Mechanism of Action of Acamprosate. Part I. Characterization of Spermidine-Sensitive Acamprosate Binding Site in Rat Brain

It has been suggested that the anticraving drug, acamprosate, acts via the glutamatergic system, but the exact mechanism of action is still unknown. The aim of this study was to characterize [³H]acam-prosate binding and establish whether this showed any relation to sites on the NMDA receptor complex. We found saturable specific binding of [³H]acamprosate to rat brain membranes with a KD of 120 μM and a B_max of 450 pmol/mg of protein. This acamprosate binding site was sensitive to inhibition by spermidine (IC₅₀: 13.32 ± 1.1 μM; Hill coefficient = 1.04), and arcaine and glutamate both potentiated the inhibitory effect of spermidine. Acamprosate binding to the acamprosate binding site was also sensitive to inhibition by divalent cations (Ca2⁺, Mg2⁺, and Sr²⁺). Conversely, acamprosate displaced [¹⁴C]spermidine binding from rat brain membranes with an IC₅₀, of 645 μM and a Hill coefficient = 1.74. This inhibitory effect of acamprosate was not affected by arcaine, and was associated with a significant reduction in B_max and binding affinity for spermidine, suggesting an allosteric interaction between acamprosate and a spermidine binding site. These data are consistent with an effect of acamprosate on the NMDA receptor protein complex, and acamprosate was also found to alter binding of [³H]dizocilpine to rat brain membranes. When no agonists were present in vitro (minimal NMDA receptor activation), acamprosate markedly potentiated [³H]dizocilpine binding at concentrations in the 5 to 200 μ range. However, under conditions of maximal receptor activation (100 μM glutamate, 30 μM glycine), acamprosate only inhibited [³H]dizocilpine binding (at concentrations concentrations > 100 μM). When these binding studies were performed in the presence of 1 μM spermidine, the enhancing effects of acamprosate on [³H]dirocilpine binding were inhibited. The results show that acamprosate binds to a specific spermidine-sensitive site that modulates the NMDA receptor in a complex way. Together, with data from al Quatari et al. (see next paper), this work suggests that acamprosate acts as “partial co-agonist“ at the NMDA receptor, so that low concentrations enhance activation when receptor activity is low, whereas higher concentrations are inhibitory to high levels of receptor activation. This may be relevant to the clinical effects of acamprosate in alcohol-dependent patients during abstinence.     

Evaluation of contraction time and recovery period as a parameter in the calcium antagonistic action on the K(+)-depolarized rat duodenum

Verapamil, nifedipine, phentolamine, tolazoline, gentamicin and neomycin inhibited calcium-induced contractions of K(+)-depolarized duodenum of rat by shifting the concentration-response curves to the right. Non-competitive inhibitions were observed with trifluoperazine, lidoflazine, procaine and tetracaine. Lanthanum behaved as a partial agonist in this preparation, while nitroprusside was ineffective. Contraction times in the presence of the antagonists and recovery time of the Ca2+ responses after the removal of the antagonists from the bathing medium were evaluated. From the findings, it is suggested that the contraction time and the time required for tissue recovery after removal of a Ca2+ antagonist are parameters making K(+)-depolarized rat duodenum a potential tool for the evaluation of the pharmacological effects of Ca2+ antagonists.     

The effects of purified recombinant murine interleukin-3 and/or purified natural murine CSF-1 in vivo on the proliferation of murine high- and low-proliferative potential colony-forming cells: demonstration of in vivo synergism

Purified natural murine L cell (macrophage) colony-stimulating factor (nCSF-1) and purified recombinant murine interleukin-3 (rIL-3) were administered to normal or lactoferrin-pretreated mice 20 to 24 hours before sacrifice. rIL-3 and nCSF-1 administered separately increased the percentage of macrophage high-proliferative potential colony- forming cells (HPP-CFC) and low-proliferative potential colony-forming cells (LPP-CFC) in active cell cycle. Endotoxin was not detected in the samples of nCSF-1 or rIL-3 with the Limulus lysate test, and the in vitro and in vivo hematopoietic stimulatory effects of both molecules were abolished or markedly reduced by 30 minutes' treatment at 100 degrees C, which demonstrates that the effects noted in vivo were not due to endotoxin. Combinations of low concentrations of rIL-3 and nCSF- 1, which by themselves were inactive, increased the percentage of HPP- CFC and LPP-CFC in active cell cycle in a synergistic fashion. No significant change in the number of HPP-CFC or LPP-CFC per femur or femoral nucleated cellularity was observed. Thus, rIL-3 and nCSF-1 can synergize to effect the proliferation of the same cell populations in vivo.     

An immunohistochemical study of mononuclear cells in meningiomas

A consecutive series of 34 meningiomas were re-examined as to subtype and presence of nuclear atypia, mitotic figures, areas of high cellularity and necrosis. Meningioma cells were epithelial membrane antigen (EMA) positive in 31 out of the 34 tumours. The presence of mononuclear cells and macrophages was assessed by immunohistochemistry using the monoclonal antibodies L26 (CD20, B cell marker), DF-T1 (CD43, T cell marker), KP1 (CD68, macrophage marker) and MAC387 (monocytes). L26 positive B cells were observed infrequently. CD43 positive mononuclear cells were infiltrating the parenchyma as individual cells and as groups of cells in 29 (87% of the tumours). CD68 positive macrophages were seen in 19 (59% of the tumours), as scattered single cells or groups of cells. There was a statistically significant association between the number of CD68 positive cells (necrotic areas excluded) and microscopic features of aggressiveness, i.e. high cellularity as well as the combination of nuclear atypia and frequent mitotic figures. MAC387 stained only a few cells; the immunopositive cells were present mainly within and around vessels. Meningioma cells displayed a diffuse immunopositivity for L26 (CD20) in 29 out of 34 meningiomas, but did not stain with macrophage markers. Mast cells were found in 9 out of 32 tumours; when present they were significantly more prevalent in the syncytial subtype. Thus, mononuclear cell infiltrates in meningiomas are mainly composed of T cells and macrophages, indicating an immune system surveillance and response to the tumour cells. The functional and prognostic significance of the presence of CD68 positive cells, macrophages, deserve further study in the search for more reliable histological criteria to predict recurrence and biological aggressiveness in meningiomas.     

Gross composition and plasma and extracellular water volumes of tissues of a reference mouse.

The laboratory mouse is a primary animal model for experimental radiation biology and pharmacology. The usefulness of the mouse for those purposes is enhanced if detailed data are available to define a Reference Mouse [weight and composition of soft tissues and bones and their in-life content of plasma and extracellular water (ECW)]. Only fragmentary data are available for wet weights and plasma volumes of soft tissues and bones of mice; there are no reports of total volume or distribution of ECW in mouse tissues. To remedy those defects, wet weight and composition of all major organs and soft tissues were measured, and measurements were made or estimates obtained for wet weights and composition of all bones of the young adult (12 to 13 wk old) female Swiss-Webster mouse. 125I-transferrin was used as a tracer for plasma, and 22Na was used as a tracer for ECW. Tissue weight and tracer measurements were conducted using the metabolic balance approach and a freezing technique that avoids blood loss during dissection. Results compare favorably with published weights and plasma volumes of tissues of mature mice of both genders and other strains. Total plasma volume (48.9 +/- 4.4 microL g-1) and Na-space (232 +/- 15 microL g-1), and the specific plasma and ECW volumes of vascular mouse tissues, exceed those of rat tissues. Applications of the data are presented: (1) interpretation of plutonium uptake kinetics in the mouse; (2) estimation of masses of mineralized bone tissue (1.92 g), bone marrow (1.2 g), and endosteal (BS) cells (0.2 g) of the mouse.     

Characterization and chromosomal assignment of a human cell surface antigen defined by the monoclonal antibody AUAI

We describe the chromosomal assignment and biochemical characterization of the genetic locus controlled by a human cell surface antigen which is defined by the monoclonal antibody (MAb) AUAI. This gene product is only expressed on epithelial cells. Therefore, human-mouse somatic cell hybrids of epithelial origin were used to assign this gene to chromosome 2. Cell surface iodination of the hybrids and parental cells followed by immunoprecipitation and polyacrylamide gel electrophoresis showed that AUAI detected a single 35-kDa protein. The MAb AUAI reacted on tissue sections with a subset of normal epithelial cells, but in tumours it showed a much wider distribution, though still only on epithelium-derived tumours.