Effects of 2-guanidinoethane sulfonate on glutamate uptake in primary astroglial cultures from the rat cerebral cortex

The competitive taurine uptake inhibitor 2-guanidinoethane sulfonate (GES) is frequently used to deplete cerebral pools of taurine. Previous work has revealed that extracellular glutamate increases during GES administration. In view of this, accumulation of glutamate in the absence or presence of 10 or 100 μM GES was measured in primary astroglial cultures from the rat cerebral cortex. At 100 μM, GES reduced K_m as well as V_max for glutamate uptake. A similar tendency was seen at 10 μM GES, but this was not statistically significant. The data suggest that GES is an uncompetitive inhibitor of reuptake of glutamate in astrocytes, which may underlie the previously noted GES-induced elevation of extracellular glutamate in vivo.     

Effects of neuroleptic agents on cyclic GMP in rat cerebral cortex.

Cyclic guanosine 3' ,5'-monophosphate(cyclic GMP) levels were increased in incubated tissue slices from rat cerebral cortex in response to added cholinomimetic agents (carbachol and choline chloride) and neuroleptic compounds (chlorpromazine, 8-hydroxychlorpromazine, 7-hydroxychlorpromazine methiodide, haloperidol, thioridazine, chlorpromazine sulfoxide and promethazine). Calcium ion was required for this effect. Moreover, selected agents namely, 7,8-dihydroxychlorpromazine, 7,8-dimethoxychlorpromazine, chlorpromazine and clozapine prevented the rise in cyclic GMP induced by carbachol.     

Vinpocetin protects against excitotoxic cell death in primary cultures of rat cerebral cortex

The protective effect of vinpocetin, a drug clinically useful in brain hypoxia/ischemia, was examined in vitro on cerebrocortical cultures treated with glutamate and related excitotoxins. The extent of cell death was quantified by measuring lactic dehydrogenase activity released from damaged cells into the culture medium. Vinpocetin partially protected the cortical cells against cell death induced by N-methyl-D-aspartate, quisqualate and kainate, indicating that the drug exerts a direct protective action on cerebrocortical cells bearing excitatory amino acid receptors.     

Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.     

Xenobiotic-metabolizing enzyme activities in primary cultures of rat type II pneumocytes and alveolar macrophages.

Because of the evidence for the involvement of xenobiotic bioactivation in pulmonary toxicity and carcinogenesis, it is important to improve our understanding of the xenobiotic-metabolizing enzymes in isolated and cultured specific pulmonary cell populations. Some phase I and phase II xenobiotic-metabolizing enzyme activities, reduced glutathione (GSH), and gamma-glutamyl transferase (gamma-GT) were studied in rat type II pneumocytes and alveolar macrophages cultured for up to 48 h and 3 h, respectively. In type II pneumocytes, 7-ethoxyresorufin activity was not detected. 7-Benzyloxyresorufin (BROD) and 7-pentoxyresorufin (PROD) O-dealkylation decreased at 24 h by 84 and 82%, respectively, and continued to decline over the next 24 h with no measurable PROD at 48 h. The activity of NADPH- and NADH-cytochrome c reductase at 48 h decreased by 31 and 67%, respectively. GST activity decreased by 25 and 42% at 24 and 48 h, respectively. A transient increase in DT-diaphorase activity was observed at 24 h (by 55%). GSH content and gamma-GT activity increased significantly with time in culture. In freshly isolated alveolar macrophages, BROD activity was the only cytochrome P450-dependent alkoxyresorufin-O-dealkylase activity measured. BROD activity decreased by 38% in 3-h-attached macrophages. There were no changes in NADPH- and NADH-cytochrome c reductase, GST, and DT-diaphorase. An increase of GSH (by 24%) was observed in attached macrophages. In conclusion, type II pneumocytes and to a lesser extent alveolar macrophages in primary cultures undergo changes in biotransformation-related enzyme activities and intracellular GSH level that may affect xenobiotic toxicity at different times in culture.     

Effects of flunarizine on rat cerebral cortex

Flunarizine was tested using a single dose (40 mg/kg IP) and multiple doses (30 mg/kg PO presurgery) in 200–250 g male Wistar rats, using physiological measures related to the rapid ionic shift seen in cerebral cortex following a sudden insult. The threshold for initiation of a spreading depression was increased by flunarizine treatment only in rats in which the cortex had previously been injured. The time to rapid ionic shift after cardiac arrest (global ischemia) was increased by both flunarizine and phenytoin. Other measures tested showed no difference in normal cortex under flunarizine treatment. Flunarizine appears to have little or no effect on normal cortical tissure, but rather seems to exert a therapeutic effect on injured cortical tissue. Flunarizine may be a useful agent in the management of cerebral trauma.     

醋酸铅对大鼠脑皮质区细胞超微结构钙分布和酶活性的影响

用电镜钙细胞化学定位法和酶组织化学亚微数字图像分析系统，观察了断奶ｄ１起，分别给饮用１０和３０ｍｇ·Ｌ－１Ｐｂ２＋水３个月的幼鼠脑组织．发现血铅浓度为对照组１．６５倍时，细胞内钙分布和超微结构无明显变化．血铅浓度为对照组２．４５倍时，脑铅含量增加，Ｃａ２＋－ＡＴＰ酶活性降低，磷脂酶活性增加，钙细胞化学观察见各种细胞的钙分布和超微结构均已发生了变化．结果显示：３０ｍｇ·Ｌ－１的铅染毒量即能引起幼鼠脑皮质区细胞钙代谢的恶性循环，如不给予阻断可能导致细胞进一步损伤．     

Accumulation of pyrethroid compounds in primary cultures from rat cortex

Recent studies have demonstrated that lipophilic compounds (e.g., methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neurons have been widely utilized to study the actions of pyrethroids, yet pyrethroid accumulation in these cells has not been studied to date. To test the hypothesis that pyrethroids rapidly accumulate in neurons in vitro, the time (0–90 min) and concentration (0.05–10 μM) dependent accumulation of [³H]-deltamethrin (DM), [³H]-bifenthrin (BF) and [¹⁴C]-cis permethrin (PM) into primary cortical cultures was examined. Accumulation of all three pyrethroids was time- and concentration-dependent, with only small differences observed between the compounds. Concentration-dependent accumulation of PM and BF were similar, achieving a of total ∼0.25 nmol in cells after 30 min in a 10 μM solution. DM accumulation was lower, reaching a maximum of 0.14 nmol after 30 min in a 10 μM solution. In 1 μM solutions, DM and PM content in cells were 0.039 and 0.038 nmol after 90 min. At all concentrations and times, pyrethroid accumulation in cells was less than 5% of the total mass applied for DM and PM, and was less than 8% for BF. However, after 90 min, accumulation of all three compounds increased to as much as ∼30–50-fold higher than the surrounding medium. The amount of compound recovered in the media at the end of incubation ranged from ∼77% to 89%; the remainder (6–15%) was presumed to bind to the plastic of the culture plates. These results demonstrate rapid time- and concentration-dependent accumulation of pyrethroids in neurons in vitro. Further, for the three pyrethroids examined, there were not statistically significant differences in their accumulation. This data will be useful for making comparisons between in vivo and in vitro studies regarding effective concentrations of pyrethroids.     

Effects of the novel cyclosporine derivative PSC833 on glucose metabolism in rat primary cultures of neuronal and glial cells

Cyclosporine A (CsA) and the cyclosporine degrees congener PSC833 are known to cause transient CNS symptoms at high dosages in animal and man. Since impaired glucose metabolism plays a fundamental role in many heriditary and drug-induced neurological disorders, it was the purpose of the present study to evaluate whether this mechanism of pathogenesis might apply to PSC833 and CsA, using neural cells from rats. PSC833 and CsA were investigated in primary cultures of rat neuronal and glial cells at the concentration of 0.1, 1, 10, and 20 microM for 24 and 48 hr. Lactate dehydrogenase was determined as a marker of cytotoxicity. Cell proliferation was determined in astrocytes. Cellular glucose metabolism was investigated by 13C-NMR using [1-13C]glucose as a substrate. Glucose and lactate concentrations in the cell culture supernatants were determined spectrophotometrically. PSC833 at 10 microM was not cytotoxic in neuronal or glial cells nor did it inhibit proliferation in astrocytes 24 hr after incubation. Under the same conditions, the determination of [1-13C]glucose and [3-13C]lactate revealed significantly increased glucose consumption and lactate production in both cell types, as well as decreased levels of Krebs cycle intermediates. In the cell culture medium of both cell types after treatment with 10 microM PSC833, the rates of glucose consumption and lactate formation increased in comparison to controls, between 60-83% and 54-78%, respectively. PSC833 (10 microM) and CsA (20 microM) resulted in nearly similar increased glucose consumption and lactate production. The major PSC833 metabolite in rats, M9, which was devoid of CNS effects, did not cause significant changes in glucose metabolism. The present data suggest that PSC833-impaired tricarboxylic acid cycle activity, resulting in decreased Krebs cycle metabolites, can cause energy depletion and acidosis, which might contribute to the transient neurological symptoms of PSC833 and CsA.     

Hepatoprotective and hepatotoxic activities of sophoradiol analogs on rat primary liver cell cultures

As a part of our studies of hepatoprotective drugs, we prepared kaikasaponin I (2), sophoradiol monoglucuronide (SoMG, 3) and sophoradiol (4) from kaikasaponin III (1). We examined the hepatoprotective effects of these analogs, using immunologically-induced liver injury in primary cultured rat hepatocytes and found that compound 1 was more effective than soyasaponin I (1a) while 2 was more effective than 1. On the other hand, 3 was less effective than 2 at 30-200 microm. Further, compound 3 was strongly cytotoxic at 500 microM while 4 exhibited hepatoprotective activity at the same dose, although less potent. When the cytotoxicity toward hepatocytes of these analogs was tested, only 3 was cytotoxic at doses of 200 and 500 microM. This is the first example of an oleanene glucuronide (OG) which is cytotoxic toward hepatocytes. Compound 3 exhibited hepatoprotective activity at 200 microM, while it was also cytotoxic at the same dose without antiserum. Therefore, the hepatoprotective activity of OG represents a balance between a hepatoprotective action and its cytotoxicity toward hepatocytes.     

Presence of hexobarbital in primary cultures of rat hepatocytes maintains cytochrome P-450 levels and drug metabolizing enzyme activities

Addition of hexobarbital (1 mM) to the culture medium of rat hepatocytes protected against the rapid decline in the level of cytochrome P-450 and the activities of various drug metabolizing enzymes. While the hepatocytes cultured for 72 hr without hexobarbital had only 30% of their original level of cytochrome P-450, the cells maintained with hexobarbital had 75% of the initial level of the hemoprotein. After 72 hr in culture, the activities of aminopyrine N-demethylase and biphenyl 4-hydroxylase were 22-24% of the original rate for the nontreated cells and 73-78% for the hexobarbital treated cells. The activities of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase in the cultures of treated cells were even higher than those of the freshly isolated hepatocytes. Additions of other substrates of hepatic mixed function oxidase to the culture medium did not protect against the loss of cytochrome P-450 and enzyme activities.     

Effects of levamisole on primary cultures of adult rat hepatocytes

Levamisole represents one of several new compounds that exhibit immunomodulating activity. Pharmacological data have documented a relationship between liver drug metabolism of levamisole and its subsequent immunomodulating activity. To directly investigate this relationship in a controlled manner, primary cultures of adult rat hepatocytes were treated with levamisole, and ultrastructural and biochemical effects were analyzed. Ultrastructurally, levamisole did not disrupt the cellular architecture of the hepatocytes. Biochemically, levamisole stimulated alkaline phosphatase activity and elevated microsomal cytochrome P-450 content after a 48-hr incubation. High pressure liquid chromatographic analysis of levamisole metabolites produced by cultured hepatocytes suggested the formation of a hepatocyte-specific metabolite(s) that may be associated with its immunological mode of action.     

Induction of monooxygenase and UDP-glucuronosyltransferase activities in primary cultures of rat hepatocytes

The inducibility of two monooxygenase and UDP-glucuronosyltransferase (GT) forms was studied in primary cultures of adult rat hepatocytes. The following enzyme activities were determined: cytochrome P-448-dependent ethoxyresorufin O-deethylase (ERDE) and cytochrome P-450-dependent aldrin epoxidase (AE), and, furthermore, the GT form(s) metabolizing 3-hydroxybenzo(a)pyrene (GT1) and the GT form(s) metabolizing 4-hydroxybiphenyl (GT2). The results were as follows. The activity of AE and GT2 decreased markedly during the first days of culture, whereas ERDE and GT1 remained stable or even increased slightly. The maintenance of ERDE activity was dependent on the presence of dexamethasone. Pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), and benz(a)anthracene (BA) induced the activity of ERDE in hepatocytes cultured in HM 84 medium by a factor of 4, 8, and 12, respectively. Similar factors of induction were obtained at the fifth day of culture using a modified Leibovitz L-15 medium. However, the time course of induction differed greatly in the two media. BA and PB had an additive effect on ERDE activity, suggesting different mechanisms of action for the two inducers. Monoclonal antibodies directed against cytochrome P-448 inhibited ERDE activities induced by BA and PB to a similar extent. Neither PB nor PCN significantly increased AE activity. However, these compounds induced GT2. BA did not affect GT2 but induced GT1. The present results show that the culture of adult rat hepatocytes changes the relative distribution of monooxygenase and GT forms. The response to inducers resembles only partially that observed in vivo.     

Effects of morphine and pentobarbitone on acetylcholine synthesis by rat cerebral cortex

1. The synthesis of carbon-14-labelled acetylcholine ((14)C-ACh) from carbon-14 uniformly labelled glucose (U-(14)C-D-glucose) under different conditions was studied.2. The ability of cerebral cortex slices and minces from morphine-treated and pentobarbitone-treated rats incubated in 4 mM K(+) medium to form (14)C-ACh was markedly reduced as compared with those from control animals.3. The ability of slices from drug-treated and control animals incubated in 31 mM K(+) medium to form (14)C-ACh was similar.4. Cerebral cortex homogenates from both groups of animals in either 4 or 31 mM K(+) medium formed similar amounts of (14)C-ACh.5. These findings add further support to the hypothesis that the concentration of ACh at the site of synthesis governs the rate of formation of the neurotransmitter.     

Effects of antidepressants on the uptake and release of norepinephrine from rat cerebral cortex

The effects of several antidepressants on the release of (³H)-norepinephrine (NE) from homogenates of rat cerebral cortex were studied. A continuous superfusion collection system was used in order to differentiate these effects from effects on reuptake. Amitriptyline, maprotiline, mianserin, and trazodone produced a statistically significant decrease in spontaneous tritium efflux when present in the superfusion medium at a concentration of 1.0 M. The other antidepressants studied had no effect. We used a buffer with the K⁺ concentration raised to 56 mM as a model of depolarization-induced release. Desipramine, fluoxetine, and iprindole (again at 1.0 M) caused a significant decrease in this measure. These results indicate that some of both the tricyclic and atypical antidepressants may alter spontaneous or depolarization-induced release of NE.This work was supported in part by a grant from the Pharmaceutical Manufactures Association Foundation-Medical Student Research Fellowship     

Effects of cocaine and norepinephrine on primary cultures of neonatal rat myocardial cells.

Sudden cardiac death associated with cocaine (Coc) abuse in healthy, physically active individuals became a grave concern in the late 1980s. It is well documented that physical activity increases circulating plasma catecholamine levels. Catecholamines as well as Coc are independently capable of inducing toxic cardiac effects. The purpose of this investigation was to evaluate the synergistic or additive toxic effects of norepinephrine (NE) and Coc in primary myocardial cell cultures obtained from 3- to 5-d-old Sprague-Dawley rats. Alterations in lactate dehydrogenase release (LDH), lysosomal neutral red retention (NR), beating activity, and morphology were evaluated after treatment of the cells for 1-24 h with 1 x 10(-3) M Coc alone, 1 x 10(-5) M Coc alone, 1 x 10(-5) M NE alone, 1 x 10(-3) M Coc with 1 x 10(-5) M NE, or 1 x 10(-5) M Coc with 1 x 10(-5) M NE. LDH release was elevated significantly after 24 h only with those cells exposed to 1 x 10(-3) M Coc alone and 1 x 10(-3) M Coc + 1 x 10(-5) M NE. Using NR retention as a score for lysosomal treatment of the cells with 1 x 10(-5) M Coc and 1 x 10(-3) M Coc alone did not decrease dye retention significantly. However, 1 x 10(-5) M NE combined with 1 x 10(-3) M Coc significantly reduced lysosomal dye retention as early as 1 h after treatment. After 24 h, 1 x 10(-5) M NE alone and 1 x 10(-5) M NE combined with 1 x 10(-5) M Coc significantly increased lysosomal fragility. Beating activity was altered in all treatment groups. Contractile activity was slow and irregular or completely absent with 1 x 10(-5) and 1 x 10(-3) M Coc, respectively. When NE (1 x 10(-5) M) was combined with both concentrations of Coc, there was distinct focalization of sharp, rapid contractions within the cells, which were asynchronous and/or arrhythmic in nature. Those cells exposed to 1 x 10(-5) M NE with 1 x 10(-5) M Coc for 24 h appeared hypercontracted with marked pseudopodia and cytoplasmic granule formation distinctly different from that exhibited by the cells exposed to 1 x 10(-5) M Coc alone. These data demonstrate that NE potentiates the adverse effects of Coc on contractile activity and morphology of spontaneously contracting neonatal myocardial cells maintained in culture.