Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

Swimming behavior regulation by GABAB receptors in Paramecium

In Paramecium, internal Ca(2+) concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca(2+) influx, and the addition of drugs that block the Ca(2+) current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABA(B) receptors in P. primaurelia swimming control. The presence of GABA(B) immunoanalogue in Paramecium was evidenced using SDS-PAGE, Western blotting, and confocal laser scanning microscopy. By applying the specific GABA(B) receptor agonist baclofen, a dose-dependent inhibition of the membrane depolarization-induced CR duration was observed. This inhibition was antagonized by phaclofen, persisted when K(+) channel blockers were applied, and disappeared after treatment with nifedipine and verapamil. Moreover, the action of baclofen on depolarization-induced CR was suppressed by treatment with pertussis toxin. Therefore, these experiments suggest that baclofen modulates CR by a G protein (G(0) or G(1)) mediated inhibition of dihydropyridine-sensible calcium channels. Finally, synthesis and release of GABA in the environment by Paramecium have been demonstrated by HPLC. Possible correlations between GABA(B) receptor activation and the regulation of intracellular Ca(2+) levels are discussed.     

Endothelins stimulate the production of stromelysin-1 in cultured rat astrocytes

The effects of endothelins (ETs) on the production of stromelysins, a sub-family of matrix metalloproteinases, were examined in cultured astrocytes. The treatment of cultured rat astrocytes with ET-1 increased stromelysin-1 mRNA levels, while stromelysin-2 and -3 mRNAs were not affected. Immunocytochemical observations showed that cultured astrocytes produced stromelysin-1 protein. ET-1 and Ala^1,3,11,15-ET-1, an ET_B receptor selective agonist, stimulated the release of stromelysin-1 from cultured astrocytes. Accompanying the increase in protein release, the peptidase activity of stromelysin-1 in the medium was also increased by ET-1. The effects of ET-1 on astrocytic stromelysin-1 expression were inhibited by PD98059, staurosporine, and Ca²⁺ chelation, but not by SB203580 or pyrrolidine dithiocarbamate. These results show that activation of astrocytic ET receptors stimulates the production of stromelysin-1, suggesting a role for ETs in stromelysin production in brain pathologies.     

The GABAB receptor associates with regulators of G-protein signaling 4 protein in the mouse prefrontal cortex and hypothalamus

Regulators of G-protein signaling (RGS) proteins regulate certain G-protein-coupled receptor (GPCR)-mediated signaling pathways. The GABA_B receptor (GABA_BR) is a GPCR that plays a role in the stress response. Previous studies indicate that acute immobilization stress (AIS) decreases RGS4 in the prefrontal cortex (PFC) and hypothalamus (HY) and suggest the possibility of a signal complex composed of RGS4 and GABA_BR. Therefore, in the present study, we tested whether RGS4 associates with GABA_BR in these brain regions. We found the co-localization of RGS4 and GABA_BR subtypes in the PFC and HY using double immunohistochemistry and confirmed a direct association between GABA_B2R and RGS4 proteins using co-immunoprecipitation. Furthermore, we found that AIS decreased the amount of RGS4 bound to GABA_B2R and the number of double-positive cells. These results indicate that GABA_BR forms a signal complex with RGS4 and suggests that RGS4 is a regulator of GABA_BR. [BMB Reports 2014; 47(6): 324-329]     

Effects of pentobarbital tolerance to and dependence on GABAB receptor binding

Effects of pentobarbital pellet implantation on [³H]baclofen binding in the frontal cortex of cerebellum of rat brains were examined. In the frontal cortex, pentobarbital tolerance caused an increase in the number of binding sites (B_max) without changing their affinity (K_D). Twenty-four hours after withdrawal of the pentobarbital pellets, there was a significant increase in the K_D and B_max values. Cerebellar binding, in contrast, was not significantly changed in any of the treatment groups. Addition of 1 mM of pentobarbital directly to binding assays using cortical membrane produced as increase in K_D without a change in B_max.In vitro, pentobarbital affected neither the K_D nor the B_max in the cerebellar [³H]baclofen binding. These results suggest that like the GABA_A receptor, [³H]baclofen binding to the GABA_B receptor in rat frontal cortex was affected by pentobarbital tolerance and dependence, and that there are regional differences in the properties of the GABA_B receptor.     

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.     

The Dictyostelium repertoire of seven transmembrane domain receptors

The availability of fully sequenced genomes allows the in silico analysis of whole gene families in a given genome. A particularly large and interesting gene family is the G-protein-coupled receptor family. These receptors detect a variety of extracellular signals and transduce them, generally via heterotrimeric G-proteins, to effector proteins inside the cell and thus elicit a physiological response. G-protein-coupled receptors are found in all eukaryotes and constitute in vertebrates 3–5% of all genes. They are also very important drug targets and approximately 25 of the top 100 selling drugs are directed against these receptors. The Dictyostelium discoideum genome contains a surprisingly high number of 55 such receptors, approximately 0.5% of the encoded genes. Besides the four well-studied cAMP receptors the genome encodes eight additional cAMP receptor-like proteins and one of these is distinguished by a novel domain structure, one secretin-like receptor, 17 GABA_B-like and 25 Frizzled-like receptors. The existence of the latter three types of receptors in D. discoideum was surprising because they had not been observed outside the animal kingdom before. Their presence suggests unprecedentedly complex and so far unknown signaling activities in this lower eukaryote.     

Functional GABA(B) receptors expressed in cultured calvarial osteoblasts

In immature and mature primary cultured rat calvarial osteoblasts, both mRNA and corresponding proteins were constitutively expressed for 2 splice variants of GABA(B) receptor (GABA(B)R) subunits but not for any known GABA(A) and GABA(C) receptor subunits. The agonist for GABA(B)R baclofen significantly inhibited cAMP formation induced by forskolin in a manner sensitive to the antagonist 2-hydroxysaclofen. Similar expression was seen with mRNA for GABA(B)R-1a and -1b splice variants in the murine calvarial osteoblast cell line MC3TC-E1 cells cultured for 7-21 days in vitro (DIV). In these MC3T3-E1 cells, baclofen not only inhibited the activity of alkaline phosphatase, but also exacerbated Ca2+ accumulation, throughout the culture period up to 28 DIV. These results suggest that GABA may play an unidentified role in mechanisms associated with cellular proliferation, differentiation, and/or development through functional GABA(B)R constitutively expressed in cultured osteoblasts.     

Cerebral GABAB receptor: Proposed mechanisms of action and purification procedures

The GABA_B receptor in brain is one of the GABA receptor subtypes, and has been found to be negatively coupled to adenylate cyclase and phosphatidylinositide turnover. This receptor easily solubilizes from cerebral synaptic membrane preparations by 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. GABA_B receptor solubilized from bovine cerebral cortex was purified using baclofen-coupled affinity beads (baclofen-coupled Toyopearl beads). Using these procedures, almost pure GABA_B receptor (80 KDa protein) was obtained in the affinity eluate. A monoclonal antibody has been also raised against the purified GABA_B receptor. The antibody recognized a protein of about 80 KDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove more than 90% of the baclofen suppressive GABA binding activity in the solubilized synaptic membrane, and this system was found to be useful for the immunoaffinity column chromatographic separation of GABA_B receptor. Preliminary studies of immunohistochemical visualization of GABA_B receptor in the rat cerebellum suggested that this receptor may be exclusively localized at the presynaptic site of GABAergic neurons.Special issue dedicated to Dr. Claude Baxter.     

GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

GABA_B receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA_B1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA_B receptors. The transgenic mice express GABA_B1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA_B1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA_B receptors via the GABA_B2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA_B2. The mice equipped with tags on GABA_B1 facilitate validation and identification of native binding partners of GABA_B receptors, providing insight into the molecular mechanisms of synaptic modulation.     

Synthesis of the Nanomolar Photoaffinity GABAB Receptor Ligand CGP 71872 Reveals Diversity in the Tissue Distribution of GABAB Receptor Forms

A radioiodinated probe, [¹²⁵I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA_B receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at 130 and 100 kDa believed to represent the long (GABA_BR1a) and short (GABA_BR1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA_B receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [¹²⁵I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (−)-baclofen, (+)-baclofen and ( )-glutamic acid with a rank order and stereospecificity characteristic of the GABA_B receptor. Photoaffinity labeling experiments revealed that the recombinant GABA_BR2 receptor does not bind [¹²⁵I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA_BR1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA_BR1a and GABA_BR1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA_BR1b receptor form was detected in kidney and liver whereas the long GABA_BR1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [¹²⁵I]-CGP 71872 and CGP 71872 GABA_BR1 ligands, and differential tissue expression of the long GABA_BR1a and short GABA_BR1b receptor forms in rat and dog.     

Enhancement of androgen receptor expression induced by (R)-methanandamide in prostate LNCaP cells

It has been recently shown that cannabinoids may regulate the growth of many cell types. In the present work we examined the effect of the anandamide analogue (R)-methanandamide (MET) on androgen-dependent prostate LNCaP cell growth. We found that 0.1 microM MET had a mitogenic effect measured by [(3)H]thymidine incorporation into DNA. The effect exerted by MET was blocked by the cannabinoid receptor antagonists SR141716 (SR1) and SR144528 (SR2) as well as by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, suggesting an involvement of cannabinoid receptors and the PI3K pathway in the mechanism of MET action. MET treatment of LNCaP cells also induced an up-regulation of androgen receptor expression that was blocked by the two cannabinoid receptor antagonists SR1 and SR2. These results show for the first time that cannabinoids may modify androgen receptor expression in an androgen-dependent cell line and by this mechanism could regulate prostate cell growth.     

Endothelin-1 stimulates glial cell line-derived neurotrophic factor expression in cultured rat astrocytes

Effects of endothelin-1 (ET-1) on glial cell line-derived neurotrophic factor (GDNF) production in cultured astrocytes were examined. Treatment of cultured astrocytes with ET-1 (100 nM) increased mRNA levels of GDNF in 1-6h. The effect of ET-1 was inhibited by BQ788, an ET(B) receptor antagonist, but not by FR139317, an ET(A) receptor antagonist. ET-1 stimulated release of GDNF into culture medium. Dexamethasone (1 microM) and pyrrolidine dithiocarbamate (PDTC, 100 microM), which inhibit activation of NFkappaB, prevented the increases in GDNF mRNA by H(2)O(2). In contrast, the effect of ET-1 was not affected by dexamethasone and PDTC. The increase of astrocytic GDNF mRNA by ET-1 was inhibited by BAPTA/AM (30 microM) and PD98059 (50 microM), but not by calphostin C, staurosporine, and cyclosporine A. These results suggest that ET-1 stimulated expression of astrocytic GDNF through ET(B) receptor-mediated increases in cytosolic Ca(2+) and ERK activation.     

Lipase-catalyzed enantioselective synthesis of (R,R)-lactide from alkyl lactate to produce PDLA (poly D-lactic acid) and stereocomplex PLA (poly lactic acid)

R-lactide, a pivotal monomer for the production of poly (D-lactic acid) (PDLA) or stereocomplex poly (lactic acid) (PLA) was synthesized from alkyl (R)-lactate through a lipase-catalyzed reaction without racemization. From among several types of lipase, only lipase B from Candida antarctica (Novozym 435; CAL-B) was effective in the reaction that synthesized (R,R)-lactide. Enantiopure (R,R)-lactide, which consisted of over 99% enantiomeric excess, was synthesized from methyl (R)-lactate through CAL-B catalysis. Removal of the methanol by-product was critical to obtain a high level of lactide conversion. The (R,R)-lactide yield was 56% in a reaction containing 100 mg of Novozym 435, 10 mM methyl (R)-lactate and 1500 mg of molecular sieve 5 A in methyl tert-butyl ether (MTBE). The important monomer (R,R)-lactide that is required for the production of the widely recognized bio-plastic PDLA and the PLA stereocomplex can be obtained using this novel synthetic method.     

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.     

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([(125)I]rUII) revealed the presence of high- and low-affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII(4-11) and [3-iodo-Tyr9]hUII(4-11) were also very potent in displacing [(125)I]rUII from its binding sites, whereas the non-cyclic analogue [Ser5,10]hUII(4-11) and somatostatin-14 could only displace [(125)I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [(125)I-Tyr0,D-Trp8]somatostatin-14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [(3)H]inositol incorporation and increased intracellular Ca(2+) concentration in a dose-dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [(125)I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high- and low-affinity UII binding sites coupled to G proteins, the high-affinity receptor exhibiting the same pharmacological and functional characteristics as UT.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.