Developmental changes in the45Ca2+ uptake byTrichoderma viride mycelium

The rate of the⁴⁵Ca²⁺ uptake by the submergedTrichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the⁴⁵Ca²⁺ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of⁴⁵Ca²⁺ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up⁴⁵Ca²⁺ proportionally with time. The autoradiography of colonies showed that⁴⁵Ca²⁺ was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of⁴⁵Ca²⁺ in conidia. This work was supported by theSwiss National Science Foundation (joint Swiss-Slovak project 75LPJ041485) andSlovak Grant Agency (grant no. 1/1158/93).     

Effect of somatostatin on glucose-induced 45Ca uptake in the pancreatic islets.

This study was designed in an attempt to elucidate a mechanism of somatostatin inhibition of glucose-induced Ca+ uptake by rat pancreatic islets. Rat pancreatic islets were perifused with Krebs-Ringer bicarbonate (KRB) buffer containing 16.7 mM of glucose with somatostatin (2 micrograms/ml) or/and diltiazem HCl (2 x 10(-5) M). Somatostatin inhibited preferentially the early phase of glucose-induced insulin release, whereas diltiazem HCl inhibited the late one. And the concomitant presence of the submaximal concentration of somatostatin (2 micrograms/ml) and diltiazem HCl (2 x 10(-5 M) provided the completely additive inhibition of glucose-induced insulin release. Rat pancreatic islets were incubated with KRB buffer supplemented with 16.7 mM of glucose and 45CaCl2 (10 muCi/ml) for 5--60 min and the biphasic 45Ca uptake by pancreatic islets was obtained. Somatostatin (500 ng/ml-4 micrograms/ml) gave the suppressive effect on the early phase of glucose-induced 45Ca uptake, but the higher concentration (2 micrograms/ml) of somatostatin did not impair the late phase of 45Ca uptake by pancreatic islets. On the other hand, diltiazem HCl did suppress the late phase of glucose-induced 45Ca uptake dose-dependently, but did not suppress the early phase (2 x 10(-5) M). These data indicate that somatostatin suppresses the early phase of glucose-induced Ca2+ uptake preferentially to the late one and has a different action mechanism from Ca antagonist on glucose-induced insulin release.     

Volatile anesthetics inhibit NMDA-stimulated45Ca uptake by rat brain microvesicles

We have previously shown that volatile anesthetics inhibit glutamate-stimulated [³H]MK-801 binding to the ionophore of NMDA receptor complexes in rat brain. In the present study, we examined the influence of enflurane and halothane on NMDA-stimulated⁴⁵Ca uptake by a microvesicle fraction isolated from rat brain. NMDA stimulated⁴⁵Ca uptake (30 sec) by rat brain microvesicles by up to 70% with an EC₅₀ of 1.4±0.5 M. The NMDA-stimulated⁴⁵Ca uptake was inhibited by MK-801 and D-AP-5 with IC₅₀'s of 10 M. Enflurane and halothane inhibited⁴⁵Ca uptake stimulated by 100 M NMDA by as much as 60–80% with IC₅₀'s of 0.2–0.3 mM, concentrations achieved during routine clinical use. Basal⁴⁵Ca uptake measured in the absence of agonist was not affected by the anesthetics. Glycine did not affect the level of NMDA-stimulated⁴⁵Ca uptake, but markedly reduced the inhibition of uptake caused by enflurane and halothane. Preincubation of microvesicles with NMDA resulted in a desensitization of NMDA-stimulated⁴⁵Ca uptake, with a t_1/2 of 20 sec. Enflurane and halothane diminished both the extent and rate of development of this desensitization, as did glycine. These findings support the idea that volatile anesthetic interference with neurotransmission at NMDA receptor complexes contributes to the development of the anesthetic state.     

Dantrolene inhibits NMDA-induced 45Ca uptake in cultured cerebellar granule neurons

Dantrolene is an inhibitor of a skeletal muscle subtype of ryanodine receptors that stabilizes intracellular calcium concentrations and exerts neuroprotective effects in neurons submitted to excitotoxic challenges. The mechanisms of dantrolene-induced neuroprotection are not clear. In this study, using a model of cultured rat cerebellar granule neurons, we demonstrated that dantrolene inhibits NMDA-evoked 45Ca uptake, indicating that this drug may inhibit the activity of NMDA receptor channels. Primary neuronal cultures were incubated for 10 min in Mg(2+)-free ionic medium with NMDA and 45Ca in the presence of different concentrations of dantrolene, then radioactivity in neurons was measured by liquid scintillation spectroscopy. The results demonstrated that dantrolene, applied at micromolar concentrations, inhibits NMDA-evoked 45Ca uptake in neurons in a dose-dependent manner. DMSO, a vehicle to dantrolene, in concentrations used in this study had no effect on NMDA-evoked 45Ca uptake. These results, indicating that dantrolene inhibits activation of the NMDA receptors, might at least partially explain the mechanisms of a dantrolene-evoked protection of neurons against excitotoxicity mediated by agonists of NMDA receptors.     

Erythropoietin stimulates 45Ca2+ uptake in Friend virus-infected erythroid cells

It has been shown previously in this laboratory that in vitro infection of mouse bone marrow cells with the anemia strain of Friend leukemia virus leads to growth of large bursts of erythroid cells which are arrested in development prior to hemoglobin synthesis but can respond to erythropoietin (EP) to complete the late stage of erythroblast differentiation. In this study, the effect of EP on the metabolism of 45Ca2+ in these cells was examined. At 4 degrees C, an increased rate of 45Ca2+ uptake and efflux as well as an increase in the steady state level of 45Ca2+ in treated cells was observed. Exchange of 45Ca2+ from preloaded cells at 4 degrees C indicated that treatment with EP increased the size of a rapidly exchanging pool of 45Ca2+ from 5 to 12% of total 45Ca2+ in the cell. The effect of treatment with EP can be seen as increased exchange of extracellular 45Ca2+ with cellular Ca2+; however, an effect of EP on the net level of Ca2+ in these cells cannot be excluded. This investigation demonstrates one of the earliest effects of EP on erythroid cells and suggests that alterations in Ca2+ metabolism may contribute to the progression of erythroid cells to their final development.     

An antihypertensive extract of erythrocytes: effects on 45Ca uptake and efflux

The present report describes some aspects of the effects of a recently described antihypertensive extract of erythrocytes (AHF) on calcium uptake and efflux in rat aortae. AHF was found to be present in the erythrocytes of both spontaneously hypertensive rats and normotensive rats. Furthermore, AHF obtained from erythrocytes of SH rats was shown to be equally effective in suppressing lanthanum-resistant calcium uptake in aortae from hypertensive and normotensive rats. AHF treatment prior to incubation of aortae with 45Ca caused an apparent increase in the total 45Ca uptake. The analysis of calcium washout curves obtained for tissue in calcium-free or lanthanum-containing media indicated that AHF had no significant effect on the rate of calcium loss from the slow component of efflux, though this compartment tended to be reduced in size. This indicated that the increase in the 45Ca content of AHF-exposed aortae prior to rinsing was confined to the rapid component of efflux. The loss of calcium from the rapidly exchanging compartment was enhanced in either of the efflux media used. The results suggest that a principal action of AHF involves an increase in the lability and exchangeability of calcium stores. In addition to its effects in resting tissue, AHF abolished the increase in lanthanum-resistant calcium uptake induced in rat aortae by the addition of high K+ or norepinephrine to the incubation media. In a second part of the study, the effect of AHF on blood pressure and in vitro calcium uptake were compared with that of phosphatidylethanolamine (PEA), the probable identity of another endogenous antihypertensive (renin preinhibitor) compound earlier shown to share important functional similarities with AHF.(ABSTRACT TRUNCATED AT 250 WORDS)     

25-Hydroxycholesterol-induced elevation in 45Ca uptake: correlation with depressed DNA synthesis

The mechanism whereby 25-hydroxycholesterol, an inhibitor of the synthesis of cholesterol, depresses DNA synthesis in cycling P815 mastocytoma cells was investigated. The uptake of 45Ca into P815 cells treated with 1 microgram/ml 25-hydroxycholesterol began to rise above control levels by 6 hours after initiation of treatment and was increased tenfold by 15 hours. Kinetic data of calcium uptake indicated the presence of at least two components of calcium uptake, fast and slow. The fast phase of calcium exchange at the cell surface was changed little by treatment with 25-hydroxycholesterol. The slow phase of calcium exchange with the intracellular compartment was markedly affected by treatment with the inhibitor, there being a large increase in the flux and half-time of uptake, and a fall in the rate constant. This resulted in a large elevation of the intracellular compartment size. Incorporation of [3H]thymidine into DNA began to decline between 9 and 12 hours posttreatment in these cultures. Uptake of calcium and depression of DNA synthesis were shown to be directly related to the dose of 25-hydroxycholesterol used. The changes in 45Ca uptake and DNA synthesis due to 25-hydroxycholesterol treatment were abolished by addition of exogenous cholesterol to the incubation medium. The results are consistent with the hypothesis that 25-hydroxycholesterol, by inhibiting cholesterol production, depresses DNA synthesis via an elevation in the uptake of calcium into the cell to a level incompatible with continued DNA replication.     

45Ca2+ uptake and phospholipid methylation in isolated rat liver microsomes

The effects of glucagon, epinephrine and insulin on hepatic phospholipid methylation were studied. Glucagon, either injected into rats or added to perfused livers, stimulated methylation in subsequently isolated microsomes. Epinephrine also increased phospholipid methylation. Insulin by itself did not influence the rate of the reaction, but, when administered prior to glucagon, it blocked the effect of the latter. The possibility that the observed stimulation of phospholipid methylation might be causally linked to the reported stimulation by glucagon of 45Ca2+ uptake in subsequently isolated liver microsomes was examined. Both the substrate and the competitive inhibitor of the methylation reaction, S-adenosylmethionine and S-adenosylhomocysteine, had profound effect on the rate of phospholipid methylation, without having comparable effects on Ca2+ uptake. S-adenosylmethionine in increasing concentration stimulated methylation four-fold, while no significant changes in 45Ca2+ uptake were seen. S-adenosylhomocysteine did not inhibit 45Ca2+ uptake even at levels causing more than 95% decrease in methylation. In conclusion, while both phospholipid methylation and 45Ca2+ uptake seem to be hormonally controlled, the correlation between these two processes was not sufficient to support the notion that the changes in 45Ca2+ uptake are caused by the changes in phospholipid methylation.     

25-Hydroxycholesterol-induced elevations in 45Ca uptake: permeability changes in P815 cells

Certain oxysterols are capable of suppressing the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. We have previously demonstrated that treatment of P815 cells with 1 microgram 25-hydroxycholesterol/ml culture results in a rapid influx of 45Ca, and supplemental cholesterol prevents this from occurring. In this paper, we report on investigations into the means whereby this influx of calcium takes place. Through the use of respiratory inhibitors which prevent mitochondrial retention of calcium it was determined that the large increase in slow phase (intracellular) calcium uptake caused by 25-hydroxycholesterol treatment was related to mitochondrial uptake. The effects of various inhibitors of calcium uptake into cells, including verapamil, diltiazem, quinidine, ruthenium red, Co++, Mn++, were tested. Of these only Co++ and ruthenium red had any effect on 45Ca uptake. 25-Hydroxycholesterol has been shown to be capable of membrane insertion and this could result in plasma membrane permeability changes. To test this hypothesis P815 cells were treated with 1 microgram 25-hydroxycholesterol/ml or 5 micrograms mevinolin/ml culture. Mevinolin, being a water soluble competitive inhibitor of HMG-CoA reductase, should be unable to disrupt membrane architecture in a manner analogous to 25-hydroxycholesterol. While both inhibitors rapidly suppressed the synthesis of digitonin-precipitable sterols, only 25-hydroxycholesterol was able to increase 45Ca influx. The implications of these findings are discussed.     

45Ca2+ uptake and peptide hormones secretion in the presence of excess Mg2+ content

Studies have been performed on the relationship between PRL and GH production and the 45Ca2+ influx in high magnesium content in vitro. The obtained data show that an elevated magnesium concentration in Krebs-Ringer solution is capable of inhibiting some hormonal function of the pituitary gland. It has been found, that PRL and GH released into the media in normal KRB solution revealed nearly two times higher concentration than in the presence of high Mg2+. Instead the cellular iPRL and iGH did not show any significant differences in control and in treated cultures. The incorporation of 4.5-3H-leucine into the prolactin and growth hormone demonstrate a significant decrease in the presence of high Mg2+ indicating that the ion is able to inhibit the secretion of newly synthesized PRL an GH. High concentration of Mg2+ abolished either the stimulation effect of releasing hormones on calcium uptake.