Genome sequencing and comparative genome analysis of 6 hypervirulent Klebsiella pneumoniae strains isolated in China.

Hypervirulent Klebsiella pneumoniae (hvKP) has been increasingly reported over the past three decades and causes severe infections. To increase our understanding of hvKP at the genome level, genome sequencing and comparative genome analysis were performed on 6 hvKPs. The whole genome DNA from 6 hvKPs with different capsular serotypes isolated in China was extracted. The genome sequencing and assembly results showed the genome size of the six hvKPs and GC content. Comparative analyses of the genomes revealed the gene homology and genome rearrangement in the 6 hvKPs compared with Klebsiella pneumonia NTUH-K2044. The phylogenetic tree based on full-genome SNPs of the 7 hvKPs showed that NTUH-K2044 formed a single clade, showing distant evolutionary distances with the other six strains, and the non-K1 hvKP strains had a relatively closer phylogenetic relationship. BLAST comparison analysis found that some selected virulence genes had different degrees of deletion in the non-K1 hvKPs. SNP-based virulence gene mutation analysis showed that some virulence genes had different degrees of SNP mutations. The whole-genome sequencing and comparative genome analysis of six hvKP strains with NTUH-K2044 provide us with a basic understanding of the genome composition, genetic polymorphism, evolution and virulence genes of hvKP and a basis for further research on these genes and the pathogenesis of hvKP.

Risk factors of bloodstream infections in recipients after liver transplantation: a meta-analysis.

PURPOSE: Bloodstream infection (BSI) is an important cause of adverse outcomes for recipients with liver transplantation (LT). This meta-analysis aimed to identify risk factors associated with post-LT BSI. METHODS: Relevant studies published up to June 2017 were searched from seven electronic databases. The studies were reviewed according to the inclusion and exclusion criteria. The Z test was used to determine the pooled odds ratio (OR) or standardized mean difference (SMD) of the risk factors. ORs and their corresponding 95% confidence intervals (CIs), or SMDs and their corresponding 95% CIs were used to identify the significant difference of risk factors. RESULTS: Seventeen studies enrolling 4410 recipients were included. Eleven risk factors were identified to be associated with BSI after LT: male recipient (OR = 1.28), ascites (OR = 1.68), model for end-stage liver disease (MELD) score (SMD = 0.20), Child-Pugh class C (OR = 1.69), operation time (SMD = 0.18), incompatible blood type (OR = 2.87), operative blood loss (SMD = 0.33), rejection (OR = 1.72), biliary complications (OR = 1.91), hemodialysis (OR = 3.37), and retransplantation (OR = 2.86). CONCLUSIONS: Although some risk factors were identified as significant factors for BSI after LT, which may provide a basis for clinical prevention, well-designed prospective studies should be done to overcome the limitations of this study.

Enhanced gap junctional channel activity between vascular smooth muscle cells in cerebral artery of spontaneously hypertensive rats.

The aim of the present study is to investigate the effects of hypertension on the gap junctions between vascular smooth muscle cells (VSMCs) in the cerebral arteries (CAs) of spontaneously hypertensive rats (SHRs). The functions of gap junctions in the CAs of VSMCs in SHRs and control normotensive Wistar-Kyoto (WKY) rats were studied using whole-cell patch clamp recordings and pressure myography, and the expression levels of connexins were analyzed using reverse transcription-quantitative polymerase chain reaction and Western blot analyses. Whole-cell patch clamp measurements revealed that the membrane capacitance and conductance of in situ VSMCs in the CAs were significantly greater in SHRs than in WKY rats, suggesting that gap junction coupling is enhanced between VSMCs in the CAs of SHRs. Application of the endothelium-independent vasoconstrictors KCl or phenylephrine (PE) stimulated a greater vasoconstriction in the CAs of SHRs than in those of WKY rats. The EC50 value of KCl was 24.9 mM (n = 14) and 36.9 mM (n=12) for SHRs and WKY rats, respectively. The EC50 value of PE was 0.9 µM (n = 7) and 2.2 µM (n = 7) for SHRs and WKY rats, respectively. Gap junction inhibitors 18ß-glycyrrhetinic acid (18ß-GA), niflumic acid (NFA), and 2-aminoethoxydiphenyl borate (2-APB) attenuated KCl-induced vasoconstriction in SHRs and WKY rats. The mRNA and protein expression levels of the gap junction protein connexin 45 (Cx45) were significantly higher in the CAs of SHRs than in those of WKY rats. Phosphorylated Cx43 protein expression was significantly higher in the CAs of SHRs than in those of WKY rats, despite the total Cx43 mRNA and protein expression levels in the cerebral artery (CA) exhibiting no significant difference between SHRs and WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may promote gap junction communication among VSMCs in the CAs of SHRs, which may enhance the contractile response of the CA to vasoconstrictors.

[Study on volatile components of Lonicerae Japonicae Flos in bud stage extended type].

Volatile components of Lonicerae Japonicae Flos in bud stage extended type Beihua 1 were determined by the headspace solid-phase micro-extraction, compared with traditional cultivar Damaohua. There are fifty-two volatile compounds were identified and the relative content of the volatiles was calculated by the area normalization method. Thirty-nine compounds were found in Beihua 1, whereas thirty-three components in Damaohua. Total twenty identical compounds existed in Beihua 1 and Damaohua. The contents of alcohols and hydrocarbons of Beihua 1 were higher significantly than that of Damaohua, while significantly lower than that of Damaohua in ketones content. Besides, twenty components were only detected in Beihua 1, such as methyl nicotinate, hexadecanoic acid, methyl ester,acetophenone, nonanoic acid.

The enhancement of Cx45 expression and function in renal interlobar artery of spontaneously hypertensive rats at different age.

BACKGROUND/AIMS: This study was designed to investigate the expression and function of gap junction protein connexin 45 (Cx45) in renal interlobar artery (RIA) of spontaneously hypertensive rats (SHR), and the association between hypertension and enhanced vasoconstrictive response in SHR. METHODS: Western blot analysis and pressure myography were used to examine the differences in expression and function of Cx45 in vascular smooth muscle cells (VSMCs) of RIA between SHR and normotensive Wistar-Kyoto (WKY) rats. RESULTS: Our results demonstrated that 1) whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ RIA VSMCs of SHR were significantly greater than those of WKY rats (p<0.05, n=6), suggesting that the coupling of gap junction between VSMCs of RIA was enhanced in SHR; 2) the KCl or phenylephrine (PE)-stimulated RIA constriction was more pronounced in SHR than that in WKY rats (p<0.05, n=10). After applying a gap junction inhibitor 18ß-glycyrrhetintic acid (18ß-GA), the inhibitory effect of 18ß-GA on KCl or PE-induced vasoconstriction was greater in SHR (p<0.05, n=10); and 3) the expression of Cx45 in RIA of SHR was greater than that in WKY rats (p<0.05, n=3) at 4, 12 and 48 wks of age. CONCLUSIONS: The hypertension-induced elevation of Cx45 may affect communication between VSMCs and coupling between VSMCs and endothelium, which results in an increased vasoconstrictive response in renal artery and might contribute to the development of hypertension.

Myoendothelial coupling is unidirectional in guinea pig spiral modiolar arteries.

Gap junctions (GJs) facilitate communication and promote transfer of signaling molecules or current between adjacent cells in various organs to coordinate cellular activity. In arteries, homocellular GJs are present between adjacent smooth muscle cells (SMCs) and between adjacent endothelial cells (ECs), whilst many arteries also exhibit heterocellular GJs between SMCs and ECs. To test the hypothesis that there is differential cell coupling in guinea pig spiral modiolar arteries (SMA), we used intracellular recording technique to record cellular activities simultaneously in ECs or SMCs in acutely isolated guinea pig SMA preparations. Cell types were identified by injection of a fluorescent dye, propidium iodide (PI), through recording microelectrodes. Stable intracellular recordings were made in 120 cells among which 61 were identified as SMCs and 28 as ECs. Dual intracellular recordings were conducted to detect the coexistence of the two distinct levels of resting potential (RP) and to estimate the intensity of electrical coupling between two cells by a current pulse of up to 0.5-1.5 nA. The electrotonic potential was detected not only in the current-injected cell, but also in the majority of non-injected cells. The electrical coupling ratios (ECRs) of homocellular cells were not significant (P>0.05) (0.084±0.032 (n=6) and 0.069±0.031 (n=7) for EC-EC and SMC-SMC pairs, respectively). By contrast, the ECRs of heterocellular cells were significantly different when a current pulse (1.5 nA, 2s) was injected into EC and SMC respectively (0.072±0.025 for EC; 0.003±0.001 for SMC, n=5, P<0.01). The putative gap junction blocker 18ß-glycyrrhetinic acid significantly attenuated electrical coupling in both homocellular and heterocellular forms. The results suggest that homocellular GJs within SMCs or ECs are well coordinated but myoendothelial couplings between ECs and SMCs are unidirectional.

[Acute hypoxia increases outward current and decreases gap junction of VSMCs in guinea-pig anterior inferior cerebellar artery].

The aim of the present study was to investigate the effects of acute hypoxia on the electrophysiological properties of vascular smooth muscle cells (VSMCs) in arteriole. Guinea-pig anterior inferior cerebellar artery (AICA) segments were isolated, and outer layer connective tissue was removed by collagenase A digestion and microforceps. By perfusion with physical saline solution containing no glucose and low oxygen, VSMC model of acute hypoxia was established. The model was studied by whole-cell patch clamp recording technique. Results were shown as below: (1) Acute hypoxia induced an outward current with amplitude of (36.4 ± 9.2) pA at holding potential of -40 mV, and the rest potential (RP) of the VSMCs was hyperpolarized from (-33.2 ± 1.9) mV to (-38.4 ± 1.5) mV. Acute hypoxia increased the outward current of VSMCs in a voltage-dependent manner, this enhancing effect being more pronounced at potentials ranging from 0 to +40 mV. The whole-cell membrane current of VSMCs induced by step command (+40 mV) increased from (650 ± 113) pA to (1 900 ± 197) pA. In the presence of 1 mmol/L tetraethylammonium (TEA), the enhancement of the VSMC membrane current by acute hypoxia was significantly reduced. (2) Acute hypoxia increased the membrane resistance (R(input)) of the VSMCs in AICA from (234 ± 63) MΩ to (1 211 ± 201) MΩ, and decreased the membrane capacitance (C(input)) from (279.3 ± 83.2) pF to (25.4 ± 1.9) pF. In the presence of 30 µmol/L 18ß-glycyrrhetinic acid (18ßGA) and 10 mmol/L TEA, the effects of acute hypoxia on the membrane current of VSMCs were nearly abolished. These results suggest that acute hypoxia causes vascular hyperpolarization and vasodilation, possibly by activating big conductance Ca(2+)-activated K(+) channels (BK(Ca)) of the VSMCs, and inhibits gap junctions between VSMCs, thus improving microcirculation and localizing the hypoxia-induced damage.

[18ß-glycyrrhetinic acid inhibits outward current of vascular smooth muscle cells of arterioles].

The aim of the present study was to investigate the effect of 18ß-glycyrrhetinic acid (18ßGA) on the membrane current of vascular smooth muscle cells (VSMCs) in arteriole. Guinea pig anterior inferior cerebellar artery (AICA) and mesenteric artery (MA) were isolated, and single VSMCs were harvested using digestion with papain and collagenase IA. Outward currents of the VSMCs were recorded by whole-cell patch clamp technique. Results were shown as below: (1) 1 mmol/L 4-AP and 1 mmol/L TEA both could partially inhibit the whole-cell current of VSMCs in arterioles. (2) 18ßGA inhibited the outward current of VSMCs in a concentration-dependent manner. The inhibitory rates of 10, 30 and 100 µmol/L 18ßGA on the membrane current of VSMCs (+40 mV) were (25.3 ± 7.1)%, (43.1 ± 10.4)% and (68.4 ± 3.9)% respectively in AICA, and (13.2 ± 5.6)%, (34.2 ± 4.0)% and (59.3 ± 7.3)% respectively in MA. There was no significant difference between the inhibitory effects of 18ßGA on AICA and MA. 18ßGA also inhibited the outward current of VSMCs in a voltage-dependent manner. 18ßGA induced a more pronounced inhibition of the outward current from 0 to +40 mV, especially at +40 mV. (3) With the pretreatment of 10 mmol/L TEA, the inhibitory effect of 18ßGA on the membrane current of VSMCs was significantly abolished. These results suggest that the outward current of VSMCs in arterioles is mediated by voltage-dependent K(+) channels (K(v)) and big conductance calcium-activated K(+) channels (BK(Ca)), which can be inhibited by 18ßGA in concentration- and voltage-dependent way.

RcsAB and Fur Coregulate the Iron-Acquisition System via entC in Klebsiella pneumoniae NTUH-K2044 in Response to Iron Availability.

The iron acquisition system is an essential virulence factor for human infection and is under tight regulatory control in a variety of pathogens. Ferric-uptake regulator (Fur) is one of Fe2+-responsive transcription factor that maintains iron homeostasis, and the regulator of capsule synthesis (Rcs) is known to regulate exopolysaccharide biosynthesis. We speculate the Rcs may involve in iron-acquisition given the identified regulator box in the upstream of entC that participated in the biosynthesis of enterobactin. To study the coregulation by RcsAB and Fur of entC, we measured the ß-galactosidase activity and relative mRNA expression of entC in WT and mutant strains. The RcsAB- and Fur-protected regions were identified by an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay. A regulatory cascade was identified with which Fur repressed rcsA expression and reduced RcsAB and entC expression. Our study demonstrated that entC was coregulated by two different transcriptional regulators, namely, RcsAB and Fur, in response to iron availability in Klebsiella pneumoniae.

CCK-8S inhibited the NMDA-activated current of cultured hippocampal neuron under normal and ethanol exposure conditions.

Whole-cell patch clamp technique was used to study the modulatory effect of Cholecystokin-8S (CCK-8S), a neuropeptide, on NMDA-activated current (I(NMDA)) in cultured rat hippocampal neurons under condition of normal and ethanol exposure. Our study showed that CCK-8S inhibited I(NMDA) in a non-concentration and non-competition dependent manner. Ethanol significantly inhibited I(NMDA), and CCK-8S further inhibited I(NMDA). Our results suggest that CCK-8S is an inhibitory modulator of NMDA receptors under normal and ethanol exposure conditions.

Niflumic acid hyperpolarizes smooth muscle cells via calcium-activated potassium channel in spiral modiolar artery of guinea pigs.

AIM: The influence of niflumic acid (NFA), a Cl(-)channel antagonist, on the membrane potentials in smooth muscle cells (SMC) of the cochlear spiral modiolar artery (SMA) in guinea pigs was examined. METHODS: The intracellular recording and whole-cell recording technique were used to record the NFA-induced response on the acutely-isolated SMA preparation. RESULTS: The SMC had 2 stable but mutually convertible levels of resting potentials (RP), that is, one was near -45 mV and the other was approximately -75 mV, termed as low and high RP, respectively. The bath application of NFA could cause a hyperpolarization in all the low RP cells, but had little effect on high RP cells. The induced responses were concentration-dependent. Large concentrations of NFA (>or=100 micromol/L) often induced a shift of a low RP to high RP in cells with an initial RP at low level, and NFA (up to 100 micromol/L) had little effect on the membrane potentials of the high RP cells. However, when the high RP cells were depolarized to a level beyond -45 mV by barium and ouabain, NFA hyperpolarized these cells with the similar effect on those cells initially being the low RP. The NFA-induced response was almost completely blocked by charybdotoxin, iberiotoxin, tetraethylammonium, 1,2-bis(2- aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester, but not by 4-aminopyridine, barium, glipizide, apamin, ouabain, and CdCl2. CONCLUSION: NFA induces a concentration-dependent reversible hyperpolarization in SMC in the cochlear SMA via activation of the Ca2+-activated potassium channels.

Transcriptional regulation of galF by RcsAB affects capsular polysaccharide formation in Klebsiella pneumoniae NTUH-K2044.

RcsAB is an atypical two-component regulatory system that can regulate exopolysaccharide biosynthesis and is involved in the virulence of K. pneumoniae. The gene galF is well known as a gene involved in the biosynthesis of capsular polysaccharide (CPS). The specific DNA identification sequence for transcriptional regulation of RcsAB was found to be present in the promoter region of galF. This study aimed to detect the function of RcsAB in virulence and in biofilm and CPS formation. In addition, the transcriptional regulation of the galF gene in K. pneumoniae was studied. To determine the function of rcsAB gene, the wild-type K. pneumoniae strain NTUH-K2044 and the rcsAB knockout and complemented strains were used. The results showed decreased virulence, biofilm formation, and CPS levels in the rcsAB knockout strain. Complementation of the knockout by introducing an rcsAB fragment on an expression plasmid partially restored the virulence, biofilm, and CPS functions of the knockout strain. It indicated that the rcsAB genes might affect CPS formation and virulence of K. pneumonia. RT-qPCR, EMSA and DNase I footprinting assays were conducted to identify the transcriptional regulation of galF by RcsAB. RcsAB was seen to bind to the galF promoter-proximal region, and the binding site was further identified to be located from -177 bp to -152 bp upstream of the galF promoter. In conclusion, RcsAB could regulate the transcription of the galF gene positively by binding to the galF promoter DNA directly, and then affects the CPS formation of K. pneumonia.

Epidemiology of pathogens and antimicrobial resistanceof catheter-associated urinary tract infections in intensivecare units: A systematic review and meta-analysis.

BACKGROUND: In the intensive care unit (ICU), catheter-associated urinary tract infection (CAUTI) is the most common urinary tract infection. Nevertheless, there is no systematic review to investigate the epidemiology of pathogens and antimicrobial resistance of CAUTIs in ICUs. METHODS: Eight electronic databases were searched for eligible studies. A meta-analysis was performed to calculate the CAUTI incidence per 1,000 catheter days, the proportion of pathogen distribution, and the resistance rate with R3.3.2 software. RESULTS: Seventy-five studies were included. The total weighted CAUTI incidence per 1,000 catheter days was 7.78. Gram-negative bacteria (47.46%), fungi (27.81%), and gram-positive bacteria (19.06%) were isolated. Candida spp (27.4%), Escherichia spp (23.41%), and Enterococcus spp (15.0%) were the most frequent pathogens. Candida albicans, Candida tropicalis, and Candida glabrata were generally resistant to itraconazole, with resistance rates of 42.5%, 53.0%, and 59.7%, respectively. Escherichia spp displayed high rates of resistance to ampicillin (87.3%), ciprofloxacin (71.7%), and norfloxacin (71.2%). Enterococcus spp showed high rates of resistance to erythromycin (83.9%), penicillin (76.7%), and levofloxacin (73.8%). CONCLUSIONS: In ICUs, the CAUTI incidence per 1,000 catheter days is high. CAUTIs were mainly caused by gram-negative bacteria that were resistant to common antibiotics. There is a pressing demand for future research into CAUTI, including effective prevention, an understanding of antimicrobial resistance mechanisms, and development of new antibiotics for patient safety.

Connexin 43 in splenic lymphocytes is involved in the regulation of CD4+CD25+ T lymphocyte proliferation and cytokine production in hypertensive inflammation.

Chronic inflammation promotes the development of hypertension and is associated with increased T cell infiltration and cytokine production in impaired organs. Gap junction protein connexin 43 (Cx43), is ubiquitously expressed in immune cells and plays an important role in T cell proliferation and activation, and cytokine production. However, the correlation between Cx43 in T cells and the hypertensive inflammatory response remains unknown. Thus, in this study, we wished to examine this correlation. First, our results revealed that hypertension caused significant thickening of the vascular wall, inflammatory cell infiltration into part of the renal interstitium and glomerular atrophy, and it increased the tubular damage scores in the kidneys of spontaneously hypertensive rats (SHRs). Moreover, the SHRs exhibited stenosis in the central artery wall ofthe spleen with increased serum levels of interleukin (IL)-2 and IL-6 compared with normotensive Wistar-Kyoto (WKY) rats. The spleens of the SHRs exhibited a significantly decreased percentage of CD4+CD25+ (Treg) T cells. However, the percentages of CD3+, CD4+ and CD8+ T cell and the levels of CD4+Cx43 and CD8+Cx43 did not differ significantly between the SHRs and WKY rats. In cultured lymphocytes from the SHRs and WKY rats, low percentages of Treg cells and reduced cytokine (IL-2 and IL-6) mRNA expression levels were observed in the lymphocytes obtained from the SHRs and WKY rats treated with the connexin blocker, Gap27, or concanavalin A (ConA) plus Gap27. The effects of ConA and Gap27 differed between the SHRs and WKY rats. On the whole, our findings demonstrate that the splenic Treg cell-mediated suppression in SHRs may be involved in hypertensive inflammatory responses. Cx43 in the gap junctional channel may regulate lymphocyte activation and inflammatory cytokine production.

A signal-decreased electrochemical immunosensor for the sensitive detection of LAG-3 protein based on a hollow nanobox-MOFs/AuPt alloy.

In this work, hollow nanobox metal-organic framework (HNM) nanocomposites were synthesised and utilised for the first time in a signal decreased electrochemical immunosensor for the ultrasensitive quantitative determination of lymphocyte activation gene-3 (LAG-3) protein, which is a newly discovered biomarker. With the aid of signal materials, namely, SiO2-tagged anti-LAG-3 antibody (SiO2-Ab2) and the biotin-streptavidin system, the sensor can achieve signal amplification. Encapsulation of tin dioxide-functionalised reduced graphene oxide (rGO-SnO2) and gold and platinum alloys (AuPt alloys) onto the surface of hollow nanobox metal-organic frameworks (MOFs) was performed to prepare rGO-SnO2/hollow nanobox-MOFs/AuPt alloys (rGO-SnO2/HNMs/AuPt) as the matrix. SiO2-Ab2, which is used as the signal-decreased label, can be utilised to enhance the distinction of the electrochemical signal after the specific recognition between antibodies and antigens, owing to its large steric hindrance property. In this sensor, this proposed sandwich immunosensor can achieve a high sensitivity, especially in the presence of low concentrations of the LAG-3 protein. Under optimal conditions, this sandwich-designed immunosensor exhibited a sensitive detection of the LAG-3 protein from concentrations of 0.01 ng mL-1 to 1 µg mL-1, with a lower detection limit of 1.1 pg mL-1 (based on 3σ). We proposed that this ultrasensitive biosensor can be utilised for the detection of the LAG-3 protein in early clinical tumour diagnosis.

Evaluation of volatile compounds from Chinese dwarf cherry (Cerasus humilis (Bge.) Sok.) germplasms by headspace solid-phase microextraction and gas chromatography-mass spectrometry.

The types and amounts of volatiles in denucleated fruit of 30 Chinese dwarf cherry germplasms were determined by headspace solid-phase microextraction and gas chromatography-mass spectrometry to assess the genotypic variation. Eighty-five volatiles were identified; hexyl acetate, 4-penten-1-yl acetate, prenyl acetate, (Z)-pent-2-enyl hexanoate, geranyl acetate, n-butyl acetate, (3Z)-3,7-dimethylocta-1,3,6-triene, geraniol, pent-2-enyl butanoate, ethyl caprylate, butyl hexanoate, and linalool were the main volatiles. The type and content of volatile varied with genotype. Red fruits had the most abundant aroma and vinicolor fruits exhibited the least. Principal component analysis clustered the 30 Chinese dwarf cherry germplasms into four groups: (1) 2 germplasms (NM2 and HN3) had high ester content, (2) 24 germplasms (BJ1-BJ6, HB1, HB2, HN1, HN2, NM1, NM3, SX1, SX2, SX4-SX6, SX8-SX10, SX12, SX13, SX15, and SX16) contained mainly esters, lactones, and terpenes, (3) 2 germplasms (SX11 and SX14) had high ester and lactone content, and (4) 2 germplasms (SX3 and SX7) had high ester and terpene content.

NSAIDs modulate GABA-activated currents via Ca2+-activated Cl- channels in rat dorsal root ganglion neurons.

The ability of non-steroidal anti-inflammatory drugs (NSAIDs) to modulate γ-aminobutyrate (GABA)-activated currents via Ca2+-activated Cl- channels in rat dorsal root ganglion neurons (DRG), was examined in the present study. During the preparation of DRG neurons harvested from Sprague-Dawley rats, the whole-cell recording technique was used to record the effect of NSAIDs on GABA-activated inward currents, and the expression levels of the TMEM16A and TMEM16B subunits were revealed. In the event that DRG neurons were pre-incubated for 20 sec with niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) prior to the administration of GABA, the GABA-induced inward currents were diminished markedly in the majority of neurons examined (96.3%). The inward currents induced by 100 µmol/l GABA were attenuated by (0±0.09%; neurons = 4), (5.32±3.51%; neurons = 6), (21.3±4.00%; neurons = 5), (33.8±5.20%; neurons = 17), (52.2±5.10%; neurons = 4) and (61.1±4.12%; neurons = 12) by 0.1, 1, 3, 10, 30 and 100 µmol/l NFA, respectively. The inward currents induced by 100 µmol/l GABA were attenuated by (13.8±6%; neurons = 6), (23.2±14.7%; neurons = 6) and (29.7±9.1%; neurons = 9) by 3, 10 and 30 µmol/l NPPB, respectively. NFA and NPPB dose-dependently inhibited GABA-activated currents with half maximal inhibitory concentration (IC50) values of 6.7 and 11 µmol/l, respectively. The inhibitory effect of 100 µmol/l NFA on the GABA-evoked inward current were also strongly inhibited by nitrendipine (NTDP; an L-type calcium channel blocker), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (a highly selective calcium chelating reagent), caffeine (a widely available Ca2+ consuming drug) and calcium-free extracellular fluid, in a concentration-dependent manner. Immunofluorescent staining indicated that TMEM16A and TMEM16B expression was widely distributed in DRG neurons. The results suggest that NSAIDs may be able to regulate Ca2+-activated chloride channels to reduce GABAA receptor-mediated inward currents in DRGs.

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats.

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells.

We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56 µM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥ 300 µM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62 µM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K(+)-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ∼300 µM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K(+)-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300µM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels.

[Comparison of membrane current of vascular smooth muscle cells in brain artery of spontaneously hypertensive rats and Wistar rats].

OBJECTIVE: To investigate the difference in membrane current of vascular smooth muscle cells (VSMCs) in brain artery (BA) of spontaneously hypertensive rats (SHR) and Wistar rats. METHODS: We compared the properties of spontaneous transient outward K+ currents (STOCs), the density and composition of current of VSMCs in BA of SHR and Wistar rats by whole-cell patch clamp technique. RESULTS: (1) When the command voltage was 0, + 20, + 40 and + 60 mV respectively, the current densities of VSMCs in BA of SHR and Wistar rats were significant different (P < 0.01). (2) The whole-cell current of VSMCs was partly inhibited by 1 mmol/L4-AP (voltage-gated K+ channel blocker) or 1 mmol/L TEA (big conductance Ca(2+)-activated K+ channel blocker) respectively. (3) The frequency and amplitude of STOCs in SHR were faster and bigger than those in Wistar rats. 1 mmol/L TEA almostly inhibited the STOCs, but not by 4-AP. CONCLUSION: These results suggest that the current densities of VSMCs in BA of SHR and Wistar rats are significant different, the outward current of VSMCs in BA of SHR and Wistar rats are composed by Kv and BK(Ca). SHR express more STOCs mediated by BK(Ca), than Wistar rats.