GABAergic signaling in the rat pineal gland

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ‐aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABA_A receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABA_B1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABA_A receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage‐gated Ca²⁺ channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48–72‐h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine‐induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found.     

γ-Aminobutyric Acid B Receptor Improves Carbon Tetrachloride-Induced Liver Fibrosis in Rats

Background

It was well known that angiotension II can inhibit hepatic stellate cell activation. The GABA_B receptor was upregulated when the hepatic stellate cell line was stimulated by angiotension II in our previous study. But the role of the GABA_B receptor in liver fibrosis has never been reported.

Aim

In the present study, we investigated the effects of this receptor on carbon tetrachloride-induced liver fibrosis in rats.

Methods

The rats were divided into four groups including GABA_B receptor agonist, antangonist, model and control group. α-smooth muscle actin (α-SMA) and GABA_B receptor expression levels were detected by immunohistochemistry and real-time polymerase chain reaction. Liver function tests were performed once blood samples was taken; Western blot analysis was used to detect protein expression level of α-SMA and TGF-β1.

Results

We found baclofen ameliorated the CCl₄-induced rats’s liver fibrosis. The highest liver enzymes and α-SMA protein levels were found in the CGP35348 group.

Conclusion

The GABA_B receptor may have a protective role in the liver.     

Acamprosate Enhances N-Methyl-D-Apartate Receptor-Mediated Neurotransmission But Inhibits Presynaptic GABAB Receptors in Nucleus Accumbens Neurons

Acamprosate (calcium acetylhomotaurine) is used therapeutically in Europe to reduce relapse in weaned alcoholics. However, the mechanisms of acamprosate action in the central nervous system are still obscure, although early studies suggested an action on GABA receptors. The nucleus accumbens (NAcc) is a brain region thought to underlie ethanol reinforcement. Recent studies from our laboratory have demonstrated that ethanol inhibits both N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamatergic synaptic transmission in the NAcc.^1,2 In the present study, we used voltage- and current-clamp intracellular recording of NAcc core neurons in a slice preparation to examine acamprosate actions on resting membrane properties and pharmacologically isolated synaptic responses. We isolated NMDA and non-NMDA receptor-mediated excitatory postsynaptic potentials or currents (EPSP/Cs) with 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovaler-ate (d-APV), respectively. Bicuculline was also included to block GABA_A receptors. Superfusion of acamprosate (5, 50, and 300 μM) did not alter the resting membrane properties of NAcc neurons. However, 300 μM acamprosate significantly increased the NMDA receptor-mediated components of EPSP/Cs (NMDA-EPSP/Cs) with recovery on washout. In contrast, 300 μM acamprosate had no significant effect on the non-NMDA receptor component of the EPSP/Cs (non-NMDA-EPSP/Cs). To test acamprosate actions on the GABA system, we superfused 60 μM d-APV and 20 μM CNQX to block glutamatergic transmission and evoked monosynaptic GABA_A receptor-mediated synaptic responses within the NAcc. Acamprosate (300 μM) did not change these monosynaptic GABA_A-IPSCs. We also used a paired-pulse paradigm to test whether acamprosate could act on presynaptic GABA_B, autoreceptors, in the presence of d-APV and CNQX to block glutamatergic transmission. Like 0.5 μM CGP 34358 (a GABA_B receptor blocker), acamprosate significantly decreased the paired-pulse inhibition (PPI) of GABA_A-IPSCs at short interstimulus intervals (ISIs). Thus, acamprosate may concomitantly enhance NMDA-EPSP/Cs while blocking presynaptic GABA_B receptor-mediated inhibition of GABA release. These results suggest that acamprosate's clinical efficacy in preventing relapse in weaned alcoholics could derive from its interactions with both the glutamatergic and GABAergic systems in the NAcc. Synaptic Transmission, Alcohol, Ethanol, EPSPs, NMDA Receptors.     

GABAA-Receptor Agonists Limit Pneumonitis and Death in Murine Coronavirus-Infected Mice

There is an urgent need for new approaches to limit the severity of coronavirus infections. Many cells of the immune system express receptors for the neurotransmitter γ-aminobutyric acid (GABA), and GABA-receptor (GABA-R) agonists have anti-inflammatory effects. Lung epithelial cells also express GABA-Rs, and GABA-R modulators have been shown to limit acute lung injuries. There is currently, however, no information on whether GABA-R agonists might impact the course of a viral infection. Here, we assessed whether clinically applicable GABA-R agonists could be repurposed for the treatment of a lethal coronavirus (murine hepatitis virus 1, MHV-1) infection in mice. We found that oral GABA administration before, or after the appearance of symptoms, very effectively limited MHV-1-induced pneumonitis, severe illness, and death. GABA treatment also reduced viral load in the lungs, suggesting that GABA-Rs may provide a new druggable target to limit coronavirus replication. Treatment with the GABA_A-R-specific agonist homotaurine, but not the GABA_B-R-specific agonist baclofen, significantly reduced the severity of pneumonitis and death rates in MHV-1-infected mice, indicating that the therapeutic effects were mediated primarily through GABA_A-Rs. Since GABA and homotaurine are safe for human consumption, they are promising candidates to help treat coronavirus infections.     

Gastroesophageal reflux disease and baclofen: Is there a light at the end of the tunnel?

Transient lower esophageal sphincter relaxations (TLESRs) are rapid and prolonged relaxations of the lower esophageal sphincter (LES) that are not associated with swallowing. They are the mechanism by which most gastroesophageal reflux episodes occur in normal people and in patients with esophagitis. Transient LES relaxations appear to be mediated by a vagovagal reflex initiated by gastric distention. Baclofen is a γ -aminobutyric acid (GABA) derivative that inhibits the production of TLESRs by acting as a GABA_B receptor agonist at one or more loci along the vagovagal reflex arc. Animal and human studies suggest that baclofen decreases the number of reflux events and amount of esophageal acid exposure. Baclofen or another GABA_B receptor agonist may be clinically useful in treatment of gastroesophageal reflux disease.     

Influence of hippocampal GABA<Subscript>B</Subscript> receptor inhibition on memory in rats with acute β-amyloid toxicity

The neurotransmitter γ-aminobutyric acid (GABA) is involved in the process of memory. It has been reported that the inhibition of GABA_B receptors has beneficial effects on cognition. The aim of this study was to investigate the role of CGP35348 (a GABA_B receptor antagonist) on dentate gyrus GABA_B receptor inhibition and its effects on learning and memory impairments that had been induced in adult male rats by microinjection of β-amyloid (Aβ). Seventy Wistar male rats were randomly divided into seven groups: control, sham (receiving the Aβ vehicle only), Aβ, Aβ + CGP35348 (1, 10, and 100 μg/μL), and CGP35348 alone (10 μg/μL). Memory impairment was induced by unilateral interventricular microinjection of Aβ (6 μg/6 μL). Rats were cannulated bilaterally in the dentate gyrus, and then, they were treated for 20 consecutive days. Learning and memory were assessed using the novel object recognition and passive avoidance learning tests. The discrimination index and the step-through latency were significantly increased in the Aβ + CGP35348 group in comparison to the Aβ only group (P?<?0.05 and P?<?0.01, respectively). Data showed that the discrimination index was decreased in the Aβ + CGP35348 group in comparison with the control group (P?<?0.05) and sham group (P?<?0.01). Moreover, the step-through latency was significantly decreased in the Aβ + CGP35348 group in comparison to the control and sham groups (P?<?0.01). Data from this study indicated that intra-hippocampal microinjection of the GABA_B receptor antagonist counteracts the learning, memory, and cognitive impairments induced by Aβ. It can be concluded that the GABA_B receptor antagonist is a possible therapeutic agent against the progression of acute Aβ toxicity-induced memory impairment.     

The GABA<Subscript>B</Subscript> Receptor Inhibits Activation of Hepatic Stellate Cells

Background/Aims Angiotensin II (Ang II) plays an important role in the activation of hepatic stellate cells (HSCs). In this study it was found that expression of the GABA_B receptor was elevated in HSCs treated with Ang II. We attempted to elucidate the mechanism of the GABA_B receptor in HSCs activation. Methods First, the target gene (GABA_B receptor) was screened by gene chip in HSCs treated with Ang II. Second, the biological function of the GABA_B receptor was analyzed by MTT, cell-cycle assay, real-time PCR, and western blot. The methods of MTT and cell-cycle assay were used to evaluate the effect of the GABA_B receptor on proliferation and DNA synthesis of HSCs. Expression of ECM, TGF-β1, and α-SMA was analyzed by real-time PCR and western blot. Results The GABA_B receptor’s specific agonist CGP35348 inhibited the activation of HSCs, which could be partially reversed by the GABA_B receptor’s antagonist. Conclusions Our in-vitro results demonstrated that the GABA_B receptor could inhibit HSCs activation.     

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1

GABA_B receptors are the G-protein–coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABA_B receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca²⁺ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABA_B-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABA_B receptors. NMDA-dependent internalization of GABA_B receptors requires activation of Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABA_B receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABA_B1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABA_B receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABA_B receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABA_B1b subunit isoform, which is the isoform that clusters with inhibitory effector K⁺ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABA_B receptors to activate K⁺ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABA_B receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABA_B receptors.     

γ-Aminobutyric acid (GABA) signalling in human pancreatic islets is altered in type 2 diabetes

Aims/hypothesis

γ-Aminobutyric acid (GABA) is a signalling molecule in the interstitial space in pancreatic islets. We examined the expression and function of the GABA signalling system components in human pancreatic islets from normoglycaemic and type 2 diabetic individuals.

Methods

Expression of GABA signalling system components was studied by microarray, quantitative PCR analysis, immunohistochemistry and patch-clamp experiments on cells in intact islets. Hormone release was measured from intact islets.

Results

The GABA signalling system was compromised in islets from type 2 diabetic individuals, where the expression of the genes encoding the α1, α2, β2 and β3 GABA_A channel subunits was downregulated. GABA originating within the islets evoked tonic currents in the cells. The currents were enhanced by pentobarbital and inhibited by the GABA_A receptor antagonist, SR95531. The effects of SR95531 on hormone release revealed that activation of GABA_A channels (GABA_A receptors) decreased both insulin and glucagon secretion. The GABA_B receptor antagonist, CPG55845, increased insulin release in islets (16.7?mmol/l glucose) from normoglycaemic and type 2 diabetic individuals.

Conclusions/interpretation

Interstitial GABA activates GABA_A channels and GABA_B receptors and effectively modulates hormone release in islets from type 2 diabetic and normoglycaemic individuals.     

GABAB autoreceptor-mediated cell type-specific reduction of inhibition in epileptic mice

GABA_B receptors (GABA_BRs) mediate slow inhibitory effects on neuronal excitability and synaptic transmission in the brain. However, the GABA_BR agonist baclofen can also promote excitability and seizure generation in human patients and animals models. Here we show that baclofen has concentration-dependent effects on the hippocampal network in a mouse model of mesial temporal lobe epilepsy. Application of baclofen at a high dose (10 mg/kg i.p.) reduced the power of γ oscillations and the frequency of pathological discharges in the Cornu Ammonis area 3 (CA3) area of freely moving epileptic mice. Unexpectedly, at a lower dose (1 mg/kg), baclofen markedly increased γ activity accompanied by a higher incidence of pathological discharges. Intracellular recordings from CA3 pyramidal cells in vitro further revealed that, although at a high concentration (10 µM), baclofen invariably resulted in hyperpolarization, at low concentrations (0.5 µM), the drug had divergent effects, producing depolarization and an increase in firing frequency in epileptic but not control mice. These excitatory effects were mediated by the selective muting of inhibitory cholecystokinin-positive basket cells (CCK⁺ BCs), through enhanced inhibition of GABA release via presynaptic GABA_BRs. We conclude that cell type–specific up-regulation of GABA_BR-mediated autoinhibition in CCK⁺ BCs promotes aberrant high frequency oscillations and hyperexcitability in hippocampal networks of chronic epileptic mice.Neuronal activity in the hippocampus shows oscillations in behavior-relevant frequency ranges including γ frequencies (30–80 Hz) (1). γ activity is prominent in the aroused brain and has been implicated in higher-level brain functions, such as sensory binding, perception (2), and storage and recall of information (3, 4). At the same time, γ frequency oscillations are also prevalent in epileptic patients and are most often observed at seizure onset during in depth EEG recordings (5). The GABAergic system plays a pivotal role in the generation of γ oscillations (6–8). However, it remains to be resolved how distinct GABAergic receptor subtypes, in particular GABA_B receptors (GABA_BRs), contribute to the generation and modulation of pathological network oscillatory activity.GABA_BRs mediate slow inhibitory effects and control synaptic transmission and the excitability of neurons in cortical networks. GABA_BRs are expressed both postsynaptically in somato-dendritic compartments and presynaptically in axon terminals, in excitatory principal cell and inhibitory interneurons (9–11). The effects of GABA_BR activation on the network are dominated by inhibition leading to an overall dampened population activity. However, if GABAergic interneurons are effected dominantly, as observed for example, during high-frequency stimulation, GABA_BR activation can produce disinhibition in principal cells (12, 13). Accordingly, the role of GABA_BRs in epilepsy and seizure generation remains ambiguous. GABA_BRs are expected to have an overall antiepileptic effect, and indeed, the receptor KO animals show an epileptic phenotype (14). However, there is also evidence that the receptor agonist baclofen can induce seizures in patients after intrathecal application (15, 16). The picture is further complicated by the fact that GABA_BR expression can be altered in both epileptic patients, e.g., in mesial temporal lobe epilepsy (mTLE) (17), and animal models (18). Thus, cell type–specific alterations in GABA_BR expression may change network excitability during the progression of mTLE.Using a chronic kainate (KA) model of mTLE, which reproduces major electrophysiological and histopathological characteristics of human mTLE (19, 20), we studied the role of GABA_BRs in altered hippocampal network activity. Our results suggest that enhanced and persistent GABA_BR activation in epileptic mice suppresses the inhibitory output from hippocampal interneurons, in particular cholecystokinin (CCK)-expressing basket cells (BCs) onto pyramidal cells (PCs). This reduction in the inhibitory output of interneurons, in turn, leads to disinhibition in hippocampal networks, enhances γ activity, and promotes the transition to pathological hyperexcitability.     

Modulation of GABAA Receptor Function by Alcohols: Effects of Subunit Composition and Differential Effects of Ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABA_A receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABA_A receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABA_A receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA_A in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 μM GABA in oocytes expressing the α₁β₂γ_2L and α₂β₂γ_2L, receptor isoforms. In the α₁β₂γ_2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABA, receptor function, but 2-butanol had a greater efficacy. In the α₂β₂ receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the a2p2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 μM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a γ_2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the α₁β₂γ_2L receptor construct. Ethanol potentiation of GABA_A receptor function was completely blocked by the benrodiazepine receptor partial inverse agonist R015-4513 at a concentration (0.5 μM) that did not alter the control GABA response. In contrast, R015-4513 did not block potentiation of GABA_A receptor activity induced by npropanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,0-diisopropylphenol). These results suggest that alcohols have specific interactions with GABA_A receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Metabotropic glutamate and GABAB receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells

Aims/hypothesis: The neurotransmitters glutamate and γ-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA_B (GABA_BRs) receptors in this process. Methods: We studied the expression of mGluRs and GABA _BRs in rat and human islets of Langerhans and in pancreatic α-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA _B R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). Results: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA _BR1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA_BR1 a/b and GABA_BR2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6–8) specific agonist inhibited insulin release at high (10–25 mmol/l) glucose concentrations. Baclofen, a GABA_BR agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. Conclusion/interpretation: These data suggest that mGluRs and GABA_BRs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca²⁺-channels, cAMP generation and G-protein-mediated modulation of K_ATP channels. [Diabetologia (2002) 45: 242–252] Received: 18 September 2001 and in revised form: 5 November 2001     

Neural Mechanisms and Delayed Gastric Emptying of Liquid Induced Through Acute Myocardial Infarction in Rats

Background

In pathological situations, such as acute myocardial infarction, disorders of motility of the proximal gut can trigger symptoms like nausea and vomiting. Acute myocardial infarction delays gastric emptying (GE) of liquid in rats.

Objective

Investigate the involvement of the vagus nerve, α 1-adrenoceptors, central nervous system GABA_B receptors and also participation of paraventricular nucleus (PVN) of the hypothalamus in GE and gastric compliance (GC) in infarcted rats.

Methods

Wistar rats, N = 8-15 in each group, were divided as INF group and sham (SH) group and subdivided. The infarction was performed through ligation of the left anterior descending coronary artery. GC was estimated with pressure-volume curves. Vagotomy was performed by sectioning the dorsal and ventral branches. To verify the action of GABA_B receptors, baclofen was injected via icv (intracerebroventricular). Intravenous prazosin was used to produce chemical sympathectomy. The lesion in the PVN of the hypothalamus was performed using a 1mA/10s electrical current and GE was determined by measuring the percentage of gastric retention (% GR) of a saline meal.

Results

No significant differences were observed regarding GC between groups; vagotomy significantly reduced % GR in INF group; icv treatment with baclofen significantly reduced %GR. GABA_B receptors were not conclusively involved in delaying GE; intravenous treatment with prazosin significantly reduced GR% in INF group. PVN lesion abolished the effect of myocardial infarction on GE.

Conclusion

Gastric emptying of liquids induced through acute myocardial infarction in rats showed the involvement of the vagus nerve, alpha1- adrenergic receptors and PVN.     

GABA uptake-dependent Ca2+ signaling in developing olfactory bulb astrocytes

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca²⁺ imaging and two-photon Na⁺ imaging. GABA evoked Ca²⁺ transients in astrocytes that persisted in the presence of GABA_A and GABA_B receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca²⁺ blocked GABA-induced Ca²⁺ transients, and depletion of Ca²⁺ stores with cyclopiazonic acid reduced Ca²⁺ transients by approximately 90%. This indicates that the Ca²⁺ transients depend on external Ca²⁺, but are mainly mediated by intracellular Ca²⁺ release, conforming with Ca²⁺-induced Ca²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca²⁺ transients, whereas the InsP₃ receptor blocker 2-APB inhibited the Ca²⁺ transients. GABA also induced Na⁺ increases in astrocytes, potentially reducing Na⁺/Ca²⁺ exchange. To test whether reduction of Na⁺/Ca²⁺ exchange induces Ca²⁺ signaling, we inhibited Na⁺/Ca²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca²⁺ transients in astrocytes. The significance of GABAergic Ca²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na⁺ rises that reduce Na⁺/Ca²⁺ exchange, thereby leading to a Ca²⁺ increase sufficient to trigger Ca²⁺-induced Ca²⁺ release via InsP₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.     

Tumor-derived extracellular vesicles containing long noncoding RNA PART1 exert oncogenic effect in hepatocellular carcinoma by polarizing macrophages into M2

AimWe explored whether tumor-derived extracellular vesicles (EVs) could deliver long noncoding RNA (lncRNA) PART1 into macrophage to orchestrate macrophage polarization in the progression of hepatocellular carcinoma (HCC).MethodThe expression patterns of PART1, microRNA (miR)-372-3p and TLR4 were detected by RT-qPCR in the HCC tissues and HCC cells. PART1 was silenced or overexpressed in HCC cells to assess its effects on the HCC cell process. EVs were isolated from PART1-overexpressed HCC cells, and co-cultured with macrophages, and gain- and loss-of-function assays were implemented in macrophages to evaluate their role in macrophage polarization. Relationship among PART1, miR-372-3p, and TLR4 was evaluated. Effect of EV-PART1 on tumorigenicity in vivo was detected by subcutaneous tumorigenicity test in nude mice.ResultPART1 and TLR4 were upregulated while miR-372-3p was downregulated in HCC tissues and cells. PART1 increased HCC cell proliferation, migration, invasion, and EMT. Mechanistically, PART1 bound to miR-372-3p to downregulate its expression, whereas TLR4 was negatively targeted by miR-372-3p in the macrophages. EVs containing PART1, TLR4 overexpression, or miR-372-3p inhibition induced M2 polarization of macrophages. Also, EVs containing PART1 promoted M2 polarization of macrophages and the occurrence of HCC by affecting miR-372-3p/TLR4 axis.ConclusionHCC cell-derived EVs might up-regulate TLR4 by inhibiting miR-372-3p via PART1 delivery to promote macrophage M2 polarization in HCC.     

Administration of growth hormone and nandrolone decanoate alters mRNA expression of the GABAB receptor subunits as well as of the GH receptor,IGF-1, and IGF-2 in rat brain

ObjectiveThe illicit use of anabolic androgenic steroids (AAS), especially among young adults, is of major concern. Among AAS users it is common to combine the AAS nandrolone decanoate (ND), with intake of growth hormone (GH) and a connection between gonadal steroids and the GH system has been suggested. Both AAS and GH affect functions in the brain, for example those associated with the hypothalamus and pituitary, and several GH actions are mediated by growth factors such as insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2). The GABAergic system is implicated in actions induced by AAS and previous studies have provided evidence for a link between GH and GABA_B receptors in the brain. Our aim was to examine the impact of AAS administration and a subsequent administration of GH, on the expression of GABA_B receptors and important GH mediators in rat brain.DesignThe aim was to investigate the CNS effects of a high-dose ND, and to study if a low, but physiological relevant, dose of GH could reverse the ND-induced effects. In the present study, male rats were administered a high dose of ND every third day during three weeks, and subsequently the rats were given recombinant human GH (rhGH) during ten days. Quantitative PCR (qPCR) was used to analyze gene expression in hypothalamus, anterior pituitary, caudate putamen, nucleus accumbens, and amygdala.ResultsIn the pituitary gland, the expression of GABA_B receptor subunits was affected differently by the steroid treatment; the GABA_B1 mRNA expression was decreased whereas a distinct elevation of the GABA_B2 expression was found. Administration of ND also caused a decrease of GHR, IGF-1, and IGF-2 mRNA expression in the pituitary while the corresponding expression in the hypothalamus, caudate putamen, nucleus accumbens, and amygdala was unaffected. The rhGH administration did not alter the GABA_B2 expression but increased the GABA_B1 gene expression in the hypothalamus as compared to the AAS treated group.ConclusionsThese results provide new insights on the impact of ND and GH on the brain and highlight the interaction of these hormones with systems influencing GABA_B receptor expression. The physiological significance of the observed effects of these hormones is discussed.     

Interactions of helminths with macrophages: therapeutic potential for inflammatory intestinal disease

ABSTRACT

Introduction: Macrophages represent a highly heterogeneous and plastic cell type found in most tissues of the body; the intestine is home to enormous numbers of these cells. Considerable interest surrounds the ‘M2 macrophage,’ as it is able to control and regulate inflammation, while promoting tissue repair.

Areas covered: As potent inducers of M2 macrophages, intestinal helminths and helminth-derived products are ideal candidates for small molecule drug design to drive M2 macrophage polarization. Several gastrointestinal helminths have been found to cause M2 macrophage-inducing infections. This review covers current knowledge of helminth products and their impact on macrophage polarization, which may in the future lead to new therapeutic strategies.

A literature search was performed using the following search terms in PubMed: M2 macrophage, alternative activation, helminth products, helminth ES, helminth therapy, nanoparticle, intestinal macrophages. Other studies were selected by using references from articles identified through our original literature search.

Expert commentary: While the immunomodulatory potential of helminth products is well established, we have yet to fully characterize many components of the intestinal helminth product library. Current work aims to identify the protein motifs responsible for modulation of macrophages and other components of the immune system.     

Sequential Co-infection of Heligmosomoides polygyrus and Mycobacterium tuberculosis Determine Lung Macrophage Polarization and Histopathological Changes

BackgroundTuberculosis is a chronic infection caused by Mycobacterium tuberculosis (M.tb), which needs proper macrophage activation for control. It has been debated whether the co-infection with helminth will affect the immune response to mycobacterial infection.ObjectiveTo determine the effect of sequential co-infection of Heligmosomoides polygyrus (H.pg) nematodes and M.tb on T cell responses, macrophages polarization and lung histopathological changes.MethodThis study used 49 mice divided into 7 treatment groups, with different sequence of infection of M.tb via inhalation and H.pg via oral ingestion for 8 and 16 weeks. T cells response in the lung, intestine, and peripheral blood were determined by flow cytometry. Cytokines (IL-4, IFN-γ, TGB-β1, and IL-10) were measured in peripheral blood using ELISA. Lung macrophage polarization were determined by the expression of iNOS (M1) or Arginase 1 (M2). Mycobacterial count were done in lung tissue. Lung histopathology were measured using Dorman’s semiquantitative score assessing peribronchiolitis, perivasculitis, alveolitis, and granuloma formation.ResultM.tb infection induced Th1 response and M1 macrophage polarization, while H.pg infection induced Th2 and M2 polarization. In sequential co-infection, the final polarization of macrophage was dictated by the sequence of co-infection. However, all groups with M.tb infection showed the same degree of mycobacterial count in lung tissues and lung tissue histopathological changes.ConclusionSequential co-infection of H.pg and M.tb induces different T cell response which leads to different macrophage polarization in lung tissue. Helminth infection induced M2 lung macrophage polarization, but did not cause different mycobacterial count nor lung histopathological changes.     

Effect of infusing gamma-aminobutyric acid receptor agonists and antagonists into the medial preoptic area and ventromedial hypothalamus on prolactin secretion in male sheep

We investigated the effects of gamma-aminobutyric acid (GABA) agonists muscimol and baclofen (GABA(A) and GABA(B) agonists, respectively) and antagonists bicuculline methiodide (BMI, GABA(A) antagonist) or 2-hydroxysaclofen (SAC) and CGP 55845A (GABA(B) antagonists) on prolactin (PRL) secretion in castrated rams. The drugs were applied by microdialysis into either the medial preoptic area (mPOA) or ventromedial hypothalamus (VMH). Dialysis of baclofen into the mPOA significantly increased mean PRL (p < 0.05), whereas SAC caused a small, but significant decrease (p < 0.01). Dialysis of either muscimol or BMI into the mPOA had no effect on prolactin. In the VMH, baclofen significantly increased (p < 0.01) mean PRL but SAC and CGP 55845A were ineffective, whereas dialysis of either muscimol or BMI increased mean prolactin (p < 0.01). These results show that infusion into the mPOA of drugs that affect GABA(B) receptor alter PRL release, whereas infusion of a GABA(A) agonists and antagonist was without effect on PRL release. In contrast, infusion of both GABA(A) and GABA(B) agonists and a GABA(A) antagonist into the VMH altered PRL secretion. This suggest that GABAergic neurons in both regions participate in regulating PRL secretion, but by different receptor systems.