Formation of the mitochondrial membrane potential (DY) depends on flux of respiratory substrates, ATP, ADP, and Pi through voltage-dependent anion channels (VDAC). As tubulin promotes single-channel closure of VDAC, we hypothesized that tubulin is a dynamic regulator of DY, which in cultured cancer cells was assessed by confocal microscopy of the potential-indicating fluorophore tetramethylrhodamine methylester (TMRM). Microtubule destabilizers, rotenone, colchicine, and nocodazole, and the microtubule stabilizer paclitaxel increased and decreased cellular free tubulin, respectively, and in parallel decreased and increased DY. Protein kinase A (PKA) activation by cAMP analogues and glycogen synthase kinase 3b (GSK-3b) inhibition decreased DY, whereas PKA inhibition hyperpolarized, consistent with reports that PKA and GSK-3b decrease and increase VDAC conductance, respectively. Plasma membrane potential assessed by DiBAC 4 (3) was not altered by any of the treatments. We propose that inhibition of VDAC by free tubulin limits mitochondrial metabolism in cancer cells.

Background: Metabolites generating mitochondrial membrane potential (⌬⌿) enter through voltage-dependent anion channels (VDAC). Results: VDAC3 contributed to ⌬⌿ formation more than VDAC1/2. VDAC3 knockdown decreased ATP and NADH/NAD ϩ .

Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT was not mediating mitochondrial ATP/ADP exchange in cancer cells [corrected]. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.

cardiac fibers also have diffusion restrictions. This is surprising because rainbow trout cardiomyocytes are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. However, results from fibers may be affected by incomplete separation of the cells. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized isolated cardiomyocytes and isolated mitochondria from rainbow trout heart. We developed a new solution specific for trout cardiomyocytes, where they retained their shape and showed stable steady state respiration rates. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. This suggests that intracellular diffusion of ADP is indeed restricted in trout cardiomyocytes. The difference between fibers and cardiomyocytes suggest that results from trout cardiac fibers were affected by incomplete separation of the cells. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respiration. These results from rainbow trout cardiomyocytes are similar to those from neonatal mammalian cardiomyocytes. Thus, it seems that metabolic regulation is related to cardiac performance. It is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in low-performance hearts. Next step will be to identify the contribution of mitochondrial outer membrane and cytosolic factors in intracellular diffusion restriction.

Ultraviolet sensitivity of two different species of Anabaena, Anabaena doliolum and Anabaena sp. was studied. These two species differ in their pigment composition; A . doliolum showed predominance of phycocyanin whereas Anabaena sp. showed predominance of phycoerythrin pigments. Survival of the two species were different after exposure to different doses of UV (A,,, 254 nm) irradiation; Anabaena sp. was more resistant to higher UV dose than A . doliolum. In both species the UV induced damage was partially repaired by white fluorescent light. A . doliolum showed a lower rate of photorecovery than Anabaena sp.. Higher UV dose (20 min) induced production of a non-nitrogen fixing strain of A. doliolum and a pigment mutant lacking phycoerythrin of Anabaena sp.Ultraviolet-induced damage and photoreactivation studies on cyanobacteria was pioneered by VAN BAALEN (1965. Since then several reports appeared on the UV sensitivity, dose modification and photoreactivation of a variety of cyanobacteria (KUMAR 1970, KASHYAP 1978, SINGH 1978. Characteristics of ultraviolet resistant and wild type strains of various cyanobacteria have been compared (KUMAR 1963, SINGH and TIWARI 1969, MISHRA and TIWARI 1986. Pigment mutants induced by UV ( VAN BAALEN 1965, MISHRA and TIWARI 1989), nitromethyl urea (SHESTAKOV and ZHENVER 1968 and N-methyl-NNitro-N-nitroso guanidine (ASATO and FOLSOME 1969) have been observed in a few species of cyanobacteria, but have not been widely investigated (STEVANS and MYERS 1976). The present work deals with the survival of two species of Anabaena after irradiation with various doses of UV. The generation of different type of mutants in different species of Anabaena by high UV dose was observed. Materials and methodsTwo different species of the filamentous nitrogen-fixing cyanobacterium Anabaena, (i) bluish green Anabaena doliolum, isolated from local rice fields (TRIPATHY et al. 1990) and (ii) brownish Anabaena sp. isolated from a distillery effluent polluted area (SAHU and ADHIKARY 1982) were used as the experimental material. The organisms were grown in the culture room in ALLEN and ARNON'S nitrogen free medium (1955) at 26 f 1 "C under continuous light (7.5 W/m2 at the surface of culture vessels) from daylight fluorescent tubes. The cells of both the organisms growing exponentially in the basal medium were concentrated and washed by repeated centrifugation with sterilized double distilled water. The cells were suspended in the same and brought into homogeneous suspension by a glass tissue homogenizer. 15 ml of this suspension (density = 0.05 absorbance at 760 nm) was poured into a 75 mm petridish. The suspension was then exposed to UV radiation under a general Electric germicidal lamp (American Ultraviolet Co., USA) emitting in the wavelength range of 325-375 nm with its main output at 254 nm and the radiation dose of about 85 ergs mm-' s-' at a distance of 22 cm. During irradiation with UV in the absence of white light, the suspension was constantly stirred by a magnetic 428 J. PATNAIK et al.stirrer an...