Aldosterone does not alter endothelin B receptor signaling in the inner medullary collecting duct

Male Sprague Dawley rats weighing ~200 g were exposed to 3 days of a normal (0.3% Na⁺, Harlan Teklad, Indianapolis, IN) or low (0.01% Na⁺, Test Diet, St. Louis, MO) salt diet, or low salt diet + the mineralocorticoid desoxycorticosterone acetate (DOCA). DOCA was administered at 7.15 mg/day subcutaneously via slow release pellets (50 mg 21‐day release pellet, 3 pellets/rat for total of 150 mg/rat, Innovative Research, Sarasota, FL). All animal studies were conducted with the approval of the University of Utah Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Rat and mouse inner medullas were removed, minced, and incubated at 37°C in 0.1% collagenase (type IV; Worthington) and 0.1% hyaluronidase (type IV, Sigma, St. Louis, MO) in Hanks Balanced Salt Solution (HBSS) containing 15 mmol/L HEPES (pH 7.4) for about 45 min. When nearly digested, 0.01% DNase (type I, Sigma) was added for 10 min. The suspension containing predominantly single cells and individual tubules was centrifuged and then subjected to two rounds of re‐suspension in 10% bovine serum albumin in HBSS and centrifuging at 400 g for 5 min. The final pellet was washed in HBSS.

The mouse IMCD cell line, mIMCD3, was used for all cell culture studies. Cells were grown to confluence in 24‐well plastic culture plates in a 5% CO₂ incubator at 37°C; 50:50 DMEM/F‐12 supplemented with 10% fetal bovine serum, 1 mg/ml penicillin and 1 mg/ml streptomycin was used as growth medium. In all studies, cells were growth arrested for 3 h prior to study by removal of serum.

Cultured IMCD3 cells were incubated with vehicle or 2.8 μmol/L aldosterone for 48 h. Cells were then exposed to the ETB‐specific agonist, sarafotoxin 6c (S6c, 100 nmol/L, Tocris Bioscience, Minneapolis, MN) or vehicle in HBSS for 15 min at 37°C. Total and phosphorylated ERK1/2 were measured by enzyme immunoassay (EIA) (Abcam, Cambridge, MA). Acutely isolated IMCD from rats fed a normal salt diet were treated with S6c or vehicle as described above and assayed for total and phosphorylated ERK. Note that a commercially supplied lysate is used to generate the standard curves for ERK and phospho‐ERK, hence the absolute values of ERK and phospho‐ERK are unknown – only relative values can be reported.

Acutely isolated IMCD from rats fed a normal, low salt or low salt diet + DOCA were incubated with 1 mmol/L isobutylmethylxanthine (IBMX) (to inhibit phosphodiesterases) for 30 min, then treated with 100 nmol/L S6c or vehicle in HBSS for 15 min at 37°C, followed by exposure to 100 nmol/L arginine vasopressin (AVP) for 10 min. Total cell cAMP was then assayed by EIA (Enzo Life Sciences, Farmingdale, NY) as previously described (Stricklett et al. 2006). Total cell protein was determined by Bradford assay (Bio‐Rad, Hercules, CA). IMCD3 cells were similarly treated with S6c and AVP, followed by determination of cAMP and total cell protein as above.

Studies were done in cultured IMCD3 and acutely isolated IMCD from rats fed a normal salt diet or low salt diet + DOCA as described above. Cells were incubated with IBMX for 30 min and then treated with 100 nmol/L S6c or vehicle in HBSS for 15 min at 37°C. Total cell cGMP was then assayed by EIA (Enzo Life Sciences) and total cell protein determined. In a separate set of studies, cells were exposed to HBSS alone or containing 100 nmol/L S6c with 1 mmol/L L‐NAME added during the IBMX pre‐incubation.

Studies were done in cultured IMCD3 and acutely isolated IMCD from rats fed a normal salt diet or low salt diet + DOCA as described above. Cells were incubated with 10 μmol/L 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate (DAF‐FM‐DA, Invitrogen, Waltham, MA) in HBSS for 30 min at 37°C, then washed and allowed to rest for 30 min in HBSS. Cells were then treated with 100 nmol/L S6c or vehicle in HBSS for 15 min at 37°C. Fluorescence was determined using 495 nm excitation and 515 nm emission with a SpectraMax Gemini EM microplate reader (Molecular Devices, Sunnyvale, CA). Total cell protein was also determined.

Data are presented as mean ± standard error. All data were analyzed by ANOVA with the post hoc Scheffe test. P < 0.05 was taken as significant.

The first series of studies were designed to test if aldosterone altered ETB signaling in the CD via Gs proteins; ERK phosphorylation was chosen for this purpose. To accomplish this, several model systems were tested. First, acutely isolated IMCD from mice and rats were tested for the ability of S6c to stimulate ERK phosphorylation. We were unable to detect any stimulatory effect of S6c at up to 100 nmol/L and for up to 15 min on ERK phosphorylation (vehicle: total ERK – 7.3 ± 0.6, phospho‐ERK – 0.66 ± 0.04, phospho/total ERK – 8.35 ± 0.6%; S6c: total ERK – 6.4 ± 0.4, phospho‐ERK – 0.49 ± 0.04, phospho/total ERK – 7.7 ± 0.3%; N = 8 each data point, values shown for rats [mice not shown but with similar results]). We next tested an IMCD cell line, IMCD3, which had been reported to have ETB‐mediated ERK phosphorylation (Hyndman et al. 2012). Notably, this cell line is derived from the mouse; rat CD cell lines are not generally available. As shown in Figure 1, exposure to 100 nmol/L S6c increased ERK phosphorylation. To test the effect of aldosterone, IMCD3 cells were pre‐incubated with 2.8 μmol/L aldosterone for 48 h; this pre‐treatment had no effect on the ability of S6c to enhance ERK phosphorylation. Notably, aldosterone alone, as has been repeatedly shown, increased ERK phosphorylation.

The second series of studies were designed to test if aldosterone altered ETB signaling in the CD via Gi proteins; AVP‐induced cAMP accumulation was chosen for this purpose. As for ERK signaling, several model systems were tested. Since S6c stimulated ERK phosphorylation in IMCD3 cells, this approach was attempted first. While AVP increased cAMP accumulation in these cells, S6c had no inhibitory effect on AVP‐induced cAMP (vehicle alone – no cAMP detected, AVP – 0.33 ± 0.06 pmoles cAMP/μg protein, AVP+S6c – 0.29 ± 0.03 pmoles cAMP/μg protein, N = 8 each data point). This finding is in accord with previous studies showing the Gi protein‐mediated effects of ET‐1 are frequently lost when CD cells are cultured (Woodcock and Land 1992). Consequently, acutely isolated IMCD were examined. Acutely isolated mouse IMCD do manifest ET‐1 inhibition of AVP‐stimulated cAMP accumulation (Strait et al. 2007), however the amount of tissue obtained, the magnitude of AVP‐stimulated cAMP, the degree of ETB‐mediated inhibition of AVP‐stimulated cAMP, and the amount of ET receptors on acutely isolated mouse IMCD are far less for mouse as compared to rat IMCD (unpublished observations by our laboratory). Hence, acutely isolated rat IMCD were examined. S6c substantially inhibited AVP‐stimulated cAMP content in acutely isolated rat IMCD (Fig. 2). Placing rats on a low salt diet for 3 days (to increase endogenous aldosterone) did not alter the magnitude of S6c inhibited AVP‐stimulated cAMP. Further, treatment with DOCA for 3 days at supraphysiological doses on top of administration of a low salt diet also did not affect the degree of S6c inhibition of AVP‐induced cAMP.

The final set of studies tested whether aldosterone altered ETB‐stimulated NO and/or cGMP accumulation. No NO signal could be detected using DAF‐FM‐DA in IMCD3 cells. As a surrogate, S6c‐stimulated cGMP levels were assessed in IMCD3 cells: S6c comparably increased cGMP quantitatively in the presence or absence of prior exposure to 2.8 μmol/L aldosterone for 48 h (Fig. 3). That S6c‐stimulated cGMP was NO‐dependent in IMCD3 cells was supported by the finding that 1 mmol/L L‐NAME prevented S6c‐stimulated cGMP accumulation (1.9 ± 0.4 pmoles cGMP/mg protein in control ± L‐NAME vs. 2.1 ± 0.3 pmoles cGMP/mg protein in S6c ± L‐NAME, N = 5). In acutely isolated rat IMCD, S6c increased both NO (as assessed by DAF‐FM‐DA) and cGMP levels (Fig. 4). Placing rats on a low salt diet plus DOCA for 3 days did not alter the magnitude of S6c induced NO or cGMP (Fig. 4).