ABSTRACT
Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. The hTRPM3 gene is comprised of 24 exons and maps to human chromosome 9q-21.12. hTRPM3 is composed of 1555 amino acids and possesses the characteristic six-transmembrane domain of the TRP family. hTRPM3 is expressed primarily in kidney and, at lesser levels, in brain, testis, and spinal cord as demonstrated by quantitative RT-PCR and Northern blotting. In situ hybridization in human kidney demonstrated that hTRPM3 mRNA expression is predominantly found in the collecting tubular epithelium. Heterologous expression of hTRPM3 in human embryonic kidney cells (HEK 293) showed that hTRPM3 is localized to the cell membrane. hTRPM3-expressing cells exhibited Ca2+ concentration-dependent Ca2+ entry. Depletion of intracellular Ca2+ stores by lowering extracellular Ca2+ concentration and treatment with the Ca2+-ATPase inhibitor thapsigargin or the muscarinic receptor agonist carbachol further augmented hTRPM3-mediated Ca2+ entry. The nonselective Ca2+ channel blocker, lanthanide gadolinium (Gd3+), partially inhibited hTRPM3-mediated Ca2+ entry. These results are consistent with the hypothesis that hTRPM3 mediates a Ca2+ entry pathway that apparently is distinct from the endogenous Ca2+ entry pathways present in HEK 293 cells.
Subject(s)
Calcium/metabolism , Chromosomes, Human, Pair 9 , Ion Channels/genetics , Ion Channels/metabolism , Kidney Tubules, Collecting/physiology , Amino Acid Sequence , Calcium Channel Blockers/pharmacology , Cell Membrane/metabolism , Cells, Cultured , Central Nervous System/physiology , Gadolinium/pharmacology , Gene Expression , Humans , Ion Channels/chemistry , Kidney Tubules, Collecting/cytology , Lanthanoid Series Elements/pharmacology , Male , Molecular Sequence Data , Protein Structure, Tertiary , RNA, Messenger/analysis , TRPM Cation Channels , Testis/physiologyABSTRACT
The biological effects of monocyte chemoattractant protein (MCP) 1 are mediated by binding to C-C chemokine receptor (CCR) 2. In the present studies, we used CCR2 knockout (CCR2-/-) mice to examine the role of MCP-1 in acetaminophen-induced macrophage accumulation in the liver, expression of inflammatory cytokines, and hepatotoxicity. We found that hepatic expression of CCR2 and MCP-1 was increased 10-fold and 20-fold, respectively, 12 to 72 hours after administration of acetaminophen to wild-type mice. Expression of these proteins was localized in centrilobular regions of the liver. Whereas MCP-1 was expressed by both hepatocytes and macrophages, CCR2 was identified in inflammatory macrophages. F4/80 is a marker of mature macrophages expressed in large quantities by Kupffer cells. In wild-type mice, a 75% decrease in F4/80-positive macrophages was observed 24 to 48 hours after administration of acetaminophen. In contrast, expression of macrosialin (CD68), a marker of activated macrophages, increased 2-fold 24 to 72 hours after administration of acetaminophen and was associated with inflammatory cells. Although there was a decrease in the overall severity of inflammation and in the number of macrosialin-positive macrophages 72 hours after administration of acetaminophen in CCR2-/- mice, the number of F4/80-positive cells did not change. Loss of CCR2 was also found to alter acetaminophen-induced expression of tumor necrosis factor alpha, monocyte chemoattractant protein 3, and KC/gro. However, the overall outcome of acetaminophen-induced hepatic injury was not affected. In conclusion, these data indicate that MCP-1 and CCR2 contribute to the recruitment of a subset of activated macrophages into the liver during acetaminophen-induced hepatotoxicity that may be important in resolution of tissue injury.
