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

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<0.05); and its effect was blocked by MK–571 (10^–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; p<0.05), and LTD₄ 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; p<0.05); however, EGTA (1 mM), a calcium chelator, partially blocked the LTD₄–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.