A comparison of the effects of chlorpromazine, 7-hydroxychlorpromazine and chlorpromazine sulfoxide on the activity of central dopaminergic neurons

Using single unit recording techniques, chlorpromazine and two of its naturally occuring metabolites in man, 7-hydroxychlorpromazine and chlorpromazine sulfoxide, were tested for their ability to reverse amphetamine-induced depression of rat dopaminergic ventral tegmental neurons (A10). Small equivalent doses of chlorpromazine and 7-hydroxychlorpromazine were found to readily reverse amphetamine-induced depression of these cells. Chlorpromazine sulfoxide was found to be 50–100 times less potent in this regard. Previous findings have demonstrated that only phenothiazines with antipsychotic properties reverse amphetamine-induced depression of A10 neurons. Thus, we would predict that 7-hydroxychlorpromazine would have anti-psychotic properties whereas chlorpromazine sulfoxide would not. A preliminary study by Sakalis et al., suggests that plasma levels of chlorpromazine and 7-hydroxychlorpromazine are possibly correlated with the therapeutic effects of chlorpromazine in schizophrenia. Chlorpromazine sulfoxide levels, on the other hand, are reported to be high in chlorpromazine treated non-responders. Thus there is a direct parallel between predictions of antipsychotic efficacy based on our test model and the possible clinical importance of these chlorpromazine metabolites. Both findings suggest that 7-hydroxychlopromazine might be a good antipsychotic agent.     

Direct Activation of RhoA by Reactive Oxygen Species Requires a Redox-Sensitive Motif

Background

Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA.

Methodology/Principal Findings

In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs.

Conclusions/Significance

Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.     

GROWTH AND DIFFERENTIATION OF AXIAL AND LATERAL FILAMENTS IN BATRACHOSPERMUM SIRODOTII (RHODOPHYTA)1

Development of the vegetative gametophyte of Batrachospermum sirodotii Skuja was examined with light and both transmission and scanning electron microscopy. Patterns of wall growth were followed using the Calcofluor White ST pulse-chase method. Thallus structure was analysed in terms of the pattern of development of the apical, periaxial and pleuridial initials that generate the axial and whorled lateral filaments characteristic of Batrachospermum. Apical cells of axial filaments elongate initially by tip growth with the nucleus maintaining a distal position. Nuclear division is horizontal. One daughter nucleus migrates basipetally and a thin, convoluted annular septum and perforate-occluded pit connection are then formed. Elongating axial cells subsequently extend by wall deposition at the base of the cell. Periaxial cells are initiated laterally and elongate primarily by tip growth while the nucleus remains within the axial cell. The nucleus then migrates to the boundary between the initial and the axial cell, divides, and one daughter nucleus moves into the initial and the other back into the axial cell. A slightly irregular annular septum and simple-occluded pit connection are then formed. Pleuridial cell initials begin as terminal to subterminal protuberances on periaxial or pleuridial cells. They first extend by tip growth and later by bipolar band growth. The nucleus remains within the parent cell as the pleuridial initial expands and a narrow septal ring is formed between the two cells. It then migrates through the septal ring into the initial and divides transversely. One nucleus passes back into the parent cell and a thick, flat septum and perforate-occluded pit connection are formed. It is concluded that the potentially indeterminate axial filaments and the determinate lateral pleuridia represent distinct developmental types in Batrachospermum.     

Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.     

CHOLINE: SELECTIVE ACCUMULATION BY CENTRAL CHOLINERGIC NEURONS

Abstract— Most of the cholinergic input to the hippocampus was destroyed by placement of lesions in the medial septal area. In animals with such lesions we found that hippocampal ChAc activity was reduced by 85–90% and endogenous acetylcholine levels were reduced by more than 80 %. When hippocampal synaptosomes from animals with lesions were incubated with [³H]choline at concentrations of 7.5 nm, 1 μm and 10 μm there was approximately a 60 % reduction in the uptake of [³H]choline, suggesting that cholinergic nerve endings were mainly responsible for [³H]choline uptake. At 0.1 mm concentrations of [³H]choline, there was only a 25 % reduction of choline uptake, suggesting that at higher concentrations of choline there was more nonspecific uptake. The uptake of radiolabelled tryptophan, glutamate and GABA were only slightly or not at all affected by the lesions. There was a significant reduction of uptake of radiolabelled serotonin and norepinephrine, since known monoaminergic tracts were disrupted. Choline uptake was reduced only in brain regions in which cholinergic input was interrupted (i.e. the cerebral cortex and hippocampus) and remained unchanged in other regions (i.e. the cerebellum and striatum). The time course of the reduction in choline uptake was similar to that of the reductions in ChAc activity and endogenous ACh levels; there was no decrease at 1 day, a significant decrease at 2 days, and the maximal decrease at 4 days postlesion. There was a close correlation among choline uptake, ChAc activity and ACh levels in the four brain regions examined (i.e. the striatum, cerebral cortex, hippocampus and cerebellum). Our results suggest that when hippocampal synaptosomes (and perhaps synaptosomes from other brain areas as well) are incubated in the presence of choline, at concentrations of 10 μm m or lower, then cholinergic nerve endings are responsible for the bulk of the choline accumulated by the tissue.