Linda S. Brunauer

Selective amphipathic nature of chlorpromazine binding to plasma membrane bilayers

Chlorpromazine (CPZ), an antipsychotic agent shown to inhibit the action of various neurophysiological receptors, also exhibits preferential association with the plasma membrane, inducing stomatocytic morphological response in red blood cells (RBC). Given the cationic nature of CPZ, fluorimetry, pH titration, and red cell morphological studies were performed to assess the associative predilection of CPZ for anionic membrane components. CPZ fluorescence intensity increased 320-370% upon addition of phosphatidylcholine (PC) small unilamellar vesicles (SUVs) to aqueous CPZ, indicating an affinity of the drug for lipidic phases. After removal of unbound drug, CPZ fluorescence increased up to 92% with increasing phosphatidylserine (PS) in the lipid phase (up to 30 mol% of total lipid), suggesting a preferential association of the drug with anionic lipids. In studies of pH titration, the pK(a) of CPZ in the presence of Triton X-100 micelles or phospholipid SUVs increased with increasing anionicity of the lipidic phase [7.8 with Triton X-100, 8.0 with PC, 8.3 with phosphatidylglycerol (PG)], lending further support to preferential drug interaction with anionic lipidic components. At 0 degrees C, CPZ-induced red cell shape change was less extensive in cells made echinocytic by adenosine triphosphate (ATP) depletion, compared to cells made echinocytic by PS treatment following vanadate preincubation. This suggests that polyphosphoinositide lipids are CPZ membrane binding sites. Since polyphosphoinositide lipids are implicated as important intermediates in a number of receptor-mediated cell signaling pathways, evidence of association with these specific lipids provides a means by which psychoactive drugs may induce neurophysiological effects through direct interaction with general membrane components.

Effects of exogenous phospholipids on platelet activation

Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and speculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457-4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406-5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.