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作者：Kay-Pong Yip作者单位：Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida
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【摘要】/ X+ G* \) t! y9 w' M. p# l' V
PKA has traditionally been thought as the binding protein of cAMP for mediating arginine vasopressin (AVP)-regulated osmotic water permeability in kidney collecting duct. It is now known that cAMP also exerts its effects via Epac (exchange protein directly activated by cAMP) and that intracellular Ca 2 mobilization is necessary for AVP-induced apical exocytosis in inner medullary collecting duct (IMCD). The role of Epac as an effector of cAMP action in addition to PKA was investigated using confocal fluorescence microscopy in perfused IMCD. PKA inhibitors (1 µM H-89 or 10 µM KT-5720) at concentrations known to inhibit aquaporin-2 (AQP2) phosphorylation did not prevent AVP-induced Ca 2 mobilization and oscillations. Epac-selective cAMP agonist (8-pCPT-2'- O -Me-cAMP) mimicked AVP in triggering Ca 2 mobilization and oscillations, which was blocked by ryanodine but not by Rp-cAMP (a competitive antagonist of cAMP binding to PKA). 8-pCPT-2'- O -Me-cAMP also triggered apical exocytosis in the presence of a PKA inhibitor. Immunolocalization of AQP2 in perfused IMCD demonstrated that 8-pCPT-2'- O -Me-cAMP induces apical targeting of AQP2 and that AQP2 is abundant in junctional regions of basolateral membrane. Immunofluorescence study also confirmed the presence of Epac (isoform I) in IMCD. These results indicate that activation of Epac by an exogenous cAMP analog triggers intracellular Ca 2 mobilization and apical exocytotic insertion of AQP2 in IMCD. - o6 D9 ]/ ^; P7 d
【关键词】 aquaporin vasopressin FM fluorescence confocal microscopy
& r$ ]3 k$ z t+ W ARGININE VASOPRESSIN INCREASES osmotic water permeability (P f ) of kidney collecting duct by triggering translocation and fusion of intracellular vesicles containing aquaporin-2 (AQP2) to the apical membrane of principal cells ( 31, 32, 45 ). The signaling process is initiated by binding of AVP to basolateral V 2 -vasopressin receptors in collecting duct cells. These receptors coupled to the heterotrimeric G protein, G s, which activates the effector enzyme adenylyl cyclase type VI ( 5 ) and increases cAMP levels in the cells. It is widely accepted that cAMP-dependent protein kinase (PKA) is the binding protein of cAMP to regulate AQP2 apical targeting through phosphorylation ( 24, 33 ). The mechanism presumably involves sorting of AQP2-containing vesicles in the Gogi complex ( 36 ). Interactions of AQP2-containing vesicles with actin and microtubule cytoskeleton ( 3, 44 ) and soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins ( 16, 32 ) are also implicated in the regulation of AQP2 apical targeting. By using confocal fluorescence microscopy to monitor intracellular Ca 2 concentration ([Ca 2 ] i ) and apical exocytosis in individual cells of perfused inner medullary collecting duct (IMCD), it was shown that physiological dose of AVP triggers intracellular Ca 2 mobilization and [Ca 2 ] i oscillations and that the Ca 2 mobilization is necessary for AVP-stimulated apical exocytosis and osmotic water permeability ( 8, 47 ). The action of AVP in triggering Ca 2 oscillations in IMCD was mimicked by cAMP ( 47 ), which suggests that both PKA activation and Ca 2 mobilization are post-cAMP events in native IMCD cells ( 8, 47 ). However, it is not known whether Ca 2 mobilization is the consequence of PKA activation or is mediated by a signaling pathway parallel to PKA activation.6 H: M, M8 f; |6 Q3 L
7 d/ W% W$ y. n2 tPKA has traditionally been thought as the effector of cAMP to mediate its cellular action in regulating osmotic water permeability in the collecting duct ( 41 ). There are recent studies indicating that cAMP acts directly on the exocytotic machinery in neuronal ( 35, 38 ) and nonneuronal ( 34, 37, 38 ) cells independently of PKA. Epac (exchange protein directly activated by cAMP, also known as cAMP-regulated guanine-nucleotide-exchange factor), is a new class of cAMP sensor proteins widely distributed in different tissues ( 12, 25 ). Epac binds cAMP and selectively activates the Ras-like small GTPase Rap1 and Rap2 ( 1, 25 ). There are two known isoforms of Epac, Epac I and Epac II ( 25 ). Epac I is abundant in kidney collecting duct and has been implicated in the regulation of H -K -ATPase activity in the cortical collecting duct via exocytosis ( 25, 26 ). Epac I also regulates the secretion of a nonamyloidogenic soluble form of amyloid precursor protein in primary culture of mouse cortical neurons ( 29 ). Epac II was shown to mediate a PKA-independent, ryanodine-sensitive, Ca 2 -induced Ca 2 release in INS-1 cells ( 19, 20, 22 ), as well as the PKA-independent insulin exocytosis in mouse pancreatic -cells ( 20, 23 ).0 Y$ }4 b; r% J% _! |' Z
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cAMP activates PKA and Epac equally ( 9 ). It is intriguing to consider that cAMP mediates AQP2 exocytosis via Epac in addition to activation of PKA. The aims of this study were to test 1 ) whether PKA inhibitors at a concentration known to inhibit AQP-2 phosphorylation abolish AVP-induced Ca 2 mobilization and 2 ) whether an agonist specific to Epac induces Ca 2 mobilization, apical exocytosis, and trafficking of AQP2 in IMCD. The results provide evidence that cAMP is capable of triggering Ca 2 mobilization and apical exocytosis of AQP2 through an Epac-dependent pathway.9 s, n, m3 U5 I
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MATERIALS AND METHODS# _( o+ E& P; h2 {8 l' P. Q0 h
) A B1 ?# J4 \, a0 BIsolation and perfusion of single IMCD segment. Experiments were carried out in accordance with guidelines for the care and use of research animals. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee at the University of South Florida, in accordance with Public Health Service Policy on Human Care and Use of Laboratory Animals. Experiments were conducted in IMCD isolated from male Sprague-Dawley rats (60-100 g body wt, Harlan). The rats were treated with furosemide intraperitoneally (5 mg) for 30 min and then killed by anesthetic overdose with 5% halothane through a Fluotec Mark-3 vaporizer. Furosemide treatment was used to wash out the medullary osmolarity gradient, and to minimize osmotic shock when medullary is placed in isotonic dissection solution ( 7 ). The kidneys were rapidly removed through a midline abdominal incision and placed in an ice-cold dissecting solution. The composition of dissection solution consisted of (in mM) 120 NaCl, 25 NaHCO 3, 2 K 2 HPO 4, 1.2 MgSO 4, 2 CaCl 2, 5.5 glucose, and 5 sodium acetate. Terminal IMCD segments were dissected from the inner half of the inner medulla ( 7 ). The isolated piece of IMCD was then transferred to a temperature-controlled perfusion chamber (Vestavia) mounted on a Zeiss Axiovert 100TV inverted microscope, which is coupled to a laser-scanning unit (Bio-Rad MRC1000) equipped with krypton-argon laser. The IMCD was then cannulated and perfused with glass concentric pipettes using the method developed by Burg ( 4 ). Luminal and bath perfusates are identical to dissecting solution. All solutions were gassed with 95% O 2 -5% CO s before use, and pH was adjusted to 7.4. For a Ca 2 -free perfusate, CaCl 2 was replaced by 2 mM EGTD.* ]6 n9 N k6 @ I2 t
9 Y, K+ n# s! I! k! }. n6 {7 t9 WMeasurement of intracellular calcium. The changes in [Ca 2 ] i in IMCD cells induced by AVP or other pharmacological agents were determined from the confocal fluorescence images of the perfused tubules as described previously ( 8, 47 ). In brief, 5 µM fluo-4/AM (Molecular Probes) was loaded into the IMCD from the peritubular solution at room temperature for 30 min. The tubule was then washed and incubated at 37°C for another 30 min for deesterification before measurements were begun. Confocal fluorescence images were acquired from the lower surface of the perfused IMCD with the 488-nm laser line. All images were collected with a Zeiss x 40 plan-apochromat objective (numerical aperture 1.2). Emission was collected with a bandpass filter 522/35 nm at 0.5 Hz and stored digitally. Residence time of the laser on the IMCD for each image was 0.4 s. The spatial and temporal variations of [Ca 2 ] i after the introduction of AVP/cAMP analogs into the peritubular perfusate were monitored in individual IMCD cell during playback of the stored fluorescence images using the software (Time Course/Ratiometric Software Module) supplied by Bio-Rad. Calibration of fluorescence emission of fluo-4 was performed using the nonfluorescent Ca 2 ionophore (4-bromo-A23187) as described previously ( 47 )., [; G' p9 W- |4 Q
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Real-time monitoring of apical exocytosis in IMCD. Apical exocytosis in perfused IMCD was monitored by using FM1-43 (Molecular Probes) as described previously ( 47 ). In brief, 2 µM FM1-43 was included in the luminal perfusate to label the apical membrane of IMCD cells. FM1-43 is a styryl dye which is nonfluorescent in aqueous solution, is impermeable to cell membrane, and is fluorescent when bound to membranes ( 11 ). The fluorescence of FM1-43 is the same whether dissolved in surface membrane or granule membrane in vesicles formed by endocytosis ( 40 ). FM1-43 fluorescence, therefore, is used as an index of accumulative exocytosis ( 11, 40 ). Confocal images were acquired at the midplane of the perfused IMCD (excited by 488-nm laser line, emission collected with a longpass filter 580LP) so that the apical membrane of each IMCD cells was clearly discerned on both sides of the lumen. The spatial and temporal variations of FM1-43 apical fluorescence induced by cAMP analogs were measured in individual IMCD cell from the stored fluorescence images.
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Immunolocalization of AQP2 and Epac in IMCD. To immunolocalize AQP2 in perfused IMCD, IMCD was fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.5% of Triton X-100 in PBS for 45 min. The tubule was then incubated with anti-AQP2 antibodies (5 µg/ml, rabbit IgG, Sigma) for 4 h, followed by incubation with corresponding cy2- or cy5-conjugated secondary antibodies (3 µg/ml; Jackson ImmunoResearch) for 2 h. Confocal optical sections were then collected from the perfused IMCD.
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; Y. c" T& i' O$ e8 j3 tTo immunolocalize AQP2 and Epac in kidney cryosections, rat kidneys were perfusion fixed with a mixture of 4% paraformaldehyde and 4% sucrose PBS by retrograde perfusion via a cannula inserted into the descending aorta distal to the renal artery. The kidneys were then removed and were postfixed overnight in the same fixative at 4°C, washed with 0.1 M aqueous NH 4 Cl solution for 30 min, and cryoprotected by incubation in 2.3 M sucrose in PBS for 1 h. Cryosections (7 µm) were cut and transferred to Fisher Superfrost Plus-charged glass slides for indirect immunofluorescence staining of AQP2 (anti-AQP2, rabbit IgG, 5 µg/ml, Sigma) and Epac (anti-EPAC I, goat IgG, 8 µg/ml; Santa Cruz Biotechnology) ( 48 ). To test the specificity of the Epac I antibody, it was preincubated with the antigenic peptide (25 µg/ml; Santa Cruz Biotechnology) at 4°C overnight before being applied to kidney cryosections.; U9 x1 f+ s9 E/ o$ j
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Chemicals. AVP was purchased form Bachem. CPT-cAMP (8-(4-Chlorophenylthio)-cAMP), Rp-cAMP (Rp-adenosine 3',5'-cyclic-monophosphorothioate triethylammonium salt), 8-pCPT-2'- O -Me-cAMP (8-(4-Chlorophenylthio)-2'- O -methy-cAMP) were purchased from Sigma. H-89 and KT-5720 were purchased from Calbiochem.
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Statistics. Results are reported as means ± SE. The BMDP statistical package (program 1R) was used for regression analysis and ANOVA of regression coefficients ( 14 )." Q4 j$ j2 t7 p% l
" f0 ]7 J3 a# DRESULTS
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Effects of PKA inhibitors on AVP-induced Ca 2 mobilization in IMCD. Previous studies demonstrated that AVP-stimulated water permeability and apical exocytosis in IMCD require intracellular Ca 2 mobilization ( 8, 47 ) and that a PKA inhibitor (H-89) inhibits cAMP-stimulated water permeability ( 41 ). The causal relationship between Ca 2 mobilization and PKA activation was investigated using two PKA inhibitors, H-89 and KT-5720. Preincubation of IMCD with 1 µM H-89 for 30 min did not inhibit AVP-induced Ca 2 mobilization and [Ca 2 ] i oscillations. Figure 1 A is the mean normalized time course of changes in fluo-4 emission intensity induced by 0.1 nM AVP in the presence of 1 µM H-89. Similarly, 10 µM KT-5720 did not inhibit AVP-induced Ca 2 mobilization and [Ca 2 ] i oscillations ( Fig. 1 B ). These two PKA inhibitors at the above concentrations are specific to PKA ( 17, 22, 27 ) and are inhibitory to PKA-dependent phosphorylation of AQP2 ( 2 ). These observations suggest that Ca 2 mobilization is not the consequence of PKA activation. Preincubation of IMCDs with 0.3 mM Rp-cAMP (an antagonist of cAMP for PKA binding) also did not inhibit AVP-induced Ca 2 mobilization, which substantiated our conclusion. However, preincubation of IMCDs with 50 µM H-89 inhibited AVP-induced Ca 2 mobilization and [Ca 2 ] i oscillations ( Fig. 1 D ).: H6 G+ M+ ~. }0 |
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Fig. 1. Normalized time course of changes in fluo-4 emission intensity in inner medullary collecting duct (IMCD) induced by 0.1 nM AVP in the presence of 1 µM H-89 (40 cells/4 tubules; A ), 10 µM KT-57201 (37 cells/4 tubules; B ), and 0.3 mM Rp-cAMP (38 cells/4 tubules; C ). D : with 50 µM H-89 (35 cells/4 tubules) and without H-89 (41 cells/3 tubules ); 0.1 nM AVP is introduced into the peritubular perfusate at time 0. Dotted lines are SE.5 F3 M! t! M6 L4 s- U d2 j
( m) p- {* Y7 G. o5 |Effects of Epac-selective agonist in IMCD [Ca 2 ] i. A cAMP analog has been shown to mimic AVP in stimulating Ca 2 mobilization ( 8 ). Epac is a newly identified effector of cAMP and is expressed in IMCD ( 26 ). To test whether Epac is the effector of cAMP to mediate Ca 2 mobilization, 8-pCPT-2'- O -Me-cAMP was used to selectively activate Epac ( 15, 22 ). In the control study, 0.2 mM CPT-cAMP (a cell-permeant cAMP analog) was used to induce Ca 2 mobilization and [Ca 2 ] i oscillations in IMCD ( Fig. 2 A ). The mean [Ca 2 ] i in the baseline and at the peak of oscillations were 69 ± 4 and 277 ± 23 nM (21 cells/3 tubules), respectively. The same concentration of 8-pCPT-2'- O -Me-cAMP also triggered similar Ca 2 mobilization and [Ca 2 ] i oscillations in IMCD ( Fig. 2 B ). However, there was a time delay between application of the agonist and the initiation of intracellular Ca 2 mobilization when the Epac-selective agonist was used. The time delay was reflected in the mean normalized time course as a gradual initial increase in [Ca 2 ] i. The mean [Ca 2 ] i in the baseline and at the peak of oscillations were 63 ± 5 and 217 ± 25 nM (22 cells/3 tubules). The [Ca 2 ] i oscillations induced by either CPT-cAMP and 8-pCPT-2'- O -Me-cAMP ( Fig. 2, C and D ) were time variant in both amplitude and frequency as observed previously ( 47 ). To test whether the Ca 2 mobilization effect of 8-pCPT-2'- O -Me-cAMP is exclusively due to the activation of Epac, IMCDs were preincubated with 0.3 mM Rp-cAMP (an antagonist of cAMP for PKA binding) for 30 min before the exposure of 8-pCPT-2'- O -Me-cAMP. Preincubation of Rp-cAMP did not inhibit 8-pCPT-2'- O -Me-cAMP-induced Ca 2 mobilization and [Ca 2 ] i oscillations ( Fig. 3 A ). These observations indicate that Epac is an effector of cAMP to mobilize Ca 2 in IMCD. To test whether 8-pCPT-2'- O -Me-cAMP-stimulated Ca 2 mobilization is ryanodine sensitive as observed in AVP-stimulated Ca 2 mobilization, IMCDs were preincubated with 0.1 mM ryanodine in the absence of extracellular Ca 2 for 30 min before the exposure to 8-pCPT-2'- O -ME-cAMP. Preincubation of ryanodine abolished 8-pCPT-2'- O -ME-cAMP-induced Ca 2 mobilization ( Fig. 3 B ) ( 47 ), suggesting that 8-pCPT-2'- O -ME-cAMP mobilizes Ca 2 from ryanodine-sensitive intracellular stores." j% R1 K# {7 J
- _7 c) z" \8 x2 OFig. 2. Mean normalized time course of changes in fluo-4 emission intensity in IMCD induced by 0.2 mM CPT-cAMP (51 cells/5 tubules; A ) and 0.2 mM 8-pCPT-2'- O -ME-cAMP (63 cells/5 tubules; B ). Representative tracing of variations in fluo-4 emission in single IMCD cell induced by 0.2 mM CPT-cAMP ( C ) and 0.2 mM 8-pCPT-2'- O -ME-cAMP ( D ). cAMP analog is introduced into the peritubular perfusate at time 0. Dotted lines are SE. Mean [Ca 2 ] i before application of cAMP analog is 65 ± 5 nM (43 cells/6 tubules). Peak [Ca 2 ] i induced by 0.2 mM CPT-cAMP and 0.2 mM 8-pCPT-2'- O -ME-cAMP are 277 ± 23 nM (21 cells/3 tubules) and 217 ± 24 nM (22 cells/3 tubules), respectively.
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" p7 F5 I1 `+ H5 o y+ {5 i+ MFig. 3. Mean normalized time course of changes in fluo-4 emission intensity in IMCD induced by 0.2 mM 8-pCPT-2'- O -ME-cAMP. IMCD was preincubated with 0.3 mM Rp-cAMP (37 cells/ 4 tubules; A ) and 0.1 mM ryanodine in Ca 2 -free solution (26 cells/3 tubules; B ). cAMP analog is introduced into the peritubular perfusate at time 0. Dotted lines are SE.
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Apical exocytosis induced by cAMP analogs. Exocytosis of IMCD at real time was monitored with FM1-43 fluorescence on the apical membrane of perfused IMCD. We previously demonstrated that 0.1 nM AVP triggered apical exocytosis within 20 s using this method ( 47 ); 0.2 mM CPT-cAMP simulated AVP in stimulating exocytosis in perfused IMCD ( Fig. 4 A ). There was a gradual increase in apical FM1-43 fluorescence in the first 15 min. The regression coefficient (slope) of the regression line was 0.83 x 10 -3 ± 0.25 x 10 -4 /s -1 ( n = 5 tubules). In the timed control, no significant increase of FM1-43 fluorescence was observed ( 47 ). These are consistent with the consensus that AVP triggers AQP2 shuttling through a cAMP-dependent pathway. Preincubation of IMCD with 1 µM H-89 attenuated but did not abolish CPT-cAMP-stimulated apical exocytosis ( Fig. 4 B ). The slope of the regression line was decreased to 0.29 x 10 -3 ± 0.15 x 10 -4 /s -1 ( n = 5 tubules), which was significantly different ( P " `9 E! ~1 }. F7 f D- j
: x3 l2 I; ^7 o G* S! hFig. 4. Mean normalized time course of changes in apical FM1-43 fluorescence intensity in IMCD induced by 0.2 mM CPT-cAMP (regression coefficient 0.83 x 10 -3 ± 0.25 x 10 -4 /s -1, n = 5 tubules; A ), 0.2 mM CPT-cAMP in the presence of 1 µM H-89 (regression coefficient 0.29 x 10 -3 ± 0.15 x 10 -4 /s -1, n = 5 tubules; B ), and 0.2 mM 8-pCPT-2'- O -ME-cAMP the presence of 1 µM H-89 (regression coefficient 0.31 x 10 -3 ± 0.1 x 10 -4 /s -1, n = 5 tubules; C ). cAMP analog is introduced into the peritubular perfusate at time 0. Dotted lines are SE. Straight lines are regression line for the first 800 s.0 Z" X; q K( h1 y! L3 C$ f: u
! S( Z1 I" R- q. A& ]; D9 pImmunolocalization of AQP2 in perfused IMCD. To test whether the apical exocytosis detected by FM1-43 fluorescence is accompanied by targeting of AQP2 to the apical membrane, AQP2 was localized in perfused IMCD with indirect immunofluorescence. IMCDs were perfused for 30 min in AVP-free buffer before fixation as a negative control. While AQP2 immunofluorescence in apical membrane was minimal, there was a distinct AQP2 immunofluorescence signal in the basolateral membrane ( Fig. 5, A and B ). These observations were consistent with the previous report that AQP2 was localized in the basolateral membrane of rat IMCD ( 10 ). In the positive controls, IMCDs were incubated with 1 nM AVP for 30 min before fixation. A distinct AQP2 immunofluorescence signal was found in both apical and basolateral membranes, confirming that it is possible to detect AQP2 apical targeting with indirect immunofluorescence in perfused IMCD ( Fig. 5, C and D ). Incubation of IMCD with 50 µM 8-pCPT-2'- O -Me-cAMP before fixation also increased apical abundance of AQP2 ( Fig. 5, E and F ). Neither AVP nor 8-pCPT-2'- O -Me-cAMP induced basolateral targeting of AQP2. These observations indicate that the apical exocytosis induced by Epac-selective agonist is associated with insertion of AQP2. Double labeling of AQP2 and Epac in kidney cryosections confirmed the expression of Epac I protein in rat IMCD ( Fig. 6 ), which is consistent with the detection of Epac I mRNA in IMCD ( 26 ). Preincubation of the Epac I antibody with the antigenic peptide attenuated the immunofluorescence of Epac I in IMCD ( Fig. 6 D ). The same antibody has been used to immunolocalize Epac I in proximal tubule of mouse kidney ( 18 ).6 a7 }% N8 g. M
& ^' J/ k- E: F3 C# ~* T% {' NFig. 5. Immunofluorescence of AQP2 in basolateral and apical surface of perfused IMCD ( A, B ) after 30 min of incubation with IMCD buffer, 1 nM AVP ( C, D ), and 50 µM 8-pCPT-2'-O-ME-cAMP ( E, F ). A, C, and E were collected from the lower surface of perfused IMCD. B, D, and F were collected in the middle section between the lower and upper surface of perfused IMCD. Arrows point to the apical membrane.
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i) l5 A( I5 I i7 ?! k7 mFig. 6. Double labeling of AQP2 ( A, C ) and Epac I ( B, D ) in kidney medulla. AQP2 is used as a marker for IMCD. D : Epac I antibody was preincubated with the antigenic peptide.- P5 I6 o0 k$ D/ s2 ?( p! {
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DISCUSSION. x5 J) j3 }3 k
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The results of the present study demonstrate that an Epac-selective cAMP analog is capable of inducing Ca 2 mobilization and apical insertion of AQP2 in rat IMCD. This work is in agreement with our previous reports that intracellular Ca 2 mobilization is necessary for AVP-stimulated water permeability in native IMCD cells ( 8, 47 ). The effect of AVP in triggering Ca 2 mobilization in IMCD is mimicked by cAMP ( 47 ), which indicates that both PKA activation and Ca 2 mobilization are post-cAMP events ( 8, 47 ). H-89 (1 µM) and KT-5720 (10 µM) at the concentrations known to be inhibitory for AQP2 phosphorylation did not inhibit AVP to mobilize intracellular Ca 2 , which implicates that Ca 2 mobilization is not the consequence of PKA activation but is mediated by a signaling pathway parallel to PKA activation. Interestingly, cAMP induces exocytosis in cultured IMCD cells without intracellular Ca 2 mobilization ( 28 ), which suggests that native IMCD cells and cultured IMCD cells are two different platforms for the study of AQP2 trafficking ( 6, 30, 46 ).3 ^& D$ w8 Z- J3 _4 V6 T
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Epac is a new class of cAMP effector which has been shown to mediate exocytosis and Ca 2 mobilization independent of PKA ( 19, 20, 22, 23, 26 ). The Ca 2 mobilization effect of CPT-cAMP was mimicked by 8-pCPT-2'- O -Me-cAMP, which is a selective agonist of Epac ( 15, 22 ). There was a significant time delay between the application of 8-pCPT-2'- O -Me-cAMP and mobilization of Ca 2 , while the Ca 2 mobilization induced by CPT-cAMP or AVP was almost instant. Similar delayed response triggered by 8-pCPT-2'- O -Me-cAMP compared with other cAMP analogs was also observed when fluorescent indicator of cAMP was used in cultured cells ( 13 ). One possible explanation is that the Epac-selective cAMP analog is less cell-permeant than other cAMP analogs. 8-pCPT-2'- O -Me-cAMP also mobilized intracellular Ca 2 in the presence of Rp-cAMP, which substantiates the notion that Epac but not PKA is responsible for the Ca 2 mobilization in IMCD. AVP-induced Ca 2 mobilization in IMCD is ryanodine sensitive ( 8, 47 ). Ca 2 mobilization triggered by the Epac-selective agonist is also ryanodine sensitive, which is consistent with the assertion that AVP-induced Ca 2 mobilization is mediated by Epac.( E1 f0 A, w! U* D4 D
, l3 M8 A+ v" w) r. `! x6 ?It has been shown that inhibition of PKA activity with 50 µM H-89 blocked 90% of an AVP-induced increase in water permeability in rabbit cortical collecting duct ( 41 ), suggesting that PKA is the sole mediator of AVP action. However, H-89 at this concentration is not selective for PKA but also inhibits other kinases such as calcium/calmodulin-dependent myosin light chain kinase (MLCK), which is a downstream target for AVP signaling in IMCD ( 6 ); 0.1 µM H-89 is sufficient to inhibit PKA activity in PC12 cells ( 27 ). H-89 at 1 µM still allows selective inhibition of PKA ( 17 ). A nonspecific inhibitory effect of H-89 on exendin-4 (a glucagons-like peptide-1 receptor agonist) induced Ca 2 mobilization was observed at 10 µM in INS-1 cells ( 19 ). Interestingly, 50 µM H-89 also attenuated AVP-induced Ca 2 mobilization in IMCD. This effect is most likely due to the nonselective inhibition of kinases by H-89. Therefore, the inhibitory effect of 50 µM H-89 on AVP hydrosmotic effect is probably mediated not only by PKA but also by a calcium-dependent mechanism.( Y. e. `! |# I1 u+ P
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Preincubation of IMCD with 1 µM H-89 only attenuated but did not abolish CPT-cAMP-induced apical exocytosis, which implicates the presence of a PKA-independent mechanism to mediate cAMP-dependent exocytosis. The Epac-selective cAMP analog could trigger apical exocytosis in IMCD in the presence of 1 µM H-89; the rate (indexed by the slope of regression line) of exocytosis is the same as that induced by CPT-cAMP in the presence of H-89. These observations suggest that both PKA-dependent and Epac-dependent mechanisms contribute to vasopressin-stimulated apical exocytosis in IMCD.
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0 l$ B+ _6 Q" j7 oActivation of Epac stimulates not only Ca 2 mobilization but also apical exocytosis in IMCD. To test whether Epac-dependent apical exocytosis is accompanied by apical targeting of AQP2, immunolocalization of AQP2 was performed in perfused IMCD. This preparation provides a unique opportunity to visualize the distribution of AQP2 from the basolateral to apical membrane through optical sectioning. By exploiting optical sectioning, we confirmed that there was an increase in AQP2 abundance in the apical membrane of IMCD after incubation with either 1 nM AVP or 50 µM 8-pCPT-2'- O -Me-cAMP. It has been shown that 50 µM 8-pCPT-2'- O -Me-cAMP does not induce PKA-dependent phosphorylation in kidney homogenate ( 18 ). Therefore, the AQP2 trafficking induced by 8-pCPT-2'- O -Me-cAMP is probably due to the activation of Epac. However, affinity precipitation of GTP-bound Rap1 is required to provide the unequivocal evidence that an Epac-dependent pathway is activated in native IMCD ( 15, 25, 42, 43 ).2 h p9 @& m- N' X5 i, X& ~4 e" R
: m1 N& H5 ?+ q! J; ^Our present study provides evidence that there are PKA-dependent and Epac-dependent components in cAMP-stimulated AQP2 trafficking. Synergistic interactions between these two signaling pathways in regulating exocytosis has been found in amylase release from rat parotid gland induced by -adrenergic stimulation ( 39 ). There are also PKA-dependent and Epac-dependent exocytosis of insulin in pancreatic -cells ( 22, 37 ). Glucagon-like peptide-1 stimulates cAMP production and Ca 2 mobilization in pancreatic -cells. Activation of either PKA or Epac is sufficient to mimic exendin-4 (a glucagons-like peptide-1 receptor agonist) in sensitizing intracellular Ca 2 release channel to stimulatory effects of cytosolic Ca 2 ( 21 ). The resulting Ca 2 -induced Ca 2 release leads to the amplification of exocytosis in pancreatic -cells ( 21 ). In cell lines transfected with AQP2, PKA-dependent phosphorylation on a seine residue at position 256 (Ser 256 ) of AQP2 is necessary for cAMP-induced exocytotic insertion ( 24, 33 ). In the present study, 1 µM H-89 impaired cAMP-induced apical exocytosis but did not block the associated Ca 2 mobilization and [Ca 2 ] i oscillations in IMCD. Buffering the changes of [Ca 2 ] i with an intracellular Ca 2 chelator inhibits AVP-stimulated exocytosis in IMCD ( 8, 47 ). These observations suggest that both PKA-dependent phosphorylation and Epac-dependent Ca 2 mobilization are integral parts of signaling events in AVP-stimulated AQP2 trafficking. The interactions and relative contribution of these two pathways in regulating AQP2 trafficking and osmotic water permeability remain to be determined." G l4 Y+ _8 y. f# k% c$ q( n) @& H
5 R( }4 r# I3 Y" X/ z" c8 aIn summary, PKA inhibitors at concentrations known to inhibit AQP2 phosphorylation did not inhibit vasopressin-induced Ca 2 mobilization in IMCD. An Epac-selective cAMP analog (8-pCPT-2'-O-Me-cAMP) stimulated AVP to trigger Ca 2 mobilization, [Ca 2 ] i oscillations, and apical exocytosis of AQP2 in IMCD. Epac-mediated Ca 2 mobilization and [Ca 2 ] i oscillations were not sensitive to PKA inhibitors. 8-pCPT-2'-O-Me-cAMP could trigger apical exocytosis in the presence of PKA inhibitor. Therefore, activation of Epac by cAMP might be an integral part of signaling event in vasopressin-induced Ca 2 mobilization and AQP2 exocytosis.6 b2 ~* c# \$ q4 C0 Z7 X. ]
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This study was supported by National Institutes of Health Grant DK-60501.
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8 O5 H6 J$ Z5 ]ACKNOWLEDGMENTS
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I acknowledge Drs. D. J. Marsh and J. S. K. Sham for helpful suggestions and C. Landon for technical assistance.
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