Effect of GABA A receptor activation on UT-coupled signaling pathways in rat cortical astrocytes

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.     

P2Y nucleotide receptor interaction with alpha integrin mediates astrocyte migration

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.     

Urotensin II protects ischemic reperfusion injury of hearts through ROS and antioxidant pathway

Urotensin II (UII) is a vasoactive peptide which is bound to a G protein-coupled receptor. UII and its receptor are upregulated in ischemic and chronic hypoxic myocardium, but the effect of UII on ischemic reperfusion (I/R) injury is still controversial. The aim of the present study was to investigate whether UII protects heart function against I/R injury. Global ischemia was performed using isolated perfused Langendorff hearts of Sprague-Dawley rats. Hearts were perfused with Krebs-Henseleit buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Pretreatment with UII (10nM) for 10min increased recovery percentage of the post-ischemic left ventricular developed pressure and ±dp/dt, and decreased post-ischemic left ventricular end-diastolic pressure as compared with I/R group. UII decreased infarct size and an increased lactate dehydrogenase level during reperfusion. Cardioprotective effects of UII were attenuated by pretreatment with UII receptor antagonist. The hydrogen peroxide activity was increased in UII-treated heart before ischemia. The Mn-SOD, catalase, heme oxygenase-1 and Bcl-2 levels were increased, and the Bax and caspase-9 levels were decreased in UII-treated hearts. These results suggest that UII has cardioprotective effects against I/R injury partly through activating antioxidant enzymes and reactive oxygen species.     

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.     

Orn5]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes

Cultured rat astrocytes, which express functional urotensin II (UII)/UII-related peptide (URP) receptors (UT), represent a very suitable model to investigate the pharmacological profile of UII and URP analogs towards native UT. We have recently designed three URP analogs [D-Trp4]URP, [Orn5]URP and [D-Tyr6]URP, that act as UT antagonists in the rat aortic ring bioassay. However, it has been previously reported that UII/URP analogs capable of inhibiting the contractile activity of UII possess agonistic activity on UT-transfected cells. In the present study, we have compared the ability of URP analogs to compete for [125 I]URP binding and to modulate cytosolic calcium concentration ([Ca2+]c) in cultured rat astrocytes. All three analogs displaced radioligand binding: [D-Trp4]URP and [D-Tyr6]URP interacted with high- and low-affinity sites whereas [Orn5]URP only bound high-affinity sites. [D-Trp4]URP and [D-Tyr6]URP both induced a robust increase in [Ca2+]c in astrocytes while [Orn5]URP was totally devoid of activity. [Orn5]URP provoked a concentration-dependent inhibition of URP- and UII-evoked [Ca2+]c increase and a rightward shift of the URP and UII dose-response curves. The present data indicate that [D-Trp4]URP and [D-Tyr6]URP, which act as UII antagonists in the rat aortic ring assay, behave as agonists in the [Ca2+]c mobilization assay in cultured astrocytes, whereas [Orn5]URP is a pure selective antagonist in both rat aortic ring contraction and astrocyte [Ca2+]c mobilization assays.     

Urotensin-II receptor blockade with SB-611812 attenuates cardiac remodeling in experimental ischemic heart disease

It is now well established that urotensin-II (UII) levels are increased in several cardiovascular diseases. We previously demonstrated that UII and the UII receptor (UT) protein levels are significantly increased in the hearts of both humans and rats with congestive heart failure (CHF). We have also recently demonstrated that UII blockade, with a selective UII antagonist, improves heart function in a rat model of ischemic CHF. Here, we evaluated the attenuation of cardiac remodeling associated with UII antagonism in the same rat model of ischemic CHF. Animals were administered a specific UT receptor antagonist, SB-611812 (30 mg/kg/day, gavage), or vehicle 30 min prior to coronary artery ligation followed by daily treatment for 8 weeks. Myocardial interstitial fibrosis was analyzed by Masson's trichrome and picrosirius red staining. RT-PCR analysis was utilized for mRNA expression studies. We used Western blotting to assess levels of collagen types I and III. Mitogenic activity of UII on cultured neonatal cardiac fibroblasts was also evaluated. Following coronary ligation, SB-611812 significantly attenuated both myocardial and endocardial interstitial fibrosis, and reduced collagen type I:III ratio (P<0.01). UII induced proliferation of cardiac fibroblasts and this mitogenic effect was significantly inhibited with 1 microM of SB-611218 (P<0.05). We demonstrate here that selective blockade of UT reduces diastolic dysfunction by decreasing myocardial fibrosis post-coronary ligation in vivo, and inhibits UII-mediated fibroblast proliferation in vitro.     

Immunolocalization of urotensin II and its receptor in human adrenal tumors and attached non-neoplastic adrenal tissues

Urotensin II (UII), first identified from goby urophysis, is a potent vasoactive peptide hormone and an endogenous ligand for an orphan G protein-coupled receptor GPR14, now named urotensin II receptor (UT-R). In addition to its vascular actions, UII has been shown to have mitogenic effects on tumor growth and some regulatory effects on adrenal steroidogenesis. In the present study, we examined expression of UII and UT-R in human adrenal tumors and attached non-neoplastic adrenal tissues by immunohistochemistry. Both UII and UT-R were immunolocalized in tumor cells of all adrenal tumors examined: 8 cases of cortisol-producing adenomas, 8 cases of aldosterone-producing adenomas, 2 cases of non-functioning adenomas, 17 cases of adrenocortical carcinomas, and 8 cases of pheochromocytomas. In attached adrenals, immunoreactivity for UII was detected in medulla, but much weaker in the cortex than in cortical tumors, suggesting that expression of UII was up-regulated in neoplastic adrenocortical tissues. No significant differences were found in the degree of immunoreactivity for UT-R between the tumors and the attached adrenal tissues. The present study showed that both UII and UT-R were expressed in the adrenal tumors and attached non-neoplastic adrenal tissues, and suggests possible roles of UII and UT-R in tumor growth and/or secretory activities of these tumors.     

GABAB receptor association with the PDZ scaffold Mupp1 alters receptor stability and function

gamma-Aminobutyric acid, type B (GABA(B)) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABA(B) receptor (GABA(B)R) functionality, we screened the GABA(B)R2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABA(B)R2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA(B)R2 in cells. Disruption of the GABA(B)R2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABA(B) receptor signaling. The effects of mutating the GABA(B)R2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABA(B) receptor stability and signaling can be modulated via GABA(B)R2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABA(B) receptors in the central nervous system.     

Effects of urotensin II on functional activity of late endothelial progenitor cells

Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10(-10), 10(-9), 10(-8), 10(-7) and 10(-6)M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.     

Elevated expression of urotensin II and its receptor in diethylnitrosamine-mediated precancerous lesions in rat liver

Urotensin II (UII) is a somatostatin-like peptide involved in cell proliferation and in tumor biology. To explore the role of liver-derived UII in the pathogenesis of precancerous liver lesions in rat, we investigated the expression of UII and its receptor, UT, in diethylnitrosamine (DEN)-induced precancerous liver lesions and the effects of UII on cell proliferation by hepatic oval cells. Radioimmunoassay, RT-PCR, immunohistochemistry and western blot were used in this study. Compared with untreated controls, rats treated with DEN showed increased UII content by 47.7% in plasma and by 164.9% in liver tissue (all P < 0.01). The expression of UII protein and of both UT mRNA and protein was significantly enhanced in the liver of treated rats. Western blot analysis revealed that the expression of phosphorylated protein kinase C (p-PKC) and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) was increased in the liver of treated animals. Treatment with UII (10⁻¹⁰-10⁻⁶ M) for 24 h significantly increased number of cultured hepatic oval cells (at 10⁻⁹-10⁻⁸ M). However, during the pre-incubation with calphostin C (inhibitor of PKC) or PD98059 (inhibitor of MEK), the proliferation was decreased by 40.1% and 25.4% respectively (both P < 0.05). In DEN-induced precancerous liver lesions, the UII/UT system was up-regulated, which may contribute to the pathogenesis of liver cancer through a PKC- or ERK1/2-dependent pro-mitogenic pathway in an autocrine/paracrine manner.     

Retrovirally mediated transfer of a G protein-coupled receptor kinase (GRK) dominant-negative mutant enhances endogenous calcitonin receptor signaling in Chinese hamster ovary cells. GRK inhibition enhances expression of receptors and receptor mRNA

G protein-coupled receptor kinases (GRKs) initiate pathways leading to agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. However, the role of GRKs in modulation of signaling properties of native receptors has not been clearly defined. Here we addressed this question by generating Chinese hamster ovary (CHO) cells stably expressing a dominant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calcitonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected control CHO cells with approximately 3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-promoted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The number of CT receptors was increased approximately 3-fold in DN-GRK2 cells, as assessed by (125)I-salmon CT-specific binding, and this was associated with increased CT receptor mRNA levels. These results indicate that DN-GRK2 has multiple consequences for CT receptor signaling, but a primary effect is an increase in CT receptor mRNA and receptor number and, in turn, enhanced CT receptor signaling. As such, our findings provide a mechanistic basis for previous observations regarding agonist-promoted down-regulation of CT receptors and for resistance and escape from response to CT in vitro and in vivo. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression and response. We speculate that GRKs play a similar role for other G protein-coupled receptors as well.     

Biological properties and functional determinants of the urotensin II receptor

The urotensin II receptor (UT) is a member of the G protein-coupled receptor (GPCR) family and binds the cyclic undecapeptide urotensin II (U-II) as well as the octapeptide urotensin II-related peptide (URP). The active UT mediates pleiotropic effects through various signal transduction pathways, including coupling to G proteins and activating the mitogen-activated protein kinase pathway. Several highly conserved residues and motifs of class A GPCRs that are important for activity are found in UT. This review highlights some of the putative roles of these motifs in the binding, activation and desensitization of UT.     

Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14

Urotensin II (UII) is a neuropeptide with potent cardiovascular effects. Its sequence is strongly conserved among different species and has structural similarity to somatostatin. No receptor for UII has been molecularly identified from any species so far. GPR14 was cloned as an orphan G protein-coupled receptor with similarity to members of the somatostatin/opioid receptor family. We have now demonstrated that GPR14 is a high affinity receptor for UII and designate it UII-R1a. HEK293 cells and COS-7 cells transfected with rat GPR14 showed strong, dose-dependent calcium mobilization in response to fish, frog, and human UII. Radioligand binding analysis showed high affinity binding of UII to membrane preparations isolated from HEK293 cells stably expressing rat GPR14. In situ hybridization analysis showed that GPR14 was expressed in motor neurons of the spinal cord, smooth muscle cells of the bladder, and muscle cells of the heart. The identification of the first receptor for UII will allow better understanding of the physiological and pharmacological roles of UII.     

Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK‐LRP6 signalling

Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c‐Jun N‐terminal kinase (JNK) and low density lipoprotein receptor‐related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal‐regulated kinase signalling, that involved in the UII‐inhibited‐ proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII‐induced‐phospho‐JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload.     

Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.     

GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L γ-aminobutyric acid type A (GABAA) receptor expression and channel gating

A GABA(A) receptor β3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three β3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of β3 subunits. The mutation had three major effects on GABA(A) receptors. First, coexpression of β3(G32R) subunits with α1 or α3 and γ2L subunits in HEK293T cells reduced surface expression of γ2L subunits and increased surface expression of β3 subunits, suggesting a partial shift from ternary αβ3γ2L receptors to binary αβ3 and homomeric β3 receptors. Second, β3(G32R) subunits were more likely than β3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, α1β3(G32R)γ2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because γ2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that α1β3(G32R)γ2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the β3 subunit N-terminal α-helix in receptor assembly and gating.     

Epidermal growth factor regulates astrocyte expression of the interleukin-4 receptor via a MAPK-independent pathway

Human astrocytes express the interleukin (IL)-4 receptor alpha chain (IL-4R alpha) in vitro and in vivo but mechanisms governing astrocyte IL-4R alpha expression have not been established. We hypothesized that epidermal growth factor (EGF) and IL-4, agents that profoundly affect astrocyte proliferation, might also alter IL-4R alpha expression. Exposure to EGF for 24 h enhanced IL-4R alpha mRNA levels; in contrast, IL-4 yielded no increase. Immunoblotting demonstrated that EGF but not IL-4 increased astrocyte IL-4R alpha protein after 2--4 days of exposure. Similarly, EGF but not IL-4 strongly activated phosphorylation of p42/p44 extracellular regulated kinase isoforms, a reaction blocked by the mitogen-activated protein kinase (MAPK) inhibitor, PD98059. PD98059 also blocked EGF-stimulated DNA synthesis but not IL-4R alpha mRNA levels, while antibody to the EGF receptor (erbB1) blocked both EGF effects. Data suggest that astrocyte IL-4R alpha expression is upregulated by EGF but not by IL-4 in an EGF-receptor-dependent manner and that mechanisms are independent of MAPK activation.     

A Role for Loop F in Modulating GABA Binding Affinity in the GABA(A) Receptor

The brain's major inhibitory neuroreceptor is the ligand-gated ion channel γ-aminobutyric acid (GABA) type A receptor (GABAR). GABARs exist in a variety of different subunit combinations that act to modulate the physiological behavior of GABAR by altering its pharmacological profile, as well as its affinity for GABA. While the α(1)β(2)γ(2) subtype is one of the most prevalent GABARs, the less populous α(6)β(3)δ subtype has much higher GABA sensitivity. Previous studies identified residues crucial for GABA binding; however, the specific molecular differences responsible for this diverse sensitivity are not known. Furthermore, the role of loop F is a divisive subject, with conflicting evidence for ligand binding function. Using homology modeling, ligand docking, and molecular dynamics simulations, we investigated the GABA binding sites of the two receptor subtypes. Simulations identified seven residues that consistently interacted with GABA in both subtypes: αF65, αR132, βL99, βE155, βR/K196, βY205, and βR207. Residue substitution at position β196 (arginine in α(6)β(3)δ, lysine in α(1)β(2)γ(2)) resulted in a shift in GABA binding. However, the major difference between the two binding sites was the magnitude of loop F involvement, with a greater contribution in the α(6)β(3)δ receptor. Free energy calculations confirm that the α(6)β(3)δ binding pocket has an increased affinity for GABA. Thus, the possible role for loop F across the GABAR family is to modulate GABA affinity.     

p75NTR Promotes Astrocyte Proliferation in Response to Cortical Stab Wound

Astrogliosis after brain trauma can have a significant impact on functional recovery. However, little is known about the mechanisms underlying astrocyte proliferation and subsequent astrogliosis. In this study, we established a cortical stab wound injury mouse model and observed dramatic astrocyte activation and nerve growth factor receptor (p75NTR) upregulation near the lesion. We also found profound alterations in the cell cycle of astrocytes near the lesion, with a switch from a mitotically quiescent (G0) phase to the G2/M and S phases. However, no changes in the level of astrocyte apoptosis were observed. Cell cycle progression to the G2/M and S phases and CDK2 protein levels in response to cortical stab wound was inhibited after p75NTR knockdown in mouse astrocytes. Conversely, p75NTR overexpression in mouse astrocytes was sufficient in promoting cell cycle progression. In conclusion, our results suggested that p75NTR upregulation in astrocytes after brain injury induces cell cycle entry by promoting CDK2 expression and promoting astrocyte proliferation. Our findings provided a better understanding of astrocytic responses after cortical stab wound injury in mice.

     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.