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Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb

Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202-2689

Submitted 25 November 2003 ; accepted in final form 22 April 2004

	ABSTRACT

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Endothelin-1 (ET-1) acutely inhibits NaCl reabsorption by the thick ascending limb (THAL) by activating the ET_B receptor, stimulating endothelial nitric oxide synthase (eNOS), and releasing nitric oxide (NO). In nonrenal tissue, chronic exposure to ET-1 stimulates eNOS expression via the ET_B receptor and activation of phosphatidylinositol 3-kinase (PI3K). We hypothesized that ET-1 increases eNOS expression in the THAL by binding to ET_B receptors and stimulating PI3K. In primary cultures of medullary THALs treated for 24 h, eNOS expression increased by 36 ± 18% with 0.01 nM ET-1, 123 ± 30% with 0.1 nM (P < 0.05; n = 5), and 71 ± 30% with 1 nM, whereas 10 nM had no effect. BQ-788, a selective ET_B receptor antagonist, completely blocked stimulation of eNOS expression caused by 0.1 nM ET-1 (12 ± 25 vs. 120 ± 40% for ET-1 alone; P < 0.05; n = 5). BQ-123, a selective ET_A receptor antagonist, did not affect the increase in eNOS caused by 0.1 nM ET-1. Sarafotoxin c (S6c; 0.1 µM), a selective ET_B receptor agonist, increased eNOS expression by 77 ± 30% (P < 0.05; n = 6). Wortmannin (0.01 µM), a PI3K inhibitor, completely blocked the stimulatory effect of 0.1 µM S6c (77 ± 30 vs. –28 ± 9%; P < 0.05; n = 6). To test whether the increase in eNOS expression heightens activity, we measured NO release in response to simultaneous treatment with L-arginine, ionomycin, and clonidine using a NO-sensitive electrode. NO release by control cells was 337 ± 61 and 690 ± 126 pA in ET-1-treated cells (P < 0.05; n = 5). Taken together, these data suggest that ET-1 stimulates THAL eNOS, activating ET_B receptors and PI3K and thereby increasing NO production.

endothelial nitric oxide synthase; phosphatidylinositol 3-kinase

ET-1 activates many signaling molecules, including protein kinase C, calcium, and phosphatidylinositol 3-kinase (PI3K) (16). Ramet et al. (25) reported that in cultured human vascular endothelial cells, stimulation of PI3K activity by high-density lipoprotein enhanced eNOS expression, but the signaling cascade that mediates the effect of ET-1 on eNOS protein levels is unknown. The renal medulla is composed of thick ascending limbs (THALs), thin limbs, IMCDs, and vasa recta and is a major site of ET production (10). The THAL expresses ET_B receptors as well as eNOS (22), and we showed that ET-1 acutely activates eNOS and increases NO release via these receptors (21). Consequently, we hypothesized that ET-1 increases eNOS expression in the THAL via activation of ET_B receptors and PI3K.

	MATERIALS AND METHODS

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Materials. The ET_B receptor antagonist BQ-788 was purchased from Peninsula (Belmont, CA), while ET-1 was obtained from either Peninsula or Bachem (Torrance, CA). The ET_A receptor antagonist BQ-123, collagenase, epidermal growth factor (EGF), wortmannin, and sarafotoxin c (S6c) were obtained from Sigma (St. Louis, MO). DMEM/F-12 medium and Hanks' balanced salt solution (HBSS) were purchased from Gibco (Carlsbad, CA), and fetal bovine serum was obtained from Hyclone (Logan, UT).

Primary cultures of medullary THALs. Six-week-old male Sprague-Dawley rats (Charles River, Kalamazoo, MI) were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip). The abdominal cavity was opened, and the kidneys were flushed with 40 ml of ice-cold 0.1% collagenase in HBSS via retrograde perfusion of the aorta. Kidneys were removed, and coronal slices were cut. The inner stripe of the outer medulla was minced into 1-mm³ fragments and digested in 0.1 mg/ml collagenase at 37°C for 30 min. During each 5-min period, the tissue was gently agitated and gassed with 100% oxygen. After continuous agitation for 30 min in cold HBSS, the tissue was filtered through a 250-µm nylon mesh and the filtered material was rinsed twice with culture medium. Cells were resuspended in DMEM/F-12 supplemented with 5% heat-inactivated fetal bovine serum, 20 ng/ml EGF, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated on collagen-coated inserts (0.4-µm pore size, 4.7-cm² area, Corning Costar, Cambridge, MA) at a concentration of 80 µg protein/insert. After 24 h, serum and EGF were removed from the medium for 12 h, after which cells were treated as indicated. Unless otherwise specified, all treatments lasted 24 h. Ninety-two percent of cells in primary cultures were THALs as evidenced by positive Tamm-Horsfall staining.

Western blot analysis. Cells were scraped and lysed in 60 µl of buffer containing 20 mmol/l HEPES (pH 7.4), 2 mmol/l EDTA, 0.3 mol/l sucrose, 1.0% NP-40, 0.1% sodium dodecyl sulfate, 5 µg/ml antipain, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 4 mmol/l benzamidine, 5 µg/ml chymostatin, 5 µg/ml pepstatin A, and 0.105 mol/l pf-block (Sigma). Debris was removed by centrifugation. Protein concentration was determined by colorimetry (Coomassie Plus protein assay, Pierce, Rockford, IL). For each experiment, equal amounts of total protein were loaded into each lane of an 8% SDS-polyacrylamide gel, separated by electrophoresis, and transferred to a PVDF membrane (Millipore, Bedford, MA). The membrane was incubated in blocking buffer containing 50 mmol/l Tris, 500 mmol/l NaCl, 5% nonfat dry milk, and 0.1% Tween 20 for 60 min and then with a 1:1,000 dilution of an eNOS-specific monoclonal antibody (BD Transduction Laboratories, San Diego, CA) in blocking buffer for 60 min at room temperature. The membrane was washed in a buffer containing 50 mmol/l Tris, 500 mmol/l NaCl, and 0.1% Tween 20 and incubated with a 1:1,000 dilution of secondary antibody against the appropriate IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL). Reaction products were detected with a chemiluminescence kit (Amersham). The signal was detected by exposure to Fuji RX film and quantified by densitometry.

eNOS mRNA. After treatment with 0.1 nM ET-1 for 24 h, cells were scraped and total RNA was isolated using a commercial kit (RNeasy, Qiagen, Valencia, CA). RT-PCR was performed using a modification of Resta's method (26). MDH was used as an internal standard. RT reactions (25 µl) contained 2.0 µg total cellular RNA, 200 U Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI), 1 µg oligo(dT)₁₅, 2 mmol/l dNTPs, 7 mmol/l MgCl₂, and 40 U rRNasin ribonuclease inhibitor. Reactions were incubated at room temperature for 10 min, 42°C for 1 h, and 94°C for 5 min. PCR reaction mixtures for eNOS and MDH were prepared according to Resta el al. (26). The mixed samples were heated to 94°C for 1 min and then cycled at 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min for 32 cycles. The final extension was 5 min at 72°C. PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide (0.5 µg/ml) for 10 min. Band density analysis of the expected 819- and 509-bp products for eNOS and MDH, respectively, was performed using Quantity One software (Bio-Rad). To validate the assay, we generated a standard curve and found that varying amounts of cDNA template resulted in a linear amount of eNOS PCR product (correlation coefficient of 0.97). All reagents were obtained from Promega.

NO measurements. Primary cultures of THALs were treated with either vehicle or 0.1 nM ET-1 for 24 h and maintained in DMEM/F-12 medium for 2 h. Basal NO levels were recorded using an amperometric electrode selective for NO (inNO measuring system, Harvard Apparatus). L-Arginine (500 µM), the substrate for NOS (Sigma), 1 nM clonidine, and 0.5 µM A23187 [GenBank] (a calcium ionophore) were added to the apical side of the cells to fully activate enzyme activity. All measurements were done inside an incubator at 37°C. NO release was measured for 1 min to establish a baseline. Then activators were added, and NO release was measured again at 2 min.

Statistics. Data are reported as means ± SE. They were evaluated by ANOVA for repetitive measurements or paired t-test as appropriate. P < 0.05 was considered significant.

	RESULTS

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First, we performed a time course analysis of treatment with 0.1 nM ET-1 for 6, 12, and 24 h. In primary cultures of medullary THALs, the addition of ET-1 increased eNOS expression by 3 ± 21% after 6-h incubation, 23 ± 13% after 12 h, and 96 ± 29% after 24 h (P < 0.05; n = 5), which is the longest duration we can test with our system (Fig. 1). Because stimulation of eNOS expression by ET-1 was maximal after 24-h incubation, all experiments were carried out at this time.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Time dependence of endothelial nitric oxide synthase (eNOS) expression in cells incubated with vehicle or 0.1 nM endothelin-1 (ET-1). *P < 0.05 vs. basal; n = 5.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Top: representative Western blot. Bottom: effect of ET-1 on eNOS expression after 24-h incubation. *P < 0.05 vs. basal; n = 5.

Because we reported that ET-1 inhibits chloride flux by binding ET_B receptors in the THAL (21), we next tested whether ET-1 stimulates eNOS expression by activating ET_B receptors. ET-1 alone (0.1 nM) increased eNOS expression by 120 ± 40% (P < 0.05; n = 5). When added 1 h after 0.1 µM BQ-788, a selective ET_B receptor antagonist, ET-1 had no significant effect on expression (change: 12 ± 25%; P < 0.05; n = 5; Fig. 3). In contrast, when added 1 h after 0.1 µM BQ-123, a selective ET_A receptor antagonist, ET-1 stimulated eNOS expression by 109 ± 27% (P < 0.05; n = 4; Fig. 4). BQ-123 or BQ-788 alone had no significant effect on basal levels of eNOS. We next tested whether S6c (an ET_B agonist) could mimic the effect of ET-1 on eNOS expression. S6c (0.1 µM) increased expression by 77 ± 30% (P < 0.05; n = 6; Fig. 5). These data suggest that ET-1 increases eNOS expression by activating the ET_B receptors in the THAL.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Top: representative Western blot. Bottom: effect of BQ-788 (a selective ETB receptor antagonist) on eNOS expression. Cells were treated with 0.1 µM BQ-788 for 1 h before stimulation with 0.1 nM ET-1 for 24 h. *P < 0.05 vs. no treatment; n = 5.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Top: representative Western blot. Bottom: effect of BQ-123 (a selective ETA receptor antagonist) on eNOS expression. Cells were treated with 0.1 µM BQ-123 for 1 h before stimulation with 0.1 nM ET-1 for 24 h. *P < 0.05 vs. no treatment; n = 4.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Top: representative Western blot. Bottom: effect of 0.1 µM sarafotoxin c (S6c; an ETB receptor agonist) on eNOS expression and effect of wortmannin (W; an irreversible PI3 kinase inhibitor) on S6c-induced eNOS expression. Cells were treated with 0.01 µM W for 30 min before stimulation with S6c for 24 h (1 µM). *P < 0.05 vs. no treatment; n = 6.

Changes in enzyme expression are often related to changes in its activity. To investigate whether the increase in eNOS protein led to enhanced activity, we measured NO release using a NO-sensitive electrode. In control cultures, the addition of 500 µM L-arginine (the substrate for NOS) along with 1 nM clonidine and 0.5 µM A-23187, a calcium ionophore (to maximize eNOS activation), increased NO release by 337 ± 61 pA. In ET-1-treated cultures, the activators increased NO release by 690 ± 126 pA, 105% more (P < 0.05; n = 5; Fig. 6). We concluded that an increase in eNOS protein augments eNOS activity.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of increased eNOS expression on NO production. Cells were treated with vehicle (control) or 0.1 nM ET-1 for 24 h. NO production was measured after the addition of 500 µM L-arginine (the substrate for NOS), 1 nM clonidine, and 0.5 µM A23187 [GenBank] , a calcium ionophore (to fully activate NOS). *P < 0.05 vs. no treatment; n = 5.

	DISCUSSION

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Our finding that ET-1 stimulates eNOS expression in the THAL is similar to studies of other tissues. Exogenous ET-1 increases eNOS protein expression in cultured IMCD cells (33) and human endothelial cells (15) as well as arterial endothelial cells (34). However, our finding that the effect of ET-1 on eNOS expression is biphasic appears to be novel. This biphasic effect of ET-1 on THAL eNOS expression could be explained by several factors, including downregulation of the receptors or activation of a negative feedback process caused by increased NO. Baldi and Dunn (2) reported dramatic downregulation of ET receptors when glomerular mesangial cells were incubated with ET, and they showed that this effect of ET is concentration dependent. Alternatively, enhanced levels of NO could mediate a negative feedback process that reduces eNOS translation or increases its degradation. Shen et al. (27) showed that the increase in eNOS expression caused by vascular endothelial growth factor (VEGF) that they observed from days 1 to 5 was prevented when the cells were treated with NO donors. In addition, they observed a decline in eNOS expression after 5 days of VEGF. This decrease was prevented by blocking NOS, suggesting that NO participates in a negative feedback mechanism that regulates eNOS expression. It is also possible that the system is rapidly downregulated in vivo during sustained increases in ET-1 expression.

Even though ET-1 increased THAL eNOS expression, we found that it did not significantly change eNOS mRNA. Recent work has made it clear that changes in the levels of specific proteins in cells are not necessarily predictable from changes in the levels of the corresponding mRNA transcripts (3, 9). It may be that for eNOS stimulation, other mechanisms are more important than transcription. In general, regulation of protein abundance by regulation of translation or protein degradation is apparently more important than previously appreciated (11).

ET-1 exerts its effects via two receptors, ET_A and ET_B (14). We found that BQ-788 completely blocked the ET-1-induced increase in eNOS expression in our primary cultures of THALs and that S6c mimicked the effect of ET-1 by binding to ET_B receptors. BQ-123, an ET_A antagonist, did not affect the increase in eNOS expression caused by the addition of ET-1. These data suggest that the increase in eNOS expression induced by ET-1 is mediated by ET_B and that ET_A is not involved. In contrast, Ye et al. (33) reported that the ET-1-induced increase in eNOS expression in IMCD cells is mediated by activation of both ET_A and ET_B. These data suggest that both ET receptors work together to trigger the postreceptor signaling cascade in this nephron segment. Other authors have also reported combined activation of ET_A and ET_B receptors on endothelial cells; for example, Wang et al. (32) reported that ET-1 activates both receptors to mediate its effects in the portal vein. The differences in receptor activation we found in the THAL compared with the IMCD could be explained by differential expression of ET receptor subtypes between the two segments. The THAL only expresses ET_B (31), whereas the IMCD expresses both receptors (13).

ET-1 activates a number of signaling molecules after binding to its receptors. These include protein tyrosine kinases, protein kinase C (PKC), calcium, phospholipases, mitogen-activated protein kinases, PI3K, and Akt (29, 30). Because PI3K mediates increases in eNOS expression in other cells (4, 19), we tested whether it mediates the effects of ET-1. The fact that wortmannin, an irreversible PI3K inhibitor, completely blocked the increase in eNOS expression induced by S6c confirmed our hypothesis that PI3K is involved. Consistent with this finding, Park et al. (19) found that PI3K is involved in the regulation of eNOS expression in ECV 304 cells. Cieslik et al. (4) reported upregulation of the eNOS promoter by a PI3K-dependent pathway. Our concentration of wortmannin was based on previous studies showing that 10 nM selectively inhibits PI3K (17) and that 100 nM is not toxic when added to the THAL (5). Other signaling molecules have been implicated in the regulation of eNOS expression in other types of cells, such as protein tyrosine kinase, PKC, and PLC. Our results do not exclude the possibility that these other pathways also mediate the effects of ET-1 on eNOS expression in the THAL in addition to PI3K.

We found that ET-1 increased eNOS expression at a concentration of 0.1 nM, whereas plasma levels of ET-1 are in the picomolar range (1). This would seem to indicate that ET-1 is not a physiological regulator of eNOS in the THAL. However, THALs and other tubular segments produce ET-1 (12), which is particularly enriched in the renal medulla (10). Moreover, clearance studies showed that urinary ET-1 is of renal origin (28), reflecting intrinsic de novo synthesis of this peptide by tubular epithelial cells. Therefore, regulation of eNOS expression in the THAL may be mediated by locally produced ET-1 in the renal medulla rather than circulating ET-1. It should also be noted that our data were obtained from cultured cells, and therefore the concentration of ET-1 required to enhance eNOS expression may not be the same as it is in vivo. When cells are cultured, there is often an upward shift in the dose-response curve for unknown reasons.

NO release was measured after simultaneous addition of L-arginine, the substrate of NOS, and two types of activators. We did this to eliminate the possibility that ET-1-stimulated NO production would lead to autoinhibition of NOS by NO. We did not use ET-1 as an activator because treatment of cells with ET-1 for 24 h may cause downregulation of ET_B and other receptors. Because an increase in calcium is the classic activator of eNOS, we used A-23187, a calcium ionophore, to increase calcium via a receptor-independent mechanism. Finally, we used clonidine to activate eNOS to be sure we were fully activating the enzyme. Our previous studies indicated that increased calcium alone may not be sufficient to fully activate eNOS in THAL cells (20).

In the THAL, ET-1 acting via activation of eNOS and release of NO is known to inhibit sodium absorption (21). Our data revealed that ET-1 not only regulates eNOS activity (21) but also eNOS expression in the THAL, suggesting that it plays an important role in the regulation of eNOS activity and expression and thus NaCl absorption by the THAL.

When animals are fed a high-salt diet, renal ET levels increase (8) and THAL eNOS expression is upregulated (18). These data suggest that in normal animals high-salt intake generates an adaptive increase of the renal ET-1 system, which promotes diuresis and natriuresis and prevents hypertension. In addition, blockade of ET_B increases arterial pressure in normal rats and this increase is much greater when animals are fed a high-salt diet (24), suggesting that ET, acting through the ET_B receptor, participates in blood pressure regulation in response to salt loading. The mechanism whereby a high-salt diet increases THAL eNOS expression has not been reported to our knowledge. It remains to be determined whether a high-salt diet increases THAL ET-1 synthesis or release by the tubular epithelium, thus stimulating THAL eNOS expression via the ET_B receptor. We believe a defect in these mechanisms could be the explanation for salt-sensitive hypertension, a disease that affects a great many people, and therefore further investigation is needed.

	GRANTS

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This work was supported by National Institutes of Health Grants HL-28982 and HL-70985 to J. Garvin.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. L. Garvin, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202-2689 (E-mail: jgarvin1{at}hfhs.org).

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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