Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide- and Catecholamine-Biosynthetic Enzymes

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of⁷⁵Se-labeled selenite (10 μmol/L) was investigated in rat jejunum. For comparison, the effect ofl-cysteine (1 mmol/L) on in vivo absorption of⁷⁵Se-labeled selenite was also studied. In the in vitro, uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH.l-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlikel-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occuring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since, GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.     

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of⁷⁵Se from⁷⁵Se-labeled selenite (conc. 10 μmol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique.l-Cysteine (l-Cys) stimulated⁷⁵Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum.d-Cys was less effective in the jejunum and similarly effective in the cecum.l-Leucine (l-Leu) andl-glutamic acid significantly reduced the stimulatory effect ofl-Cys on Se uptake in the distal jejunum, whereas the respective effect ofd-Cys was not diminished byl-Leu. Cysteamine stimulated mucosal⁷⁵Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced⁷⁵Se uptake in the distal jejunum. The stimulatory effects ofl-Cys, mercaptopyruvate, and thiologlycolate were Na⁺-dependent, whereas the effect of cysteamine also occurred in the absence of Na⁺. Mercaptosuccinate,d-penicillamine, ergothioneine, and thiosulfate did not enhance mucosa⁷⁵Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na⁺-dependent and Na⁺-independent, mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.     

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

Summary The Ca⁺⁺-mediated increase in K⁺-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca⁺⁺ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K⁺ concentration. In K⁺-free solutions the concentration of quinine to achieve 50% inhibition (K ₅₀) was 5 m, but at 5mm K⁺ the required concentration was increased 20-fold to 100 m. An increase in internal Na⁺ had the opposite effect, allowing a high potency of quinine despite the presence of external K⁺. Alterations in the internal K⁺ level, on the other hand, were without effect on theK ₅₀, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl^– by NO ₃ ^– , a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K⁺ from an external binding site, the reported K⁺-activation site for the Ca⁺⁺-mediated K⁺-permeability.     

Terpenes from pittosporaceae

The monoterpenes of the fruits of Pittosporum resiniferum and P. undulatum have been identified. The essential oil of P. resiniferum contai     

Reactivity and fate of benzene and formaldehyde in culture medium with and without fetal calf serum; relevance to in vitro mutagenicity testing

Gas chromatographic-mass spectrometric analyses were performed to determine the reactivity and fate of benzene (BEN) and formaldehyde (FA) in culture medium. BEN (solubility in water: 500 ppm) does not react with culture medium, either with or without fetal calf serum, but its volatility, even in closed vials, is so great that 90% of a 250-ppm solution is lost to the head space after 1 h at 24°C. FA, as a 37% aqueous solution, is a complex mixture that changes composition after 15-min incubation at 38°C. FA is extremely reactive in culture medium containing fetal calf serum, and is much less reactive with medium components in the absence of serum. There is a dramatic increase in the number of daughter products in FA-treated medium over time, such that those seen immediately after FA is added to medium have been replaced after 60-min incubation (38°C in closed vials) by many other interaction products. Methods ensuring maximum solubilization and minimal volatilization of BEN during exposure are essential for obtaining reproducible data on the mutagenic potential of BEN. The volatilization of FA from stock formalin solutions, and, more importantly, the interaction product(s) formed by this highly reactive compound with medium components, especially those in serum, are probably the critical aspects of an effective testing protocol for FA.