Certain aspects of uncoupling due to mitochondrial uncoupling proteins in vitro and in vivo.

Thermogenic uncoupling has been proven only for UCP1 in brown adipose tissue. All other isoforms of UCPs are potentially acting in suppression of mitochondrial reactive oxygen species (ROS) production. In this contribution we show that BAT mitochondria can be uncoupled by lauric acid in the range of approximately 100 nM when endogenous fatty acids are combusted by carnitine cycle and beta-oxidation is properly separated from the uncoupling effect. Respiration increased up to 3 times when related to the lowest fatty acid content (BSA present plus carnitine cycle). We also illustrated that any effect leading to more coupled states leads to enhanced H2O2 generation and any effect resulting in uncoupling gives reduced H2O2 generation in BAT mitochondria. Finally, we report doubling of plant UCP transcript in cells as well as amount of protein detected by 3H-GTP-binding sites in mitochondria of shoots and roots of maize seedlings subjected to the salt stress.

Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction.

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.

Activating omega-6 polyunsaturated fatty acids and inhibitory purine nucleotides are high affinity ligands for novel mitochondrial uncoupling proteins UCP2 and UCP3.

UCP2 (the lowest Km values: 20 and 29 microm, respectively) for omega-6 polyunsaturated FAs (PUFAs), all-cis-8,11,14-eicosatrienoic and all-cis-6,9,12-octadecatrienoic acids, which are also the most potent agonists of the nuclear PPARbeta receptor in the activation of UCP2 transcription. omega-3 PUFA, cis-5,8,11,14,17-eicosapentaenoic acid had lower affinity (Km, 50 microm), although as an omega-6 PUFA, arachidonic acid exhibited the same low affinity as lauric acid (Km, approximately 200 microm). These findings suggest a possible dual role of some PUFAs in activating both UCPn expression and uncoupling activity. UCP2 (UCP3)-dependent H+ translocation activated by all tested FAs was inhibited by purine nucleotides with apparent affinity to UCP2 (reciprocal Ki) decreasing in order: ADP > ATP approximately GTP > GDP >> AMP. Also [3H]GTP ([3H]ATP) binding to isolated Escherichia coli (Kd, approximately 5 microm) or yeast-expressed UCP2 (Kd, approximately 1.5 microm) or UCP3 exhibited high affinity, similar to UCP1. The estimated number of [3H]GTP high affinity (Kd, <0.4 microm) binding sites was (in pmol/mg of protein) 182 in lung mitochondria, 74 in kidney, 28 in skeletal muscle, and approximately 20 in liver mitochondria. We conclude that purine nucleotides must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.

Substitutional mutations in the uncoupling protein-specific sequences of mitochondrial uncoupling protein UCP1 lead to the reduction of fatty acid-induced H+ uniport.

Mutants were constructed for mitochondrial uncoupling protein UCP1, with single or multiple substitutions within or nearby the UCP-signatures located in the first alpha-helix and second matrix-segment, using the QuickChange site directed mutagenesis protocol (Stratagene), and were assayed fluorometrically for kinetics of fatty acid (FA)-induced H+ uniport and for Cl- uniport. Their ability to bind 3H-GTP was also evaluated. The wild type UCP1 was associated with the FA-induced H+ uniport proportional to the added protein with a Km for lauric acid of 43 micro M and Vmax of 18 micro molmin(-1)(mg protein)(-1). Neutralization of Arg152 (in the second matrix-segment UCP-signature) led to approximately 50% reduction of FA affinity (reciprocal Km) and of Vmax for FA-induced H+ uniport. Halved FA affinity and 70% reduction of Vmax was found for the double His substitution outside the signature (H145L and H147L mutant). Neutralization of Asp27 in the first alpha-helix UCP-signature (D27V mutant) resulted in 75% reduction of FA affinity and approximately 50% reduction of Vmax, whereas the triple C24A and D27V and T30A mutant was fully non-functional (Vmax reduced by 90%). Interestingly, the T30A mutant exhibited only the approximately 50% reduced FA affinity but not Vmax. Cl- uniport and 3H-GTP binding were preserved in all studied mutants. We conclude that amino acid residues of the first alpha-helix UCP signature may be required to hold the intact UCP1 transport conformation. This could be valid also for the positive charge of Arg152 (second matrix-segment UCP signature), which may alternatively mediate FA interaction with the native protein.

Experimental photodynamic therapy with MESO-tetrakisphenylporphyrin (TPP) in liposomes leads to disintegration of human amelanotic melanoma implanted to nude mice.

Liposomal meso-tetrakis-phenylporphyrin (TPP) was tested for photodynamic therapy (PDT) of human amelanotic melanomas implanted in nude mice. After intratumoural TPP application (15 mg x kg(-1)) followed by PDT lamp irradiation (600-700 nm, 635 nm peak), tumours retained their original volume up to the 23rd day post-PDT, whereas volumes increased 6 times in controls. PDT with intravenously (i.v.) administered liposomal (3.2 mg x kg(-1)) TPP mostly disintegrated tumours to zero volumes. Melanoma remissions were accompanied by tumour surface necroses and were documented by the appearance of nontumourous cells with nonpycnotic nuclei. Spatial arrangement of capillaries in remissing tumour was the same as in healthy surrounding tissue. Lower TPP doses (1, 0.3 and 0.1 mg x kg(-1)) were more or equally efficient than hydrophilic TPPS(4) (3.2 mg x kg(-1), i.e., sulfonated TPP), i.v. administered also in liposomes. Liposomal TPPS(4) only delayed the onset of subsequent tumour growth. Commercial Photosan 3 disintegrated tumours only in doses of approx. 7.5 mg x kg(-1); in lower doses it was less efficient than TPPS(4). The second PDT cycle (3.2 mg x kg(-1) TPP or 7.5 mg x kg(-1) Photosan 3), performed in a few unsuccessfully cured mice, predominantly led again to tumour remissions. Since the measured TPP and TPPS(4) content in melanomas was similar, these results demonstrate the advantage of PDT with a hydrophobic photosensitizer such as TPP. Photophysical properties of TPP and TPPS(4) are equal, but TPP has probably more favorable intracellular distribution, as documented by our studies, which leads to more efficient PDT. Consequently, liposomal TPP is suggested as a potentially suitable efficient preparation for PDT.