Factors affecting in vivo electrochemistry: electrode-tissue interaction and the ascorbate amplification effect

While in vivo electrochemistry has been shown to be useful for discovering new neurophysiological phenomena, there is still considerable controversy about the identity of the compounds being measured and the concentration of those compounds in extracellular fluid in brain. We have found that carbon paste electrodes undergo changes in sensitivity and specificity for dopamine and other compounds after being implanted in brain. We have also examined the effect of ascorbate on the selective enhancement of catecholamine peaks to provide an explanation for the apparently very high concentrations of dopamine measured in the extracellular fluid space. After temporary brain implantation (20 min), carbon paste electrodes tested in vitro showed increased sensitivity and lower oxidation potentials for dopamine, norepinephrine and serotonin. These brain-treated electrodes also detected 3,4-dihydroxyphenylacetic acid (DOPAC) as a distinct peak at +0.16 V, although the electrode sensitivity for DOPAC was some 25 times lower than that for dopamine. Brain treatment did not alter electrode sensitivity or oxidation potential for 5-HIAA. The oxidation current for ascorbic acid when processed as the semiderivative showed no distinct peak in the potential range -0.2 to +0.4V for either untreated or brain-treated electrodes. However ascorbic acid amplified the electrochemical peaks of catechols in direct proportion to the ratio of the concentration of ascorbate to the concentration of the catechol. In the physiologic concentration range of 300 microM ascorbate, the electrochemical signal for 1 microM dopamine was amplified 4250%. While ascorbate amplification improves detectability of dopamine and norepinephrine, it also introduces ambiguity since changing catechol concentrations cannot be distinguished from changing ascorbate concentrations.     

Genotype-treatment interaction between amantadine and chlorpromazine in the mouse.

1. Repeated administration of amantadine prior to chlorpromazine to two different strains of mice altered both locomotor activity, concentrations of brain biogenic amines and selected major metabolites as a function of mouse strain. 2. Amantadine antagonized chlorpromazine effect on motility which was associated with increases in whole brain levels of homovanillic acid in the CDF-1 but not C57BL/6 mice. 3. Conversely, the treatment with amantadine prior to chlorpromazine reduced whole brain normetanephrine and 5-hydroxyindoleacetic acid levels from respective controls in the C57BL/6 and CDF-1 mice, respectively. 4. The results suggest that genetic factors underly differential alteration of brain dopamine and serotonin which may underly the mechanism of amantadine efficacy in neuroleptic-induced extrapyramidal disorders and to the variable responses to amantadine therapy.     

The influence of N-acetylneuraminic acid on the properties of human orosomucoid.

Little is known of the relationships that may exist among the three principal functionalities of glycoproteins. Orosomucoids of closely defined N-acetylneuraminic acid content were examined for evidence of influence of N-acetylneuraminic acid content on the physical properties of the glycoprotein. Fluorescence spectroscopy gave no indication of conformational change in the protein core upon desialylation. Small changes in the chromatographic partition coefficient, sigma, and thermal stability, Td, are interpreted to reflect loss of water of hydration and increased glycan stem-protein interaction without a major repositioning of the chains. Ligand-binding measurements indicate no alteration in the hydrophobic binding domain and a possible interaction between chlorpromazine and N-acetylneuraminic acid. All changes seen are progressive and occur through a region where changes in biological activity are not found. It is suggested that the dependence of biological activity on N-acetylneuraminic acid content in orosomucoid reflects, not coupled changes in protein conformation, but a charge-density-related interaction such that, below a contribution of four or five N-acetylneuraminic acid residues, activity is modified.     

A new method for studying the interaction between chlorpromazine and phospholipid bilayer

The spectroscopic and transmission electron microscopy (TEM) studies of interaction between chlorpromazine (CPZ) and dimyristoyl phosphatidylglycerol (DMPG) bilayer by using gold nanoparticles (AuNPs) as probes are reported. The DMPG bilayer-protected AuNPs were prepared by a simple one-step method. The DMPG bilayer tethered on the AuNPs was considered as a biomembrane model. The addition of CPZ affected the surface plasmon resonance (SPR) and morphology of the prepared AuNPs, and this effect was monitored by UV-vis spectroscopy and TEM. The interaction between CPZ and DMPG bialyer was CPZ concentration-dependent, and the possible mechanism was discussed. This simple and facile method may be quite general and work for other surface active drug-biomembrane or protein-biomembrane interactions.     

Interaction of chlorpromazine with the human erythrocyte membrane

The interaction of the amphipath chlorpromazine (CPZ) with the human erythrocyte membrane was evaluated. The partition coefficient of CPZ between the membrane bilayer and the aqueous compartment, measured spectrophotometrically, ranged between 1 and 3 X 10(3). An independent estimate, 4.6 X 10(3), was obtained by a novel method which avoided the measurement of binding and determined instead the variation of the hemolytic potency of the amphipath with the ratio of buffer volume to membrane volume. The maximal uptake of CPZ exceeded 2 X 10(9) molecules/red cell, corresponding to a volume greater than that of the bilayer itself. Such heavily loaded membranes were increased in thickness more than 2-fold, suggesting the formation of a CPZ-rich zone at the center of the bilayer. Ghosts loaded with massive levels of CPZ condensed approximately 20-fold in surface area and increased proportionately in thickness, suggesting the formation of a novel CPZ-lipid solution. CPZ caused hemolysis by a colloid-osmotic mechanism. By measuring the simultaneous uptake of mannitol and sucrose, we determined that CPZ induced holes of constant size but variable number. If circular, the holes would have had a diameter of approximately 14 A. The time-averaged number of holes ranged from 0.09 per cell (signifying intermittency) to 16. Freeze-fracture electron microscopy of CPZ-treated red cells revealed multiple round patches of nearly particle-free bilayer up to 0.3 micron in diameter with crowding of the intramembrane particles into the surrounding membrane. We interpret these images to signify lateral phase separation within the CPZ-treated bilayer. Hemolysis could, therefore, result from the intermittent opening of weak seams at phase boundaries; these could then be fluctuating slits approximately 14 A in width and of variable length, rather than simple circular holes.     

Sequestration: causes and consequences

Adhesion of Plasmodium falciparum-infected erythrocytes to endothelial cells and to syncytiotrophoblasts lining the placenta is a key feature of malaria pathogenesis. P. falciparum erythrocyte membrane protein 1, a family of variable proteins, mediates adhesion to CD36 and intercellular adhesion molecule 1 in the systemic vasculature, and to chondroitin sulphate A and hyaluronic acid in the placenta. Recent studies of the pathology of fatal cerebral malaria and of placental malaria that follow such sequestration suggest that coagulation disturbances may have a greater role in pathogenesis than previously realized, and that monocyte infiltrates in response to malaria may initiate some of these changes. Chemokines such as macrophage inflammatory protein 1 alpha and beta and monocyte chemoattractant protein 1 may play a key role in attracting monocytes to the placenta and other organs, but the stimulus to chemokine secretion is not presently known.     

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.     

Electrophoretic and immunoelectrophoretic study of the enzymatic degradation of human orosomucoid (alpha 1 -acid glycoprotein)

Purified, intact orosomucoid (alpha 1-acid glycoprotein) derived from whole human plasma was incubated with a number of proteolytic and saccharolytic enzymes under a variety of conditions. The unfractionated digests were immediately examined by both agar gel- and immunoelectrophoresis for the presence of antigenically active (precipitating) and/or inactive macrofragments. Despite otherwise clear evidence of rapid degradation by several proteases, no antigenic subunits were detected. Among the glycosidases, almond emulsin produced a suggestion of modification of the carbohydrate moiety of a sialic acid-poor orosomucoid preparation obtained from Cohn Fr. VI, but had no effect on antigenic properties. These results are presented as further evidence for a lack of involvement of the extensive carbohydrate component of orosomucoid in its antigenic reactions, and support previous data implicating the polypeptide chain as solely responsible for its antigenicity.     

Sequestration of acetylated LDL and cholesterol crystals by human monocyte-derived macrophages

Monocyte-derived macrophages accumulate and process cholesterol in atherosclerotic lesions. Because of the importance of this process, we examined the interaction of cholesterol crystals and acetylated low density lipoprotein (AcLDL) with human monocyte-macrophages in a combined chemical and morphological study. These two forms of cholesterol induced extensive compartmentalization of the macrophage cytoplasm. Unexpectedly, the compartments maintained a physical connection to the extracellular space as demonstrated with ruthenium red staining. The compartments formed through invagination of the top surface of the macrophage plasma membrane. Some cholesterol crystals and AcLDL were sequestered within these surface-connected compartments for up to five days in the case of the crystals and for one day in the case of AcLDL. Pulse-chase studies of fractionated macrophages indicated that [3H]cholesterol redistributed from the surface-connected compartments into lysosomes (where the cholesterol remained unesterified) and into lipid droplets (where the cholesterol was stored as cholesteryl ester). Intracellular uptake and esterification of cholesterol was blocked by cytochalasin D. However, once cholesterol was sequestered in the surface-connected compartments, subsequent esterification of the cholesterol could not be inhibited by cytochalasin D. Apolipoprotein E was localized within the surface- connected compartments by immunogold labeling suggesting a possible function for this protein in the processing of lipid taken up through the sequestration pathway. Removal of microcrystalline cholesterol from the medium resulted in release of most of the accumulated cholesterol microcrystals from the macrophages, as well as disappearance of the surface-connected compartments. Thus, sequestration is a novel endocytic mechanism in which endocytic compartments remain connected to the extracellular space. This differs from phagocytosis where endocytic vacuoles rapidly pinch off from the plasma membrane. Sequestration provides a means for macrophages to remove substances from the extracellular space and later release them.     

Cleavage of human orosomucoid by a chromium(V) species: relevance in biotoxicity of chromium

A chromium(V) complex, CrO(salen)+, was generated in situ and its interaction with human orosomucoid (alpha1-acid glycoprotein) has been evaluated. The chromium(V) species has been found to oxidize the protein rapidly. A second order rate constant of 5 +/- 0.4 x 10(4) M(-1) s(-1) has been obtained for the redox process. Gel electrophoresis pattern of AGP in the presence of metal ion clearly reveals the decrease in the intensity of the AGP band with the subsequent formation of protein fragments of lower molecular weight. At higher metal ion concentration a continuous smear is observed which indicates the nonselective cleavage of the glycoprotein. Cleavage of AGP is through the direct pathway of oxidation by a highly reactive chromium(V) species.     

ESR studies on the effect of cholesterol on chlorpromazine interaction with saturated and unsaturated liposome membranes

In this study, the effects of chlorpromazine (CPZ) on lipid order and motion in saturated (DMPC, DMPG) and unsaturated (SOPC) liposome membranes were investigated by electron spin resonance (ESR) spin labeling technique. We have shown that above the main phase transition temperature of membrane lipids (T(M)), CPZ slightly increases lipid order in membranes without cholesterol, whereas below T(M) it has a strong opposite effect. Addition of 30 mol% of cholesterol into DMPC and SOPC membranes changes significantly the CPZ effects both above and below T(M). Additionally, above T(M), the ordering effect of CPZ on pure SOPC membrane is stronger at pH 7.4 than at pH 9.0, whereas below T(M), as well as in the presence of cholesterol, pH does not seem to play a role in CPZ effect on both membranes. Because of the strong influence of membrane composition on CPZ effect on membranes, the use of cholesterol as a marker of CPZ photosensitized reactions has been discussed.     

氯丙嗪生态毒理效应与人体健康影响研究与展望

氯丙嗪作为一种镇静类兽药被广泛使用,其主要在肝脏中代谢,并随尿液逐渐排出;但因排出过程缓慢,容易产生药物残留。随着人们的生活质量和对动物性食品安全要求的不断提高,氯丙嗪药物残留问题受到越来越多的关注。氯丙嗪随尿液排出部分可经多种途径进入环境和人体,对生态系统和人类健康存在着潜在的威胁。本文对氯丙嗪的药物残留原因进行了分析,概述了环境中氯丙嗪的生态毒理学效应以及对人体健康的潜在影响,说明了氯丙嗪的残留与环境和人类健康有着十分密切的联系,对深入研究具有重要的现实意义。