Concentration-dependent binding of the chiral beta-blocker oxprenolol to isoelectric or negatively charged unilamellar vesicles.

Large unilamellar vesicles (LUVs) of different lipid compositions were used to study the type of binding of the beta-blocking cationic agent oxprenolol to the lipid matrix of biological membranes at a physiologic pH value of 7.4. When isoelectric membranes of pure egg lecithin or egg lecithin/cholesterol (7:3 mol/mol) were used, a linear relationship between membrane-bound and free oxprenolol indicated a constant molar partition coefficient of 54 or 44 between the liposomal and the aqueous phase over a wide concentration range of the drug up to 25 mM. This pointed to deep insertion of the drug molecules into the hydrophobic membrane interior. Drug binding to membranes of negatively charged phosphatidylserine from bovine brain was cooperative with a Hill coefficient h of 3.4 at concentrations below 0.5 mM and a molar ratio Re of bound drug per lipid of 1:10. Above drug concentrations of 2.5 mM and Re = 1:5, a constant molar partition coefficient of 33 could be estimated. R-oxprenolol or S-oxprenolol, as well as the racemic drug, showed no differences in membrane binding, even with egg lecithin LUVs containing 20 mol% of the negatively charged (2S, 4R)-N-(hexadecanoyl)-4-hydroxyproline, which has a pronounced chiral headgroup. Our results suggest that enantioselective interactions of the chiral oxprenolol with the chiral lipids of biological membranes can be excluded. Furthermore, surface adsorption of the drug is probable only on the negatively charged cytosolic side of biological plasma membranes, whereas on the isoelectric exterior the cationic drug is inserted deeply into the membrane.

Energy-dependent extrusion of cyclic 3',5'-adenosine-monophosphate. A drug-sensitive regulatory mechanism for the intracellular nucleotide concentration in rat erythrocytes.

In reticulocyte-rich suspensions of red blood cells from rats extrusion of cAMP as a regulatory mechanism of intracellular cAMP was investigated. In response to isoprenaline and/or the phosphodiesterase inhibitors Ro 20-1724 and rolipram extrusion of cAMP increases dependent on the concentration of the drugs and time of exposure. However, these drugs exert their effects on the extrusion of cAMP only indirectly, i.e. via increased intracellular levels of cAMP, since the respective EC50-values of the drugs for intracellular accumulation and extrusion of cAMP are identical (isoprenaline: approximately 50 nM; rolipram: approximately 1 microM; Ro 20-1724: 15 microM). The dependence of the rate of extrusion on intracellular levels of cAMP is characterized by a typical concentration-effect relationship from which a maximal capacity of cAMP extrusion of 3-6 nmol/10 min/10(9) cells and a half maximal effective intracellular cAMP concentration of 40-50 nmol/10(9) cells can be derived. This relationship has been inferred from either kinetic or steady-state approaches. At rapidly changing intracellular levels of cAMP an apparent time lag of extracellular cAMP accumulation is obligatorily conditioned by this relationship. Vasodilating drugs which lower the ATP content of the cells as well as the uncoupler of oxidative phosphorylation, FCCP, inhibit the extrusive process (papaverine greater than FCCP greater than dipyridamole greater than dilazep much greater than hexobendine greater than or equal to carbocromene) leading to a 3-5-fold increase of the intrato extracellular concentration gradient of cAMP. It is concluded that extrusion of cAMP is a saturable and energy-dependent process which regulates the intracellular cAMP concentration independent of the activities of adenylate cyclase and phosphodiesterase.