Biomedical subjects

G Szabo

Publications and source records attributed to G Szabo.

At least 19 recordsLinked to original sources

PubMed1995

Unitary anion currents through phospholemman channel molecules.

Phospholemman (PLM) is a 72-amino-acid peptide with a single transmembrane domain, the expression of which induces chloride currents in Xenopus oocytes. It has remained unknown whether PLM is an ion channel or acts as a channel regulator. Here we show, by measuring unitary anion currents across planar phospholipid bilayers to which immunoaffinity-purified recombinant PLM was added, that it does indeed form ion channels. Excised patches of oocytes expressing PLM had similar currents. Of the ions tested, the sulphonic amino acid taurine was the most permeant, and expression of PLM increased fluxes of radiolabelled taurine in oocytes. Phospholemman is the smallest protein in cell membranes known to form an ion channel and the taurine selectivity suggests that it is involved in cell volume regulation.

Animals↗

PubMed1995

Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1).

An epitope-tagged form of an inwardly rectifying and G protein-coupled K+ channel (GIRK1-cp) was expressed at high levels in transfected mammalian cells. Immunoblot analysis of transfected human embryonic kidney cells (HEK293) and mouse insulinoma cells (beta TC3) revealed several GIRK1-cp polypeptides, including the major 59-kDa band, corresponding to the predicted mass of the GIRK1 polypeptide plus the epitope tag. Immunohistochemical staining using two anti-tag antibodies showed abundant immunoreactive material, which was predominantly concentrated in the perinuclear area in both transfected cell types. While functional GIRK1-cp message was present in poly(A)+ RNA prepared from HEK293 cells expressing GIRK1-cp protein, appropriate K+ currents could not be detected. In contrast, whole cell recordings made directly from transfected beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of beta TC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 microM guanosine 5'-O-(thiotriphosphate), with a slope conductance of 39.1 +/- 1.0 picosiemens. This is the first report of stable heterologous expression of a functional G protein-coupled K+ channel in mammalian cells. The activity of an epitope-tagged channel in insulinoma cells demonstrates the utility of this system for further biochemical and biophysical analyses of G protein-K+ channel interactions.

Animals↗

PubMed1995

Membrane depolarization prevents cell invasion by Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase toxin from Bordetella pertussis is a 177-kDa calmodulin-activated enzyme that has the ability to enter eukaryotic cells and convert endogenous ATP into cAMP. Little is known, however, about the mechanism of cell entry. We now demonstrate that intoxication of cardiac myocytes by adenylate cyclase toxin is driven and controlled by the electrical potential across the plasma membrane. The steepness of the voltage dependence of intoxication is comparable with that previously observed for the activation of K+ and Na+ channels of excitable membranes. The voltage-sensitive process is downstream from toxin binding to the cell surface and appears to correspond to the translocation of the catalytic domain across the membrane.

Adenylate Cyclase Toxin↗

PubMed1995

Anesthetics reduce the magnitude of the membrane dipole potential. Measurements in lipid vesicles using voltage-sensitive spin probes.

Lipid membranes possess a large internal dipole potential that greatly exceeds the magnitude of typical transmembrane or surface potentials. The volatile general anesthetics, halothane, isoflurane and enflurane were tested by the use of positively and negatively charged hydrophobic ion spin labels in lipid bilayer vesicles for their ability to modulate the membrane dipole potential. These anesthetics decreased the binding of negatively charged hydrophobic ion spin probes based on trinitrophenol, but increased the binding of positively charged hydrophobic ion probes based on triphenylalkylphosphoniums. They also enhanced the transit rates for both hydrophobic anions and cations; however, translocation rates were enhanced to a greater extent for the cation probes compared to the anion probes. The changes in binding constant for cations versus anions could be accurately accounted for using a simple model for the free energy profile for hydrophobic ions across membranes, and indicate that these anesthetics decrease the membrane dipole potential. From a fit of the experimental data to this model, anesthetics could promote a decrease in the dipole potential in two ways. First, anesthetics appear to modify the effective dipole moment in the membrane interface and may accomplish this by orienting their molecular dipole antiparallel to the intrinsic dipoles at the interface. Second, they modify the membrane dielectric constant, leading to a decrease in the field across the interface. At equivalent membrane concentrations, isoflurane, enflurane, and halothane produced similar changes in the dipole potential and decreased the dipole potential as much as 65 mV at a membrane mole fraction of 0.20.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1995

Inhibition of superantigen-induced T cell proliferation and monocyte IL-1 beta, TNF-alpha, and IL-6 production by acute ethanol treatment.

Alcohol use has been shown to decrease monocyte antigen presentation capacity and inflammatory cytokine production, thereby increasing susceptibility to infections. Here, we demonstrate that in vitro acute treatment of normal monocytes with pharmacological doses of ethanol can decrease superantigen [Staphylococcus enterotoxins B (SEB) and A (SEA)]-induced T cell proliferation. Furthermore, ethanol treatment (25-100 mM) significantly inhibited SEA- or SEB-induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 in monocytes. Ethanol-induced down-regulation of monocyte TNF-alpha, IL-1 beta, and IL-6 occurred at both the protein and mRNA levels. Additional data suggest that ethanol can decrease IL-1 beta mRNA stability. Furthermore, experiments using cycloheximide indicate that de novo protein synthesis is required for the inhibitory effect of ethanol on SEB-induced IL-1 beta mRNA production. Finally, ethanol treatment decreased HLA-DR expression in monocytes, suggesting that ethanol treatment can compromise monocyte stimulation by down-regulating the SEB-binding capacity of monocytes. These results suggest that acute ethanol treatment can interfere with monocyte activation by SEB at multiple steps. Consequently, decreased superantigen-induced polyclonal T cell activation and inflammatory monokine production would contribute to an impaired immune response to bacterial challenge with superantigens after acute alcohol intake.

Adult↗

PubMed1994

Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium- and polarity-dependent manner.

Adenylate cyclase toxin (AC toxin) from Bordetella pertussis enters target cells to produce supraphysiologic levels of cAMP and, by a cAMP-independent process, is hemolytic. In the present study, we show for the first time that this toxin also produces ion-permeable, cation-selective pores in phospholipid bilayers. The resulting membrane conductance is absolutely calcium-dependent, as are the intoxication and hemolytic activities. It is strongly affected by the polarity and magnitude of the membrane potential and enhanced by the presence of negatively charged phospholipid. AC toxins from two mutants, BPDE386 and BPD377, which are defective in toxin activity, produce little or no conductance. Finally, evaluation of the current-voltage relationships and the concentration dependence of pore formation and of hemolysis reveal a greater than 3rd power dependence, suggesting that a multimer of AC toxin, probably consisting of three or more holotoxin molecules, is involved in pore formation.

Adenylate Cyclase Toxin↗

PubMed1994

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor alpha production late postinjury.

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor alpha (TNF alpha) production during the very early postinjury (0-3 days) period. However, TNF alpha production by these alcohol-exposed patients' monocytes (M0) became hyperelevated late postinjury (> 9 days). Consequently, these massively elevated M0 TNF alpha levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen, Staphylococcus enterotoxin B (SEB), results in a preferential induction of cell-associated M0 TNF alpha production, described as characteristic of immunosuppressed trauma patients. Acute in vitro ethanol treatment down-regulated the elevated TNF alpha production by trauma patients' M0 after either SEB, muramyl-dipeptide (MDP), interferon-gamma plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF alpha mRNA induction was inhibited by acute alcohol treatment in normal M0, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor beta production in trauma patients' activated M0.

Adult↗

PubMed1994

The L-selectin (Leu8) molecule is associated with the TcR/CD3 receptor; fluorescence energy transfer measurements on live cells.

Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-Leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecules.

CD3 Complex↗

PubMed1994

Differential effects of pertussis toxin on the muscarinic regulation of Ca2+ and K+ currents in frog cardiac myocytes.

The ability of acetylcholine (ACh) to inhibit beta-agonist stimulated calcium current was compared to its ability to activate the inwardly rectifying potassium current IK(ACh) in frog atrial myocytes. As suggested by previous studies, ACh inhibited the calcium current at concentrations (EC50 = 8 nM) significantly lower than those required for the activation of IK(ACh) (EC50 = 101 nM). The pharmacological profiles of the two responses suggest that despite the differences in agonist sensitivity, both are mediated by the same (m2) type of muscarinic receptors. Intracellular application of GDP beta S, an inhibitor of G protein function, completely abolished both responses, implying that both actions of ACh are coupled to effectors by G proteins. In contrast, intracellular application of pertussis toxin (PTX) shifted to higher concentrations (EC50 = 170 nM) but did not abolish inhibition of the calcium current by ACh even though the block of the IK(ACh) response was complete. Increasingly large PTX concentrations and/or prolonged PTX treatments revealed a limiting, PTX-resistant inhibitory component that appears to be mediated by a PTX-insensitive G protein distinct from that mediating IK(ACh). For the PTX-sensitive components, the different agonist dependencies of IK(ACh) activation and calcium current inhibition may imply that different G proteins mediate each response although alternate possibilities involving the same G protein either functionally sequestered and/or differentially affected by interactions with effectors, can not be ruled out.

Acetylcholine↗

PubMed1994

Regulatory potential of ethanol and retinoic acid on human monocyte functions.

Retinoic acid (RA), a metabolic product of vitamin A, has been shown to affect a variety of immune functions, including monocytes. Monocyte functions and mediator production are also modulated by ethanol exposure. This study demonstrates that therapeutic doses of RA (0.1-10 microM) significantly increase transforming growth factor-beta (TGF beta) production both in THP-1, human myelomonocytic cells, and in human peripheral blood monocytes. We have previously reported TGF beta induction by ethanol in human M theta. Combination of RA stimulation with acute in vitro ethanol treatment, however, resulted in significantly lower M theta TGF beta production than TGF beta levels induced by RA alone (p < 0.003). Down-regulation of M theta TGF beta production by ethanol was tested at the concentration range of 25-150 mM and occurred both at high and low RA concentrations (10-0.1 microM). In contrast to its inhibitory effect on RA-induced M theta TGF beta production, ethanol augmented TGF beta production induced by muramyl dipeptide (20 micrograms/ml), suggesting that ethanol can either up- or down-regulate M theta TGF beta production, depending on the costimulatory factors. RA also induced a moderate increase in M theta tumor necrosis factor-alpha (TNF alpha) production, which was down-regulated by ethanol both at the level of secreted and cell-associated TNF alpha. In addition to regulation of cytokine production, both RA and ethanol decreased expression of CD4 on THP-1 cells. The degree of inhibition of CD4 expression by RA was more significant than by ethanol, but RA-induced decrease in CD4 expression was not significantly affected by the combined stimulation with ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

CD4 Antigens↗

PubMed1994

Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development.

It has been shown that the enzyme glutamic acid decarboxylase (GAD; EC 4.1.1.15), which catalyzes the conversion of L-glutamate to gamma-aminobutyric acid in the central nervous system of vertebrates, can be first detected in rodents at late embryonic stages. In contrast, we have found that the gene coding for the 67-kDa form of GAD is already transcriptionally active at embryonic day E10.5 in the mouse. In addition to the 3.5-kb adult-type mRNA, we have detected two 2-kb embryonic messages that contain alternatively spliced exons of 80 (I-80) and 86 (I-86) bp, respectively. The overlapping stop-start codon TGATG, found in the embryonic exons, converts the monocistronic adult-type transcript into a bicistronic one, coding for a 25-kDa leader peptide and a 44-kDa enzymatically active truncated GAD. A second stop codon at the 3' end of the 86-bp exon abolishes the expression of truncated GAD. The products of the two embryonic mRNAs were identified in a rabbit reticulocyte in vitro translation system, COS cells, and mouse embryos. The two GAD embryonic forms represent distinct functional domains and display characteristic developmental patterns, consistent with a possible role in the formation of the gamma-aminobutyric acid-ergic inhibitory synapses.

Alternative Splicing↗

PubMed1993

The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drug transporter.

The MDRI protein is an energy-dependent transport protein responsible for the multi-drug resistance seen in many tumors. A variety of drugs have been shown to inhibit the function of this pump, including compounds known to block various ion channels. The mouse lymphoma cell line L5178Y has been transduced with the human mdrI gene. Using this cell line, we have tested a number of compounds to determine whether there is a correlation between the ability to block a specific type of ion channel, or shift membrane potential, and the ability to act as an MDR-reversing agent using the fluorescent substrates Rhodamine 123 and daunorubicin as test compounds. Our results show no apparent correlation between the ability to block a specific ion channel and reversal of MDR transport ability. We have found active MDR inhibitors in compounds that affect K+, Na+, Ca++, H+, but not Cl- channels. Our data suggest that Cl- channel activity may be distinct from MDR activity. Several immunosuppressive compounds and analogs were also tested and found to be active reversing agents. Measurements suggest a significant difference in resting membrane potential between the L5178YvMDR line and the L5178Y parental cell line used in these experiments. No correlation was found between the ability of drugs to alter membrane potential and to inhibit MDR transport activity. Our results suggest that MDR transport function may be independent of the physiological movement of ions and show that a wide variety of compounds can inhibit MDR transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Selective inhibition of antigen-specific T lymphocyte proliferation by acute ethanol exposure: the role of impaired monocyte antigen presentation capacity and mediator production.

Ethanol consumption is associated with impaired immunity. Our data demonstrate that even a single dose of a biologically relevant concentration (25-150 mM) of ethanol can down-regulate antigen-specific T lymphocyte proliferation. In contrast, ethanol augmented mitogen-induced T cell proliferation, suggesting that its inhibitory effect on antigen-specific T cell proliferation was due to its effects on monocytes (m phi s) rather than on T cells. The immunodepressive effects of ethanol on m phi antigen-presenting cell (APC) capacity were manifested whether alcohol treatment was limited to the antigen uptake-processing period only or was present during the entire period of antigen presentation. These inhibitory effects of ethanol were also evident on both the high-antigen-presenting, Fc gamma RI-negative (-31 +/- 17%), and low-antigen-presenting, Fc gamma RI-positive (-42 +/- 15%) m phi subpopulations. Further analysis demonstrated that ethanol inhibits the production of interleukin-1 beta (IL-1 beta) and induces transforming growth factor beta (TGF-beta) and prostaglandin E2 (PGE2), monocyte-derived mediators that can affect T cell proliferation. Ethanol resulted in a dose-dependent down-regulation of secreted and cell-associated IL-1 beta protein as well as IL-1 beta mRNA levels induced by adherence or bacterial stimulation. The causal relationship between decreased m phi IL-1 beta production, elevated TGF-beta levels, and the decreased m phi APC capacity was further substantiated when exogenous IL-1 beta protein or anti-TGF-beta neutralizing antibody prevented the down-regulatory effect of ethanol on antigen-specific T cell proliferation. Utilizing a cyclooxygenase inhibitor, we also demonstrated that the ethanol-induced decrease in m phi APCs is not mediated by enhanced PGE2 production.

Adult↗

PubMed1992

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes.

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of -90 mV but not from -60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.

Calcium Channels↗

PubMed1992

Induction of transforming growth factor-beta and prostaglandin E2 production by ethanol in human monocytes.

To test our hypothesis that monocytes (M phi) and their mediators are major contributors to ethanol-related immunodepression, the modulating capacity of acute ethanol treatment was assessed on the production of transforming growth factor-beta (TGF beta) and prostaglandin E2 (PGE2) by human peripheral blood M phi. We demonstrate that acute in vitro treatment of adherent M phi with either 50 or 150 mM ethanol induced a significant increase in the production of TGF beta (P < 0.045 and P < 0.001, respectively). Furthermore, M phi pretreatment with both 50 and 150 mM ethanol augmented TGF beta production in response to subsequent stimulation with the synthetic bacterial analog, muramyl dipeptide (MDP) (P < 0.05 and P < 0.001, respectively). Ethanol also increased TGF beta production in interferon gamma (IFN gamma-activated M phi in response to MDP stimulus (P < 0.05). M phi TGF beta levels, however, were always lower in IFN gamma-activated than in non-IFN gamma-activated M phi after the same stimulation with ethanol plus MDP, suggesting that M phi preactivation by IFN gamma can partially counteract the TGF beta inducing potential of ethanol. Similar to its TGF beta-inducing potential, ethanol (150 mM) had the capacity to induce PGE2 production in adherent human M phi (P < 0.045). However, ethanol failed to augment M phi PGE2 production induced by the PGE2 secretagogue, MDP. TGF beta induction by ethanol was unaffected by the presence of cyclooxygenase inhibitor, suggesting that ethanol-induced M phi TGF beta production does not require M phi PGE2 production. These results indicate that ethanol is a potent inducer for inhibitory M phi mediators, TGF beta and PGE2, and also has the capacity to augment M phi TGF beta production in response to subsequent stimulation. Thus, ethanol-induced elevation of M phi TGF beta and PGE2 production might contribute to decreased T cell proliferation and abnormal M phi functions after alcohol exposure, resulting in a depressed immune response.

Cell Line↗

PubMed1991

Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation.

The positive inotropic effect of the sympathetic nervous system on the heart is partly mediated by an increase in the voltage-gated Ca2+ current (ICa). This increase is generally attributed to beta-adrenergic receptor-stimulated cyclic AMP-dependent phosphorylation of the Ca2+ channel. It has been suggested that cAMP-dependent phosphorylation cannot explain all the effects of beta-adrenergic agonists on ICa and that a parallel membrane-delimited pathway involving the 'direct' action of the G protein Gs also stimulates ICa. A precedent exists for such a membrane-delimited pathway in the activation of a K+ channel by acetylcholine in heart. A membrane-delimited pathway for stimulation of ICa might be important in rapid beat-to-beat regulation of contraction by the sympathetic nervous system, because isoproterenol may produce a biphasic increase in ICa with the rapid phase (tau = 150 ms) putatively mediated by the direct pathway and the slow phase (tau = 35 s) by cAMP-dependent phosphorylation. Here we report that in frog, rat, and guinea pig ventricular myocytes ICa increases slowly and monophasically in response to isoproterenol. The increase is completely blocked by inhibitors of cAMP-dependent phosphorylation. Furthermore, the time course of the increase in ICa closely parallels the increase in contractile force produced by sympathetic nerve stimulation. These data refute earlier suggestions that regulation of Ca2+ channels by the sympathetic nervous system involves or requires a direct G-protein pathway.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1991

Receptor-mediated deactivation of Gk in cardiac myocytes.

The muscarinic potassium current IK(ACh) of atrial myocytes can be evoked in the absence of agonists by intracellular application of stable GTP analogs (GXP). This receptor-independent opening of K(ACh) channels is a consequence of the direct activation of the guanyl nucleotide binding protein Gk that couples muscarinic receptors to K(ACh) channels, and was previously thought to be unaffected by subsequent application of agonist. We report here that in the presence of GTP, application of a pulse of muscarinic agonist to atrial cells can abolish the GXP-induced IK(ACh). The results imply that in intact cells the agonist-bound receptor can interact with Gk not only in its inactive, GDP-bound form, but also in its active, GXP-bound form in a process that promotes the release of guanine nucleotide from its binding site.

Acetylcholine↗

PubMed1991

Role of elevated monocyte transforming growth factor beta (TGF beta) production in posttrauma immunosuppression.

We previously reported that increased production of prostaglandin E2 by monocytes is a pivotal mechanism in posttrauma immunopathology. Here we characterize monocyte levels of transforming growth factor beta and examine the effects of elevated transforming growth factor beta on prostaglandin E2 release by patients' monocytes. Trauma patients' and normals' monocyte supernates (+/- stimulation with muramyl dipeptide) were acid treated and assayed for transforming growth factor beta using the mink lung-cell bioassay. Alternatively, human transforming growth factor beta was added to patients' and normals' monocytes and prostaglandin E2 production assayed. Significantly elevated transforming growth factor beta levels (median = 181.7 pmol/10(6) monocytes) were detected in immunosuppressed patients' monocytes but not immunocompetent trauma patients' (median = 32.0 pM) or normals' (median = 20.4 pM) monocytes. Adding transforming growth factor beta to monocytes resulted in a significant elevation of prostaglandin E2 levels. Elevated monocyte transforming growth factor beta levels in trauma patients could be both suppressing T-lymphocyte functions and maintaining elevated monocyte prostaglandin E2 synthesis.

Acetylmuramyl-Alanyl-Isoglutamine↗