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.