Tong Mook Kang

Loss of Autophagy Diminishes Pancreatic β Cell Mass and Function with Resultant Hyperglycemia

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells.

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.

Effects of hypoxia and mitochondrial inhibition on the capacitative calcium entry in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia and mitochondria inhibitors on the capacitative Ca(2+) entry (CCE) in cultured smooth muscle cells from rabbit small pulmonary arteries. Cyclopiazonic acid (CPA) depleted Ca(2+) from sarcoplasmic reticulum (SR) in Ca(2+)-free medium and subsequent addition of Ca(2+) led to the nifedipine-insensitive, La(3+)-sensitive Ca(2+) influx. The presence of CCE was further verified by the measurement of unidirectional Mn(2+) influx. During the decay phase of the CCE-induced [Ca(2+)]c transients, hypoxia (P(O2) < 50 mmHg) and the mitochondria inhibitor FCCP reversibly increased [Ca(2+)]c, that is La(3+)-sensitive. Once SR is depleted by CPA, subsequent treatment of FCCP slowed the decay of CCE-induced [Ca(2+)]c transients but it did not attenuate Mn(2+) influx. Mitochondrial uptake of incoming Ca(2+) through CCE was demonstrated by additional increase in [Ca(2+)]c with Ca(2+) ionophore after terminating CCE. Together, it is suggested that the augmentation of CCE-induced [Ca(2+)]c transients by hypoxia and FCCP reflects a net gain of [Ca(2+)]c by the inhibition of mitochondrial Ca(2+) uptake.

CA2+ INFLUX THROUGH CARBACHOL-ACTIVATED NON-SELECTIVE CATION CHANNELS IN GUINEA-PIG GASTRIC MYOCYTES

1 Ca2+ microfluorometry (100 μm K5 fura‐2) and the voltage‐clamp technique were combined to study the effect of carbachol (CCh, 50 μm) in inducing currents (ICCh) through non‐selective cation channels (NSCCCh) and increments in global cytosolic Ca2+ concentration (Δ[Ca2+]c). 2 In Na+‐containing bath solution, ICCh fell from an initial phasic to a subsequent small (5 %) tonic component; Δ[Ca2+]c fell to zero. Tonic ICCh and [Ca2+]c became prominent after substitution of extracellular 140 mm Na+ by 140 mm Cs+. Tonic ICCh and Δ[Ca2+]c were insensitive to intracellular heparin (3 mg ml−1) and ryanodine (4 μm), i.e. they did not depend on Ca2+ release from sarcoplasmic reticulum (SR). 3 Single channel currents of NSCCCh could be resolved in whole‐cell recordings. Substitution of Na+ by Cs+ increased NSCCCh activity by one order of magnitude and slope conductance from 22 to 30 pS. Extracellular quinidine (3 μm) reversibly blocked the NSCCCh activity. 4 Both tonic ICCh and tonic Δ[Ca2+]c (a) followed a similar time course of activation, desensitization and facilitation, (b) were reversibly blocked by 3 μm quinidine, and (c) persisted upon block of SR Ca2+ release. 5 A Ca2+ fractional current of tonic ICCh (fCa) of 0.009 was calculated by comparing the ratio Δ[Ca2+]c (corrected for simultaneous Ca2+ redistribution) over ICCh with depolarization‐induced *Δ[Ca2+]c (Δ[Ca2+]c calculated from ICa induced by a 400 ms depolarization from −60 to 0 mV at 2 mm[Ca2+]o, 145 mm[Cs+]o) over ICa. fCa was 0.023 at [Ca2+]o= 4 mm. 6 With 110 mm extracellular CaCl2 and 145 mm intracellular CsCl, ICCh reversed at +19.5 mV suggesting a permeability ratio PCa/PCs of 2.8. 7 We conclude that Ca2+ influx through NSCCCh under physiological [Ca2+]o could induce Δ[Ca2+]c. The fCa was, however, much smaller than the one calculated from the reversal potential.

DNA Hydrogel Fiber with Self‐Entanglement Prepared by Using an Ionic Liquid

DNA hydrogels have a wide range of biomedical applications in tissue engineering and drug-delivery systems. There are two ways to create hydrogel structures: one is enzymecatalyzed assembly of synthetic DNA and the other is by crosslinking natural DNA chemically. For natural DNA, formaldehyde and metal compounds such as arsenic, chromate, and nickel are widely used as crosslinkers. However, these modified DNA hydrogels are unsafe to apply in biological systems because the crosslinkers have potentially adverse side effects, with some being carcinogens. Besides this, these DNA hydrogels are difficult to form into hydrogel fibers by using conventional spinning methods in the absence of chemical crosslinking. In solution, DNA resembles modular proteins such as titin, silk, and polysaccharides. The very flexible linear DNA strands and their noncovalent assemblies can form compacted interwound supercoils in bulk aqueous solution with cationic salts. Alternatively, they can roll into soluble clusters of toroids. In concentrated DNA solutions in poor solvents, rodlike multiple-chain bundling occurs and, simultaneously, single or multiple loops form knots with themselves or with adjacent loops through a nucleation-growth pathway. Thus, these condensates are seen primarily as intertwined aggregates of toroids. We were inspired by the spinning processes used by insects (for example, silkworms and spiders) to develop spinning conditions to create the desired DNA hydrogel fibers. It is known that the last process to occur in insect spinning is the formation of a dragline in air. It can be considered that the air effectively removes water through evaporation to produce dense, dried fibers. To replicate this process in wet spinning, we need to ensure that the coagulation solvent does not fill the space created in the spinning droplet by the exiting water. In addition, the diffusion rates of the coagulation solvent and water must be controlled to prevent the formation of a dense skin on the fiber, which could trap water and create a porous structure. If the coagulation solvent also contains crosslinking cations, then the concentrated DNA solution can form hydrogel fibers with intertwined toroidal entanglements. We have found that room-temperature hydrophilic ionic liquids (RTILs) can produce suitable conditions. The feasibility of using RTILs was suggested by our previous work, which showed that 100% of RTILs will absorb water even when bound to a polymer network. Moreover, some RTILs can create low-pH conditions when in contact with water, and such acidic conditions have been used to promote coagulation in the wet spinning of DNA fibers. The cations present in RTILs condense the DNA as a matter of course. In this work, we prepared a DNA hydrogel fiber in a single step by injecting aqueous DNA solution into a coagulation bath of an RTIL.

Tough Supersoft Sponge Fibers with Tunable Stiffness from a DNA Self-Assembly Technique

Tough and soft: Highly porous, spongelike materials self-assemble by calcium ion condensation of DNA-wrapped carbon nanotubes (SWNTs-DNA; see picture, IL = ionic liquid). The toughness, modulus, and swellability of the electrically conductive sponges can be tuned by controlling the density and strength of interfiber junctions. The sponges have compliances similar to the softest natural tissue, while robust interfiber junctions give high toughness.

Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phosphatidylinositol 4,5-bisphosphate in B lymphocytes

In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5‐bisphosphate (PIP2) is not known yet. The mouse B cells express large conductance background K+ channels (LKbg) that are inhibited by PIP2. In inside‐out patch clamp studies, the application of MgATP (1 mm) also inhibited LKbg due to the generation of PIP2 by phosphoinositide (PI)‐kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LKbg, which was antagonized by PIP2 (> 1 μm) or higher concentration of MgATP (5 mm). The inhibition by PIP2 was partially reversible. However, the application of methyl‐β‐cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LKbg activity and facilitated the activation by stretch. In cell‐attached patches, LKbg were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano‐activation of LKbg was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m‐3M3FBS or by cross‐linking IgM‐type B cell receptors activated LKbg. Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI‐231 induces tyrosine phosphorylation of PLCγ2 and PIP2 hydrolysis of plasma membrane. The time dependence of PIP2 hydrolysis and LKbg activation were similar. The presence of LKbg and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch‐dependent ion channel activation, namely, that the degradation of PIP2 caused by stretch‐activated PLC releases LKbg from the tonic inhibition by PIP2.

Ion Fluxes in Giant Excised Cardiac Membrane Patches Detected and Quantified with Ion-selective Microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2–3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10–15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion–coupled transporters in giant patches.

Positive feedback control between STIM1 and NFATc3 is required for C2C12 myoblast differentiation.

Up-regulation of STIM1-mediated store-operated Ca(2+) entry (SOCE) and Ca(2+)-dependent NFAT signaling is important for myogenic differentiation. However, the molecular mechanisms for differentiation specific up-regulation of STIM1/SOCE-mediated signaling are poorly understood. This study explored whether functional crosstalk between STIM1 and a member of NFAT transcription factor is important for C2C12 myoblast differentiation. Transient increase of NFATc3 expression was observed in the initial phase of differentiation, and the increased activity of NFATc3 isoform was correlated with up-regulation of STIM1 expression. Overexpression of NFATc3 increased STIM1 expression, SOCE activity, and myotube formation, whereas NFATc3 knockdown showed the opposite effects. Overexpression of STIM1 increased the activity and expression level of NFATc3, and enhanced myotube formation, whereas STIM1 knockdown resulted in the opposite effects. Taken together, our findings suggest that a positive feedback control between STIM1/SOCE and NFATc3 is required for efficient induction and progression of myoblast differentiation.