Type II cAMP-dependent protein kinase regulates electrogenic ion transport in rabbit collecting duct

Zhonghua Qi, Chuan-Ming Hao, Kelli Salter, Reyadh Redha, and Matthew D. Breyer

Division of Nephrology, Departments of Medicine and Molecular Physiology and Biophysics, Veterans Affairs Medical Center, and Vanderbilt University School of Medicine, Nashville, Tennessee 37212


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cAMP mediates many of the effects of vasopressin, prostaglandin E2, and beta -adrenergic agents upon salt and water transport in the renal collecting duct. The present studies examined the role of cAMP-dependent protein kinase (PKA) in mediating these effects. PKA is a heterotetramer comprised of two regulatory (R) subunits and two catalytic (C) subunits. The four PKA isoforms may be distinguished by their R subunits that have been designated RIalpha , RIbeta , RIIalpha , and RIIbeta . Three regulatory subunits, RIalpha , RIIalpha , and RIIbeta , were detected by immunoblot and ribonuclease protection in both primary cultures and fresh isolates of rabbit cortical collecting ducts (CCDs). Monolayers of cultured CCDs grown on semipermeable supports were mounted in an Ussing chamber, and combinations of cAMP analogs that selectively activate PKA type I vs. PKA type II were tested for their effect on electrogenic ion transport. Short-circuit current (Isc) was significantly increased by the PKA type II-selective analog pairs N6-monobutyryl-cAMP plus 8-(4-chlorophenylthio)-cAMP or N6-monobutyryl-cAMP plus 8-chloro-cAMP. In contrast the PKA type I-selective cAMP analog pair [N6-monobutyryl-cAMP plus 8-(6-aminohexyl)-amino-cAMP] had no effect on Isc. These results suggest PKA type II is the major isozyme regulating electrogenic ion transport in the rabbit collecting duct.

epithelium; sodium; regulatory subunit; short-circuit current


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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THE RENAL COLLECTING DUCT is a major site of hormonally regulated salt and water transport (5). cAMP serves as an important signaling mechanism in this nephron segment, mediating effects of several hormones on water transport and electrogenic Na+ and Cl- absorption, including arginine vasopressin, beta -adrenergic agents, and prostaglandin E2 (18, 19, 34, 37, 40). cAMP also regulates numerous other cellular functions including intermediary metabolism, secretion, cell proliferation and differentiation, and gene transcription (20, 31, 37, 47, 49). These diverse actions of cAMP may be differentially mediated by distinct cAMP-dependent protein kinase (PKA) isoforms (4, 11, 14, 21).

PKA is a heterotetramer comprised of two cAMP binding regulatory (R) subunits and two catalytic (C) subunits. The four known R subunits, derived from distinct genes, have been designated RIalpha , RIbeta , RIIalpha , and RIIbeta (9, 22, 36, 44). On the basis of their R subunit composition, PKAs can be subdivided into two major isoforms, type I and type II PKA (44). Type I PKAs utilize either RIalpha or RIbeta subunits, whereas type II PKAs utilize RIIalpha or RIIbeta subunits (29). Most tissues coexpress both PKA isoforms, although the relative amount of each isoform may vary between tissues or within the same tissue at different developmental stages (33, 35). The significance of the existence of these multiple PKA isoforms remains uncertain.

The PKA subtypes responsible for regulating salt and water transport in the collecting duct remain uncharacterized. Selective activation of PKA type I versus type II may be accomplished by taking advantage of the differing affinity of these PKA R subunits for cAMP analogs (3, 29, 39). Each R subunit possesses two cAMP binding sites, designated site A and site B (15, 44). Occupation of all four cAMP binding sites induces dissociation of the two R subunits from the two C subunits, releasing the catalytic subunits from the inhibitory effect of the R subunits (44). Selective activation of type I versus type II PKA in cells can be achieved using pairs of cAMP analogs that preferentially activate the A and B sites of RI subunits or RII subunits (3, 29, 39). The purpose of the present studies was to examine the expression of the PKA isoforms in freshly isolated and cultured rabbit cortical collecting ducts (CCDs) and determine their role(s) in regulating electrogenic ion transport in this epithelium.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Collecting duct cell culture. Female New Zealand White rabbits were anesthetized with intramuscular ketamine and xylazine (44 and 10 mg/kg, respectively) and killed by decapitation. Both kidneys were perfused with Krebs-Ringer and harvested. Rabbit CCD cells were immunodissected as previously described (12, 26). The renal cortex was separated from the capsule and medulla via gross dissection and passed through a tissue press. The dispersed tissue was digested with collagenase (0.1%), hyaluronidase (0.1%), DNase (100 U/ml), and soybean trypsin inhibitor (1,000 U/ml, 37°C) in Krebs-Ringer. This suspension was then poured over plates precoated with monoclonal antibody specific for rabbit CCD (3G10) as previously described (12, 26).

Kidney cortex cells were allowed to adhere to sterile polystyrene, 100 × 20-mm plastic culture dishes precoated with 3G10 antibody (150 µg of purified monoclonal antibody in 5 ml PBS for 14 h, 4°C) for 5 min. Nonadherent cells were removed by gentle aspiration. The adherent CCD cells were then knocked off the plates with a sharp mechanical blow. Cells were plated onto 4.52 cm2 collagen-coated semipermeable supports (Costar, Cambridge, MA) at seeding densities of 1.5-2.5 × 106 cells per well. Cells were grown to confluence in DMEM (GIBCO-BRL; Life Technologies, Gaithersburg, MD) plus 1% penicillin-streptomycin-neomycin and 10% heat-inactivated FBS, at 37°C in a humidified 5% CO2-21% O2 atmosphere. Culture medium was replaced every 48 h. Confluence was assessed both by microscopic inspection and by measurements of transepithelial voltage (Vt) and resistance (Rt) using an epithelial volt-ohmmeter (EVOM; World Precision Instruments, Sarasota, FL; see below). For studies using the EVOM, the polarity of the Vt readings was given using the basolateral compartment as reference. Rabbit CCDs cultured on 4.52-cm2 Transwell inserts reached confluence 6-7 days after plating. Rt increased from <400 to >1,500-2,000 Omega  · cm2 as confluence was achieved. Vt typically exceeded -20 mV (basolateral reference) after confluence. Both Vt and Rt reached plateau values after 7-10 days in culture and were stable for approximately an additional week. Only CCD monolayers exhibiting apical negative voltages of -20 to -80 mV (as measured by the EVOM) with resistance of at least 1,000 Omega  · cm2 were used for experimentation.

Preparation of cell and tissue extracts for immunoblot analysis. Freshly immunodissected or cultured CCD cells were homogenized in SDS sample buffer [final concentration: 62.5 mM Tris (pH 6.8), 2% SDS, and 10% glycerol] and equal volumes of 10 mM potassium phosphate buffer (pH 6.8), containing 10 mM EDTA, 10 mM sodium fluoride, 20 mM benzamidine, and 4 mM 2-mercaptoethanol, followed by heating in boiling water for 4 min. For tissue extracts, tissue was homogenized in 4 vol (wt/vol) of 10 mM potassium phosphate buffer at pH 6.8. The homogenate was centrifuged at 15,000 g for 15 min at 4°C. The supernatant was mixed with equal volume of SDS sample buffer and heated in boiling water for 4 min.

Immunoblotting. Cell or tissue extracts (50 µg/lane) were loaded onto a 10% polyacrylamide gel (1.0 mm × 8.0 cm × 7.3 cm) and electrophoresed at 200 V for 45 min using mini-Protein II electrophoresis cell (Bio-Rad Laboratories). The proteins were transferred to nitrocellulose paper at 100 V for 1 h at 4°C. Nitrocellulose paper was blocked (blocking buffer: Tris-buffered saline containing 150 mM NaCl, 50 mM Tris, 0.05% Tween 20 detergent, and 5% nonfat dry milk, pH 7.5) for 1 h at room temperature, then incubated with primary antibodies [mouse anti-RIalpha , anti-RIIalpha , and/or anti-catalytic subunit monoclonal antibodies (Transduction Laboratories), diluted 1:250 in blocking buffer; rabbit anti-RIbeta and -RIIbeta polyclonal antibodies (Chemicon International), diluted 1:5,000 in blocking buffer] for another 1 h at room temperature. This was followed by three washings with TBST (50 mM Tris base, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) at 5-min intervals. The nitrocellulose paper was further incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (diluted 1:5,000 in blocking buffer) for 1 h at room temperature. Following three additional washes with TBST for 10-min intervals, the antigen-IgG complexes were visualized using enhanced chemiluminescence Western blotting detection reagents (ECL, Amersham Life Science). Signals were recorded on Kodak X-ray film (X-OMAT-AR; Eastman Kodak, Rochester, NY).

RNA isolation and RT-PCR. The cDNA sequences of all four subtypes of rabbit PKA regulatory subunits have not been previously reported. We therefore designed primers to amplify fragments of these regulatory subunit cDNAs. Total RNA was isolated from freshly immunodissected and cultured CCD cells using TRIzol (Life Technologies) according to the manufacturer's recommendations. The RNA samples were subjected to reverse transcription using GeneAmp RNA PCR kit (Perkin-Elmer Cetus, Norwalk, CT). Reverse transcription reaction was carried out in 20-µl volume containing 4 µg total RNA, 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 5 mM MgCl2, 1 mM dNTPs, 2.5 µM random hexamers, 1 U/µl RNase inhibitor, and 2.5 U/µl reverse transcriptase.

Degenerate primers were designed according to reported gene sequences for PKA regulatory subunits of other species. Based on mouse PKA RIbeta subunit cDNA sequence, we synthesized degenerate primers: pair 1, 5'-G(A/C)G G(C/T)G TGA GTG C(C/T)G A(A/G)G T(C/G)T-3'; and 5'-ACA TCT TGC GTT TCC TCA G(C/T)G-3'. Based on human PKA RIIalpha subunit cDNA sequence, we synthesized pair 2 of PCR primers: 5'-ACG GTG GAG GTG (C/G)(G/T)(C/G) CGA CAG-3'; and 5'-AGA (A/G)CG (C/G)GT TTG ATT ATC TTT-3'. A final pair of primers (pair 3) based on the rat PKA RIIbeta subunit cDNA sequence were designed as follows: 5'-CCC AG(C/T) AAG GG(G/T) GTC AAC TTC-3'; and 5'-(C/T)GC C(A/G)A AAC TCC CAC GAT T(A/G)T-3'. PCR was performed in 50 µl of total reaction volume containing 10 µl of reverse transcription product mentioned above and the final concentration of 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 2-3 mM MgCl2, 0.3 µM of each primer, and 1.25 U/50 µl of Taq polymerase (Perkin-Elmer Cetus). The cDNA amplification was performed using a Perkin-Elmer DNA Thermal Cycler (model 2400) set to the following cycler protocol: 94°C initial melt for 4 min followed by 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, 32-40 cycles, with a 7-min extension at 72°C.

The fragments of RIalpha , RIbeta , and RIIalpha were subcloned into PCR2.1 vector (Invitrogen) and then transferred into PCRII vector (Invitrogen). RIIbeta fragment was subcloned into PCR2.1 vector (Invitrogen) and then transferred into pBluescript SK(-) vector (Stratagene, La Jolla, CA). The fragments were subjected to sequence with forward and reverse primers at least three times. Sequence comparisons were performed using the BLAST programs provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST) server.

Ribonuclease protection assay. Nuclease protection assays were performed using a commercially available kit according to the manufacturer's directions (In Vitro Transcription Kit and Ribonuclease Protection Assay Kit, Ambion). Briefly, 32P-labeled anti-sense RNA probes were synthesized for each of the four isoforms for rabbit PKA R subunits and for glyceraldehyde-3-phosphate dehydrogenase using either T3, or T7, or SP6 RNA polymerase, as required. The riboprobes were purified using 1 M ammonium acetate and ethanol precipitation, and the radioactivity was determined. Antisense RNA probes, 3 × 105 cpm, were hybridized to 30 µg of sample total RNA in hybridization buffer at 42°C for overnight. After digestion with a mixture of RNase A and RNase T1 (250 U/ml RNase A and 10,000 U/ml RNase T1), the protected fragments were separated on a 5% polyacrylamide gel and visualized by autoradiography.

Electrophysiology. Electrophysiological studies were performed using confluent monolayers of collecting duct cells cultured on semipermeable supports and studied in an Ussing chamber as previously described (12). The Ussing chamber was specifically designed to house 4.52-cm2 Costar Transwells (Jim's Instruments, Iowa City, IA). Each half of the Ussing chamber was filled with 19 ml DMEM (300 mosmol/kgH2O) and continuously bubbled with 95% O2-5% CO2, yielding a pH of 7.30-7.40. The temperature was maintained at 37°C using recirculating heated water bath and a thermistor probe (Yellow Springs Instrument, Yellow Springs, OH) to continuously determine the temperature. Each half-chamber was connected to a voltage-current clamp amplifier (model VCC600; Physiologic Instruments, San Diego, CA) by two agarose/KCl electrodes (3% agar/1 M KCl), one to measure Vt and the other to pass current. The agarose bridges were connected to the amplifier via a 1 M KCl reservoir, and an Ag-AgCl electrode was connected to the amplifier head-stage device. Open-circuit Vt, transepithelial conductance (Gt, mS/cm2), and short-circuit current (Isc, µA/cm2) were determined. Isc was measured when Vt was clamped to 0 mV. The clamp was zeroed before each experiment by inserting a blank Transwell as described above and placing sterile Ringer solution into each half-chamber. After transferring the cells into the Ussing chamber, a 20- to 40-min equilibration was allowed for stabilization of Isc.

Statistics. Changes in Isc were compared by Student's t-test or one-way ANOVA. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of cDNA fragments of rabbit PKA regulatory subunits. Using primer pair 1, a 445-bp product was obtained from rabbit CCD RNA that was 93% identical to the corresponding region of human RIalpha cDNA nucleotide sequence (32). A 440-bp fragment that was 90% identical to the corresponding region of human RIbeta cDNA (41) nucleotide sequence was also amplified from rabbit ovary RNA using primer pair 1. Primer pair 2 amplified a 373-bp fragment from rabbit testis, which was 92% identical to the human RIIalpha cDNA fragment nucleotide sequence (30). Primer pair 3 amplified a 469-bp fragment from rabbit CCD mRNA, which was 93% identical to human RIIbeta cDNA nucleotide sequence (23). These sequences have been deposited in GenBank (accession nos. AF089893, AF093005, AF089894, and AF089895)

Characterization of PKA regulatory subunits in CCDs. Immunoblots using antibodies specific for RIalpha , RIbeta , RIIalpha , and RIIbeta subunits detected proteins migrating at the expected size for RIalpha (49 kDa), RIIalpha (51-54 kDa), and RIIbeta (52-53 kDa) (38) in both cultured and freshly immunodissected CCD cells (Fig. 1A, n = 6). In contrast, RIbeta subunit (55 kDa) (9) was not detected in either fresh or cultured CCDs, although as expected, it was detected in brain (n = 6). Simultaneous immunodetection of PKA catalytic (Calpha ) subunit protein expression showed it was present in comparable amounts in all lanes. In contrast, RIIbeta subunit protein appeared to be more abundant than either RIIalpha or RIalpha subunit by immunoblot (Fig. 1B, n = 6).


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Fig. 1.   Immunoblots of cAMP-dependent protein kinase (PKA) regulatory (R) subunit isoforms in rabbit cortical collecting duct (CCD) cells. A: immunodetection of RIalpha , RIIalpha , and RIIbeta subunits in freshly immunodissected rabbit CCD cells (f-CCD) and cultured CCD cells (c-CCD). Protein loading was equal (50 µg/lane), except for brain tissue (10 µg/lane). B: comparison of protein expression levels of PKA R subunits in cultured rabbit CCD cells. Simultaneous immunodetection of catalytic (C) subunit was performed using an anti-catalytic subunit of PKA antibody (anti-Calpha monoclonal antibody).

The presence of RIalpha , RIIalpha , and RIIbeta subunits in CCDs was confirmed by nuclease protection assay using antisense RNA probes specific for rabbit R subunits generated as described above. RIalpha , RIIalpha , and RIIbeta subunit mRNAs were detected in both cultured and freshly immunodissected CCDs (Fig. 2, n = 4). Furthermore, although RIbeta subunit signal was detected in rabbit brain, it was not detected in CCDs (n = 4).


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Fig. 2.   Nuclease protection assays of CCD cell mRNA for the four isoforms of PKA regulatory subunits. RNAs from both fresh (f-CCD) and cultured CCD cells (c-CCD) were studied. Expected lengths of protected fragments for RIalpha , RIbeta , RIIalpha , and RIIbeta subunits were 403 bp, 396 bp, 333 bp, and 427 bp, respectively. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe was used simultaneously to normalize for RNA loading.

Type II PKA regulates ion transport in CCDs. The effect of increasing concentrations of site-selective cAMP analogs on Isc in cultured CCDs was determined to establish dose-response curves for individual analogs (Fig. 3). Site-selective cAMP analogs displayed different threshold concentrations above which they increased Isc, presumably by nonspecifically binding to both site A and site B of PKA regulatory subunit. The two site A-selective analogs, which are active at either PKA type I or type II, displayed markedly different potency. N6-monobutyryl-cAMP required 100 µM to increase Isc, whereas N6-phenyl-cAMP increased Isc at concentrations above 3 µM. Type II site B-selective analogs displayed similar potency with 8-chloro-cAMP (8-Cl-cAMP) and 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) increasing Isc at concentrations above 3 µM when administered as single agents. In contrast, the type I site B-selective analog 8-(6-aminohexyl)-amino-cAMP (8-AHA-cAMP) only increased Isc at concentrations above 100 µM.


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Fig. 3.   Dose-dependent effects of site-selective cAMP analogs on short-circuit current (Isc) in cultured rabbit CCDs. Vertical axis depicts the change in Isc (Delta Isc) before and after addition of each analog at the indicated concentration. Effects of N6-phenyl-cAMP (, n = 4), 8-Cl-cAMP (triangle , n = 4), 8-CPT-cAMP (, n = 4), N6-monobutyryl-cAMP (black-diamond , n = 4), and 8-(6-aminohexyl)-amino-cAMP (8-AHA-cAMP, ; n = 4) were studied. Error bars represent ±SE.

Combinations of PKA site-selective cAMP analogs were chosen to activate either PKA type I or type II. Subthreshold concentrations of these site-selective analogs were used so as to selectively bind to site A or site B on PKA R subunits, as follows: N6-phenyl-cAMP (1 µM), 8-Cl-cAMP (1 µM), 8-CPT-cAMP (1 µM), N6-monobutyryl-cAMP (50 µM), and 8-AHA-cAMP (100 µM). Only combinations of a type II site A plus site B-selective analogs increased Isc (Figs. 4 and 5). An example of synergy between a PKA type II-selective combination is shown for the pair N6-monobutyryl-cAMP + 8-CPT-cAMP in Fig. 4A. N6-monobutyryl-cAMP + 8-CPT-cAMP changed Isc by +5.22 ± 1.05 µA/cm2 (n = 4, P < 0.001, compared with N6-monobutyryl-cAMP + N6-phenyl-cAMP). Similarly, N6-monobutyryl-cAMP + 8-Cl-cAMP changed Isc by +4.62 ± 0.76 µA/cm2 (n = 4, P < 0.001, compared with that of N6-monobutyryl-cAMP + N6-phenyl-cAMP). Synergy was not observed using combinations of two site B-selective analogs (8-CPT-cAMP + 8-Cl-cAMP, Delta Isc = +0.35 ± 0.64 µA/cm2, n = 4) or two site A-selective analogs (N6-monobutyryl-cAMP + N6-phenyl-cAMP, Delta Isc = +0.43 ± 0.25 µA/cm2, n = 4) (Fig. 5). Similar studies using the type I-selective cAMP analog pair N6-monobutyryl-cAMP + 8-AHA-cAMP failed to significantly increase Isc, changing Isc by -0.168 ± 0.46 µA/cm2 (n = 4). In these latter studies, subsequent addition of 8-CPT-cAMP (5 µM) resulted in the expected synergy, increasing Isc by +5.69 ± 0.22 µA/cm2 (Figs. 4B and 5).


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Fig. 4.   A: Isc trace before and after addition of the type II-selective cAMP analog pair N6-monobutyryl-cAMP (N6mono, 50 µM) plus 8-CPT-cAMP (8CPT, 1 µM). The subsequent effect of the epithelial Na+ channel blocker, amiloride (10 µM), on cAMP-stimulated Isc is also shown. B: Isc trace in response to type I-selective cAMP analogs. After adding the PKA type I-selective pair [8-AHA-cAMP (8AHA), 100 µM; plus N6-monobutyryl-cAMP, 50 µM], the PKA type II active analog 8-CPT-cAMP (8CPT), 5 µM, was added, followed by the addition of amiloride (10 µM).


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Fig. 5.   Effects of PKA isoform-selective cAMP analog pairs on Isc. Type II-selective analog pairs [N6-monobutyryl-cAMP (50 µM) + 8-CPT-cAMP (1 µM), n = 4; N6-monobutyryl-cAMP (50 mM) + 8-Cl-cAMP (1 µM), n = 4] increased Isc significantly. * P < 0.001, compared with Isc change of N6-monobutyryl-cAMP (50 µM) + N6-phenyl-cAMP (1 µM) pair (n = 4). Type I-selective analog pair [N6-monobutyryl-cAMP (50 µM) + 8-AHA-cAMP (100 µM), n = 4] had no effect on Isc. Subsequent addition of 8-CPT-cAMP (5 µM, n = 4) induced a significant increase in Isc. * P < 0.001, compared with Isc change after N6-monobutyryl-cAMP + N6-phenyl-cAMP pair. Error lines represent ±SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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The present studies characterized the PKA isoforms in the rabbit CCD. We amplified and sequenced the fragments of regulatory subunits for all known PKA R subunit isoforms from rabbit. These subunits showed high homology to those of other species. Immunoblot and ribonuclease protection assays yielded mutually supporting results, detecting RIalpha , RIIalpha , and RIIbeta subunits in both freshly immunodissected and cultured rabbit CCDs. RIbeta subunit was not detected in either fresh or cultured CCDs. Cultured CCDs grown on semipermeable support display many functional similarities to freshly isolated CCD cells (7, 12, 17, 18, 26). The present study provides evidence that the expression pattern of PKA regulatory subunits in cultured CCD cells and freshly isolated CCDs represents another such similarity.

Immunoblots also suggest that RII subunits (alpha  and beta ) are the predominant subunits expressed in cultured collecting ducts. There appears to be a preferred order of assembly of RII subunits with catalytic subunits versus a lower rate of incorporation of RI subunits (2, 10, 11, 28, 29). Overexpression of RIIalpha subunit in ras-transformed NIH3T3 (R3T3) cells, which normally contains approximately equal amounts of type I and type II holoenzymes, eliminated type I PKA isozyme and reduced RIIbeta -isozyme expression (29). Similarly overexpression of RIIbeta subunit in R3T3 cells eliminated the type I PKA holoenzyme (29). These data suggest that assembly of PKA type II holoenzyme is positively correlated with the expression of type II regulatory subunit protein. Taken together with the predominance of the type II alpha - and beta -subunits in CCDs, these data suggest that type II is the major holoenzyme present in the collecting duct.

The present studies also provide information regarding the functional role of specific PKA isoforms regulating ion transport in the collecting duct. The cAMP analogs used in this experiment are well characterized with respect to their binding selectivity for the two cAMP sites on R subunits of PKA (Table 1) (1, 27). For example, N6-monobutyryl-cAMP and N6-phenyl-cAMP display a higher affinity for site A on either PKA type I or PKA type II than for site B (27). Conversely, 8-CPT-cAMP is selective for PKA type II site B, whereas 8-AHA-cAMP displays approximately a 15-fold selectivity for type I site B versus site A. The selectivity of these cAMP analogs is maintained only within a certain concentration range, above which selectivity is lost, with the analog occupying both cAMP binding sites, thereby activating the holoenzyme. It is therefore important that the present studies identified threshold concentrations for these single analogs, below which no significant change in CCD Isc could be detected. Studies using combinations of analogs selective for site A and/or site B, at concentrations below these threshold values, demonstrated that only pairs of site A + B active compounds (N6-monobutyryl-cAMP + 8-Cl-cAMP, or N6-monobutyryl-cAMP + 8-CPT-cAMP), induced a significant increase in Isc. The lack of similar synergy between subthreshold concentrations of two site A-selective analogs (N6-monobutyryl-cAMP + N6-phenyl-cAMP) or two site B-selective analogs (8-CPT-cAMP + 8-Cl-cAMP), provides evidence that PKA mediates the increased Isc elicited by cAMP. The demonstration of synergy makes alternative mechanisms including direct gating of ion channels by cAMP (13) less likely.

                              
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Table 1.   Affinity of cAMP analogs for site A and site B of PKA regulatory subunits

The present studies also suggest that PKA type II is the primary isozyme mediating changes in CCD electrogenic ion transport, rather than PKA type I. Although PKA RIalpha and RIIalpha and RIIbeta subunits were all detected in the CCD, only the PKA type II-selective analog pairs, N6-monobutyryl-cAMP + 8-Cl-cAMP or N6-monobutyryl-cAMP + 8-CPT-cAMP, affected Isc. In contrast the type I-selective analog combination N6-monobutyryl-cAMP + 8-AHA-cAMP failed to affect Isc. Were type I PKA involved in the regulation of ion transport in CCDs, synergy should have been seen using this analog pair, since selective activation of PKA type I by 8-AHA-cAMP has been well demonstrated in other cells (39, 42, 48). These findings make it unlikely that PKA type I regulates ion transport in rabbit CCDs; rather, it seems likely that type II holoenzyme is the primary isozyme regulating ion transport in the CCD.

There are multiple effects of cAMP on electrogenic ion transport in both freshly microdissected collecting ducts and cultured collecting ducts. These include increased Cl- transport (19, 24, 34) and a biphasic change in amiloride-sensitive Na+ absorption (6, 7). Although the present studies do not address which ion transport process is activated by cAMP in cultured CCDs, the studies using amiloride (e.g., Fig. 4) suggest electrogenic Na+ transport comprises a major fraction of the Isc in these cells and may be one target of PKA type II isozyme effects. These studies do not exclude additional effects of type II PKA on electrogenic Cl- transport. Cystic fibrosis transmembrane conductance regulator (CFTR) may interact with amiloride-sensitive epithelial sodium channel and has been found expressed in CCD cells (43, 45, 46). Type II PKA has also been suggested to regulate CFTR function in human pancreatic adenocarcinoma cells (42).

The precise roles for different PKA isozymes in regulating cell function remains unclear, but accumulating data indicate that PKA holoenzymes are differentially compartmentalized and activate distinct cellular processes. Steagall et al. (42) demonstrated that the type I PKA-selective combination 8-AHA-cAMP + 8-piperidino-cAMP stimulated soluble PKA activity, but not the insoluble PKA activity in a human pancreatic adenocarcinoma cell line. Other data indicate that RI subunits play a special role in cell proliferation and growth, since this expression increases with cellular transformation (8, 25). Conversely, differentiation of mouse ST13 preadipocytes to adipocytes is associated with a marked increase in PKA type II isozyme (33). An important role for PKA type II in fat metabolism has also been suggested in mouse white adipose tissue, which predominantly expresses RIIbeta subunit. Knockout of the RIIbeta gene produces lean mice with altered fat metabolism (11). These differences may relate to differential association of R subunits with distinct A-kinase-anchoring proteins (AKAPs) (16, 21). AKAPs may serve to coordinate the interaction of PKA with other signaling pathways, as well as to localize the different PKA isoforms close to their downstream targets (21). The identification of the PKA isoforms involved in regulating collecting duct ion transport should facilitate characterization of the phosphorylation targets immediately downstream of PKA which control salt transport in the collecting duct.


    ACKNOWLEDGEMENTS

These studies were supported by a Dept. of Veterans Affairs Merit Award (to M. D. Breyer). M. D. Breyer is the recipient of a Veterans Affairs Clinical Investigator Award. We also acknowledge the use of the Vanderbilt Cancer Center DNA sequencing resource and support from National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-37097 (to M. D. Breyer).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. D. Breyer, F-427 ACRE Bldg., Veterans Affairs Medical Center, Nashville, TN 37212 (E-mail: Matthew.Breyer{at}mcmail.vanderbilt.edu).

Received 10 November 1998; accepted in final form 15 January 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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