Biomedical subjects

I Holen

Publications and source records attributed to I Holen.

26 records · Page 2Linked to original sources

PubMed1993

Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors.

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.

Animals↗

PubMed1992

Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity.

The protein phosphatase inhibitor okadaic acid suppressed autophagy completely in isolated rat hepatocytes, as measured by the sequestration of electroinjected [3H]raffinose into sedimentable autophagic vacuoles. Okadaic acid was effectively antagonized by the general protein kinase inhibitors K-252a and KT-5926, the calmodulin antagonist W-7, and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK-II). These inhibitors also antagonized a cytoskeleton-disruptive effect of okadaic acid, manifested as the disintegration of cell corpses after breakage of the plasma membrane. CaMK-II, or a closely related enzyme, would thus seem to play a role in the control of autophagy as well as in the control of cytoskeletal organization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Hepatocytic autophagy.

Autophagy is a non-selective bulk process for degradation of cytoplasm, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lifes. The initial autophagic sequestration step is subject to feedback inhibition by amino acids, an effect which is potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive, alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy in a differential (ligand-dependent) manner, and that amphisomes may play a central role as collecting stations for material destined for lysosomal degradation.

Amino Acids↗

PubMed1991

Role of cyclic nucleotides in the control of hepatic autophagy.

Using isolated hepatocytes as a model system we have investigated whether the cyclic nucleotides cAMP and cGMP are involved in the regulation of the autophagic process. The dibutyryl-cyclic nucleotide analogues db-cAMP and db-cGMP both inhibited autophagic sequestration, suggesting that cAMP and cGMP may be of significance for this step. The adenylate cyclase stimulator deacetyl-forskolin both raised the level of intracellular cAMP and reduced sequestration markedly. In contrast, the guanylate cyclase stimulating agent atriopeptin did not affect sequestration although, it effectively elevated, the level of cGMP. Several inhibitors of cyclic nucleotide phosphodiesterases strongly suppressed autophagy and elevated the level of both cAMP and cGMP. However, one inhibitor, milrinone, raised the cAMP level 3-4 x while having no significant effect on cGMP. These results suggest that cAMP may be involved in the control of hepatic autophagy, whereas the role of cGMP, if any, remains unclear.

Animals↗

PubMed1991

Effects of adrenergic agonists and antagonists on autophagic activity in isolated rat liver cells.

The effect of various adrenergic agonists on autophagic sequestration--measured as the transfer of electroinjected [3H]raffinose from cytosol to vacuoles of the autophagic pathway--was investigated. Epinephrine and other agonists with alpha-effects inhibited sequestration through a specific alpha 1-adrenergic, i.e. prazosin-sensitive, mechanism. The beta-adrenergic agonist isoproterenol also inhibited sequestration, but by a non-beta-specific (propranolol-insensitive) mechanism. All sequestration-inhibitory agents suppressed overall autophagic-lysosomal proteolysis. The inhibitory action of the adrenergic agonists on protein metabolism was not specific to the autophagic pathway since protein synthesis was suppressed as well. However, intracellular levels of ATP were not adversely affected, ruling out the possibility that the agonists might be generally cytotoxic.

Adenosine Triphosphate↗

PubMed1990

Morphological transformation and catalase activity of Syrian hamster embryo cells treated with hepatic peroxisome proliferators, TPA and nickel sulphate.

The abilities of the hepatic peroxisome proliferators (HPPs) clofibrate, di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)-phthalate (MEHP), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and tiadenol to induce morphological transformation and to increase the catalase activity of Syrian hamster embryo (SHE) cells were studied. DEHP, MEHP, clofibrate and tiadenol induced morphological transformation of SHE cells and increased the catalase activity. DEHP was more potent than clofibrate and tiadenol in both inducing catalase and morphological transformation, while MEHP seemed more potent than DEHP in inducing catalase, but not morphological transformation, 2,4,5-T and 2,4-D did not induce morphological transformation, but 2,4,5-T was more potent than clofibrate in increasing the catalase activity. These results show that several HPPs induce morphological transformation of SHE cells and an increase in the catalase activity. There is, however, no direct connection between these two parameters, as seen from the results of 2,4,5-T. The tumor promoter TPA, and the metal salt nickel sulphate, induced morphological transformation of SHE cells without any appreciable increase in the catalase activity. These results further corroborate the dissociation between induction of morphological transformation and the increase in catalase activity.

2,4,5-Trichlorophenoxyacetic Acid↗

PubMed1990

Effects of dinitrotoluenes on morphological cell transformation and intercellular communication in Syrian hamster embryo cells.

The effects of four isomers of dinitrotoluene (DNT) and technical DNT (a mixture of DNT isomers and other compounds, with 2,4-DNT as the major constituent) were studied in two short-term in vitro assays. None of the isomers or technical DNT induced an increase in morphological transformation of Syrian hamster embryo (SHE) cells. Four DNT metabolites (2,4-diaminotoluene, 2-amino-4-nitrotoluene, 2-amino-6-nitrotoluene, and 2,4-dinitobenzoic acid), representing different stages in reduction or oxidation of DNT isomers, were also negative for induction of morphological transformation. The DNT isomers were tested in an intercellular communication assay based on dye transfer. 2,4-DNT, 2,6-DNT, and technical DNT inhibited intercellular communication in the SHE cell line BPNi at toxic concentrations. This may be reminiscent of in vivo data showing promoting activity of these compound. 2,3-DNT and 3,4-DNT did not inhibit communication.

Animals↗

PubMed1990

Non-selective autophagy.

Autophagy is the major process by which cells degrade their own cytoplasm. Autophagy begins with the sequestration of a portion of the cytoplasm by a membraneous organelle called a phagophore. The resulting vacuole (autophagosome) can fuse with an endocytic vacuole to form am amphisome, which subsequently fuses with a lysosome to have its mixed autophagic/endocytic content degraded by lysosomal enzymes. Autophagy is a non-selective bulk process as indicated by the fact that hepatocytic cytosol enzymes with widely different half-lives are sequestered at the same rate. Regulation of autophagy is exerted at the sequestration step by amino acids, purines, ATP-depleting metabolites, cyclic nucleotides, phosphorylation, and hormones like insulin, glucagon and alpha-adrenergic agonists.

Amino Acids↗