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Abstract

Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent K_i values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E₂, and prostaglandin D₂ was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. K_i,PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. K_i, _PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N¹-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit _PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [³H]cAMP and [³H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: K_m = 1.5 mmol/l, Jmax = 0.34 pmol S^-1 cm^-1, r (extracellular/intracellular amount at steady state) = 0.91; for cGMP: K_m = 0.29 mmol/l, Jmax = 0.31 pmol S^-1 cm^-1, r = 0.55. Comparison of app. K_i,cGMP with app. K_i, _PAH of ten substrates gave a linear relation with a ratio of 1.83 +/- 0.5. All prostanoids applied inhibited the contraluminal _PAH transport; the prostaglandins E₁, F₁ alpha, A₁, B₁, E₂, F₂ alpha, D₂, A₂ and B₂ with an app. K_i, _PAH between 0.08 and 0.18 mmol/l. The app. K_i of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1 alpha (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. K_i,_PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1 alpha (0.77 mmol/l) and 2,3-dinor-6-keto-PGF1 alpha (0.57 mmol/l) as well as that of thromboxane B₂ (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB₁, E₂ and D₂ inhibited contraluminal sulphate transport (app. K_i,SO₄(^2-) 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE₂ showed complex transport kinetics with a mixed K_m = 0.61 mmol/l and Jmax of 4.26 pmol S^-1 cm^-1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD₂, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E₂ a significant uptake through the sulphate transporter occurs in addition