The impact of pharmacist practice of medication therapy management in ambulatory care: an experience from a comprehensive Chinese hospital.

BACKGROUD: With the reform of medical system in China, Beijing municipal hospitals explored a new pharmaceutical care model and set up medication therapy management services (MTMs) in ambulatory care since 2019. We were one of the first hospitals to set up this service in China. At the present, there were relatively few reports about the effect of MTMs in China. In this study, we summarized the implementation of MTMs in our hospital, explore the feasibility of pharmacist-led MTMs in ambulatory care and the impact of MTMs on patients' medical costs. METHODS: A retrospective study was conducted in a university-affiliated, tertiary comprehensive hospital in Beijing, China. The patients who received at least one MTMs and with complete medical records and pharmaceutical documents from May 2019 to February 2020 were included. Pharmacists provided pharmaceutical care for patients according to the MTMs standards issued by the American Pharmacists Association, identified the numbers and classification of the patients' perceived medication-related demands, identified medication-related problems (MRPs), and developed the medication-related action plans (MAPs). All MRPs found by pharmacists, pharmaceutical interventions, and resolving recommendations were documented, and calculate the cost of treatment drugs that patients can reduce. RESULTS: A total of 112 patients received MTMs in ambulatory care, among them 81 cases with the completed record were included in this study. 67.9% of patients had five or more diseases, 83% of them co-took over 5 drugs. While performing MTMs, 128 patients' perceived medication-related demands were recorded in all, monitoring and judgment of adverse drug reaction (ADR) (17.19%) was the most common demand. 181 MRPs were found, with an average of 2.55 MPRs per patient. Nonadherence (38%), excessive drug treatment (20%), and adverse drug events (17.12%) were the top three MRPs. Pharmaceutical care (29.77%), adjustment of drug treatment plan (29.10%) and referral to the clinical department (23.41%) were the top three MAPs. Whereby the MTMs provided by pharmacists, the cost-saving of each patient was about $ 43.2 monthly. CONCLUSION: By participating in the MTMs of outpatients, the pharmacists could identify more MRPs and develop personalized MAPs timely for patients, thereby promoting rational drug use and reducing medical expenses.

Association between the polymorphism of HLA and ESRD in Dalian Han population located in north of China.

BACKGROUND: End-stage renal disease (ESRD), the last stage of chronic renal failure, is a global health problem. The number of ESRD patients worldwide is increasing faster than the number of kidneys available per year for renal transplantation. Most of the ESRD patients are awaiting renal transplantation. The immune response to the transplanted kidney is directed mainly against mismatched human leukocyte antigen (HLA) glycoproteins expressed on donor tissues. Thus, the analysis of HLA allele and haplotype polymorphisms is valuable not only for identifying ESRD susceptibility factors but also to improve graft survival. METHODS: In this study, 163 Han ESRD patients were recruited to participate. The blood samples were genotyped by sequence-specific oligonucleotide method. A group of 14,529 healthy Chinese Han individuals registered at the Dalian Blood Center as bone marrow donors, living in the same region and of the same ethnicity, were used as controls. RESULTS: We found that only one allele, HLA-DRB1*12, showed a positive association with ESRD (p = 0.004, pc = 0.028, odds ratio = 1.530, 95% confidence interval = 1.147-2.041); A*02-B*40-DRB1*09, A*02-B*40-DRB1*12, A*24-B*15-DRB1*12, and B*40-DRB1*12 were significantly more frequent in ESRD patients after Bonferroni correction (pc < 0.05). CONCLUSION: They were potentially valuable predictors for evaluating the risk of ESRD in the Dalian Han population.

Incidence and Outcomes of Inferior Vena Cava Filter Thrombus during Catheter-directed Thrombolysis for Proximal Deep Venous Thrombosis.

BACKGROUND: The aim of the study was to retrospectively evaluate the incidence and outcomes of inferior vena cava (IVC) filter thrombus during catheter-directed thrombolysis (CDT) for acute proximal deep venous thrombosis (DVT). METHODS: From October 2006 to June 2015, patients diagnosed with acute proximal DVT and received CDT after a retrievable IVC filter was placed were included. The incidence, treatment, and outcomes of IVC filter thrombus during CDT were recorded and analyzed. RESULTS: A total of 189 patients (91 women, 98 men; mean age, 57.6 ± 9.8 years; range, 24-85 years) were included in this study. Among the 189 cases, the DVTs involved popliteal iliofemoral veins in 54 patients, iliofemoral veins in 113 patients, and iliac veins in 22 patients, of which 18 patients had thrombus extended into the IVC. Of the 189 patients, a total of 8 (4.2%, 8 of 189) patients were identified with IVC filter thrombus during CDT. The IVC filter thrombus was detected on a median of 2 days (range, 2-4 days) of CDT therapy, including small-size (n = 6) and large-size (n = 2) filter thrombus. Of the 8 patients, CDTs were performed with a mean 7.6 ± 1.1 days (range, 6-11 days) after the presence of symptoms for the treatment of proximal DVT, and all the IVC filter thrombi were lysed during CDT for the proximal DVT. All the IVC filters were removed successfully with a mean of 12.8 ± 0.93 days from placement. There were no procedure- or thrombolysis-related major complications, and no symptomatic pulmonary embolism breakthrough was seen in any of the patients after the filter placement. CONCLUSIONS: IVC filter thrombus during CDT for the acute proximal DVT is uncommon, and all of them did not need any additional treatment.

Identification of growth trait related genes in a Yorkshire purebred pig population by genome-wide association studies.

OBJECTIVE: The aim of this study is to identify genomic regions or genes controlling growth traits in pigs. METHODS: Using a panel of 54,148 single nucleotide polymorphisms (SNPs), we performed a genome-wide Association (GWA) study in 562 pure Yorshire pigs with four growth traits: average daily gain from 30 kg to 100 kg or 115 kg, and days to 100 kg or 115 kg. Fixed and random model Circulating Probability Unification method was used to identify the associations between 54,148 SNPs and these four traits. SNP annotations were performed through the Sus scrofa data set from Ensembl. Bioinformatics analysis, including gene ontology analysis, pathway analysis and network analysis, was used to identify the candidate genes. RESULTS: We detected 6 significant and 12 suggestive SNPs, and identified 9 candidate genes in close proximity to them (suppressor of glucose by autophagy [SOGA1], R-Spondin 2 [RSPO2], mitogen activated protein kinase kinase 6 [MAP2K6], phospholipase C beta 1 [PLCB1], rho GTPASE activating protein 24 [ARHGAP24], cytoplasmic polyadenylation element binding protein 4 [CPEB4], GLI family zinc finger 2 [GLI2], neuronal tyrosine-phosphorylated phosphoinositide-3-kinase adaptor 2 [NYAP2], and zinc finger protein multitype 2 [ZFPM2]). Gene ontology analysis and literature mining indicated that the candidate genes are involved in bone, muscle, fat, and lung development. Pathway analysis revealed that PLCB1 and MAP2K6 participate in the gonadotropin signaling pathway and suggests that these two genes contribute to growth at the onset of puberty. CONCLUSION: Our results provide new clues for understanding the genetic mechanisms underlying growth traits, and may help improve these traits in future breeding programs.

Mechanotransduction of trigeminal ganglion neurons innervating inner walls of rat anterior eye chambers.

To address mechanoreceptive roles of trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior eye chambers, we investigated the mechanotransduction process and mechanosensitive (MS) channel on somata of TG neurons innervating this area in vitro. Rat TG neurons innervating inner walls of anterior chambers were labeled by anterior chamber injection of 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI). The neuronal cell bodies were voltage clamped using a whole cell patch-clamp technique, while it was deformed by ejection of bath solution to verify mechanotransduction. Immunofluorescence staining was performed on sections of TG ganglia to determine the specific MS channel proteins. Mechanical stimuli induced MS currents in 55 out of 96 FAST DiI-labeled TG neurons. The MS currents exhibited mechanical intensity-dependent and clamp voltage-dependent characteristics. Mechanical stimulation further enhanced the membrane potential and increased the frequency of action potentials. Transient receptor potential ankyrin 1 (TRPA1), TRP vanilloid 4 (TRPV4), acid-sensing ion channel (ASIC) 2 and ASIC3 channel proteins were expressed in FAST DiI-labeled TG neurons. The inhibitory effect of HC-030031, a specific inhibitor of TRPA1, on MS currents demonstrated that TRPA1 was an essential MS channel protein. Taken together, our results show that mechanical stimuli induce MS currents via MS channels such as TRPA1 to trigger mechanotransduction in TG neurons innervating inner walls of anterior chambers. Our results indicate the existence of mechanoreceptive TG nerve endings in inner walls of anterior chambers. Whether the mechanoreceptive TG nerve endings play a role in intraocular pressure sensation warrants further investigation.

Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

BACKGROUND: Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect. RESULTS: In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication. CONCLUSIONS: These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

FAT1 inhibits AML autophagy and proliferation via downregulating ATG4B expression.

BACKGROUND: Emerging studies have shown that FAT atypical cadherin 1 (FAT1) and autophagy separately inhibits and promotes acute myeloid leukemia (AML) proliferation. However, it is unknown whether FAT1 were associated with autophagy in regulating AML proliferation. METHODS: AML cell lines, 6-week-old male nude mice and AML patient samples were used in this study. qPCR/Western blot and cell viability/3H-TdR incorporation assays were separately used to detect mRNA/protein levels and cell activity/proliferation. Luciferase reporter assay was used to examine gene promoter activity. Co-IP analysis was used to detect the binding of proteins. RESULTS: In this study, we for the first time demonstrated that FAT1 inhibited AML proliferation by decreasing AML autophagy level. Moreover, FAT1 weakened AML autophagy level via decreasing autophagy related 4B (ATG4B) expression. Mechanistically, we found that FAT1 reduced the phosphorylated and intranuclear SMAD family member 2/3 (smad2/3) protein levels, thus decreasing the activity of ATG4B gene promoter. Furthermore, we found that FAT1 competitively bound to TGF-ßR II which decreased the binding of TGF-ßR II to TGF-ßR I and the subsequent phosphorylation of TGF-ßR I, thus reducing the phosphorylation and intranuclear smad2/3. The experiments in nude mice showed that knockdown of FAT1 promoted AML autophagy and proliferation in vivo. CONCLUSIONS: Collectively, these results revealed that FAT1 downregulates ATG4B expression via inhibiting TGFß-smad2/3 signaling activity, thus decreasing the autophagy level and proliferation activity of AML cells. GENERAL SIGNIFICANCE: Our study suggested that the "FAT1-TGFß-smad2/3-ATG4B-autophagy" pathway may be a novel target for developing new targeted drugs to AML treatment.

Carbamazepine cutaneous adverse reactions and HLA gene variation in the Chinese population: a systematic review and meta-analysis.

Aim: Examining the association between HLA-A/B alleles and different carbamazepine (CBZ)-induced cutaneous adverse reactions in the Chinese population. Methods: A systematic review and meta-analysis of case-control studies was conducted. A systematic search was conducted of PubMed, Embase, the Cochrane Library, National Knowledge Infrastructure, the Chinese Biomedical Literature database and Wanfang Digital Periodicals. Results: 23 studies with a total of 1174 patients were included. In the Han population, HLA-B*15:02 is significantly associated with the increased risk of CBZ-related Stevens-Johnson syndrome/toxic epidermal necrolysis, and this correlation was not related to geographic distribution. HLA-A*31:01, B*38:02 are associated with CBZ-related maculopapular eruption in South Han population. HLA-A*31:01 is associated with CBZ-DRESS in Taiwan Han population. Conclusion: HLA-B*15:02, A*31:01 and B*38:02 genes were found to be involved in the occurrence of CBZ cutaneous adverse reactions in Han Chinese.

Case report: Successful treatment of Chidamide in a refractory/recurrent SPTCL with ARID1A mutation on the basis of CHOP plus auto-HSCT.

RATIONALE: Subcutaneous panniculitis like T-cell lymphoma (SPTCL) is a rare primary cutaneous lymphoma that belongs to peripheral T cell lymphomas, of which the overall prognosis is poor. Chidamide, a deacetylase inhibitor, has been approved for the treatment of peripheral T cell lymphomas. However, due to the rare occurrence of SPTCL, it is currently unknown whether Chidamide is effective for all SPTCL patients and whether there are molecular markers that can predict its therapeutic effect on SPTCL. PATIENT CONCERNS AND DIAGNOSES: The patient was a sixteen-year-old male and underwent subcutaneous nodule biopsy which showed SPTCL. Next-generation sequencing revealed AT-rich interaction domain 1A (ARID1A) mutation, and positron emission tomography/computed tomography showed scattered subcutaneous fluorodeoxyglucose metabolic lesions throughout the body. INTERVENTIONS AND OUTCOMES: During the first 3 CHOP (cyclophosphamide, doxorubicin, vindesine, and prednisone) treatment, the patient relapsed again after remission, and the successive addition of methotrexate and cyclosporine did not make the patient relapsing again. Then, after adding Chidamide to the last 3 CHOP treatment, the patient was relieved again. The patient underwent autologous hematopoietic stem cell transplantation (auto-HSCT) after completing a total of 8 cycles of chemotherapy, and continued maintenance therapy with Chidamide after auto-HSCT. Currently, the patient has been in continuous remission for 35 months. LESSONS SUBSECTIONS: This case is the first report of a refractory/recurrent SPTCL with ARID1A mutation treated with Chidamide. The treatment of Chidamide on the basis of CHOP plus auto-HSCT therapy achieved good results, suggesting that ARID1A may act as a molecular marker to predict the therapeutic effect of Chidamide on SPTCL patients, which helps to improve the precision of SPTCL treatment and the overall prognosis of SPTCL patients.

[A Bw12 blood type caused by 278C>T mutation of ABO gene].

OBJECTIVE: To determine the serotype and genotype of a sample with ABO blood group discrepancies. METHODS: Serotype was determined with serological method. Sequence specific primer polymerase chain reaction (SSP-PCR) was carried out based on the serotype. Sequences of exons 6 and 7 of ABO gene was analyzed by sequence-based testing (SBT). RESULTS: Completely agglutinated A antigen, half agglutinated B antigen and weak agglutinated anti-B antibody were detected in both erythrocytes and serum, which suggested presence of a ABw serotype. An A/Bw12 genotype was revealed by B subgroup detection. Sequences of exons 6 and 7 were 278CT, 297GA and 467CT, 526CG, 657CT, 703GA, 796CA, 803GC, 930GA, respectively. The genotype fit with A102/B101 except for a nt278 C>T mutation. Blood group antigen gene mutation database (BGMUT) search has confirmed the mutant allele to be Bw12. CONCLUSION: An A102/Bw12 genotype has been found in the Chinese population.

Genome-Wide Assessment Characteristics of Genes Overlapping Copy Number Variation Regions in Duroc Purebred Population.

Copy number variations (CNVs) are important structural variations that can cause significant phenotypic diversity. Reliable CNVs mapping can be achieved by identification of CNVs from different genetic backgrounds. Investigations on the characteristics of overlapping between CNV regions (CNVRs) and protein-coding genes (CNV genes) or miRNAs (CNV-miRNAs) can reveal the potential mechanisms of their regulation. In this study, we used 50 K SNP arrays to detect CNVs in Duroc purebred pig. A total number of 211 CNVRs were detected with a total length of 118.48 Mb, accounting for 5.23% of the autosomal genome sequence. Of these CNVRs, 32 were gains, 175 losses, and four contained both types (loss and gain within the same region). The CNVRs we detected were non-randomly distributed in the swine genome and were significantly enriched in the segmental duplication and gene density region. Additionally, these CNVRs were overlapping with 1,096 protein-coding genes (CNV-genes), and 39 miRNAs (CNV-miRNAs), respectively. The CNV-genes were enriched in terms of dosage-sensitive gene list. The expression of the CNV genes was significantly higher than that of the non-CNV genes in the adult Duroc prostate. Of all detected CNV genes, 22.99% genes were tissue-specific (TSI > 0.9). Strong negative selection had been underway in the CNV-genes as the ones that were located entirely within the loss CNVRs appeared to be evolving rapidly as determined by the median dN plus dS values. Non-CNV genes tended to be miRNA target than CNV-genes. Furthermore, CNV-miRNAs tended to target more genes compared to non-CNV-miRNAs, and a combination of two CNV-miRNAs preferentially synergistically regulated the same target genes. We also focused our efforts on examining CNV genes and CNV-miRNAs functions, which were also involved in the lipid metabolism, including DGAT1, DGAT2, MOGAT2, miR143, miR335, and miRLET7. Further molecular experiments and independent large studies are needed to confirm our findings.

Trigemino-cardiac reflex: the trigeminal depressor responses during skull base surgery.

OBJECTIVE: To observe and analyze the occurrence and management of the trigemino-cardiac reflex (TCR) defined as the phenomenon of abrupt drops in heart rate (HR) and blood pressure during skull base surgery. METHOD: One hundred patients underwent skull base surgery for various lesions were recruited and great attention was paid to heart rate and blood pressure throughout the surgical procedure to screen intraoperative TCR. RESULT: Twelve patients had TCR intro-operatively, all patients showed abrupt drops in HR of 38% from a mean of 78 beats/min to a mean of 49 beats/min, mean arterial blood pressure (MABP) decreased 33% from a mean of 93 mmHg to a mean of 60 mmHg, respectively. TCR was resolved spontaneously in eight patients, but had to be offset by intraoperative administration of relatively higher dose atropine in another four patients. CONCLUSION: (1) Manipulation at or near the trigeminal nerve during the skull base surgery may cause TCR, even if premedication with anticholinergic drug is used; (2) cessation of irritation from surgical manipulation to disrupt the reflex is the most important step to offset TCR; (3) continuous, especially repeated TCR in some rare cases occasionally necessitates the administration of high dose atropine.

Effects of chlorpromazine on sleep quality, clinical and emotional measures among patients with schizophrenia.

OBJECTIVES: The present study was aimed to examine the effects of chlorpromazine on sleep quality, clinical and emotional measures in people suffering from schizophrenia. PATIENTS AND METHODS: Twenty-five patients with schizophrenia or schizoaffective disorder were enrolled in this study. Our study included a one-week running-in no-treatment period and two-month experimental period. Patients received chlorpromazine during the experimental period. The baseline and treatment outcome were recorded. The objective and subjective sleep were respectively measured by a wrist actigraph and two sleep questionnaires (Mini Sleep Questionnaire (MSQ) and Pittsburgh Sleep Quality Index Hebrew Translation (PSQI-H)). Besides, Brief Psychiatric Rating Scale (BPRS) and Positive and Negative Syndrome Scale (PANSS) were performed to assess the clinical psychopathology levels, and while Calgary Depression Scale for schizophrenia (CDSS) and Hamilton Anxiety Rating Scale (HAS) had been carried out to examine the emotional changes. RESULTS: There was a significant effect of chlorpromazine treatment on four objective sleep variables: longest wake episode, sleep onset latency, sleep percentage, and mean activity level (all P < 0.05). However, no significant differences were found in the subjective sleep measures. Likewise, psychopathology levels and emotional measures (depression level and anxiety level) were statistically improved by chlorpromazine treatment compared to the baseline (P < 0.05 or P < 0.01). CONCLUSION: Our results demonstrate that chlorpromazine could improve the insomnia and psychopathology symptoms of patients with schizophrenia.

Bimatoprost Increases Mechanosensitivity of Trigeminal Ganglion Neurons Innervating the Inner Walls of Rat Anterior Chambers via Activation of TRPA1.

PURPOSE: Our previous study found that some trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior chambers were mechanosensitive, and transient receptor potential ankyrin 1 (TRPA1) was an essential mechanosensitive channel in the membrane. To address the effect of bimatoprost on the mechanosensitive TG nerve endings in the inner walls of rat anterior chambers, we investigated its effect on their cell bodies in vitro. METHODS: Rat TG neurons innervating the inner walls of the anterior chambers were labeled by anterior chamber injection of 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI). Calcium imaging and whole cell patch clamp were used on neuronal cell bodies to detect the activation effect of TRPA1 channels. Whole cell patch clamp was performed to record the currents induced by drugs and mechanical stimulation. Mechanical stimulation was applied to the neurons by buffer ejection. RESULTS: Bimatoprost mimicked the effect of TRPA1 agonists, allyl isothiocyanate (AITC), and (R)-(+)-WIN55, 212-2 mesylate salt (WIN) in the TG neurons. Bimatoprost induced Ca(2+) influx in HEK293 cells stably transfected with human TRPA1, but not in untransfected cells as AITC and WIN. Moreover, bimatoprost evoked inward currents via TRPA1 activation in FAST DiI-labeled TG neurons as WIN. Bimatoprost also enhanced mechanosensitivity of FAST DiI-labeled TG neurons via TRPA1 activation. CONCLUSIONS: Our results indicate that bimatoprost is a novel agonist of TRPA1, and it can enhance mechanosensitivity of TG nerve endings in the inner walls of anterior eye chambers via TRPA1 activation in rats.

Cannabinoids increase mechanosensitivity of trigeminal ganglion neurons innervating the inner walls of rat anterior chambers via activation of TRPA1.

Our previous study found that some trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior chambers were mechanosensitive, and transient receptor potential ankyrin 1 (TRPA1) was an essential mechanosensitive channel in the membrane. To address the effect of cannabinoids on the mechanosensitive TG nerve endings in the inner walls of anterior chambers of rat eye, we investigated the effect of the (R)-(+)-WIN55, 212-2 mesylate salt (WIN), a synthetic cannabinoid on their cell bodies in vitro. Rat TG neurons innervating the inner walls of the anterior chambers were labeled by 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfona (FAST DiI). Whole cell patch clamp was performed to record the currents induced by drugs and mechanical stimulation. Mechanical stimulation was applied to the neurons by buffer ejection. WIN evoked inward currents via TRPA1 activation in FAST DiI-labeled TG neurons. WIN enhanced mechanosensitive currents via TRPA1 activation in FAST DiI-labeled TG neurons. Our results indicate that cannabinoids can enhance the mechanosensitivity of TG endings in the inner walls of anterior chambers of rat eye via TRPA1 activation.

Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes.

BACKGROUND: It has been found that gap junction-associated intracellular Ca(2+) [Ca(2+)](i) disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca(2+) signaling, in particular the basal [Ca(2+)](i) activities, is unclear. METHODS AND RESULTS: Global and local Ca(2+) signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca(2+) transients and local Ca(2+) sparks in monolayer NRVMs and Ca(2+) sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP(3) butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca(2+) signal and LY uptake by gap uncouplers, whereas blockade of IP(3) receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca(2+) signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca(2+) signaling regulation in cardiomyocytes. CONCLUSIONS: These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca(2+) signaling in normal ventricular myocytes, in which IP(3)/IP(3) receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca(2+) activities in cardiomyocytes.

Possible mechanisms underlying the biphasic regulatory effects of arachidonic acid on Ca2+ signaling in HEK293 cells.

Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca(2+) ([Ca(2+)](i)) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca(2+)](i) and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 µM, reduced the PM fluidity by activating PKCα and PKCßII translocation to PM and also the ER fluidity directly. In accordance, 3 µM AA did not impact the basal [Ca(2+)](i) but significantly suppressed the thapsigargin-induced Ca(2+) release and Ca(2+) influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCß using shRNA significantly attenuated the inhibitory effects of 3 µM AA on PM fluidity and agonist-induced Ca(2+) signal. However, AA at high concentration, 30 µM, caused robust release and entry of Ca(2+) accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCßI and PKCßII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30 µM AA-induced Ca(2+) influx by 45%, Gd(3+) at concentration of 10 µM could completely abolish both release and entry of Ca(2+) induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca(2+) signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca(2+)](i) response, while Gd(3+)-sensitive 'pores' in PM/ER play an important role in AA-induced Ca(2+) signal in HEK293 cells.

Protective Properties of Laggera alata Extract and its Principle Components Against d-Galactosamine-Injured Hepatocytes.

Laggera alata extract (LAE) was quantitatively analyzed, and its principle components isochlorogenic acids were isolated and authenticated. Protective properties of LAE were studied using a d-galactosamine (d-GalN)-induced injury model in neonatal rat hepatocytes and a d-GalN-induced acute liver damage model in mice. Meanwhile, the effect of isochlorogenic acids derived from LAE on d-GalN-induced hepatocyte injury were also measured in vitro. LAE at concentrations of 10-100 µg/ml significantly reduced cellular leakage of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and improved cell viability. The isochlorogenic acids (4,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 3,4-O-dicaffeoylquinic acid) at concentrations of 1-100 µg/ml also remarkably improved viability of hepatocytes. The oral treatment of LAE at doses of 50, 100 and 200 mg/kg markedly reduced the serum AST and ALT activity of mice and resulted in significant recovery of hepatocytes in liver sections.

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCß using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCßII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCßI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux.