Spermine, another specific allosteric activator of calcium uptake in rat liver mitochondria

Calcium uptake in rat liver mitochondria is accelerated by spermine. At a concentration of 2 microM Ca2+ and 1 mM Mg2+ a maximal, 10-fold activation by 1.2 mM spermidine was obtained; a half-maximal activation was attained with 0.2 mM spermine. Spermidine was far less effective than spermine whereas putrescine was ineffective. The acceleration of Ca uptake at low, physiological Ca2+ concentrations is related to the altered kinetics of the Ca uniporter. Corresponding to the alteration by high Ca2+ concentrations previously described, the kinetics changed from sigmoidal in the absence to nearly hyperbolic in the presence of spermine. Mg2+ behaves as an allosteric inhibitor. This phenomenon of the allosteric activation of Ca uptake could not be observed in heart mitochondria.     

Dynamics of calcium release and uptake by the internal calcium stores in rat sensory neurons

Calcium dynamics in the endoplasmic reticulum of dorsal root ganglion neurons of rats during Ca²⁺ release induced by caffeine and subsequent Ca²⁺ uptake were studied. Calcium release is shown to include two (a short transient and a prolonged slow) phases. We suggest that the transient phase reflects release of free Ca from the calcium store, while the slow phase reflects transition of Ca from a bound form to a free one. The process of Ca²⁺ uptake is characterized by exponential recovery of the calcium level in the store due to the SERCA activity. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 361–363, July–August, 2006.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Relationships between hormone-induced calcium release and 86rubidium uptake stimulation in starfish oocytes

86Rubidium+ uptake, but not 86Rubidium efflux, is strongly stimulated after addition of the meiosis inducing hormone 1-methyladenine (1-MeAde) to prophase blocked oocytes of the starfish Marthasterias glacialis. This stimulation is a transient process which does not require the continuous presence of 1-MeAde and is elicited within 1 minute of contact. 1-MeAde and its biologically active structural analogs fully stimulate Rb+ uptake at concentrations which are about two orders of magnitude lower than those required to trigger meiosis reinitiation but which already release underthreshold levels of Ca2+ from the inner part of the plasma membrane. External Ca2+ concentrations effective in triggering meiosis reinitiation also stimulate Rb+ influx, while drugs like D600, theophyllin and caffein which suppress the hormone induced Ca2+ release, simultaneously preclude the stimulation of Rb+ uptake. Dithiothreitol (DTT) which mimicks 1-MeAde action in releasing Ca2+ and inducing meiosis acts both on the efflux and on active and passive Rb+ influxes. Ouabain, the classical inhibitor of the Na+, K+ pump does not preclude meiosis reinitiation under the influence of 1-MeAde, its agonists of mimetics. It suppresses the active component of Rb+ uptake both in control or stimulate oocytes. When applied only in preincubation before starting the hormone treatment, it cannot however inhibit the stimulation of Rb+ uptake, while basal pump inhibition is preserved. These results demonstrate that stimulation of the active Rb+ or K+ transport is not indispensable to meiosis reinitiation. They suggest moreover that the hormone induced Ca2+ release from the plasma membrane may be responsible for unmasking new ouabain sensitive transport sites.     

Intestinal uptake and retention of copper in the suckling rat, Rattus rattus--II. Copper accumulation in the ileum and distal jejunum

High concentrations of copper were demonstrated histochemically in the enterocytes lining the ileum and distal jejunum of suckling rats. Copper was not detected in cells from the duodenum or proximal jejunum of these rats or from any region of the small intestine of rats in which "closure" had taken place. X-Ray microanalysis demonstrated copper, in equi-atomic association with sulphur, within discrete vesicles in the supranuclear cytoplasm of the enterocytes. Despite the high concentrations of these two elements no biochemical evidence was found for the presence of significant amounts of copper-binding metallothionein. The highest concentrations of copper (226 +/- 48 mg atoms/kg dry wt: +/- SD) were found in vesicles adjacent to the nucleus, which did not accumulate particulate tracers, or calcium from the lumen of the intestine. These vesicles probably result from the coalescence of Golgi-derived primary lysosomes followed by fusion with endocytic vacuoles. They may provide a mechanism of copper excretion from the neonatal rat.     

Biosynthesis, release, and uptake of carnosine in primary cultures

Biosynthesis, release, and uptake of carnosine (beta-alanyl-L-histidine) in highly enriched primary cell cultures of skeletal muscle and CNS tissue have been investigated. The synthesis is restricted to muscle cells, oligodendrocytes, and ensheathing cells of olfactory bulb and increases during differentiation of these cells. Astrocytes, in contrast, do not synthesize carnosine but are equipped with a dipeptide transporter by which carnosine is taken up very efficiently.     

Inactivation of the early calcium uptake and noradrenaline release evoked by potassium in cultured chromaffin cells

Upon stimulation with a 59 mM K solution (59K), 45Ca uptake into cultured bovine adrenal chromaffin cells quickly enhanced to reach a plateau within 60 sec. 45Ca transients could be clearly measured with a time resolution (10 sec) and a net Ca uptake (75 times the basal uptake) that considerably improve data reported in other recent papers; this experimental design allows the direct comparison of 45Ca transient data with electrophysiological measurements of chromaffin cell Ca currents. In addition, it is shown that upon sustained depolarization with 59K both, the rates of 45Ca uptake and 3H-noradrenaline release decline in a parallel manner, suggesting that the voltage-dependent Ca channel activity modulates the kinetics of the early secretory response.     

Oestradiol uptake and retention, and high-affinity binding sites in cultured rabbit uterus

Oestradiol uptake and turnover was examined in rabbit uterus maintained in organ culture for up to 3 days. Serum decreased the uptake of [(3)H]oestradiol, whereas insulin had no significant effect. During the first 24h of culture unoccupied high-affinity receptors for oestradiol were markedly depleted in the cytosol. Nuclear binding sites remained high during the first day of culture, and were still present after 3 days. The stability of nuclear-bound oestradiol was confirmed by examining the turnover of radioactivity during culture of uteri of rabbits injected with [(3)H]oestradiol 6h before death. Over half of the radioactivity was retained for as long as 3 days in tissue cultured in the absence of oestrogen. In tissue cultured for 24h with unlabelled oestrogen, there was a progressive increase in the displacement of [(3)H]oestradiol as the concentration of unlabelled hormone in the medium was increased from 0.1 to 5nm. Higher concentrations of oestradiol had little additional effect. The oestradiol involved in this displacement reaction was associated with macromolecules, characterized by Sephadex G-25 chromatography and sucrose-density-gradient ultracentrifugation of the 0.4m-KCl extract of the nuclear pellet.     

Ultrastructural changes in muscle mitochondria in situ,including the apparent development of internal septa,associated with the uptake and release of calcium

Treatment of mammalian muscle with the divalent cation ionophore A23187 causes the release of Ca2+ from the sarcoplasmic reticulum and allows the ultrastructural changes of the mitochondria during Ca2+-uptake to be demonstrated in situ. Electron micrographs reveal that the mitochondria swell dramatically during uptake, before contracting again when the accumulated Ca2+ is released once more into the cytoplasm. When maximally swollen, the mitochondria are apparently subdivided and internal "septa" are formed. The ultrastructural details concerning these internal membranous structures are shown in detail and their significance is discussed.     

Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention

Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.     

Images of Ca flux in astrocytes: evidence for spatially distinct sites of Ca release and uptake

In this study, we have developed a mathematical method to derive the Ca²⁺ fluxes underlying agonist-evoked Ca²⁺ waves in cultured rat cortical astrocytes. Astrocytes were stimulated with norepinephrine (100 nM) to evoke Ca²⁺ waves, which were recorded by measuring FIuo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (ΔF/F) was analyzed in discrete cellular spaces in a series of successive slices along the length of the cell. From these data, Ca²⁺ flux was then calculated using a one dimensional reaction-diffusion equation which utilizes the temporal and spatial derivatives of the fluorescence data and the diffusion coefficient of Ca²⁺ in the cytosol. This method identified distinct sites of positive flux (Ca²⁺ release into the cytosol) and of negative flux (Ca²⁺ removal from cytosol) and showed that in astrocytes, sites of Ca²⁺ release from stores regularly alternate with sites of Ca²⁺ removal from the cytosol. Cross correlation analysis of the two distribution patterns gave positive correlation at 2 μm out of phase and a negative correlation in phase. Thapsigargin-induced Ca²⁺ waves were analyzed to determine if the negative flux was due to Ca²⁺ uptake via thapsigargin-sensitive Ca²⁺ pumps. Negative flux sites were still found under these conditions, suggesting that multiple mechanisms of Ca²⁺ removal from the cytosol may contribute to negative flux sites. This method of calculation of flux may serve as a means to describe the distribution of functional ion channels and pumps participating in cellular Ca²⁺ signalling.     

Copper: uptake,toxicity and tolerance in plants and management of Cu-contaminated soil

BioMetals - Copper (Cu) is an essential mineral nutrient for the proper growth and development of plants; it is involved in myriad morphological, physiological, and biochemical processes. Copper...     

Effects of male sex hormones on specific uptake and release of 3H-serotonin in the rat hypothalamus in vitro]

With the use of "isotopic method" a study was made of the main parameters of functional activity of serotoninergic elements of hypothalamus--the specific uptake and release of 5-OT. The animals used were sexually mature rats castrated on the first postnatal day. In sexually mature intact males the specific uptake of 3H-5-OT by serotoninergic structures of the anterior hypothalamus was significantly lower than in females. Castration of animals on the first day of life resulted in the increase of specific 5-OT uptake in sexually mature males up to that observed in females. There were no differences between the sexes in the rate of spontaneous release of 5-OT. However, response to K(+)-depolarization in the anterior hypothalamus of intact males was significantly lower than that in females. In the hypothalamus of males castrated neonatally the amplitude of the response to the effect of the depolarizing agent was increase up to the level observed in females. By the results obtained it is indicated that elimination of the effect of male hormones on the first postnatal day results in the increase of 5-OT uptake and release in the hypothalamus of sexually mature rat males.     

Kinetics of intracellular calcium release by inositol 1,4,5-trisphosphate and extracellular ATP in porcine cultured aortic endothelial cells.

Quantitative, time-resolved measurements have been made of intracellular Ca ion release by inositol 1,4,5-trisphosphate (InsP3) and extracellular ATP in porcine aortic endothelial cells in tissue culture. Intracellular free [Ca] was detected with the calcium dye fluo-3 and InsP3 released intracellularly by photolysis of 'caged' InsP3 in whole-cell voltage-clamped aortic endothelial cells. A rise of [Ca] was recorded at InsP3 concentrations greater than 0.2 microM. The timecourse at low InsP3 concentrations comprised a delay of mean 300 ms (range 266-330 ms), a peak in 2-3 s before declining with a half-time of 5-10 s. The delay and time-to-peak decreased with increasing concentrations of InsP3 over the range 0.2-5 microM. At very high concentrations of InsP3 (> 5 microM), the delay in the Ca response was short, always less than 20 ms. The results are consistent with a direct binding and gating action of InsP3 on the Ca channel of the cellular store. Following InsP3 action there is a refractoriness of the InsP3 Ca release process which recovers with a timecourse of half-time about 30 s. A comparison can be made between the timecourse of InsP3 and extracellular ATP actions. High concentrations of ATP (500 microM) acted with a delay of mean 1.8 s (range 1.2-2.5 s), whereas even moderate concentrations of InsP3 acted much more quickly, suggesting that there are slow coupling steps before or during the production of InsP3 in response to extracellular ATP. Both ATP and InsP3 evoked an increase in membrane conductance to K+, probably via Ca.     

Receptors, sparks and waves in a fire-diffuse-fire framework for calcium release

Calcium ions are an important second messenger in living cells. Indeed calcium signals in the form of waves have been the subject of much recent experimental interest. It is now well established that these waves are composed of elementary stochastic release events (calcium puffs or sparks) from spatially localised calcium stores. The aim of this paper is to analyse how the stochastic nature of individual receptors within these stores combines to create stochastic behaviour on long time-scales that may ultimately lead to waves of activity in a spatially extended cell model. Techniques from asymptotic analysis and stochastic phase–plane analysis are used to show that a large cluster of receptor channels leads to a release probability with a sigmoidal dependence on calcium density. This release probability is incorporated into a computationally inexpensive model of calcium release based upon a stochastic generalisation of the fire-diffuse-fire (FDF) threshold model. Numerical simulations of the model in one and two dimensions (with stores arranged on both regular and disordered lattices) illustrate that stochastic calcium release leads to the spontaneous production of calcium sparks that may merge to form saltatory waves. Illustrations of spreading circular waves, spirals and more irregular waves are presented. Furthermore, receptor noise is shown to generate a form of array enhanced coherence resonance whereby all calcium stores release periodically and simultaneously.