 0.7% higher than the starting volume (n = 42), is much lower than predicted for osmometric swelling. If we es2600
-
2400
-
. g2200
-
"0 2000
-
o
1800
-
1600
-
V E > z
I
0
I 10
I 20
I 30
I 40
1 50
min.
1. Cell volume measurements (in fl) in hyposmotic and isosmotic solutions. Medullary thick ascending limb (MTAL) cells were suspended and used for measurements of cell volume as described in MATERIALS AND METHODS. At time 0, 2 ml of suspension of cells were exposed to isosmotic (315 mosmol/kgH20, q) and hyposmotic solutions (180 mosmol/kgH20, n) at normal K’ concentrations (4.5 mM) or to isosmotic and hyposmotic media containing a higher concentration of K’ (60 mM) (0, l , respectively). Because of dilution factors, final osmolarity of hyposmotic solutions was -190 mosmol/kgH20. Although initial rate of RVD was completely inhibited by high-K+ media in experiment presented here, in 7 different experiments inhibition of RVD was 75.3 & 7% compared w ith hyposmotic media at normal K’ concentration. FIG.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 10, 2019.
F404 TABLE
CAM+-ACTIVATED
K-
CHANNELS
1. Initial rate of RVD in hypotonic conditions Hyposmotic Medium
RVD
Normal K’ (4.5 mM) High K’ (60 mM) Control Quinine (1 mM) Control Barium (5 mM)
Rates, fl/s
0.6OkO.09 0.13kO.04 0.62t0.10 0.29kO.05 0.62t0.16 0.25kO.04
n
7 7 6 6 3 3
P
co.05
calcium in volume regulation thick ascending limb cells
CHAHRZAD MONTROSE-RAFIZADEH AND WILLIAM B. GUGGINO Physiology Department, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205
CHAHRZAD, AND WILLIAM B. GUGcalcium in volume regulation by rabbit medullary thick ascending limb cells. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F402-F409, 1991.-Previous studies demonstrated that Ca2+-activated K’ channels in luminal membrane of rabbit medullary thick ascending limb cells (MTAL) are activated on exposure of the cells to hyposmotic solutions [J. Taniguchi and W. B. Guggino. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F347-F352, 19891. In this study, we investigated the mechanism of activation of Ca2+activated K’ channels in MTAL cells exposed to hyposmotic solutions. MTAL cells swell in hyposmotic medium and regulate volume back toward the starting volume. This regulatory volume decrease (RVD) is inhibited at high medium K’ concentrations or by presence of quinine or Ba2’ in extracellular medium, suggesting involvement of K’ channels. Measurements of intracellular Ca2+ concentrations with furashow that intracellular Ca2’ rises in hyposmotic solutions and that this rise does not occur in absence of extracellular Ca2+. Nifedipine and verapamil also inhibit rise in intracellular Ca2+. Decreasing intracellular Ca2’ by removal of external Ca2’ in presence of EDTA or by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) inhibits RVD. We conclude that hypotonic solutions activate K+ efflux probably via K’ channels and Ca2+ influx via a nifedipineand verapamil-sensitive pathway. Lowering intracellular Ca2+ removes the ability of MTAL cells to regulate volume in hyposmotic solutions. MONTROSE-RAFIZADEH, GINO. Role of intracellular
intracellular stores. In either case, an increase in cellular Ca2+ results in an increase in Ca2+-activated K+ channel open probability. Taken together, these observations suggest an involvement of maxi-K+ channels in regulatory volume decrease (RVD). This study evaluates the mechanisms causing hyposmotic activation of Ca2+-activated K+ channels in MTAL cells. We have examined whether intracellular Ca2’ increases during cell swelling and have determined whether increases in intracellular Ca2+ are caused by an increase in Ca2+ entry or by a release from intracellular stores. MATERIALS
AND
METHODS
Cell culture and cell suspension. The clone, A3, of MTAL cells (7), originally derived from rabbit MTAL cells (GRB-Mali), was cultured as described previously (9). Cells were grown to confluency, usually 3-5 days before use. Cell suspensions were created by lo- to 15 min incubation of the cell monolayer with 2 ml of a Ca2+free, Mg2+-free medium containing (in mM) 145 K-gluconate, 4.5 NaCl, 17 glucose, 0.5 Na2HP04, 0.5 NaH2P04, and 20 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-tris(hydroxymethyl)aminomethane (Tris), pH 7.4. Cells were then diluted into 10 ml of an isosmotic solution (see below for composition) and cenkidney; regulatory volume decrease; furatrifuged at 200 g for 12 s, and then the cell pellet was resuspended in 6 ml of isosmotic solution. Suspended cells were gently shaken for 2 h at 25°C to allow the cells CALCIUM-ACTIVATED potassium channels are present in to recover. After the recovery period, the cells had a K+the luminal membrane of many renal cells including the to-Na+ ratio of 7.8 t 0.3 (mean t SE, n = 15). K+ and following: a medullary thick ascending limb (MTAL) cell Na+ content were measured by flame photometry as described previously (19). After the 2-h recovery period line derived from rabbit kidney (9), the cortical collecting ratios were stable for 4 h after suspension tubule of rat kidney (6), a canine kidney cell line (l), an K’-to-Na’ opossum kidney cell line (24), and the distal tubule of (data not shown). Solutions. Cells were suspended in a control solution Amphiuma kidney (13). These Ca’+-activated K+ channels probably do not contribute to the resting apical cell containing (in mM) 145 NaCl, 4.5 KCl, 1 CaC12,1 MgC12, membrane conductance in these tissues, because the 0.5 NaH2P04, 0.5 Na2HP04, 17 glucose, and 20 HEPESopen probability is very low in unstimulated cells. Re- Tris (pH 7.4), as well as 1 mg/ml of bovine serum cently, studies by Taniguchi and Guggino (23) have albumin (BSA). The osmolality of this solution was 315 shown that the open probability of maxi-K+ channels in mosmol/kgH20, as measured by vapor-pressure osmothe luminal membrane of cultured MTAL cells increases meter (model 5lOOC; Wescor, Logan, UT), and is referred when cells are exposed to hyposmotic solutions or mem- to as the control osmolality. Except where noted, hyposwere identical to the control solution, brane stretch. Activation of Ca”+-activated K+ channels motic solutions except, that the NaCl concentration was reduced to 60 by stretch has also been demonstrated in opossum kidney mM to obtain an osmolality of 180 mosmol/kgH20. For cells (24), in choroid plexus (3), and in Necturus proximal high-K+ solutions at control osmolality, 60 mM NaCl tubule cells (5). In choroid plexus and Necturus proximal tubule, stretch is thought to act directly to increase Ca2+ was omitted and KC1 concentration was increased to 60 influx, whereas in opossum kidney, Ca”+ is released from mM. To obtain high K+ at low osmolality, the NaCl F402
0363-6127/91
$1.50 Copyright
0 1991 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 10, 2019.
CA’+-ACTIVATED
K’
concentration was reduced to 4.5 mM and KC1 concentration was maintained at 60 mM. Ca”+-free media were prepared by omitting CaC12 from the media and by addition of either 0.1-l mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) or 10 mM EDTA. Electronic cell sizing. Measurements of cell volume were made using a model ZM Coulter Counter (Hialeah, FL) coupled to a pulse-height analyzer (The Nucleus, Oak Ridge, TN) and interfaced to an IBM-AT computer. At time 0, 2 ml of cells were diluted in 20 ml of experimental medium. Cells in the diluted suspension were gently mixed and maintained at 37OC. Cells were sized in the instrument by flow through a loo-pm orifice. Each volume measurement included 15-30 s of data collection. The average size of single cells was determined by cornputer. The centroid (weighted mean) of the cell size distribution of 20,000-30,000 cells was calculated and is reported in femtoliters. The instrument was calibrated with spherical latex beads of defined size (Coulter). The calibration was independent of the composition of the medium or of the different osmolarities used in this study (190-315 mosmol/kgH20). The average size of the single cells in suspension in the isosmotic solutions was 1,961 t 40 (SE) fl (n = 47). The initial rate of change in volume was measured by linear regression fit of 3-5 measurements and is given in femtoliters per second. Because of the dilution factors in the experiments (e.g., 2 ml of cells at 315 mosmol/kgH20 were added to 20 ml of media at 180 mosmol/kgH20), the final osmolarity of each solution was measured at the end of the experiment. Intracellular Ca2’ measurements. To measure intracellular Ca”+, cells were exposed to 2 PM acetoxy methylester of fura- (fura-2/AM; Molecular Probes, Eugene, OR) for 1 h at 25°C. Cells were pelleted by gentle centrifugation for 12 s using a microcentrifuge (2,000 g), and the supernatant was aspirated. The cell pellet (l-3 x 10" cells) was resuspended in 50 ,ul of isosmotic media without BSA and injected into a prewarmed (37°C) cuvette containing 3 ml of experimental media. Using a fluorescence microscope, we observed that cellular dye fluorescence was homogenous at 360 nm (Ca”+-insensitive excitation wavelength). For experiments, fluorescence was measured by a model SPF-500C SLM Aminco Spectrofluorometer (Urbana, IL) using alternating excitation wavelengths of 340 and 380 nm (1-nm bandpass) and an emission wavelength of 505 nm (20-nm bandpass). Ratios of fluorescence (R = 340/380) were measured every 2 s, automatically corrected for autofluorescence, and plotted graphically. Cellular autofluorescence was measured in an identical aliquot of cells not exposed to fura-2. Values of autofluorescence were ~5% of the fluorescence from dye-loaded cells and were measured for every experiment. To calculate free Ca2+ concentrations, the dye was calibrated using the Tsien equation (8) [Ca2+] = & x B - R), where B is the ratio of 380-nm (R ~rnin)I(~rnax fluorescence in the absence of Ca2+ to 380-nm fluorescence in the presence of saturating concentrations of Ca2’ and & is the dissociation constant. We assumed & of 224 nM (8) and estimated R,;,, R,,,, and B from two separate protocols. In the protocol used routinely, R,,;, was determined by using 0.02% digitonin (Fluka,
CHANNELS
IN
F403
RVD
Ronkonkoma, NY) to release dye from furaloaded cells into a Ca2+ -free medium plus EGTA. Subsequent addition of saturating concentrations of CaC12 (l-2 mM) from 1 M stock solution allowed the R,,, to be determined. In a second protocol, cells loaded with fura- were exposed to saturating concentrations of ionomycin (6 PM) in a Ca2+-containing medium to obtain the R,,,. After release of the dye using digitonin (0.02%), the Rmin was determined by addition of EGTA (10 mM). The R,,, and Rmin obtained with these two different protocols were similar, and for convenience the first protocol was used routinely. In the protocol used routinely, cells loaded with fura- were resuspended in 50 ~1 of Ca2+-free isosmotic media (plus 1 mM EGTA) and injected in a cuvette containing 3 ml of Ca”+ -free isosmotic media (plus 1 mM EGTA). In this protocol Rmin was 0.963 t 0.034 (SE), R,,, was 12.323 t 1.375, and B was 4.524 t 0.523 (n = 9). The initial rate of increase of intracellular Ca”+ concentration was measured by linear regression fit between the first 20-30 measurements of intracellular C a 2+ after exposure of the cells to the experimental solutions. Statistical analysis. Data are presented as means t SE. Statistical significance was evaluated in paired t tests. RESULTS
Volume regulation in hyposmotic solutions. A typical response of MTAL cells exposed to hyposmotic medium is shown in Fig. 1. In hyposmotic medium at 37°C MTAL cells swell, initially reaching a maximum cell volume within 2-4 min. The maximal increase of volume, 25 t 0.7% higher than the starting volume (n = 42), is much lower than predicted for osmometric swelling. If we es2600
-
2400
-
. g2200
-
"0 2000
-
o
1800
-
1600
-
V E > z
I
0
I 10
I 20
I 30
I 40
1 50
min.
1. Cell volume measurements (in fl) in hyposmotic and isosmotic solutions. Medullary thick ascending limb (MTAL) cells were suspended and used for measurements of cell volume as described in MATERIALS AND METHODS. At time 0, 2 ml of suspension of cells were exposed to isosmotic (315 mosmol/kgH20, q) and hyposmotic solutions (180 mosmol/kgH20, n) at normal K’ concentrations (4.5 mM) or to isosmotic and hyposmotic media containing a higher concentration of K’ (60 mM) (0, l , respectively). Because of dilution factors, final osmolarity of hyposmotic solutions was -190 mosmol/kgH20. Although initial rate of RVD was completely inhibited by high-K+ media in experiment presented here, in 7 different experiments inhibition of RVD was 75.3 & 7% compared w ith hyposmotic media at normal K’ concentration. FIG.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 10, 2019.
F404 TABLE
CAM+-ACTIVATED
K-
CHANNELS
1. Initial rate of RVD in hypotonic conditions Hyposmotic Medium
RVD
Normal K’ (4.5 mM) High K’ (60 mM) Control Quinine (1 mM) Control Barium (5 mM)
Rates, fl/s
0.6OkO.09 0.13kO.04 0.62t0.10 0.29kO.05 0.62t0.16 0.25kO.04
n
7 7 6 6 3 3
P
co.05
