Brain norepinephrine and convulsions in the genetically epilepsy-prone rat: sex-dependent responses to Ro 4-1284 treatment

Seizure predisposition in the Genetically Epilepsy-Prone Rat (GEPR) is at least partially dependent on central nervous system noradrenergic deficits. We have previously shown that moderate seizure GEPRs (GEPR-3) experience an increase in seizure severity after receiving Ro 4-1284, a monoamine vesicle inactivating drug. We are now reporting the effect of this drug on severe seizure GEPRs (GEPR-9). Motives for this study were: (a) to determine the effects of further depletion of innately deficient monoaminergic stores on seizure latencies and (b) to investigate whether a previously documented seizure severity difference between the sexes is related to the defective monoaminergic system in these subjects. GEPR-9s with known seizure history were tested for latency to onset of running phase and convulsion 45 minutes after Ro 4-1284 or saline administration. Brain norepinephrine levels were also determined. Ro 4-1284 caused severe depletion of monoamines in all brain areas assayed in both sexes of GEPR-9s and also caused a reduction in the latencies for onset of running and convulsion. The drug-induced norepinephrine depletion across the brain areas surveyed was significantly greater in females than in their male littermates. These observations prompt us to postulate that noradrenergic neurons in female GEPR-9s are functionally different from those in males and that this difference is detected in the differential effectiveness of Ro 4-1284 between the two sexes. Also, the influence of gonadal hormones on seizure predisposition and on the neurochemical actions of Ro 4-1284 may be different in GEPR-9 males and females.     

Effect of Ro 4-1284 on audiogenic seizure susceptibility and intensity in epilepsy-prone rats

The effects of CNS monoamine depletion on audiogenic seizure (AGS) susceptibility and intensity were studied in two types of Sprague-Dawley derived rats: (1) the progeny of a nonsusceptible strain (controls); and (2) the nonsusceptible progeny of epilepsy-prone (audiogenic seizure susceptible) parents (NSPSP). Forty-five minutes after injection of the benzoquinolizine Ro 4-1284, a significant fraction of the NSPSP developed AGS susceptibility, whereas the incidence in controls was not significant. AGS intensity was also significantly elevated 45 minutes and 19 days following Ro 4-1284 in NSPSP. In controls, there was a smaller, but significant, elevation of seizure intensity only at the earlier time period. Both types of rats exhibited a marked depletion of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in all of the six different areas of the CNS which were examined. In the NSPSP, a significant incidence of seizure susceptibility was retained as long as 19 days after Ro 4-1284 administration, despite the complete repletion of monoamine stores. These observations suggest that CNS monoaminergic neurons function as determinants of AGS susceptibility and intensity in animals which also carry some other genetically determined susceptibility factor(s). A deficiency in monoaminergic transmission is insufficient to cause susceptibility in animals not carrying the other trait(s). Also, although a monoaminergic deficit may initially cause the appearance of susceptibility, the presence of the deficit may not be necessary for the continuation of susceptibility once an animal has actually sustained an AGS in the presence of the monoaminergic deficit.     

外周苯二氮(艹卓)受体激动剂Ro5-4864抑制大鼠心肌细胞线粒体通透性转换

线粒体通透性转换（mitochondrial permeability transition,MPT）导致线粒体氧化应激性损伤。近年研究认为,位于线粒体外膜的外周苯二氮节受体（peripheral benzodiazepine receptor,PBR）参与了线粒体的重要生理功能。本研究在心肌细胞线粒体水平探讨激动PBR能否抑制Ca^2＋诱发的MPT。分离Sprague—Dawley大鼠心肌细胞线粒体,将PBR激动剂Ro5-4864（50、100、200μmol／L）和线粒体孵育,利用150μmol／L Ca^2＋诱发MPT,部分线粒体在与100μmol／L Ro5-4864孵育前5min加入MPT孔道开放剂苍术苷（atractyloside,ATR）。采用分光光度法观察线粒体膨胀情况：Westernblot检测线粒体细胞色素C（cytochrome C,CytoC）释放;利用荧光探针JC-1在激光共聚集显微镜下观察线粒体膜电位的变化。50、100、200μmol／L Ro5-4864均显著抑制Ca^2＋诱发的520nm处线粒体吸光度的下降,而且抑制Ca^2＋引起的线粒体CytoC释放和线粒体膜电位下降,但ATR可阻断R05—4864的上述作用。结果提示,PBR激动剂可抑制大鼠心肌MPT,保持线粒体CytoC含量和稳定线粒体膜电位,减轻线粒体损伤。PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。     

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.     

Lipid peroxidation of rat spleen and thymus lymphocytes treated with x-rays (0-5-1.0 Gy)

It is established, that low doses of X-ray irradiation have affected activation of lipid peroxidation (LPO) in immunocompetent cells of the spleen and thymus. The amount of malonic dialdehyde (MDA) in lymphocytes of spleen and thymocytes increases 2 times twenty-four hours after animals' irradiation by X-rays in a dose of 0.5 Gy; when a dose grows to 1.0 Gy, the MDA content in the spleen lymphocytes increases from the 1st to the 6th days and in thymocytes from the 1st to the 3d days reaching its maximum at the 3d day. MDA accumulation in the immunocompetent cells of irradiated animals varies depending on the method of lipid peroxidation initiation.     

The structure of Ro 09-0198 in different environments.

The constitution and configuration of Ro 09-0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09-0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.     

The effect of the electron affinic sensitizers Ro 07-0582 and Ro 03-6156 on the survival of several E. coli mutants.

Concentration versus radiosensitizing effect curves have been determined for four E. coli strains with two nitroaromatic sensitizers. The data are consistent with a simple competition kinetic model of sensitization, and K values for Ro 07-0582 are reported. An empirical relationship, DMF=alpha o.e.r., relating the dose-modifying factor produced by a given concentration of sensitizer to the oxygen-enhancement ratio of a bacterium was found to hold for all the mutants tested for values of alpha greater than 1/o.e.r. The limiting value of alpha at high concentrations of sensitizer was 0.75. Some implications of this relationship and the limiting value of alpha are discussed.     

Effect of Ro 5-4864 and PK11195 on protein phosphorylation in mitochondria isolated from primary cultures of rat astrocytes

Astrocytes are the most numerous cell type within the central nervous system. Earlier, high-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of astrocytes. TSPO/PBR was shown to be localized in mitochondria. Recently, we showed that TSPO/PBR ligands, Ro 5-4864 and PK11195, were able to modulate the function of non-specific pore (PTP) in brain and liver mitochondria as well as protein phosphorylation in the presence of threshold calcium concentrations. In the present study for the first time the function of astrocyte mitochondria were studied under condition of PTP opening. Parameters of PTP induction were measured by means of simultaneous registrations of the membrane potential, calcium accumulation and calcium release as well as detection of the oxygen consumption with selective electrodes. Four phosphorylated proteins in range of 67 kDa, 46 kDa, 48 kDa and 3.5 kDa have been found under these conditions. It was established that in astrocyte mitochondria TSPO/PBR exists in monomer form (18 kDa). The phosphorylation level of these proteins was found to be modulated by TSPO/PBR ligands, Ro 5-4864 and PK11195, in a range of concentrations from 0.01 to 1 μM, in the same way as it was earlier described for brain mitochondria [Azarashvili et al., J Neurochem., 2005].     

Interaction of a cyclic peptide, Ro09-0198, with phosphatidylethanolamine in liposomal membranes

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum. This peptide caused permeability increase and aggregation of liposomes containing phosphatidylethanolamine. Liposomes containing phosphatidylserine, phosphatidylinositol or cardiolipin instead of phosphatidylethanolamine were, however, not appreciably reactive with the peptide. Among the structural analogs of phosphatidylethanolamine, dialkylphosphatidylethanolamine and 1-acylglycerophosphoethanolamine incorporated into liposomes could interact with Ro09-0198 to cause a permeability increase, whereas liposomes consisting of alkylphosphoethanolamine or phosphatidyl-N-monomethylethanolamine were insensitive to the peptide. These findings indicate that a glycerol backbone and a primary amino group of phosphatidylethanolamine are necessary for interaction with Ro09-0198 to cause membrane damage. Ro09-0198 induced a selective permeability change on liposomes. Glucose and umbelliferyl phosphate were effluxed significantly, but sucrose was only slightly permeable and inulin could not be released. Consequently, the permeability increase induced by Ro09-0198 is rather specific to molecules smaller than sucrose. Line broadening of electron spin resonance signals of spin-labeled phosphatidylethanolamine was observed upon treatment of liposomes with Ro09-0198. It was suggested from these results that Ro09-0198 can alter the physical organization of phosphatidylethanolamine in membranes, thus providing a basis for changes in membrane permeability.     

The ATP4- receptor of rat mast cells.

The concentration-dependence on exogenous ATP of activation and inhibition of mast-cell histamine secretion, phosphatidylinositol labelling and leakage of metabolites shows that all these functions are regulated by the free acid ATP4-. Maximal histamine secretion and phosphatidylinositol labelling occur with ATP4- at approx. 2 microM, but higher concentrations, which cause inhibition of secretion and phosphatidylinositol labelling, are required to maximize leakage of 32P-labelled metabolites. Both enhancement and inhibition of phosphatidylinositol labelling (due to low and high concentrations of ATP4- respectively) are rapid in onset; histamine secretion is characterized by a delay, especially at low concentrations of ATP4- (approx. 1 microM). Phosphatidylinositol labelling and histamine secretion are dependent on extracellular Ca2+. Metabolite leakage due to the presence of exogenous ATP4- is slow and does not require Ca2+. Of 18 analogues of ATP that were tested, only four were agonists for secretion, and only these four permitted leakage of 32P-labelled metabolites. It is argued that activation and inhibition of histamine secretion, phosphatidylinositol labelling and metabolite leakage are all initiated by ATP4- acting at the same receptor. For mast cells stimulated with ATP4- enhancement of phosphatidylinositol metabolism is not sufficient by itself to cause Ca2+-dependent secretion.     

In vitro interactions of the aromatic retinoids Ro 10-9359 (etretinate) and Ro 10-1670 (acitretin), its main metabolite, with human serum lipoproteins and albumin

Serum lipoproteins are good carriers for the aromatic retinoid Ro 10-9359 (etretinate) and to a lesser extent for its main metabolite in human Ro 10-1670 (acitretin). Up to about 200 and 130 etretinate molecules and 200 and 70 acitretin molecules can bind to one LDL and one HDL, respectively. In contrast human serum albumin only binds about 10 etretinate or 30 acitretin molecules. In whole human serum loaded with the retinoids, lipoproteins carry approx. 67% of total etretinate or approx. 37% of total acitretin. In the particular case of etretinate, low density lipoproteins account for about 30% of the lipoprotein-carried etretinate.     

Gene expression profile of isolated rat adipocytes treated with anthocyanins

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and adipocyte gene expression in adipocytes. In this study, we have shown for the first time the gene expression profile in isolated rat adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The rat adipocytes were treated with 100 muM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. A total of 633 or 427 genes was up-regulated (>1.5-fold) by the treatment of adipocytes with C3G or Cy, respectively. The up-regulated genes include lipid metabolism and signal transduction-related genes, however, the altered genes were partly different between the C3G- and Cy-treated groups. Based on the gene expression profile, we demonstrated the up-regulation of hormone sensitive lipase and enhancement of the lipolytic activity by the treatment of adipocytes with C3G or Cy. These data have provided an overview of the gene expression profiles in adipocytes treated with anthocyanins and identified new responsive genes with potentially important functions in adipocytes related with obesity and diabetes that merit further investigation.