Choi S et al. (2015), RNS60, a charge-stabilized nanostructure saline...

XB-ART-50213

Physiol Rep 2015 Mar 01;33:. doi: 10.14814/phy2.12261.

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RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

Choi S , Yu E , Kim DS , Sugimori M , Llinás RR .

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We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

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Species referenced: Xenopus laevis
Genes referenced: gnl3

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	Figure 1. Intracellular recordings from Xenopus laevis oocytes in the presence of RNS60- (A), NS- (B), RNS10.3- (C), and ONS60- (D)-based Ringer's solutions. Note that solutions based on RNS60, but not those based on normal saline (NS), RNS10.3 (TCP-modified saline without excess oxygen), or ONS60 (saline containing comparable level of oxygen without TCP modification), increased (hyperpolarized) resting membrane potential. Also, step pulse transmembrane current injection-induced membrane potential changes were increased only in the presence of RNS60. (E) Intracellular recording with serial applications of NS, RNS10.3, RNS60, or ONS60 with measurement of oxygen level in recording solutions.
	Figure 2. Membrane potential changes as a function of current injection (I–V curve) in the presence of RNS60 (A, average of 16 experiments), NS (B, average of 13 experiments), RNS10.3 (C, 14 experiments) and ONS60 (D, 10 experiments). P < 0.05, P < 0.01 and **P < 0.001 two-way ANOVA/Tukey's post-hoc test.
	Figure 3. RNS60 effect on Xenopus laevis oocyte membrane potential and input resistance following ouabain Na+/K+ ATPs block. In (A) Control, left set of recordings (in black), resting potential and input resistance recordings before ouabain administration. In red similar set of recordings 10 min following 100 μmol/L ouabain administration. The oocyte resting potential was reduced by nearly 10 mV (P < 0.001 by paired t-test). In addition, a significant decrease in membrane input resistance was also registered (P < 0.05 by paired t-test). In green, following ouabain addition of RNS60 did not modify X. laevis oocyte resting potential or membrane resistance (results from 13 different oocyte experiments). In (B) a similar set of recordings as in (A). The last set in purple demonstrates no change of membrane properties in normal saline solution with ouabain (results from 10 different oocyte experiments).
	Figure 4. Effects of RNS60 on membrane potential (A) and membrane input resistance (B) following block of intracellular “usable ATP” inhibition. Note that RNS60 effect is absent after both the inhibition of electron transport chain in mitochondria by rotenone and after oligomycin A, an inhibitor of ATP synthase. P < 0.01 and *P < 0.001 by paired t-test, indicating that the target for RNS60 action is mitochondrial ATP synthesis (n = 7 in RNS60 with rotenone, 6 in RNS60 with oligomycin A, 8 in NS with rotenone, and 6 in NS with oligomycin A).
	Figure 5. Intracellular ATP levels determined by luciferin/luciferase photon emission in Xenopus laevis oocytes. ATP levels increased 5 min following RNS60-based Ringer's superfusion, but not so following RNS-based Riger's with intracellular ATP blockages (with rotenone and oligomycin A) as well as NS-based Ringer's superfusion (A). Photon ATP images were obtained by photon accumulation for 1 min. Scale bar = 0.5 mm. Statistical difference is shown in (B). *P < 0.05 by one-way ANOVA. (n = 8 in RNS60, 6 in NS, 4 in RNS60 with rotenone, and 5 in RNS60 with oligomycin A).
	Figure 6. Ultrastructural analysis of mitochondria in response to RNS60-based Ringer's, compared to air oxygenated standard NS-based Ringer's. (A) Electron microscopy of cortical cytoplasm in Xenopus laevis oocytes. (B) The increased number of longer length mitochondria in response to RNS60. Total 36 areas (area size: 170 μm2, 6 areas per X. laevis oocyte) in cortical cytoplasm were analyzed in each group, YP: light yolk platelet. (C) The comparison of averaged mitochondrial diameter. Statistically significant differences marked as P < 0.05 and **P < 0.001 by one-way ANOVA.

References [+] :

Allen, A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM). 2007, Pubmed , Xenbase
Bach, Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. A novel regulatory mechanism altered in obesity. 2003, Pubmed
Bahima, Endogenous hemichannels play a role in the release of ATP from Xenopus oocytes. 2006, Pubmed , Xenbase
Baud, Sodium channels induced by depolarization of the Xenopus laevis oocyte. 1982, Pubmed , Xenbase
Bauer, An endogenous inactivating inward-rectifying potassium current in oocytes of Xenopus laevis. 1996, Pubmed , Xenbase
Choi, Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline. 2014, Pubmed
Dascal, Xenopus oocyte resting potential, muscarinic responses and the role of calcium and guanosine 3',5'-cyclic monophosphate. 1984, Pubmed , Xenbase
Dumollard, The role of mitochondrial function in the oocyte and embryo. 2007, Pubmed
Heasman, The mitochondrial cloud of Xenopus oocytes: the source of germinal granule material. 1984, Pubmed , Xenbase
Khasnavis, Suppression of nuclear factor-κB activation and inflammation in microglia by physically modified saline. 2012, Pubmed
Khasnavis, Protection of dopaminergic neurons in a mouse model of Parkinson's disease by a physically-modified saline containing charge-stabilized nanobubbles. 2014, Pubmed
Lafaire, Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis. 1986, Pubmed , Xenbase
Lagouge, Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. 2006, Pubmed
Marinos, Mitochondrial number, cytochrome oxidase and succinic dehydrogenase activity in Xenopus laevis oocytes. 1981, Pubmed , Xenbase
McElroy, The Energy Source for Bioluminescence in an Isolated System. 1947, Pubmed
Mondal, Protection of Tregs, suppression of Th1 and Th17 cells, and amelioration of experimental allergic encephalomyelitis by a physically-modified saline. 2012, Pubmed
Ninomiya, Electrical responses of smooth muscle cells of the mouse uterus to adenosine triphosphate. 1983, Pubmed
Nisoli, Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals. 2004, Pubmed
Paulmichl, Cellular mechanisms of ATP-induced hyperpolarization in renal epitheloid MDCK-cells. 1991, Pubmed
STREHLER, Purification of firefly luciferin. 1949, Pubmed
Xie, Na(+)/K(+)-ATPase as a signal transducer. 2002, Pubmed