Leukotriene D4 Inhibits Na+ Uptake through cAMP and PLC Pathways in Primary Cultured Renal Proximal Tubular Cells

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Abstract
Leukotriene D4 (LTD4) is one of the slow–reacting substances of anaphylaxis and is reported to have a diverse response including the mediation of glomerular nephritis. However, little is known about the functions of LTD4 and its mechanisms of action in primary cultured rabbit renal proximal tubular cells (PTCs). The purpose of this study is to investigate the effect of LTD4 on Na+ uptake and its related signal transduction pathways in PTCs. LTD4 (>10–9 M) significantly inhibited the Na+ uptake after 15 min (in nmol/mg protein: controls 431.7±11.4 vs. LTD4 (10–9 M) 355.0±23.6; p–6 M), a leukotriene receptor antagonist, in PTCs. Preincubation with cilastatin, a renal dipeptidase inhibitor, and polyclonal antibody against renal dipeptidase potentiated the inhibitory effect of LTD4 on Na+ uptake. SQ 22536 (10–6 M), an adenylate cyclase inhibitor, and the myristoylated protein kinase A inhibitor amide 14–22 (PKI; 10–5 M) blocked the effect of LTD4 on Na+ uptake (in nmol/mg protein: LTD4 349.9±18.5 vs. SQ 22536+LTD4 476.5±22.0 and PKI+LTD4 440.3±19.3; p4 induced an increase in cyclic adenosine monophosphate (cAMP), suggesting the involvement of cAMP in the inhibition of Na+ uptake. In addition, U 73122 (10–6 M) and neomycin (10–4 M), phospholipase C (PLC) inhibitors, W–7 (10–4 M), a calmodulin antagonist, and bisindolylmaleimide I, a protein kinase C (PKC) inhibitor, blocked the LTD4–induced inhibition of Na+ uptake, strongly suggesting involvement of the PLC–PKC signal pathways in the effect of LTD4. LTD4 significantly increased [Ca2]i by 49±7% as compared with baseline. TMB–8 (10–5 M) and BAPTA/AM (10–5 M), intracellular calcium mobilization blockers, completely blocked the LTD4–induced inhibition of Na+ uptake (in nmol/mg protein: LTD4 347.6±19.0 vs. TMB–8+LTD4 436.4±22.3 and BAPTA/AM+LTD4 419.9±14.3; p4–induced inhibition of Na+ uptake. In conclusion, LTD4–induced inhibition of Na+ uptake may be involved in both cAMP and PLC–PKC signal pathways in PTCs. In addition, Ca2+, which comes from the intracellular Ca2+ mobilization, is primarily responsible for the LTD4–induced inhibition of Na+ uptake.