Cellular Cytosol Research Articles

Evidence for a DeltapH-driven Ca2+ uptake in EGTA-treated bovine spermatozoa

Calcium efflux from ejaculated bovine spermatozoa occurred upon incubation in Ca2+/EGTA buffers with Ca2+ ion concentrations ranging from 0.1 microM to 1 nM. Both total cellular calcium and cytosol free Ca2+ concentrations, the latter measured with Quin 2, were inversely correlated with the Ca2+ activity of the medium. An influx of radioactive 45Ca2+ parallel to a net efflux of calcium took place in spermatozoa incubated in 45Ca2+/EGTA buffers with 45Ca2+ activity of 0.01 microM or 0.1 microM. The uptake of the radioactive isotope was higher in spermatozoa incubated at pH 7.8 than that found at pH 6.8, increased in the presence of acetate or amiloride but decreased when ammonium chloride or monensin was added to the incubation mixture. Addition of acetate produced a decrease of the cytoplasmic pH, determined with the indicator carboxyfluorescein, whereas addition of NH4Cl or monensin caused a pH increase. Addition of either nigericin or monensin to spermatozoa suspended in a choline medium containing low concentrations of Na+, K+ and Ca2+ produced a cytosolic acidification, the subsequent addition of Ca2+ caused a cytosolic alkalinization parallel to an increase of the cytosolic free Ca2+. Addition of CaCl2 to EGTA-pretreated spermatozoa resuspended in a poorly buffered medium induced an evident decrease of extracellular pH suggesting a cellular proton extrusion. Both monensin and nigericin caused an increase of the calcium transport in spermatozoa suspended in a choline medium containing a physiological concentration of 1.5 mM CaCl2. Taken together the present results indicate that, under the experimental conditions used, a delta pH-driven Ca2+ uptake occurs in ejaculated bovine spermatozoa and suggest that Ca2+ is taken up in exchange with H+.

Effect of tunicamycin on synthesis and secretion of carcinoembryonic antigen by human colonic adenocarcinoma cells.

The effect of the glycosylation inhibitor, tunicamycin, on synthesis and secretion of the membrane-associated glycoprotein carcinoembryonic antigen (CEA), was studied in the LS174T human colon cancer cell line. Tunicamycin treatment inhibited total cellular glycoprotein synthesis but did not affect CEA levels of cellular homogenate, membrane or cytosol fractions as determined by enzyme immunoassay. Control cells metabolically labelled with 3H-glucosamine, 3H-leucine or 35S-cysteine exhibited membranous and extracellular (i.e. secreted) CEA with an MW of 200 kDa as judged by SDS-gel electrophoresis following immunoprecipitation. However, in the tunicamycin-treated cells several forms of CEA with lower MWs and representing molecules with decreased glycosylation could be detected in addition to the original CEA molecule of 200 kDa present in control cells. The rates of synthesis, secretion and turnover of the lower-molecular-weight forms of poorly glycosylated CEA that appear after tunicamycin treatment are similar to those of CEA in control cells. These data suggest that the carbohydrate portion of the CEA molecule is not essential in synthesis, incorporation into the membrane, and secretion of CEA by colon cancer cells in vitro.

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells.

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.

Alternate putrescine metabolites: Quantitative analysis of Δ′-pyrroline oxidation to 2-pyrrolidone in tissue homogenates by high-pressure liquid chromatography

A sensitive analytical procedure for following the oxidation of δ′-pyrroline to 2-pyrrolidone in tissue homogenates is deseribed. Homogenates are extracted with chloroform/acetonitrile and fractionated by high-performance liquid chromatography, and 2-pyrrolidone is quantitated by monitoring the column effluent at 200 nm. The lower limit of 2-pyrrolidone that can be accurately (±5%) quantitated is approximately 100 pmol. Phenazine methosulfate significantly enhances the rate of 2-pyrrolidone biosynthesis from Δ′-pyrroline. Phenazine methosulfate and reduced glutathione are required to obtain proportionality between 2-pyrrolidone formation and incubation time. Formation of 2-pyrrolidone as a function of protein concentration is linear and 2-pyrrolidone biosynthesis as a function of Δ′-pyrroline concentration is characterized by hyperbolic kinetics. Based on analysis of enzyme activity in different tissue, liver appears to play the dominant role in 2-pyrrolidone biosynthesis. The metabolic step from Δ′-pyrroline to 2-pyrrolidone was localized in the cellular cytosol. These results demonstrate that the oxidation of Δ′-pyrroline to 2-pyrrolidone is enzyme mediated and provide a useful method for further characterization of this metabolic step.

Relation between steroid receptor levels and prolactin level in the decidua of early human pregnancy

To clarify the relation between steroids and prolactin (PRL) production in human decidua, the levels of estrogen receptors (ERs), progesterone receptors (PRs), and androgen receptors (ARs) and the level of PRL in the decidua were determined simultaneously during early pregnancy. Cytosol, nuclear, and total ER levels and the cytosol PRL level were found to increase with the advance of gestation; whereas levels of PRs and ARs remained constant. Multiple regression analysis showed that the multiple correlation coefficient in the relationships of cellular ERs and cytosol PRL with gestational age was 0.722 and relatively high among the others. These results suggest an association between estrogen and decidual growth. Significant correlations between the cytosol PRL level and cellular PR or AR levels were seen during early pregnancy, suggesting that progestin and androgen may regulate PRL synthesis.

Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex

Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, γ-aminobutyric acid, aspartate, glutamate and catecholamines were 4–8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca-Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the [ATP] [ADP] ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the [NADH] [NAD +] ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD + couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the [ATP] [ADP] ratio brought about by glucagon may be secondary to the increase in the [NADH] [NAD +] ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the [NADH] [NAD +] and [ATP] [ADP] ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD + system.