Differential localization of G-proteins Gi and Go in the accessory olfactory bulb of the rat.

To clarify the functional differences among G-proteins, we investigated the localization of Gi and Go in the olfactory bulb of rats by both immunohistochemical and immunochemical techniques, using purified antibodies specific to the alpha-subunits of Gi1 (Gi1 alpha), Gi2 (Gi2 alpha), and Go (Go alpha), respectively. We found that Gi2 alpha is localized exclusively in the accessory olfactory bulb, but it is present at only low levels in the main olfactory bulb. The unique pattern of immunoreactivity specific for Gi2 alpha and Go alpha within the glomeruli of the accessory olfactory bulb and the results of immunoassays indicate that the accessory olfactory bulb is divided into two parts: the anterior region is rich in Gi2, while the posterior region is rich in Go. These findings suggest that the accessory olfactory bulb has two different functions. In addition, we found that the concentration of Gi2 alpha in the accessory olfactory bulb increases during puberty and reaches the adult level at 12 weeks after birth, while that in the main olfactory bulb remains constant. By contrast, the concentrations of Go alpha in the accessory olfactory bulb and the main olfactory bulb increase with similar kinetics. These findings suggest that Gi2 is a key protein in signal transduction in the accessory olfactory bulb, and increases in its level seem to be related to sexual maturation.     

Expression of mRNA for Gs alpha and Gi2 alpha in primary cultured mouse cerebral cortical neurons.

Developmental changes of mRNAs for alpha-subunits of GTP binding protein (G-protein) such as Gs alpha and Gi2 alpha during neuronal development were examined in both primary cultured cerebral cortical neurons and cerebral cortices obtained from age-matched mice. The expression of mRNAs for both G-protein alpha-subunits in primary cultured neurons showed a development pattern similar to that in the cerebral cortex in vivo. Both mRNAs were expressed at an early stage of neuronal development, and the expression patterns of mRNA for both G-protein alpha-subunits were found to be only slightly changed during development. These results suggest that the G-protein appears at an early stage of neuronal development and may play an important role therein.     

Selective regulation of Gi alpha 1 expression and function in PC12 cells by cAMP.

Hormone and neutrotransmitter receptor systems regulate both the activity and expression of GTP-binding proteins (G-proteins). However, relatively little is known about the mechanism by which this regulation occurs. One G-protein subtype, Gi alpha 1, is expressed primarily in neuronal cells. Here, we demonstrate the selective regulation of Gi alpha 1 mRNA and protein levels by cAMP. Treatment of PC12 cells with forskolin increases Gi alpha protein levels. Similarly, incubation of PC12 cells with agents that increase intracellular levels of cAMP, including forskolin, dibutyryl-cAMP, and 8-bromo-cAMP, induce a two- to fourfold increase in Gi alpha 1 mRNA levels. Furthermore, the effect of increased intracellular cAMP is specific for Gi alpha 1 mRNA expression; the levels of mRNA encoding other G-protein subtypes remain unaltered. cAMP-stimulated Gi alpha 1 expression occurs within hours of treatment and is sustained for days. Increasing intracellular cAMP by activation of cell surface adenosine receptors also increases Gi alpha 1 mRNA levels. Treatment of PC12 cells with phorbol esters, NGF, or depolarizing concentrations of KCl did not increase Gi alpha 1 mRNA expression, demonstrating that Gi alpha 1 expression is specifically regulated by cAMP. Guanine nucleotide-mediated inhibition of adenylate cyclase activity was measured in order to determine if the change in Gi alpha protein expression was accompanied by a change in G-protein function. Adenylate cyclase activity in PC12 cells treated with an adenosine analog and therefore expressing higher levels of Gi alpha protein is more sensitive to inhibition by guanine nucleotides than in nontreated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonists determine the pattern of G-protein activation in mu-opioid receptor-mediated supraspinal analgesia

The opioids heroin, methadone, buprenorphine, and morphine produce supraspinal antinociception in CD-1 mice that is antagonized by Cys(2), Tyr(3), Orn(5), Pen(7)-amide but not by naltrindole or nor-binaltorphimine. The patterns of GTP-binding regulatory proteins (G-proteins) activation exhibited by these agonists at mu-opioid receptors were characterized. The expression of alpha-subunits of Gi-protein classes, Gi1, Gi2, Gi3, Go1, Go2 and Gz, and those of the Gq-protein family, Gq and G11, was reduced by administration of antisense oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. The ODN treatments demonstrated differences in the analgesic profiles of these opioids. Though the knock-down of G(i2)alpha or G(z)alpha subunits diminished the analgesic effects of the four opioids, impairment of G(i3)alpha did not modify the potency of morphine. In mice with reduced G(i1)alpha, G(o1)alpha or G(11)alpha levels, antinociception induced by heroin and methadone was diminished, but buprenorphine and morphine showed no change in their effects. Also, antinociception induced by heroin and buprenorphine, but neither morphine nor methadone, required intact G(o2)alpha or G(q)alpha levels. Thus, morphine, heroin, methadone, and buprenorphine showed different patterns of G-protein activation in evoking mu-opioid receptor-mediated supraspinal antinociception. Therefore, after binding identical receptors, each agonist determines the classes of GTP-binding regulatory transducer proteins to be activated.     

Potentiation of thromboxane A2-induced platelet secretion by Gi signaling through the phosphoinositide-3 kinase pathway

Platelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3'phosphate-5'-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T(AC) receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T(AC) receptor or the alpha2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2T(AC) receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.     

Immunolocalization of G protein alpha-subunits in the Drosophila CNS

In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, we have begun a multidisciplinary study of the G proteins present in the fruit fly, Drosophila melanogaster. In this report, we describe the distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit. Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning (Quan et al., 1989; Thambi et al., 1989). The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA (Provost et al., 1988). However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E. coli. Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina contained low levels of these alpha-subunits in the CNS. Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina. Little or no Gi alpha was detected in other brain regions or in the thoracic ganglion. Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila.     

Deciphering the role of Gi2 in opioid-induced adenylyl cyclase supersensitization

Prolonged opioid treatment of HEK 293 cells expressing opioid receptors are known to induce adenylyl cyclase supersensitization, a process that requires pertussis toxin (PTX)-sensitive G(i/o) proteins. Here, the role of Gi2 in adenylyl cyclase supersensitization was investigated. A PTX-insensitive G alpha(i2)/z chimera was stably co-expressed with mu-, kappa- or delta-opioid receptors in HEK 293 cells. Functional coupling of G alpha(i2)/z to the opioid receptors was demonstrated by opioid-induced inhibition of adenylyl cyclase and stimulation of ERK1/2 phosphorylation in PTX-treated cells. Chronic opioid treatment of each cell line led to adenylyl cyclase supersensitization but this response was blocked by PTX. Our results demonstrated that although PTX-sensitive G proteins are obligatory for opioid-induced adenylyl cyclase supersensitization, Gi2 alone was insufficient to mediate this response.     

Platelet alpha2A-adrenoceptor function in major depression: Gi coupling, effects of imipramine and relationship to treatment outcome

Studies suggest alpha2A-adrenoceptors (alpha(2A)AR) dysregulation in major depressive disorder (MDD). Platelet alpha(2A)ARs exist in high- and low-conformational states that are regulated by Gi protein. Although alpha(2A)AR coupling to Gi protein plays an important role in signal transduction and is modulated by antidepressants, it has not been previously investigated. Alpha2AR density in the high- and low-conformational states, agonist affinity and coupling efficiency were investigated in 27 healthy control subjects, 23 drug-free MDD patients and 16 patients after imipramine treatment using [3H]yohimbine saturation and norepinephrine displacement of [3H]yohimbine binding experiments. Coupling measures were derived from NE-displacement experiments. Patients had significantly higher alpha(2A)AR density, particularly in the high-conformational state, than control subjects. Coupling indices were normal in patients. High pre-treatment agonist affinity to the receptor in the high-conformational state and normal coupling predicted positive treatment outcome. Decreased coupling to Gi predicted a negative treatment outcome. Imipramine induced uncoupling (-11%) and redistribution of receptor density in treatment responders only, but had no effect on alpha(2A)AR coupling or density in treatment non-responders. Increased alpha(2A)AR density may represent a trait marker in MDD. The results provide indirect evidence for abnormal protein kinase A (PKA) and protein kinase C (PKC) in MDD which may be pursued in future investigations.     

Light and electron microscopic localization of alpha subunits of GTP-binding proteins, G(o) and Gi, in the cerebral cortex and hippocampus of rat brain.

Antibodies that recognize alpha subunits of G(o), Gi2 and Gi3 were used to evaluate their association with synaptic junctions. G(o), but not Gi, was concentrated within perikaryal and dendritic cytoplasm of a small population of bipolar neurons. All three G-proteins were associated with the intracellular surface of dendritic, axonal and astrocytic plasma membranes and postsynaptic densities (PSDs). However, association with PSDs was more prevalent for the two Gi's than for G(o) while the association with terminals forming putatively excitatory synapses was more prevalent for G(o) and Gi3 than for Gi2. Thus, neuromodulators may modulate the release of excitatory transmitters via activation of presynaptic Gi3 and G(o) and also regulate the opening of Ca2+ and/or K+ channels via activation of Gi's and G(o) at PSDs.     

The subcellular localization of the G-protein Gi alpha in the basal ganglia reveals its potential role in both signal transduction and vesicle trafficking.

The G-protein Gi is known to mediate signal transduction in cells by coupling its 41 kDa alpha-subunit to plasma membrane-bound receptors and inhibiting adenylyl cyclase or affecting ion channel function. Although this G-protein has been functionally associated with D2/dopamine and mu-opioid receptors in striatal membranes, its localization to neurons of the neostriatum, a brain region rich in adenylyl cyclase activity, has not been established. Light and electron microscopic study of the basal ganglia was conducted using the immunoperoxidase method and an antiserum directed against the alpha-subunit of Gi. In the neostriatum, immunoreactivity was localized to medium-sized spiny and aspiny neurons and axon terminals that formed symmetric synapses. Some astrocytes and glial processes that encapsulated axospinous complexes were also labeled. Immunoreactive axon terminals were numerous in the globus pallidus and substantia nigra, where they exhibited a dense pattern of distribution characteristic of neostriatal spiny projection neurons. Gi alpha immunoreactivity was distributed to multiple subcellular compartments. In neostriatal somata and dendrites, labeling was present intermittently along plasma membranes, and on rough and smooth endoplasmic reticulum and microtubules. In axon terminals, reaction product appeared on plasma membranes and heavily labeled the membranes of synaptic vesicles. The presence of Gi alpha in axon terminals was confirmed in purified synaptosome preparations. G-proteins consistent with the masses of Go alpha and Gi alpha, respectively, were ADP-ribosylated in the presence of pertussis toxin in striatal synaptosomes. Western blot analysis in purified synaptosome preparations of the neostriatum, globus pallidus, and substantia nigra with the same antiserum used in the immunohistochemistry demonstrated a predominant 41 kDa protein corresponding to the molecular mass of Gi alpha. Immunohistochemical localization of Gi alpha with the immunogold method in a crude striatal synaptosome preparation showed gold particles associated with synaptic vesicles and plasma membranes. Results provide the first direct evidence that Gi alpha is localized to medium-sized neostriatal projection neurons and interneurons, where it is likely to function in membrane-bound signal transduction at the postsynaptic and presynaptic level. The presence of Gi alpha in synaptic vesicle membranes points to another potentially important role for this G-protein in vesicle trafficking, such as that recently shown for smaller-molecular-mass G-proteins.     

Pertussis toxin or 8-bromo-cAMP block inhibition of the acoustic startle response by the alpha 2-adrenergic agonist ST-91

Stimulation of supraspinal alpha 2-adrenergic receptors by intraventricular infusion of the alpha 2-adrenergic agonist ST-91 depresses a simple vertebrate behavior, the acoustic startle response. Intraventricular pretreatment with pertussis toxin, an agent known to inactivate the inhibitory guanine nucleotide binding protein (Gi) which can inhibit adenylate cyclase, completely prevented the depressant behavioral effect of ST-91. In contrast, pertussis toxin did not alter the depressant effect of intraventricular infusion of the 5-HT 1B agonist 1-m-chlorophenylpiperazine (mCPP). Intraventricular infusion of the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP also reversed the depressant effect of ST-91 without altering the effect of mCPP. These data suggest that inhibition of adenylate cyclase may be involved in the effect of activation of central alpha 2-adrenergic receptors.     

The Identification and Localization of the Guanine Nucleotide Binding Protein G0 in the Auditory System

The identification of guanine nucleotide binding proteins (G proteins) in guinea-pig tissues was assessed by the adenosine diphosphate-ribosylation of the alpha subunit by Bordetella pertussis toxin using [alpha32P]nicotinamide adenine dinucleotide as the substrate followed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis and autoradiography. Three tissues (inferior colliculus, neuroblastoma cells, and the organ of Corti) contained G0alpha (39 kD), as well as Gi2alpha (40 kD) and Gi1alpha and/or Gi3alpha (41 kD). The stria vascularis and the VIIIth nerve contained mainly Gi2alpha, Gi1alpha and/or Gi3alpha, but G0alpha was barely detectable. A purified preparation of outer hair cells from the organ of Corti contained all three pertussis toxin substrates including G0alpha, with the Gi2alpha (40 kD) subunit being the most prominent. The immunocytochemical localization of the G0alpha subunit was determined by light microscopy after incubating isolated outer hair cells, Hensen cells and the stria vascularis with affinity-purified anti-G0alpha antibodies. In hair cells a positive reaction was observed along the plasma membrane and around the perimeter of the cuticular plate (zona adherens). Positive reaction was also observed within the infracuticular network extending from the cuticular plate towards the nucleus in outer hair cells. Finally, the base of the outer hair cells also contained G0alpha. However, it is likely that the G0alpha that is present in this cell region is not within the hair cell itself, but rather in nerve terminals which remained attached during dissection.     

Maturational and aging effects on guanine nucleotide binding protein immunoreactivity in human brain.

Age-related changes in transmembrane signal transduction have been reported for a number of hormonal receptors in human tissues. Guanine nucleotide binding proteins (G-proteins) are major regulatory components in the signal transduction processes for numerous receptors. Developmental changes in the abundance of specific G-protein alpha subunits, especially for Go, have already been shown in rat brain and in neuronal cell lines. In this study, immunoblotting analyses were performed with specific rabbit polyclonal antisera to Gs alpha, Gi alpha, Go alpha, and G beta subunits to estimate semi-quantitatively these G-protein subunits in samples of parietal cortex obtained postmortem from 18 subjects free of neurologic or psychiatric disease whose ages ranged from 3 days to 92 years. The Gs and Gi alpha subunit immunoreactivities were correlated significantly and inversely with age. The Gi alpha immunoreactivity declined markedly (46%) after the age of 40. As other G-protein subunit concentrations showed no age-dependent changes, the observed relationship between Gs and Gi alpha subunits and age is not likely due to cell loss occurring with advancing age. Of particular interest, the ratio of 52 to 45 kDa Gs alpha subunit immunoreactivities was significantly higher in infants than that found for adult parietal cortex. Given that changes in G-protein subunit abundance directly affect receptor-G-protein-effector functionality and response, these age-related alterations may be of importance in cerebral dysfunction and the development of neuropsychiatric disease in the later years of life.     

Differential expression of thyroid hormone receptor isoforms by thyroid hormone and lithium in rat GH3 and B103 cells.

BACKGROUND: The interaction between lithium, a mood stabilizer, and the thyroid axis has been extensively studied; however, the regulation of thyroid hormone receptors by lithium is yet to be investigated. METHODS: To test whether lithium affects thyroid hormones at the receptor level, we examined the effects of lithium in combination with triiodothyronine (T3) on gene expression of thyroid hormone receptor isoforms in GH3 and B103 cells. RESULTS: The pattern of expression as well as the magnitude of regulation of the different thyroid hormone receptor isoforms appeared to be cell line specific. Whereas T3 regulated all four isoforms in GH3 cells at both time points, T3 did not alter thyroid hormone receptor TR alpha 1 and TR alpha 2 mRNA in B103 cells. Addition of lithium to thyroid hormone-deficient GH3 cells decreased TR alpha 1, alpha 2, and beta 2 expression without affecting TR beta 1 expression at 2 but not 5 days. Addition of lithium to T3-treated GH3 cells did not further modulate gene expression of TR alpha 1, alpha 2, beta 1, or beta 2 when compared to cells treated with T3 alone. The effects of lithium in B103 cells appeared to be isoform specific as well as time dependent, since TR alpha 1 expression was selectively decreased in B103 cells, when treated with T3 in the presence of lithium. CONCLUSIONS: The present study provides direct evidence that T3 and/or lithium regulate TR gene expression in vitro in a both time-dependent and cell line-specific manner.     

Involvement of activated integrin alpha2beta1 in the firm adhesion of platelets onto a surface of immobilized collagen under flow conditions

Recently, we demonstrated that agonist-induced activation of the platelet surface collagen-receptor integrin alpha1beta2 converts it to an active form that can bind soluble collagen with high affinity (Jung, SM, Moroi, M: J Biol Chem 1998; 273: 14827-37). Here, the involvement of alpha2beta1 activation and the high affinity binding property of activated alpha2beta1 in platelet adhesion to a collagen surface under flow conditions were analyzed. Platelet adhesion to immobilized collagen was measured in the presence of TS2/16, an activating anti-integrin alpha2beta1 antibody, and inhibiting antibodies, Gi9 and 6F1. TS2/16 decreased the moving velocity of platelets on the collagen surface, but Gi9 and 6F1 increased it, indicating that alpha2beta1 activation induces the tight binding of platelets to immobilized collagen under flow. Platelet adhesion, expressed as the surface area occupied by adhered platelets, in the presence of TS2/16 was similar to that in its absence. In contrast, adding Gi9 or 6F1 caused biphasic adhesion composed of a first phase, a lag phase whose length differed in each experiment, and a second phase adhesion with a rate similar to that of the control. This biphasic adhesion indicates that alpha2beta1 activity is inhibited and also suggests that some other factor(s) may contribute to the adhesion under flow. At concentrations where neither 6F1 nor Gi9 affected collagen-induced aggregation, these antibodies inhibited soluble collagen binding to thrombin-activated platelets. Only at much higher concentration did 6F1 inhibit collagen-induced aggregation. TS2/16 had no effect on the aggregation. The present results are evidence against the major involvement of integrin alpha2beta1 in platelet aggregation; instead, they indicate that integrin alpha2beta1 would be mainly associated with the tight binding of platelets to collagen.     

Ischemia of rat brain decreases pertussis toxin-catalyzed [32P]ADP ribosylation of GTP-binding proteins (Gi1 and G0) in membranes

As an approach to understanding the molecular basis of the pathophysiology of cerebral ischemia, we examined qualitative and quantitative changes in pertussis toxin substrates, Gi1 and G0, in the membrane of rat cerebral cortex after decapitation. Within 1 min after decapitation, the extent of pertussis toxin-catalyzed [32P]ADP ribosylation of the G proteins in the cerebral cortex membrane was significantly decreased and the magnitude of the decrease became slightly larger upon further incubation of the decapitated brain. Addition of guanine nucleotides, GTP and GDP, or the purified beta gamma subunits of transducin to the membranes of control and ischemic cerebral cortex stimulated [32P]ADP ribosylation of the G proteins. The stimulation of [32P]ADP ribosylation in the control situation by guanine nucleotides was almost to the same extent as that in ischemia. However, the stimulation by transducin beta gamma subunits was different; the control stimulation was greater than that in ischemia. In immunoblots probed with antibodies against Gi1 alpha, G0 alpha, and T beta, the immunoreactivity of the corresponding proteins in ischemia was similar to that in control, suggesting that the amounts of G proteins were not changed in ischemia. These results suggest that ischemia accelerates the dissociation of alpha-GDP-beta gamma to alpha-GDP and free beta gamma and causes the denaturation of the dissociated alpha-GDP, thereby decreasing [32P]ADP ribosylation.     

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.     

Alpha2-adrenoreceptor activation inhibits LTP and LTD in the basolateral amygdala: involvement of Gi/o-protein-mediated modulation of Ca2+-channels and inwardly rectifying K+-channels in LTD

Activation of adrenoreceptors modulates synaptic transmission in the basolateral amygdala. Here, we investigated the effects of alpha2-adrenoreceptor activation on long-term depression and long-term potentiation in an in vitro slice preparation of the mouse basolateral amygdala. Field potentials and excitatory postsynaptic currents were evoked in the basolateral amygdala by stimulating the lateral amygdala. Norepinephrine (20 micro m) reduced synaptic transmission and completely blocked the induction of long-term potentiation and long-term depression. The alpha2-adrenoreceptor antagonist yohimbine (2 micro m) reversed this effect. The alpha2-adrenoreceptor agonist clonidine (10 micro m) mimicked the effects of norepinephrine. The Gi/o-protein inhibitor pertussis toxin (5 micro g/mL) reversed the effect of clonidine. Long-term depression was blocked in the presence of omega-conotoxin GVIA, but not omega-agatoxin IVA. Clonidine inhibited voltage-activated Ca2+ currents mediated via N- or P/Q-type Ca2+-channels. The inhibitory action of clonidine on long-term depression was reversed when inwardly rectifying K+-channels were blocked by Ba2+ (300 micro m). The present data suggest that alpha2-adrenoreceptor activation impairs the induction of long-term depression in the basolateral amygdala by a Gi/o-protein-mediated inhibition of presynaptic N-type Ca2+-channels and activation of inwardly-rectifying K+-channels.     

Mutually exclusive expression of Gαia and Gα14 reveals diversification of taste receptor cells in zebrafish

A comprehensive reevaluation of the G protein alpha subunit genes specifically expressed in taste buds in the tongue epithelium of rodents revealed that Gq and G14 of the Gq class and Gi2 and Ggust (Gt3, also known as gustducin) of the Gi class are expressed in mammalian taste buds. Meanwhile, a database search of fish genomes revealed the absence of a gene encoding an ortholog of the mammalian Ggust gene, which mediates sweet, umami, and bitter taste signals in mammalian taste receptor cells (TRCs). Histochemical screening identified two G protein alpha subunit genes, zfGia and zfG14, expressed in subsets of TRCs in zebrafish. The expression patterns of zfGia and zfG14 in taste buds were mutually exclusive, and the expression of known T1R and T2R genes in zebrafish was restricted to a subset of zfGia-expressing TRCs. These findings highlight the existence of a novel subset of TRCs in zebrafish that is absent in mammals and suggest that unidentified G protein-coupled receptors are expressed in zfG14-expressing TRCs and in zfGia-expressing TRCs where known T1R and T2R genes were not expressed in zebrafish. The existence of not only generalized but also specialized subsets of TRCs may imply a strong connection between the evolution of the peripheral gustatory system and the evolution of particular species.     

Altered expression levels of G protein subclass mRNAs in various seizure stages of the kindling model

The expressions of mRNAs encoding G protein alpha subunits were analyzed in the cerebral cortex of amygdaloid kindled rats. A remarkable increase in Gsalpha mRNA were observed on the bilateral cerebral cortex at 24 h after the last generalized seizure and persisted 3 weeks on the unstimulated side. Gi2alpha mRNA level was also increased on the stimulated side at 24 h and persisted 3 weeks. These result suggest that dysfunction of Gs and Gi2 might relate to the basic mechanisms of seizure generation and the maintenance of epileptogenesis.