Enhanced astroglial GABA uptake attenuates tonic GABAA inhibition of the presympathetic hypothalamic paraventricular nucleus neurons in heart failure.

γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents (Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure.

Single-nucleotide polymorphism PCR for the detection of Mycoplasma pneumoniae and determination of macrolide resistance in respiratory samples.

The aim of this study was to develop a single-nucleotide polymorphism (SNP) PCR assay to be performed directly on respiratory samples for the simultaneous detection of Mycoplasma pneumoniae and its 23S rRNA gene mutations, which are responsible for macrolide resistance. For multiplex SNP PCR, two outer primers for amplification of the 23S rRNA gene and two mutant-specific primers for the discrimination of single base changes were designed. A total of 73M. pneumoniae-positive samples and 100M. pneumoniae-negative samples were analyzed using this assay. By SNP PCR, we detected two mutations conferring high-level macrolide resistance in 22 samples (A2063G from 20 and A2064G from 2 samples); these results are identical to those produced by the 23S rRNA gene sequencing of M. pneumoniae-positive samples. Thus, this assay can be used as a practical method for the simultaneous detection of M. pneumoniae and mutations associated with macrolide resistance directly from respiratory samples.

Dual mechanisms diminishing tonic GABAA inhibition of dentate gyrus granule cells in Noda epileptic rats.

The Noda epileptic rat (NER), a Wistar colony mutant, spontaneously has tonic-clonic convulsions with paroxysmal discharges. In the present study, we measured phasic and tonic γ-aminobutyric acid A (GABAA) current (I tonic) in NER hippocampal dentate gyrus granule cells and compared the results with those of normal parent strain Wistar rats (WIS). I tonic, revealed by a bicuculline-induced outward shift in holding current, was significantly smaller in NER than in WIS (P < 0.01). The frequency of inhibitory postsynaptic currents (IPSCs) was also significantly lower in NER than in WIS (P < 0.05), without significant differences in the IPSC amplitude or decay time between WIS and NER. I tonic attenuation in NER was further confirmed in the presence of GABA transporter blockers, NO-711 and nipecotic acid, with no difference in neuronal GABA transporter expression between WIS and NER. I tonic responses to extrasynaptic GABAA receptor agonists (THIP and DS-2) were significantly reduced in NER compared with WIS (P < 0.05). Allopregnanolone caused less I tonic increase in NER than in WIS, while it prolonged the IPSC decay time to a similar rate in the two groups. Expression of the GABAA receptor δ-subunit was decreased in the dentate gyrus of NER relative to that of WIS. Taken together, our results showed that a combination of attenuated presynaptic GABA release and extrasynaptic GABAA receptor expression reduced I tonic amplitude and its sensitivity to neurosteroids, which likely diminishes the gating function of dentate gyrus granule cells and renders NER more susceptible to seizure propagation.

Collagen triple helix repeat containing-1 promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells.

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein involved in vascular remodeling, bone formation and developmental morphogenesis. CTHRC1 has recently been shown to be expressed in human cancers such as breast cancer and melanoma. In this study, we show that CTHRC1 is highly expressed in human pancreatic cancer tissues and plays a role in the progression and metastasis of the disease. CTHRC1 promoted primary tumor growth and metastatic spread of cancer cells to distant organs in orthotopic xenograft tumor mouse models. Overexpression of CTHRC1 in cancer cells resulted in increased motility and adhesiveness, whereas these cellular activities were diminished by down-regulation of the protein. CTHRC1 activated several key signaling molecules, including Src, focal adhesion kinase, paxillin, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase and Rac1. Treatment with chemical inhibitors of Src, MEK or Rac1 and expression of dominant-negative Rac1 attenuated CTHRC1-induced cell migration and adhesion. Collectively, our results suggest that CTHRC1 has a role in pancreatic cancer progression and metastasis by regulating migration and adhesion activities of cancer cells.

Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells.

Elafin, a serine protease inhibitor, induces the intrinsic apoptotic pathway in human melanoma cells, where its expression is transcriptionally silenced. However, it remains unknown how the elafin gene is repressed in melanoma cells. We here demonstrate that elafin expression is modulated via epigenetically regulated expression of the transcription factor Foxa2. Treatment of melanoma cells with a DNA methyltransferase inhibitor induced elafin expression, which was specifically responsible for reduced proliferation and increased apoptosis. Suppression of Foxa2 transcription, mediated by DNA hypermethylation in its promoter region, was released in melanoma cells upon treatment with the demethylating agent. Luciferase reporter assays indicated that the Foxa2 binding site in the elafin promoter was critical for the activation of the promoter. Chromatin immunoprecipitation assays further showed that Foxa2 bound to the elafin promoter in vivo. Analyses of melanoma cells with varied levels of Foxa2 revealed a correlated expression between Foxa2 and elafin and the ability of Foxa2 to induce apoptosis. Our results collectively suggest that, in melanoma cells, Foxa2 expression is silenced and therefore elafin is maintained unexpressed to facilitate cell proliferation in the disease melanoma.

Neurosteroid modulation of benzodiazepine-sensitive GABAA tonic inhibition in supraoptic magnocellular neurons.

Interactions between neurosteroids and GABA receptors have attracted particular attention in the supraoptic nucleus (SON). Although GABA(A) receptors (GABA(A)R) mediate a sustained tonic inhibitory current (I(tonic)), as well as conventional phasic inhibitory postsynaptic currents (IPSCs, I(phasic)) in the SON, whether the steroid modulation on I(tonic) is present in SON magnocelluar neurosecretory cells (MNCs) is unknown. Here, we addressed this question and gained insights into the potential molecular configuration of GABA(A) receptors mediating I(tonic) and conferring its neurosteroids sensitivity in SON MNCs. 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) (1 µM), a relatively selective extrasynaptic GABA(A)R agonist, facilitated I(tonic) without affecting the main characteristics of IPSCs, while DS-2, a relatively selective modulator of GABA(A)R δ-subunits, caused minimal changes in I(tonic) of SON MNCs. l-655,708, a relatively selective GABA(A)R α(5)-subunit inverse agonist, blocked ∼35% of the total I(tonic) both under basal and elevated ambient GABA concentration (3 µM). Facilitation of I(tonic) by benzodiazepines further supported the role of GABA(A)R γ(2)-subunit in I(tonic) of SON MNCs. Quantitative RT-PCR analysis showed much lesser expression of GABA(A)R δ-subunit than the α(5) or γ(2)-subunit in the SON. Allopregnanolone and 3α,5α-tetrahydrodeoxycorticosterone increased both I(tonic) and I(phasic) in SON MNCs, respectively, although more than 90% of the current increase was mediated by I(tonic) during the neurosteroid facilitation. Finally, l-655,708 attenuated the neurosteroid facilitation of I(tonic) but not of I(phasic). Altogether, our results suggest that I(tonic), mediated mainly by benzodiazepine-sensitive GABA(A)Rs containing α(5)-, ß-, and γ(2)-, and to a lesser extent, δ-subunits, is a potential target of neurosteroid modulation in SON neurons.

PAUF promotes adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase.

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.

Major role of GABA(A)-receptor mediated tonic inhibition in propofol suppression of supraoptic magnocellular neurons.

Using slice patch clamp recording, we examined the effects of general anesthetic propofol (2,6-diisoprophlphenol) on dual modality of GABA(A) inhibition in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs): conventional quantal synaptic transmission (IPSCs, I(phasic)) and persistent tonic form of inhibitory current (I(tonic)). Propofol (10 µM) enhanced I(tonic) as shown by an inward shift in I(holding) (16.46±2.93 pA, n=27) and RMS increase (from 3.37±0.21 pA to 4.68±0.33 pA, n=27) in SON MNCs. Propofol also prolonged the decay time of IPSCs with decreased IPSCs frequency but no significant changes in IPSCs amplitude. Overall, propofol (1-10 µM) caused much smaller increase in mean I(phasic) than mean I(tonic) at all tested concentrations. In consistent with the enhancement of GABA(A) currents, propofol attenuated ongoing firing activities of SON MNCs by ∼65% of control. Selective inhibition of I(phasic) by a GABA(A) antagonist, gabazine (1 µM), failed to block the propofol suppression of the firing activities, while inhibition of I(tonic) and I(phasic) by bicuculline (20 µM) efficiently blocked the propofol-induced neurodepression in SON MNCs. Taken together, our results showed that propofol facilitated I(tonic) with marginal increase in mean I(phasic), and this could be a mechanism reducing the intrinsic SON MNCs excitability during propofol anesthesia.

Regulation of tonic GABA inhibitory function, presympathetic neuronal activity and sympathetic outflow from the paraventricular nucleus by astroglial GABA transporters.

Neuronal activity in the hypothalamic paraventricular nucleus (PVN), as well as sympathetic outflow from the PVN, is basally restrained by a GABAergic inhibitory tone. We recently showed that two complementary GABA(A) receptor-mediated modalities underlie inhibition of PVN neuronal activity: a synaptic, quantal inhibitory modality (IPSCs, I(phasic)) and a sustained, non-inactivating modality (I(tonic)). Here, we investigated the role of neuronal and/or glial GABA transporters (GATs) in modulating these inhibitory modalities, and assessed their impact on the activity of RVLM-projecting PVN neurons (PVN-RVLM neurons), and on PVN influence of renal sympathetic nerve activity (RSNA). Patch-clamp recordings were obtained from retrogradely labelled PVN-RVLM neurons in a slice preparation. The non-selective GAT blocker nipecotic acid (100-300 microM) caused a large increase in GABA(A)I(tonic), and reduced IPSC frequency. These effects were replicated by beta-alanine (100 microM), but not by SKF 89976A (30 microM), relatively selective blockers of GAT3 and GAT1 isoforms, respectively. Similar effects were evoked by the gliotoxin L-alpha-aminodipic acid (2 mM). GAT blockade attenuated the firing activity of PVN-RVLM neurons. Moreover, PVN microinjections of nipecotic acid in the whole animal diminished ongoing RSNA. A robust GAT3 immunoreactivity was observed in the PVN, which partially colocalized with the glial marker GFAP. Altogether, our results indicate that by modulating ambient GABA levels and the efficacy of GABA(A)I(tonic), PVN GATs, of a likely glial location, contribute to setting a basal tone of PVN-RVLM firing activity, and PVN-driven RSNA.

Serotonin inhibits GABA synaptic transmission in presympathetic paraventricular nucleus neurons.

Activation of serotonin (5-hydroxytryptamine, 5-HT) receptors produces various autonomic and neuroendocrine responses in the hypothalamic paraventricular nucleus (PVN), including increased blood pressure and heart rate. However, the role(s) of 5-HT on the local GABA synaptic circuit have not been well understood in the PVN, where the inhibitory neurotransmitter GABA plays a key role in the modulation of sympathoexcitatory outflow. In the present study, we examined the effects of 5-HT on GABA synaptic transmission in presympathetic PVN neurons projecting to spinal cord using patch-clamp electrophysiology combined with tract-tracing techniques. Bath application of 5-HT (0.01-100 microM) reversibly decreased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (sIPSC) in a concentration dependent manner (IC50, 0.07 microM), with no significant changes in the amplitudes and decay kinetics of sIPSC. The sIPSC inhibition of 5-HT was mimicked by 5-HT1A agonist, 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin, 10 microM), and blocked by 5-HT1A antagonist WAY-100635 but not by 5-HT1B antagonist SB224289. 5-HT also reduced the frequency of miniature IPSC (mIPSC) (2.59+/-0.51 Hz, control vs. 1.25+/-0.31 Hz, 5-HT, n=16) in similar extent with 5-HT induced reduction of sIPSC frequency (sIPSCs, 55.8+/-6.2%, n=11 vs. mIPSCs, 52.30+/-5.85%, n=16; p>0.5). All together, our results indicate that 5-HT can inhibit presynaptic GABA release via presynaptic 5-HT1A receptors in presympathetic PVN neurons projecting to spinal cord.