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Low dietary calcium intake and absorption may increase the risk of hypocalcaemia disease states. Reducing the particle size of calcium-containing powders and increasing the specific surface area (SSA), may have high oral calcium bioavailability. The absorption of a single dose of different sized calcium carbonate nanoparticles was traced in Sprague-Dawley rats with radioactive calcium-45 (half-life = 162.6 days, β - endpoint = 258 keV; 100%). Four calcium carbonate formulations (calcium-45) were administered to Sprague-Dawley rodents (6 per treatment; n = 24). The groups were [ 45 Ca]CaCO 3 SSA 3 m 2 /g, [ 45 Ca]CaCO 3 36 m 2 /g, [ 45 Ca]CaCO 3 64 m 2 /g and a separate [ 45 Ca]CaCO 3 36 m 2 /g formulation produced by flame assisted pyrolysis. Blood and urine were sampled periodically, and organs collected and analysed after euthanasia. No changes in SSA or crystallinity were observed when powders before or after irradiation were compared. The [ 45 Ca]CaCO 3 64 m 2 /g formulation presented with higher levels in blood 2 h after administration and a higher liver and femur concentration. These findings suggest [ 45 Ca]CaCO 3 64 m 2 /g could lead to increased oral bioavailability., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Previous work with isolated outer membrane vesicles of lobster branchiostegite epithelial cells has shown that 45 Ca 2+ uptake by these structures is significantly (p < 0.02) reduced by an incremental decrease in saline pH (increased proton concentration) and that this decrease is due to competitive inhibition between carrier-mediated transport of 45 Ca 2+ and hydrogen ions. The present paper extends these previous findings and describes the combined effects of pH and cationic heavy metals on branchiostegite uptake of 45 Ca 2+ . Partially purified membrane vesicles of branchiostegite cells were produced by a homogenization/centrifugation method and were loaded with mannitol at pH 7.0. The time course of 1 mM 45 Ca 2+ uptake in a mannitol medium at pH 8.5 containing 100 µM verapamil (Ca 2+ channel blocker) was hyperbolic and approached equilibrium at 30 min. This uptake was either significantly reduced (p < 0.05) by the addition of 5 µM Zn 2+ or essentially abolished with the addition of 5 µM Cu 2+ . Increasing zinc concentrations (5-500 µM) reduced 1 mM 45 Ca 2+ uptake at pH 8.5 or 7.5 in a hyperbolic fashion with the remaining non-inhibited uptake due to apparent non-specific binding. Uptake of 1 mM 45 Ca 2+ at pH 8.5, 7.5, 7.5 + Zn 2+ , and 7.5 + Zn 2+  + Cu 2+  + Cd 2+ in the presence of 100 µM verapamil displayed a stepwise reduction of 45 Ca 2+ uptake with the addition of each treatment until only non-specific isotope binding occurred with all cation inhibitors. 45 Ca 2+ influxes (15 s uptakes; 0.25-5.0 mM calcium + 100 µM verapamil) in the presence and absence of 10 µM Zn 2+ were both hyperbolic functions of calcium concentration. The curve with Zn 2+ displayed a transport K m twice that of the control (p < 0.05), while inhibitor and control curve J max values were not significantly different (p > 0.05), suggesting competitive inhibition between 45 Ca 2+ and Zn 2+ influxes. Analysis of the relative inhibitory effects of increased proton or heavy metal interaction with 45 Ca 2+ uptake suggests that divalent metals may reduce the calcium transport about twice as much as a drop in pH, but together, they appear to abolish carrier-mediated transport.

Introduction: With an objective to develop a cost-effective radiochemical formulation for palliation of pain due to skeletal metastases, we have demonstrated a viable method for large-scale production of (45)Ca (t½=163 days, Eβmax=0.3MeV) using moderate flux research reactor, its purification from radionuclidic impurities adopting electrochemical approach and preclinical evaluation of (45)CaCl2., Methods: Irradiation parameters were optimized by theoretical calculations for production of (45)Ca with highest possible specific activity along with minimum radionuclidic impurity burden. Based on this, the radioisotope was produced in reactor by irradiation of isotopically enriched (98% in (44)Ca) CaO target at a thermal neutron flux of ~1 × 10(14) n.cm(-2).s(-1) for 4 months. Scandium-46 impurity co-produced along with (45)Ca was efficiently removed adopting an electrochemical separation approach. The bone specificity of (45)CaCl2 was established by in vitro studies involving its uptake in hydroxyapatite (HA) particles and also evaluating its biodistribution pattern over a period of 2 weeks after in vivo administration in Wistar rats., Results: Thermal neutron irradiation of 100mg of enriched (98% in (44)Ca) CaO target followed by radiochemical processing and electrochemical purification procedure yielded ~37 GBq of (45)Ca with a specific activity of ~370 MBq/mg and radionuclidic purity>99.99%. The reliability and reproducibility of this approach were amply demonstrated by process demonstration in several batches. In vitro studies indicated significant uptake of (45)CaCl2 (up to 65%) in HA particles. In vivo biodistribution studies in Wistar rats showed specific skeletal accumulation (40-46%ID) with good retention over a period of 2 weeks., Conclusions: To the best of our knowledge, this is the first study on utilization of (45)CaCl2 in the context of nuclear medicine. The results obtained in this study hold promise and warrant further investigations for future translation of (45)CaCl2 to the clinics, thereby potentially enabling a cost-effective approach for metastatic bone pain palliation especially in developing countries., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the present study, a method was developed for determining the alimentary tract Ca absorption capacity of ruminant animals by measuring the absorption rate of Sr after the administration of an oral dose of strontium chloride acting as a tracer analogue of Ca. A close correlation between the absorption rates of the two tracers was observed upon simultaneous administration of an oral dose of stable Sr and radioactive calcium (r 0·98). The Ca absorption capacity of the rumen and small intestine was determined separately by either directing the solution into the rumen or by diverting it into the post-ruminal tract by vasopressin-induced closure of the ruminoreticular groove. The animals were treated with 1α-hydroxyvitamin D3 administered via subcutaneously implanted mini-osmotic pumps. The effect of elevated plasma 1,25-dihydroxycholecalciferol concentrations on the Ca absorption capacity of the alimentary tract was then determined. An increased rate of Sr absorption was observed in both the rumen and small intestine of sheep after treatment, although it is unclear whether the rumen possesses the same vitamin D-dependent Ca absorption pathway as the small intestine.

Background: Although inotropic stimulation is considered harmful in the presence of myocardial ischaemia, both calcium sensitisers and phosphodiesterase inhibitors may offer cardioprotection. We hypothesise that these cardioprotective effects are related to an acute alteration of myocardial metabolism. We studied in vivo effects of milrinone and levosimendan on calcium overload and ischaemic markers using left ventricular microdialysis in pigs with acute myocardial ischaemia., Methods: Anaesthetised juvenile pigs, average weight 36 kg, were randomised to one of three intravenous treatment groups: milrinone 50 μg/kg bolus plus infusion 0.5 μg/kg/min (n = 7), levosimendan 24 μg/kg plus infusion 0.2 μg/kg/min (n = 7), or placebo (n = 6) for 60 min prior to and during a 45 min acute regional coronary occlusion. Systemic and myocardial haemodynamics were assessed, and microdialysis was performed with catheters positioned in the left ventricular wall. (45) Ca(2+) was included in the microperfusate in order to assess local calcium uptake into myocardial cells. The microdialysate was analysed for glucose, lactate, pyruvate, glycerol, and for (45) Ca(2+) recovery., Results: During ischaemia, there were no differences in microdialysate-measured parameters between control animals and milrinone- or levosimendan-treated groups. In the pre-ischaemic period, arterial blood pressure decreased in all groups while myocardial oxygen consumption remained stable., Conclusions: These findings reject the hypothesis of an immediate energy-conserving effect of milrinone and levosimendan during acute myocardial ischaemia. On the other hand, the data show that inotropic support with milrinone and levosimendan does not worsen the metabolic parameters that were measured in the ischaemic myocardium., (© 2013 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Acute and prolonged bone complications associated with radiation and chemotherapy in cancer survivors underscore the importance of establishing a laboratory-based complementary dual-isotope tool to evaluate short- as well as long-term bone remodeling in an in vivo model. To address this need, a liquid scintillation dual-label method was investigated using different scintillation cocktails for quantitative measurement of (3)H-tetracycline ((3)H-TC) and (45)Ca as markers of bone turnover in mice. Individual samples were prepared over a wide range of known (45)Ca/(3)H activity ratios. Results showed that (45)Ca/(3)H activity ratios determined experimentally by the dual-label method were comparable to the known activity ratios (percentage difference ∼2%), but large variations were found in samples with (45)Ca/(3)H activity ratios in range of 2-10 (percentage difference ∼20-30%). Urine and fecal samples from mice administered with both (3)H-TC and (45)Ca were analyzed with the dual-label method. Positive correlations between (3)H and (45)Ca in urine (R=0.93) and feces (R=0.83) indicate that (3)H-TC and (45)Ca can be interchangeably used to monitor longitudinal in vivo skeletal remodeling., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Nanotechnology has been employed in attempts to enhance bone incorporation of dental implants. Often, nanoparticles are applied to the implant surface as particle coatings. However, the same properties that may increase the functionality may also lead to undiscovered negative effects, such as instability of the nanocoating. The aim of this study was to investigate the stability/instability of the nanoparticles using a radiolabeling technique., Materials and Methods: Twenty threaded and turned titanium microimplants were inserted in 10 rats. All 20 implants were coated with nanometer-sized hydroxyapatite (HA) particles. In order to trace the HA nanoparticles, the particles for 16 implants were labeled with calcium 45 (45Ca). After 1, 2, 4, and 8 weeks, the implants and surrounding bone were retrieved and analyzed using autoradiography with respect to particle migration from the implant surface. Samples from the brain, liver, thymus, kidney, and blood, as well as wooden shavings from the rats' cages, were also retrieved and analyzed using liquid scintillation counting., Results: The radioactivity representing the localization of 45Ca decreased over time from the vicinity of the implant. The amounts of 45Ca found in the blood and in the rats' excretions decreased with time and corresponded well to each other. After 8 weeks, the only trace of 45Ca was found in the liver., Conclusion: The results indicated that released particles leave the body through the natural cleaning system, and the probability that the nanocoating will assemble in vital organs and thus become a potential biologic risk factor is unlikely.

Unlabelled: Urinary excretion of tritiated tetracycline ((3)H-TC) and (41)Ca tracers was validated as reflecting skeletal disappearance of these bone-seeking tracers as a direct measure of bone turnover following ovariectomy in rats., Introduction: Tritiated tetracycline ((3)H-TC) and Ca tracers have been used to measure bone resorption in animal models, but urinary excretion of these labels has not been directly compared to skeletal turnover. We aimed to evaluate the use of bone-seeking labels by comparing label release into urine with label in the skeleton when bone turnover was perturbed following ovariectomy., Methods: Sixty-four 6-month-old ovariectomized (OVX) rats were randomized to one of eight groups in a 2 × 4 design that differed in time of (3)H-TC and (41)Ca administration following ovariectomy (1 month, when bone turnover would be accelerated following estrogen depletion or 3 months when bone loss due to OVX had slowed down) and time of euthanasia (1 week, 1 month, 3 months, and 6 months post-dose). Twenty-four-hour urine pools over two to four consecutive days and total skeleton were collected and recovered for the assessment of (3)H-TC and (41)Ca., Results: Urinary (3)H-TC levels reflected skeletal (3)H-TC levels (r = 0.93; p < 0.0001) over a wide range of bone turnover rates in response to an intervention. Urinary (41)Ca and (3)H-TC excretion were highly correlated (r = 0.95, p < 0.0001)., Conclusion: This study confirms that bone-seeking label excretion into the urine directly measures bone turnover.

Nifedipine has a high incidence of neurologic adverse reactions as compared with other dihydropyridine Cav1 (L-type) channel blockers used for treating cardiovascular diseases. The mechanism mediating neuronal excitation by nifedipine is still in debate. Nifedipine caused a dual role on veratridine-induced 45Ca uptake by rat hippocampal synaptosomes. In the nanomolar range (0.001-0.3 microM), nifedipine decreased 45Ca uptake in a cadmium-sensitive manner. In contrast with nitrendipine (0.001-10 microM), nifedipine consistently facilitated 45Ca accumulation when used in low micromolar concentrations (0.3-10 microM). The cadmium-insensitive nifedipine facilitation became less evident upon increasing veratridine concentration from 5 to 20 microM and was not detected when the synaptosomes where depolarised with 30 mM KCl. Na+ substitution by N-methyl-D-glucamine (132 mM) or blockade of Na+ currents with tetrodotoxin (1 microM) both prevented nifedipine excitation. The Na+/Ca2+-exchanger inhibitor, KB-R7943 (3-50 microM), did not reproduce nifedipine actions. Data suggest that tetrodotoxin-sensitive Na+ channels may operate paradoxical nifedipine facilitation of 45Ca uptake by rat hippocampal synaptosomes.

The effect of interleukin-1beta on presynaptic NMDA receptors was evaluated by studying NMDA-induced 45Ca2+ uptake by synaptosomes from rat brain cortex. Interleukin-1beta inhibited 45Ca2+ uptake by synaptosomes. Our results indicate that interleukin-1beta modulates presynaptic NMDA receptors and is probably involved in the regulation of synaptic transmission in the central nervous system.

The effect of moult stage on cadmium accumulation and distribution was investigated in vivo in male shore crabs Carcinus maenas exposed to 1 mg Cd l(-1) for 7 days. The accumulation of cadmium in all tissues examined was markedly higher in postmoult (A(1-2) and B(1-2)) compared to intermoult (C1, C3 and C4) and premoult (D(0-3)). In addition, elevated levels of cadmium were found in gills of late premoult (D(2-3)) animals. The total amount of cadmium accumulated in the tissues (haemolymph, gills, midgut gland and muscle) increased from 43 microg Cd in early premoult (D(0-1)) to 391 microg Cd in late postmoult (B(1-2)). Gills and midgut gland were the primary cadmium accumulating tissues in C4-intermoult and premoult (D(0-3)); in early postmoult (A(1-2)) haemolymph and midgut gland were the main cadmium containing tissues, while midgut gland dominated in late postmoult (B(1-2)) and early intermoult (C1 and C3). A detailed account of calcium distribution in haemolymph, gills, midgut gland, muscle and exoskeleton during the moult cycle is presented. Mechanistic links between cadmium and calcium uptake in posterior gills of C4-intermoult and early postmoult (A(1-2)) crabs were explored using an in vitro gill perfusion technique. Calcium and cadmium influxes were markedly higher in postmoult compared to intermoult. No differences between intermoult and postmoult effluxes were found for either calcium or cadmium. From intermoult to postmoult net influx increased from 2.4 to 29 micromol Ca2+ g(-1) ww(gill) h(-1) and from 0.24 to 25 nmol Cd2+ g(-1) ww(gill) h(-1). The results indicate that the postmoult increase in cadmium influx is due to increased active transport of cadmium, at least partly, by accidental uptake via calcium transporting proteins. The in vitro net influx rates corresponded accurately to the observed in vivo accumulation of both cadmium and calcium. Although cadmium accumulation and distribution are clearly linked to changes in calcium requirements, cadmium did not interfere with calcium accumulation or distribution at any stage during the moult cycle.

The objective of this study was to examine the effects of chronically elevated dietary Ca2+ (as CaCO3), alone and in combination with elevated dietary Cd, on survival, growth, and Cd and Ca2+ accumulation in several internal compartments in juvenile rainbow trout (Oncorhynchus mykiss). In addition, effects on short-term branchial uptake and internal distribution of newly accumulated waterborne Ca2+ and Cd during acute waterborne Cd exposure (50 microg/L as CdNO3 for 3 h) were monitored using radiotracers (45Ca, 65Cd). Fish were fed with four diets: 20 mg Ca2+/g food (control), 50 mg Ca2+/g food, 300 microg Cd/g food, and 50 mg Ca2+/g + 300 microg Cd/g food for 30 days. There were no significant effects on growth, mortality, or total body Ca2+ accumulation. The presence of elevated Ca2+, Cd, or Ca2+ + Cd in the diet all reduced waterborne Ca2+ uptake in a short-term experiment (3 h), though the inhibitory mechanisms appeared to differ. The effects were marked after 15 days of feeding, but attenuated by 30 days, except when the diet was elevated in both Ca2+ and Cd. The presence of elevated Ca2+ in the diet had only modest influence on Cd uptake from the water during acute Cd challenges but greatly depressed Cd uptake from the diet and accumulation in most internal tissues. None of the treatment diets prevented the decreases in waterborne Ca2+ uptake and new Ca2+ accumulation in internal tissues caused by acute exposure to waterborne Cd. In conclusion, there are complex interactions between waterborne and dietary effects of Ca2+ and Cd. Elevated dietary Ca2+ protects against both dietary and waterborne Cd uptake, whereas both waterborne and dietary Cd elevations cause reduced waterborne Ca2+ uptake.

Net influxes into the haemolymph and tissue distribution of 45Ca and 109Cd were studied in vivo in female Carcinus maenas at different moult stages. Net influxes of 45Ca and 109Cd from water were higher in postmoult (A and B) C. maenas than in C3- and C4-intermoult crabs and the net influx of calcium was higher in C3-intermoult crabs than in C4-intermoult crabs. The net influxes of 45Ca and 109Cd increased in postmoult C. maenas with decreasing external calcium concentrations at constant salinity. At all external calcium concentrations a significant correlation existed between 45Ca and 109Cd accumulated in the haemolymph of individual animals. In vivo exposure of postmoult C. maenas to external lanthanum decreased the 45Ca and 109Cd uptake rates to 30 and 10%, respectively, of the control values. About 30% of injected 109Cd were found in the midgut gland, 10-20% in the gills and only a few (1-2) percent was lost to the seawater 24 h after injection. No major variations in tissue distribution of 109Cd were observed between moult stages in these tissues. Premoult crabs retained more cadmium in the haemolymph 24 h after injection than other moult stages, and postmoult crabs retained more in muscle. Between 20 and 40% of the injected 45Ca were excreted to the water, while only a few percent of the injected 45Ca were found in the soft tissues 24 h after injection. Large moult stage variations, however, were observed in the tissue distribution of internalised 45Ca. This study demonstrates that cadmium and calcium uptakes are elevated in postmoult C. maenas. The results indicate that cadmium and calcium in this stage are taken up via Ca2+-channels located in the apical membrane of gill epithelium cells. When internalised, however, cadmium and calcium are metabolised in fundamentally different ways, determined by the chemical properties and biological significance of the two metals.

Introduction: Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction., Materials and Methods: The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of 45Ca, radiographically and histologically 3 weeks after the implantation., Results: None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. 45Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, 45Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups., Conclusions: Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.

We investigated the effect of zooplankton feeding on tissue and skeletal growth of the scleractinian coral Stylophora pistillata. Microcolonies were divided into two groups: starved corals (SC), which were not fed during the experiment, and fed corals (FC), which were abundantly fed with Artemia salina nauplii and freshly collected zooplankton. Changes in tissue growth, photosynthesis and calcification rates were measured after 3 and 8 weeks of incubation. Calcification is the deposition of both an organic matrix and a calcium carbonate layer, so we measured the effect of feeding on both these parameters, using incorporation of (14)C-aspartic acid and (45)Ca, respectively. Aspartic acid is one of the major components of the organic matrix in scleractinian corals. For both sampling times, protein concentrations were twice as high in FC than in SC (0.73 vs 0.42 mg P(-1) cm(-2) skeleton) and chlorophyll c(2) concentrations were 3-4 times higher in fed corals (2.1+/-0.3 micro g cm(-2)). Cell specific density (CSD), which corresponds to the number of algal cells inside a host cell, was also significantly higher in FC (1.416+/-0.028) than in SC (1.316+/-0.015). Fed corals therefore displayed a higher rate of photosynthesis per unit area (P(g)(max)= 570+/-60 nmol O(2) cm(-2) h(-1) and I(k)=403+/-27 micro mol photons m(-2) s(-1)). After 8 weeks, both light and dark calcification rates were twofold greater in FC (3323+/-508 and 416+/-58 nmol Ca(2+) 2 h(-1) g(-1) dry skeletal mass) compared to SC (1560+/-217 and 225+/-35 nmol Ca(2+) 2 h(-1) g(-1) dry skeletal mass, respectively, under light and dark conditions). Aspartic acid incorporation rates were also significantly higher in FC (10.44+/-0.69 and 1.36+/- 0.26%RAV 2 h(-1) g(-1) dry skeletal mass, where RAV is total radioactivity initially present in the external medium) than in SC (6.51+/-0.45 and 0.44+/-0.02%RAV 2 h(-1) g(-1) dry skeletal mass under dark and light conditions, respectively). Rates of dark aspartic acid incorporation were lower than the rates measured in the light. Our results suggest that the increase in the rates of calcification in fed corals might be induced by a feeding-stimulation of organic matrix synthesis.

Intestinal calcium absorption accounts for 60% of the variance in calcium balance and is therefore a potentially very important determinant of bone status. Whether measured by the balance technique or with radiocalcium, it is known to be significantly reduced in postmenopausal women with vertebral and hip fractures. By contrast, there is very little information about calcium absorption in other types of postmenopausal fracture. We now report a series of 549 untreated, Caucasian postmenopausal women in whom we recorded prevalent fractures, measured radiocalcium absorption, and obtained radiographs of the lateral thoracic and lumbar spine. Of these women, 172 had no prevalent fractures, showed normal spine radiographs, and served as controls; 72 had one or more peripheral fractures but normal spine radiographs; 147 had one or more wedged or crushed vertebrae but no peripheral fractures; and 158 had a history of peripheral fracture and one or more fractured vertebrae. Age-adjusted radiocalcium absorption was significantly lower in the two groups with spinal fractures than in the controls ( P<0.001) but not in the group with peripheral fractures only. It was also lower in the cases with more than two spinal fractures than in those with two or less (P<0.001). In respect of peripheral fractures, the greatest age-adjusted absorption deficit was found in fractures of the humerus (35%) followed by hip (32%), spine (21%), wrist (19%), and rib 17% (all significant but not significantly different from each other). Lesser deficits in tibia, ankle and foot fractures were not significant but type 2 errors could not be excluded. We conclude that impaired calcium absorption is particularly associated with those fractures for which osteoporosis is a significant risk factor.

Strong, K+ -induced contractions of rat aorta in Ca-free, Mg-free media were not accompanied by increased intracellular calcium concentration, [Ca2+](i), whereas such contractions in the presence of the divalent cations were correlated with rising [Ca2+](i) as assessed by fura-2. At the same time, calcium channel blockers, a modulator of Ca2+-binding proteins, and a modulator of actin polymerization, inhibited all types of K+ -induced contractions. Increasing the K+ in isotonic medium evoked a rise of (45)Ca2+ binding to the plasma membrane of freshly isolated aortic cells. Although Ca2+ -dependent events underlie the mechanism of K+ -induced vascular contractions in both the presence and absence of Ca2+, in contrast to the view that [Ca2+](i) is a key regulator of excitation-contraction coupling in smooth muscle, we suggest that the modulation of Mg2+ -dependent Ca2+ binding, probably within/at the L-type calcium channel by K+, is a trigger for aortic contraction. This Ca2+ binding may then activate actin-myosin interaction.

Objective: To explore the mechanism of cytotoxic effects of the organophosphates (OPs) with delayed neurotoxicity on human neuroblastoma cells., Methods: The proliferation of neuroblastoma SH-SY5Y cells was determined by MTT spectrometry. (45)Ca uptake was determined by adding (45)CaCl(2) and tri-o-cresyl phosphate (TOCP) or methamidophos into the cultured medium for the SH-SY5Y cells. The cells were incubated and then lysed and finally counted in a Beckman LS 6000 liquid scintillation spectrometer., Results: Methamidophos stimulated the cell proliferation of SH-SY5Y at its lower concentrations (7 x 10(-7) mol/L to 7 x 10(-6) mol/L), with an increase by 28% at 7 x 10(-7) mol/L; however, it inhibited the proliferation at higher ones (7 x 10(-4) mol/L to 7 x 10(-3) mol/L) with 62% inhibition at 7 x 10(-3) mol/L. TOCP only inhibited the cell proliferation at high concentration (with 34% inhibition at 7 x 10(-3) mol/L) and markedly inhibited calcium uptake of the cells up to 55% at higher concentrations (1 x 10(-6) mol/L to 1 x 10(-4) mol/L); while the uptake was stimulated by OPs up to 241% of increase at lower concentrations (1 x 10(-9) mol/L to 1 x 10(-7) mol/L)., Conclusion: The interference of growth in nerve cells and disturbance of calcium homeostasis may be involved in the mechanisms of neurotoxicity of OPs.

The first criticality accident in Japan occurred in a uranium processing plant in Tokai-mura on September 30, 1999. The accident, which occurred while a large amount of enriched uranyl nitrate solution was being loaded into a tank, led to a chain reaction that continued for 20 h. Two workers who were pouring the uranium solution into the tank at the time were heterogeneously exposed to neutrons and gamma rays produced by nuclear fission. Analysis of dose distributions was essential for the understanding of the clinical course observed in the skin and organs of these workers. We developed a numerical simulation system, which consists of mathematical human models and Monte Carlo radiation transport programs, for analyzing dose distributions in various postures and applied the system to the dose analysis for the two workers. This analysis revealed the extreme heterogeneity of the doses from neutrons and gamma rays in the skin and body, which depended on the positions and postures of the workers. The detailed dose analysis presented here using color maps is indispensable for an understanding of the biological effects of high-dose exposure to a mixed field of neutrons and gamma rays as well as for the development of emergency treatments for victims of radiation exposure.

An international programme of work is currently under way to develop methods for calculating doses to infants from ingestion of radionuclides present in mothers' milk. This paper considers the special case of the alkaline earth elements. Models have been developed for 45Ca, 90Sr and 226Ra and the sensitivity of results to various changes in parameter values is discussed. A complication when calculating doses from intakes of radium is that the International Commission on Radiological Protection has previously recommended that doses from decay products of radium should be calculated using element-specific biokinetic models (so-called independent biokinetics). An extension of this method to the models for breastfeeding is proposed. Preliminary estimates of the doses received by the infant for a number of maternal intake scenarios show that doses to the infant can exceed the corresponding adult dose, such as for 45Ca (ratio = 3.1) while, in other cases such as 90Sr, the infant dose can be a significant fraction of the adult dose.

Numerous isoforms of plasmalemmal K-dependent (NCKX) and K-independent (NCX) Na+/Ca2+ exchangers are expressed in the brain. The physiological functions of each isoform are presently unknown. Therefore, in this study, we compared expression of NCKX and NCX transcripts between primary cultures of cerebellar granule cells, and astrocytes. Northern blot analysis showed that granule cells expressed NCKX2, NCKX3, NCKX4 and NCX3, whereas astrocytes expressed primarily NCX1. Consistent with this molecular characterization, a significant fraction of 45Ca2+ accumulation in Na-loaded granule cells, but not in astrocytes, depended on external K+. This is the first demonstration of native NCKX activity in neurons derived from the central nervous system. Our data suggest that NCKX isoform expression may correspond to the unique Ca2+ homeostasis requirements of neurons., (Copyright 2002 Lippincott Williams & Wilkins.)

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs), play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. They are composed of the GluRzeta subunit (NR1) combined with any one of four GluRepsilon subunits (GluRepsilon1-GluRepsilon4; NR2A-NR2D). Although the GluRzeta subunit exists in the majority of the CNS throughout all stages of development, the GluRepsilon subunits are expressed in distinct temporal and spatial patterns. In the present study, we investigated neuronal functions in mice lacking the embryonic GluRepsilon4 subunit. GluRepsilon4 mutant mice exhibited reductions of [(3)H]MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] binding and (45)Ca(2+) uptake through the NMDA receptors. The expression of GluRzeta subunit protein, but not GluRepsilon1 and GluRepsilon2 subunit proteins, was reduced in the frontal cortex and striatum of the mutant mice. A postmortem examination in GluRepsilon4 mutant mice revealed that tissue contents of norepinephrine, dopamine, serotonin, and their metabolites were reduced in the hippocampus and that dopamine, as well as serotonin, metabolism was upregulated in the frontal cortex, striatum, hippocampus, and thalamus. To clarify the phenotypical influences of the alteration in neuronal functions, performances in various behavioral tests were examined. GluRepsilon4 mutant mice showed reduced spontaneous locomotor activity in a novel environment and less sensitivity to stress induced by the elevated plus-maze, light-dark box, and forced swimming tests. These findings suggest that GluRepsilon4 mutant mice have dysfunctional NMDA receptors and altered emotional behavior probably caused by changes in monoaminergic neuronal activities in adulthood.

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures on filters, an acute transient increase (phase I response; triggered by P2Y(2) receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated by augmented calcium influx and protein kinase C-dependent increase in tight junctional resistance. The objective of the study was to determine the role of P2X(4) receptor as a mediator of phase II response. Human cervical epithelial cells express P2X(4) receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms by Northern blot analysis) and P2X(4) protein. Depletion of vitamin A reversibly downregulated P2X(4) receptor mRNA and protein and ATP-induced calcium influx. Depletion of vitamin A abrogated phase II response, and the effect could be partially reversed only with retinoic acid receptor (RAR)-selective retinoids but not retinoid X receptor (RXR) agonists. Depletion of vitamin A also abrogated protein kinase C increase in tight junctional resistance, and the effect could not be reversed with retinoids. Depletion of vitamin A also abrogated phase I increase in permeability and reversibly downregulated P2Y(2) receptor mRNA and ATP-induced calcium mobilization. However, in contrast to phase II response, both RAR and RXR agonists could fully reverse those effects. These results suggest that phase II response is mediated by a P2X(4) receptor mechanism.

This paper describes the development of a functional assay system to express crustacean epithelial electrogenic 2Na(+)/1H(+) antiporters in Xenopus laevis oocytes. Subsequent publications will use this assay method to establish nucleotide and amino acid sequence information about this transporter by functionally screening an hepatopancreatic cDNA library. In this method, oocytes were injected with hepatopancreatic mRNA (50 ng) isolated from Homarus americanus, while control oocytes received injections of an equivalent volume of distilled water. Three to five days post-injection, oocytes were incubated in media containing either (22)Na(+) or (45)Ca(2+) for specific time intervals and the rates of ion transfer into the oocytes were monitored under a variety of experimental conditions. Uptakes of both radiolabelled cations were stimulated by mRNA injection. mRNA-stimulated (22)Na(+) uptake was significantly (P < 0.05) inhibited by addition of calcium, amiloride, or by an antiporter-specific monoclonal antibody to the external medium. mRNA-stimulated (45)Ca(2+) uptake was significantly (P < 0.05) inhibited by addition of sodium, amiloride, cadmium, zinc, or by the antiporter-specific monoclonal antibody (also inhibitory for (22)Na(+) transport) to the external medium. The kinetics of (22)Na(+) influx in mRNA-injected oocytes were sigmoidal functions of external sodium concentration, exhibiting a Hill Coefficient (n) of approximately 3.0. Both calcium and amiloride significantly (P < 0.05) reduced sigmoidal sodium influx kinetics by alterations in the J(max) (amiloride) or K(Na) (calcium) of the transporter. Size fractionation of hepatopancreatic mRNA resulted in a single fraction that was most stimulatory for sodium and calcium transport and which likely contains the antiporter transcript. The results of this study provide the basis for using (22)Na(+) and (45)Ca(2+) transport assays of lobster mRNA-injected oocytes to functionally screen an hepatopancreatic cDNA library for clones that will provide full length nucleotide and amino acid sequences of the invertebrate electrogenic 2Na(+)/1H(+) antiporter protein., (Copyright 2001 Wiley-Liss, Inc.)

The NMDA subtype of glutamate receptor plays an important role in the molecular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calcium-dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cotransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cellular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immunoblot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C-terminal region. In vitro calpain digestion of fusion protein constructs containing the C-terminal region of NR2A yielded two cleavage sites at amino acids 1279 and 1330. Although it has been suggested that calpain cleavage of the NMDA receptor may act as a negative feedback mechanism, the current findings demonstrated that calpain cleavage did not alter [(125)I]MK801 binding and that receptors truncated to the identified cleavage sites had peak intracellular calcium levels, (45)Ca uptake rates and basal electrophysiological properties similar to wild type.

1,25(OH)(2)D(3) enhances parathyroid hormone (PTH)-induced Ca(2+) signaling in osteoblasts by activating plasma membrane voltage-sensitive Ca(2+) channels (VSCCs). The ability of 1,25(OH)(2)D(3) or the VSCC-activating analog AT (25-hydroxy-16-ene-23-yne-D(3)) to enhance parathyroid hormone-stimulated (45)Ca(2+) release from cultured new-born rat calvaria was measured. Analog BT (1,24-dihydroxy-22-ene-24-cyclopropyl-D(3)), that does not mobilize Ca(2+), also was tested along with PTH. Control experiments were performed with and without PTH and with and without serum. Individual calvaria labeled in utero with (45)Ca(2+) were cultivated in serum-free medium on filters at the medium/air interface of 24-well culture plates and (45)Ca(2+) release followed over 72 h. The results demonstrated that 1,25(OH)(2)D(3) and the Ca(2+)-mobilizing analog, AT, but not the nuclear receptor-binding analog, BT, enhanced PTH-stimulated (45)Ca(2+) release under serum-free conditions. This enhancement effect of the seco-steroids was not evident in the presence of 10% fetal calf serum. The effect of analog AT was faster than that of 1,25(OH)(2)D(3). Nitrendipine, a specific L-type VSCC blocker, attenuated enhancement by vitamin D compounds, indicating that the high-threshold L-type VSCC is a molecular transducer of costimulation. These results emphasize the synergy between the calcitropic hormones 1,25(OH)(2)D(3) and PTH in cultures containing osteoblasts and osteoclasts, and suggest that the Ca(2+)-mobilizing activity of 1,25(OH)(2)D(3) enhances Ca(2+) release from bone.

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium-calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called 'simplified analogues', where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium-calcium exchanger was screened on both cell types by measuring their effect on 45Ca(2+) uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N'-(1-naphtylmethyl)guanidine (IC(50)=3.4 microM) was found more active than amiloride (IC(50)=690 microM) and 3,4-dichlorobenzamil (IC(50)=15.2 microM), the reference inhibitor.

We examined the Ca2+-buffering function of the sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents that inhibit SR Ca2+-ATPase, and of ryanodine, which depletes SR Ca2+, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 4-week-old SHR and normotensive Wistar-Kyoto rats (WKY). Addition of CPA, thapsigargin or ryanodine to the resting state of the strips caused an elevation of cytosolic Ca2+ level and a contraction in both WKY and SHR. These responses were larger in SHR than in WKY. The contractions were inhibited strongly by 100 nM nifedipine or 3 microM verapamil and were abolished by Ca2+-free solution. Nifedipine, verapamil or Ca2+-free solution itself caused a relaxation from the resting state of SHR strips, but not from that of WKY strips. The resting Ca2+ influx in arteries measured by a 5-min incubation with 45Ca was significantly larger in SHR than in WKY. This influx was decreased by 10 microM CPA or 10 microM ryanodine in both WKY and SHR. These results suggest that in the resting state of the femoral artery from 4-week-old SHR, the greater part of the increased Ca2+ influx via L-type Ca2+ channels is buffered by Ca2+ uptake into the SR, while some Ca2+ reaches the myofilaments, resulting in the maintenance of resting tone.

To test the hypothesis that diabetes can selectively affect the intracellular and extracellular components of the noradrenergic vascular response in rats, we studied changes in blood pressure, in vitro vascular contraction and (45) Ca(2+) uptake in experimental diabetes induced by injection of 60 mg/kg of streptozotocin (STZ). One week after induction of diabetes mean blood pressure decreased significantly from 106 +/- 3 mmHg to 89 +/- 2 mmHg. After incubation in Ca(2+) =1.6 mM, contraction of STZ aortic rings to 10(- 7) M of norepinephrine was preserved in its intracellular component (Control: 231 +/- 28, STZ: 274 +/- 22 mgForce/mgTissue, NS) but depressed in its extracellular component (Control: 277 +/- 24, STZ: 133 +/- 33 mgForce/mgTissue, P<0.05). Uptake of (45) Ca(2+) in the same rings was depressed in both components. Norepinephrine contractions due to extracellular Ca(2+) (prior depletion of norepinephrine-sensitive Ca(2+) stores) unexpectedly exhibited a initial component whose magnitude in control rings was similar to the response due to intracellular Ca(2+) (extra: 503 +/- 65 mg, intra: 411 +/- 30 mgForce/mgTissue), and was not depressed in STZ preparations (399 +/- 62 mgForce/mgTissue). The sustained contraction to norepinephrine in extracellular Ca(2+) was significantly reduced in STZ aortas (1163 +/- 92 vs. 528 +/- 95 mgForce/mgTissue). We conclude that: 1) Short-term streptozotocin-induced diabetes features reduced blood pressure along with deficient aortic (45) Ca uptake and contraction to norepinephrine, and 2) Only the sustained phase of the norepinephrine contraction, dependent on extracellular Ca(2+), was depressed in the diabetic rats and could possibly be associated with the observed fall in blood pressure.

The rat is a nocturnal animal and uses its vibrissae extensively to navigate its environment. The vibrissae are linked to a highly organized part of the sensory cortex, called the barrel cortex which contains spiny neurons that receive whisker specific thalamic input and distribute their output mainly within the cortical column. The aim of the present study was to develop a method to evaluate glutamate receptor function in the rat barrel cortex. Long Evans rats (90-160 g) were killed by cervical dislocation and decapitated. The brain was rapidly removed, cooled in a continuously oxygenated, ice-cold Hepes buffer (pH 7.4) and sliced using a vibratome to produce 0.35 mm slices. The barrel cortex was dissected from slices corresponding to 8.6 to 4.8 mm anterior to the interaural line and divided into rostral, middle and caudal regions. Depolarization-induced uptake of 45Ca2+ was achieved by incubating test slices in a high K+ (62.5 mM) buffer for 2 minutes at 35 degrees C. Potassium-stimulated uptake of 45Ca2+ into the rostral region was significantly lower than into middle and caudal regions of the barrel cortex. Glutamate had no effect. NMDA significantly increased uptake of 45Ca2+ into all regions of the barrel cortex. The technique is useful in determining NMDA receptor function and will be applied to study differences between spontaneously hypertensive rats (SHR) that are used as a model for attention deficit disorder and their normotensive control rats.

We investigated the mechanisms underlying the increase in diazepam binding inhibitor (DBI) and its mRNA expression induced by nicotine (0.1 microM) exposure for 24 h using mouse cerebral cortical neurons in primary culture. Nicotine-induced (0.1 microM) increases in DBI mRNA expression were abolished by hexamethonium, a nicotinic acetylcholine (nACh) receptor antagonist. Agents that stabilize the neuronal membrane, including tetrodotoxin (TTX), procainamide (a Na(+) channel inhibitor), and local anesthetics (dibucaine and lidocaine), dose-dependently inhibited the increased expression of DBI mRNA by nicotine. The nicotine-induced increase in DBI mRNA expression was inhibited by L-type voltage-dependent Ca(2+) channel (VDCC) inhibitors such as verapamil, calmodulin antagonist (W-7), and Ca(2+)/calmodulin-dependent protein kinase II (CAM II kinase) inhibitor (KN-62), whereas P/Q- and N-type VDCC inhibitors showed no effects. In addition, nicotine exposure for 24 h induced [3H]nicotine binding to the particulate fractions of the neurons with an increased B(max) value and no changes in K(d). Under these conditions, the 30 mM KCl- and nicotine-induced 45Ca(2+) influx into the nicotine-treated neurons was significantly higher than those into non-treated neurons. These results suggest that the nicotine-stimulated increase in DBI mRNA expression is mediated by CAM II kinase activation resulting from the increase in intracellular Ca(2+) through L-type VDCCs subsequent to the neuronal membrane depolarization associated with nACh receptor activation.

Polyethylene wear debris generated at the bearing surfaces of total artificial hip joints is thought to play an important role in the periprosthetic osteolysis and ultimately the aseptic loosening of these prostheses. The macrophage is believed to be central to this process by releasing various cytokines and other mediators of osteolysis upon phagocytosis of the polyethylene wear debris. This study evaluated the in vitro bone resorption response of C3H murine peritoneal macrophages to clinically relevant GUR 1120 polyethylene particles. Macrophages were co-cultured in vitro with GUR 1120 particles with a mean size of 0.24, 0.45, 1.71, and 7.62, and GUR 1120 polyethylene resin with a mean size of 88 microm at various particle volume (microm)(3): macrophage ratios (0.1:1; 1:1; 10:1; and 100:1). The conditioned supernatants were incubated with (45)calcium radio-labeled mouse calvariae, and bone resorption was measured as (45)calcium release. The results showed that the 0.24 microm particles stimulated the macrophages to generate bone resorbing activity at a ratio of 10(microm)(3) per macrophage. The 0.45 and 1.71 microm particles were active at a ratio of 100( microm)(3) per macrophage, and the 7.62 and 88 microm particles were inactive at all the doses tested. The co-culture supernatants were also assayed for TNF-alpha, IL-1beta, IL-6, and PGE(2). The results followed the same trend for particle size and volume dose to that observed for the bone resorbing activity. This study has demonstrated, for the first time, the importance of size and dose of clinically relevant polyethylene particles on the osteolytic response of macrophages in vitro.

Retention of intravenously or orally administered 47Ca in the human body are described by a two-parameter function. It is then sufficient to make only a few whole-body measurements to determine the retention function, avoiding faeces sampling and stool markers. Seven days after intake the non-absorbed calcium was excreted and the model agreed with the measured relative retention. Absorption of calcium could then, in some cases (e.g. comparative studies), be described by relative retention at the 7th day after intake.

Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphate (A2P4), were recently proposed to participate in the stimulus-secretion coupling for nutrient-stimulated insulin release. Since NaF, an inhibitor of inorganic pyrophosphatase, was reported to lower A2P3 and A2P4 content in glucose-stimulated pancreatic islets, its effects upon metabolic, cationic, biosynthetic and secretory variables in rat pancreatic islets were investigated in the present study. Up to a concentration close to 0.1 mM, NaF failed to affect most of these variables, except for a decrease in 45Ca net uptake. Much higher concentrations of NaF (e.g. 5.0 mM) were required to cause inhibition of the metabolic, ionic, biosynthetic and secretory responses of the islets to nutrient secretagogues. Yet, even at this high concentration, NaF failed to lower the islet content in tritiated A2P3 and A2P4 in islets prelabelled with [2,8-3H]adenosine and failed to prevent the glucose-induced increase in such a content. It is concluded, therefore, that NaF may not represent a suitable tool to assess the participation of diadenosine polyphosphates in the process of nutrient-induced insulin secretion.

The crosstalk between 3', 5'-cyclic adenosine monophosphate (cAMP), intracellular calcium, and histamine release in rat mast cells using the stimulatory effect of three different drugs, thapsigargin, sodium fluoride (NaF), and compound 48/80 were studied. Each of these drugs induces histamine release by different mechanisms. The transducting pathways modulating cAMP and intracellular calcium levels were modified by using, cholera toxin (CTX) which ADP-rybosylates Gs-protein, pertussis toxin (PTX) which ADP-rybosylates Gi-protein, and okadaic acid (OA) which inhibits phosphatases 1 and 2a. Our results show that CTX increased cAMP levels and inhibited histamine release elicited by thapsigargin and compound 48/80. The inhibitory effect of CTX on histamine release was potentiated by OA in the presence of compound 48/80 but was decreased in the presence of thapsigargin. Calcium uptake was stimulated by NaF and compound 48/80. The previous treatment with OA increased calcium uptake when combined with compound 48/80 but not with NaF. Treatment with NaF highly stimulated calcium uptake and cAMP levels only when combined with OA and CTX. These results suggest that the modulatory effect of intracellular calcium and cAMP on histamine release depend more on the crosstalk of the activated signal transducting pathway than on the final level of calcium or cAMP, further supporting the theory that rat mast cells are divided into functionally distinct compartments.

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.

In the absence of any other exogenous nutrient, D-fructose stimulates insulin release from rat pancreatic islets provided that it is tested at high concentrations in excess of a threshold value close to 80 mM, an optimal secretory response being recorded at 240 mM. In the present study, the cationic determinants of the insulinotropic action of D-fructose, used at the latter high concentration, were explored in perifused rat islets that had been prelabelled with either 86Rb or 45Ca. The changes in 86Rb outflow and 45Ca efflux evoked by 240 mM D-fructose were comparable to those caused by 11.1 mM D-glucose in that both hexoses inhibited 86Rb and 45Ca outflow and, at normal Ca2+ concentration, caused a secondary rise in 45Ca efflux. These cationic changes coincided with stimulation of insulin release. The major differences between the two series of experiments consisted, in the islets exposed to D-fructose, in the occurrence of an early and transient increase in 45Ca efflux at normal extracellular Ca2+ concentration, a secondary reascension in 86Rb outflow and a dramatic off-response in both 86Rb and 45Ca outflow as well as insulin release. These phenomena were also observed in islets exposed to 240 mM 3-O-methyl-D-glucose, suggesting that they may be linked to the massive influx (or efflux) of monosaccharides, as possibly accompanied by Na+ inward co-transport, mobilization of Ca2+ from intracellular stores and activation of voltage- and/or Ca(2+)-sensitive K+ channels. This interpretation was supported by the finding that, at high concentrations (80.0 mM) of D-glucose or D-mannose, the aldohexoses also provoked a reascension in 86Rb outflow and off-response in insulin release. The cationic determinants of the insulinotropic action of D-fructose, in high concentration (240 mM), thus appear similar, if not identical, to those currently incriminated in the stimulation of insulin release by D-glucose.

Unlabelled: 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.

Washing is usually used to reduce the risk of a skin contamination. Skin biopsies were used and contaminations were realised with 1) soluble radioactive calcium 2) insoluble particles of uranium. The result obtained with different wash liquids are presented here. Water and soap had no effect but hypertonic DTPA was the better therapy to remove uranium from skin.

We studied the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultures enriched (96.4+/-0.4%) in rat cholinergic amacrine-like neurons, as determined by labeling with an antibody against choline acetyltransferase. A small population of these cells also contained GABA. Using these cultures we observed that both [3H]ACh release, which was largely Ca2+-dependent, and 45Ca2+ influx, evoked by depolarization with 50 mM KCl, were increased when adenosine A1 receptor activation was prevented by removal of endogenous adenosine with adenosine deaminase, or by application of the A1 receptor antagonist DPCPX. Our results indicate that, in cultured rat amacrine-like neurons, the activation of A1 receptors decreases calcium influx and, thereby, inhibits [3H]ACh release.

We investigated the effect of domoate, kainate and AMPA on 45Ca2+ uptake and on metabolic activity of cultured chick amacrine-like cells, as measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Domoate and kainate stimulated 45Ca2+ uptake and decreased MTT reduction, in a LY 303070-sensitive manner. AMPA caused a small increase on 45Ca2+ uptake, but it was without effect on MTT reduction. AMPA reduced both the 45Ca2+ entry and neurotoxicity induced by kainate, and cyclothiazide enhanced both the 45Ca2+ entry and neurotoxicity induced by AMPA. The results indicate that the AMPA receptors are the non-NMDA glutamate receptors involved in excitotoxicity.

Diffusion in the extracellular space (ECS) is important in physiologic and pathologic brain processes but remains poorly understood. To learn more about factors influencing tissue diffusion and the role of diffusion in solute-tissue interactions, particularly during cerebral ischemia, we have studied the kinetics of several radiotracers in control and hypoxic 450-microm hippocampal slices and in 1,050-microm thick slices that model the ischemic penumbra. Kinetics were analyzed by nonlinear least squares methods using models that combine extracellular diffusion with tissue compartments in series or in parallel. Studies with 14C-polyethylene glycol confirmed prior measurements of extracellular volume and that ECS shrinks during ischemia. Separating diffusion from transport also revealed large amounts of 45Ca that bind to or enter brain as well as demonstrating a small, irreversibly bound compartment during ischemia. The rapidity of 3H2O entry into cells made it impossible for us to distinguish intracellular from extracellular diffusion. The diffusion-compartment analysis of 3-O-methylglucose data appears to indicate that 5 mmol/L glucose is inadequate to support glycolysis fully in thick slices. Unexpectedly, the diffusion coefficient for all four tracers rose in thick slices compared with thin slices, suggesting that ECS becomes less tortuous in the penumbra.

Pool sizes and internal fluxes of minerals can be quantitatively determined by tracer-kinetic investigations and compartmental analysis. Investigations performed in 252 patients revealed characteristic patterns for calcium pools and fluxes with respect to disease. The method provides a useful diagnostic tool in the early detection of metabolic bone disorders and in problems of differential diagnostics.

Disturbance of calcium homeostasis and unregulated increase in intracellular calcium have been implicated in cell damage and cell death in the central nervous system in particular. To determine the specific pathway(s) of cerebral Ca transport of importance in a pathophysiological situation, we have measured long term Ca flux in brain in vivo in rat, and developed a kinetic model incorporating physiologically relevant pathways of cerebral Ca transport. 45Ca was injected into a tail vein in conscious rats. Plasma 45Ca was monitored up to 4 days post-injection and 45Ca uptake determined in samples of cerebrospinal fluid (CSF) and at several sites of brain at euthanasia. Uptake of the tracer by tissues peaked after 1 h, isotopic equilibration taking longer. The uptake at hippocampus was the highest. Computer simulation of the kinetics of the plasma, CSF and tissue data was performed using a compartmental model, which included two subcompartments (intra- and extra-cellular) and two pathways from plasma to the brain: directly across the blood-brain barrier and via CSF, which included a delay. The analysis based on this model enabled estimation of the fractional rates of transport of Ca to cerebral and noncerebral tissues across all the barriers of the model.

In an attempt to evaluate the acute effect of PRL on bone turnover, experiments were performed in sexually mature female Wistar rats which were given an intraperitoneal (i.p.) administration of 1.25 mM CaCl2 solution containing 6 microCi 45Ca. Dose response study showed a two fold higher rate of 45Ca uptake in 30 min by control trabecular bones (sternum and vertebrae 5-6) compared with control compact bones (femur and tibia). Femur exhibited a dose dependent increase in 45Ca uptake in response to pharmacological doses of 0.01 and 0.02 mg PRL/100 g body weight but was less responsive to 0.04 mg PRL/100 g body weight. Tibia only responded with increased 45Ca uptake to 0.02 and 0.04 mg PRL/100 g body weight while trabecular bones were unresponsive. By varying the intervals between administration of 45Ca (0 min) and PRL (0, 30 or 60 min) and bone harvesting (30, 60 or 90 min), it was found that 0.01 mg PRL/100 g body weight had a biphasic action on 45Ca movement. It initially enhanced 45Ca influx into femur by 30 min followed by an accelerated 45Ca efflux from femur back to the circulation. It could be concluded that PRL acutely stimulated calcium turnover in compact bone.

Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on 45Ca2+ uptake and glutamate release. 45Ca2+ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK-801. K+ depolarization-induced 45Ca2+ uptake was also reduced by MK-801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the 45Ca2+ uptake induced by depolarization. Opioid agonists, selective for mu-, kappa-, and delta-receptors, inhibited the 45Ca2+ uptake induced by K+ depolarization. The combination of low concentrations of MK-801 and opioid agonists resulted in additive inhibition of K(+)-induced 45Ca2+ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca2+ uptake under acute, and perhaps also chronic, opiate treatment.

The effects of the imidazole compound SK&F 96365 on Ca2+ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca2+ gradient, 30 microM SK&F 96365 doubled [Ca2+]i suggesting a Ca2+ release from intracellular stores. The SK&F 96365-induced [Ca2+]i rise was not accompanied by detectable NO and vWF production, while 1 microM thapsigargin enhanced [Ca2+]i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 +/- 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca2+]i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca2+ from internal pools, its effect and that of thapsigargin on the ATP-dependent 45Ca2+ uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of 45Ca2+ uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from the extracellular space, the [Ca2+]i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca2+ influx in endothelial secretion processes.

The infusion of physiological salt solution into the lumen of ring segments of the isolated rabbit facial vein induces an increase in vessel wall tone. Changes in 22Na+ and 45Ca2+ net uptake as well as 22Na+ unidirectional efflux in response to flow in this vessel were compared to the response to histamine (1 microM) and to angiotensin II (0.1 microM). 22Na+ net uptake per unit force developed in response to flow was 7.9-fold greater than that for histamine (1 microM) and twice that for angiotensin II (0.1 microM). In comparison, 45Ca2+ net uptake per unit force in response to flow, histamine and K+ were similar. Flow-induced contraction and the related 45Ca2+ and 22Na+ net uptake were decreased by amiloride (30 microM) and methyl isobutyl amiloride (30 microM) without changes in 45Ca2+ or 22Na+ net uptake per unit force. Ouabain (10 microM) enhanced flow-induced contraction and the related 22Na+ net uptake. Both ouabain (10 microM) and Bay K 8644 (1 microM) increased flow-induced contraction and the related 45Ca2+ net uptake whereas they decreased the 45Ca2+ net uptake per unit force. Flow, histamine and angiotensin II increased the unidirectional efflux of 22Na+. This increase in 22Na+ unidirectional efflux was attenuated by ouabain (10 microM). These experiments demonstrate that the increases in wall force due to flow, histamine and angiotensin II are associated with increases in 22Na+ net uptake and unidirectional efflux. Flow preferentially promotes the entry of Na+, compared to histamine and angiotensin II.

An in vivo bioassay based on 45Ca uptake from the ambient medium was used to test the efficacy of serum from rabbits immunized against trout stanniocalcin to passively immunize trout, tilapia, American eel, and guppy against endogenous stanniocalcin. The passive immunization was effective in all species. The fact that this procedure worked under both homologous and heterologous conditions, and in fish from different taxonomic infradivisions, is consistent with the view that the stanniocalcins in the four species examined share common antigenic determinants. The trout stanniocalcin antiserum had no effect on whole body calcium uptake (inCa2+) in stanniectomized eels, indicating that the effect of the antiserum was dependent on the presence of functional Stannius corpuscles. The technique was then used to show that the inhibitory effects that calcium loading and the injection of the cholinoreceptor agonist carbachol have on inCa2+ probably involve a catecholamine-induced release of endogenous stanniocalcin from the Stannius corpuscles.