ATP-DEPENDENT TRANSPORT OF THE LINEAR RENIN-INHIBITING PEPTIDE EMD-51921 BY CANALICULAR PLASMA-MEMBRANE VESICLES OF RAT-LIVER - EVIDENCE OFDRUG-STIMULATABLE ATP-HYDROLYSIS

Certain peptide drugs, such as the linear hydrophobic renin-inhibitor EMD 51921, are rapidly eliminated via the bile. At the sinosoidal memb rane of liver cells EMD 51921 is taken up via a sodium-independent car rier-mediated mechanism, competing for the uptake of bile acids. Until now, the mechanisms of biliary excretion of EMD 51921 were unknown. I n this study we describe an ATP-dependent transport system for the enz ymatically and metabolically stable hydrophobic linear renin-inhibitin g peptide EMD 51921. The ATP-dependent uptake into the osmotic reactiv e intravesicular space is saturable (K-m 12 mu M, V-max 663 pmol/min p er mg protein), temperature dependent and specifically requires ATP. T ransport is inhibited by vanadate but not by ouabain, EGTA or NaN3, an d does not function in basolateral plasma membrane vesicles. Transport is not altered in canalicular membrane vesicles isolated from Tr(-) r ats lacking the canalicular ATP-dependent transport of cysteinyl leuko trienes and related anions. Transport is inhibited by taurocholate, a typical substrate of the candicular ATP-dependent bile acid transporte r, but also by vincristine and daunomycin, substrates of P-grycoprotei ns. EMD 51921, however, only inhibits the uptake of taurocholate, wher eas the transport of daunomycin is not influenced. Taurocholate and EM D 51921 are mutually non- or un-competitive transport inhibitors. Incu bation of rat liver canalicular membranes with micromolar concentratio ns of EMD 51921 resulted in a 1.8-2.5-fold increase in the rate of ATP -hydrolysis. In contrast, ATP-hydrolysis was not affected by fragments of the peptide that are not transported in an ATP-dependent manner. T he apparent K-m value (EMD) for ATP-hydrolysis is 68 mu M. V-max, is 0 .032 U/mg protein. ATPase activity is pH dependent. Stimulation of ATP -hydrolysis is inhibited by vanadate, NEM, hydroxymercuribenzoate and ascorbate, but is not affected by ouabain, EGTA or NaN3. EMD 51921 doe s not stimulate the ATPase activity of the Na+/K+-ATPase isolated from kidney medulla. The EMD-stimulatable ATPase seems to be distinct from the glutathione-S-conjugate stimulatable ATPase and the mdr 1a/b gene products and differs in its characteristics from that of the canalicu lar ecto-ATPase.