Yoshinobu Nakamura

Organic anion-transporting polypeptide transporters (SLCO/OATPs) function as cellular gatekeepers, regulating intestinal absorption, hepatic and renal clearance, and the tissue distribution of drugs and metabolites in the human body. However, the mechanisms underlying substrate selection within the SLCO superfamily remain unclear, hampering efforts to rationalize the interaction of drugs and metabolites with these transporters. SLCO2A1 (also known as OATP2A1) is responsible for the distribution of eicosanoids, including prostaglandins (PGs) and thromboxanes, throughout the body, in addition to several families of nonsteroidal anti-inflammatory drugs (NSAIDs). Here, we present cryogenic electron microscopy structures of SLCO2A1 bound to endogenous PGs and to four widely prescribed medications for treating inflammation, chronic asthma, and Parkinson’s disease (PD). Complementary molecular dynamics and in vivo cellular assays elucidate the molecular basis for PG and drug recognition. Our study reports essential mechanistic details that underpin substrate selection and subfamily adaptation within the broader SLCO superfamily of drug and metabolite transporters.

A previous study indicated that the ANXA2/S100A10 complex represents the regulatory component of SLCO2A1-mediated Maxi-Cl channel activity. The present study showed that apparent PGE₂ uptake by C127 cells was osmoinsensitive and uncompetitively inhibited by loss of ANXA2 expression, demonstrating that ANXA2 is a regulatory factor of SLCO2A1-mediated PG transport activity.

Loss-of-function mutations in the solute carrier organic anion transporter family, member 2a1 gene (SLCO2A1), which encodes a prostaglandin (PG) transporter, have been identified as causes of chronic nonspecific multiple ulcers in the small intestine; however, the underlying mechanisms have not been revealed. We, therefore, evaluated the effects of systemic knockout of Slco2a1 (Slco2a1^−/−) and conditional knockout in intestinal epithelial cells (Slco2a1^ΔIEC) and macrophages (Slco2a1^ΔMP) in mice with dextran sodium sulphate (DSS)-induced acute colitis. Slco2a^−/− mice were more susceptible to DSS-induced colitis than wild-type (WT) mice, but did not spontaneously develop enteritis or colitis. The nucleotide-binding domain, leucine-rich repeats containing family, pyrin domain-containing-3 (NLRP3) inflammasome was more strongly upregulated in colon tissues of Slco2a^−/− mice administered DSS and in macrophages isolated from Slco2a1^−/− mice than in the WT counterparts. Slco2a1^ΔMP, but not Slco2a1^ΔIEC mice, were more susceptible to DSS-induced colitis than WT mice, partly phenocopying Slco2a^−/− mice. Concentrations of PGE₂ in colon tissues and macrophages from Slco2a1^−/− mice were significantly higher than those of WT mice. Blockade of inflammasome activation suppressed the exacerbation of colitis. These results indicated that Slco2a1-deficiency increases the PGE₂ concentration, resulting in NLRP3 inflammasome activation in macrophages, thus exacerbating intestinal inflammation.

Prostaglandin E2 (PGE2) is a well-established chemical mediator for the generation of the fever at the hypothalamus of the brain. PGE2 mediates fever generation via PGE receptor 3 (i.e., EP3) on neurons in the preoptic area. The role of PGE2 has been analyzed by measuring PGE2 concentration in cerebrospinal fluid (Ccsf); however, local PGE2 concentration at the hypothalamus may not necessarily be consistent with Ccsf. In this protocol, we introduce our method to measure directly the alteration in PGE2 concentration in interstitial fluid in the hypothalamus (Cisf) of awake (free-moving) mice using a microdialysis technique. Male mice (c57BL/6J) were anesthetized and fixed in the stereotaxic instrument, and a microdialysis probe was inserted into the hypothalamus through a guide cannula. On the fifth postoperative day, Cisf was monitored in free-moving mice that were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS). PGE2 and other eicosanoids recovered in Krebs-Ringer phosphate buffer and defused through a microdialysis probe were extracted into ethyl acetate/formic acid and then quantified with LC-MS/MS. Our method is useful to understand the role of key regulators of prostaglandin concentration such as those of transporters, which have been unappreciated in inflammation-based brain diseases.

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Prostaglandin E₂ (PGE₂) in the hypothalamus is a principal mediator of the febrile response. However, the role of organic anion transporting polypeptide 2A1 (OATP2A1/SLCO2A1), a prostaglandin transporter, in facilitating this response is unknown. Here, we investigated the effect of Slco2a1 deficiency on the body core temperature (Tc) and on the PGE₂ concentration in hypothalamus interstitial fluid (C_isf) and CSF (C_csf) of lipopolysaccharide (LPS; 100 μg/kg, i.p.)-treated mice of both sexes. Slco2a1^−/− mice did not develop a febrile response. C_csf was increased in Slco2a1^+/+ and Slco2a1^−/− mice, and C_csf of Slco2a1^−/− mice was well maintained at 5 h after LPS injection (1160 pg/ml) compared with Slco2a1^+/+ mice (316 pg/ml). A microdialysis study revealed that C_isf peaked at 2 h after LPS injection in Slco2a1^+/+ mice (841 pg/ml), whereas the increase in C_isf was negligible in Slco2a1^−/− mice. The PGE₂ plasma concentration in Slco2a1^−/− mice (201 pg/ml) was significantly higher than that in Slco2a1^+/+ mice (54 pg/ml) at 1 h after LPS injection, whereas the two groups showed similar PGE₂ concentrations in the hypothalamus. Strong Oatp2a1 immunoreactivity was observed in F4/80-positive microglia and perivascular cells and in brain capillary endothelial cells. The changes in Tc and C_isf seen in LPS-injected Slco2a1^+/+ mice were partially attenuated in monocyte-/macrophage-specific Slco2a1^−/− (Slco2a1^Fl/Fl/LysM^Cre/+) mice. Thus, OATP2A1 facilitates the LPS-induced febrile response by maintaining a high level of C_isf, possibly by regulating PGE₂ secretion from F4/80-positive glial cells and/or facilitating PGE₂ transport across the blood–brain barrier. These findings suggest that OATP2A1 is a useful therapeutic target for neuroinflammation. SIGNIFICANCE STATEMENT Fever is a physiological response caused by pyrogen-induced release of prostaglandin E₂ (PGE₂) in the hypothalamus, which plays a central role in regulating the set-point of body temperature. However, it is unclear whether the prostaglandin transporter OATP2A1/SLCO2A1 is involved in this response. We show here that LPS-induced fever is associated with increased PGE₂ concentration in hypothalamus interstitial fluid (C_isf), but not in CSF (C_csf), by means of a microdialysis study in global Slco2a1-knock-out mice and monocyte-/macrophage-specific Slco2a1-knock-out mice. The results suggest that OATP2A1 serves as a regulator of C_isf in F4/80-positive glial cells. OATP2A1 was detected immunohistochemically in brain capillary endothelial cells and, therefore, may also play a role in PGE₂ transport across the blood–brain barrier.