Neurosteroid transport by the organic solute transporter OSTα-OSTβ

Abstract

A variety of steroids, including pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) are synthesized by specific brain cells, and are then delivered to their target sites, where they exert potent effects on neuronal excitability. The present results demonstrate that [(3)H]DHEAS and [(3)H]PREGS are relatively high affinity substrates for the organic solute transporter, OSTα-OSTβ, and that the two proteins that constitute this transporter are selectively localized to steroidogenic cells in the cerebellum and hippocampus, namely the Purkinje cells and cells in the cornu ammonis region in both mouse and human brain. Analysis of Ostα and Ostβ mRNA levels in mouse Purkinje and hippocampal cells isolated via laser capture microdissection supported these findings. In addition, Ostα-deficient mice exhibited changes in serum DHEA and DHEAS levels, and in tissue distribution of administered [(3)H]DHEAS. OSTα and OSTβ proteins were also localized to the zona reticularis of human adrenal gland, the major region for DHEAS production in the periphery. These results demonstrate that OSTα-OSTβ is localized to steroidogenic cells of the brain and adrenal gland, and that it modulates DHEA/DHEAS homeostasis, suggesting that it may contribute to neurosteroid action.

Figure 1. Cis-inhibition of Ostα-Ostβ- and OSTα-OSTβ-mediated [³H]estrone 3-sulfate uptake by conjugated steroids

Effects of different steroids on mouse Ostα-Ostβ (A) and human OSTα-OSTβ (B) mediated transport of [³H]estrone 3-sulfate. Uptake of 50 nM [³H]estrone 3-sulfate was measured for 5 min in the absence (control) and presence of 12.5% ethanol and/or 500 µM of the indicated compounds. Data are expressed as a percent of the control values ± S.E. (n = 3–6 different oocyte preparations, each performed in triplicate), * P<0.05. (-S= sulfated; -G= glucuronidated; -2S= 2 sulfate groups; -S-G= sulfated and glucuronidated)

Figure 2. Human OSTα-OSTβ-mediated transport of [³H]DHEAS and [³H]PREGS

Uptake of 1 µM of either [³H]DHEAS and [³H]DHEA (A), or of [³H]PREGS and [³H]PREG (B) was measured for 5 min at 25°C. To define transport kinetics, oocytes were incubated with [³H]PREGS (C) or [³H]DHEAS (D) concentrations of 0.1, 0.33, 1, 3.3, 10, 50, 100, 200, 400, and 800 µM, and uptake was measured for 5 min at 25°C. Values are means ± S.E. of 4 experiments in distinct oocyte preparations, each performed in triplicate. For DHEAS, the Km (µM), Vmax (fmol/oocyte.5min), and Vmax/Km (nL/oocyte.5min) values of the high-affinity component were 1.5±0.4, 0.8±0.2, and 0.6±0.2, whereas for the low-affinity component they were 532±58, 101±17, and 0.19±0.03, respectively. For PREGS, the Km (µM), Vmax (fmol/oocyte.5min), and Vmax/Km (nL/oocyte.5min) values of the high-affinity component were 6.9±2.1, 9.4±1.5, and 1.5±0.2, whereas for the low-affinity component they were 909±72, 643±34, and 0.71±0.04, respectively.

Figure 3. Human OSTα and OSTβ and mouse Ostα and Ostβ mRNA expression in brain and steroidogenic tissues

Total RNA isolated from different human (A,B) or mouse tissues (C) were subjected to quantitative real time RT-PCR analysis. Ostα and Ostβ (C) and IP3R1 (E) mRNA levels were also measured in specific mouse cells captured by laser capture microdissection. Data are reported relative to GAPDH expression for each tissue and cell. Values are means ± S.E., n=4 separate experiments.

Figure 4. Immunolocalization of Ostα and Ostβ in mouse brain

Brain sections from Ostα^+/+ and Ostα^−/− mice were labeled with anti-Ostα (panels 1–3 in rows A–F) or anti-Ostβ antibodies (panels 4–6 in rows A–F), along with antibodies to either IP3R1, a Purkinje cell marker (panels 2, 3, 5, and 6 in rows A–C), or NeuN, a neuronal marker (panels 2, 3, 5, and 6 in rows D–F).

Figure 5. Immunolocalization of OSTα and OSTβ in human brain and adrenal gland

Human tissues were labeled with anti-OSTα (A–C, G–I, M, N) or anti-OSTβ (D–F, J–L, O, P) antibodies. In the adrenal gland, signals for OSTα and OSTβ were relatively strong in the zona reticularis (ZR) of the adrenal cortex, whereas the zona glomerulosa (ZG) and zona fasciculata (ZF) had weaker staining (M, O). Higher magnification showed that OSTα and OSTβ are expressed in cells of the zona reticularis (N, P). Mu = medulla; scale bar represents 100 µm.

Figure 6. Serum DHEA and DHEAS levels are altered in Ostα-deficient mice

Serum DHEA (A) and DHEAS (B) levels were measured in male and female mice. DHEA/DHEAS ratios were also calculated (C). Values are means ± SE, n = 5–6 mice in each group; *Significantly different from Ostα^+/+ animals, P < 0.05.

Figure 7. Altered distribution of [³H]DHEAS in Ostα-deficient mice

Tissue distribution of radioactivity after administration of [³H]DHEAS in male (A, B) and female (C, D). Mice were injected intraperitoneally with 0.4 µmol in 400 µl, and tissues collected after 2 h. Values are means ± SE, n = 3–4; *Significantly different from Ostα^+/+ animals, P < 0.05.

Figure 8. Hepatic expression of some of the key genes involved in cholesterol and steroid homeostasis in wild type and Ostα-deficient mice

Total RNA isolated from male (A) and female (B) mouse livers was subjected to quantitative real time RT-PCR analysis. Data were assessed relative to Gapdh expression, and are reported as a percent of the value in the Ostα^+/+ mice. Values are means ± S.E., n=4–5 mice per group. *Significantly different from Ostα^+/+ animals, P < 0.05.