Departments of 1Medicine and 2Physiology, University of Maryland School of Medicine; and 3Department of Veterans Affairs Medical Center, Baltimore, Maryland
Submitted 24 August 2004 ; accepted in final form 6 December 2004
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ABSTRACT |
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PDZ proteins; calcium channels; medulla; pericytes; endothelium; microcirculation
In prior experiments, we investigated Ca2+ signaling pathways and the channel architecture of DVR pericytes and endothelia. Angiotensin II depolarizes pericytes by activating a Ca2+-dependent Cl conductance, leading to Ca2+ entry into the cytoplasm and DVR contraction (13, 15, 29). Interestingly, angiotensin II inhibits Ca2+ signaling in DVR endothelial cells (18). Vasodilators such as acetylcholine and bradykinin as well as wall stress increase the cytosolic concentration of Ca2+ and NO production in endothelial cells (15, 27). The pathways that mediate Ca2+ entry into the cytoplasm of these cells is, at present, poorly defined.
The two isoforms of the Na+/H+ exchanger regulatory factor (NHERF-1 and NHERF-2) are scaffolding proteins that facilitate protein-protein interactions through their tandem PDZ-binding domains and their COOH-terminal ezrin-radixin-moesin-merlin binding domain (19, 22, 26). We previously identified the presence of NHERF-2 in the DVR of the rat kidney, although its explicit function is unknown (24). Recent studies have suggested that some members of the transient receptor potential (TRP) protein family function as nonselective cation channels that conduct Ca2+ and Na+ ions into the cytoplasm (5, 9, 10). Two members of the canonical subclass of the TRP family of proteins, TRP channels (TRPC) TRPC4 and TRPC5, have a COOH-terminal amino acid sequence, TRL, that represents a class 1 PDZ-binding domain. With the use of a heterologous expression model, recent studies have indicated that NHERF binds TRPC4 and TRPC5 and directs its expression in the plasma membrane (20). Given NHERF-2 expression in the DVR and the important physiological role of Ca2+ in the function of DVR, we initiated experiments to explore the possibility that TRPC4 and/or TRPC5 are expressed in DVR pericytes and/or endothelial cells and to determine whether these Ca2+ channels interact with NHERF. The results show that both the pericytes and endothelia of DVR express TRPC4 and NHERF-2 but not TRPC5. Confocal microscopy and coimmunoprecipitation experiments verified that TRPC4 and NHERF-2 are physically associated, findings that suggest a functional interaction.
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MATERIALS AND METHODS |
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RT-PCR. RT-PCR was performed using a PCR and cDNA synthesis kit (Invitrogen). Reaction tubes were centrifuged for a few seconds at 10,000 rpm, and the supernatant was discarded. Samples were incubated for 15 min on ice with 2% Triton X-100 containing RNase inhibitor and 5 mM DTT to permeabilize the cells. First-strand synthesis was accomplished by incubating the samples with a RT mixture containing random primers at 42°C for 60 min. The reaction was stopped by heating the sample to 70°C for 15 min. The samples were stored at 4°C until PCR.
PCR for TRPC4 and NHERF-2 was performed using degenerative primers pairs. For TRPC4, the sense primer was 5'-GCCCCCTACCGAGACCGCATCCC-3' (bp 173206) and the antisense primer was 5'-CCCCACGAGGTCCGCTGTAACTGTG-3' (bp 512536). For the nested PCR of TRPC4, the sense primer was 5'-CTCTCACCATCAGAGAAAGCCTAC-3' (bp 235258). TRPC4 antisense primers were 5'-ACTCTTGGTCCAGAAGGGTGTCTC-3' (bp 628651) and 5'-GGTGCCTCCCATCCTCCTTGACAAA-3' (bp 529553). For the nested PCR of TRPC5, the sense primer was 5'-CATGGAGCTACTGCTGAACCACAG-3' (bp 255278) and 5'-GTATGTGGGCGATGCATTACTCTATGCC-3' (bp 281309); the antisense primers were 5'-GATGACCACAGCGAAGAACTTGACCC-3' (bp 817842) and 5'-GCTCGGAGCTCC c/a G a/g GAACTGGAG-3' (bp 775798). The sense primer for NHERF-2 was 5'-CCACAGGATCAAGGCTGTGGAGGGACAG-3' (bp 381408), and the antisense primer was 5'-GGTCTCCACCTGAGCCCCACAGCAG-3' (bp 1,0571,081). Nested PCR for NHERF-2 used the sense primer 5'-GCCGGCGGCAACTGACCTGCACTGAGG-3' (bp 452478) and the antisense primer 5'-GGACAATGAGGATGGCAGTGCTTGG-3' (bp 1,0081,032). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using 5'-TCCCTCAAGATTGTCAGCAA-3' (bp 1,2691,289) as the forward primer and 5'-AGATCCACAACGGATACATT-3' (bp 1,5581,577) as the reverse primer. Each primer (100 µl) was used at 0.2 pmol/µl. The annealing temperature was 56°C for 1 min and the final extension was at 72°C for 7 min.
PCR product analysis. The amplified products were ethanol precipitated and separated by electrophoresis on 1.5% agarose gels. DNA bands were visualized under ultraviolet light after staining with ethidium bromide. The PCR products were subcloned into the pCR II-TOPO vector (Invitrogen) and sequenced (Bio Polymer Lab).
Immunoblotting and immunoprecipitation. Cortical and medullary lysates were prepared from kidneys of the rat. Kidneys were decapsulated, cleaned of connective tissue, and separated into cortex and medulla by dissection. Homogenization and centrifugation were performed to solubilize proteins for Western blot analysis. The protein concentration of the lysates was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce). Aliquots (30 µg) of total lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Proteins were transferred to a nitrocellulose membrane and then exposed to primary antibody. Antibodies for the immunoblots included anti-TRPC4 (1:200 dilution; Chemicon), anti-NHERF-2 (1:500 dilution). Secondary antibodies for immunoblots were conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories) at 1:5,000 dilution. Blots were developed with the enhanced chemiluminescence (ECL) kit (Amersham Biosciences) according to the manufacturer's instructions. Coimmunoprecipitation experiments were performed using cortical and medullary lysates. Aliquots (1 mg of protein) of the lysates were reacted with 1 µg of anti-NHERF-2 antibody for 1 h at 4°C. Immune complexes were separated by binding to protein A-Sepharose resin followed by five washes with buffer containing 0.5% Nonidet P-40. Proteins were separated on 10% SDS-PAGE using Tris-glycine-SDS running buffer (Bio-Rad) at 100 V for 2 h, electrophoretically transferred to nitrocellulose membranes, maintained at 30 V overnight at 4°C, and then probed with secondary antibodies as specified above. The reverse experiment also was performed, in which anti-TRPC4 was used for the immunoprecipitation step (1 mg lysate protein) and the immunoprecipitates were probed for TRPC4 (1:200 dilution; Chemicon) and NHERF-2 (1:500 dilution) after separation and transfer.
Immunocytochemical staining of renal medulla and isolated DVR.
Tissue sections of the renal medulla of rat kidney were prepared as described previously (23, 24). For direct study of endothelial cells and pericytes, hand-dissected DVR were placed on glass coverslips, fixed with 3% paraformaldehyde in 0.1 M cacodylate for 5 min at 25°C, and washed with microdissection solution. Samples were then incubated with 5% BSA and 0.1% Triton X-100 in microdissection solution for 30 min at 25°C, reacted with primary antibody overnight at 4°C, and washed three times. Reaction with secondary antibodies proceeded for 1 h at room temperature. In some experiments, the primary TRPC4 antibody was the rabbit polyclonal anti-TRPC4 (1:50 dilution; Chemicon). To generate a more specific antibody that functioned at higher titers, a synthetic peptide corresponding to amino acids 946961 of rat TRPC4 was used to immunize chickens. Serum was affinity purified using the synthetic peptide coupled to keyhole limpet hemocyanine cross linked to agarose beads. Immunostaining of NHERF-2 was performed with a previously characterized rabbit polyclonal antibody (1:100 dilution) (23, 24). Pericytes were identified with monoclonal antibody against -smooth muscle actin (1:500 dilution; Sigma). Endothelium was identified using antibody against aquaporin-1 (1:100 dilution; Alomone Laboratories). The secondary antibody was Alexa 488-conjugated donkey anti-rabbit or anti-chicken IgG (1:200 dilution; Molecular Probes). Negative controls were performed by omitting the incubation with primary antibody. Immunofluorescent images were captured with a Zeiss LSM410 confocal fluorescence microscope that uses Coherent INNOVA Enterprise Model 653 and Zeiss He/Ne internal lasers. Images were captured at 512 x 512-pixel resolution and z-axis sectioning at 0.5-µm intervals.
Materials. TRPC4 and TRPC5 antibodies were purchased from Chemicon. Aquaporin-1 antibody was obtained from Alomone Laboratories. Secondary HRP-conjugated antibodies for immunoblotting were purchased from Jackson ImmunoResearch Laboratory. Secondary Alexa-conjugated antibodies for immunofluorescence were obtained from Molecular Probes. Tris-glycine-SDS running buffer was purchased from Bio-Rad, and the BCA protein assay kit was obtained from Pierce Biotechnology. ECL solution and protein A-Sepharose resin were purchased from Amersham. RNase inhibitor was obtained from Clontech. RT-PCR was performed using a kit purchased from Invitrogen.
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RESULTS |
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DISCUSSION |
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The adaptor proteins NHERF-1 and NHERF-2 are expressed in the kidney in selected cells of the nephron, where they are thought to modulate the function of other proteins with which they interact (19, 2224, 26). One remarkable finding, initially observed in the medulla of the rat kidney and recently confirmed in the mouse, was prominent NHERF-2 staining of DVR (1, 23, 24). In the medulla, antibodies directed against NHERF-2 recognized only DVR. NHERF-1 was not detected in DVR or any other structures within the renal medulla. The NHERF proteins contain two PDZ protein-interactive domains and a COOH-terminal sequence that binds to structural proteins in the cell. More than 50 proteins that interact with NHERF isoforms have been identified, including transporters, ion channels, receptors, and signaling proteins (22). Specific binding targets of NHERF-2 in DVR previously were unknown.
The large TRP family of proteins is divided into several subfamilies. The function of many of the individual members is either unknown or poorly characterized. Two members of the canonical subfamily, TRPC4 and TRPC5, have been suggested to function as nonselective cation channels. They have COOH-terminal sequences that posses class 1 PDZ-binding domains (9, 10, 11, 21). The latter is of considerable interest because TRP channels in the Drosophila eye interact with the PDZ domain of INAD (protein responsible for the inactivation no afterpotential Drosophila mutant D) (10, 11). A human homolog of INAD has not been identified, and it has been speculated that NHERF-1 and NHERF-2 might serve as their functional analog in mammals (8, 20). Mery et al. (8) provided evidence that the PDZ-interacting domain of TRPC4 controlled its localization and surface expression in human embryonic kidney HEK-293 cells. Tang et al. (20) demonstrated that TRPC4 and TRPC5, as well as phospholipase C-1 and phospholipase C-
2, interacted with the first PDZ domain of NHERF-1, forming a multiprotein signaling complex. Given the presence of NHERF-2 in DVR and the role of Ca2+ in the function of its constituent cells, we initiated experiments to determine whether either TRPC4 or TRPC5 is expressed in DVR and, if so, whether a specific association with NHERF-2 exists.
Our studies demonstrate that DVR express TRPC4 and NHERF-2 message and protein. Using hand-dissected DVR and RT-PCR, mRNA for both proteins was identified (Fig. 1). Using RT-PCR, we did not detect TRPC5 in isolated DVR. We detected TRPC5 using Western immunoblot analysis of medullary lysates (Fig. 2), so it remains possible that TRPC5 is expressed in another structure within the renal medulla. Detailed confocal microscopic studies indicated that both TRPC4 and NHERF-2 colocalize in some cells of the DVR wall (Figs. 47). The physical interaction between these proteins was confirmed by performing immunoprecipitation experiments (Fig. 8). Antibody directed against NHERF-2 immunoprecipitates TRPC4 from lysates of the renal medulla. Using an antibody against NHERF-1, we failed to detect either TRPC4 or TRPC5 in immunoprecipitate derived from lysates of the cortex or medulla. Taken together, these findings suggest that TRPC4 is present in the cells of the DVR and associates with NHERF-2. To our knowledge, this represents the first in vivo demonstration of a TRPC4-NHERF-2 interaction. Through its PDZ binding domain, TRPC5 also has the potential to interact with NHERF-2. We were unable, however, to detect TRPC5 message using RT-PCR or protein using immunochemistry, which implies that such an association does not exist in the cells of the DVR. We recognize, however, that the quality of the available TRPC5 antibody may have limited detection and may require reevaluation when better reagents are available.
The function of TRPC4 and the significance of its binding to NHERF-2 are unknown at present. TRPC4 and TRPC5 of the TRPC subfamily have been proposed to function as nonselective cation channels (5, 9, 11, 21, 25). Freichel et al. (5) reported their studies in TRPC4/ mice. A striking observation in those experiments was the marked attenuation of agonist-induced increases in intracellular Ca2+ in endothelial cells deficient in TRPC4. Freichel et al. also demonstrated impaired agonist-induced vasorelaxation in preconstricted aortic rings from TRPC4-null mice. They interpreted their results as showing that TRPC4 functions as a store-operated Ca2+ channel that mediates capacitive Ca2+ entry, at least in endothelia of a large conduit vessel. While these data are intriguing, we think it is important to emphasize that it has not firmly been established that TRPC4 is the only store-operated Ca2+ channel or that all endothelial cells, especially microvascular endothelial cells, use similar mechanisms. Recently, Sansom et al. (25) reported that TRPC4 forms store-operated Ca2+ channels in mouse kidney mesangial cells. As shown in Fig. 4B, we also observed immunological evidence of TRPC4 in glomeruli, where it might also be associated with NHERF-2.
Study of DVR is difficult because this structure is inaccessible in the intact kidney. A major advance has been the development of ex vivo methods that permit delineation of DVR responses to hormones, activation of specific ion channels, and modulation of cytoplasmic Ca2+ concentrations in both pericytes and endothelia. Investigations have repeatedly demonstrated that activation of Ca2+-mediated signaling pathways plays a vital role in the regulation of vasoactivity and the release of NO (1215, 18, 2729). Those functional studies have not permitted delineation of the importance of interactions between specific signaling proteins, receptors, and ion channels. The present study indicates that TRPC4 and NHERF-2 associate with one another in DVR, which provides a rationale for investigations of the physiological role of these proteins in the regulation of medullary blood flow.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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