Department of Medicine, University of British Colombia, Vancouver, British Columbia, Canada V6T 1Z3
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ABSTRACT |
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Previous studies have shown that endothelin (ET) antagonizes the actions of arginine vasopressin (AVP) in the renal collecting ducts. On the other hand, the effects of AVP on ET function within the collecting ducts of the kidney have not been investigated extensively. Using isolated inner medullary collecting ducts (IMCD), we examined the possibility that a decrease in ETB receptor mRNA accompanied AVP-induced downregulation of ETB receptors. Binding studies revealed that overnight incubation of rat IMCD cells with AVP significantly reduced the maximal binding capacity (Bmax) of ET. Activation of adenylate cyclase by forskolin decreased the total ETB receptor density by ~42% but did not affect the density of ETA receptors. The Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothionate, Rp-cAMPS (a specific inhibitor of protein kinase A), blocked the AVP-induced reduction in ET receptor density. Using competitive PCR method, we also observed downregulation of ETB receptor mRNA in IMCD treated with AVP. Additional studies were done with IMCD to determine whether AVP inhibited the ET-induced accumulation of cGMP. We saw a reduction in ET-induced cGMP accumulation when IMCD was incubated overnight with AVP. This inhibition of ET-induced accumulation of cGMP was blocked by Rp-cAMPS. These results suggest that AVP regulates ETB receptor expression in IMCD.
cAMP; protein kinase A; endothelin-B receptor mRNA
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INTRODUCTION |
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ENDOTHELIN (ET) is a 21-amino-acid peptide and has a potent vasoconstrictor activity. It exists in three isoforms, namely ET-1, ET-2, and ET-3 (9, 21). Two subtypes of ET receptors, ETA and ETB, have been identified in the mammalian kidney. Both receptor subtypes are present in the inner medullary collecting duct (IMCD) cells (1, 13, 17). It is now recognized that ET plays an important role in many organ systems and in a variety of physiological events. In the kidney, the IMCD is the main site of ET synthesis, suggesting that this peptide plays an autacoid role in this segment of the nephron (11). Supporting this notion are studies done in rat IMCD that showed ET inhibits arginine vasopressin (AVP)-stimulated water flux through the ETB receptors (7).
Several studies have implicated the involvement of protein kinases in ET receptor regulation. Durieu-Traumann et al. (6) demonstrated that forskolin and dibutyryl-cAMP decrease the cell surface ET receptor number in rat astrocytoma C6 cells. Asada et al. (2) concluded that downregulation of ETB receptor mRNA in ROS 17/2 rat osteosarcoma cells involves a protein kinase A (PKA)-dependent pathway. Their findings suggest that ET induces homologous regulation of its receptors by accumulating cAMP directly through adenylate cyclase activation or indirectly through a PGE-dependent pathway. Studies with the rat cortical collecting duct cells showed that PKA is involved in the downregulation of ETB receptor (19). Roubert et al. (16) showed that ANG II and AVP downregulate ET receptors in vascular smooth muscle cells and suggested the existence of a PKC-dependent regulatory pathway. Furthermore, Cozza and Gomez-Sanchez (4) have reported that PKC activation decreases the numbers of surface ET receptors, by receptor internalization. The observation that PKA and PKC modulate ET receptors suggests the existence of multiple regulatory mechanisms that may be specific to cell type or receptor subtypes.
We have shown previously that the density of ETB receptors in the IMCD of heart failure hamsters is significantly reduced (20). It is known that AVP levels are elevated in advanced stages of heart failure. This reduction in ETB receptor density may be related to the increased circulating AVP levels. One piece of supporting evidence comes from preliminary study that showed a V2 receptor antagonist could prevent the reduction in ETB receptor density. These data suggest that AVP regulates the density of ETB receptors in the IMCD.
The present study investigates the mechanisms by which AVP regulates ET receptor density. Our results provide evidence that a cAMP pathway is involved in the regulation of ETB receptors in the IMCD.
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MATERIALS AND METHODS |
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Isolation of IMCD Cells
Male Wistar rats weighing 250-300 g were obtained from Charles River Breeding Laboratories (Wilmington, MA). The animals were anesthetized with phenobarbital sodium (50 mg/kg ip), and their kidneys were removed and bisected with a scalpel in ice-cold PBS. The papillary tissues were isolated and minced in 1 ml of 37°C RPMI-1640 medium containing 1.5 mg of collagenase. The tissues were further digested for 30 min in 4 ml of 37°C RPMI-1640 medium containing collagenase (1.5 mg/ml). The digestion process was stopped by adding 4 ml of RPMI-1640 medium containing 10% FCS. The sample was then centrifuged for 3 min at 1,000 rpm, and the supernatant was discarded. The pellet was resuspended in 2 ml of RPMI-1640 containing 10% FCS. This was further centrifuged for another 5 min at 1,000 rpm. The pellet was resuspended in 10 ml of RPMI-1640 containing 10% FCS. The resulting papillary collecting duct cell suspension was fractionated in Percoll (sp. gravity 1.07) for 20 min at 2,000 rpm. Papillary collecting duct cells were found at the top of the Percoll layer. These cells were stained with hematoxylin and eosin and periodic acid-Schiff. They formed low columnar tubular structures with distinct cell borders, but they lacked brush borders. They also stained positively for an epithelial membrane antigen (EMA; Dakao, Santa Barbara, CA) and low-molecular-weight cytokeratin (Enzo, New York) but negative for high-molecular-weight cytokeratin (8, 15). Biochemical studies showed that these cells could be stimulated by AVP and atrial natriuretic factor (ANF) to generate cAMP and cGMP. These observations confirmed that the cells were of collecting duct origin.Receptor Binding Studies
The IMCD cells obtained were homogenized with a Caframo Stirrer (Wiatron, Ontario) for 2 min at 4°C. Low-speed (1,000 rpm) centrifugation for 5 min at 4°C sedimented the cellular debris, unbroken cells, and nuclei discarded. The remaining supernatant was kept in Tris-Tyrode buffer (1%) and subjected to a centrifugation of 15,000 rpm for 20 min at 4°C. The resulting supernatant was discarded, and the pellet was resuspended with 8.5 ml of Tris-Tyrode (1%). This suspension was sonicated at low speed for 5 s while on ice. A 20-µl aliquot of this suspension was taken for protein assays by the Lowry method.The binding reactions began by adding 100 µl of IMCD homogenate with increasing concentration of ET-1 (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 ng/20 µl) and 100 µl of 125I-labeled ET-1 (~100,000 cpm). BQ-123 was used to block the ETA receptor to reveal the distribution of ETB receptor. Nonspecific binding was defined as binding in the presence of 1 µM of unlabeled ET-1. At the end of the reaction, bound and unbound labeled peptides were separated by double antibody precipitation: 50 µl of 10% normal rabbit serum, 100 µl of goat anti-rabbit IgG, and 1 ml of 5% polyethylene glycol 8000 were added sequentially. The mixture was left at room temperature for 15 min followed by centrifugation at 300 rpm for 30 min at 4°C. The supernatant was aspirated by vacuum suction, and the pellet radioactivity was counted by an LKB mini gamma counter (Wallac, Turku, Finland). The Ligand Program (15a) was used to calculate the dissociation constant (Kd) and maximum binding capacity (Bmax) of ET receptors for ET-1.
cGMP Accumulation Studies
cGMP accumulation studies were done by a method described before (14). In brief, 100 µl of isolated IMCD was incubated at 37°C for 15 min in 400 µl of Tris buffer. Fifty microliters of IBMX (1 mM) was added 2 min later with ET-1 (10Quantitative RT-PCR
Competitive RT-PCR was used to quantify the levels of mRNA for ETB receptors. Known amounts of competitor DNA molecules are added to each PCR, and the amount of target cDNA present in the sample is determined from the added competitor, which gives an equimolar amount of PCR products as the target cDNA. A single set of primers to amplify both target cDNA and an added competitor of known concentration were used. Competitors for ETB were synthesized with the sense primer 5'-TTA CAA GAC AGC CAA AGA CT-3' and the antisense primer 5'-CAC GAT GAG GAC AAT GAG ATA GCA GCA CAA ACA CGA CTT A-3', producing a 427-bp fragment. The competitive PCR was done with the sense primer 5'-TTA CAA GAC AGC CAA AGA CT-3' and the antisense primer 5'-CAC GAT GAG GAC AAT GAG AT-3', and the length of the amplicon was 564 bp. PCR was carried out with GeneAMP PCR System 2400 (Perkin-Elmer, Norwalk, CT) in a total volume of 50 µl containing 2 µl of cDNA, 2 µl of competitor, 20 pmol of each primer, 100 µmol/l dNTPs, 10 mmol/l Tris · HCl, 0.75 mmol/l MgCl2, and 1.25 U of Taq DNA polymerase (GIBCO-BRL). Amplification was carried out as follows: step 1, 95°C for 2 min; step 2, 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min; step 3, 72°C for 2 min. After completion of PCR, an aliquot of the reaction mixtures was electrophoresed on 2% agarose gels followed by staining with ethidium bromide. The appropriate bands were scanned and quantitated by computer densitometry.Experimental Protocol
Time control experiments were done to be sure that ET-1 binding was not affected by overnight incubation and that any changes detected in ET binding with treated IMCD cells were drug induced. In these studies, IMCD cells isolated from both kidneys of each rat were incubated for 17 h in RPMI-1640 medium containing 10% FCS at 37°C. After incubation, binding studies were done with IMCD from both kidneys to ensure that no differences were detected between control (right) and experimental (left) kidney with overnight incubation. In all the studies involving agonists and antagonists, IMCD cells from the left kidney of each animal were treated with AVP (10Data Analysis
All data are expressed as means ± SE. Analysis of variance and Student's t-tests were used to compare differences between groups. P < 0.05 was accepted as significant. ![]() |
RESULTS |
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Binding Studies
Time control studies. ET-1 binding to isolated IMCD cells from the right and left kidneys of each rat was not affected after being incubated at 37°C for 17 h. Total ET receptor density between each pair of kidneys was similar (3,171 ± 506 vs. 3,671 ± 466 fmol/mg protein, n = 4). Similarly, no change was seen in the densities of ET receptor subtypes (ETA, 1,255 ± 400 vs. 1,430 ± 319 fmol/mg protein; ETB, 1,917 ± 202 vs. 2,241 ± 367 fmol/mg protein). Receptor affinity (Kd) was also stable after overnight incubation (total ET, Kd = 2.0 ± 0.7 vs. 2.5 ± 0.7 nM; ETB, Kd = 1.0 ± 0.7 vs. 1.3 ± 0.1 nM).Effects of AVP on ET-1 binding to IMCD. Overnight
incubation with AVP (106 M) caused a
decrease in ET-1 binding in IMCD cells (Fig.
1). AVP incubation reduced the
Bmax of ET receptors from 5,398 ± 1,562 to 3,589 ± 884 fmol/mg protein (P < 0.05, n = 6). With the use of
BQ-123, we showed that ETB receptor density fell in
the presence of AVP from 4,375 ± 1,415 to 2,966 ± 829 fmol/mg protein (P < 0.05), whereas the change
in ETA receptor density was not statistically significant (1,023 ± 237 to 623 ± 179 fmol/mg protein, not
significant).
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Effects of forskolin on ET-1 binding to IMCD. Studies were done
in IMCD cells to explore the possible role of PKA in downregulating ET
receptor density (Fig. 2). IMCD cells were
incubated with forskolin (106 M). The
results of these studies showed that forskolin mimicked the effects of
AVP in reducing ET receptor density (6,099 ± 1,319 to 3,939 ± 672 fmol/mg protein, P < 0.05, n = 6). These data also showed that forskolin treatment downregulated ETB receptor
density from 5,029 ± 998 to 2,941 ± 674 fmol/mg protein (P < 0.01), but did not affect ETA receptors (1,070 ± 393 to 998 ± 224 fmol/mg protein).
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Effects of Rp-cAMPS on ET-1 binding to IMCD. The involvement of
PKA pathways in downregulating ET receptors was further
examined with a cAMP analog, Rp-cAMPS (Fig.
3). In cells incubated with Rp-cAMPS and
AVP, the effects of AVP on ET receptor density were prevented. ET
receptor densities were comparable to those seen in the control group
(Rp-cAMPS, 5,889 ± 840 fmol/mg protein vs. AVP, 4,114 ± 800 fmol/mg
protein). In additional studies, Rp-cAMPS alone in the incubation
medium did not affect ET-1 binding to IMCD cells.
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cGMP Accumulation Studies
The results of ET-induced cGMP accumulation are shown in Fig. 4. The baseline cGMP accumulations were similar in all three groups examined (control, 675 ± 41; AVP, 691 ± 42; Rp-cAMPS: 593 ± 25 fmol · µg
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Effect of AVP on Expression of ETB Receptor mRNA in IMCD
The effect of AVP on the expression of ETB receptor mRNA in the IMCD was evaluated by competitive RT-PCR (Fig. 5). The expression of ETB receptor mRNA was significantly decreased in IMCD incubated with AVP (13.18 ± 1.34 vs. 8.65 ± 0.82 amol/µg total RNA, P < 0.01). However, when IMCD was incubated with both AVP and Rp-cAMPS, the expression of ETB receptor mRNA did not differ between the control and the experimental groups.
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DISCUSSION |
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Previous studies showed that cAMP downregulates ETB receptors (2, 19). Heterologous regulation by ET by other neurohumoral factors that activate the cAMP-PKA axis has been examined. Takemoto and colleagues (19) showed that AVP downregulates ETB receptors in the rat cortical collecting ducts by reducing Kd through a PKA-dependent pathway. Our present report showed that overnight incubation of rat IMCD with AVP consistently reduced ET-1 binding to the ETB receptors, but as a result of the reduction in Bmax. The differences between our studies and that reported by Takemoto and colleagues can be due to differences in the segment of the nephron studied and also the incubation time with AVP. Studies by Asada et al. (2) showed that cAMP began to decrease ETB mRNA in rat osteosarcoma cells at 6 h and reached its maximum effect in 24 h. Our result is consistent with previous reports. In addition, our ligand binding studies also suggest the important role of cAMP in AVP-induced downregulation of ETB receptors, as shown by the forskolin studies. Activation of adenylate cyclase with forskolin caused a significant downregulation of ETB but did not affect ETA receptor density. In our studies, Rp-cAMPS, a competitive cAMP analog, prevented the AVP-induced downregulation of ETB receptors. These data support that downregulation of ETB receptors after AVP treatment involved the cAMP-PKA axis. The ability of the cAMP-PKA axis to reduce ETB receptor density suggests that distinct regulatory mechanisms exist for the two ET receptor subtypes. It is known that the ETB receptor subtypes possess a PKA-activated phosphorylation sequence site in the third intracellular loop (19). The correspondence domain in the ETA receptor is occupied by two consensus sequences for PKC phosphorylation. The differences in amino acid sequences between the two receptor subtypes provide a different regulatory pathway and may explain the observations of AVP- and forskolin-induced downregulation of ETB receptors but not ETA receptors.
The AVP-induced changes in ETB receptors may be attributed to both short- and long-term mechanisms. We visualize the following scenario with short-term exposure to an agonist. When cells are exposed to AVP for a brief period, it activates PKA, which leads to phosphorylation of the ETB receptors at serine and threonine residues in the third intracellular loop, which is involved in AVP-induced ETB receptor desensitization (12). Koshimizu and colleagues (12) have postulated that phosphorylation of the intracellular loops of the ETB receptor is important in the desensitization of the human ETB receptors when cells are exposed to an agonist for 5 min. This is confirmed by recent studies of Crammer and colleagues (5), who showed that ETA receptor is not phosphorylated in response to ligand stimulation and desensitizes slowly, whereas the ETB receptor becomes phosphorylated on serine and threonine residues immediately after ligand stimulation and desensitizes swiftly.
In the present studies, in which we incubated the IMCD cells with AVP or forskolin for 17 h, long-term regulation of ET receptors involving degradation, inactivation, and internalization, and transcriptional control can be important. These observations agree with results reported with rat mesangial cells that showed ET receptor downregulation when exposed to ET-1 for 18 h (3). Furthermore, AVP might have influenced ETB gene expression. Asada et al. (2) showed that cAMP began to decrease ETB mRNA in rat osteosarcoma cells in 6 h and reached its maximum effects in 24 h. Exposure to isoproterenol and forskolin downregulated ET receptors in a similar biphasic manner within the same time span in rat astrocytoma cells (6). We also examine the possible effects of AVP on ETB receptor mRNA expression in the IMCD cells. Using competitive PCR, we showed a reduction in ETB receptor mRNA expression when cells were preincubated with AVP. Interestingly, when IMCD cells were preincubated with AVP and Rp-cAMPS, the expression of ETB receptor mRNA was the same as in the untreated cells. The inhibitory effect of AVP on ETB receptor mRNA may be mediated by cAMP as reported by Asada and colleagues (2). AVP receptors can be classified into V1 and V2 subtypes. The main subtype found in the kidney is the V2 subtype, whose second message is the cAMP. In the present study, one visualizes the activation of V2 receptors by AVP leads to the generation of cAMP that decreases ETB receptor mRNA expression. Rp-cAMPS, which is a PKA-specific inhibitor, eliminated the inhibitory effects of AVP on ETB receptor mRNA expression. These results suggested that vasoactive peptides modulate each other's actions by regulating gene expression and protein production. Another possibility for the lower expression of ETB receptor mRNA is due to the decrease in the intracellular stability of the ETB receptor mRNA induced by AVP. It can be seen from these studies that the cAMP-PKA axis plays a role in regulating ETB mRNA expression.
The stimulating effects of ETs on cGMP accumulations have been reported in kidney epithelial cells (10) and freshly isolated glomeruli (18). ET-induced cGMP generation depends on binding of ET-1 to its receptors, guanylate cyclase activity, phosphodiesterase activity, and substrate availability. In the present report, we measured cGMP accumulations in IMCD cells preincubated with and without AVP. Results obtained from these studies complemented our binding studies. It showed that preincubation with AVP attenuated the ET-1-induced cGMP accumulation, but in the presence of Rp-cAMPS the attenuated response was not seen. There is a possibility that AVP prevents binding of ET-1 to its receptors, which could account for the attenuated response of the AVP-treated IMCD to ET-1-induced cGMP generation. However, this is unlikely, because the response was normalized in the presence of Rp-cAMPS. A more logical explanation would be that of phosphorylation of the ETB receptors leading to receptor inactivation. Another possibility is that ET stimulates phosphodiesterase activity, but this is also unlikely, because the studies were done in the presence of IBMX, a phosphodiesterase inhibitor. Our results are consistent with the notion that cAMP is involved in AVP-induced downregulation of ETB receptors.
In summary, IMCD cells exposed to AVP caused a downregulation of ETB receptors. The downregulation of ETB receptors by AVP as shown in the present study and reports that showed the inhibitory effects of ET on the action of AVP in the collecting ducts suggested that these two hormones work together in this part of the nephron to modulate sodium and water excretion by the kidney. The balance between these two hormones may play an important role in regulating salt and water handling by the kidney. Under pathological conditions, the imbalance between these two hormones leads to salt and water retention in congestive heart failure or, conversely, an increase in water excretion in chronic renal failure.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of Alice Fok.
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FOOTNOTES |
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This study was supported by a grant from the Heart and Stroke Foundation of British Columbia and Yukon to N. L. M. Wong.
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: N. L. M. Wong, Dept. of Medicine, Vancouver Hospital, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3.
Received 12 February 1999; accepted in final form 12 October 1999.
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