Experiments were designed to measure Jlwb, Pf, and transepithelial voltage ( VT) .... Na, 5 K, 1.5 Ca, 1.0 Mg, 140 Cl, 25 HC03, 2 sodium phosphate. (pH 7.4), 5 ... and another agar bridge in the bath which was the reference. A high-impedance ...
Intracellular Ca2+ and PKC activation do not inhibit Na+ and water transport ALEXANDER J. ROUCH, LU CHEN, LUCIA BETH C. FOWLER, BEVERLY D. CORBITT,
in rat CCD
H. KUDO, P. DARWIN BELL, AND JAMES A. SCHAFER
Departments of Physiology and Biophysics, and Medicine, Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 Rouch, Alexander J., Lu Chen, Lticia H. Kudo, P. Darwin Bell, Beth C. Fowler, Beverly D. Corbitt, and James A. Schafer. Intracellular Ca2+and PKC activation do
not inhibit Na+ and water transport in rat CCD. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F569-F577, 1993.Experiments examined the effects of elevation of intracellular calcium concentration ([Ca’+]i) or activation of protein kinase C (PKC) on Na+ and water transport in the rat cortical collecting duct (CCD). We measuredthe lumen-to-bath 22Na+ flux (Jlwb), transepithelial voltage ( VT), and water permeability (P,) in CCD from deoxycorticosterone (DOC)-treated rats. Ionomytin (0.5 and 1 PM) and thapsigargin (1 and 2 PM) were usedto increase [Ca2+]i. Phorbol 12-myristate 13-acetate (PMA; 0.3 and 1 PM) and oleoyl-acetyl-glycerol (OAG; 100PM) were used asactivators of PKC. [Ca”‘] i wasmeasuredin isolatedperfused tubules using the fluorescent dye fura 2. When added to the bathing solution, 220 pM arginine vasopressin(AVP) failed to affect [Ca’+]i, whereas 1 PM ionomycin increased [Ca’+]i by 103t 15%and 2 PM thapsigarginincreased[Ca2+]i by 24 & 4%. In flux studies, neither ionomycin nor thapsigargin affected Jlbb or Pf, although ionomycin causedmarked morphological changes. Ionomycin also failed to alter either parameter in tubules from non-DOC-treated rats. Neither 100PM OAG nor 1 PM PMA affected J 1-b or Pf. OAG at 50 PM had no effect on VT or transepithelial resistance,indicating no inhibition of conductive Na+ transport. We concludethat increased[Ca”+]i and PKC activation do not affect J1-b or Pf in the rat CCD. These findings may account for the sustainedincrease in C&-b produced in the rat CCD by AVP. vasopressin;arginine vasopressin;mineralocorticoid; cortical collecting duct; protein kinase C; thapsigargin; ionomycin; phorbol 12-myristate 13-acetate; oleoyl-acetyl-glycerol; intracellular secondmessengers; sodiumchannel
of phospholipase C generates two products of phosphatidylinositol hydrolysis, i.e., inositol trisphosphate, which increases intracellular calcium ([ Ca2+] i), and diacylglycerol, which activates protein kinase C (PKC). Both of these products have been shown to play roles in the regulation of Na+ and water transport in the isolated perfused rabbit cortical collecting duct (CCD). Basal rates of Na+ transport are inhibited by an increase in [Ca2+]i (4,5,10,17), and the activation of PKC has been shown to inhibit the transport of Na+ and K+ (15). Similarly, the increase in water permeability (P,) produced by vasopressin (AVP) in the rabbit CCD is inhibited both by PKC activation (2,3,5,16,34) and by increases in [Ca2+]i (3, 5, 20, 21). It has also been suggested that a rise in [Ca2+]; produced by AVP might be responsible for the fact that AVP stimulation of Na+ transport is transient in the rabbit CCD (4, 10). AVP has been shown to produce a modest initial increase in Na+ transport, but this initial stimulation is followed by a fall in transport to or below control levels within lo-20 min (5, 7, 8, 18). Breyer (4) has reported that AVP concentrations in the physiologi-
STIMULATION
0363-6127/93
cal range also increase [Ca2+]i in the rabbit CCD, and Breyer (4) and Frindt and Windhager (10) have suggested that this [Ca2+]i increase could account for the reduction in Na+ transport subsequent to the initial increase produced by AVP. Furthermore, Holt and Lechene (18) have shown that the reduction in Na+ transport to less than control levels after the initial AVP stimulation is associated with prostaglandin production, and the inhibition of Na+ and water transport in the rabbit CCD by prostaglandin E2 (PGE2) has also been linked to a rise in [Ca2+]i (16, 17). It is also interesting that pretreatment of the rabbit with deoxycorticosterone (DOC) increases basal Na+ transport in the CCD and prevents any change in the transport rate on AVP addition (7) and that this DOC pretreatment also decreases the degree of Na+ transport inhibition produced by ionomycin (10). In contrast to the rabbit CCD, when AVP is added to the rat CCD, Na+ transport increases and remains elevated for at least 3-5 h and Pf rises to levels considerably higher than those reported in the rabbit CCD (7,24, 25, 37). DOC pretreatment in the rat elevates the basal Na+ transport rate, and the subsequent addition of AVP produces an even greater stimulation. Thus the two hormones act synergistically, and this response is also sustained for at least 3 h (7, 25, 37). Furthermore, although PGE2 inhibits Na+ and water transport in the rabbit CCD, it has no inhibitory effect on either parameter in the rat CCD (6). The mechanisms underlying these species-related differences are not understood, but it is certainly possible that differences in transport regulation by [Ca2+]i and/or PKC are involved. Studies of the type that showed inhibition of AVPdependent Na+ and water transport in the rabbit CCD with increases in [Ca2+]; or with PKC activation have not as yet been conducted in the rat CCD. However, Silver et al. (33) have shown that the application of ionomycin to isolated rat CCD segments inhibits Na+ channel activity in cell-attached patches. They suggested that a rise in [Ca2+]i inhibited Na+ channels indirectly by a mechanism that requires some component(s) of the intact cell, possibly by increased prostaglandin production as suggested by Jones et al. (20). The purpose of the present study was to determine whether either [Ca2+]i elevation or PKC activation inhibits AVP-dependent Na+ transport and Pf in isolated perfused CCD segments from DOC-treated rats. In these studies we treated the CCD segments with AVP in addition to the DOC conditioning because there is no net Na+ transport in the absence of either hormone (25), and inhibitory effects could be observed more easily under conditions of maximal stimulation (6). We found that the high rates of AVP-dependent Na+ and water
$2.00 Copyright 0 1993 the American Physiological
Society
F569
F570
CA’+
AND
PKC
transport were maintained in the rat CCD despite elevations of [Ca’+]i or the addition of PKC activators. Thus the lack of effect of these products of phospholipase C activation could be responsible for the sustained increase in Na+ transport produced by AVP in the rat CCD and might provide at least a partial explanation for the intriguing differences in the hormonal regulation of Na+ and water transport in the rat and rabbit CCD. METHODS
The animals used in these studies were male Sprague-Dawley rats, weighing 50-100 g, obtained from a barrier-maintained colony [strain Hsd(N):SD] at Harlan Sprague Dawley (Indianapolis, IN). They were housed in our animal resources facility and were maintained on a standard pelleted diet (Prolab RMH 1000; Agway) and tap water ad libitum. This diet contains +70-180 meq/kg feed of both Na+ and K+. In all but one set of experiments rats were treated with DOC pivalate (5 or 2.5 mg im) 4-7 days before the experiment. Flux studies. These experiments were conducted to determine whether thapsigargin, ionomycin, phorbol 12-myristate 13-acetate (PMA), or oleoyl-acetyl-glycerol (OAG) affected lumento-bath 22Na+ flux (J& and Pf. The first two agents are known to increase [ Ca2+]i, and the latter two are known to stimulate PKC. Experiments were designed to measure Jlwb, Pf, and transepithelial voltage ( VT) simultaneously in the isolated rat CCD aspreviously describedin detail (7, 24, 25, 28, 31, 38). Rats were killed by decapitation, and CCD segmentswere dissectedat 15-20°C in the bathing solution, describedbelow, to which 6 g/d1 bovine serum albumin had been addedto prevent adherence of tubule segmentsto the dissection forceps and glassware.Tubule segmentswere transferred to a perfusion chamber and mounted on concentric pipettes. CCD segments averaged0.49 t 0.16 mm in length and 19.6 t 2.0 pm inside diameter. The bathing solution contained (in mM) 122NaCl, 25 NaHCOs, 5 sodium acetate, 5 KCl, 1.5 CaCl,, 0.5 MgC12, 8 glucose,4 L-alanine, 6 urea, and 2 sodiumphosphate (pH 7.4). This solution was equilibrated with 95% 02-5% CO, at 38”C, and the pH was adjustedto 7.4. The measuredosmolality was 316 t 2 (SD) mosmol/kgH,O. The perfusate was hypotonic to the bath and contained (in mM) 88 NaCl, 5 KCl, 2 sodium phosphate (pH 6.6), 1.5 CaCl,, 0.5 MgCl,, and 50 urea. The measuredosmolality was 228k 2 (SD) mosmol/kgH,O. The pH was adjusted to 6.6, and the solution was equilibrated with a 95% 02-5% Co, gas mixture by bubbling for 30 min at 38°C. The perfusate contained 25 &i/ml 22Na+ for Jlbb measurement and 50 &i/ml of exhaustively dialyzed [methoxy-3H]inulin, which served as the volume marker. The perfusion rate was calculated as (X,/X,) J&, where XL and X0 are the [methoxy-3H]inulin concentrations (counts min-l l l-l) in the collected and perfused luminal solutions, respectively, and V, is the collection rate as measured directly with a calibrated constant-bore samplingpipette. J1-b (pm01min-l *mm+) was calculated as Jlwb = C$/(L+S), where Cz is the total counts per minute of 22Na+(corrected for bulk fluid leakage)collected in the bath, t is the collection time in minutes, L is the tubule length in millimeters, and S is the specific activity of 22Na+(countsmin-l l pmol-‘) in the original perfusate (31). We have calculated previously that, at a perfusion rate of lo-15 nl/min asusedin these studies,there is no significant change in S due to the very low bath-to-lumen flux of unlabeled Na+ (7, 25). Pf was calculated as described previously (7, 38). VT (mV, lumen with respect to bathing solution) was measuredbetweenAg-AgCl electrodesconnectedvia 0.9% NaCl-4% agar bridges inserted into the perfusate and the bathing solutions, with VT being continuously recordedwith a strip-chart recorder. The ionic composition of perfusion and bathing solul
l
IN RAT
CCD
tions remained constant throughout each experiment, and thus the net liquid-junction potential did not changefrom period to period within an experiment. After the tubules were dissectedand mounted on concentric pipettes, the bathing solution temperature was raised to 38”C, the isotopeswere added to the perfusate, and an equilibration period of 20-30 min precededthe first collection period. Bathing fluid sampleswere collected for Jlbb and leak determinations at lo-min intervals, as wereperfusate fluid samplesfor Pf determinations, as describedabove. A 20-min equilibration period alsofollowed eachexperimental manipulation, and a minimum of four collections were taken subsequently.&, and Pf were determined for eachcollection, and the reported valuesfor a given experimental period were calculated as averagesof the individual collections taken during that period. Bath flow rate and luminal perfusion flow rate were constant at 0.3 ml/min and lo- 15 nl/min, respectively. Each experimental agent was addedto the bathing solution. Five protocols were conducted in separate groups of perfused CCD segmentsas follows: 1) with 1 or 2 PM thapsigargin, 2) with 0.5 or 1.0 PM ionomycin, 3) with ionomycin in CCD segments from rats not treated with DOC, 4) with 0.3 or l$M PMA, and 5) with 100PM OAG. All studies,with the exception of protocol 3 above,wereconductedwith DOC-treated rats. AVP at 220 pM was continuously present in the bathing solution or it wasadded to the bath after an initial control period without AVP and remainedin the bath throughout the remainderof the experiment. Electrophysiology experiments. In a separate set of experiments, we tested the effect of OAG on VT and transepithelial resistance(R,) in the DOC-treated CCD. Methods usedin this laboratory to determine VT and RT have been describedpreviously (29, 30, 32). Rats were killed by decapitation, and CCD segmentswere dissectedat 15-20°C in the samedissectionsolution asusedin the flux studies.Tubule segmentswereisolated, transferred to a perfusion chamber,and mounted on concentric pipettes by useof the Luigs-Neumanin vitro perfusion system (Ratingen, Germany). The bathing and perfusion solutionswere identical unlessotherwiseindicated and contained (in mM) 160 Na, 5 K, 1.5 Ca, 1.0 Mg, 140 Cl, 25 HC03, 2 sodiumphosphate (pH 7.4), 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), and 5 glucose.The measuredosmolality of the solutions was 320 & 2 (SD) mosmol/kgH,O, and the solutions were equilibrated with 95% 02-5% CO, before and during the experiment. Perfusion pipettes were made from glasscapillaries with a septum (“theta glass”). VT was measuredby placing an agar bridge in the luminal perfusate via one of the pipette channels and another agar bridge in the bath which wasthe reference.A high-impedanceelectrometer was usedto completethe circuit. Current pulsesof 30-60 nA and 800-msduration were passed (via the other perfusion pipette channel) into the lumen every 8 s, and current-induced voltage deflections weremeasuredat the perfusion and collection ends of the tubule as describedpreviously (32). VT and the voltage deflections were continuously monitored on a strip-chart recorder. The bathing solution flowed continuously through the chamber at 20-40 ml/min, and the luminal perfusion rate waskept at lo-20 nl/min. After the tubules weremounted, the temperature of the bathing solution was raised to 38”C, and steady-state measurementsof VT and &r were recordedin the control condition. AVP wasthen addedto the bathing solution, and after new steady-state values were taken, 50 PM OAG wasaddedto the bathing solution and new valueswere recordedover a 15- to 30-min period. [CC?+/~ measurements. [Ca2+]i was measuredto determine whether it waselevated by AVP, ionomycin, or thapsigargin in
AND PKC IN RAT CCD
cA2+
F571
the bathing solution. Tubules were mounted on concentric pipettes using the sameperfusion and bathing solutions as described above for the flux studies. The acetoxymethyl ester of fura 2 (fura 2-AM, 7.1 PM) wasaddedto the perfusate for 30 to 40 min to load the tubules with the dye. Fura 2-AM was then removed and the tubule wasperfused with the control solution for an additional 15-25 min to ensure maximal conversion of fura 2-AM to the nonesterified form fura 2. Fura 2 fluorescence was measuredwith a dual excitation wavelength system (Photon Technologies, New Brunswick, NJ) connected to a Leitz inverted microscope equipped with quartz optics. Excitation monochrometerswere set at 350 and 380 nm, and light entering the microscopewas alternated between these two wavelengths using a computer-controlled chopper at a rate of 60 Hz. Fluorescent emissionswere restricted by a lo-nm band-passfilter centered at 510 nm. Fluorescencemeasurementswere obtained usinga variable rectangular diaphragmplacedover a segmentof the collecting duct, during observation at ~400 using an Olympus x40 UVF lens. After the equilibration period, a baseline reading of 250350 s duration was taken with the control bathing solution. Subsequentmeasurementswere taken in three sequentialperiodsasfollows: 1) with 220 pM AVP only in bath, 2) with 2 PM thapsigarginand AVP in bath, and 3) with 1 PM ionomycin and AVP in bath. Each experimental period lasted 5-10 min. Calculationsof [Ca2+]i were madeaccordingto the method of Grynkiewicz et al. (11) from the ratio of emissioncounts (R) recorded at 350 nm to those at the 380 nm excitation wavelength, using the equation
any lossin initial experiments in which the calibration solution containing 5 PM ionomycin and 3 mM Ca2+ was added to obtain Rmax.However, removal of Ca2+in an EGTA-containing solution (to obtain Rmi,) led to a rapid lossof fura 2 and obvious deterioration of the CCD segments.Becauseof this problem, calibration was conducted in vitro using a solution containing 120 mM KCl, 10 mM HEPES, 1 PM fura 2 (free acid), and either 3 mM EGTA or 3 mM CaCl,, to obtain Rmaxand Rmi,, respectively. All measurementswere corrected for background and autofluorescence.We (P. D. Bell and B. C. Fowler) have found previously in several types of cells that differences in estimating [Ca2+]i using in vitro rather than in situ calibrations rangedfrom only 10 to 15%. Thus, although the absoluteaccuracy of the [Ca”+]i measurementsmay be compromisedby the in vitro calibration procedure, the relative changesin [Ca”+]i with treatments should not be affected (1). Sourceof biochemicals. Synthetic AVP from Sigma Chemical (St. Louis, MO) was addedto the bathing solution from a stock solution of 100 mu/ml in deionized water to a final concentration of 100 pU/ml (-220 PM). DOC pivalate was from CibaGeigy Animal Health (Memphis, TN). Ionomycin from Calbiothem (San Diego, CA) was added to the bath from a stock solution of 5 mM in dimethyl sulfoxide (DMSO). Thapsigargin and PMA from LC Services (Woburn, MA) were added to the bath from stock solutionsof 10 mM and 100PM, respectively, in DMSO, respectively. OAG and fura 2 from Molecular Probes (Eugene,OR) wereaddedto the bath from a stock solution of 25 mM and 1 mM in DMSO, respectively. In all experiments, the DMSO concentration in all control and experimental collection periods was the same. Statistics. Results were examined by analysis of variance (ANOVA) and the significanceof treatment effects was evaluwhere & is the fura 2 dissociation constant at 37”C, 224 nM ated using the Scheffe F ratio test. The significance level was (11), Rmin is the minimum ratio obtained in a solution (see chosento be P < 0.05. below) containing 3 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) in place of Ca2+, RESULTS Rmaxis the maximum ratio obtained in the solution containing3 Measurement of intracellular Ca2+ in isolated perfused mM Ca2+,and 38O,i, and 380,,, are the emissionsobtained at rat CCD. Figure 1 presents the results of a representative the 380-nm excitation during the measurementof Rmin and experiment in which [Ca2+]i was measured in an isolated Rmax9 respectively. After loading with fura 2-AM, emissionphotometer countsat perfused CCD from a DOC-treated rat. During the conboth excitation wavelengths exceeded400,000 counts/s in all trol period, [ Ca2+]i was very stable in all experiments. In experiments. During the courseof an experiment there was no the experiment shown in Fig. 1, within 20 s following the indication of fura 2 lossfrom the cells, even in the presenceof addition of 220 pM AVP there was a slight fall in [Ca2+]i, 1 PM ionomycin in the normal bathing solution, nor wasthere which stabilized within 100 s. The subsequent addition of 450
0 450
50
100
150
200
250
300 *
C
350z .c
250-
-
150-
+ %I 0
5
350 450
400
450
500
550
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650
.--
?oo
t
D
3502IrMmap 1
50 1300
I 1400
I 1500
Time
I 1600
I 1700
(sets)
I 1800
I 1900
50 2400
1 2600
I 2800
Time
(sets)
I 3000
3200
Fig. 1. Effect of arginine vasopressin (AVP), thapsigargin, and ionomycin on intracellular Ca2+ concentration ( [Ca2+]J in a rat cortical collecting duct (CCD); realtime results for a single isolated CCD. Tubules were loaded with fura 2, and [Ca2+]i was determined by dual-wavelength fluorescence spectrometry as described in METHODS. Time 0 was taken to be the beginning of control period (A), 15-25 min after fura 2 had been removed from lumen. AVP (220 PM) was added to bath at -490 s (B), followed by 2 PM thapsigargin (Thap) at -1,375 s (C), and 1 PM ionomycin (Iono) at -2,540 s (D). Interruptions in the record are because of the storage limits of the sampling software and hardware in use at time of experiments.
F572
cA2+
AND PKC IN RAT CCD
2 PM thapsigargin led to a slow rise in [Ca2+]i, reaching a peak level within 400-600 s and then falling slightly, although not back to previous levels, over the subsequent 200-300 s. Addition of ionomycin led to a more rapid and sustained increase of [Ca2+]i. The averaged results for seven such experiments in CCD segments from DOCtreated rats are presented in Fig. 2. During the control period, [Ca2+]i ranged from 144 to 264 nM and averaged 213 t 22 nM. Alth ough a fall in [Ca2+]i was observed following the addition of 220 pM AVP in four of seven experiments, the average [Ca2+]i of 201 t 20 nM was not significantly different from the control level by ANOVA. Addition of 2 PM thapsigargin to the AVP-containing bathing solution significantly increased [Ca2+]i to 253 t 31 nM (P < 0.01 compared with control, averaged during 500-600 s after thapsigargin addition). Removal of thapsigargin followed by addition of 1 PM ionomycin produced a steady-state [Ca2+]i of 399 t 40 nM (P < 0.001) at 500-600 s after addition of the ionophore. Effect of increased intracellular Ca2+ on salt and water transport. We measured Pf, VT, and Jlbb in isolated per-
fused CCD segments before and after the application of thapsigargin 0; ionomycin to the bathing solution to determine whether an elevation of [Ca2+]i was inhibitory to salt and/or water transport . In our first two experiments with thapsigargin ,, we used a conce ntration of 1 PM and switched to 2 PM in six additional experiments when we observed no effect on the parameters measured. As shown in Fig. 3, neither dose produced any significant effect on nor was VT altered. pf or JLb, The addition of ionomycin to the bathing solution also failed to produce any significant changes in Pf or J1-b (Table 1; Fig. 4). However, there was morphological evidence of considerable structural damage to the tubules after application of this agent. We found that, within 10 min after addition of 1 PM ionomycin to the bathing solution, granules in the cytoplasm of CCD cells (observed by bright-field light microscopy at ~200 magnification) exhibited a swirling or spinninglike activity. Subsequently, usually within 15 min, some of the cells began to dislodge from the epithelium. This sometimes resulted in a precipitous depolarization of VT and a rise in the 120
r
100 0
80
-20
1
AVP
AVP
Thap
+
AVP
+
Ion0
Fig. 2. Average effects of AVP, thapsigargin, and ionomycin on [Ca2+]i in 7 experiments. Methods of measurement and addition of test agents were conducted as described for Fig. 1. Changes in [Ca2+]i with treatments are given as % of control; estimated absolute [Ca2+]i concentrations and comparisons are given in text. * P < 0.01 and t P < 0.001, significance of difference compared with control [Ca”+]i for 7 experiments. AVP alone did not significantly alter [Ca2+]i.
250
0 E a
100 50
'f < -
0 t
Control
AVP
AVP + Thap
Fig. 3. Thapsigargin has no significant effect on lumen-to-bath 22Na+ flux (JI-b, top) or water permeability (P, bottom). Addition of 220 pM AVP significantly increased J 1-b and Pf (P < 0.001). Subsequent addition of thapsigargin at 1 PM (solid thin lines, n = 2) or 2 PM (dashed lines, n = 6) to bath did not affect either parameter. Thick solid lines, means of pooled experiments with 1 and-2 PM thapsigargin (vertical lines are &SE).
[methoxy-3H]inulin leak to the bathing solution, in which case the experiment was discontinued and the data were not included with those summarized in Fig. 4 and Table 1. In two additional experiments, we reduced the ionomycin concentration to 0.5 PM, which produced less obvious morphological changes, but there was no significant effect on the measured transport parameters. The results of the 0.5 and 1 PM ionomycin experiments have been combined and are presented in Fig. 4 and Table 1. As might be expected if the morphological changes observed compromised the barrier properties of the junctional complexes, ionomycin produced a reversible depolarization of VT. Surprisingly, there was no statistically significant effect on either Jl-b or pf; however, both parameters declined continuously throughout the course of the experiment after ionomycin addition, and we attributed this to the progressive structural deterioration. Because previous studies conducted in the rabbit CCD had shown that DOC pretreatment decreased the extent of Na+ transport inhibition produced by ionomycin (lo), we conducted an additional series of experiments with CCD segments from rats that had not been pretreated with DOC. As shown in Fig. 5 and Table 1, there was no statistically significant effect of 1 PM ionomycin on J1-b or Pf, although VT reversibly depolarized. However, as observed in the CCD segments from DOC-treated rats, there was a progressive decline in Pf and J1-b during the second and third experimental periods, which we attributed to nonspecific deterioration of the tubules. Effect of activators of PKC on salt and water transport.
We first examined PMA at a concentration of 0.3 PM as an activator of PKC. Although this dose was slightly higher than that producing maximal inhibition of J1-b (0.27 PM) in the rabbit CCD (15), we observed no significant effect. Therefore, we increased the dose to 1 PM, but again with no significant effect. Figure 6 and Table 1
CA”
Table 1. Summary of data from flux studies Experimental Protocol
J
Thupsigargin Control AVP AVP + Thap
1 -.b,
pm01 - min-
l - mm-
l
Pf9
VT,
m/s
mV
at 1 PM (2) and 2 PM (6) 49.2t9.7 20t9
133.9t10.1* 129.7rt11.9”
1,125+186* 1,248+264*
-5.4t0.9 -18.2t1.4* -19.0t1.6*
Ionomycin at 0.5 PM (2) and 1 PM (3) AVP AVP + Iono AVP
183.5t21.4 173.0t19.8 136.9zk12.8
665t227 543t106 460t148
F573
AND PKC IN RAT CCD
-14.2t1.8 -6.4k1.41 -10.8*1.8?
although AVP produced a decrease in RT from 42 t 10 to 27 + 8 Q-cm2 which was comparable to that observed previously (32), the further addition of 50 PM OAG produced no change in R T, which averaged 27 t 7 %cm2. DISCUSSION
The results presented above show that neither increases in [Ca2+]i nor activation of PKC inhibits Na+ or water transport in the rat CCD. These results are quite different from those observed in the rabbit CCD. Considering first the effects of elevated [ Ca2+]i, Frindt and
Ionomycin (non-DOC rats) at 1 PM (6) AVP AVP + Iono AVP
66.6k22.3 63.2k22.7 24.1k13.7.t
1,378+496 1,325+,471 1,064+595
-8.Ot1.6 -5.0*1.0-f -4.0+1.1$
PMA at 300 nM (3) and 1 PM (3) Control
47.3t6.4
84t55
-4.7tl.l
AVP 142.2t19.3” 802t197’ -23.0t4.2* AVP + PMA 148.7t26.3’ 994t201” -25.4t4.0* AVP 135.3k20.7” 885t188” -23.4t3.6* OAG at 100 PM (5) without and with 1 PM ionomycin AVP 163.5t26.1 1,208+93.2 - 18.8t2.4 AVP + OAG 138.5k21.6 1,759f331 -9.6+1.9? AVP + Iono + OAG 144.0*19.1 2,133+312$ -5.5+2.5$ Values are means t SE; numbers of experiments for each condition are in parentheses. All experiments were conducted in cortical collecting ducts (CCD) from deoxycorticosterone (DOC)-treated rats, with the exception of the 3rd group, in which the rats were not DOC treated. Experimental agents were added to bathing solution at indicated concentration; when two concentrations are indicated, no effect was observed with either concentration and results have been pooled to obtain mean values. PMA, phorbol 12-myristate 13-acetate; OAG, oleoyl-acetyl-glycerol; AVP, arginine vasopressin; Iono, ionomycin; thap, thapsigargin. Jldb, lumen-to-bath 22Na flux; Pf, water permeability; VT, transepithelial voltage. Significance of differences by ANOVA is indicated for P < 0.05 or better as follows: * significantly different from control period; 7 significantly different from preceding experimental period; $ significiantly different from first experimental period.
show the combined results with both PMA concentrations. Neither dose affected Pf, VT, or J1+b. In a separate study in one of our laboratories, Runquist et al. (27) confirmed that the same PMA used in the present studies was active, because it was able to enhance Na+/Ca2+ exchange in cultured mesangial cells when added at 100 nM (P. D. Bell, personal communication). It has been shown previously that 25 PM OAG is sufficient to inhibit conductive Na+ transport in LLC-PK1 cells by 93% (23). We tested 100 PM OAG in the rat CCD and found that it had no significant effect on JIYb; however, VT depolarized significantly and Pf increased in three of five experiments, although the average change was not statistically significant (Fig. 7; Table 1). We subsequently added 1 PM ionomycin to see whether the combined effects of PKC activation and elevated [Ca2+]i might produce a more convincing inhibitory effect. With this combination of agents there was a statistically significant increase in Pf, and VT further depolarized, which appeared to be due to nonselective damage to the tubules (see above). Despite the changes in Pf and VT, there was no statistically significant change in Jldb. Although OAG produced no significant increase in the [methoxy-3H]inulin leak, we wished to determine whether there might be a nonspecific increase in the ionic permeability of the epithelium by measuring the transepithelial resistance, R Tm However, as shown in Fig. 8,
t \ \ \
\ \ \ /’
\
-0 -5 -0 -
AVP
AVP
lonomycin
+
-
0.
AVP
Fig. 4. Ionomycin did not affect J 1-b (top) or Pf (bottom) in deoxycorticosterone (DOC)-treated rat CCD. Ionomycin was added to bath at 0.5 (thin solid lines, n = 2) or 1 PM (dashed lines, n = 3). Thick solid lines, means of pooled experiments with 0.5 and 1 PM ionomycin (vertical lines are &SE).
_1 FL
3000 2500
---0 -0
-0 -9
---0.
- 0.
--a
0
AVP
AVP +
lonomycin
AVP
Fig. 5. Ionomycin did not affect J 1-b (top) or Pf (bottom) in CCD segments from rats not treated with DOC. Ionomycin added to bath at 1 PM did not affect either parameter. Dashed lines, individual experiments; solid lines, means (vertical lines are &SE).
F574
cA2+ 250
AND PKC IN RAT CCD
r
\ -25
\ -0-C
-30 80 7 iiiv)
r
1600 1200
I
E s
- -.
t '
800
cc 400
Control
AVP
AVP+PMA
AVP
Fig. 6. Phorbol 12-myristate 13-acetate (PMA) did not affect &, (top) or Pf (bottom). Addition of AVP to bath significantly increased Jldb (P c 0.001) and Pf (P < 0.02) above control values. However, subsequent addition of PMA at 300 nM (thin solid lines, n = 3) or 1 PM (dashed lines, n = 3) did not affect either parameter. Thick solid lines, means of pooled values for both experiments with both PMA concentrations (vertical lines are &SE). n
i E 7 .Ic
250 200 150
i 100 I
i! 5ot 5 0'
n 7 ki
L
3. ‘= e
”
Control
AVP
AVP + OAG
Fig. 8. OAG did not affect transepithelial voltage (VT, top) or resistance (RT, bottom) in CCD segments from DOC-treated rats. Addition of AVP to bath significantly reduced RT and hyperpolarized VT (P < 0.05) from control levels. Addition of OAG at 50 PM to bath did not affect either parameter. Dashed lines, individual experiments (n = 4); solid lines, means (vertical lines are &SE).
r
E = E La
n
2500
2000
-
1500
-
AVP
AVP + OAG
AVP + OAG + lonomycin
Fig. 7. Effect of OAG on J 1-b (top) and Pf (bottom). Addition of 100 PM oleoyl-acetyl-glycerol (OAG) to bath resulted in a slight but not statistically significant reduction in J l-b. Further addition of 1 PM ionomytin in 3rd period did not affect J 1-b. Pf in 3rd period was significantly different from AVP period (P < 0.05). Dashed lines, individual experiments (n = 5); solid lines, means (vertical lines are *SE).
Windhager (10) showed that 1 PM ionomycin in the bathing solution of CCD segments from non-DOC-treated rabbits produced a 45% reduction in J1-b through inhibition of the luminal membrane Na+ permeability. Jones et al. (20) conducted an extensive study which documented that elevations in [Ca2+]i also inhibited AVPstimulated Pf in the rabbit CCD. Furthermore, ionomytin reduced the increase in Pf produced by 8-( pchlorophenylthio)-adenosine 3’,5’-cyclic monophosphate (a nonhydrolyzable analogue of CAMP), although the
magnitude of the reduction was not as large as that with AVP-stimulated Pf (20). Jones et al. (20) concluded that the [Ca2+]i-induced inhibition of Pf could occur via both pre- or post-CAMP events (20). To investigate the effects of [Ca2+]i in the rat CCD, we used 1 and 2 PM thapsigargin, a sesquiterpene lactone known to increase [Ca2+]i (19), and 0.5 and 1 PM ionomycin, a widely used Ca2+ ionophore. Neither agent produced any significant change in Jl+b or Pf (Figs. 3 and 4; Table l), despite our experiments which demonstrated that both agents significantly increased [ Ca2+]i. In light of the data reported by Frindt and Windhager (lo), in which DOC pretreatment in the rabbit lowered the [Ca2+];-induced inhibition of Jl+b, we conducted an additional set of flux studies to test the effect of ionomycin in tubules from non-DOC-treated rats. Figure 5 shows that ionomycin also failed to produce inhibition under these conditions. It should also be noted that in our studies ionomycin produced striking morphological changes in the tubule within 3-5 min after its addition to the bath. There appeared to be a rapid swirling of particles in the cytoplasm of a majority of the tubular cells; this is a unique observation for our laboratory, which has extensively studied the isolated rat CCD for the past several years. This activity was followed by generalized disruption of the tissue evidenced by the loss of some cells from the epithelium and an elevated leak rate measured by the appearance of 3H in the bathing solution in some experiments (see METHODS). Ionomycin clearly induced irreversible cell injury at these concentrations, and future experiments with this agent should be conducted and interpreted with caution. Nevertheless, we believe our conclusion that [Ca2+]i elevation did not affect J1-b or Pf is valid because it is based on the results from ionomycin experiments in which 3H leak rates were cl% of the
CA”+
ANDPKCINRATCCD
perfusion rate (Figs. 4 and 5); also, the data show that thapsigargin increased [Ca’+]i (Figs. 1 and 2) but had no effect on J 1-b or Pf (Fig. 3). Interestingly, a reversible decrease in VT also occurred with ionomycin (Table l), which could be associated with the morphological changes noted above. On the other hand, thapsigargin did not affect VT. Our decision to exclude those ionomycin experiments in which the leak rate was greater than 1% might be criticized; however, this is our uniform criterion for accepting any of our experimental data; i.e., when leak rates are much higher than this we feel the data have little meaning, and in several of the discarded experiments the leak exceeded 10%. However, even in those experiments with high leak rates, we could observe no inhibitory effect of ionomycin. In the absence of any effect of even the 0.5 PM dose of ionomycin, we were forced to conclude that even increases in [ Ca2+]i that compromise the structural integrity of the epithelium were not inhibitory to Na+ or water transport in the rat CCD. Our results contrast with those of Silver et al. (33), who observed that increases in [ Ca2+] i produced by ouabain or 5 PM ionomycin in the presence of 0.1 mM extracellular Ca2+ produced a decrease in Na+ channel activity measured by patch clamp of the rat CCD apical membrane. However, Silver et al. (33) maintained the rats for at least 1 wk on a low-Na+ diet. We have found that, although plasma aldosterone levels in such Na+-depleted rats are elevated as expected, the CCD segments have a very low Na+ transport rate (15-20% of that observed in the present studies) and a blunted response of Na+ and water transport to AVP in comparison with rats given either high levels of DOC or high but physiological levels of aldosterone in vivo (unpublished observations from this laboratory). The most likely reason for the lower Na+ transport rate in CCD segments from the Na+-depleted rats is the decreased Na+ load delivered to the CCD in vivo, which has been shown to have an important influence on Na+ reabsorption in the distal tubule irrespective of plasma mineralocorticoid levels (35). Thus it is not clear that the Na+ channels studied in the patch clamp studies of Silver et al. (33) are either the identical channel or under the same regulation as those mediating the high Na+ fluxes in the present experiments, in which CCD segments were stimulated by both DOC and AVP. With regard to our measurements of [Ca2+];, despite the fact that we were forced to use a solution rather than an in situ calibration procedure (see METHODS), the control [Ca2+]i concentration (213 t 22 nM) was in reasonable agreement with the average of 209 t 6 nM observed in the rat CCD by Frindt et al. (9), although it is higher than that reported in another study from the same laboratory (33). Basal [Ca2+]; concentrations reported in the rabbit CCD also vary from 74 to 260 nM (5,34). Irrespective of the accuracy of the absolute [Ca”+]; measurements in these experiments, the relative effects of the various treatments on [Ca2+]i should not be compromised. As shown in Fig. 2, AVP did not increase [Ca2+]i; however, even in the rabbit CCD, AVP concentrations above the physiological level may be required to produce such an increase. Ando et al. (1) observed no change in [Ca2+]i with AVP doses of 230 pM or less, but a dose of 23 nM produced a blunted hydrosmotic response as well as a
F575
significant elevation of [Ca2+]i. On the other hand, Breyer (4) observed that 23-230 pM AVP produced a transient spike of [Ca2+]i and a significantly elevated steady-state level. This effect appeared to be due to increased Ca2+ entry from the extracellular fluid, whereas the much larger increment in [Ca2+]i produced by 23 nM AVP appeared to require Ca2+ release from intracellular stores. AVP has also been shown to elevate [Ca2+]i in rat inner medullary collecting duct (IMCD) cells, but again at AVP concentrations above the physiological range (12, 36), and it has been suggested that these effects are mediated by V1 or oxytocin receptors (12, 22). In any case, our results show that physiological concentrations of AVP do not elevate [Ca2+]i, whereas both thapsigargin and ionomycin do and yet have no effect on Pf or &b; however, again it should be noted that our results come from rat CCD, which differs markedly from rabbit CCD (7, 25, 37), as discussed below, and probably also differs from the rat IMCD in its hormonal response patterns. Considering now the second arm of phospholipase C action, i.e., PKC activation, Hays et al. (15) reported that &b in the rabbit CCD was significantly reduced by the PKC activators PMA and dioctanoylglycerol in a dosedependent fashion with the maximum inhibition occurring at 270 nM and 75 PM, respectively. Ando et al. (2) reported that 0.1 PM PMA significantly inhibited AVPstimulated Pf. Their findings nicely demonstrated that the PMA-induced inhibition occurred by a post-CAMP mechanism, because the dose-dependent inhibition by PMA was equivalent when Pf was stimulated by AVP or by CAMP. Consistent with these findings, Hebert et al. (16) demonstrated that the PGE2-induced inhibition of Pf in the rabbit CCD occurred by a post-CAMP mechanism and depended on PKC activation. Because Jl-b is not different from zero and AVP produces only a modest increase in Jl,b in CCD segments from non-DOC rats (25, 37), we conducted our studies under conditions of high C&-b induced by the synergism of AVP and DOC effects so that inhibition by [Ca2+]i or PKC would be more easily observed. To activate PKC we used the phorbol ester PMA and the synthetic diacylglycerol OAG. PMA failed to alter J1 b or Pf (Fig. 6; Table 1). In three of these experiments, we used 300 nM PMA, which was slightly higher than the concentration used by Hays et al. (15) to maximally decrease Jl-b in the rabbit CCD. Because of the negative results, we increased the concentration to 1 PM in three additional experiments and again observed no effect. We did observe a slight decrease in &b when OAG was added to the bath in four of the five experiments, but the average change was not statistically significant (Fig. 7). As a final attempt to provoke an inhibitory effect, ionomycin was added to the bathing solution with OAG to optimize the conditions for the stimulation of PKC, a Ca2+-dependent enzyme. Ionomycin was chosen for this experiment rather than thapsigargin because of its relatively larger effect on [Ca2+]i, and, as always, we discarded any experiments in which the leak rate exceeded 1%. Addition of ionomycin in the third period induced no further effect on J l-b, but Pf tended to increase. Despite only a small effect on Jl,b, a progressive decrease in VT was observed in these experiments (Table 1). Such findings could indicate the development of a conductive leak
F576
cA2+
AND PKC IN RAT CCD
pathway, but there was no significant leak of [methoxy3H] inulin in the experiments reported. Because Mohrmann et al. (23) demonstrated that OAG at 10 pg/ml(25 PM) reduced the amiloride-sensitive, conductive Na+ transport by 93% in cultured LLC-PK1 cells, we performed one set of experiments designed to determine whether OAG would affect ionic conductance in the rat CCD. Previous results from our laboratory indicated that net Na+ absorption in the rat CCD occurs exclusively via a conductive mechanism whereby Na+ moves across the luminal membrane only through amiloridesensitive Na+ channels (26, 32). If OAG inhibited Na+ transport in the rat CCD as it did in LLC-PK1 cells, VT should depolarize and Z&r should increase; yet neither effect was observed (Fig. 8). In contrast to the previous flux experiments (Table l), OAG did not depolarize VT in these electrophysiological experiments. Thus we have no explanation for the depolarization of VT with OAG in the flux experiments. Despite the apparent absence of any inhibitory regulation of Na+ or water transport by increased [Ca2+]i or PKC activation, we have shown that a2-adrenergic agonists can markedly inhibit transport. In CCD segments from DOC-treated Sprague-Dawley and Dahl salt-sensitive rats (Rapp strain, %/Jr), clonidine inhibited Jl-b, VT, and pf, each by 20-40% (14, 26). In CCD segments from Sprague-Dawley, SS/Jr, and Dahl salt-resistant rats (SR/Jr), 100 nM epinephrine reduced J1+b to levels not significantly different from the backflux from bath to lumen, i.e., it eliminated net Na+ transport (13). The same concentration of epinephrine also inhibited Pf to levels not significantly different from zero. The inhibitory actions of epinephrine are mediated primarily by an a2-adrenergic receptor that inhibits CAMP production; however, epinephrine also appears to have a secondary route of inhibitory action (13). The present results would suggest that this other pathway does not involve either increases in [Ca2+]i or PKC activation. In summary, our findings in the rat CCD indicate that physiological concentrations of AVP do not increase [Ca2+]; (Fig. 2), nor does an artificially imposed [ Ca2+]; elevation or PKC activation inhibit Pf or Jl-b (Figs. 3-5). The absence of these inhibitory mechanisms in the rat CCD may thus permit the AVP-induced increase in J1-b to be sustained. In agreement with this conclusion, although Hebert et al. (17) reported that PGE2 inhibited Na+ absorption in the rabbit CCD by increasing [Ca2+];, we have found no effect of PGE2 on Pf or J1-b in the rat CCD (6), as would be expected if its effect depended on an increase in [Ca2+]i. Our experiments do not, however, address the possibility that much higher AVP concentrations, which might be expected to affect V1 and oxytocin receptors, might have inhibitory actions via [Ca2+]i- or PKC-dependent pathways. NOTE ADDED
IN
PROOF
Since this manuscript was accepted for publication, additional reports have appeared that show that AVP, oxytocin, and their analogues elevate [Ca2+]i in the rat collecting duct. In the isolated perfused IMCD, after administration of AVP at a concentration out of the physiological range (10 nM), spikes in [Ca2+]; were observed followed bv a return of lCa2+1; to or below baseline levels
[see: Y. Maeda, J. S. Han, C. C. Gibson, and M. A. Knepper, Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F15-F25, 1993; A. Champigneulle, E. Siga, G. Vassant, and M. Imbert-Teboul. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F35-F45, 1993.1 More recent studies in the rat CCD by M. Imbert-Teboul, A. Champigneulle, and E. Siga. [Fourth International Vasopressin Conference; P. Gross, D. Richter, and G. Robertson (editors); Paris: Libbey, In press] have also revealed a transient [Ca2+]i spike after administration of 10 nM AVP and various analogues. For this reason, we undertook additional experiments in the rat CCD to determine whether a high AVP concentration would produce a [Ca2+3; spike. In intial experiments conducted in collaboration with Dr. M. D. Breyer (Vanderbilt University; Nashville, TN), we found that even 23 nM AVP produced, if anything, only a small decline in [Ca2+]i. However, in a limited number of additional studies, we altered our method of AVP delivery and were able to demonstrate a [Ca2+]i spike in the rat CCD in 1 experiment with 23 nM AVP. We then attempted additional experiments with 220 pM AVP (as used in the experiments reported here), and in 2 of 5 experiments we saw convincing spikes in [Ca2+]i. After very careful reexamination of our previous data (summarized in Fig. 2), we are convinced that, under the conditions in which those experiments were conducted, there was no significant change in [Ca2+]i on the administration of 220 pM AVP. At present, we have no data that would allow us to speculate about the reasons for these disparate findings, but we are exploring these questions in ongoing experiments. Despite these caveats, based on the other results in this paper, as well as unpublished data, we feel that it is unlikely that increases in [Ca2+]; are a significant regulator of either Na+ or water transport in the rat CCD at physiological AVP concentrations. However, regardless of whether AVP may transiently elevate [ Ca2+]; under some conditions, our experiments with ionomycin and thapsigargin have shown that such increases are not inhibitory to Na+ or water transport in the rat CCD. We gratefully acknowledge the competent technical assistance of Katy Gonder and the assistance of Dr. Lawrence D. Nelson in the preparation of Fig. 1. Support for this study was provided by National Institute of Diabetes and Digestive and Kidney Diseases Postdoctoral Training Grant DK07545 and by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants DK-25519, DK-39258, and DK-32032. L. H. Kudo was the recipient of a postdoctoral fellowship training award from the State of Sgo Paulo, Brazil (FAPESP 90/049-2). Some of the data presented in this paper have been reported in abstract form (J. Am. Sot. Nephrol. 2: 749, 1991; and J. Am. Sot. Nephrol. 3: 488, 1992). Current address of A. J. Rouch: Dept. of Physiology and Pharmacology, College of Osteopathic Medicine, Oklahoma State Univ., 1111 W. Seventeenth, Tulsa, OK 74107-1898. Current address of L. H. Kudo: Faculdade de Medicina da USP, Av. Dr. Arnaldo 455, CEP 01246, Sgo Paulo, SP, Brazil. Address for reprint requests: J. A. Schafer, Rm. 958 BHS, 1918 University Ave., Birmingham, AL 35294. Received 8 February 1993; accepted in final form 14 June 1993. REFERENCES 1. Ando, Y., M. D. Breyer, and H. R. Jacobson. Dose-dependent heterogenous actions of vasopressin in rabbit cortical collecting ducts. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F556-F562. 1989.
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