Nerve growth factor (NGF) stimulates rat pheochromocytoma cells (PC12) to differentiate into a neuronal-like cell that exhibits neurite extensions. The role of protein kinase C in signal transduction has been examined in PC12 cells treated with phorbol 12-myristate 13-acetate (PMA) and bryostatin, a macrocyclic lactone that activates protein kinase C at both the nuclear and the plasma membranes [Hocevar, B. A., & Fields, A. P. (1991) J. Biol. Chem. 266, 28-33]. In contrast to PMA down-regulation [Reinhold, D. S., & Neet, K. E. (1989) J. Biol. Chem. 264, 3538-3544], chronic (24 h) treatment with bryostatin blocked the formation of neurites in response to NGF or basic fibroblast-derived growth factor stimulation, but, like PMA, bryostatin did not block the induction of c-fos or c-jun protooncogenes by NGF. Chronic bryostatin treatment down-regulated protein kinase C activity in the cytosolic, membrane, and nuclear fractions. Acute (60 min) bryostatin or NGF treatment activated cytosolic and nuclear protein kinase C activity, suggesting possible translocation to the nucleus. Bryostatin did not induce neurite outgrowth, either alone or in combination with PMA. Thus, the bryostatin-sensitive protein kinase C is distinct from PMA- or K252a-sensitive kinases previously described. The bryostatin-sensitive protein kinase C is necessary, but not sufficient, for neurite outgrowth and acts in the nucleus in a manner independent of c-fos and c-jun transcription.

ABSTRACT. Reports indicate that L-carnitine administration before 100% lethal dose of ammonium acetate suppresses the symptoms of ammonia toxicity and prevents death in mice. However, we have been unable to confirm this observation. The cause of discrepancy between our results and the results of others was investigated with two models of hyperammonemia in mice: I) that induced by intraperitoneal injection of urease and 2) that induced by intraperitoneal injection of ammonium acetate. L-Carnitine administration failed to protect mice against ammonia toxicity induced by intraperitoneal injection of urease. Mortality in mice treated with L-carnitine 30 min before injection of ammonium acetate was similar to that of controls pretreated with saline. Ammonia and urea levels in plasma, liver, and brain were also similar in both groups. However, the values were significantly lower than those in mice denied either pretreatment before the ammonium acetate challenge. These results indicate that pretreatment acts to reduce blood and tissue ammonia simply by diminishing the rate of absorption of the challenge, owing to the dilution of ammonium acetate upon mixing with the contents of the peritoneal cavity. Thus, any protocol that does not compare results of a putative protective agent with those obtained with an equal volume of solvents or saline runs the risk of ascribing protective property to the agent when the protection may, in fact, have been afforded by the solvent. (Pediatr Res 28: [256][257][258][259][260] 1990) Abbreviations Hyperammonemia occurs in several pathologic conditions such as cirrhosis of the liver, inborn errors of the urea cycle, and other hereditary disorders such as organic acidemia and hyperlysinemia. Elevated ammonia is toxic to the brain and leads to convulsions, coma, and death. Several approaches have been recommended to combat hyperamrnonemia. These include limiting nitrogen intake, improving protein quality, supplying dietary metabolites such as arginine, and increasing nitrogen excretion in the form of derived conjugates (1,2). It has been reported that L-carnitine administration to mice prevents the lethal effects of acute hyperammonemia induced by ammonium acetate injection (3, 4). These authors proposed that L-carnitine decreases the ammonia in blood and brain by inducing ureagenesis. Another group of investigators carried out similar studies using an identical protocol and also reported that L-carnitine protected mice against ammonia toxicity (5). However, when we attempted to repeat this work, we were unable to demonstrate the eficacy of L-carnitine in reducing hyperammonemia (6). Hearn et al. (7) recently reported that the protective effect of L-carnitine was short lived and was not consistently reproducible. They observed that in some experiments 100% of rats treated with L-carnitine survived an ammonium acetate challenge, whereas in other experiments more than 60% of the animals died after an identical treatment (7).The first objective of our study was to investigate the cause for ...

The nuclei of isolated rat hepatocytes were separable into three receptor compartments based upon their differential salt extractabilities: nucleoplasmic receptors (NP) extractable with 0.15 M KCl, high-salt extractable receptors (HSE) extractable with 0.4 M KCl, and salt-resistant receptors (SR) extractable with 0.4 M KCl/5 mM dithiothreitol. The receptor distribution among the three compartments was approximately NP, 45%; HSE, 30%; SR, 25%. The mean percent occupancy with endogenous T3 of the SR receptors (86%) was higher than the occupancies of the NP receptors (68%) and the HSE receptors (63%). When hepatocytes were pulsed with 3 nM [125I]T3 at 37 degrees C for brief intervals, receptor-[125I]T3 complexes were detectable in all three nuclear compartments within 15 sec. With increasing pulse intervals up to 120 sec, the receptor content of each nuclear compartment increased progressively and without evidence of preferential accumulation in any of the three compartments. To determine the life span and intercompartmental "migration" pattern of nuclear receptors, hepatocytes were pulsed with 3 nM [125I]T3 at 37 degrees C for 2.5 min or 5 min, followed by a chase with a 500-fold excess of nonlabeled T3. The population of receptor-[125I]T3 complexes generated during the pulse was serially recovered at increasing intervals after the chase. The complexes of each compartment dissociated with a half-life of approximately 3 min and manifested no predilection to accumulate in any of the compartments. Exposure of isolated hepatocytes to 3 nM T3 for 5 min or 10 min at 37 degrees C induced no change in the gross intercompartmental distribution of receptors compared to control hepatocytes incubated without T3.(ABSTRACT TRUNCATED AT 250 WORDS)