Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

gamma-Aminobutyric acid (GABA) is a neurotransmitter that also occurs in a few non-neuronal cell types, where it may serve as a paracrine modulator. GABA is biosynthesized from glutamate by glutamate decarboxylase (GAD) and from putrescine via diamine oxidase (DAO). GAD is demonstrable in several GABA-positive cell types but is undetectable in the GABA-containing gastrin cells and somatostatin cells of the antropyloric mucosa of the stomach. Using two antisera raised against synthetic peptides corresponding to two different regions of rat DAO, we now demonstrate strong reactivity for DAO in gastrin-positive cells of the rat antropyloric mucosa, whereas somatostatin-positive cells as well as other structures of the antrum are unreactive. Western blotting analysis of antrum and colon demonstrate that both antisera react with a single band of 85 kD, consistent with the predicted molecular weight of DAO. Expression of DAO mRNA in the antrum is demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Our results strongly indicate that gastrin cells produce GABA via DAO-catalyzed oxidation of putrescine, and experimental data moreover suggest that the biosynthesis of GABA is regulated by the prandial state. Because GABA modulates release of somatostatin, these results point to a new mechanism of paracrine interaction between gastrin cells and somatostatin cells.
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PMID:Immunocytochemical evidence suggesting that diamine oxidase catalyzes biosynthesis of gamma-aminobutyric acid in antropyloric gastrin cells. 1082 Jan 57

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.
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PMID:The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues. 1099 66

To examine the direct effects of neurosteroids on gamma-aminobutyric acid type A (GABA(A)) receptor expression, we exposed developing neuronal cells (P19) in vitro to 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP, allopregnanolone). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed a concentration-dependent decrease in GABA(A) receptor alpha4 subunit mRNA expression that reversed 24 h after steroid withdrawal. These data suggest that variations in neurosteroid levels regulate the pattern of GABA(A) receptor subunit expression and may alter the trophic effects of GABA.
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PMID:3alpha-hydroxy-5alpha-pregnan-20-one exposure reduces GABA(A) receptor alpha4 subunit mRNA levels. 1110 37

Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic gamma-aminobutyric acid type A (GABA(A)) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei-the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABA(A) channels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 +/- 2.5 vs. 3.8 +/- 0.7 (SE) ms, respectively]. GABA(A) receptors in nRt and VB cells were pharmacologically distinct. Zn(2+) (100 microM) reduced GABA(A) channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed alpha1-alpha3, alpha5, beta1-3, gamma2-3, and delta, while nRt cells expressed alpha3, alpha5, gamma2-3, and delta. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABA(A) receptor heterogeneity within individual thalamic neurons. beta subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABA(A) receptors on beta subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding beta subunits, our findings do support alpha subunits, possibly alpha1 versus alpha3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the gamma2 and delta subunits previously implicated in Zn(2+) sensitivity are both expressed in each cell type, the observed differential Zn(2+) actions at VB versus nRt GABA(A) receptors may involve other subunit differences.
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PMID:Kinetic and pharmacological properties of GABA(A) receptors in single thalamic neurons and GABA(A) subunit expression. 1169 21

The development of synthetic enzymes in the GABAergic system (GAD(67) and GAD(65)) of the rat retina was analyzed from birth to the 4th postnatal week by the reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. As previously observed for GABA, immunoreactive GAD(67) profiles are seen clearly in the inner retinal layers at birth. At the end of the 1st week of postnatal life, immunolabeling is detected in amacrine and/or ganglion cells and in horizontal cells. GAD(67) immunoreactivity is transiently expressed in horizontal cells and disappears during the 3rd postnatal week. GAD(65) however does not develop until the 5th postnatal day. Immunolabeling is detected in the processes layering the inner plexiform layer (IPL) before being detected in the amacrine and/or ganglion cell bodies. The appearance of transcripts for GAD coincided with the appearance of the proteins. A transient form of mRNA transcripts of the GAD(67) gene containing an extra exon (ES-exon) is also observed which disappears progressively from birth to the 4th postnatal week. This form synthesizes a truncated, enzymatically inactive protein, which could participate in the regulation of GABA synthesis from glutamate present at high levels during retinogenesis.
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PMID:Differential expression of GAD(65) and GAD(67) during the development of the rat retina. 1170 Nov 36

The effects of GABA in the CNS are mediated by three different GABA receptors: GABA(A), GABA(B) and GABA(C) receptors. GABA(A) and GABA(B) receptors, but not yet GABA(C) receptors, have been demonstrated in the enteric nervous system, where GABA has been proposed to be a transmitter. The purpose of this study was to determine whether GABA(C) receptors are present and thus may play a role in mediating the effects of GABA in the myenteric plexus of the rat gastrointestinal tract. We examined the expression of the three known GABA(C) receptor subunits, rho1, rho2 and rho3, in the rat duodenum, ileum and colon using the reverse transcriptase-polymerase chain reaction. We determined the localization of GABA(C) receptors in the myenteric plexus of these regions using two different antisera directed against GABA(C) receptor subunits. The polymerase chain reaction revealed that all three subunits were expressed in the gastrointestinal tract. When the layers of the intestine were separated and the layer containing myenteric neurons was assayed, the rho3 subunit was found in the ileum and colon, whereas rho1 was expressed in the duodenum and weakly in the colon and rho2 was expressed in the ileum. Immunocytochemistry revealed numerous labeled neurons in the myenteric plexus of each region. Colocalization showed that a large proportion of calbindin plus calretinin immunoreactive neurons (intrinsic primary afferent neurons) were immunoreactive for the GABA(C) receptor, and that 56% of nitric oxide synthase immunoreactive neurons (inhibitory motor neurons) exhibited the receptor. These results indicate that GABA(C) receptors of differing subunit compositions are expressed by neurons in the rat gastrointestinal tract. The effects of GABA on intrinsic sensory and on inhibitory motor neurons are likely to be mediated in part through GABA(C) receptors.
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PMID:Gene expression and localization of GABA(C) receptors in neurons of the rat gastrointestinal tract. 1174 57

It has been proposed that the antimitogenic action of progesterone (P(4)) is mediated through a membrane receptor that has GABA(A) receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P(4) with its gamma amino butyric acid(A) (GABA(A)) receptor-activating metabolite, 5alpha-pregnane-3alpha-21-diol-20-one (5alpha3alpha). These studies revealed that P(4) was more effective than 5alpha3alpha in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P(4) bound to SIGCs with an apparent dissociation constant (K(d)) of 0.32 +/- 0.09 microM, whereas 5alpha3alpha bound with an apparent K(d) of 40 +/- 19 microM. Further, the GABA(A) antagonist, bicuculline, did not attenuate P(4)'s antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known alpha chains of the GABA(A) receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P(4) does not mediate its action via a GABA(A)-like receptor. Additional studies revealed that P(4) regulated intracellular free calcium levels ([Ca(2+)](i)) as part of its antimitotic action. Specifically, P(4) maintained a basal [Ca(2+)](i) level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca(2+)](i) but also attenuated P(4)'s antimitogenic effect. P(4)'s actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P(4) (HP-P(4)), which is cell impermeable, was as effective in blocking mitosis as P(4). Progesterone receptor (PR) mRNA was not detected in SIGCs by RT-PCR analysis, which is consistent with the findings in GCs. However, a 60-kDa protein was detected within crude membrane fractions of both GCs and SIGCs using an antibody directed against the ligand binding domain of the PR (C-262). This antibody was also used in immunocytochemical studies to detect a protein that was associated with the plasma membrane of SIGCs. It is proposed that this 60-kDa protein mediates P(4)'s membrane-initiated actions.
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PMID:Membrane-initiated events account for progesterone's ability to regulate intracellular free calcium levels and inhibit rat granulosa cell mitosis. 1213 70

Long-term horizontal optokinetic stimulation (HOKS) decreases the gain of the horizontal optokinetic reflex and evokes the second phase of optokinetic afternystagmus (OKAN-II). We investigated the possible molecular constituents of this adaptation. We used a differential display reverse transcriptase-PCR screen for mRNAs isolated from retinas of rabbits that received HOKS. In each rabbit, we compared mRNAs from the retina stimulated in the posterior-->anterior (preferred) direction with mRNAs from the retina stimulated in the anterior-->posterior (null) direction. Acyl-CoA-binding protein (ACBP) mRNA was one of four mRNAs selected by this screen, the proteins of which interact with GABA receptors. HOKS in the preferred direction increased ACBP mRNA transcription and ACBP protein expression. ACBP was localized to Muller glial cells by hybridization histochemistry and by immunohistochemistry. ACBP interacts with the alpha1-subunit of the GABA(A) receptor, as determined by a yeast two-hybrid technique. This interaction was confirmed by coimmunoprecipitation of ACBP and the alpha1-subunit of the GABA(A) receptor using an antibody to GABA(A)alpha1. The interaction was also confirmed by a "pull-down" assay in which histidine-tagged ACBP was used to pull down the GABA(A)alpha1. ACBP does not cross the blood-brain barrier. However, smaller truncated proteolytic fragments of ACBP do, increasing the excitability of central cortical neurons. Muller cells may secrete ACBP in the inner plexiform layer, thereby decreasing the sensitivity of GABA(A) receptors expressed on the surface of ganglion cell dendrites. Because retinal directional sensitivity is linked to GABAergic transmission, HOKS-induced expression of ACBP could provide a molecular basis for adaptation to HOKS and for the genesis of OKAN-II.
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PMID:Activity-dependent expression of acyl-coenzyme a-binding protein in retinal muller glial cells evoked by optokinetic stimulation. 1476 20

Hypoxia causes dysfunction of excitatory and inhibitory neurotransmission, often resulting in encephalopathy, seizures or myoclonus. We evaluated the effects of hypoxia on GABAA receptor (GABAAR) function and expression in an in vitro model of neuronal hypoxia. NT2-N cells, derived from the human NT2 teratocarcinoma cell line, were exposed to < or =1% O2 for 8 h and then used immediately for experiments or allowed to recover under normoxic conditions (95% air/5% CO2) for 24, 48 or 96 h. Hypoxic treatment did not cause obvious morphological changes or cell death. In whole-cell patch-clamp recordings, the GABA current EC50 was unchanged, however, maximal GABA-evoked currents changed in a biphasic manner. Maximal GABA currents were significantly increased immediately after hypoxia, but were significantly reduced after 48 h normoxic recovery, and then returned to baseline after 96 h recovery. Maximal potentiation of 10 microM GABA currents by diazepam was increased 48 h after hypoxia, but potentiation by zolpidem was decreased. Barbiturate enhancement and zinc inhibition of GABA currents were unchanged. Semiquantitative reverse transcriptase (RT)-PCR showed decreased alpha1, alpha5, beta2 and gamma2 subunit mRNA after hypoxia. Hypoxic exposure altered GABAAR physiology and subunit mRNA expression, which may correlate with symptoms observed after hypoxia in vivo.
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PMID:Hypoxia alters GABAA receptor function and subunit expression in NT2-N neurons. 1497 87

The role of interneurons in neurovascular coupling was investigated by patch-clamp recordings in acute rat cortical slices, followed by single-cell reverse transcriptase-multiplex PCR (RT-mPCR) and confocal observation of biocytin-filled neurons, laminin-stained microvessels, and immunodetection of their afferents by vasoactive subcortical cholinergic (ACh) and serotonergic (5-HT) pathways. The evoked firing of single interneurons in whole-cell recordings was sufficient to either dilate or constrict neighboring microvessels. Identification of vasomotor interneurons by single-cell RT-mPCR revealed expression of vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS) in interneurons inducing dilatation and somatostatin (SOM) in those eliciting contraction. Constrictions appeared spatially restricted, maximal at the level of neurite apposition, and were associated with contraction of surrounding smooth muscle cells, providing the first evidence for neural regulation of vascular sphincters. Direct perfusion of VIP and NO donor onto the slices dilated microvessels, whereas neuropeptide Y (NPY) and SOM induced vasoconstriction. RT-PCR analyses revealed expression of specific subtypes of neuropeptide receptors in smooth muscle cells from intracortical microvessels, compatible with the vasomotor responses they elicited. By triple and quadruple immunofluorescence, the identified vasomotor interneurons established contacts with local microvessels and received, albeit to a different extent depending on interneuron subtypes, somatic and dendritic afferents from ACh and 5-HT pathways. Our results demonstrate the ability of specific subsets of cortical GABA interneurons to transmute neuronal signals into vascular responses and further suggest that they could act as local integrators of neurovascular coupling for subcortical vasoactive pathways.
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PMID:Cortical GABA interneurons in neurovascular coupling: relays for subcortical vasoactive pathways. 1548 13


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