Sex differences in ET-1 receptor expression and Ca2+ signaling in the IMCD

Abstract

The inner medullary collecting duct (IMCD) is the nephron segment with the highest production of endothelin-1 (ET-1) and the greatest expression of ET-1 receptors that function to adjust Na(+) and water balance. We have reported that male rats have reduced natriuresis in response to direct intramedullary infusion of ET-1 compared with female rats. Our aim was to determine whether alterations of ET-1 receptor expression and downstream intracellular Ca(2+) signaling within the IMCD could account for these sex differences. IMCDs from male and female rats were isolated for radioligand binding or microdissected for intracellular Ca(2+) ([Ca(2+)]i) measurement by fluorescence imaging of fura-2 AM. IMCD from male and female rats had similar ETB expression (655 ± 201 vs. 567 ± 39 fmol/mg protein, respectively), whereas male rats had significantly higher ETA expression (436 ± 162 vs. 47 ± 29 fmol/mg protein, respectively; P < 0.05). The [Ca(2+)]i response to ET-1 was significantly greater in IMCDs from male compared with female rats (288 ± 52 vs. 118 ± 32 AUC, nM × 3 min, respectively; P < 0.05). In IMCDs from male rats, the [Ca(2+)]i response to ET-1 was significantly blunted by the ETA antagonist BQ-123 but not by the ETB antagonist BQ-788 (control: 137 ± 27; BQ-123: 53 ± 11; BQ-788: 84 ± 25 AUC, nM × 3 min; P < 0.05), consistent with greater ETA receptor function in male rats. These data demonstrate a sex difference in ETA receptor expression that results in differences in ET-1 Ca(2+) signaling in IMCD. Since activation of ETA receptors is thought to oppose ETB receptor activation, enhanced ETA function in male rats could limit the natriuretic effects of ETB receptor activation.

Keywords: endothelin; inner medullary collecting duct.

Fig. 1.

Maximum binding (B_max) for ET_A receptors (A) and ET_B receptors (B) measured by [I¹²⁵]-ET-1 and [I¹²⁵]-ET-3 in the IMCD of male and female rats (n = 4–5 rats). *Significant difference (P < 0.05).

Fig. 2.

Typical intracellular Ca²⁺ concentration ([Ca²⁺]_i) tracing before and after treatment with endothelin-1 (ET-1; 1 nM) in freshly isolated inner medullary collecting duct (IMCD) from male (A) and female (B) rats. Baseline [Ca²⁺]_i is measured for 2 min, followed by treatment with ET-1. Shaded area represents the area under the curve used to quantify [Ca²⁺]_i response to ET-1. C: an image of an IMCD loaded with fura 2. Boxes represent fields from which [Ca²⁺]_i was measured.

Fig. 3.

Represents [Ca²⁺]_i response as area under the curve (AUC) to either vehicle (open bars) or ET-1 (filled bars) in freshly isolated IMCD from male and female rats (n = 6–9 rats per group). *Significant difference by two-way ANOVA (P < 0.05).

Fig. 4.

Represents [Ca²⁺]_i response as AUC in freshly isolated IMCD from male and female rats in response to the ET_B agonist sarafotoxin 6c.

Fig. 5.

[Ca²⁺]_i response to ET-1 (1 nM) in the presence or absence of an ET_A antagonist (BQ-123; 1 μM) or an ET_B antagonist (BQ-788; 1 μM) in IMCD of male (A; n = 8) or female (B; n = 6) rats. *Significant difference by one-way ANOVA (P < 0.05).

Fig. 6.

[Ca²⁺]_i response in freshly isolated IMCD of male or female rats to ET-1 (1 nM) in the presence or absence of a PLC inhibitor (U-73122; 3 μM).