Sigma Receptor Antagonists Inhibit Cholesterol Synthesis in HepG2 Cells

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We examined the effects of sigma (σ) receptor ligands on the synthesis of cholesterol from ^3H-labeled acetate in HepG2 cells derived from human hepatoblastoma. Putative σ_1, antagonists, but not agonists, effectively inhibited the cholesterol synthesis, resulting in decreased distribution of newly synthesized cholesterol in the Triton X-100 (TX-100) -resistant fraction which contained lipid raft domains of plasma membrane. The inhibitory potency of σ_1 antagonists was as follows: haloperidol > reduced haloperidol > ifenprodil > GBR-12909 > trifluoperazine > progesterone. This rank order correlated well (r = 0.89, p < 0.05) with that of binding affinity for σ_1 receptors of HepG2 cells, which were detected for the first time in the present study. Unlikecholesterol synthesis, the synthesis of sphingomyelin from ^3H-labeled choline was not affected by the σ_1, antagonists. The σ_1, agonists also had no effect on sphingomyelin synthesis. These results suggest that the σ_1, antagonists may inhibit cholesterol synthesis via binding to σ_1, receptors at the smooth endoplasmic reticulum, eventually disrupting the lipid raft architecture of the plasma membrane and impairing subcellular signal transductions. Sigma (σ) receptors which are localized primarily at the smooth endoplasmic reticulum (ER)^<1-4)> mediate diverse biological functions, in particular neuroregulatory effects^<4-6)>. Radioli-gand-binding and pharmacological studies have identified at least two subtypes of σ receptors, σ_1, and σ_2, with the molecular wei-ghts of 25-29 kDa and 18-22 kDa, respec-tively^<6,7> . Recently, σ_1, receptors have been cloned and shown to be highly conserved among mammals^<2,8,9)>. It has been shown that the σ_1, receptors at the smooth ER or translocated to the plasma membrane are involved in the modulation or production of cGMP, inositol phosphates, protein kinases and calcium, eventually participating in plasma membrane-mediated signal transductions^<3-6)>. On the other hand, recent studies have revealed that various plasma membraneme-diated signal transductions can be triggered on the lipid rafts, liquid-ordered microdomains enriched in cholesterol and sphingolipids such as sphingomyelin. Thus, depletion of cholesterol from the plasma membrane impairs signal transductions, probably due to disruption of lipid raft architecture^<10,11)>. Various polarized epithelial cells possess lipid raft-like structures at apical plasma membrane and depletion of cholesterol from these structures may perturb apical transport of related lipids and proteins including glycosylphosphatidylinositol (GPI)-anchored proteins^<12)>. Memb-ranes of both biliary canaliculi of hepato-cytes^<13)> and vacuoli formed between HepG2 cells^<14)> correspond to the apical plasma membrane of other cells, possessing a large amount of GPI-anchored proteins, such as 5' -nucleotidase, alkaline phosphatase and Ca^<2+>-ATPase^<13)>. These results show that cholesterol plays a crucial role in various signal transductions at lipid rafts and in apical transport of GPI-anchored proteins. The smooth ER of hepatocytes is rich in σ receptors^<1,8,15)> and active in cholesterol synthesis. Two σ ligands, haloperidol and ifenpro-dil, inhibit yeast sterol Δ8-Δ7 isomerase involved in cholesterol synthesis^<16)>. This enzyme possesses 66% similarity in amino acid sequence with the guinea pig liver σ_1 receptor^<8)> Ifenprodil and trifluoperazine, but not haloperidol, inhibit mammalian sterol Δ8-Δ7 iso-merase possessing little similarity in amino acidsequence with yeast sterol Δ8-Δ7 isomerase^<17,18)>. In addition, progesterone, a steroid hormone which is also regarded as σ ligand, impairs cholesterol synthesis by inhibiting sterol Δ24-reductase activityi^<9)> and/or intracellular transport of sterol substrates in mammalian cells^<20)>. These findings strongly suggest that σ receptors and their ligands may play a role in the regulation of cholesterol synthesis at the smooth ER, and thereby contribute to the structure and function of lipid rafts in the plasma membrane. In the present study, we investigated the effects of various σ ligands on the synthesis of cholesterol and sphingomyelin in HepG2 cells. HepG2 is a cell line derived from human hepatoblastoma and highly active in the synthesis of cholesterol and sphingomyelin^<19,21)>. The results show that putative σ_1 antagonists effectively inhibited the synthesis of cholesterol, but not sphingomyelin, and the inhibition of cholesterol synthesis by the σ_1 antagonists correlated well with their binding affinities for σ_1, receptors.
2005-04-25

Sigma Receptor Antagonists Inhibit Cholesterol Synthesis in HepG2 Cells

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