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.     

Higher GABA Concentrations in Fallopian Tube Than in Brain of the Rat

Abstract: The GABA content was determined simultaneously in two peripheral organs, i.e., ovary and Fallopian tube. Moreover, the effects of inhibitors of glutamate decarboxylase or γ-aminobutyrate transaminase (GABA-T) on the GABA concentrations of the two organs were examined, to point out similarities and differences between central and peripheral pathways of GABA biosynthesis and degradation. In ovary, GABA concentration was found to be about 30% of that in total brain tissue. Furthermore, isoniazid and thiosemicarbazide caused significant reduction of GABA levels in peripheral organs. In contrast to the CNS, aminooxyacetic acid failed to increase, but even produced a significant diminution in peripheral GABA content. Gabaculine did not change GABA levels. In conclusion, it has been demonstrated for the first time that a peripheral organ, i.e. fallopian tube, contained higher GABA concentrations than the CNS. On the other hand, in the organs examined GABA seemed to be synthesized similarly, but metabolized by a pathway different from that in the brian.     

Comparative Characterization of Glutamate Decarboxylase in Crude Homogenates of Oviduct, Ovary, and Hypothalamus

Some biochemical characteristics of L-glutamate decarboxylase (GAD) were compared using crude homogenates of the rat oviduct, ovary, and hypothalamus. As estimated by the measurement of CO2 production, the specific activities of oviductal and ovarian GAD were about 10 and 3% of the hypothalamic value, respectively. Stoichiometric formation of gamma-aminobutyric acid (GABA) and CO2 from L-glutamate could be observed in oviduct and hypothalamus, while in ovarian homogenates the production of CO2 was more than nine times that of GABA. Hypothalamic and tubal GAD showed similar time course, temperature dependence, and pH dependence. The dependence on protein concentration and on exogenous cofactor supply was also similar in these two tissues. The kinetic constant for L-glutamate as a substrate was nearly the same in oviduct (1.30 mM) and hypothalamus (1.64 mM). The responsiveness of tubal and hypothalamic GAD to various inhibitors was also similar. The present findings indicate that the oviductal and the hypothalamic GAD may be identical, and they suggest a possible GABAergic innervation of the Fallopian tube.     

Gamma-aminobutyric acid in the genital tract of the rat during the oestrous cycle

The highest values of gamma-aminobutyric acid (GABA) in the genital tract of the rat at different stages of the oestrous cycle were found in the oviduct (3.5-7 micrograms/mg protein) and the lowest in the ovary (50-100 ng/mg protein). The values for uterus and vagina ranged between 80 and 150 ng/mg protein. GABA (10-30 ng/microliter) was also found in fluid in the ovarian bursa. At 11:00 h, on the day of oestrus, GABA content increased in the ovaries but values in the oviducts were maximal at 11:00 h on the day of pro-oestrus. Variations in GABA content of the vagina were also found. Uterine cervix or uterine horn showed no changes during the oestrous cycle. The GABA content was not uniform throughout the oviduct: the highest values were found in the portion next to the ovary. At 10 days after removal of the right oviduct, GABA values in the ovary and ovarian bursa fluid decreased on the operated side. At 1 month after surgery, the values in ovary were normal but the values in ovarian bursa fluid were still low, suggesting that the source of ovarian GABA was not the oviduct. The variations observed in the present paper suggest an involvement of GABA in reproductive physiology.     

Correlation of ovarian binding of 125I-HCG with formation of cAMP, estradiol and progesterone during pregnancy.

The content of ovarian gonadotropin receptors in rat was found to change during pregnancy. The specific binding of 125I-HCG to ovarian homogenates rose to its maximal values on days 13 and 16. Thereafter, the binding declined as parturition approached. No consistent changes in responsiveness of rat ovary to LH in synthesis of cAMP and estradiol were observed during pregnancy. Plasma estradiol concentration increased on day 16 and remained high throught days 21 and 22. Progesterone levels increase steadily during the first half of pregnancy and then fall, especially sharply on day 21 and 22. The results demonstrate that the secretion of progesterone correlates with gonadotropin receptors during pregnancy.     

Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy

Levels of preprorelaxin mRNA in the rat ovary during pregnancy were determined by cell-free translation and by hybridization analyses with cloned preprorelaxin cDNA. Translation of poly(A+) RNA from rat ovaries taken at different stages of pregnancy resulted in the incorporation of [35S]cysteine into two peptides, of Mr 17,500 and 20,500, that were specifically bound by anti-relaxin IgG. Both peptides also were demonstrated by translation of ovarian poly(A+) RNA that was hybrid-selected with cloned preprorelaxin cDNA, the sequence of which corresponds to the Mr 20,500 peptide. The origin of the Mr 17,500 putative precursor is not presently known. Preprorelaxin mRNA translational activities corresponded to previously reported concentrations of relaxin in rat ovaries during pregnancy. The results of hybridization analyses, both by Northern blotting of poly(A+) RNA and dot blotting of unfractionated RNA, agreed with those of translation assays. Preprorelaxin mRNA activity/concentration was low in early pregnancy, rose markedly and reached a plateau on days 15-20 (about 1-2% of total translation activity), and then fell to low levels again by day 23, the time of parturition. These findings indicate that the concentration of relaxin in the rat ovary is directly dependent on preprorelaxin mRNA levels.     

High-Affinity, Sodium-Dependent Γ-Aminobutyric Acid Uptake by Slices of Rat Ovary

The high concentration of gamma-aminobutyric acid (GABA) recently demonstrated in rat ovary prompted us to examine the capacity of ovarian slices to take up [3H]GABA. Active uptake, dependent on temperature and sodium concentration, was observed and a kinetic constant (Km) of 1.0 microM found for the uptake process. Ouabain (100 microM) reduced the rate of accumulation of [3H]GABA. Uptake was inhibited only partially by 100 microM d,l-nipecotic acid, but more strongly by 100 microM beta-alanine. These results suggest that the uptake system in ovary possesses properties similar to those of high-affinity GABA transport systems in the brain.     

The disappearance of CD34-positive and alpha-smooth muscle actin-positive stromal cells associated with human intra-uterine and tubal pregnancies

In order to elucidate the change in alpha-smooth muscle actin (ASMA)-positive and CD34-positive stromal cells associated with pregnancy, we examined endometrial and Fallopian tube tissues from 40 patients including normal endometrium (n=10), intra-uterine pregnancy (n=10), normal Fallopian tube (n=10), and tubal pregnancy (n=10), using immunohistochemistry. In normal endometrium, only a few ASMA-positive cells were focally observed. Additionally, a wide range of CD34-positive stromal cell abundance was observed. In normal Fallopian tube mucosa, a small to moderate number of both ASMA-positive and CD34-positive stromal cells was observed. Neither ASMA-positive nor CD34-positive stromal cells were observed anywhere in the decidual stroma during both intra-uterine and tubal pregnancies. Likewise, a varying abundance of ASMA-positive cells but no CD34-positive stromal cells were observed at the fetal side during both intra-uterine and tubal pregnancies. In conclusion, the disappearance of CD34-positive and ASMA-positive stromal cells may be an indicator of decidualisation induced change in the stroma during both intra-uterine and tubal pregnancies. ASMA-positive stromal cells at the fetal side associated with pregnancy may play a role in the production of villous extracellular matrix or regulation of blood flow.     

Distribution of γ-Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20-microns thick freeze-dried sections (0.2-1.5 micrograms dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10-fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.     

Presence of γ-Aminobutyric Acid in Rat Ovary

Abstract: As γ-aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non-neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.     

Alterations in γ-aminobutyric acid content in the rat superior cervical ganglion and pineal gland

The endogenous γ-aminobutyric acid (GABA) content of the rat pineal gland and superior cervical ganglion (SCG) was measured by high pressure liquid chromatography. It was found that GABA levels in both tissues increased after decapitation of the animals. The GABA content of tissues frozen within 20 seconds after decapitation was the same as that of tissues removed from animals killed by microwave irradiation. Amino-oxyacetic acid, a GABA-transaminase inhibitor, increased the endogenous GABA content of both of these tissues. Dimethylphenylpiperizinium or isoniazid administration did not alter GABA levels in these tissues. Isoproterenol increased the GABA content of the SCG but did not change the pineal gland GABA levels. The ability of the pineal gland to take up and accumulate ³H-GABA was significantly reduced in rats that had been ganglionectomized. A fluctuation in endogenous GABA levels in the pineal gland was seen to occur when measures were taken at different times of the day. These results tend to suggest that GABA may have some functional role in the pineal gland and the superior cervical ganglion.     

Evidence for an extended duration of GABA-mediated excitation in the developing male versus female hippocampus

Gamma-aminobutyric acid (GABA) is as an excitatory neurotransmitter during brain development. Activation of GABA(A) receptors in neonatal rat hippocampus results in chloride efflux and membrane depolarization sufficient to open voltage sensitive calcium channels. As development progresses, there is a decline in the magnitude of calcium influx subsequent to GABA(A) receptor activation and the number of cells that respond to GABA with excitation. By the second postnatal week in the rat, GABA action in the hippocampus is predominantly inhibitory. The functional consequences and endogenous regulation of developmental GABA-mediated excitation remains under-explored. Hippocampal neurons in the newborn male and female rat respond to GABA(A) receptor activation with increased intracellular calcium and are susceptible to GABA-mediated damage -- both being indicative of the excitatory nature of GABA. In the present study we observed that by postnatal day 7, only males are susceptible to GABA(A) agonist-induced damage and respond to GABA(A) agonist administration with elevated levels of intracellular calcium in cultured hippocampal neurons. By postnatal day 14, GABA(A) agonist administration was without effect on intracellular calcium in both males and females. The age-related sex difference in the impact of GABA(A) receptor activation correlates with a sex difference in chloride co-transporter expression. Males have elevated protein levels of pNKCC1 on PN0 and PN7, with no sex difference by PN14. In contrast, females displayed elevated levels of KCC2 on PN7. This converging evidence infers that sex affects the duration of GABA(A) receptor-mediated excitation during normal hippocampal development, and provides a mechanism by which the effect is mediated.     

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.     

Histamine content and mast cells distribution in mouse uterus: the effect of sexual hormones, gestation and labor

The histamine content of uteri from mice was analyzed in terms of both concentration and total amount per uterine horn a) at two stages of the estrous cycle (estrous and diestrous), b) under sex hormone treatment, c) during pregnancy and after delivery. Histamine concentration and mast cell density were greater during diestrous and in mice treated with progesterone (p less than 0.001). This effect was attributed to a reduction in uterine mass weight, since the amount of histamine per uterine horn remained constant throughout the estrous cycle. During pregnancy, both concentration and amount of histamine per uterine horn were increased, values were significantly higher than in estrous (p less than 0.001) from day 14-17 until day 21 when labor occurred. After six to eight hours post-partum an abrupt reduction on histamine content was observed. Mast cells were more abundant in myometrium than in endometrium, their density followed the same pattern as histamine concentration throughout the estrous cycle.     

mtDNA m.3635G>A may be classified as a common primary mutation for Leber hereditary optic neuropathy in the Chinese population

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via ionotropic (GABA_A and GABA_C) and metabotropic (GABA_B) receptors. The GABA_B receptor is a dimer composed of R1 and R2 components. In addition to their location on neurons, GABA and functional GABA_B receptors also have been detected in some peripheral tissues. In the present study, we combined immunohistochemistry, immunoblot and tension recording to determine if the human fallopian tube express glutamic acid decarboxylase (GAD65/67), two isoforms for synthesis of GABA and functional GABA_B receptors. Immunoblots showed that the human fallopian tube tissue contained GABA_BR1 protein which was localized in the epithelial cells and smooth muscle cells by immunohistochemistry. In addition, epithelial cells also expressed GAD65/67. Tension recording found that both GABA and baclofen, a GABA_B receptor agonist increased the spontaneous activity of human fallopian tube. The expressions of GABA_BR and GAD65/67 were significantly upregulated in the ectopic pregnancy group than in the intrauterine pregnancy group. We conclude that the human fallopian tube is capable of synthesizing GABA and expresses functionally active GABA_B receptors. An upregulation of GABA synthesis and corresponding GABA_B receptors may involve in ectopic pregnancy.     

Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets

OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

Evidence for Feedback Regulation of Glutamate Decarboxylase by γ-Aminobutyric Acid

Abstract: The concentration of γ-aminobutyric acid (GABA) in the human ovary and the capacity of a membrane preparation from the same organ to bind [³H]GABA specifically were examined. The GABA concentration in the ovary was found to be 214 ± 66 nmol/g frozen tissue (mean ± SEM of six independent determinations). Moreover, a single population of high-affinity GABA binding sites has been identified in the ovarian membranes. The apparent dissociation constant ( K _d) and maximum binding capacity ( B _max) were 38.3 n M and 676 fmol/mg protein, respectively. The specific binding of [³H]GABA was displaced by muscimol, unlabelled GABA, or (+)bicuculline, but was unaffected by (±)baclofen and picrotoxin. The present results show that GABA and an extremely high density of GABA_A receptor binding sites are present in the human ovary, indicating a physiological significance of this amino acid in the female reproductive system.     

BIOCHEMICAL EFFECTS IN MAN and RAT OF THREE DRUGS WHICH CAN INCREASE BRAIN GABA CONTENT

Abstract— Brain amino acids were measured in rats given aminooxyacetic acid (AOAA) by mouth, and in rats given sodium dipropylacetate (DPA) both orally and by intraperitoneal injection. Brain GABA content was significantly elevated by AOAA doses of 10mg/kg/day, but not by 5mg/kg/day. Approximately 4 times as much AOAA is required by mouth as by parenteral injection to raise brain GABA content in the rat. DPA (400mg/kg) increased brain GABA and lowered brain aspartate content significantly 1 h after a single injection. However, DPA given orally (350 mg/kg/day) produced no alterations of any amino acids in rat brain.
Amino acids were measured in plasma and urine from patients treated orally with isonicotinic acid hydrazide (INH) or DPA, and from a volunteer who took AOAA. INH (10–21 mg/kg/day) increased concentrations of β -alanine and ornithine in plasma, as well as urinary excretion of β -alanine. DPA had no such effect. AOAA in oral doses ranging from 1.25 to 5.0 mg/kg/day increased plasma concentrations of β -alanine, ornithine, β -aminoisobutyric acid, proline and hydroxyproline, and produced massive urinary excretion of β -alanine, β -aminoisobutyric acid, and taurine.
Both INH and AOAA, given in doses practical for human use, inhibit the transamination of β -alanine and ornithine in liver, and may also inhibit the transamination of GABA in brain. In addition, AOAA interferes with the catabolism of β -aminoisobutyric acid, proline, and hydroxyproline. AOAA, in the lowest dose employed, appeared more effective than INH as an inhibitor of GABA aminotransferase in man, and might therefore be useful in the treatment of neurological diseases in which brain GABA is deficient.     

Identification of gamma-aminobutyric acid and its binding sites in the rat ovary

Gamma-aminobutyric acid (GABA), GABA synthesizing enzyme and GABA binding sites were measured in rat ovaries. The concentration of GABA in the ovary (0.56 μg/mg protein) was less than that in the brain (1.2–3.4 μg/mg protein), but was six-fold higher than any other non-neuronal tissue examined. Glutamate decarboxylase, the GABA synthesizing enzyme was also found in high concentrations in whole ovarian homogenate but not in enriched ovarian granulosa cells, testis, anterior pituitary or muscles. Furthermore, high affinity (Kd = 15–21 nM), specific GABA binding sites were identified in the ovaries by specific [³H]muscimol binding and the majority of GABA binding sites were associated with the granulosa cells. These data suggest a possible role of GABA in the regulation of ovarian functions.