title: Identification and transmembrane signaling of leukotriene D4 receptors in human mesangial cells.
abstract: Although peptidoleukotriene (LTC4, LTD4) receptors have been characterized by radioligand binding studies, pathways of transmembrane signaling by activated leukotriene receptors remain obscure. We employed [3H]LTD4 binding studies and fluorescent measurements of intracellular Ca2+ concentration ([Ca2+]) and pH to identify LTD4 receptors and mechanisms of transmembrane signaling in cultured human mesangial cells. Mesangial cells expressed a single class of saturable, specific binding sites for [3H]LTD4. Kinetic, competition, and saturation analyses gave an average KD of approximately 12.0 nM with a Bmax of 987 fmol/mg protein. LTC4 competed with high affinity for [3H]LTD4 binding sites, as did LTB4 but with much lower affinity. [3H]LTD4 binding was blocked by a specific LTD4 receptor antagonist, SKF 102922. LTD4 and LTC4 also evoked a rapid (2-3 s), transient increase in intracellular [Ca2+], followed by a second, sustained increase. The transient phase was independent of extracellular Ca2+, whereas the sustained phase was dependent on extracellular Ca2+. Intracellular [Ca2+] was unaffected by LTB4. The LTD4-stimulated Ca2+ transients were dose dependent (1 nM-1 microM) and, similar to [3H]LTD4 binding, Ca2+ transients were inhibited by LTD4 receptor antagonists. We also report evidence that LTD4 affects intracellular pH and activates Na+-H+ exchange. Specifically, LTD4 induced an initial acidification within 1-2 min, followed by net alkalinization at 5 min. Alkalinization was due to activation of an amiloride-inhibitable Na+-H+ exchanger. LTD4 receptors were apparently not coupled to adenylate cyclase or phospholipase A2 as we detected no changes of adenosine 3',5'-cyclic monophosphate (cAMP) or prostanoids. Thus we conclude that [3H]LTD4 binding sites on human mesangial cells are coupled to a Ca2+-signaling system and Na+-H+ exchange. Moreover LTD4, a potent inflammatory mediator, failed to stimulate cAMP or prostaglandin E2/prostaglandin I2, two counterregulatory autacoids that preserve normal mesangial function.
authors.affiliation: Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio.
authors.forename: M S
authors.initials: MS
authors.lastname: Simonson
authors.forename: P
authors.initials: P
authors.lastname: Mené
authors.forename: G R
authors.initials: GR
authors.lastname: Dubyak
authors.forename: M J
authors.initials: MJ
authors.lastname: Dunn
mesh_terms: D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011453:Prostaglandins / Q000096:biosynthesis; D011971:Receptors, Immunologic / Q000378:metabolism*; D018077:Receptors, Leukotriene; D013189:SRS-A / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction; D013929:Thromboxane B2 / Q000096:biosynthesis
chemical_list: D011453:Prostaglandins; D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; D013189:SRS-A; D013929:Thromboxane B2; D000242:Cyclic AMP; D002118:Calcium