Enhancement of Lanthanum (III) on Sodium Currents in Acutely Isolated Hippocampal CA1 Neurons of Rat

The effects of lanthanum (III) (La³⁺) on voltage-gated sodium channel currents (I _Na) in freshly dissociated rat hippocampal CA1 neurons were studied using the whole-cell patch clamp techniques. La³⁺ reversibly enhanced I _Na in a concentration- and voltage-dependent manner. The 50% enhancement concentration (EC₅₀) of La³⁺ on I _Na was 9.93 μM. In addition, 10 μM La³⁺ shifted the steady state activation curve of I _Na towards positive potential and the steady state inactivation curve towards negative potential without changing the slope factor. These results indicated that La³⁺ could increase the amplitudes of I _Na and change the activation and inactivation courses of I _Na even in very low concentration.     

不同信号分子参与M胆碱能受体激动剂对不同胚胎发育阶段小鼠心肌细胞起搏电流的调控

应用全细胞膜片钳技术,研究了M胆碱能对不同孕期的胚胎小鼠心肌细胞的起搏电流(If)的调节。我们发现,在胚胎发育的早期阶段,M胆碱能受体激动剂(muscarinic agonist carbachol,CCh)明显抑制If,但在胚胎发育的晚期阶段,CCh对If的抑制作用消失。腺苷酸环化酶(adeinylate cyclase,AC)激动剂毛喉素Forskolin和非选择性磷酸二酯酶(PDE)抑制剂IBMX均可增强发育早期阶段和晚期阶段的If。但有趣的是,尽管,Forskolin和IBMX可增加基础If,它们对CCh抑制的If的作用却大不相同。在胚胎发育的早期阶段,Forskolin不能拮抗CCh对If的抑制作用,但IBMX可以。在发育的中期阶段Forskolin可以拮抗CCh的抑制作用,但IBMX不可以。因此,我们推断,CCh可能是通过调控细胞内的CAMP水平来调节If的。但是在胚胎发育的早期阶段和中期阶段,CCh可能通过不同的信号转导通路来实现对胞内cAMP的水平调控。在发育的早期阶段,CCh主要是通过增强PDE的活性,加速cAMP的降解而实现对f的调控。而在发育的中期阶段,CCh则主要通过与AC耦联,抑制其活性,通过减慢cAMP的合成而实现对If的调控。     

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap₅A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl⁻ channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.     

Tonic modulation of anxiety-like behavior by corticotropin-releasing factor (CRF) type 1 receptor (CRF1) within the medial prefrontal cortex (mPFC) in male mice: Role of protein kinase A (PKA)

The medial prefrontal cortex (mPFC) and the neuropeptide corticotropin-releasing factor (CRF) have recently been receiving more attention from those interested in the neurobiology of anxiety. Here, we investigated the CRF pathway in the modulation of anxiety-like behaviors in male mice exposed to the elevated plus-maze (EPM), through intra-mPFC injections of CRF, CP376395 [N-(1-ethylpropyl)-3,6-dimethyl-2-(2,4,6-trimethylphenoxy)-4-pyridinamine hydrochloride, a CRF type 1 receptor antagonist (CR F1)] or H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase (PKA) inhibitor]. We also investigated the effects of intra-mPFC injections of H-89 on the behavioral effects induced by CRF. Mice received bilateral intra-mPFC injections of CRF (0, 37.5, 75 or 150 pmol), CP376395 (0, 0.75, 1.5 or 3 nmol) or H-89 (0, 1.25, 2.5 or 5 nmol) and were exposed to the EPM, to record conventional and complementary measures of anxiety for 5 min. Results showed that while CRF (75 and 150 pmol) produced an anxiogenic-like effect, CP376395 (all doses) and H-89 (5 nmol) attenuated anxiety-like behavior. When injected before CRF (150 pmol), intra-mPFC H-89 (2.5 nmol, a dose devoid of intrinsic effects on anxiety) completely blocked the anxiogenic-like effects of CRF. These results suggest that (i) CRF plays a tonic anxiogenic-like role at CRF1 receptors within the mPFC, since their blockade per se attenuated anxiety indices and (ii) the anxiogenic-like effects following CRF1 receptor activation depend on cAMP/PKA cascade activation in this limbic forebrain area.     

Mechanisms for modulation of mouse gastrointestinal motility by proteinase-activated receptor (PAR)-1 and -2 in vitro

Proteinase-activated receptor (PAR)-1 or -2 modulates gastrointestinal transit in vivo. To clarify the underlying mechanisms, we characterized contraction/relaxation caused by TFLLR-NH2 and SLIGRL-NH2, PAR-1- and -2-activating peptides, respectively, in gastric and small intestinal (duodenal, jejunal and ileal) smooth muscle isolated from wild-type and PAR-2-knockout mice. Either SLIGRL-NH2 or TFLLR-NH2 caused both relaxation and contraction in the gastrointestinal preparations from wild-type animals. Apamin, a K+ channel inhibitor, tended to enhance the peptide-evoked contraction in some of the gastrointestinal preparations, whereas it inhibited relaxation responses to either peptide completely in the stomach, but only partially in the small intestine. Indomethacin reduced the contraction caused by SLIGRL-NH2 or TFLLR-NH2 in both gastric and ileal preparations, but unaffected apamin-insensitive relaxant effect of either peptide in ileal preparations. Repeated treatment with capsaicin suppressed the contractile effect of either peptide in the stomach, but not clearly in the ileum, whereas it enhanced the apamin-insensitive relaxant effect in ileal preparations. In any gastrointestinal preparations from PAR-2-knockout mice, SLIGRL-NH2 produced no responses. Thus, the inhibitory component in tension modulation by PAR-1 and -2 involves both apamin-sensitive and -insensitive mechanisms in the small intestine, but is predominantly attributable to the former mechanism in the stomach. The excitatory component in the PAR-1 and -2 modulation may be mediated, in part, by activation of capsaicin-sensitive sensory nerves and/or endogenous prostaglandin formation. Our study thus clarifies the multiple mechanisms for gastrointestinal motility modulation by PAR-1 and -2, and also provides ultimate evidence for involvement of PAR-2.     

Anti-microRNA-222 (anti-miR-222) and -181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and modulating mitogenic signal

We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHT(R)) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHT(R) cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHT(R) cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHT(R) cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHT(R) cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHT(R) cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.