Biochemical and Immunochemical Studies on the GABAergic System in the Rat Fallopian Tube and Ovary

gamma-Aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) activities were measured in the ovary and the Fallopian tube of rats and compared with brain values. GABA levels in the Fallopian tube were about twice as high as in the brain, while in the ovary they represented only about 5% of the amino acid content of the CNS. In vitro decarboxylation of glutamate, measured via CO2 formation, occurred both in the Fallopian tube and in the ovary. These two organs contained, respectively, 10% and 1% of brain GAD activity. However, the actual formation of GABA from glutamate in a high-speed supernatant was detectable only in the Fallopian tube, where it represented about 5% of brain GAD activity. In contrast with the enzyme present in ovary, liver, anterior pituitary, and kidney, that in the Fallopian tube was quantitatively precipitated by a specific antiserum directed against rat neuronal GAD. Moreover, subcutaneous transplantation resulted in a quantitative decrease of both GABA levels and GAD activity in the Fallopian tube while no change occurred in the ovary, and vagus nerve section induced a 50% decrease of GAD activity in the Fallopian tube, although GABA levels were not significantly altered. The findings suggest an extrinsic GABAergic innervation in the rat Fallopian tube but not in the ovary.     

Alteration of GABA levels in ovary and fallopian tube of the pregnant rat

The concentration and the total quantity of gamma-aminobutyric acid (GABA) were determined in ovary and Fallopian tube of the rat on day 3, 7, 14 and 21 of pregnancy and the values were compared with a diestrous control group. In ovary, both GABA concentration and total GABA content gradually elevated up to day 14 of gestation but, thereafter, the diminution of the two parameters was observed. The GABA concentrations in Fallopian tube were found to be strongly reduced during pregnancy, and the total amount of the amino acid in this organ also decreased markedly. The significant alterations of GABA levels in both organs suggest a possible correlation between the functional status and GABA content of ovary and Fallopian tube of the pregnant rat.     

IMMUNOCHEMICAL AND IMMUNOFLUORESCENCE STUDIES OF BETA-TRACE PROTEIN IN DIFFERENT SPECIES AND ORGANS, WITH SPECIAL REFERENCE TO THE CENTRAL NERVOUS SYSTEM

—The localization of beta-trace protein in different human organs and in the brains of other mammals has been investigated with immunochemical and immunofluorescence methods. With the double immunodiffusion technique, the immunological identity between beta-trace protein in human CSF, brain, femoral nerve, testis, epididymis, spermatic cord, ovary and fallopian tube and in monkey brain could be demonstrated. By means of single radial immunodiffusion on agar, the highest concentrations of beta-trace protein were found in preparations of water-soluble proteins of brain and genital organs, especially in white CNS matter, testis and epididymis. With the indirect immunofluorescence technique, bright fluorescence was found in CNS, especially in white matter, and in the stroma of epididymis. The content of beta-trace protein in femoral nerve was found to be considerably lower than that in the CNS. The results indicate a connexion between beta-trace protein and the CNS, probably in the myelin or glial cells, and also between beta-trace protein and the genital system, possibly to cells involved in steroid metabolism. A specificity for highly developed mammals, i.e. man and monkey, is proposed for beta-trace protein, as no precipitation could be found when brain extracts from calves, rabbits or mice were incubated with anti-serum against human beta-trace protein.     

Immunohistochemical localization of glutamate decarboxylase in the rat oviduct and ovary: Further evidence for non-neural GABA systems

Summary The distribution of L-glutamate decarboxylase (GAD), a major biosynthetic enzyme for gamma-aminobutyric acid (GABA), was examined in the oviduct and ovary of the rat by means of an immunohistochemical technique. The polyclonal antiserum raised against brain GAD showed specific immunoreaction in some non-neuronal elements of the sex organs. In the oviduct, the inner layer of the mucosa was predominantly labelled. The selective distribution of GAD immunoreactivity in epithelial cells of the oviduct is consistent with former findings for GABA-like immunoreactivity in the same organ, indicating that the GAD-catalyzed reaction may be a major biosynthetic pathway for GABA even in these cells. In the ovary, vacuole-like formations within the follicular fluid and oocytes showed intense, specific staining. The occurrence of GAD immunoreactivity inside developing ovarian follicles including the oocyte may suggest a role for GABA related to follicular development and certain functions concerning the ovum.     

The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by manganese

In the central nervous system (CNS), extracellular concentrations of amino acids (e.g., aspartate, glutamate) and divalent metals (e.g., zinc, copper, manganese) are primarily regulated by astrocytes. Adequate glutamate homeostasis and control over extracellular concentrations of these excitotoxic amino acids are essential for the normal functioning of the brain. Not only is glutamate of central importance for nitrogen metabolism but, along with aspartate, it is the primary mediator of excitatory pathways in the brain. Similarly, the maintenance of proper Mn levels is important for normal brain function. Brain glutamate is removed from the extracellular fluid mainly by astrocytes via high affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of Mn on specific glutamate transporters have yet to be determined. As a first step in this process, we examined the effects of Mn on the transport of [D-2, 3-3H]D-aspartate, a non-metabolizable glutamate analog, in Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) or GLT-1 (EAAT2). Mn-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was pronounced in both the GLT-1 and GLAST transfected cells. This resulted in a statistically significant inhibition (p<0.05) of glutamate uptake compared with transfected control in the absence of Mn treatment. These studies suggest that Mn accumulation in the CNS might contribute to dysregulation of glutamate homeostasis.     

Diazepam Increases γ-Aminobutyric Acid in Human Cerebrospinal Fluid

In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.     

Ontogenesis of histamine in the chick nervous system

Tissues from the central and peripheral nervous systems of the chick were analyzed for concentration of histamine (Hm) during development. Of the three CNS organs examined, cerebral hemispheres had the highest Hm content. Expressed on the bases of wet weight, protein, and DNA concentrations, sciatic nerve and the pineal gland had the highest levels of this biogenic amine of the five tissues investigated. The concentration of Hm was higher in the cerebellum, cerebral hemispheres, and thalamus of adult animals than in the 15 to 17-day-old embryos. The level of Hm rose markedly in the sciatic nerve and pineal gland after the 15th day of embryonic development. These data might indicate a possible involvement of Hm in controlling the course of maturation of certain organs in the nervous system.     

Further Characterization of In Vitro Conditions Appropriate for GABA Determination in Human CSF: Impact of Acid Deproteinization and Freeze/Thaw

Recently established standardized protocols for collection, handling, and storage of CSF for measurement of gamma-aminobutyric acid (GABA) have proven valuable in the characterization of various CNS disorders. In response to two recent reports which may have an impact on certain widely used protocols, we have, using the confirmed ion-exchange/fluorometric procedure, systematically evaluated the effects of deproteinization with various concentrations of sulfosalicylic acid (SSA) ranging from 0 to 10% (100 mg/ml), as well as the effects of freeze/thaw (F/T) on CSF GABA levels. Results of F/T studies documented that levels are stable to freezing and thawing. Acid deproteinization studies revealed the presence of an equilibrium between strictly free GABA, demonstrable only in acid-free CSF, and a very loosely bound form of GABA, fully demonstrable only in CSF deproteinized with concentrations of SSA above 1% (10 mg/ml). The relationship between GABA concentrations in undeproteinized and acid-deproteinized CSF revealed a highly significant (p less than .001) correlation, suggesting that alterations of central GABAergic activity would be reflected by either the level of strictly free GABA or free plus loosely bound GABA. This hypothesis was upheld in studies of patients with Parkinson's disease (PD) and Huntington's disease (HD), two neurologic disorders in which dysfunctions of the GABA system have been implicated. Results indicated that CSF GABA levels are significantly reduced in both PD and HD patients compared with neurologically normal controls, whether the measurement is of free GABA or free plus loosely bound GABA. Thus, we conclude that the level of strictly free GABA is stable to freezing and thawing and can only be accurately determined in nonacidified CSF; however, existing protocols employing deproteinization in 5% SSA yield data that provide an equally good reflection of central GABAergic transmission.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depletion of Brain Glutathione in Preweanling Mice by L-Buthionine Sulfoximine

Previous studies indicated that DL-buthionine sulfoximine (DL-BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of L-BSO with marked declines (79.6-86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8-29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L-BSO represents a tool for studying the role of GSH in the CNS.     

Surgical ablation of oviductal extrinsic innervation changes GABA levels in the rat fallopian tube

The origin of gamma-aminobutyric acid (GABA) in the rat oviduct was investigated by measuring GABA levels in the oviduct after selective ablation of the extrinsic oviductal innervation. Rats killed 20 days after ablation of nerves connected with the left oviduct showed no differences in GABA levels in the left vs the right oviduct. Rats killed 50 days after ablation of the left ovarian vascular nerve bundle showed a decreased GABA content in the left vs the right (intact) oviduct which was more pronounced in rats killed 90 days after ablation. In contrast, GABA levels were unchanged 50 days after ligation of either the suspensory ligament of the ovary or the uterine artery. Our results indicate the involvement of a GABAergic component in the extrinsic innervation of the rat oviduct.     

Changes in polyamine levels in various organs of Bombyx mori during its life cycle

Polyamines in various organs of larval, pupal, and moth stages of Bombyx mori, were assayed by high-performance ion-exchange chromatography and paper and thin-layer chromatography. Putrescine and spermidine were especially abundant in the silk gland, gonads, mucous gland, and sucking stomach; spermine was also present in them, but at much lower concentrations. Both norspermidine and norspermine were detected in almost all organs examined, while their precursor 1,3-diaminopropane was found only in a limited number of organs. Low concentrations of sym-homospermidine were observed in the silk gland and ovary. Cadaverine content was particularly high in the mucous gland which contained diapause eggs and the sucking stomach. Diapause eggs contained much higher levels of cadaverine than non-diapause eggs. The concentrations of most polyamines in the silk glands remained rather constant during the larval stage, and decreased markedly at the pupal stage. Polyamines in gonads, in contrast, did not decrease at the pupal stage, but putrescine, diaminopropane, and norspermidine rather increased during the pupal and moth stages.     

The intraovarian progesterone modulation of follicle development in the rabbit ovary

Intraovarian progesterone levels were manipulated by surgically adjusting the number of corpora lutea (CL) present in rabbit ovaries and this model was used to study the local effect of luteal progesterone on growth of follicles. The results show that when a single CL or several CL were present, follicle growth was inhibited. However, when all CL on one ovary were removed, increased numbers of follicles grew even when a single CL was present in the contralateral ovary. These findings show that progesterone inhibits follicle growth and that at least part of its action is local, i.e., exerted within the ovary. Additionally, ovarian blood vessels and periovarian lymph ducts were cannulated, and samples were collected and analyzed for steroid and protein content. The results show that when CL were present, ovarian vein progesterone levels were elevated 10-30-fold over levels in ovaries without CL; this high concentration points to the blood vascular system as the principal carrier of the steroid within the ovary. Analysis of lymph showed that protein content was consistently high and that the progesterone concentration was not significantly altered with the presence of CL; these two findings show that ovarian capillaries are extremely permeable to proteins, but the unexpectedly low concentrations of progesterone in lymph may signal an intraovarian countercurrent mechanism by which it is returned to the blood.     

Peripheral injection of lipopolysaccharide enhances expression of inflammatory cytokines in murine locus coeruleus: possible role of increased norepinephrine turnover

Cytokines and catecholamines are known to constitute a significant portion of the regulatory neuroimmune networks involved in maintaining homeostasis in the central nervous system (CNS). Although we have already reported an increase in norepinephrine (NE) turnover within the locus coeruleus (LC) at 2 and 4 h after the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), the implication of this increase remains unclear. In view of evidence that norepinephrine (NE) acts in an anti-inflammatory manner by way of negatively regulating pro-inflammatory cytokine expression, we examined the inflammatory cytokine expression levels in the LC of C3H/HeN mice (male, 8 weeks old) after an i.p. LPS injection. The mRNA expression levels of the genes encoding IL-1beta and TNF-alpha within the LC increased during the first 2 h, and showed two peaks, the first at 4 h and the second lesser one at 15 h after the LPS injection. Microglia, which are one of the major cell types that produce pro-inflammatory cytokines in the CNS, were isolated from mouse neonate brains in order to clarify more precisely the relationship between the changes in NE content and the up-regulation of inflammatory cytokines in the LC. Simultaneous incubation of microglia with LPS and NE enhanced the expression of IL-1beta at both mRNA and protein levels, but reduced the mRNA and protein levels of TNF-alpha. These data support the hypothesis that NE negatively regulates the expression of pro-inflammatory cytokine expression, at least in the case of TNF-alpha, which action could contribute to the observed anti-inflammatory properties of NE. This report, based on the results of both in vivo and in vitro experiments, is the first to suggest a relationship between NE content and cytokine expression levels in the CNS.     

The Use of Inhibitors of GABA-Transaminase for the Determination of GABA Turnover in Mouse Brain Regions: An Evaluation of Aminooxyacetic Acid and Gabaculine

Abstract: The accumulation of γ -aminobutyric acid (GABA) after inhibition of GABA-T (4-aminobutyrate: 2-oxoglutamate aminotransferase, EC 2.6.1.19) by various doses of aminooxyacetic acid (AOAA) and gabaculine was studied in four different regions of the mouse brain. The dose-response curve for GABA accumulation after treatment with AOAA was linear up to 10 mg/kg i.p., and then leveled off. The increase in GABA accumulation after gabaculine treatment was linear up to 100 mg/kg i.p. No further increase was observed with doses up to 300 mg/kg i.p. The selectivity of both GABA-T inhibitors was assessed by measuring their effects on the content of free amino acids in mouse brain. Apart from the substantial increase in the GABA concentration, there were significant decreases in the content of glutamic acid, aspartic acid, alanine and glutamine, and an increase in ornithine content after administration of gabaculine. The same changes in amino acid content were observed after treatment with AOAA, but the level of lysine was also increased and the change in alanine level was biphasic. All these changes, however, were very small compared with the large increase in GABA level. A method for estimating the rate of the GABA turnover in vivo by measuring the initial rate of GABA accumulation after administration of AOAA or gabaculine is proposed, and the validity of the two techniques is discussed. The effect of diazepam on GABA levels and on the gabaculine-induced accumulation of GABA was studied. The results obtained with diazepam show that this method can provide valuable insight into the effects of drugs on GABAergic mechanisms in vivo.     

Enzymatic and metabolic evidence for a region specific mitochondrial dysfunction in brains of murine succinic semialdehyde dehydrogenase deficiency (Aldh5a1-/- mice)

Succinic semialdehyde dehydrogenase deficiency, a rare inherited defect of gamma-aminobutyrate (GABA) catabolism, presents with characteristic biochemical abnormalities in the central nervous system (CNS). These include elevated concentrations of GABA, gamma-hydroxybutyrate (GHB), succinic semialdehyde (SSA), 4,5-dihydroxyhexanoic acid (DHHA) and alanine as well as decreased concentrations of glutamine. GABA degradation is coupled to Krebs cycle function in mammalian CNS ("GABA shunt") through succinate and alpha-ketoglutarate. Accordingly, we hypothesized that disruption of Krebs cycle and respiratory chain function in the CNS is involved in the neuropathogenesis of this disease. For this purpose, we investigated cerebral activities of Krebs cycle and respiratory chain enzymes as well as the glutathione content in Aldh5a1(-/-) mice, a recently generated mouse model for this disease. In CNS tissue of Aldh5a1(-/-) mice, we found a significantly decreased glutathione content (hippocampus, cortex) and decreased activities of complexes I-IV (hippocampus) suggesting increased oxidative stress and mitochondrial dysfunction. However, specific activities of Krebs cycle and respiratory chain were not affected by GABA, GHB, SSA, or DHHA (up to 1 mmol/L). Although our results suggest hippocampal and cortical dysfunction in Aldh5a1(-/-) brain, we found no evidence that accumulating key metabolites of SSADH deficiency directly induce impairment of energy metabolism.     

Immunohistochemical study of arachidonate 12-lipoxygenase in porcine tissues

12-Lipoxygenase oxygenates the 12 position of arachidonic acid and produces its 12-hydroperoxy derivative. The enzyme is found in greatest amounts in porcine leukocytes and is distributed widely in various other tissues. An anti-12-lipoxygenase antibody was raised in rabbits with the immunoaffinity-purified enzyme as an antigen and was used in immunohisto- and cytochemical studies on the enzyme, the physiological significance of which remains to be clarified. When peripheral blood cells were examined by immunoelectron microscopy, the enzyme was found in neutrophils and monocytes but was not detected in lymphocytes, platelets, and erythrocytes. In immunostained neutrophils and monocytes the enzyme was localized in the cytosol but was not clearly detected in the plasma membrane, nuclear membrane, endoplasmic reticulum, and other organelles. Several other organs known to contain considerable amounts of 12-lipoxygenase were also investigated immunohistochemically, i.e., alimentary tract (ileum and jejunum), lymphatic organs (spleen, lymph node, and thymus), ovary, lung, liver, and others. In these organs, resident mast cells and granulocytes infiltrating the interstitial tissues were positively immunostained. The enzyme was not detected in parenchymal cells of these organs under our experimental conditions.     

Self‐pollination and parthenocarpic ability in developing ovaries of self‐incompatible Clementine mandarins (Citrus clementina)

This study aimed to determine if self‐pollination is needed to trigger facultative parthenocarpy in self‐incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). ‘Marisol’ and ‘Clemenules’ mandarins were selected, and self‐pollinated and un‐pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self‐pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time‐course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA₁ and GA₄), auxin (IAA) and abscisic acid (ABA) content in the ovary. ‘Clemenules’ showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self‐pollination did not stimulate parthenocarpy, whereas both un‐pollinated and self‐pollinated ovaries set fruit regardless of the cultivar. On the other hand, ‘Marisol’ un‐pollinated flowers showed greater parthenocarpic ovary growth than ‘Clemenules’ un‐pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of ‘Marisol’ paralleled higher levels of GA₁ in the ovary (+34% at anthesis). ‘Marisol’ ovary also showed higher hexoses and starch mobilization, but lower ABA levels (?64% at anthesis). Self‐pollination did not modify carbohydrates or GA content in the ovary compared to un‐pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination‐independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self‐incompatible mandarins is constitutively regulated.