In vitro effects of inorganic lead on isolated rat brain mitochondrial respiration

The effects of lead acetate on respiration in cerebral and cerebellar mitochondria from immature and adult rats were studied polarographically. With all substrates low lead concentrations produced an increase in respiration. Higher concentrations produced an inhibition of both this lead-induced respiration and ADP-dependent (State 3) respiration. Lead-induced respiration required inorganic phosphate and was inhibited by oligomycin, suggesting a coupling to oxidative phosphorylation. Inhibition of respiration was produced by much lower lead concentrations with NAD-linked citric acid cycle substrates than with succinate or -glycerophosphate. In partially disrupted mitochondria, NAD-linked substrate oxidation was inhibited at lead concentrations which did not affect NADH oxidation. Thus, in brain mitochondria the NAD-linked dehydrogenases, located in the matrix space, were more sensitive to inhibition by lead than were inner membrane enzymes. All in vitro lead effects on mitochondrial respiration were comparable in cerebral and cerebellar mitochondria isolated from both immature and adult rats.     

In vitro study of microwave effects on calcium efflux in rat brain tissue

In this study we investigated the prospect of microwave-induced alteration of ⁴⁵Ca²⁺ efflux from rat neural tissue at low pulse repetition frequencies and low power densities under in vitro conditions. Rat cerebral tissue, preloaded with ⁴⁵Ca²⁺, was exposed to pulsed-microwave radiation (1-GHz carrier frequency) according to one of several PRF-power density exposure schemes: 16 Hz at 0.5, 1.0, 2.0, or 15 mW/cm², or 32 Hz at 1.0 or 2.0 mW/cm² average power density. Measurements of radioactivity in the efflux medium and in the tissue sample were used to calculate an efflux value for each sample. The results indicate that the radiation conditions used did not alter calcium efflux in rat brain tissue.     

In vitro response of rat gastrointestinal segments to cholecystokinin and bombesin

In previous studies of the rat gastrointestinal (GI) tract, we have demonstrated specific binding of cholecystokinin (CCK) to the pylorus and of bombesin (BN) to the gastric fundus, gastric antrum, duodenum, and ileum. We now present the results of an investigation of the in vitro response of the same regions of the rat GI tract to CCK-8 (the active octapeptide of CCK) and BN. Sections of rat fundus, antrum, pylorus, duodenum, and ileum were suspended in a Tyrode buffer and attached to an isometric pressure transducer in a longitudinal orientation. Dose-response curves to CCK-8 and BN were generated for each tissue. CCK-8 consistently induced a change only in pylorus, while BN induced a response from fundus, antrum and duodenum. With the exception of the lack of ileal response to BN, the regions of the rat GI tract which biologically respond (i.e., contract or relax) to CCK-8 or BN were the same regions in which we have located BN and CCK-8 binding sites. This correlation supports the hypothesis that GI function is modified by specified hormone-receptor interactions with these peptides.     

In vitro synthesis of rat brain hexokinase

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)⁺ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipatation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical ‘soluble’ enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that (1) it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and (2) limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.     

In vitro synthesis of rat brain hexokinase

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)+ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipitation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical 'soluble' enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.     

In vitro and ex vivo effects of antidepressants on rat brain membrane-bound phosphatidylinositol synthetase activity

The in vitro and ex vivo effects of antidepressant drugs on membrane-bound phosphatidylinositol (PI) synthetase and PI: myo-inositol exchange enzyme activities were examined. In rat brain subcellular fractions, PI synthetase occurred exclusively in the microsomes. In comparison, the activity of CDP-diglyceride independent PI: myo-inositol exchange enzyme was low (3%). Of the various CDP-diglycerides tested for the activation of PI synthetase, CDP-dipalmitin was the most active. Addition of 1 mM of desipramine, amitriptyline, imipramine, iprindole, clomipramine and mianserin in vitro significantly inhibited (30–60%) PI synthetase activity, whereas the same concentration of zimelidine and fluoxetine had no effect. At low liponucleotide concentrations, PI synthetase activity was significantly enhanced by imipramine (1 mM), whereas the enzyme activity was inhibited at higher liponucleotide concentrations (>0.3 mM). In contrast, imipramine had no effect on the PI: myo-inositol exchange enzyme activity. No significant alteration in the PI synthetase activity was found following either acute (2 h) or chronic (21 d) treatment of rats with imipramine. The above results indicate that the de novo synthesis of PI is inhibited in vitro but not ex vivo by some antidepressant drugs. However, in view of the high concentration of the drugs required, the pharmacological significance of this inhibitory action with respect to their therapeutic effects is doubtful.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of prolactin in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of prolactin (PRL) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma PRL level after 10 and 20 min. CCK-8 at concentrations of 10(-11) M to 10(-7) M also caused dose-dependent stimulation of PRL release from dispersed cells of rat anterior pituitary. On the other hand, dopamine inhibited PRL release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-8) M to 10(-6) M. Release of PRL from the cells was increased by addition of K+ at high concentration (53 mM) in a Ca++-dependent manner. Addition of 10(-3) M verapamil to the incubation medium inhibited CCK-8-induced PRL release from the cells. Addition of dopamine (10(-7) M) to the incubation medium inhibited PRL release from the cells induced by CCK-8 or high K+ (53 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate PRL release and that calcium ion is involved in the mechanism of this effect.     

Dopamine effects on identified rat vagal motoneurons

Catecholaminergic neurons of the A2 area play a prominent role in brain stem vagal circuits. It is not clear, however, whether these neurons are noradrenergic or adrenergic, i.e., display tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH) immunoreactivity (-IR) or dopaminergic (i.e., TH- but not DbetaH-IR). Our aims were to investigate whether a subpopulation of neurons in the A2 area was dopaminergic and, if so, to investigate the effects of dopamine (DA) on the membrane of gastric-projecting vagal motoneurons. We observed that although the majority of A2 neurons were both TH- and DbetaH-IR, a small percentage of nucleus tractus solitarius neurons were TH-IR only, suggesting that DA itself may play role in these circuits. Whole cell recordings from thin brain stem slices showed that 71% of identified gastric-projecting motoneurons responded to DA (1-300 microM) with either an excitation (28%) or an inhibition (43%) of the membrane; the remaining 29% of the neurons were unresponsive. The DA-induced depolarization was mimicked by SK 38393 and prevented by pretreatment with SCH 23390. Conversely, the DA-induced inhibition was mimicked by bromoergocryptine and prevented by pretreatment with L741626. When tested on the same neuron, the effects of DA and NE were not always similar. In fact, in neurons in which DA induced a membrane depolarization, 77% were inhibited by NE, whereas 75% of neurons unresponsive to DA were inhibited by NE. Our data suggest that DA modulates the membrane properties of gastric-projecting motoneurons via D1- and D2-like receptors, and DA may play different roles than norepinephrine in brain stem vagal circuits.     

In vitro effects of organophosphate pesticides on rat erythrocytes

In vitro effects of various organophosphate pesticides (dimethoate, chlorpyrifos, ethion and monocrotophos) were studied on hemolysis, K+ leakage and lipid peroxidation in rat erythrocytes. All the four pesticides increased hemolysis and K+ leakage from erythrocytes, that was concentration and time dependent. On the contrary, there was decrease in lipid peroxidation in erythrocyte membrane. Effect of pesticides on lipid peroxidation could be due to pesticide itself abstracting protons or interacting with free radicals rather than polyunsaturated fatty acids (PUFA), thereby protecting the latter against peroxidation.     

In vitro effects of cannabinoids on follicular function in the rat

delta 1-Tetrahydrocannabinol (delta 1-TCH), the major psychoactive constituent of marihuana, was found to suppress the preovulatory surge of gonadotropins and thereby to prevent ovulation in rats, rabbits and rhesus monkeys. These studies suggested that the drug acts primarily on the hypothalamus to suppress luteinizing hormone releasing hormone (LHRH) secretion. The aim of the present study was to examine the direct effect of delta 1-THC, the psychoactive constituent of marihuana and cannabidiol (CBD), one of its nonpsychoactive constituents, on preovulatory rat follicles in vitro. Both cannabinoids inhibited follicular steroidogenesis in a dose-dependent manner. Basal accumulation of progesterone (P), testosterone (T) and estradiol-17 beta (E2) was reduced up to 60% by the highest doses examined (100-200 microM). The luteinizing hormone (LH)-stimulated increase in P and T was inhibited by 75-88% by the highest doses of both cannabinoids (50-200 microM), while E2, accumulation was inhibited by only 40%. It appears that the inhibitory action of cannabinoids is exerted beyond LH binding and activation of adenylate cyclase and prior to pregnenolone formation in the gonadal steroidogenic pathway. In addition to this anti-steroidogenic effect, both cannabinoids induced resumption of meiosis in follicle-enclosed oocytes cultured in hormone-free medium; 200 microM delta 1-THC resulted in 80% maturation and CBD in 75%. It seems that the action of cannabinoids on rat follicles in vitro is unrelated to their psychotropic activity.     

In vivo and in vitro effects of cholecystokinin octapeptide on the release of growth hormone in rats

The effect of cholecystokinin octapeptide (CCK-8) on the release of growth hormone (GH) in rats was studied in vivo and in vitro. Intravenous injection of 5 micrograms/100 g BW of CCK-8 resulted in significant increase in the plasma GH level after 10 and 20 min. CCK-8 at concentrations of 10(-11)M to 10(-7)M also caused dose-dependent stimulation of GH release from dispersed cells of rat anterior pituitary. On the other hand, somatostatin (SRIF) inhibited GH release from dispersed cells of rat anterior pituitary in a dose-related manner at concentrations of 10(-7)M to 10(-9)M. Release of GH from the cells was increased by addition of K+ at high concentration (50 mM) in a Ca++-dependent manner. Addition of 10(-3)M verapamil to the incubation medium inhibited CCK-8-induced GH release from the cells. Addition of SRIF (10(-7)M) to the incubation medium inhibited GH release from the cells induced by CCK-8 or high K+ (50 mM). These results indicate that CCK-8 acts directly on the anterior pituitary cells to stimulate GH release and that calcium ion is involved in the mechanism of this effect.     

In vitro study of cysteine oxidase in rat brain

Effects of some cysteine analogs and other compounds on in vitro cysteine oxidase were studied in rat brain microsomes. Among the tested compounds, maximum inhibition of microsomal cysteine oxidase was by alpha,alpha'-dipyridyl and the least inhibition by dithiothreitol. Kinetic and dialysis studies found L-homocysteine to be a competitive and reversible inhibitor of cysteine oxidase. Epinephrine was shown to inhibit cysteine oxidase, whereas pyridoxal HCl activated cysteine oxidase at the same concentration. Except for the Mg2+ ion, other metallic ions inhibited cysteine oxidase activity in the following order: Zn2+ greater than Cu2+ greater than Li+ greater than Ca2+ greater than Co2+ greater than K+ greater than Mn2+. A 12 mM concentration of Mg2+ ion was required to obtain maximum cysteine oxidase activity.     

In vitro effects of microwave radiation on rat liver mitochondria

Liver mitochondria were exposed in vitro at 30°C to microwave radiation (2.45 GHz) during the following states of respiraton: resting, state 1; substrate dependent, state 2; ADP stimulated, state 3; and ADP depleted, state 4. At 10 or 100 mW/g, with succinate as substrate, no effect of exposure was observed on states 1–4 or the respiratory control index (state 3/state 4) of either tightly or loosely coupled mitochondria. When glutamate was used as substrate, no effects were observed at 10 mW/g. However, in the loosely coupled mitochondria the 100 mW/g exposure produced an increase in states 2 and 4 and a decrease in the respiratory control index. The results suggest that the function of loosely coupled mitochondria can be affected at high power levels of microwave radiation.     

In vitro studies on the methylation of histones in rat brain nuclei.

When isolated nuclei from 12-day-old rat brains were incubated with S-adenosyl-L-[methyl-3H]methionine, significant amounts of 3H-methyl were incorporated into lysyl residues in histones H3 and H4. About 0.024% of the total methylation sites on histone H3 and 0.013% of the sites on histone H4 were unmethylated at the time the nuclei were isolated. Methylation of these sites proceeded stepwise, progressing to a stable ratio of 0.93:1.0:0.17 for N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysine in histone H3 and 0.19:1.0 for N epsilon-mono- and N epsilon-dimethyllysine in histone H4. The Km values of the enzyme for S-adenosyl-L-methionine were 11.5 +/- 1.1 micron and 12.5 +/- 1.3 micron with histones H3 and H4 as methyl acceptors, respectively. The Vmax values were 11.1 and 5.3 pmol of 3H-methyl incorporated/min/mg of histone H3 and H4, respectively. Since histone H3 contains 2 mol of N epsilon-methyllysine/mol and histone H4 contains 1 mol/mol, no difference in the overall rates of methylation can be deduced from the data. S-Adenosyl-L-homocysteine, one of the products of the reaction, was a competitive inhibitor with respect to S-adenosyl-L-methionine. The Ki values for S-adenosyl-L-homocysteine were 5.5 +/- 0.4 micron and 5.9 +/- 0.5 micron with histones H3 and H4 as methyl acceptors, respectively.     

In vitro characterization of subventricular zone isolated neural stem cells,from adult monkey and rat brain

Neural stem cells (NSCs) are multipotent, self-renewable cells who are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside at the subventricular zone (SVZ) of the adult brain permanently to guarantee a lifelong neurogenesis during neural network plasticity or undesirable injuries. Although the specious inaccessibility of adult NSCs niche hampers their in vivo identification, researchers have been seeking ways to optimize adult NSCs isolation, expansion, and differentiation, in vitro. NSCs were isolated from rhesus monkey SVZ, expanded in vitro and then characterized for NSCs-specific markers expression by immunostaining, real-time PCR, flow cytometry, and cell differentiation assessments. Moreover, cell survival as well as self-renewal capacity were evaluated by TUNEL, Live/Dead and colony assays, respectively. In the next step, to validate SVZ-NSCs identity in other species, a similar protocol was applied to isolate NSCs from adult rat’s SVZ as well. Our findings revealed that isolated SVZ-NSCs from both monkey and rat preserve proliferation capacity in at least nine passages as confirmed by Ki67 expression. Additionally, both SVZ-NSCs sources are capable of self-renewal in addition to NESTIN, SOX2, and GFAP expression. The mortality was measured meager with over 95% viability according to TUNEL and Live/Dead assay results. Eventually, the multipotency of SVZ-NSCs appraised authentic after their differentiation into neurons, astrocytes, and oligodendrocytes. In this study, we proposed a reliable method for SVZ-NSCs in vitro maintenance and identification, which, we believe is a promising cell source for therapeutic approach to recover neurological disorders and injuries condition.

     

In vitro RNA synthesis by intact rat brain nuclei

The characteristics of DNA-dependent RNA polymerase activity in intact rat cerebral cortex nuclei tested at low ionic strength are presented. The system was most dependent on the presence of spermidine and an ATP-generating system and to a lesser extent on Mg2+ and K+ for maximal incorporation. Substitution of Mg2+ by Mn2+ or of K+ by Na+ resulted in substantially less activity than under the optimum conditions described. Maximal incorporation was about 10 per cent that of brain nuclear systems of high ionic strength. In addition, the labelling patterns of the in vitro RNA products were shown to be very similar to those found in vivo. The stability of isolated nuclei toward degradation of RNA synthetic capacity and products formed was much greater than that of a similar liver system.     

Effect of cholecystokinin octapeptide sulphate ester on brain monoamines in the rat

The effect of different doses of intracerebro-ventricularly administered cholecystokinin octapeptide sulphate ester (CCK-8-SE) was studied on dopamine (DA), norepinephrine (NE) and serotonin (5-HT) contents in the hypothalamus, mesencephalon, amygdala, septum and striatum, 10, 20 and 60 min following administration. The DA and NE content increased and the 5-HT content decreased in the hypothalamus and mesencephalon. A biphasic action was observed in the amygdala of DA, NE and 5-HT depending upon the time and doses used. Similar action was seen on DA and NE in the septum. In the striatum, the DA and 5-HT content decreased while the NE level first increased and then decreased. The data indicate that the CCK-8-SE is able to modify the activity of DA, NE and 5-HT in different brain regions in a time and dose-dependent manner, with a local specific action.