Fibroblast growth factor-16 is a growth factor for embryonic brown adipocytes. (57/1057)

In rat embryos, fibroblast growth factor (FGF)-16 is predominantly expressed in brown adipose tissue. To elucidate the role of FGF-16, we examined the expression of FGF-16 mRNA in rat embryonic brown adipose tissue at different developmental stages by Northern blotting analysis and in situ hybridization. FGF-16 mRNA was expressed abundantly in brown adipose tissue during embryonic day 17. 5, embryonic days 17.5-19.5, and thereafter at lower levels into the neonatal period. The expression profile of FGF-16 mRNA well corresponds to the proliferative profile of embryonic brown adipose tissue reported. We also examined the mitogenic activity of recombinant rat FGF-16 for primary brown adipocytes prepared from rat embryonic brown adipose tissue. FGF-16 showed significant mitogenic activity for primary brown adipocytes. The mitogenic activity was found to be exerted by binding and activating FGF receptor-4 in the brown adipose tissue. As a great induction of proliferation of rat brown adipose tissue during cold acclimation was reported, we also examined the expression of FGF-16 mRNA in the brown adipose tissue during cold acclimation by Northern blotting analysis. The expression of FGF-16 mRNA was not increased, but rather decreased. The expression profile of FGF-16 mRNA and the mitogenic activity of FGF-16 reported here indicate that FGF-16 is a unique growth factor involved in proliferation of embryonic brown adipose tissue.  (+info)

Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue. (58/1057)

Calcium-dependent uncoupling of liver mitochondrial oxidative phosphorylation by a non-metabolizable long chain fatty acyl analogue was compared with uncoupling induced by in vivo thyroid hormone treatment. beta,beta'-Methyl-substituted hexadecane alpha, omega-dioic acid (Medica 16) is reported here to induce a saturable 20-30% decrease in liver mitochondrial DeltaPsi, DeltapH and protonmotive force which proceeds in the presence of added Ca(2+) to cyclosporin A-sensitive mitochondrial permeabilization. Ca(2+)-dependent uncoupling by Medica 16 was accompanied by atractylate-enhanced, bongkrekic-inhibited activation of mitochondrial Ca(2+) efflux. The direct mitochondrial effect exerted in vitro by Medica 16 is similar to that induced by in vivo thyroid hormone treatment. Hence, the thyromimetic protonophoric activity of Medica 16 and the uncoupling activity of TH converge onto components of the mitochondrial permeabilization transition pore.  (+info)

Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP. (59/1057)

Ca(2+) transport and UTP hydrolysis catalyzed by sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle was studied. A passive Ca(2+) load inside microsomal vesicles clearly decreased the net uptake rate and the final accumulation of Ca(2+) but not the UTP hydrolysis rate, causing energy uncoupling. In the absence of passive leak, the Ca(2+)/P(i) coupling ratio was 0.7-0.8. UTP hydrolysis did not maintain a rapid component of Ca(2+) exchange between the cytoplasmic and lumenal compartments as occurs with ATP. The uncoupling process in the presence of UTP is associated with: (i) the absence of a steady state accumulation of ADP-insensitive phosphoenzyme; (ii) the cytoplasmic dissociation of Ca(2+) bound to the ADP-sensitive phosphoenzyme; and (iii) the absence of enzyme inhibition by cyclopiazonic acid. All these characteristics confirm the lack of enzyme conformations with low Ca(2+) affinity and point to the existence of an uncoupling mechanism mediated by a phosphorylated form of the enzyme. Suboptimal coupling values can be explained in molecular terms by the proposed functional model.  (+info)

A novel particulate form of Ca(2+)/calmodulin-dependent [correction of Ca(2+)/CaMKII-dependent] protein kinase II in neurons. (60/1057)

Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of approximately 100 nm and, in distinction to postsynaptic densities, do not immunolabel for PSD-95. These structures were purified to near homogeneity by extraction with the detergent N-lauryl sarcosinate. Biochemical analysis revealed that CaMKII accounts for virtually all of the protein in the purified preparation, suggesting that spherical structures are clusters of self-associated CaMKII. Exposure of cultured hippocampal neurons to a mitochondrial uncoupler in glucose-free medium promotes the formation of numerous CaMKII-immunopositive structures identical in size and shape to the CaMKII clusters observed in subcellular fractions. Clustering of CaMKII would reduce its kinase function by preventing its access to fixed substrates. On the other hand, clustering would not affect the ability of the large cellular pool of CaMKII to act as a calmodulin sink, as demonstrated by the Ca(2+)-dependent binding of gold-conjugated calmodulin to CaMKII clusters. We propose that the observed clustering of CaMKII into spherical structures is a protective mechanism preventing excessive protein phosphorylation upon loss of Ca(2+) homeostasis, without compromising calmodulin regulation.  (+info)

Annexins V and XII alter the properties of planar lipid bilayers seen by conductance probes. (61/1057)

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to approximately 30% of its original value. Both negative lipid and approximately 30 microM Ca(2+) are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.  (+info)

Mg(2+) induces intermembrane electron transport by cytochrome c desorption in mitochondria with the ruptured outer membrane. (62/1057)

The mechanism of external NADH oxidation in rat liver mitochondria is not clear yet, but it evidently includes the intermembrane electron transport (IMET). We showed that Mg(2+) significantly stimulated IMET insensitive to rotenone-antimycin A-myxothiazol in mitochondria with the hypotonically damaged outer membrane, even in the absence of exogenous cytochrome c. This effect was more specific than simply ionic strength increase. Direct evidence for cytochrome c desorption from mitochondrial membranes under 4 mM Mg(2+) influence was obtained to explain the stimulatory effect of Mg(2+) on external NADH oxidation that was coupled to the inner membrane potential generation. Obtained data suggest that cytochrome b(5) is normally oriented towards the cytosol in the outer membrane, and can be accessible for endogenous cytochrome c reduction only through the outer membrane rupture or permeabilization, to activate external NADH oxidation.  (+info)

Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat. (63/1057)

BACKGROUND: The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation. AIMS: To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat. METHODS: We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control. RESULTS: Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin. CONCLUSIONS: These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.  (+info)

Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. (64/1057)

The effect of inhibiting ATP production via oxidative phosphorylation during pericompaction of in vitro produced bovine embryos was investigated. This was achieved by: (i) varying the atmospheric O2 concentration (0, 1, 2, 4 and 7%); (ii) addition of oxidative phosphorylation inhibitors, NaN3 and antimycin A; and (iii) addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation from electron transport. The development of embryos under various O2 concentrations from day 5 to day 7 of development indicated that an optimal concentration occurred at about 2%. Addition of NaN3 revealed that doses above 100 mumol l-1 were toxic to embryo development, but that concentrations of 5-10 mumol l-1 stimulated embryo development by 10-25%. A similar result was observed after addition of 2,4-dinitrophenol, whereas antimycin A was inhibitory at doses as low as 1 mumol l-1. At concentrations of NaN3 or 2,4-dinitrophenol that stimulated embryo development, the number of cells of the resulting blastocysts was also significantly increased. Addition of NaN3 from day 1 of development inhibited subsequent development. Metabolic data of NaN3-treated embryos revealed that O2 uptake was significantly lower at inhibitory doses (100 mumol l-1). A significant (P < 0.05) log linear increase in glucose uptake was measured between the three concentrations of NaN3 (0, 10 and 100 mumol l-1). These results demonstrate that ATP production via oxidative phosphorylation is essential for bovine embryo development in vitro. However, transient (subacute) inhibition appears to be beneficial to embryo development and the number of cells, perhaps by creating a more favourable intracellular environment.  (+info)