ABSTRACT:The purpose of this study was to characterize blood-brain barrier (BBB) transport of oxycodone, a cationic opioid agonist, via the pyrilamine transporter, a putative organic cation transporter, using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13). Oxycodone and [ 3 H]pyrilamine were both transported into TR-BBB13 cells in a temperature-and concentration-dependent manner with K m values of 89 and 28 M, respectively. The initial uptake of oxycodone was significantly enhanced by preloading with pyrilamine and vice versa. Furthermore, mutual uptake inhibition by oxycodone and pyrilamine suggests that a common mechanism is involved in their transport. Transport of both substrates was inhibited by type II cations (quinidine, verapamil, and amantadine), but not by classic organic cation transporter (OCT) substrates and/or inhibitors (tetraethylammonium, 1-methyl-4-phenylpyridinium, and corticosterone), substrates of OCTN1 (ergothioneine) and OCTN2 (L-carnitine), or organic anions. The transport was inhibited by metabolic inhibitors (rotenone and sodium azide) but was insensitive to extracellular sodium and membrane potential for both substrates. Furthermore, the transport of both substrates was increased at alkaline extracellular pH and decreased in the presence of a protonophore (carbonyl cyanide-ptrifluoromethoxyphenylhydrazone). Intracellular acidification induced with ammonium chloride enhanced the uptakes, suggesting that the transport is driven by an oppositely directed proton gradient. The brain uptake of oxycodone measured by in situ rat brain perfusion was increased in alkaline perfusate and was significantly inhibited by pyrilamine. These results suggest that blood-brain barrier transport of oxycodone is at least partly mediated by a common transporter with pyrilamine, and this transporter is an energy-dependent, proton-coupled antiporter.

Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L–P complex from parainfluenza virus 5 (PIV5) at 4.3-Å resolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Å resolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5′ end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.

BackgroundKnowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model.MethodsDiphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions.ResultsDiphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB.ConclusionsThe present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.

No abstract

No abstract

No abstract

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn’t an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

No abstract