Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro

Summary The objective of this in-vitro study was to examine whether the diencephalic floor or the mesenchyme is involved in differentiation of LH cells in the developing rat adenohypophysis. Overall growth of the adenohypophysial tissue was retarded when the adenohypophysial primordium was cultivated after enzymatic removal of the diencephalic floor on days 11.5 and 12.5 of gestation. This malgrowth was more marked when the brain was separated on day 11.5; most expiants retained a simple cystiform structure that consisted of a few layers of undifferentiated cells. Removal of the brain also caused a highly significant decrease (P < 0.001) in the number of immunoreactive LH cells, if it was performed on day 11.5 but not day 12.5. Mesenchyme had little effect on the adenohypophysial growth or the number of immunopositive cells. Cultivation of the adenohypophysial primordium with the diencephalic floor resulted in the appearance of many immunoreactive LH cells. The number of LH cells significantly decreased, however, when the co-cultivated brain completely surrounded the adenohypophysial tissue.These results indicate that in 11.5-day-old fetal rats the diencephalic floor is indispensable for the initial proliferation of adenohypophysial primordial cells and for the early determinating process of LH cells. Once determined, the development of LH cells may proceed without the surrounding tissues. The cytodifferentiation seems to be rather inhibited when in contact with the brain. The significance of the intimate spatial relationship between developing LH cells and the surrounding mesenchyme is also discussed.     

In vitro interaction of premazepam with benzodiazepine receptors in rat brain regions

Premazepam (PRZ) in vitro competitively displaced 3H-diazepam (DIA), 3H-flunitrazepam (FLU) and 3H-RO 15-1788 from their binding sites on rat brain synaptosomes, with a potency intermediate to other benzodiazepines (BDZs), and Hill coefficients near 1 in different brain regions. Incubation at 37 degrees C reduced premazepam's affinity for BDZ receptors to a lower extent than other benzodiazepines and had no effect on the Hill coefficient. The IC50 of PRZ on 3H-RO 15-1788 and 3H-FLU binding was markedly reduced by GABA in rat cortex, like those of reference classical BDZs, but was GABA-independent in the cerebellum. The IC50 of the BDZ antagonist, RO 15-1788 was unaffected by GABA in both brain areas. The possibility that PRZ behaves as a partial agonist in the cortex and as an antagonist in the cerebellum is discussed.     

Gonadotropin-releasing hormone increases melatonin release in the pineal gland of the female rat in vitro.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin ( N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 -12, 10 -10, or 10 -8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 -10 and 10 -8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 -12 to 10 -8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole- O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.     

In vitro response of relaxin-treated rat uterus to prostaglandins and oxytocin

Paired segments of rat uterus were treated in vitro with relaxin (W1164-3, 150 GPU/mg) until the amplitude of contraction was reduced to at least 50% of the pre-treatment amplitude. Test segments then received 100 ng of either PGE1, PGE2, PGF2alpha or 250 uU of oxytocin. Control segments remained untreated. There was a significant increase in contraction amplitude in response to the spasmogens (P less than 0.05) but no increase was seen in controls.     

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.     

Neurotransmitter accumulation and calcium-dependent release from different regions of rat brain.

High affinity accumulation and calcium-dependent release of ³H-NE, ³H-dopamine, ¹⁴C-GABA and ¹⁴C-choline/ACh were investigated in 12 regions of the rat brain. Regional differences in accumulation and fractional calcium-dependent release were observed for all four substances. The corpus striatum exhibited particularly high accumulation values and high release rates for all four substances. Caudal structures exhibited low accumulation and release values. The differences are discussed in terms of possible differences in neurotransmitter dynamics in the different regions.     

Kinetic analysis of 3H-serotonin accumulation in four regions of rat brain after lesions in the medial forebrain bundle

Kinetic analysis of ³H-serotonin accumulation by crude synaptosomal suspensions of neocortex, hippocampus and caudate or by whole homogenates of cerebellum revealed the presence of a high affinity uptake component having an apparent K_m for serotonin which ranged from 2.8 to 6.0 × 10^?8 M. A second, low affinity, uptake component with an apparent K_m of 7 × 10^?6 M was present in caudate. A comparable low affinity uptake component for serotonin was not observed in neocortex, hippocampus or cerebellum. Lesions in the medial forebrain bundle produced significant decreases in serotonin comtent of neocortes, hippocampus and caudate (66 to 75%) and a significant increase in serotonin content of cerebellum (25%). The lesions did not affect the apparent K_m of the high affinity uptake system but did produce change in V_max which paralleled the changes in content of serotonin. The lesions also produced decreases in dopamine and norepinephrine content of caudate and a comparable decrease in the V_max of the low affinity uptake system with no change in the apparent K_m. There was a correlation of 0.97 between the endogenous content of serotonin and the V_max of the high affinity uptake system. These results support the view that the high and low affinity components of serotonin uptake represent accumulation into serotonergic and catecholaminergic neurons, respectively.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

The effect of isatin (tribulin) on metabolism of indoles in the rat brain and pineal: In vitro and in vivo studies

Isatin (Tribulin) produced a dose-dependent inhibition of both MAO A and MAO B in broken cell preparations from rat brain and pineal. However, isatin administered in vivo (80–160 mg/kg) to the intact animal significantly increased brain, but not pineal, serotonin and did not affect 5HIAA or other indoles in either brain or pineal. Further, in vivo administration did not produce detectable MAO inhibition in either tissue. In pineal organ culture, addition of isatin up to 1mM had no influence on the concentrations of pineal indoles or the activities of monoamine oxidase or serotonin N-acetyltransferase. However, the diazepam augmentation of beta adrenergic induction of serotonin N-acetyltransferase activity was blocked by isatin. The results of these studies call into question the proposed role of isatin as an endogenous monoamine oxidase inhibitor but support a possible role as a benzodiazepine receptor blocker.     

In vitro study of rat uterus after chronic formaldehyde exposure.

To measure cholinergic, adrenergic and tryptaminergic receptor activity of formaldehyde (HCHO) in rat uterus, albino rats were treated with 5 and 10 mg/kg, ip HCHO for 30 days. Acetylcholine (ACh) in doses 1.33, 2 and 3 micrograms/ml produced mild to moderate contraction of isolated rat uterus in control group. HCHO had no effect on isolated rat uterus per se, however it reduced ACh and carbachol induced contraction and presence of adrenaline influences in respect of ACh and carbachol activity. Adrenaline per se had no effect in control preparations, but reduced carbachol induced contraction. Propranolol had no effect on rat uterus; but its presence in the bathing medium increased activity of adrenaline. 5-Hydroxytryptamine (5-HT) had no effect of its own on isolated rat uterus but its presence in the bathing medium enhanced contractions of carbachol and oxytocin.     

Developmental changes in choline acetyltransferase and glutamate decarboxylase activity in various regions of the brain of the male,female, and neonatally androgenized female rat

In an attempt to discern effects of sex hormones on the development of neurotransmitter systems in the rat brain, choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) have been measured at postnatal days 8, 12, 25, and 60 in five regions (amygdala, anterior hypothalamus, hippocampus, olfactory bulbs, and cerebral cortex) of the brains of normal male, normal female, and neonatally androgen-treated female rats. Essentially no associations between sex or of neonatal androgenization on either enzyme were found. The data, however, provide new information on the relative rates of development of ChAT and GAD in five regions of the rat brain which supplement the limited information already available in the literature. ChAT activity was highest in amygdala and hypothalamus, but developed most rapidly in hippocampus and cerebral cortex. The relative activities and patterns of development of GAD activity were similar to those of ChAT.     

Release of endogenous norepinephrine and dopamine from rat brain regions in vitro

Using radioenzymatic assay procedures, we have measured picomolar amounts of endogenous norepinephrine (NE) and dopamine (DA) released $\text{in vitro}$. The release of NE and DA in response to KCl stimulation was examined in 6 brain regions: cortex, hippocampus, hypothalamus, striatum, combined accumbens-olfactory tubercle, and substantia nigra. NE release was detectable in all regions except striatum. Amounts of NE released by 55mM KCl (expressed as % control) were: cortex (313%), hippocampus (227%), hypothalamus (225%), accumbens-tubercle (278%), s. nigra (155%). KCl stimulated release of DA was detected in 3 regions: striatum (414%), accumbenstubercle (282%), and hypothalamus (312%). DA was measurable in filtrates from the s. nigra but levels in control and KCl stimulated samples were equal. Release of NE and DA was also measured in 12 brain regions after incubation of tissue $\text{in vitro}$ with 10^?4M d-amphetamine sulfate. d-Amphetamine stimulated NE outflow when compared to controls in all regions examined. DA outflow was markedly increased in most regions, especially striatum (287%), hypothalamus (387%) and accumbens-tubercle (670%). d-Amphetamine doubled endogenous DA outflow from the s. nigra.     

In vivo morphine decreases [3H]nimodipine receptor binding in rat brain regions

Thein vivo effect of the mu agonist morphine and antagonist naloxone on [³H]nimodipine receptor binding in rat brain regions has been investigated. Morphine administration (15 mg/s.c.) for thirty minutes produced a 19% decrease in [³H]nimodipine receptor binding (B _max 158.2 fmol to 128.9 fmol) in cortex and 29% decrease in cerebellum (65.3 fmol to 46.0 fmol). Lesser changes were observed in hippocampal and striatal regions with no changes in hypothalamus and brain stem. All effects were completely antagonized by naloxone pretreatment (1 mg/kg). The studies suggest that opiates in vivo can alter [³H]nimodipine binding to the Ca²⁺ channel receptor protein. These findings agree with the previously observed decreases in Ca²⁺ influx in nerve ending preparations and inhibition of I_Ca ²⁺ following opiate treatment and suggest opiates reduce Ca²⁺-dependent neurotransmitter release by altering the Ca²⁺ channel receptor protein in an allosteric fashion.     

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 effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland

1. To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10-1,000 nM NPY in the presence or absence of 10 microM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA). 2. NPY (10-1,000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1,000 nM), enhanced NE stimulation of melatonin production. 3. NPY (1,000 nM) impaired 3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with 3H-NE. 4. These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.     

Hormone accumulation in song regions of the canary brain

[³H]Testosterone (T) was injected into male and female canaries (Serinus canarius), a species in which females are able to sing but do so more rarely and more simply than males. Autoradiographic analysis revealed that males and females have equal proportions of cells labeled by T or its metabolites in four song control nuclei: the high vocal center (HVC), the lateral portion of the magnocellular nucleus of the anterior neostriatum (IMAN), the robust nucleus of the archistriatum (RA), and the hypoglossal motor nucleus (nXII). Labeled cells were also observed in both sexes in the medial portion of MAN, and in hypothalamic nuclei. In both sexes, labeled cells in HVC, IMAN, RA, and nXII were larger than unlabeled cells. There were no sex differences in the size of either labeled or unlabeled cells in these song nuclei. The density of labeled cells per unit volume of tissue did not differ between the sexes in any song nucleus analyzed. However, because males have larger HVC and RA than females, males have a greater total number of hormone-sensitive cells in these regions than do females. Comparison of these results with measures of hormone accumulation in zebra finches and tropical duetting wrens suggests that the complexity of song that a bird can produce is correlated with the total number of hormone-sensitive cells in song nuclei. © 1992 John Wiley & Sons, Inc.