Rab6A GTPase contributes to phenotypic modulation in pulmonary artery smooth muscle cells under hypoxia

Previous studies have demonstrated that hypoxia can induce phenotypic modulation of pulmonary smooth muscle cells; however, the mechanisms remain unclear. The present study aimed to investigate the effect of the GTPase Rab6A-mediated phenotypic modulation and other activities of rat pulmonary artery smooth muscle cells (RPASMCs). We revealed that Rab6A was induced by hypoxia (1% O₂) and was involved in a hypoxia-induced phenotypic switch and endoplasmic reticulum stress (ERS) in RPASMCs. After 48 hours of hypoxia, the expression of the phenotype marker protein smooth muscle actin was downregulated and vimentin (VIM) expression was upregulated. Rab6A was upregulated after 48 hours of hypoxia, and the level of glucose-regulated protein, 78 kDa (GRP78) after 12 hours of hypoxic stimulation was also increased. After transfection with a Rab6A short interfering RNA under hypoxic conditions, the expression levels of GRP78 and VIM in RPASMCs were downregulated. Overall, hypoxia-induced RPASMCs to undergo ERS followed by phenotypic transformation. Rab6A is involved in this hypoxia-induced phenotypic modulation and ERS in RPASMCs.     

Inorganic polyphosphates and phosphohydrolases inHalobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 μmol P_i/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content ofH. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities ofH. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase ofH. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective K_m of this enzyme with respect to pyrophosphate was 115 μM.     

The influence of lipoic acid on adriamycin induced nephrotoxicity in rats

Adriamycin, which is widely used in the treatment of various neoplastic conditions, exerts toxic effects in several organs. Adriamycin nephrotoxicity has been recently documented in a variety of animal species. The present study was designed to investigate the effect of lipoic acid on the nephrotoxic potential of adriamycin. The study was carried out with adult male albino rats of Wistar strain. Test animals were divided into four groups of six rats each as follows: Group I (control) received only normal saline throughout the course of the experiment. Group II (ADR) received intravenous injections of adriamycin through the tail vein (1 mg kg^–1 body wt day^–1) once a week for a period of 12 weeks. Group III (LA) received lipoic acid (35 mg kg^–1 body wt day^–1) intraperitoneally once a week for a period of 12 weeks. Group IV (ADR + LA) received a single injection of lipoic acid intraperitoneally 24 h prior to the administration of adriamycin through the tail vein once a week for a period of 12 weeks. Intravenous injections of adriamycin resulted in decreased activities of the glycolytic enzymes; hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase in the rat renal tissue. The gluconeogenic enzymes; glucose-6-phosphatase and fructose-1,6-diphosphatase, showed a decline in their activities on adriamycin administration. The transmembrane enzymes namely the Na⁺,K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brush-border enzyme alkaline phosphatase also showed a decrease in their activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush-border membrane damage. Decreased activities of the TCA cycle enzymes isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, suggest a loss in mitochondrial function and integrity. Nephrotoxicity was evident from the increased excretions of N-acetyl--D-glucosaminidase and -glutamyl transferase in the urine of adriamycin administered rats. These biochemical disturbances were effectively counteracted on pretreatment with lipoic acid, which brought about an increase in the activities of glycolytic enzymes, ATPases and the TCA cycle enzymes. On the other hand, the gluconeogenic enzymes showed a further decrease in their activities on lipoic acid pretreatment. LA pretreatment also restored the activities of the urinary enzymes to normal. These observations shed light on the nephroprotective action of lipoic acid rendered against experimental aminoglycoside toxicity.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg²⁺ requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.