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CONTEXT: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic and neurologic diseases. OBJECTIVE: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein. MATERIALS AND METHODS: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity. RESULTS: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity. DISCUSSION: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases. CONCLUSION: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.

Whole-tree harvest (WTH) may compromise tree productivity and health and lead to soil and surface water acidification. The aim of this study was to assess the long-term change (up to circa 40 years) in soil exchangeable calcium (Ca 2+ ) pools following conventional (CH) and WTH at three Swedish coniferous sites. A second aim was to evaluate how well the results could be reproduced by the dynamic model MAGIC (Model of Acidification of Groundwater in Catchments). Soil Ca 2+ pools (down to 20 cm) decreased at all three sites from stand age 15–16 to 37–38 years. The depletion ranged from 2.6 to 8.6 kEq ha −1 (26.5–52.7%) and 0.2 to 5.0 kEq ha −1 (2.3–49.1%) in the CH and WTH treatment, respectively. The presence of an interaction effect indicated that the main effect of time was not statistically significant at all three sites. Over the course of time, soil Ca 2+ pools have also become more similar between the CH and WTH treatments, but the Ca 2+ pools were still significantly lower ( p 2+ pools were generally greater than what has been found in other studies and were largely explained by high soil Ca 2+ availability and high tree Ca 2+ uptake, especially in the CH-plots, as indicated by the MAGIC mass balance budgets. Model simulations by MAGIC partly agreed with the measured data. However, the model exaggerated the soil Ca 2+ losses between 1990 and 2013 (CH = 3.6–9.9 kEq ha −1 ; WTH = 3.0–8.3 kEq ha −1 ), especially at the spruce sites. Furthermore, MAGIC could not reproduce the rapid diminishing differences between CH and WTH. Uncertainties in model parameters, underestimated soil Ca 2+ pools or biological feed-back mechanisms could explain this discrepancy. Until these have been resolved, interpretations of Ca 2+ changes related to CH or WTH using dynamic modelling or mass balance budget calculations should be done with caution.

In modern laying hybrids, calcium (Ca) homeostasis is immensely challenged by daily eggshell calcification. However, excessive mobilization of Ca from bones may lead to osteoporosis, which then manifests in a high incidence of poor bone quality. The aim of this study was to characterize the hens&rsquo, adaptation response to an alternating dietary Ca restriction. The animal model consisted of four purebred layer lines, differing in laying performance (high vs. moderately performing lines) and phylogenetic origin (white- vs. brown-egg lines). According to the resource allocation theory, hens selected for high egg production were assumed to show a different response pattern to cope with this nutritive challenge compared to moderately performing lines. Data collected included egg number, egg quality traits, body weight and bone characteristics. The Ca depletion led to a temporary drop in egg production and shell quality and a loss of bone stability due to Ca mobilization. The white-egg lines response was more pronounced, whereas the brown-egg lines were less sensitive towards reduced Ca supply. Our study shows that the hens&rsquo, responsiveness to coping with a nutritive Ca depletion is not ultimately linked to genetic selection for increased egg production but rather to phylogenetic origin.

Gammarus fossarum is an often-abundant crustacean detritivore that contributes importantly to leaf litter breakdown in oligotrophic, mainly heterotrophic, headwater streams. This species requires large amounts of Ca to moult, thus allowing growth and reproduction. Because resource quality is tightly coupled to the organism’s growth and physiological status, we hypothesised that low Ca concentration [Ca] and low food resource quality (low phosphorus [P] and/or reduced highly unsaturated fatty acid [HUFA] contents) would interactively impair molecular responses (gene expression) and reproduction of G. fossarum. To investigate the effects of food resources quality, we experimentally manipulated the P content of sycamore leaves and also used diatoms because they contain high amounts of HUFAs. Three resource quality treatments were tested: low quality (LQ, unmanipulated leaves: low P content), high quality 1 (HQ1; P-manipulated leaves: high P content), and high quality 2 (unmanipulated leaves supplemented with a pellet containing diatoms: high P and HUFA content). Naturally, demineralised stream water was supplemented with CaSO4 to obtain three Ca concentrations (2, 3.5, and 10.5 mg Ca L−1). For 21 days, pairs of G. fossarum were individually exposed to one of the nine treatments (3 [Ca] × 3 resource qualities). At the individual level, strong and significant delays in moult stage were observed in gammarids exposed to lower [Ca] and to lower resource quality, with additive effects lengthening the duration of the reproductive cycle. Effects at the molecular level were investigated by measuring expression of 12 genes involved in energy production, translation, or Ca or P homeostasis. Expression of ATP synthase beta (higher in HQ2), calcified cuticle protein (higher in HQ1 and HQ2), and tropomyosin (higher in HQ2 compared to HQ1) was significantly affected by resource quality, and significant additive effects on Ca transporting ATPase expression were induced by [Ca] and resource quality (higher for low [Ca] and higher resource quality). These results highlight the potential drastic deleterious effects of water [Ca] depletion on G. fossarum physiology, populations, and ecosystem functioning, especially in oligotrophic environments.

Flax seedlings grown in the absence of environmental stimuli, stresses and injuries do not form epidermal meristems in their hypocotyls. Such meristems do form when the stimuli are combined with a transient depletion of calcium. These stimuli include the "manipulation stimulus" resulting from transferring the seedlings from germination to growth conditions. If, after a stimulus, calcium depletion is delayed, meristem production is also delayed; in other words, the meristem-production instruction can be memorised. Memorisation includes both storage and recall of information. Here, we focus on information recall. We show that if the first transient calcium depletion is followed by a second transient depletion there is a new round of meristem production. We also show that if an excess of calcium follows calcium depletion, meristem production is blocked; but if the excess of calcium is in turn followed by another calcium depletion, again there is a new round of meristem production. The same stored information can thus be recalled repeatedly (at least twice). We describe a conceptual model that takes into account these findings.

Removal of Ca2+ from the incubation medium of cultured rat cardiac fibroblasts causes cellular morphological changes, such as the formation of blebs, the ballooning of the cell membrane and the detachment from the culture dish. A 24 hr preincubation with 20 mM taurine blocked the Ca2+ depletion-induced detachment of the cardiac fibroblasts. However, taurine treatment did not prevent other morphological changes induced by Ca2+ depletion. The data suggest that taurine plays an important role in cell adhesion in the heart.

The structural consequences of calcium depletion of Photosystem II (PS II) by treatment at pH 3.0 in the presence of citrate has been determined by Mn K-edge X-ray absorption spectroscopy. X-ray absorption edge spectroscopy of Ca-depleted samples in the S1′, S2′, and S3′ oxidation states reveals that there is Mn oxidation on the S1′–S2′ transition, although no evidence of Mn oxidation was found for the S2′–S3′ transition. This result is in keeping with the results from EPR studies where it has been found that the species oxidized to give the S3′ broad radical signal found in Ca-depleted PS II is tyrosine Yz. The S2′ state can be prepared by two methods: illumination followed by dark adaptation and illumination in the presence of DCMU to limit to one turnover. Illumination followed by dark adaptation was found to yield a lower Mn K-edge inflection-point energy than illumination with DCMU, indicating vulnerability to reduction of the Mn complex, even over the relatively short times used for dark adaptation (~15 min). EXAFS measurements of Ca-depleted samples in the three modified S states (referred to here as S′ states) reveals that the Fourier peak due to scatterers at ~3.3 Å from Mn is strongly diminished, consistent with our previous assignment of a Ca-scattering contribution at this distance. Even after Ca depletion, there is still significant amplitude in the third peak, further supporting our conclusions from earlier studies that the third peak in native samples is comprised of both Mn and Ca scattering. The Mn–Mn contributions making up the second Fourier peak at ~2.7 Å are largely undisturbed by Ca-depletion, but there is some evidence that S1′-state samples contain significant amounts of reduced Mn(II), which is then photooxidized in the preparation of higher S′ states.

A 5 min exposure of photosystem II to a pH 3 citric acid solution is a simple method for selective removal of Ca(2+) from the O(2)-evolving complex. The resulting preparation retains the 23 and 17 kDa extrinsic polypeptides, but the activity of this material is only 10-20% of that of an untreated control sample. Biochemical characterization of citrate-treated photosystem II reveals that some reaction centers lose the extrinsic proteins during citrate treatment. Furthermore, a comparison of photosystem II preparations treated with citrate, or depleted of 23 and 17 kDa extrinsic polypeptides by high-salt treatment, shows that low concentrations of a small reductant, NH(2)OH, which has little effect on the activity of intact photosystem II, can reduce and inhibit the Mn cluster in both types of preparations. In contrast, a large reductant, hydroquinone, cannot access the majority of O(2)-evolving centers in citrate-treated preparations, while 23 and 17 kDa-depleted material is rapidly inactivated by the reductant. Incubation of the citrate-treated samples in high ( approximately 60 mM) concentrations of CaCl(2) restores 50% of the lost activity; this Ca(2+)-reconstituted activity is chelator-insensitive, indicating that rebinding of Ca(2+) restores the structural integrity of the O(2)-evolving complex. A characterization of Ca(2+) and Cl(-) affinities in steady-state activity assays shows that citrate-treated preparations exhibit a Cl(-) requirement similar to that of polypeptide-depleted photosystem II, while Ca(2+) reactivation of O(2) evolution appears to occur at two structurally distinct sites. One site exhibits a high Ca(2+) affinity, similar to that found in polypeptide-depleted samples, but a second, lower-affinity site also exists, with a K(M) that is approximately 10 times greater than that of the high-affinity site, which is associated with centers that retain the extrinsic polypeptides. These data indicate that citrate-induced Ca(2+) depletion causes release of the 23 and 17 kDa extrinsic polypeptides from some photosystem II reaction centers, and also modifies the structure of the polypeptide-retaining O(2)-evolving centers so that the Mn cluster is exposed to small, but not large, reductants. This change may be due to subtle modifications to the structure of the photosystem II extrinsic proteins that produces a new pathway between the solvent and the Mn cluster or, alternatively, to the opening of an existing channel in the intrinsic lumenal polypeptide domain, between the solvent and the Mn cluster, that is normally occluded by a bound Ca(2+) atom.

Numerous anthropogenic factors can deplete calcium (Ca) from forest ecosystems. Because an adequate supply of Ca is needed to support fundamental biological functions, including cell membrane stability and stress response, the potential for Ca deficiency following the individual, cumulative, or potentially synergistic, influences of anthropogenic factors raises important questions concerning organism and ecosystem health. Past work has shown that one Ca-depleting factor (foliar acid mist exposure) reduces concentrations of biologically important membrane-associated Ca (mCa) from red spruce foliar cells, destabilizes these cells, and results in their increased susceptibility to the freezing injury responsible for red spruce decline in northeastern U.S. montane ecosystems. Data presented here indicate that these same disruptions can occur for other tree species and that soilbased Ca manipulation can also alter critical mCa pools. Considering the unique role Ca plays in the physiological response of cells to environmental change and stress, we hypothesize that depletion of biologically available Ca (e.g., mCa) could result in a scenario similar to recognized immune deficiency syndromes in animals. A hypothetical pathway through which anthropogenically induced Ca deficiencies could predispose plants, and possibly animals, to exaggerated injury following exposure to environmental stress is presented, and the potential implications of this scenario to ecosystem health are discussed.

Photosystem II (PS II) evolves oxygen from two bound water molecules in a four-stepped reaction that is driven by four quanta of light, each oxidizing the chlorophyll moiety P680 to yield P+680. When starting from its dark equilibrium (mainly state S1), the catalytic center can be clocked through its redox states (S0ellipsisS4) by a series of short flashes of light. The center involves at least a Mn4-cluster and a special tyrosine residue, named YZ, as redox cofactors plus two essential ionic cofactors, Cl- and Ca2+. Centers which have lost Ca2+ do not evolve oxygen. We investigated the stepped progression in dark-adapted PS II core particles after the removal of Ca2+. YZ was oxidized from the first flash on. The difference spectrum of YZ-->YoxZ differed from the one in competent centers, where it has been ascribed to a hydrogen-bonded tyrosinate. The rate of the electron transfer from YZ to P+680 was slowed down by three orders of magnitude and its kinetic isotope effect rose up from 1.1 to 2.5. Proton release into the bulk was now a prerequisite for the electron transfer from YZ to P+680. On the basis of these results and similar effects in Mn-(plus Ca2+-)depleted PS II (M. Haumann et al., Biochemistry, 38 (1999) 1258-1267) we conclude that the presence of Ca2+ in the catalytic center is required to tune the apparent pK of a base cluster, B, to which YZ is linked by hydrogen bonds. The deposition of a proton on B within close proximity of YZ (not its release into the bulk!) is a necessary condition for the reduction in nanoseconds of P+680 and for the functioning of water oxidation. The removal of Ca2+ rises the pK of B, thereby disturbing the hydrogen bonded structure of YZB.

Aim: Cardiac basal metabolism upon extracellular calcium removal and its relationship with intracellular sodium and calcium homeostasis was evaluated. Methods: A mechano-calorimetric technique was used that allowed the simultaneous and continuous measurement of both heat rate and resting pressure in arterially perfused quiescent adult rat hearts. Using pharmacological tools, the possible underlying mechanisms related to sodium and calcium movements were investigated. Results: Resting heat rate (expressed in mW g-1 dry wt) increased upon calcium withdrawal (+4.4 ± 0.2). This response was: (1) unaffected by the presence of tetrodotoxin (+4.3 ± 0.6), (2) fully blocked by both, the decrease in extracellular sodium concentration and the increase in extracellular magnesium concentration, (3) partially blocked by the presence of either nifedipine (+2.8 ± 0.4), KB-R7943 (KBR; +2.5 ± 0.2), clonazepam (CLO; +3.1 ± 0.3) or EGTA (+1.9 ± 0.3). The steady heat rate under Ca2+-free conditions was partially reduced by the addition of Ru360 (-1.1 ± 0.2) but not CLO in the presence of EGTA, KBR or Ru360. Conclusion: Energy expenditure for resting state maintenance upon calcium withdrawal depends on the intracellular rise in both sodium and calcium. Our data are consistent with a mitochondrial Ca2+ cycling, not detectable under normal calcium diastolic levels. The experimental condition here analysed, partially simulates findings reported under certain pathological situations including heart failure in which mildly increased levels of both diastolic sodium and calcium have also been found. Therefore, under such pathological conditions, hearts should distract chemical energy to fuel processes associated with sodium and calcium handling, making more expensive the maintenance of their functions. Fil: Bonazzola, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina Fil: Takara, Delia. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Male Lewis rats (n = 27) were fed a nonpurified diet containing 0.9% calcium, 0.7% phosphorus, and 0.005% zinc until 8 wk of age. At this time rats were assigned randomly to one of two groups. Both groups were fed a low calcium, low zinc, purified diet (0.2% calcium, 0.4% phosphorus, less than 0.00007% zinc), but one group was fed 1.78 mg Zn/(animal.d). The zinc-replete animals were individually matched by weight to the zinc-depleted animals and pari-fed. Balances and plasma concentrations of zinc, calcium, and phosphorus and parathyroid hormone, 25 hydroxycholecalciferol [25(OH)D] and 1,25-dihydroxycholecalciferol [1,25(OH)2D] were determined at the start of calcium depletion and 2 wk later. Calcium and 25(OH)D levels were lower in both groups after calcium depletion. Dietary zinc had no significant effect on calcium or 25(OH)D levels. Phosphorus concentrations were lower after calcium depletion, but phosphorus concentration was higher in the zinc-depleted compared with the zinc-replete group at the end of the experiment. 1,25(OH)2D increased in both groups, but was higher in the zinc-replete than the zinc-depleted group at the end of the experiment. Calcium and phosphorus balances were greater in the zinc-depleted group at the end of the experiment. We conclude zinc depletion diminishes the response of 1,25(OH)2D to calcium depletion in rats. The mechanism is unknown, but may involve nonhormonally mediated changes in gastrointestinal absorption of calcium and phosphorus or an affect of zinc on extraintestinal processes.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 μM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.Key words: calcium paradox, adenosine, frog heart.

Commentary to: Stathopulos PB, Zheng L, Li G-Y, Plevin MJ, Ikura M. Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry. Cell 2008; 135:110-22.AndLuik RM, Wang B, Prakriya M, Wu MM, Lewis RS. Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. Nature 2008; 454:538-42.AndPenna A, Demuro A, Yeromin AV, Zhang SL, Safrina O, Parker I, Cahalan MD. The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 2008; 456:116-20.

Previous studies in a weanling rat model indicated that dietary calcium depletion not only stimulated osteoclastic resorption but also inhibited bone formation. The present study sought to test whether the depletion-associated inhibition of bone formation is related to a reduction in serum insulin-like growth factor-I (IGF-I) and/or an increase in its binding proteins (IGFBPs). Twenty male weanling rats were divided into two weight-matched groups. The study group was subjected to a semisynthetic diet deficient in calcium (0.02% calcium) for 28 days, while the control group was pair-weighed on the same diet but containing 0.62% calcium. After the depletion phase, all rats were fed the same calcium-containing diet for an additional 14 days. Serum samples were obtained from each animal on a weekly basis and assayed for IGF-I and IGFBPs. During depletion, there was no statistically significant difference in serum IGF-I level between the study group and the control group. In contrast, the study group showed a statistically significant increase in several serum IGFBPs with apparent molecular size of 30-38 kD (IGFBP-3), 26-28 kD (IGFBP-1, -2, -5, and/or -6), and 24-25 kD (IGFBP-4), respectively, compared to the control group. There was no difference in nutritional intakes between the two groups of rats during depletion. During repletion, there was also no significant difference in serum IGF-I level between the control and study group. However, during the first 7 days of repletion, serum IGFBP-3 and the 26-28 kD IGFBP of the study group was significantly less than those of the control group, which then returned to the control level after 2 weeks of repletion. In summary: (1) calcium depletion in weanling rats increased several serum IGFBPs without an effect on IGF-I; and (2) calcium repletion induced an acute reduction in serum IGFBP-3. In conclusion, these findings represent the first evidence that the depletion-related inhibition of bone formation in the rat may be associated with an increase in several serum IGFBPs, which may act to inhibit the osteogenic actions of IGFs.

Female Wistar rats were separated into 9 groups, and 9 different synthetic diets (each diet contains different level of protein and calcium) were given to each group. After 5 weeks of these dietary regimens, all rats were sacrificed and calcium, magnesium, and phospho-rus levels in plasma and various tissues were determined. In calcium-deficient groups, calcium, magnesium, and phosphorus level in bone decreased, plasma calcium level decreased and there was a tendency that magnesium levels in brain and liver and phosphorus level in brain in-creased. When comparison was made among the calcium-deficient groups, calcium, magnesium, and phosphorus levels in bone were higher in low protein/calcium-deficient group than higher protein/calcium-deficient groups. It is probable that protein deficiency inhibits calcium depletion and consequently the influence of calcium deficiency is less significant in the condition of deficiency in both protein and calcium.

Intact perfused hearts subjected to calcium depletion and subsequent repletion undergo irreversible structural damage characterized by sarcolemmal disruption, hypercontracture, and the appearance of large electron dense deposits (thought to be calcium phosphate) within mitochondria. This structural damage is accompanied by increased intracellular Na+ and Ca++. Cultured embryonic chick heart cells subjected to the same conditions do not exhibit ultrastructural damage but do undergo similar ionic changes. This study characterizes the intracellular ionic changes associated with calcium repletion and assesses the role of mitochondria in the buffering of calcium.METHODS: Heart cells were obtained from 11-day-old chicken embryos and cultured as spherical aggregates for 3-4 days. The aggregates were then incubated at 37°C in HEPES buffered solution containing 1.3 mM Ca for 10 minutes (control period). Following this control period, the aggregates were incubated for 10 minutes in Ca-free HEPES buffer containing 1mM EGTA (depletion period). Finally, the aggregates were returned to normal, Ca-containing, HEPES buffer (repletion period).

Among patients exposed to hydrofluoric acid the potentially lethal effect of calcium depletion induced by binding with fluoride ion has not been well reported. Three patients exposed to hydrofluoric acid had acute fluoride poisoning with serum calcium levels equal to or below 4.1 mgm/dl. Treatment included administration of large amounts of calcium, both intravenously and by subsechar injection, to replenish the biologically active calcium and to bind fluoride. This report describes successful treatment of two survivors, apparently the first two, of severe hypocalcemia caused by hydrofluoric acid.

The role of Ca2+ in the in situ secretory response of rat colon and pig ileum was studied by chelation depletion of Ca2+ and by treatment with chlorpromazine. The effect of depleting lumenal Ca2+ by chelation and the effect of intraperitoneal administration of chlorpromazine were determined relative to colonic permeability and net fluid flux measured across the rat colon or pig ileum. Replacement of Ca2+ in the perfusate by 1.0 mM ethyleneglycol-(bis-beta-ethylaminoether) (EGTA) did not produce significant changes in the net absorptive fluid flux measured in the control state or in the net secretory fluid flux caused by secretory detergent agents. The concentration of EGTA used in the perfusate did not alter mucosal permeability. Nonsecretory bile acids or A23187 had no effect on net colonic fluid flux or on colonic permeability to mannitol in the rat. The known correlation between net fluid flux and increased colonic permeability to polar molecules has been confirmed for the secretory detergent compounds. Chlorpromazine pretreatment caused a partial reversal of net secretory fluid fluxes induced by deoxycholate and high concentrations (6.0 mM) of ricinolate and dioctyl sulfosuccinate without significantly altering mucosal permeability to mannitol. We conclude that depletion of lumenal Ca2+ is not an effective method for determining the possible Ca2+ dependence of these intestinal secretory events. The antisecretory actions of chlorpromazine may provide some indirect evidence for Ca2+ involvement in the secretory effects of the detergent class of laxative compounds. Permeability may be essential for secretion caused by these agents, but the driving force would appear to be provided by the active transfer of electrolytes from the blood to the lumen of the colon.

Epidemiologically, there is a remarkably consistent picture of the relationship between calcium depletion and the development of hypertension—apparently independent of weight, cigarette smoking, alcohol intake, and other risk factors. Typically, the inverse correlation between dietary calcium and blood pressure is more evident with systolic than with diastolic pressures.

In smooth muscle of guinea pig taenia coli, showing spontaneous activity, the curves of rhythmic changes in muscle tension appears parallel to the curves of changes in Po2 level in the medium (1-3). On the other hand, Saito et al. (3) reported that an application of high K (isotonically, 40 mM) caused an increase in oxygen consumption accompanying increase in tension change in taenia coli, and that this was in accordance with the concept (4, 5) that the K-induced tonic response resulted from a transmembrane transport of Ca that depended on the aerobic breakdown of carbohydrates. In the present paper, effects of Ca depletion on the oxygen consumption accompanying muscle tension change both spontaneous and that induced by high K were studied, and a dissociation of the changes in oxygen consumption and muscle tension change was attempted by substituting strontium (Sr) for Ca in the external medium. Some of the results have already been reported (6, 7).

Skin from Rana catesbeiana adults and tapoles was found to contain an unusually high concentration of inorganic phosphate which was chemically identified and histologically localized as dense granules of calcium phosphate deposited as a discrete layer at the junction between the stratum spongiosum and the stratum compactum of the dermis. Electron microscopic studies revealed that the granules were composed of fine needle-like crystals and were not associated with cells which seemed obviously related to their genesis. Although no definite function was established for this apparently normal structure of bullfrog skin, its possible role as a reservoir of readily available calcium was suggested. As such, it would represent an important factor to be considered in studies of the biochemistry and physiology of frog skin, especially those which deal with the effects of calcium depletion.

THE EFFECTS OF CALCIUM DEPLETION OF CARDIAC MUSCLE UPON THE RELATION BETWEEN STIMULUS PATTERN AND ACTION POTENTIAL, ANALYSED IN TERMS OF A MATHEMATICAL MODEL

THE retention of radiocalcium or radiostrontium in bones of humans (Spencer et al., 1961), rats (Cohn et al., 1961) and non-laying pullets (Hurwitz, 1965a), following a single intravenous dose, is reduced by the feeding of high-calcium diets. However, in the laying hen, the severe calcium depletion from bone during calcium restriction (Taylor and Moore, 1954; Hurwitz and Griminger, 1961) would suggest the possibility of reducing the body burden of bone-seeking radiosotopes by calcium restriction. It was the purpose of the present study to investigate the effects of variation in dietary calcium level and sources, and the timing of this variation, on the retention of calcium-45 and strontium-85 in various bone segments, and the deposition of these nuclides in the egg shell. METHODS AND RESULTS General procedure: The method for preparation of samples for analysis was given in detail in the preceding paper (Hurwitz and Bar, 1966). For calcium-45 determinations, calcium …

Calcium in rats' teeth was labeled with radioactive Ca-45 by feeding the animals a calcium-free diet and administering calcium as calcium-45 levulinate biweekly for eight weeks starting with a two weeks' old animal. The radioactive teeth were removed and the calcium deletion which resulted from immersion of the teeth in various liquids for twenty-Pour hours was determined by tracer methods. The liquids tested were distilled water, commercial mouthwashes, citric and tartaric acid solutions, beverages, milk, and human saliva.

Excised mouse thyroids incubated in Ca++-free medium were stimulated to release increased amounts of stable thyroxine. This stimulation of thyroxine release by incubated thyroid tissue was not additive with TSH or (Bu)2cAMP. It was reversed by norepinephrine through an alpha adrenergic receptor, similar to TSH or (Bu)2cAMP stimulation. Depletion of Ca++ did not result, however, in an increase in the concentration of cAMP in incubated thyroid glands, suggesting that its locus of action was subsequent to TSH stimulation or cAMP production.

The change in responsiveness to a-adrenergic stimulation and to calcium ions in amylase release from parotid glands of hypothyroid rats was compared with that in euthyroid rats. Calcium depletion and addition of verapamil or W-7 in the medium caused a decrease in methoxamine-induced amylase release in both eu- and hypothyroid rats. Moreover, addition of calcium ions to Ca2+-free medium markedly increased the methoxamine-induced amylase release in proportion to its concentration in hypothyroid rats. These results suggest that calcium ions play an important role in methoxamine-induced amylase release from parotid glands of hypothyroid rats as well as those of the euthyroid ones.

1 Administration of a single dose of morphine sulphate (25 mg/kg) induces tolerance to calcium depletion lasting seven days. 2 There are no apparent changes in calcium content in any of eight discrete brain regions throughout this seven day period. 3 Pretreatment with reserpine (5 mg/kg) did not alter the ability of morphine to induce tolerance. Reserpine alone produced no tolerance to its own calcium depleting action. 4 Cycloheximide (500 mug/kg) but not chloramphenicol (200 mg/kg) effectively prevented development of tolerance. 5 It is concluded that the induction of tolerance to calcium depletion seen after morphine may involve changes in various proteins in membranes of synaptic origin.

Our interest in calcium currents arose from our intention to approach an understanding of excitation-contraction coupling in muscle fibers from Balanus (Bacigalupo et al., 1979; Hidalgo et al., 1979; Luxoro and Nassar-Gentina, 1984). For a full account of recent developments on the subject, the student is referred to recent reviews (Reuter, 1979; Hagiwara and Byerly, 1981; Tsien, 1983; Kostyuk, 1984).

Lowering extracellular calcium in cultures of human diploid fibroblast-like cells caused a rapid depletion of NAD pools. This loss of NAD was reversed by restoring extracellular Ca2+ and was inhibited by 3-aminobenzamide, an inhibitor of ADP-ribosyl transfer reactions. The concentrations of 3-aminobenzamide needed to inhibit the loss of NAD were consistent with those required to inhibit cellular mono(ADP-ribosyl) rather than poly(ADP-ribosyl) reactions. Calcium depletion did not inhibit the biosynthesis of NAD. These results suggest that mono(ADP-ribosyl)ation is involved in the regulation of cellular Ca2+ levels.

This contribution defines the ultrastructural characteristics of two very different conditions which can both lead to very extensive, and in some ways rather similar, myocardial injury. The two conditions are oxygen depletion and repletion and calcium depletion and repletion. Both conditions are of clinical relevance and both have been studied extensively in the laboratory, revealing in the process a great deal of valuable information about cellular injury and its prevention.

Scanning electron microscopy (SEM) coupled with energy-dispersive x-ray detection was used to study fragments of fine pottery excavated on the archaeological site at Locri Epizephiri (Southern Italy). The fragments analysed were specimens of black coated ware and included pieces suspected to have been produced on the site in the course of the fourth and third centuries BC and suspected imports from foreign centres of production. Comparison between the composition of slip and body of the samples indicated that selection and refinement of clays led to silicon and calcium depletion and aluminium, iron and potassium enrichment in the coating. In addition, pronounced depletion of magnesium was observed in the slip of the imported wares and the earlier local products, but not of the later local wares; this might account for the low quality of some 3rd century products with respect to the earlier pieces. Examination of SEM images indicated that the thickness of the surface slip reaches up to about 20-25 μm on the imported sherds, but measures ca. 10-20 μm on most of the local products.

Calcium-free incubation followed by exposure to Ca produces ultrastructural damage in the intact myocardium—a phenomenon known as the Ca paradox. Controversy exists as to whether the observed Ca overload during Ca repletion is a primary cause for, or a secondary consequence of, sarcolemmal disruption and cell injury. In these experiments cultured embryonic chick heart cells have been subjected to Ca depletion/repletion to explore the structural, transport and metabolic mechanisms by which cells may be irreversibly damaged.Monolayer muscle enriched cell cultures were prepared by serial trypsin-disaggregation of 11-day old chick embryo hearts. Cells were incubated for 10 or 120 min in Ca-free (1 mM EGTA) Hepes-buffered salt solution (pH 7.4) followed by incubation for 60 or 180 min in the same solution containing 1 mM Ca. In another series of experiments, the mitochondrial uncoupling agent carbonyl cyanide chlorophenylhydrazone (CCCP, 10 μM ) was added during depletion and repletion.