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Publications and source records attributed to S Grisolía.
We have previously reported that inhibition of protein kinase C induces differentiation of neuroblastoma cells in culture. It is shown now that actinomycin D, a well known inhibitor of DNA synthesis, reduces selectively the content of protein kinase C and induces neuritogenesis in Neuro 2a cells in culture.
Rats were fed a standard diet (20% protein) or a protein-free diet for up to 65 days. After 20 days on the protein-free diet some rats were refed the standard diet. By the 20th day the rats fed the protein-free diet showed a blood ammonia level approximately 70% higher than controls and urea excretion decreased approximately 20-fold. At this time the liver acetylglutamate decreased to approximately one-fifth of the initial and control levels, returning to normal after 3 days of refeeding the standard diet, with a concomitant increase in urea excretion. The protein-deficient diet resulted in decreased activities of liver enzymes related to ammonia metabolism. All enzyme activities assayed returned to normal values rapidly upon refeeding the standard diet, except hepatic carbamylphosphate synthetase, glutamine synthetase, and glutaminase, which took approximately 1 month to return to control values. The findings presented here are consistent with the view that urea production is controlled, at least under certain conditions, by acetylglutamate, the physiological activator of carbamylphosphate synthetase.
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Carbamoyl phosphate synthetase (CPS) from rat liver is proteolitically inactivated at acid pH by broken lysosomes. Inactivation increases when lysosomes are previously incubated with inner mitochondrial membrane, although this mitochondrial fraction does not inactivate CPS 'per se'. The increased degradation is due to membrane factor(s), most probably mitochondrial proteinase(s), solubilized by lysosomal matrix proteinases, after incubation of the inner mitochondrial membrane fraction with broken lysosomes. This (these ) factor(s) degrade(s) CPS and other proteins in the absence of lysosomal proteinases or when these are inhibited by leupeptin, chymostatin and pepstatin. We have also tested the possible regulation of this degradation and found that ATP and, particularly, acetyl glutamate accelerate the degradation of CPS by the factor(s) liberated from the inner mitochondrial membrane.
Recent reports suggest that protein kinase C is involved in neural differentiation. We show that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), the more specific inhibitor of protein kinase C known, induces morphological and functional differentiation of neuro 2a cells, as indicated by the marked increase in the number of neurites/cell and in acetylcholinesterase activity. HA 1004 does not induce differentiation of neural cells. The induction of differentiation by H7 was very rapid; 3 h after addition of H7 the percentages of differentiated cells were 17, 33, 37, 55, and 75% for 17, 50, 85, 250, and 500 microM H7, respectively, while for controls it was 9%. When 500 microM H7 was added to the culture medium, protein kinase C was inhibited by 72 and 62% in cytosol and membrane, respectively. Also, acetylcholinesterase activity (a marker of functional differentiation) increased with time, reaching a 7-fold increase after 48 h.
The synthesis of urea in the liver is the main mechanism for the elimination of excess ammonia. Rapid stimulation of the synthesis of urea (e.g. by administration of carbamyl glutamate, the analog of the physiological activator of carbamyl phosphate synthetase I) protects animals given lethal doses of ammonia. Since ammonia enhances the activity of the urea cycle, we tested and show here that administration of small doses of ammonium acetate supresses the mortality induced by a series of repeated LD100 of ammonium acetate separated by one hour, when the first LD100 is injected i.p. starting from 30 min to 5 hours after the initial smaller dose of ammonium acetate. Under these conditions, the levels of ammonia in blood are elevated more than ten times, but in spite of the greater amount of ammonia administered, the ammonemia is much lower than in mice dying after a single LD100. The enhanced synthesis of urea observed is correlated with an increase in the intramitochondrial content of N-acetyl glutamate. These findings are of interest as far as the short-term regulation of urea cycle, the mechanism of ammonia toxicity and have clinical implications.
We have used a new approach to test the possible participation of lysosomes in the degradation of long-lived proteins. Rat liver lysosomal proteins were introduced, via multilamellar liposomes, into L-132 cells. Viability and protein synthesis were not impaired by this treatment. The liposomal content was released into the lysosomes of the cultured cells, as revealed by ferritin uptake and electron microscopy. Degradation rates of long-lived proteins increased with the uptake of lysosomal proteases. However, the increased protein degradation of chloroquine and leupeptin, in contrast to the inhibition by these reagents of the increased protein degradation of cells 'starved' of serum (step-down conditions). This approach opens a new way of investigating the degradation of intracellular proteins in cultured cells.
We have developed an animal model of hyperammonemia consisting of feeding rats a diet containing ammonium acetate. Using this model we have found that hyperammonemia induces tubulin synthesis in brain. Initially tubulin accumulates rapidly (28% after 2 days on diet) and continues increasing but at a slower rate, reaching a 50% increase after 100 days on the diet. The effect is reversible, rats fed the ammonium diet return to normal levels of tubulin two days after withdrawal of the ammonium diet. In contrast to the effect on brain, hyperammonemia did not increase tubulin content in liver or kidney. Moreover, the effect on brain is selective, with maximum increases of tubulin content in hippocampus, septum and reticular formation while other areas such as locus coeruleus and mammillary nucleus are not affected at all. The results presented show that the induction of tubulin is a consequence of an increased polymerization of microtubules which in turn is due to an altered phosphorylation of microtubule-associated proteins.
Rats were made hyperammonemic by feeding them a diet containing ammonium acetate. The tubulin content in their brain increased greater than or equal to 30% after 20 days on the diet. All the increase was found in polymerized tubulin; no increase in free tubulin was noted. When rats on the ammonium diet were then fed the standard diet, the tubulin increased slightly on the first day but decreased markedly on the second day, reaching control values on the third day. It should be noted that brain tubulin synthesis, was not reduced on the first day of feeding the standard diet but was markedly inhibited (to approximately 40% of control) on the second day, returning to control values on the third day. On the first day of refeeding there is a remarkable disassembly of microtubules with a large, proportional increase (approximately 50%) of free tubulin. Both free and polymerized tubulin levels returned to control values on the third day. These results indicate that in hyperammonemia changes in the degree of polymerization of tubulin preceded those in tubulin synthesis.
1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 microM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 microM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased approximately 7-fold after 48 hr with 500 microM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed.
The precursor of ornithine carbamoyltransferase can be transported in vitro into rat liver mitochondria using the postmitochondrial supernatant from rat liver, a more homologous medium than the commonly used rabbit reticulocyte lysate. The transport of the precursor in the case of reticulocyte lysate requires a standard translation mixture. In the presence of the postmitochondrial supernatant the same is true. However, when the components of the translation mixture were added individually to the postmitochondrial supernatant, it was found that spermidine or spermine, at physiological concentrations, sufficed for the transport of the precursor of ornithine carbamoyltransferase. The activity of the postmitochondrial supernatant was inactivated by trypsin and slightly decreased by RNase treatment; it was not lost by dialysis or by heating at 100 degrees C.
It is shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a specific inhibitor of protein kinase C, induces neuritogenesis in neuro 2a cells. The percentage of differentiated cells was 9%, 20%, 59% and 85% at 0, 17, 85 and 500 microM H7, respectively. The number of neurites cell increased 2-, 8- and 14-fold over the controls for 17, 85 and 500 microM H7, respectively. These results indicate that protein kinase C plays a key role in the control of differentiation of neural cells and that its specific inhibition may be of basic as well as of practical importance.
Carnitine deficiency associated with impairment of muscle function has been described in infants and young adults as a result of inherited or acquired disease but there are no reports on carnitine levels in healthy aging people. In this paper we show a marked reduction of carnitine and its derivatives in muscle, and of long-chain acyl carnitine in heart of 6-month-old mice, when compared to animals of 6 weeks of age. Analysis of muscle samples of healthy humans of different ages showed a drastic reduction of carnitine and acetyl carnitine in the older subjects with a strong reverse correlation between age and carnitine levels.
We have developed an animal model of hyperammonemia consisting of feeding rats a diet containing 20% (w/w) ammonium acetate. Ingestion of this diet markedly affects carcass composition, with a 46% reduction in lipid content. The ammonium diet alters levels of several key compounds involved in lipid metabolism. Long-chain acylcarnitine is increased in liver by approx. 60% while free carnitine and acetylcarnitine are unaffected. The hepatic content of acetyl-CoA increases by approx. 50%. The level of ketone bodies in blood increases by 32% but remains unchanged in liver. Our data indicate that hyperammonemia alters lipid metabolism and results in a significant decrease in body lipid content.
The polyamines spermidine, spermine and putrescine, by themselves, at physiological concentrations, induce the transport of the precursor of ornithine carbamoyltransferase into isolated rat liver mitochondria. The presence of polyamines in the transport medium results in the approach of both mitochondrial membranes, suggesting a possible role of these molecules in the transport of the precursor of ornithine carbamoyltransferase into mitochondria, by the formation and/or stabilization of mitochondrial structures involved in the transport system.
Rats were fed a standard diet or the standard diet supplemented with ammonium acetate (20% w/w) for up to 100 days. The effect of the ingestion of the high-ammonium diet on some aspects of nitrogen metabolism in rats was studied. Ammonia levels in blood increased approximately 3-fold; in brain, liver and muscle the increases were 36, 34 and 50%, respectively. Urea levels in blood and urea excretion increased approximately 2-fold. There was no increase of carbamyl phosphate synthase. Liver glutamine synthase activity increased by 58% and glutamate dehydrogenase by 40%, whereas glutaminase was not affected. Glutamine content in brain was twice that of controls. This new animal model to study hyperammonemia offers several advantages over others: it is simpler, is bloodless, requires no animal manipulation and permits long-term studies.
Sodium benzoate has been recommended for the treatment of hyperammonaemia in humans. However, benzoate potentiates ammonia toxicity and reduces urea synthesis in vitro and in vivo by decreasing the intramitochondrial levels of N-acetyl glutamate. Pretreatment of mice with carbamyl glutamate, a structural analogue of N-acetyl glutamate, decreases mortality induced by ammonium acetate and sodium benzoate administration. The protective effect of carbamyl glutamate is accompanied by an increase in urea production and of carbamyl phosphate synthetase activity.
Synaptosomes were isolated from cerebrums of rats fed standard (20% protein) or protein-free diets for 30 days. Arrhenius plots of their (Na+/K+)ATPase activities revealed a transition temperature of 25.5 degrees C for control rats and 23.4 degrees C for rats on protein-free diet, indicating that the latter increases synaptosomal membrane fluidity. The only change observed in the composition of the synaptosomal membranes was a 26% decrease of sialic acid. In synaptosomes from rats on protein-free diet the uptake of tyrosine was slightly reduced while that of glutamate was not affected. However, the exit of glutamate was reduced.