Ultrasound-Induced Calcium Oscillations and Waves in Chinese Hamster Ovary Cells in the Presence of Microbubbles

This study investigated the effects of ultrasound on the intracellular [Ca2+] of Chinese hamster ovary cells in the presence of albumin-encapsulated Optison microbubbles. Cells were exposed to 1 MHz ultrasound (tone burst of 0.2 s duration, 0.45 MPa peak pressure) while immersed in solution of 0.9 mM Ca2+. Calcium imaging of the cells was performed using digital video fluorescence microscopy and Ca2+-indicator dye fura-2AM. Experimental evidence indicated that ultrasound caused a direct microbubble-cell interaction resulting in the breaking and eventual dissolution of the microbubble and concomitant permeabilization of the cells to Ca2+. These cells exhibited a large influx of Ca2+ over 3–4 s and did not return to their equilibrium levels. Subsequently, some cells exhibited one or more Ca2+ oscillations with the onset of oscillations delayed by 10–80 s after the ultrasound pulse. A variety of oscillations were observed including decaying oscillations returning to the baseline value over 35– 100 s, oscillations superimposed on a more gradual recovery over 150–200 s, and oscillations continued with increased amplitude caused by a second ultrasound tone burst. The delays in onset appeared to result from calcium waves that propagated across the cells after the application of the ultrasound pulse.NIH grant R01CA116592American Cancer Society Institutional Research Grant to Case Western Reserve UniversityPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/84354/1/L29.pd

Spatiotemporal Effects of Sonoporation Measured by Real-Time Calcium Imaging

Published in PubMed Central on 01 March 2010To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca2+]i) was determined using real time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison™ microbubbles. At extracellular [Ca2+]o of 0.9 mM, ultrasound application generated both non-oscillating and oscillating (periods 12–30 s) transients (changes of [Ca2+]i in time) with durations of 100–180 s. Immediate [Ca2+]i transients after ultrasound application were induced by ultrasound-mediated microbubble–cell interactions. In some cases, the immediately-affected cells did not return to pre-ultrasound equilibrium [Ca2+]i levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca2+]i in different cells formed a calcium wave and was observed to propagate outward from the immediately-affected cells at 7–20 μm/s over a distance greater than 200 μm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca2+ for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison™ microbubbles can generate transient [Ca2+]i changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membraneNational Institutes of Health R01CA116592Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/84355/1/nihms99796.pd

Molecular Mechanisms and Clinical Implications of Reversible Protein S-Glutathionylation

Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol–disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein–SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein–SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein–SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein–thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases. Antioxid. Redox Signal, 10, 1941–1988