Biomedical subjects

Z Su

Publications and source records attributed to Z Su.

At least 19 recordsLinked to original sources

PubMed2006

Angiotensin-(1-7) inhibits angiotensin II-stimulated phosphorylation of MAP kinases in proximal tubular cells.

Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE, which is not blocked by ACE inhibitors. High amounts of ACE2 are present in the proximal tubule, and ACE2 catalyzes generation of angiotensin 1-7 (Ang-(1-7)) by this segment. Ang-(1-7) binds to a receptor distinct from the AT1 or AT2 Ang II receptor, identified as the mas receptor. We studied the effects of Ang-(1-7) on Ang II-mediated cell signaling pathways in proximal tubule. In primary cultures of rat proximal tubular cells, activation of mitogen-activated protein kinases (MAPK) was detected by immunoblotting, in the presence or absence of agonists/antagonists. Transforming growth factor-beta1 (TGF-beta1) was measured by enzyme-linked immunosorbent assay. Ang II (5 min, 10(-7) M) stimulated phosphorylation of the three MAPK (p38, extracellular signal-related kinase (ERK 1/2), and c-Jun N-terminal kinase (JNK)). While incubation of proximal tubular cells with Ang-(1-7) alone did not significantly affect MAPK phosphorylation, Ang-(1-7) (10(-7) M) completely inhibited Ang II-stimulated phosphorylation of p38, ERK 1/2, and JNK. This inhibitory effect was reversed by the Ang-(1-7) receptor antagonist, D-Ala7-Ang-(1-7). Ang II significantly increased production of TGF-beta1 in proximal tubular cells, an effect that was partly inhibited by Ang-(1-7). Ang-(1-7) had no significant effect on cyclic 3',5'-adenosine monophosphate production in these cells. In summary, Ang-(1-7) inhibits Ang II-stimulated MAPK phosphorylation in proximal tubular cells. Generation of Ang-(1-7) by proximal tubular ACE2 could thereby serve a protective role by counteracting the effects of locally generated Ang II.

Angiotensin I↗

PubMed2006

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Adenoviridae↗

PubMed2004

Operon prediction by comparative genomics: an application to the Synechococcus sp. WH8102 genome.

We present a computational method for operon prediction based on a comparative genomics approach. A group of consecutive genes is considered as a candidate operon if both their gene sequences and functions are conserved across several phylogenetically related genomes. In addition, various supporting data for operons are also collected through the application of public domain computer programs, and used in our prediction method. These include the prediction of conserved gene functions, promoter motifs and terminators. An apparent advantage of our approach over other operon prediction methods is that it does not require many experimental data (such as gene expression data and pathway data) as input. This feature makes it applicable to many newly sequenced genomes that do not have extensive experimental information. In order to validate our prediction, we have tested the method on Escherichia coli K12, in which operon structures have been extensively studied, through a comparative analysis against Haemophilus influenzae Rd and Salmonella typhimurium LT2. Our method successfully predicted most of the 237 known operons. After this initial validation, we then applied the method to a newly sequenced and annotated microbial genome, Synechococcus sp. WH8102, through a comparative genome analysis with two other cyanobacterial genomes, Prochlorococcus marinus sp. MED4 and P.marinus sp. MIT9313. Our results are consistent with previously reported results and statistics on operons in the literature.

ATP-Binding Cassette Transporters↗

PubMed2004

Association between apolipoprotein CI HpaI polymorphism and sporadic Alzheimer's disease in Chinese.

OBJECTIVE: To investigate into the relationship of apolipoprotein CI (ApoCI) polymorphism with sporadic Alzheimer's disease (AD) in Chinese. SUBJECTS AND METHODS: A total of 257 AD patients and 242 age-matched elderly individuals were genotyped for the ApoCI HpaI and apolipoprotein E (ApoE) HhaI polymorphisms. RESULTS: The ApoCI A allele was associated with AD of moderate to severe dementia when patients were divided into two subgroups according to Clinical Dementia Rating scale, and the AA genotype was strongly associated with moderate to severe AD in ApoE epsilon4 allele carriers [odds ratio (OR) = 8.19, 95% confidential interval: 1.28-52.30, after adjusting for age and gender by logistic regression analysis], although in total no significant differences of allele or genotype frequency between patients and controls were found. CONCLUSION: The present study partially confirmed the previous findings, suggesting that the ApoCI A allele might contribute to the susceptibility to moderate to severe sporadic AD in Chinese.

Aged↗

PubMed2003

Anti-oxidant effects of estrogen reduce [Ca2+]i during metabolic inhibition.

We previously reported that 17beta-estradiol (betaE2) inhibits the rise in [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in mouse cardiomyocytes, but the mechanism has not yet been clarified. Estrogen has been reported to have anti-oxidant properties. We, therefore, have investigated whether interaction with the estrogen receptor (ER) is involved, or whether estrogen reduces free-radical-induced impairment of Na(+)-K(+) ATPase in cardiac myocytes, and whether this effect reduces [Ca(2+)](i) rise. Male mouse ventricular myocytes were studied. Flow cytometry was used with fluo-3 for [Ca(2+)](i) measurement. Dead cells were excluded from analysis by propidium iodide fluorescence. betaE2 reduced the increase in [Ca(2+)](i) during MI even in the presence of the ER blocker tamoxifen. A similar effect on [Ca(2+)](i) was produced by its non-estrogenic isomer, betaE2-estradiol. Other hormones (estrone and estriol) with a phenolic structure also inhibited Ca(2+) overload during MI, but testosterone without the structure did not. The betaE2 effect was attenuated by inhibition of Na(+)-Ca(2+) exchanger (KB-R7943) or Na(+)-K(+) ATPase (low K(+) or ouabain), but not by block of L-type Ca(2+) channel (nifedipine). Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid), a superoxide scavenger, decreased the rise in [Ca(2+)](i) and abolished the betaE2 effect during MI. We conclude that the acute cardioprotective effect of estrogen during MI may be mediated by an ER-independent anti-oxidant action, which results in improved function of Na(+)-K(+) ATPase.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2002

Abnormal optical waveform profiles in coagulation assays from patients with antiphospholipid antibodies.

Transmittance waveforms are the optical data generated during clot formation on photo-optical coagulation analyzers and are used to define specific events of the clotting reactions. Thus, a prothrombin time (PT) or an activated partial thromboplastin time (aPTT) can be divided into a pre-coagulation phase, a coagulation phase, and a post-coagulation phase. These phases are further characterized by parameters that define the timing, the rate, the 'slope', and the magnitude of the signal change of the reactions. We investigated the transmittance waveform parameters obtained during PT and aPTT of patients with antiphospholipid antibodies (APLA) who were or were not taking warfarin, normal donors, and non-APLA patients taking warfarin. An abnormal deflection in the pre-coagulation phase of the PT (called slope 1) was observed in 61.5% of the patients with APLA, in contrast to 5.9% of non-APLA patients taking warfarin (P= 0.0015). The presence of an abnormal PT slope 1 was reagent specific and was inversely correlated with the anticardiolipin antibody immunoglobulin G (IgG) level, which suggests that the abnormal PT slope 1 may reflect interactions between patient IgG and components from the thromboplastin, possibly phospholipids. The abnormal PT slope 1 values may be of diagnostic utility in the identification of patients with antiphospholipid syndromes.

Antibodies, Anticardiolipin↗

PubMed2001

Myocyte function and [Ca 2+ ]i homeostasis during early allogenic heart transplant rejection.

BACKGROUND: Recent studies from our laboratory have demonstrated that in vivo contractile function of rejecting mouse heterotopic abdominal heart allografts 5 days after transplantation is depressed to 40% of that of syngenic controls, and that this depression of function is prevented by the nitric oxide synthase (NOS) inhibitor NG-monomethyl-l-arginine. However, the mechanisms of altered myocyte function caused by nitric oxide production in this setting are not established. METHODS: We measured intracellular calcium concentration ([Ca2+]i) transients (fluo-3, confocal microscopy), fractional shortening (video motion), and L-type Ca2+ currents (whole-cell patch clamp) 5 days after transplantation in ventricular myocytes freshly isolated from syngenic (Balb/C into Balb/C) and allogenic (Balb/C into C3H) transplants. RESULTS: L-type Ca2+ currents, [Ca2+]i transient amplitudes, and fractional shortening did not differ between nonrejecting, syngenic and rejecting, allogenic transplants. Catecholamine responsiveness as analyzed by the change in the peak [Ca2+]i transient induced by 100 nM isoproterenol was also similar. Superfusion with l-arginine, an NOS substrate, caused decreased shortening with no change in [Ca2+]i transients in allogenic myocytes, but had no effect in syngenic myocytes. CONCLUSIONS: Depressed contractile function of rejecting allogenic heart transplants in vivo appears to be caused in part by an NOS-dependent decrease in myofilament Ca2+ sensitivity.

Animals↗

PubMed2001

Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

Antigens, Neoplasm↗

PubMed2001

Gender influences [Ca(2+)](i) during metabolic inhibition in myocytes overexpressing the Na(+)-Ca(2+) exchanger.

BACKGROUND: The Na(+)-Ca(2+) exchanger (NCX) may contribute to Ca(2+) overload and injury in ischemic cardiomyocytes. Recently, NCX overexpression was reported to increase ischemia/reperfusion injury in male and oophorectomized female but not in female mice. We therefore measured the effects of gender and estrogen on [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in myocytes from wild-type (WT) and transgenic (TG) mice overexpressing NCX. METHODS AND RESULTS: Flow cytometry was used with fluo 3 for [Ca(2+)](i) and sodium green for [Na(+)](i) measurements. Male TG mouse myocytes had higher [Ca(2+)](i) after 30 minutes of MI (1086+/-160 nmol/L, n=8) than male WT (688+/-104 nmol/L, n=9, P=0.01). The increase in [Ca(2+)](i) during MI induced by NCX overexpression in female myocytes was not significant, however (TG 552+/-62 nmol/L, n=9; WT 426+/-44 nmol/L, n=7). The magnitude of rise in [Ca(2+)](i) during MI was greater in male than female myocytes. KB-R7943, an NCX inhibitor, abolished the effect of NCX overexpression but did not totally eliminate the effect of gender on [Ca(2+)](i) during MI. NCX current density and basal Na(+) pump function were not influenced by gender. The rise in [Na(+)](i) during MI was greater in male than in female myocytes. Estrogen attenuated the increase in [Ca(2+)](i) and [Na(+)](i) in male myocytes during MI and abolished the gender difference in [Na(+)](i) during MI. CONCLUSIONS: Increased expression of NCX results in a more marked rise in [Ca(2+)](i) during MI in male than in female mouse myocytes. This gender difference appears to be mediated in part by an inhibitory effect of estrogen on the rise in [Na(+)](i), an NCX modifier, during MI.

Animals↗

PubMed2001

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells.

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.

Adenoviridae↗

PubMed2001

Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding.

Protein refolding is still a bottleneck for large-scale production of valuable proteins expressed as inclusion bodies in Escherichia coli. Usually biologically active proteins cannot be obtained with high yield at a high concentration after refolding. In order to meet the challenge of protein refolding a urea gradient gel filtration-refolding system was developed in this article. A Superdex 75 column was pre-equilibrated with a linear decreased urea gradient, the denatured protein experienced the gradual decrease in urea concentration as it went through the column. The refolding of denatured lysozyme showed this method could significantly increase the activity recovery of denatured lysozyme at high protein concentration. The activity recovery of 90% was obtained from the initial protein concentration up to 17 mg/ml within 40 min.

Chromatography, Gel↗

PubMed2001

Cloning of differentially expressed genes in an HIV-1 resistant T cell clone by rapid subtraction hybridization, RaSH.

An HIV-1 resistant T cell clone R1c2 has been generated that carries mutant, latent HIV-1 in a minority of the cell population. Resistant cells express HIV-1 receptors CD4 and CXCR4 and display resistance to infection by wild type (wt) HIV-1 at the level of virus transcription. To begin to define the repertoire of genes modulated in R1c2 cells that correlate with and potentially control expression of the HIV-1 resistance phenotype we have employed a rapid subtraction hybridization (RaSH) technique. For this approach, cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified followed by incubation of tester and driver PCR fragments. The RaSH scheme resulted in the cloning of genes displaying differential expression between HIV-1 resistant (R1c2) and susceptible (SupT1) cells, including known genes and those not described in current DNA databases. Analysis of the pattern of expression of the differentially expressed genes documented eleven genes with enhanced (HR clones) and six genes with reduced (HS clones) expression in HIV-1 resistant versus HIV-1 susceptible T-cell clones.

Cell Line↗

PubMed2001

PEA3 sites within the progression elevated gene-3 (PEG-3) promoter and mitogen-activated protein kinase contribute to differential PEG-3 expression in Ha-ras and v-raf oncogene transformed rat embryo cells.

Transformation of normal cloned rat embryo fibroblast (CREF) cells with cellular oncogenes results in acquisition of anchorage-independent growth and oncogenic potential in nude mice. These cellular changes correlate with an induction in the expression of a cancer progression-promoting gene, progression elevated gene-3 (PEG-3). To define the mechanism of activation of PEG-3 as a function of transformation by the Ha-ras and v-raf oncogenes, evaluations of the signaling and transcriptional regulation of the approximately 2.0 kb promoter region of the PEG-3 gene, PEG-Prom, was undertaken. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and tested for promoter activity in CREF and oncogene-transformed CREF cells. An analysis was also performed using CREF cells doubly transformed with Ha-ras and the Ha-ras specific suppressor gene Krev-1, which inhibits the transformed phenotype in vitro. These assays document an association between expression of the transcription regulator PEA3 and PEG-3. The levels of PEA3 and PEG-3 RNA and proteins are elevated in the oncogenically transformed CREF cells, and reduced in transformation and tumorigenic suppressed Ha-ras/Krev-1 doubly transformed CREF cells. Enhanced tumorigenic behavior, PEG-3 promoter function and PEG-3 expression in Ha-ras transformed cells were all dependent upon increased activity within the mitogen-activated protein kinase (MAPK) pathway. Electrophoretic mobility shift assays and DNase I footprinting experiments indicate that PEA3 binds to sites within the PEG-Prom in transformed rodent cells in an area adjacent to the TATA box in a MAPK-dependent fashion. These findings demonstrate an association between Ha-ras and v-raf transformation of CREF cells with elevated PEA3 and PEG-3 expression, and they implicate MAPK signaling via PEA3 as a signaling cascade involved in activation of the PEG-Prom.

Animals↗

PubMed2001

Protein kinase C phosphorylates nonmuscle myosin-II heavy chain from Drosophila but regulation of myosin function by this enzyme is not required for viability in flies.

Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this regulation in a complex eukaryote, we purified the Drosophila myosin-II tail expressed in Escherichia coli and showed that it was phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using a 341 amino acid tail fragment, we show that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, we generated transgenic flies that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way.

Amino Acid Sequence↗

PubMed2001

Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis.

A new program to characterize polyethylene glycol-modified (PEGylated) proteins is outlined using capillary zone electrophoresis (CZE). PEGylated ribonuclease A and lysozyme were selected as examples. Five separation procedures were compared to select out the mixed buffer of acetonitrile-water (1:1, v/v) at pH 2.5 as the best to characterize the PEGylated proteins without sample pretreatment. Polyethylene oxide (PEO) with a high molecular mass of 8 x 10(6) was applied to rinse the capillary to form a dynamic coating which would decrease the undesirable proteins adsorbed to the inner wall of the silica. The electroosmotic flow (EOF) mobility of the five procedures was determined, respectively. It is found that acetonitrile is mainly responsible for the good resolution of PEGylated proteins with the help of PEO coating in the semi-aqueous system. The low EOF mobility and current in the semi-aqueous system might also have some responsibility for the high resolution. The semi-aqueous procedure described in this paper also demonstrates higher resolution of natural proteins than aqueous ones.

Electrophoresis, Capillary↗

PubMed2001

The generation of LMP2a-specific cytotoxic T lymphocytes for the treatment of patients with Epstein-Barr virus-positive Hodgkin disease.

Based upon the success of using polyclonal, Epstein-Barr virus (EBV)-specific CTL lines for the prophylaxis and treatment of patients with post-transplant lymphoproliferative disease (PTLPD), there is now considerable incentive to develop CTL directed against the sub-dominant EBV antigens EBNA1, LMP1 and LMP2, which are expressed by the tumor cells of Hodgkin disease and nasopharyngeal carcinoma. To develop a system for generating LMP2a-specific CTL in vitro, we transfected autologous immature dendritic cells (DC), which had been grown in the absence of serum, with LMP2a RNA in the presence of the cationic lipid DOTAP. This transfection method did not adversely affect the DC in terms of immunophenotyping and they expressed high levels of HLA class I and II and critical costimulatory molecules. These LMP2a(+) DC, as compared to DC which had been transfected with irrelevant RNA, were shown to be highly immunostimulatory in autologous mixed lymphocyte reactions and, importantly, could stimulate the generation of CD8(+) and CD4(+) CTL which exclusively recognized LMP2a-expressing targets. This specific cytotoxicity was confirmed using antibody blocking experiments and cytotoxicity assays of the separated T cell subsets. Using this DC-based system we could also reactivate LMP2a-specific memory in EBV-seropositive donors whose polyclonal CTL response to LCL stimulation did not contain a LMP2a-specific component.

Antigens, Viral↗

PubMed2001

Activation of connexin-43 hemichannels can elevate [Ca(2+)]i and [Na(+)]i in rabbit ventricular myocytes during metabolic inhibition.

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na(+) and Ca(2+) accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca(2+)]i and [Na(+)]i in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 microM fluo-3 AM for 30 min, or with 5 microM sodium green AM for 60 min at 37 degrees C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI+H (0.95 mM, 4.7 mM) for 60 min. Propidium iodide (PI, 25 microM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca(2+)]i and [Na(+)]i were then calculated by calibration. In some experiments, the effect of 10 microM tetrodotoxin (TTX) and 20 microM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca(2+)]i and [Na(+)]i in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca(2+)]i and [Na(+)]i in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca(2+)]i and [Na(+)]i overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na(+) and Ca(2+) channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.

Anesthetics, Inhalation↗

PubMed2001

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes.

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.

Animals↗