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Biomedical subjects

B Grinde

Publications and source records attributed to B Grinde.

68 records · Page 4Linked to original sources
PubMed1983

Effects of prostaglandins and divalent cations on cAMP production in isolated rat hepatocytes.

In intact hepatocytes, prostaglandin E2 (PGE2) inhibits up to 60% of the cyclic AMP (cAMP) formation in response to glucagon. PGE2 was found also to inhibit cAMP production in response to Mn2+, and Mn2+ counteracted the effect of PGE2 on the response to glucagon. However, when added to cells with ruptured plasma membranes, PGE2 had no effect on cAMP production, even though the ruptured cells had an equally strong response to glucagon as did intact cells. Only when cells were ruptured in the presence of PGE2 and incubated as very dense suspensions did PGE2 inhibit a response to glucagon up to 20%. These results may be explained by assuming the existence of a cytosolic factor necessary for transmitting the inhibitory effect of PGE2. The role of divalent cations in cAMP formation in intact cells was investigated. After extraction of cations with ethylenediamine-tetraacetic acid (EDTA), cAMP production in response to glucagon was reduced by more than 60%. Addition of Mn2+ restored cAMP formation completely, whereas Mg2+ was somewhat less effective, and Ca2+ could not restore any activity. Even low concentrations of Ca2+ appeared under certain conditions to repress adenylate cyclase activity.

Animals↗
PubMed1983

Effect of carboxylic ionophores on lysosomal protein degradation in rat hepatocytes.

Three different carboxylic ionophores (monensin, nigericin and lasalocid) were each found capable of causing a relatively complete block of the lysosomal (i.e., methylamine-sensitive) protein degradation in isolated rat hepatocytes. Monensin was found to be the most specific in action, as it had no effect on non-lysosomal degradation and did not bring about any substantial inhibition of protein synthesis. Morphometric examination of electron micrographs revealed that monensin causes an accumulation of early forms of autophagic vacuoles and blocks the swelling of lysosomes seen in the presence of methylamine. The results indicate that monensin inhibits lysosomal protein degradation by affecting lysosomal pH.

Animals↗
PubMed1982

Selective inhibition of lysosomal protein degradation by the thiol proteinase inhibitors E-64, Ep-459 and Ep-457 in isolated rat hepatocytes.

The effects on protein degradation of the thiol proteinase inhibitor E-64 of fungal original, and its two synthetic analogs Ep-459 and Ep-475, were examined, using isolated rat hepatocytes. All three inhibitors were found to act selectively on lysosomal protein degradation. i.e., their effects were not additive to the lysosomotropic weak base propylamine. Such weak bases appear to be relatively complete and selective inhibitors of lysosomal protein degradation. Ep-475 and E-64 were found to be the most potent of the three, inhibiting as much as 50% of the total degradation (i.e., approx. 70% of the lysosomal degradation) at concentrations at which they did not disturb protein synthesis. Their lack of additivity to the lysosomotropic weak base propylamine further testifies to the usefulness of weak bases differentiating between lysosomal and non-lysosomal protein degradation.

Animals↗
PubMed1982

Effects of protein-degradation inhibitors on the inactivation of tyrosine aminotransferase, tryptophan oxygenase and benzopyrene hydroxylase in isolated rat hepatocytes.

The following three potent inhibitors of hepatocytic proteolysis were investigated to see if they would inhibit the intracellular inactivation of enzymes: chymostatin and leupeptin (proteinase inhibitors) and methylamine (a lysosomotropic weak base). Chymostatin inhibited the inactivation of two of the three enzymes tested: tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (tryptophan 2,3-dioxygenase, EC 1.13.11.11). Leupeptin had no effect on any of the enzymes, whereas methylamine had only a weak inhibitory effect on tyrosine aminotransferase inactivation. Apparently proteolytic cleavage (probably by a non-lysosomal proteinase, since only chymostatin is effective) is involved in the inactivation of tyrosine aminotransferase and tryptophan oxygenase. The third enzyme, benzopyrene hydroxylase (flavoprotein-linked mono-oxygenase, EC 1.14.14.1), is probably inactivated by a non-proteolytic mechanism.

Animals↗
PubMed1981

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes.

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help to elucidate the biochemical mechanisms for control of this important process.

Amino Acids↗
PubMed1981

Effects of insulin and anchorage on hepatocytic protein metabolism and amino acid transport.

Insulin partially inhibits endogenous protein degradation in isolated hepatocytes. The inhibition seems to specifically affect the lysosomal pathway of degradation, since it is not additive to the effects of lysosome inhibitors such as propylamine and leupeptin. The insulin effect is potentiated by intermediate concentrations of amino acids, but is largely abolished at high amino acid concentrations which suppress degradation maximally, suggesting that the hormone may exert its effect indirectly by acting upon the more basal amino acid control mechanism. Glucagon, which stimulates protein degradation, similarly displays its effect only in the presence of intermediate amino acid concentrations. The insulin inhibition is not affected by the aminotransferase inhibitor, aminooxyacetate, indicating that it is not due to interference with amino acid metabolism. Protein synthesis furthermore does not seem to be required, since a significant insulin effect can be seen in the presence of the protein synthesis inhibitor, cycloheximide. The issue is, however, complicated by the fact that cycloheximide itself inhibits protein degradation to approximately the same extent as does insulin. Insulin stimulates uptake of the amino acid alpha-aminoisobutyrate (AIB), but not the uptake of valine, indicating a specific stimulation of 'A'-type transport. Cycloheximide similarly stimulates AIB uptake, without completely obfuscating the transport effect of insulin. Neither protein synthesis, protein degradation, amino acid transport, nor the effects of insulin were affected by cell-to-substratum anchorage (attachment and spreading) in any detectable way.

Amino Acids↗
PubMed1981

Role of microtubuli in the lysosomal degradation of endogenous and exogenous protein in isolated rat hepatocytes.

Colchicine and vinblastine, two well known inhibitors of microtubular function, inhibited endogenous protein degradation as well as protein secretion and the degradation of an exogenous, internalized protein (asialo fetuin), in isolated rat hepatocytes. The inhibitors appeared to selectively affect the lysosomal pathway of degradation, since their effects were not additive to that of ammonia, a lysosomotropic degradation inhibitor.

Ammonium Chloride↗
PubMed1981

Effects of amino acids and amino acid analogues on lysosomal protein degradation in isolated rat hepatocytes.

An amino acid mixture, specially designed to improve the protein balance in isolated hepatocytes, inhibited lysosomal (propylamine-sensitive) degradation of endogenous proteins by 80-90%. The amino acids had no effect on the degradation of the endocytosed protein asialofetuin, the conclusion being that amino acids are involved in the regulation of autophagy. Furthermore, the effect of two amino acids was tested individually, at high concentrations, in an electron microscopic analysis. Leucine caused a 79% decrease in the early forms of autophagic vacuoles, while asparagine had no effect. Leucine appears to be important in the regulation of the bulk autophagy observable in the electron microscope but this type of autophagy possibly contributes only about one-half of the total amount of protein degraded in lysosomes, since leucine only inhibits some 30% of the lysosomal protein degradation. Certain branched-chain amino acids related to leucine inhibited lysosomal protein degradation to the same extent as did leucine.

Amino Acids↗
PubMed1981

Inhibitors and pathways of hepatocytic protein degradation.

On the basis of experiments using amino acids and various inhibitors (lysosomotropic amines, leupeptin, chymostatin, vanadate, vinblastine, anoxia, methylaminopurines), five different modes of endogenous protein degradation in isolated rat hepatocytes can be distinguished. The two non-lysosomal (amine-resistant) mechanisms preferentially degrade relatively labile (short-lived) proteins: one of these mechanisms is energy-dependent and chymostatin-sensitive, the other is not. Of the three lysosomal (amine-sensitive) mechanisms, one--quantitatively minor--is amino acid-resistant and preferentially degrades labile proteins. The two amino acid-sensitive mechanisms each seen account for about one-half of the degradation of relatively stable (long-lived) proteins; one of them is suppressed by leucine and apparently corresponds to the formation of electron microscopically visible autophagosomes; the other may represent a different type of autophagy, inhibited by asparagine and glutamine. A new class of inhibitors, the purine derivatives (methylated 6-aminopurines, and 6-mercaptopurines) appear to specifically suppress autophagic/lysosomal protein degradation, and may help to further elucidate the mechanisms of autophagy.

Adenine↗
PubMed1980

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes.

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70---80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation. Leupeptin was found to inhibit lysosomal protein degradation by 80---85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstatin, bestatin and aprotinin (Trasylol) produced minor inhibitory effects (possibly on both degradation pathways), whereas bacitracin and soybean trypsin inhibitor were ineffective. Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway. The effects of the various inhibitors were additive to the extent expected on the basis of their known actions only sosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptin and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance. The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosomes. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.

Amines↗