Changes In Transmembrane Potential Research Articles

Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity.

The resolution of neutrophil (PMN)-mediated inflammation occurs through the apoptosis, or programmed cell death, of the neutrophil. PMN apoptosis is inhibited by a variety of inflammatory stimuli; moreover, PMN from critically ill septic patients show profoundly delayed rates of apoptosis in vitro. Since apoptosis is effected through the activity of intracellular cysteine proteases (caspases), we evaluated caspase expression and activity in neutrophils from septic patients and compared them with caspase expression and activity of resting or lipopolysaccharide-activated neutrophils from healthy volunteers. Prospective observational cohort study. Tertiary level intensive care unit and associated research laboratory. Thirty-six intensive care unit patients with sepsis; ten healthy laboratory controls. Collection of up to 10 mL of whole blood for in vitro study of rates of apoptosis, expression and activity of caspases-1, -3, and -9, activation of nuclear factor-kappaB, and change in mitochondrial transmembrane potential. Following 24 hrs of in vitro culture, 52 +/- 7.8% of control neutrophils, but only 29 +/- 5.4% of lipopolysaccharide-stimulated (1 microg/mL) PMN, showed nuclear changes of apoptosis. Only 6.2 +/- 1.1% of neutrophils from septic patients were apoptotic after 24 hrs. Significant nuclear translocation of nuclear factor-kappaB was evident in septic PMN, and inhibition of apoptosis was partially abrogated by prevention of nuclear factor-kappaB dissociation with pyrrolidine dithiocarbamate. Caspase-3 transcription and catalytic activity were significantly reduced in both patients' and lipopolysaccharide-treated PMN; caspase-1 transcription and activity were increased by lipopolysaccharide but reduced in septic patients. In contrast, caspase-9 transcription and activity were reduced in septic patients but not in lipopolysaccharide-treated PMN. Decreased caspase-9 activity was associated with sustained maintenance of mitochondrial transmembrane potential and reduced translocation of cytochrome c from the mitochondria to the cytosol. Apoptosis of circulating neutrophils from patients with clinical sepsis is profoundly suppressed, through a mechanism that involves activation of nuclear factor-kappaB that is associated with reduced activity of caspases-9 and -3 and maintenance of mitochondrial transmembrane potential and that differs in important respects from the inhibitory effects seen following the exposure of healthy neutrophils to inflammatory stimuli.

MIZORIBINE-MEDIATED APOPTOTIC SIGNALING PATHWAY IN HUMAN T CELL LINE

P778 Propose: Mizoribine (MZR), an inhibitor of IMP dehydrogenase which depletes cellular GTP, is used clinically as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. Methods: The data demonstrated that treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis characterized by chromatin condensation and H2AX phosphorylation. Results: MZR increased the catalytic activity of caspase-3 protease -8 protease and -9 proteases. Activation of caspase-3 protease was further confirmed by degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat cells. Furthermore, MZR induced the changes of mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from mitochondria. In addition, MZR induced the decrease of Bcl-XL expression whereas increase of Bcl-XS, Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis with consistent suppression of activity of caspase-8 protease, upstream caspase among caspase family, H2AX phosphorylaion and PARP cleavage in MZR-treated cells. Also, we have screened the expression profile of proteins in Jurkat cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in 2-D gels, the comparison of control versus apoptotic cells revealed that signal intensity of 10 spots was decreased and 5 spots was increased. Conclusions: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.

Mitochondrial Thioredoxin System: EFFECTS OF TrxR2 OVEREXPRESSION ON REDOX BALANCE, CELL GROWTH, AND APOPTOSIS

Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (DeltaPsi(m)), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of DeltaPsi(m) dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against beta-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells.

Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves

The rate of cyclic electron flow measured in dark-adapted leaves under aerobic conditions submitted to a saturating illumination has been performed by the analysis of the transmembrane potential changes induced by a light to dark transfer. Using a new highly sensitive spectrophotometric technique, a rate of the cyclic flow of ∼130 s −1 has been measured in the presence or absence of 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU). This value is ∼1.5 times larger than that previously reported [Proc. Natl. Acad. Sci. U. S. A. 99 (2001) 10209]. We have characterized in the presence or absence of DCMU charge recombination process ( t 1/2∼60 μs) that involves P 700 + and very likely the reduced form of the iron sulfur acceptor F X. This led to conclude that, under saturating illumination, the PSI centers involved in the cyclic pathway have most of the iron sulfur acceptors F A and F B reduced. In the proposed mechanism, electrons are transferred from a ferredoxin bound to a site localized on the stromal side of the cytochrome b 6 f complex to the Q i site. Two possible models of the organization of the membrane complexes are discussed, in which the cyclic and linear electron transfer chains are isolated one from the other.

Intramural virtual electrodes in ventricular wall: effects on epicardial polarizations.

Intramural virtual electrodes (IVEs) are believed to play an important role in defibrillation, but their existence in intact myocardium remains unproven. Here, IVEs were detected by use of optical recordings of shock-induced transmembrane potential (V(m)) changes (DeltaV(m)) measured from the intact epicardial heart surface. To detect IVEs, isolated porcine left ventricles were sequentially stained with a V(m)-sensitive dye by 2 methods: (1) surface staining (SS) and (2) global staining (GS) via coronary perfusion. Shocks (2 to 50 V/cm) were applied across the ventricular wall in an epicardial-to-endocardial direction during the action potential plateau via transparent mesh electrodes, and shock-induced DeltaV(m) were measured optically from the same epicardial locations after SS and GS. Optical recordings revealed significant differences between DeltaV(m) of 2 types that became more prominent with increasing shock strength: (1) for weak shocks, SS-DeltaV(m) were larger and faster than GS-DeltaV(m); (2) for intermediate shocks, cathodal GS-DeltaV(m) became multiphasic, whereas SS-DeltaV(m) remained monophasic; and (3) for strong shocks, cathodal GS-DeltaV(m) became uniformly negative, whereas SS-DeltaV(m) typically remained positive. The radical differences in the shape and polarity of SS and GS polarizations can be explained by the contribution of subepicardial IVEs to optical signals. Histological examination revealed a dense network of collagen septa in the subepicardium, which could form the IVE substrate. Intramural virtual electrodes are reflected in optical measurements of shock-induced DeltaV(m) on the intact epicardial surface. These IVEs could be a result of microscopic resistive discontinuities formed by collagen septa.

An analytical model for the calculation of the change in transmembrane potential produced by an ultrawideband electromagnetic pulse.

The electric field pulse shape and change in transmembrane potential produced at various points within a sphere by an intense, ultrawideband pulse are calculated in a four stage, analytical procedure. Spheres of two sizes are used to represent the head of a human and the head of a rat. In the first stage, the pulse is decomposed into its Fourier components. In the second stage, Mie scattering analysis (MSA) is performed for a particular point in the sphere on each of the Fourier components, and the resulting electric field pulse shape is obtained for that point. In the third stage, the long wavelength approximation (LWA) is used to obtain the change in transmembrane potential in a cell at that point. In the final stage, an energy analysis is performed. These calculations are performed at 45 points within each sphere. Large electric fields and transmembrane potential changes on the order of a millivolt are produced within the brain, but on a time scale on the order of nanoseconds. The pulse shape within the brain differs considerably from that of the incident pulse. Comparison of the results for spheres of different sizes indicates that scaling of such pulses across species is complicated.

Bcl-2 overexpression inhibits tetrachlorohydroquinone-induced apoptosis in NIH3T3 cells: a possible mechanism for tumor promotion.

TCHQ is a major carcinogenic metabolite of the widely used wood preservative PCP. Recently, we found that TCHQ was a promoter in a mouse skin carcinogenesis model. However, the mechanism is still not clear. In this study, we showed that overexpression of Bcl-2 effectively suppressed TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphological changes and DNA fragmentation. Although production of ROS contributes to TCHQ-induced apoptosis, Bcl-2 failed to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, overexpressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. We also found that TCHQ induced a change in mitochondrial transmembrane potential, and that caspase-9 and subsequent caspase-3 can be activated during TCHQ-induced acute apoptosis. Interestingly, TCHQ induced a significant upregulation of Bcl-2 expression, and over-expressed Bcl-2 can dramatically inhibit the change of mitochondria membrane potential and activation of both caspase-9 and -3. Thus, our results suggest TCHQ-induced tumor promotion may be through a mechanism of upregulation of Bcl-2 protein and subsequent apoptosis inhibition.

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation.

We previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, beta-actin, tubulin-alpha-1-chain and enolase-alpha. Interestingly, all carbonylated proteins except calreticulin and enolase-alpha showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

Use of SYBR14, 7-amino-actinomycin D, and JC-1 in assessing sperm damage from rats with spinal cord injury.

Although fluorescent dyes combined with flow cytometry have been used to confirm the viability of sperm in the past, methods to detect damage to spermatozoa following injury have been limited to use of dyes, which are often difficult to adequately compensate for in a single laser system. In this article, we present what we believe is a better method to assess damage to sperm secondary to spinal cord injury in an in vivo model, for use with a standard Ar laser and flow cell. In this rat model of spinal cord injury leading to sperm damage, the spinal cords of the rats were injured, but the reproductive organs were not. To understand the origins of sperm injury, and to develop ways to overcome the loss of fertility, we used the viability dye SYBR-14 along with 7-amino actinomycin D to detect apoptosis. Additionally, we used the dye JC-1 to measure the changes in mitochondrial transmembrane potential that accompany the damage. We found that SYBR-14 plus 7-amino actinomycin D was a useful method for quantifying apoptosis, particularly when another dye, such as JC-1, was used simultaneously. By using these dyes in concert with motility studies, we were able to quantify the extent of damage to sperm and correlate it to the decrease in motility of sperm (r(2) = 0.99 for SYBR14 versus motility and r(2) = 0.98 for JC-1 versus motility by regression analysis). With a method established to measure injury to sperm, we hope to determine which treatment regimens of ones we will test are effective in restoring sperm to a more fertile state, in the future.

Depletion of Endogenous Zinc Stores Induces Oxidative Stress and Cell Death in Human Melanoma Cells

The role of intracellular free zinc and its chelation by TPEN (N,N,N',N'- tetrakis(2-pyridylmethyl)ethylene-diamine) was studied in Bowes human melanoma cells. The content of free Zn pools was determined by fluorescent probe Zinquin. Depletion of zinc triggered apoptosis confirmed by cell blebbing, changes in mitochondrial transmembrane potential and GSH levels, caspase-3 activation and nuclear fragmentation. Apoptosis was only partially prevented by cyclosporin A or N-acetylcystein, suggesting various independent but likely interrelated mechanisms participating in this process.

Nonlinear changes of transmembrane potential during electrical shocks: role of membrane electroporation.

Defibrillation shocks induce nonlinear changes of transmembrane potential (DeltaVm) that determine the outcome of defibrillation. As shown earlier, strong shocks applied during action potential plateau cause nonmonotonic negative DeltaVm, where an initial hyperpolarization is followed by Vm shift to a more positive level. The biphasic negative DeltaVm can be attributable to (1) an inward ionic current or (2) membrane electroporation. These hypotheses were tested in cell cultures by measuring the effects of ionic channel blockers on DeltaVm and measuring uptake of membrane-impermeable dye. Experiments were performed in cell strands (width approximately 0.8 mm) produced using a technique of patterned cell growth. Uniform-field shocks were applied during the action potential plateau, and DeltaVm was measured by optical mapping. Shock-induced negative DeltaVm exhibited a biphasic shape starting at a shock strength of approximately 15 V/cm when estimated peak DeltaV-m was approximately -180 mV; positive DeltaVm remained monophasic. Application of a series of shocks with a strength of 23+/-1 V/cm resulted in uptake of membrane-impermeable dye propidium iodide. Dye uptake was restricted to the anodal side of strands with the largest negative DeltaVm, indicating the occurrence of membrane electroporation at these locations. The occurrence of biphasic negative DeltaVm was also paralleled with after-shock elevation of diastolic Vm. Inhibition of I(f) and I(K1) currents that are active at large negative potentials by CsCl and BaCl2, respectively, did not affect DeltaVm, indicating that these currents were not responsible for biphasic DeltaVm. These results provide evidence that the biphasic shape of DeltaVm at sites of shock-induced hyperpolarization is caused by membrane electroporation.

Effects of weak electric fields on the activity of neurons and neuronal networks.

Electric fields applied to brain tissue will affect cellular properties. They will hyperpolarise the ends of cells closest to the positive part of the field, and depolarise ends closest to the negative. In the case of neurons this affects excitability. How these changes in transmembrane potential are distributed depends on the length constant of the neuron, and on its geometry; if the neuron is electrically compact, the change in transmembrane potential becomes an almost linear function of distance in the direction of the field. Neurons from the mammalian hippocampus, maintained in tissue slices in vitro, are significantly affected by fields of around 1-5 V m(-1).

Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes.

Fluorescent dyes have been widely employed as optical indicators of the membrane potential difference in cells, isolated organelles and lipid vesicles that are too small to make microelectrode measurements feasible. We describe here the application of a carbocyanine dye, 3,3'-dipropylthiodicarbocyanine iodide [DiS-C3-(5)], to monitor the transmembrane potential changes induced by a variation of the K+ concentration for the cells of Escherichia (E.) coli and photosynthetic bacterium Rhodospirillum (R.) rubrum. The cells were first incubated in buffers containing DiS-C3-(5) and K+ ions of various concentrations until the fluorescence intensity reached a constant value. Valinomycin was then added to the solution, which caused changes in the fluorescence intensity, depending on the K+ concentrations. The membrane potential is shown to have a linear relationship with the fluorescence intensity of DiS-C3-(5). The results demonstrate that the K+ concentrations inside intact cells are 4.6 mM and 5.3 mM for E. coli and R. rubrum, respectively. The diffusion potentials of K+ ions were determined using the Nernst equation over the range of -1.3 mV to 44 mV, corresponding to K+ concentrations of 5 mM -25 mM outside of the cells.

Curcumin: a potential vaginal contraceptive

The purpose of this investigation was to evaluate the sperm-immobilizing effects of curcumin, a plant-derived diferuloylmethane compound. Washed human healthy sperm were suspended in Ham's F10 and exposed to varying concentrations of curcumin. Sperm motility was evaluated and changes in sperm mitochondrial transmembrane potential (MTP) was quantified by flow cytometry. Incubation of normal human sperm with curcumin resulted in a dose- and time-dependent loss of sperm motility. At lower concentrations (30 g/mL), curcumin produced a significant (20%) decrease in sperm motility within 30 min without significant effects on sperm viability. An instantaneous (>50%) loss of sperm motility was observed with higher concentrations (300 g/mL) of curcumin and a total loss of sperm motility was achieved within 60 min. A significant reduction in sperm MTP was found with all doses of curcumin tested. Our results indicate that curcumin has a selective sperm-immobilizing effect, in addition to a previously studied anti-HIV property. This compound may have potential clinical applications as a novel intravaginal spermicidal agent for contraception and HIV prevention.

The Effect of Asymmetric Surface Potentials on the Intramembrane Electric Field Measured with Voltage-Sensitive Dyes

Ratiometric imaging of styryl potentiometric dyes can be used to measure the potential gradient inside the membrane (intramembrane potential), which is the sum of contributions from transmembrane potential, dipole potential, and the difference in the surface potentials at both sides of the membrane. Here changes in intramembrane potential of the bilayer membranes in two different preparations, lipid vesicles and individual N1E-115 neuroblastoma cells, are calculated from the fluorescence ratios of di-4-ANEPPS and di-8-ANEPPS as a function of divalent cation concentration. In lipid vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) or from a mixture of the negatively charged lipid phosphatidylserine (PS) and PC, di-4-ANEPPS produces similar spectral changes in response to both divalent cation-induced changes in intramembrane potential and transmembrane potential. The changes in potential on addition of divalent cations measured by the fluorescence ratios of di-4-ANEPPS are consistent with a change in surface potential that can be modeled with the Gouy-Chapman-Stern theory. The derived intrinsic 1:1 association constants of Ba and Mg with PC are 1.0 and 0.4 M −1; the intrinsic 1:1 association constants of Ba and Mg with PS are 1.9 and 1.8 M −1. Ratiometric measurements of voltage sensitive dyes also allow monitoring of intramembrane potentials in living cells. In neuroblastoma cells, a tenfold increase of concentration of Ba, Mg, and Ca gives a decrease in intramembrane potential of 22 to 24 mV. The observed changes in potential could also be described by Gouy-Chapman theory. A surface charge density of 1 e −/115 Å 2 provides the best fit and the intrinsic 1:1 association constants of Ba, Mg, and Ca with acidic group in the surface are 1.7, 6.1, and 25.3 M −1.

Long QT syndrome and anaesthesia

Long QT syndrome (LQTS) is an arrhythmogenic cardiovascular disorder resulting from mutations in cardiac ion channels. LQTS is characterized by prolonged ventricular repolarization and frequently manifests itself as QT interval prolongation on the electrocardiogram (ECG). The age at presentation varies from in utero to adulthood. The majority of symptomatic events are related to physical activity and emotional stress. Although LQTS is characterized by recurrent syncope, cardiac arrest, and seizure-like episodes, only about 60% of patients are symptomatic at the time of diagnosis. 3 The clinical features of LQTS result from a peculiar episodic ventricular tachyarrhythmia called ‘torsade de pointes’. ‘Twisting of the points’ describes the typical sinusoidal twisting of the QRS axis around the isoelectric line of the ECG. Usually torsade de pointes start with a premature ventricular depolarization, followed by a compensatory pause. The next sinus beat often has a markedly prolonged QT interval and abnormal T wave. This is followed by a ventricular tachycardia that is characterized by variation in the QRS morphology, and a constantly changing R-R interval (Fig. 1). The ‘short-long-short’ cycle length sequence heralding torsade de pointes is a hallmark of LQTS. Commonly, the episode of torsade de pointes is self-terminating, producing a syncopal episode or pseudoseizure, secondary to the abrupt decrease in cerebral blood flow. The majority of episodes of sudden death in LQTS result from ventricular fibrillation triggered by torsade de pointes, although the mechanism of this deterioration is unknown. Traditionally, LQTS has been classified as either familial (inherited) or acquired. However, it is likely that many patients with previously labelled acquired LQTS carry a silent mutation in one of the genes responsible for congenital LQTS. 22 119 The evidence for this hypothesis has been gradually emerging over the past few years. It is important for anaesthetists to be aware of this concept, as it means that a much higher proportion of the general population may be affected by asymptomatic mutations in genes encoding cardiac ion channels than was thought previously. The prevalence of LQTS in developed countries may be as high as 1 per 1100‐3000 of the population. 32 119 About 30% of congenital LQTS carriers have an apparently normal phenotype, and thus a normal QT interval, and remain undiagnosed until an initiating event. 105 Fatal arrhythmias associated with primary electrical disease of the heart such as the Brugada and LQTS, probably account for 19% of sudden deaths in children between 1 and 13 yr of age, and 30% of sudden deaths that occur between 14 and 21 yr of age. 10 Furthermore, there is a strong association between prolonged corrected QT interval (QTc) in the first week of life and risk of sudden infant death syndrome. 86

Gamma-Tocopheryl quinone induces apoptosis in cancer cells via caspase-9 activation and cytochrome c release

Recently, it was suggested the potential role of gamma-tocopheryl quinone (gamma-TQ), an oxidative metabolite of gamma-tocopherol, as a powerful chemotherapeutic agent, since it was shown that this molecule exerts powerful cytotoxic effects, induces apoptosis and escapes drug resistance in human acute lymphoblastic leukemia and promyelocytic leukemia cells. We have studied the apoptogenic potential of gamma-TQ in cultured human leukemia HL-60 and colon adenocarcinoma WiDr cells, and in murine thymoma cells growing in vivo in ascites form. The cells were treated with gamma-TQ and apoptosis was evaluated morphologically by acridine-orange staining and cytofluorimetrically by Annexin V binding assay. gamma-TQ-induced apoptosis in a dose- and time-dependent manner in all the cell types tested, although HL-60 and thymoma cells were much more sensitive than WiDr cells. In HL-60 cells apoptosis was mediated by the activation of the caspase-3 cascade. In particular, we observed a time- and dose-dependent increase in the activities of the upstream caspase-9 and caspase-8 and of the downstream caspase-3. The activation of caspase-9 preceded that of caspase-8 and its specific inhibition completely prevented apoptosis. These findings and data showing the precocious release of cytochrome c from mitochondria, a decrease in Bcl-2, and a change in mitochondrial transmembrane potential (Delta psi(m)), all suggest that the intrinsic mitochondrial pathway is primarily involved in the development of gamma-TQ-induced apoptosis. The late activation of caspase-8 and data showing the partial cleavage of pro-apoptotic protein BID suggest that the initial activation of caspase-9 may be potentiated by a feedback amplification loop involving the caspase-8/BID pathway.

Monensin-mediated growth inhibition of SNU-C1 colon cancer cells via cell cycle arrest and apoptosis

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we demonstrate that monensin inhibited the proliferation of solid tumor cells with IC50 of about 2.5 micro M. Monensin induced a G1 or a G2-M phase arrest in these cells. When we examined the effects of this drug on SNU-C1 cells, monensin decreased the levels of CDK2, CDK4, CDK6, cyclin D1 and cyclin A proteins. While p27 was increased by monensin, p21 was not. In addition, monensin markedly enhanced the binding of p27 with CDK2, CDK4 and CDK6. Furthermore, the activities of CDK2-, CDK4- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Monensin also induced apoptosis in solid tumor cells. Apoptotic process of SNU-C1 cells was associated with the changes of Bax, caspase-3 and mitochondria transmembrane potential (deltapsim). Taken together, these results demonstrated for the first time that monensin inhibited the growth of solid tumor cells, especially SNU-C1 cells, via cell cycle arrest and apoptosis.

Effects of membrane lipids on ion channel structure and function.

Biologic membranes are not simply inert physical barriers, but complex and dynamic environments that affect membrane protein structure and function. Residing within these environments, ion channels control the flux of ions across the membrane through conformational changes that allow transient ion flux through a central pore. These conformational changes may be modulated by changes in transmembrane electrochemical potential, the binding of small ligands or other proteins, or changes in the local lipid environment. Ion channels play fundamental roles in cellular function and, in higher eukaryotes, are the primary means of intercellular signaling, especially between excitable cells such as neurons. The focus of this review is to examine how the composition of the bilayer affects ion channel structure and function. This is an important consideration because the bilayer composition varies greatly in different cell types and in different organellar membranes. Even within a membrane, the lipid composition differs between the inner and outer leaflets, and the composition within a given leaflet is both heterogeneous and highly dynamic. Differential packing of lipids (and proteins) leads to the formation of microdomains, and lateral diffusion of these microdomains or "lipid rafts" serve as mobile platforms for the clustering and organization of bilayer constituents including ion channels. The structure and function of these channels are sensitive to specific chemical interactions with neighboring components of the membrane and also to the biophysical properties of their membrane microenvironment (e.g., fluidity, lateral pressure profile, and bilayer thickness). As specific examples, we have focused on the K+ ion channels and the ligand-gated nicotinicoid receptors, two classes of ion channels that have been well-characterized structurally and functionally. The responsiveness of these ion channels to changes in the lipid environment illustrate how ion channels, and more generally, any membrane protein, may be regulated via cellular control of membrane composition.

Pulled Sideways

Bacteria lack rigid cell walls and must respond rapidly to salt-induced gradients in osmotic pressure. In Escherichia coli, the mechanosensitive channel proteins MscL and MscS open in response to pressure applied perpendicularly to the membrane, which has the effect of pulling laterally on the membrane-embedded proteins. Bass et al. (see the Perspective by Bezanilla and Perozo) provide the crystal structure of MscS and describe how the transmembrane helices might reorient themselves as a result of being pulled sideways. The authors also propose how changes in transmembrane electrical potential might trigger movement of positively charged arginines and voltage-gate the osmolyte flux.R. B. Bass, P. Strop, M. Barclay, D. C. Rees, Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science 298, 1582-1587 (2002). [Abstract] [Full Text]F. Bezanilla, E. Perozo, Force and voltage sensors in one structure. Science 298, 1562-1563 (2002). [Summary] [Full Text]