Comparison of the effects of divalent cations on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells.
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1. The facilitatory effects of external Ca2+, Sr2+ and Ba2+ (Ca2+o, Sr2+o and Ba2+o) on the noradrenaline-evoked non-selective cation current (Icat) were compared in rabbit portal vein smooth muscle cells using patch pipette techniques. 2. All divalent cations tested potentiated the amplitude of Icat and the potency sequence was Ca2+o > Sr2+o > Ba2+o. Ca2+o and Sr2+o increased the amplitude of Icat by about eight times whereas Ba2+o produced only a threefold facilitation. 3. The current-voltage relationship of Icat was not changed by Ca2+o, Sr2+o or Ba2+o. 4. From noise analysis the single channel conductance (gamma) was approximately 10 pS in divalent cation-free solution but was about 20 pS with Ca2+o, Sr2+o and Ba2+o. 5. From noise and voltage-jump experiments it was apparent that at least three kinetically resolvable channel states are associated with Icat in divalent cation-free solution. Ca2+o and Sr2+o produced marked changes in the characteristics of the power spectrum and relaxations of Icat in response to voltage steps, consistent with a shift in the equilibrium between the channel states, whereas Ba2+o produced minimal effects. 6. The data show that Ca2+o, Sr2+o and Ba2+o increase the amplitude of Icat, which results in part from an increase in the single channel conductance. In addition the results suggest that Ca2+o and Sr2+o alter the kinetic behaviour of the single channels whereas Ba2+o has little effect on the equilibrium between the channel states. (+info)
Increased serum strontium levels in dialysis patients: an epidemiological survey.
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BACKGROUND: We previously reported on increased bone strontium levels in dialysis patients with osteomalacia versus those presenting other types of renal osteodystrophy. A causal role of strontium in the development of osteomalacia was established in a chronic renal failure rat model. METHODS: To further elucidate the latter issue and to find out whether dialysis patients from particular centers/countries are at an increased risk for strontium accumulation, a worldwide multicenter study was established. In total, 834 patients from 34 dialysis centers in 23 countries were included. In each of the patients, a serum sample was taken for strontium determination, and water and dialysate samples were taken at the various steps of the water purification process. For each patient clinical data and for each center dialysis modalities were recorded. RESULTS: Strontium levels in serum of dialysis patients showed major differences between the various centers, ranging from mean values of 25 +/- 8 microgram/liter in the center with the lowest level up to 466 +/- 90 microgram/liter in the center with the highest concentration. It is of interest that these high levels were mainly found in developing countries. Furthermore, our data point toward a role of the final dialysate in the accumulation of the element, as indicated by the strong correlation (r = 0.74, P < 0.001) between mean serum and dialysate strontium levels. As the high tap water concentration of strontium was adequately reduced during the water purification process, contamination of the final dialysis fluid occurred by the addition of concentrates contaminated with strontium. Besides the dialysate, other factors, such as duration of dialysis, vitamin D supplements, or types of phosphate binders, played a less important role in the accumulation of the element. CONCLUSIONS: Data of this multicenter study indicate patients of particular dialysis centers to be at an increased risk for strontium accumulation, the clinical consequence of which is under current investigation. (+info)
alpha-latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction.
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alpha-latrocrustatoxin (alpha-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which mEPSP frequency returned toward control levels. EPSPs were increased in amplitude during periods of enhanced mEPSP discharge. alpha-LCTX had no effect when applied in Ca(2+)-free saline, but subsequent addition of Ca(2+) caused an immediate enhancement of mEPSP frequency even when alpha-LCTX was previously washed out of the bath with Ca(2+)-free saline. Furthermore removal of Ca(2+) from the saline after alpha-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus alpha-LCTX binding is insensitive to Ca(2+), but toxin action requires extracellular Ca(2+) ions. Preincubation with wheat germ agglutinin prevented the effect of alpha-LCTX but not its binding. These binding characteristics suggest that the toxin may bind to a crustacean homologue of latrophilin/calcium-independent receptor for latrotoxin, a G-protein-coupled receptor for alpha-latrotoxin (alpha-LTX) found in vertebrates. alpha-LCTX caused "prefacilitation" of EPSP amplitudes, i.e., the first EPSP in a train was enhanced in amplitude to a greater degree than subsequent EPSPs. A similar alteration in the pattern of facilitation was observed after application of the Ca(2+) ionophore, A23187, indicating that influx of Ca(2+) may mediate the action of alpha-LCTX. In nerve terminals filled with the Ca(2+) indicator, calcium green 1, alpha-LCTX caused increases in the fluorescence of the indicator that lasted for several minutes before returning to rest. Neither fluorescence changes nor toxin action on mEPSP frequency were affected by the Ca(2+) channel blockers omega-agatoxin IVA or Cd(2+), demonstrating that Ca(2+) influx does not occur via Ca(2+) channels normally coupled to transmitter release in this preparation. The actions of alpha-LCTX could be reduced dramatically by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid. We conclude that induction of extracellular Ca(2+) influx into nerve terminals is sufficient to explain the action of alpha-LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions. (+info)
Dual responses of CNS mitochondria to elevated calcium.
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Isolated brain mitochondria were examined for their responses to calcium challenges under varying conditions. Mitochondrial membrane potential was monitored by following the distribution of tetraphenylphosphonium ions in the mitochondrial suspension, mitochondrial swelling by observing absorbance changes, calcium accumulation by an external calcium electrode, and oxygen consumption with an oxygen electrode. Both the extent and rate of calcium-induced mitochondrial swelling and depolarization varied greatly depending on the energy source provided to the mitochondria. When energized with succinate plus glutamate, after a calcium challenge, CNS mitochondria depolarized transiently, accumulated substantial calcium, and increased in volume, characteristic of a mitochondrial permeability transition. When energized with 3 mM succinate, CNS mitochondria maintained a sustained calcium-induced depolarization without appreciable swelling and were slow to accumulate calcium. Maximal oxygen consumption was also restricted under these conditions, preventing the electron transport chain from compensating for this increased proton permeability. In 3 mM succinate, cyclosporin A and ADP plus oligomycin restored potential and calcium uptake. This low conductance permeability was not effected by bongkrekic acid or carboxyatractylate, suggesting that the adenine nucleotide translocator was not directly involved. Fura-2FF measurements of [Ca(2+)](i) suggest that in cultured hippocampal neurons glutamate-induced increases reached tens of micromolar levels, approaching those used with mitochondria. We propose that in the restricted substrate environment, Ca(2+) activated a low-conductance permeability pathway responsible for the sustained mitochondrial depolarization. (+info)
Biologic mechanisms of 89SrCl2 incorporation into type I collagen during bone mineralization.
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89SrCl2 is currently used as a palliative treatment for painful osseous metastases associated with an osteoblastic reaction in bone. However, the underlying biologic mechanism by which 89SrCl2 accumulates at these lesions and mediates palliation remains unclear. The aim of this study was therefore to elucidate this mechanism. METHODS: An in vitro cell biologic model, incorporating the MC3T3-E1 murine osteoblast cell line, was established to replicate the process of collagen production and mineralization. Experiments were performed to investigate the cellular association of 89SrCl2 and 45CaCl2 with both MC3T3-E1 cells and the PC-3 human prostate adenocarcinoma cell line. RESULTS: No evidence of intracellular localization of 89SrCl2 or 45CaCl2 was found for either cell line. Localization of radiolabel was seen to be associated with MC3T3-E1 cells but only in cultures that had undergone both differentiation and mineralization. The association of 89SrCl2 was inhibited by the alkaline phosphatase inhibitor levamisole, and extracellular localization of 89SrCl2 was confirmed by microautoradiography. CONCLUSION: 89SrCl2 acts as a calcium mimic and, as such, becomes associated with the collagen matrix produced by the MC3T3-E1 cells during collagen mineralization. (+info)
Strontium as a marker for intestinal calcium absorption: the stimulatory effect of calcitriol.
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BACKGROUND: Intestinal strontium absorption is becoming accepted as a clinical and diagnostic tool for assessing intestinal calcium absorption in humans. However, little is known about whether intestinal strontium absorption, like that of calcium, is stimulated by calcitriol in healthy humans. METHODS: The effect of calcitriol on intestinal strontium absorption was measured in eight healthy men, ages 20-60 years. Before administration of calcitriol, two tests were performed with an interval of 10 days for calculating the within-subject variation (SE(R)). Before the third test, 0.5 microg of calcitriol was given twice daily for 3 days. In each test, the fractional strontium absorption (Fc(240)) and the area under the concentration-time curve (AUC(0-240)) 4 h after an oral strontium load of 2.5 mmol were calculated. RESULTS: The within-subject SE(R) of Fc(240) and AUC(0-240) was 1.7 +/- 0.7 and 0.83 +/- 0.1, respectively. The stimulatory effect of calcitriol on Fc(240) and AUC(0-240) was 35% (21.8 +/- 2.0 to 28.8 +/- 2.4; P = 0.003) and 61% (8.97 +/- 0.97 to 14.4 +/- 1.3 mmol. L(-1). min; P = 0.001), respectively. CONCLUSIONS: Although the reproducibility of AUC(0-240) and its sensitivity to calcitriol were better than those of Fc(240), the Fc(240) of strontium is preferred for a clinical test because of its simplicity, requiring only two instead of five blood samples. (+info)
Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.
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The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs. (+info)
Ca(2+), Sr(2+), and Ba(2+) identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca(2+)-sensitive ACs.
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Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations. (+info)