Membrane shape changes of human erythrocytes induced by each of five cationic and two anionic phenothiazines were observed semiquantitatively, and the amounts of the drugs incorporated into erythrocytes or ghosts were determined. Cationic and anionic phenothiazines induced membrane invagination and exvagination on human erythrocytes, respectively. The former drugs were incorporated mainly into the membrane of cells, whereas only a small portion of the latter drugs was incorporated into the membrane and the remainder into the cytosol. The initial concentrations of cationic phenothiazines needed to induce the same extent of shape change decreased in the order promethazine, chlorpromazine, perazine, prochlorperazine, and trifluoperazine, indicating that their shape-transforming activities increase in the same order. The phenothiazines with a halogen atom at position C-2 of the phenothiazine nucleus exhibited higher affinity for the membrane than those with a hydrogen atom. Phenothiazines with the N-methylpiperazinyl group in the side chain showed a stronger effect on membrane shape change than those with the dimethylamino group and with the same nonpolar moiety. The introduction of halogen atom(s) appears to increase the affinity of drugs for the plasma membrane, and the polar head group in the side chain seems mainly responsible for perturbation of the membrane, which leads to induction of a shape change.
Calcium ions were found to stimulate the Na+-K+-sensitive ATPase as well as Mg2+-ATPase (Na+-K+-insensitive ATPase) activities of human erythrocyte membrane in a concentration between approximately 0.04-0.4mM, and to inhibit them in a concentration higher than 4 mM. In the presence of Ca ions, the Na+-K+-ATPase activity dose not correspond to the ouabain-sensitive ATPase activity anymore which is apparently inhibited with increasing concentration of Ca ions. The effects of Ca2+ on the Mg2+-ATPase seems to be dependent on the ratio of Ca2+/Mg2+ concentrations. Erythrocyte stroma previously in contact with CaCl2 and then washed, showed increased Mg2+-ATPase activity when assayed in the absence of added Ca2+ in the incubation mixture, with the maximal level of the activity almost equivalent to the maximal Ca2+-Mg2+-ATPase activity. This would mean that the Ca ions absorbed on the outer surface of the erythrocyte membrane is well capable of stimulating the ATPase activity. Normal values of these ATPase activities of human erythrocyte stroma were established. The ATPase of porcine erythrocyte membrane were similarly stimulated by Caions, whereas those of bovine erythrocyte were only slightly stimulated by this divalent cation.
The effect of cholesterol-enrichment in platelet membranes on U46619 binding to the specific receptor and phospholipase A2 and C activities was studied using cholesterol-loaded human platelets prepared by in vitro incubation with cholesterol-rich liposomes. The cholesterol-enriched platelets, having a higher cholesterol/phospholipid molar ratio, were hyperaggregable to collagen, arachidonic acid, the thromboxane mimetic U46619 or thrombin. The number of binding sites for U46619, but not the affinity, was significantly increased. Arachidonic acid liberation from membrane phospholipids in response to collagen, thrombin or A23187 was also markedly increased. Furthermore, GTPγS-induced stimulation of the platelet membranes isolated from the cholesterol-enriched platelets, caused significantly increased arachidonic acid liberation but not increased diacyglycerol formation, as compared with the membranes from normal platelets.These results suggest that a certain physical change in cholesterol-loaded membranes brings about a hyperresponsiveness of the endoperoxide receptor and a hyperreactivity of phospholipase A2, probably through enhancement of the coupling efficiency of the corresponding GTP-binding protein to the enzyme, thereby resulting in the increased aggregability to collagen or arachidonic acid. The increased sensitivity to thrombin may be due to a factor such as an increase in thrombin binding, since phospholipase C activity was not enhanced in response to stimuli without intervention of the receptor.
Plasma membranes (PM) were isolated from island-forming types of rat ascites hepatoma (AH 130, AH 602, and AH 7974) and from their free-cell sublines (AH 130FN and AH 7974F), and were characterized in terms of electron-microscopic morphology, marker enzyme activities, and lipid contents. The results were compared with those of the PM isolated in a similar way from newborn, regenerating, and adult livers. The marker enzyme activities, such as Na+, K+-insensitive Mg2+-ATPase [EC 3.6.1.3] (Mg2+-ATPase) and 5'-nucleotidase [EC 3.1.3.5], as well as the phospholipid composition of the PM isolated from hepatomas by Wallach's nitrogen gas cavitation method were similar to those obtained with the PM isolated by a modification of Emmelot's method, although the former method gave a much lower yield in terms of protein than the latter. Based on the modified Emmelot method, sufficiently pure PM preparations could be obtained from the hepatomas in the form of large membrane sheets without any contamination by other identifiable components, as determined with an electron microscope, and with high specific activities of the marker enzymes, such as Na+, K+-sensitive ATPase [EC 3.6.1.3] (Na+, K+ -ATPase), Mg2+ -ATPase, and 5'-nucleotidase. As for the characteristics of the hepatoma PM, lower specific activity of 5'-nucleotidase and higher fatty aldehyde molar percentages in total phospholipids were noted in all the PM from the hepatomas in comparison with normal liver PM of various origins. The PM from the hepatomas showed an increased amount of cholesterol (mumole per mg protein), whereas actively growing newborn and regenerating livers gave rather lower amounts in comparison with that of normal adult liver.
The inhibitory effects of several contrast media on acetylcholinesterase (AchE) activity were studied in vitro. Their inhibiting power was in the order: iothalamate > ioxaglate > iopamidol > iohexol. Because both the Km and maximal velocity (V max) values of the enzyme were altered on adding ioxaglate, the inhibition mode seemed to be of a mixed type. Complete recovery from such inhibition was observed by removing the contrast media from the enzyme. Adding albumin (1%) to the reaction mixture led to a decrease in the inhibitory effect of ioxaglate. These results suggest that the contrast media, which have an inhibitory effect on AchE activity in vitro, may have no influence on its activity in blood which contains 4–5% of albumin under physiological conditions. Radiological iodinated contrast media are relatively non-toxic, but they are employed in such large doses that mild toxic effects are common in clinical practice, and severe reactions sometimes occur (Labbe & Peyroux, 1984; Dawson, 1983, 1985). Since some symptoms of their toxicity such as peripheral vasodilation, bronchospasm and disturbances of cardiac rhythm are similar to those produced by acetylcholine, their inhibitory effects on AchE (acetylcholine acetylhydrolase, EC 3117) have been studied (Lasser & Lang, 1966; Dawson & Edgerton, 1983; Howell & Dawson, 1985; Guidollet et al, 1988). The published studies, however, have yielded conflicting results. In this short communication, we report more precisely the nature of the inhibition of AchE by some representative ionic and non-ionic contrast media.
