Mitochondrial coupling in vivo in mouse skeletal muscle.
Mitochondrial coupling in vivo in mouse skeletal muscle.
Marcinek DJ, Schenkman KA, Ciesielski WA, Conley KE.
Department of Radiology, University of Washington Medical Center, Seattle, WA 98195, USA. dmarc@u.washington.edu
The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with 31P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 +/- 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 +/- 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought.
PMID: 14522819 [PubMed - indexed for MEDLINE]
Role of ATP in the sensitivity to heat and the induction of apoptosis in mammalian ce
Role of ATP in the sensitivity to heat and the induction of apoptosis in mammalian cells.
Miyazaki N, Kurihara K, Nakano H, Shinohara K.
Tokyo Metropolitan Industrial Technology Research Institute, Komazawa Office, 2-11-1, Fukazawa, Setagaya-ku, Tokyo 158-0081, Japan. miyazaki.noriyuki@iri.metro.toyko.jp
Heat-induced cell death and apoptosis were studied with respect to intracellular ATP. Studies on the relationship between hyperthermic cell-killing at 44 degrees C and cellular ATP levels in four cell lines grown as monolayers and six cell lines grown in suspension showed good correlations between cellular ATP levels and the sensitivity to heat. D(0) values (the dose required to reduce survival in the linear portion of the response by 63%) linearly increased with an increase in cellular ATP levels. No such changes in sensitivity to heat were observed between the cells cultured at different cell densities, regardless of the change in the cellular ATP level. These results suggest that cellular intrinsic ability to supply ATP rather than the level of pooled ATP per se is responsible for the thermal response. Heat-induced apoptosis in L5178Y cells was observed following treatment at 42 degrees C for 70 min, 44 degrees C for 20 min or 47 degrees C for 3 min, which corresponded to surviving fractions of 25, 0.6 and 0.8%, respectively, but not at 47 degrees C for 20 min, indicating that mild heat shock induced apoptosis. 2-deoxyglucose (2DG) and 2,4-dinitrophenol (DNP) increased the sensitivity to heat and affected the mode of cell death. Cells treated with 2DG and DNP (2DG/DNP) were heated at 42 degrees C for 20 min, and then incubated at 37 degrees C for up to 2h in the presence or absence of 2DG/DNP. In the absence of 2DG/DNP, the cellular ATP level recovered to 76% of the control level and DNA ladder formation was observed, whereas in the presence of 2DG/DNP, the cellular ATP level was further decreased (3-7% of the control) and no DNA fragmentation was detected. These results suggest that the inhibition of ATP synthesis is closely associated with the enhancement of sensitivity to heat and that ATP is required for the induction of apoptosis.
PMID: 12079587 [PubMed - indexed for MEDLINE]
2,4-Dinitrophenol pharmacologically promotes retinal detachment in rabbits.
2,4-Dinitrophenol pharmacologically promotes retinal detachment in rabbits.
Deen A, Benner JD, Kramer T, Bassichis-Saland K, Purohit A, Steidl SM.
Department of Ophthalmology, University of Maryland, Baltimore, USA.
PURPOSE: The most difficult and unpredictable step of macular translocation surgery is creating the retinal detachment. The authors evaluated the efficacy of 2,4-dinitrophenol (2,4-DNP) to promote retinal detachment in the rabbit. METHODS: A vitrectomy was performed in each eye of a Dutch-belted rabbit. One eye was injected with 0.1 cc of a 5 mmol/L 2,4-DNP, the other eye with 0.1 cc of BSS+. After 30 minutes, the minimum aspiration pressure required to visibly elevate the retina was recorded. Four nonvitrectomized eyes received an intravitreal injection of either 0.1 cc of BSS+ or 5 mmol/L 2,4-DNP, and were enucleated and fixated for histopathologic examination. RESULTS: In the 12 masked eyes, the mean aspiration pressure decreased from 217 +/- 20 mmHg in the six BSS+ eyes to 117 +/- 20 mmHg in the six 2,4-DNP treated eyes (P = 0.0022). A retinal detachment was present in three of six masked and two of two unmasked 2,4-DNP treated eyes and none of eight BSS+ treated eyes. There was no short-term toxicity to the retina at the light microscope level. CONCLUSION: Intravitreal injection of 2,4-DNP reduced the retinal adhesive force by over 50% when compared to the BSS+ treated control eyes, without any short-term retinal toxicity.
PMID: 15805912 [PubMed - indexed for MEDLINE]
Probing actomyosin interactions with 2,4-dinitrophenol.
Probing actomyosin interactions with 2,4-dinitrophenol.
Ribeiro AS, Salerno VP, Sorenson M.
Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 RJ, Brazil.
Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.