Ambient Calcium Concentration Research Articles

Effects of Multi-stage Procurement on the Viability and Function of Human Donor Parathyroid Glands.

Parathyroid allotransplantation is an emerging treatment for severe hypoparathyroidism. Ensuring the viability and functional integrity of donor parathyroid glands following procurement is essential for optimal transplantation outcomes. Cellular viability, calcium-responsive hormone secretion, and gland xenograft survival were assessed in a series of deceased donor parathyroid glands following a two-stage procurement procedure recently developed by our group (en bloc cadaveric dissection with subsequent gland isolation after transport to the laboratory). Parathyroid glands resected in this manner and stored up to 48h in 4°C University of Wisconsin (UW) media retained invitro viability with no induction of hypoxic stress (HIF-1α) or apoptotic (caspase-3) markers. Exvivo storage did not significantly affect parathyroid gland calcium sensing capacity, with comparable calcium EC50 values and suppression of parathyroid hormone secretion at high ambient calcium concentrations. The isolated glands engrafted readily, vascularizing rapidly invivo following transplantation into mice. Parathyroid tissue retains viability, calcium-sensing capacity, and invivo engraftment capability after en bloc cadaveric resection, exvivo dissection, and extended cold storage.

Comparison of calcium concentration in scales and vertebral column of a cyprinid from calcium-limited environments in the Lake Victoria Basin, Uganda

Despite dramatic differences in calcium concentrations within aquatic systems, very few studies have explored the relationships between ambient calcium concentration and the calcium concentration of resident fish under natural conditions. This study compares calcium concentration in the water to that of the scales and vertebral column of the African cyprinid, Rastrineobola argentea from lakes Nabugabo and Victoria, East Africa. The concentration of calcium in Lake Nabugabo, which averaged 1.50 mg l−1, was much lower than the average of literature-derived values for the Ugandan portion of Lake Victoria (6.96 mg l−1). Rastrineobola argentea from Lake Victoria were characterised by higher levels of calcium in the scales than that of conspecifics from Lake Nabugabo, whereas there was no difference in vertebral column calcium concentration between the two populations. Within Lake Nabugabo, calcium concentration was lower in the scales than in the vertebral column of R. argentea, whereas no difference was detected between the scalar and vertebral calcium concentrations of conspecifics from Lake Victoria. These results suggest that ambient calcium concentration may affects tissue levels and that fish in Lake Nabugabo may remobilise calcium from their scales to maintain skeletal growth and development.

Role of Calcium in Modulating the Conformational Landscape and Peptide Binding Induced Closing of Calmodulin.

Metal ions play an essential role in several cellular functions. Calcium is a ubiquitous regulator and is involved in numerous physiological processes. A class of proteins have evolved that sense calcium levels inside cells and act as effector molecules. Calmodulin is one such protein that gets activated after binding to calcium and thereafter interacts with its many targets. Calmodulin comprises two homologous domains that are connected by a flexible linker. The calcium-dependent flexibility of the linker results in numerous conformations of calmodulin. In this work using microsecond long MD simulations and well-tempered metadynamics, we explore how the calcium induced conformation dynamics of calmodulin is different from the inherent fluctuations of apocalmodulin and whether it has any role in preparing calmodulin for its interaction with its target-smooth muscle myosin light chain kinase (smMLCK). We have observed that calcium bound calmodulin could explore states that are predisposed toward peptide binding. We also found that though the binding of calmodulin to smMLCK peptide is calcium-independent, calcium regulates the domain to which the peptide will be bound. On the basis of our findings, we have proposed two alternate pathways for smMLCK peptide binding to calmodulin as directed by the ambient calcium concentrations. Our work proposes how calmodulin functions under physiologically dynamic calcium concentrations.

Lanthanum and Cerium Toxicity to the Freshwater Green Alga Chlorella fusca: Applicability of the Biotic Ligand Model.

The environmental risk assessment of rare earth elements (REEs) requires data on their potential toxicity. In the present study, the toxicity of lanthanum (La) and cerium (Ce) was studied in relation to metal speciation in solution. For both La and Ce, the use of organic ligands demonstrated that the calculated free ion concentration was a good indicator of toxicity. Whether in the absence or presence of organic ligands, when based on free ion concentrations, the obtained half-maximal effective concentrations were similar. When all generated data were pooled, Ce and La showed identical toxicity thresholds after 120 h of exposure with free ion concentration-based median effective concentration values (95% confidence intervals) of 0.48 (0.38-0.60) µM and 0.47 (0.36-0.61) µM for La3+ and Ce3+ , respectively. The inhibition of algal growth was also correlated with the intracellular lanthanide concentrations, regardless of the ligand used. Finally, increasing the ambient calcium concentration protected the test algae by reducing the amount of lanthanide internalized into the cells. These results suggest that, at constant pH (5.5), REE accumulation and toxicity are linked to the free ion concentration and ambient calcium concentration, as predicted by the biotic ligand model. Environ Toxicol Chem 2020;39:996-1005. © 2020 SETAC.

De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that ‘intrinsic component of membrane’ was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in ‘secondary metabolites biosynthesis, transport and catabolism’ and ‘signal transduction mechanisms’ in the KOG clusters and ‘processing in endoplasmic reticulum’, and ‘ABC transporters’ in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.

Calcium-induced differentiation in normal human colonoid cultures: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity.

Background and aimsThe goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue.MethodsColonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5–3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy.ResultsUnlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 in the absence of intervention (i.e., in control condition). Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention.ConclusionsThese findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.

Exoskeleton calcification in Norwegian populations of the crayfish Astacus astacus (Linnaeus, 1758) (Decapoda: Astacidae) varies with size, gender, and ambient calcium concentration

Declining ambient calcium (Ca 2+ ) concentrations in boreal, soft-water lakes of North America and Europe is one of many threats facing their biotic assemblages such as crayfish populations. We examined the specific exoskeleton calcium (Ca) concentration in Astacus astacus (Linnaeus, 1758) populations from a wide range of ambient Ca 2+ concentrations to determine a possible correlation between the amount of Ca accumulated in their carapaces and the ambient Ca 2+ concentrations. Exoskeleton Ca was the major constituent of the crayfish A. astacus carapaces in this survey (21.2 to 25.8% Ca of dry weight (DW)), whereas magnesium (Mg) displayed a disproportionately low constituent. The strong correlation between mineral contents of dry weight (DW) and ash weight (AW) ( r = 0 . 98 ) allowed us to refer mineral contents consequently to DW. A linear model using gender, length and ambient Ca 2+ concentration (log transformed) explained 82% of the variation in carapace Ca content (as % DW). Astacus astacus females were slightly more calcified than males (0.4% of DW, when adjusted for ambient Ca 2+ and body length). Large-bodied populations were slightly, but significantly more heavily calcified than those with smaller bodies: carapace Ca content increased by 0.2% DW for each cm increase in body length. The strong logarithmic effect of ambient Ca 2+ implies that carapace Ca content increases by 1.7 × log(2) = 1.2% DW for every doubling of the Ca 2+ concentration in the water.

Manganese Bioconcentration in Aquatic Insects: Mn Oxide Coatings, Molting Loss, and Mn(II) Thiol Scavenging

Streams below mountaintop removal-valley fill coal mining operations often have elevated Mn concentrations, but it remains unclear if Mn plays a role in biodiversity reduction. We examined various aspects of aqueous Mn interactions with aquatic insects exposed to environmentally relevant Mn concentrations, revealing complex behavior. First, Mn accumulation rates varied widely among 9 species. A significant percentage of total Mn accrued (mean 74%, range 24-95%) was associated with the cuticle, predominantly in the form of Mn-oxides, and to a lesser degree Mn(II). Mn II is also absorbed into tissues, possibly through calcium transporters. Increased ambient calcium concentrations decreased both adsorbed and absorbed Mn accumulation from solution. Though species showed similar Mn efflux rate constants (0.032-0.072 d(-1)), the primary mode of Mn loss was through molting. Both adsorbed and absorbed Mn is lost during the molt. Subcellular compartmentalization studies revealed an overwhelming tendency for internalized Mn to associate with the heat stable cytosolic protein fraction. After short dissolved Mn exposures, intracellular glutathione and cysteine levels were markedly reduced relative to controls. These findings suggest that Mn exposure results in transient physiological stress in aquatic insects which is likely relieved, in part, during the molting process.

Voltage-gated calcium channels are involved in the regulation of calcium oscillations in vascular smooth muscle cells from isolated porcine retinal arterioles

Disturbances in the regulation of retinal arteriolar tone are involved in the pathophysiology of major vision threatening diseases. Therefore, improving the treatment of these diseases requires knowledge about how the contraction of retinal arterioles is controlled. The present study hypothesized that in addition to calcium release from sarcoplasmic reticulum, calcium entry from the extracellular space is also involved in the generation of calcium oscillations as a basis for initiation of contraction in retinal arterioles. Isolated porcine retinal arterioles were mounted in a confocal microvascular myograph and loaded with the calcium sensitive fluorophore Oregon Green allowing simultaneous measurements of oscillations in the intracellular calcium concentration in individual smooth muscle cells and changes in the vascular tone. Recordings were made after pre-contraction with the thromboxane analogue U46619 in a low and a high ambient calcium concentration, and after subsequent blocking of voltage-gated calcium channels using nifedipine or the sarcoplasmic reticulum dependent calcium recruitment using ryanodine, caffeine, thapsigargin and cyclopiazonic acid. U46619 increased the number of oscillations in a low but not in physiological extracellular calcium concentration. In contrast to mesenteric arterioles, blocking of sarcoplasmic reticulum Ca 2+ channels with ryanodine was without effect on both retinal arteriolar tone and calcium oscillations. Conversely, blocking of the sarcoplasmic reticulum Ca 2+ pump with cyclopiazonic acid or thapsigargin significantly reduced calcium oscillations, but not arteriolar tone. At a physiological extracellular calcium concentration, addition of nifedipine, an L-type calcium channel blocker, reduced vascular tone but increased the number of cells displaying calcium oscillations. Fast oscillations in the intracellular calcium concentration in retinal smooth muscle cells depend on calcium transport by both the SR and the sarcolemma. However, both sites of calcium recruitment seem to have unique properties in porcine retinal arterioles as compared to other organs.

Acquisition of Ca2+ and HCO3 −/CO3 2− for shell formation in embryos of the common pond snail Lymnaea stagnalis

Embryos of the freshwater common pond snail Lymnaea stagnalis develop to hatch within 10 days under control conditions (22°C, Miami-Dade tap water) and this development is impaired by removal of ambient calcium. In contrast, embryos did not exhibit dependence upon an ambient HCO3 −/CO3 2− source, developing and hatching in HCO3 −/CO3 2−-free water at rates comparable to controls. Post-metamorphic, shell-laying embryos exhibited a significant saturation-type calcium uptake as a function of increasing ambient calcium concentration. However, changes in ambient bicarbonate concentration did not influence calcium or apparent titratable alkalinity uptake. There was a distinct shift from no significant flux in pre-metamorphic embryos to net uptake of calcium in post-metamorphic stages as indicated by an increased uptake from the micro-environment surrounding the egg mass and increased net uptake in 24-h, whole egg mass flux measurements. Furthermore, HCO3 −/CO3 2− acquisition as measured by titratable alkalinity flux is at least partially attributable to an endogenous carbonate source that is associated with acid extrusion. Thus, calcium requirements for embryonic shell formation are met via uptake but HCO3 −/CO3 2−, which is also necessary for shell formation is acquired in part from endogenous sources with no detectable correlation to ambient HCO3 −/CO3 2− availability.

A review of the influence of low ambient calcium concentrations on freshwater daphniids, gammarids, and crayfish

The widespread decline in aqueous calcium (Ca) is emerging as a newly recognised stressor in freshwater ecosystems in regions with historically high acid deposition, especially when coupled with multiple logging cycles. Currently, Ca and other base cations are being depleted in the soils of acid-sensitive watersheds of eastern North America and western Europe, resulting in falling Ca levels in streams and lakes. Freshwater crustaceans have high Ca demands due to their heavily calcified exoskeleton and regular moult cycle. Because they rely on Ca in the external environment for the majority of their Ca uptake, aquatic crustaceans are restricted to waters at or above the Ca concentration needed to satisfy their demands. The zoogeographic distributions along ambient Ca gradients, and Ca requirements and metabolism of three groups of freshwater crustaceans — daphniids, crayfish and gammarids — have been relatively well studied. Here we have four objectives: (1) We briefly review biological features of the above three taxa that relate to Ca metabolism. (2) We review the literature regarding minimum Ca thresholds permitting survival, Ca saturation points, and the individual and population scale costs of existence at suboptimal Ca concentrations. (3) Using Daphnia as an example, we explore the ecological consequences of falling environmental Ca concentrations. (4) We identify gaps and weaknesses in the literature that may inhibit the development of environmental risk assessments for Ca decline for these three crustacean groups. We conclude that crayfish are especially vulnerable to ongoing Ca decline, and that all three taxa are probably living under suboptimal conditions in areas of Ca decline, and are therefore likely under chronic metabolic stress.

The influences of ambient and body calcium on cadmium and zinc accumulation in Daphnia magna

The dissolved uptake rate constant (k(u)), dietary assimilation efficiency (AE), and efflux rate constant (k(e)) of cadmium (Cd) and zinc (Zn) were quantified in a freshwater zooplankton Daphnia magna cultured at different ambient calcium (Ca) concentrations. The animals were first acclimated to different ambient Ca levels for 7 d from birth before the biokinetic measurements at corresponding Ca levels. With increasing ambient Ca level from 0.5 to 200 mg/L, the body Ca content increased from 0.91% (as tissue dry wt) to 3.75%. The k(u) for Cd decreased by nine times; for Zn it decreased by 2.6 times; and the AE decreased from 62 to 19% and from 46 to 24% for Cd and Zn, respectively. In contrast, Ca levels did not affect significantly the efflux rates of Cd and Zn. The effects of ambient and body Ca levels were separated by measuring the biokinetic parameters in both low--(0.5 mg/L) and high--(50 mg/L) Ca environments using the daphnids containing different body Ca levels. Ambient and body Ca levels had synergistic inhibitory effects on the AEs of Cd and Zn; however, the protective effects against dissolved uptake of Cd and Zn were explained fully by the effects of ambient Ca. The body Ca either had no significant effect (for Cd) or stimulative effects (for Zn). Multiphase biokinetic modeling using the measured parameters gave reasonable predictions of the body burdens of Cd and Zn in different Ca environments. Our results better explain the role of ambient and body Ca in the accumulation of Cd and Zn in D. magna.

Cadmium biodynamics in the oligochaete Lumbriculus variegatus and its implications for trophic transfer

It has become increasingly apparent that diet can be a major source of trace metal bioaccumulation in aquatic organisms. In this study, we examined cadmium uptake, efflux, and subcellular compartmentalization dynamics in the freshwater oligochaete Lumbriculus variegatus. L. variegatus is an important component of freshwater food webs in Europe and North America and is potentially useful as a standard food source for laboratory-based trophic transfer studies. Cadmium accumulation and depuration were each followed for 10 days. Rate constants of uptake ( k u) and efflux ( k e) were estimated and subcellular Cd compartmentalization was followed over the course of uptake and efflux. The partitioning of Cd into operationally-defined subcellular compartments was relatively consistent throughout the 20-day experiment, with the majority of Cd accumulating in the cytosol. No major changes in Cd compartmentalization were observed over uptake or depuration, but there appeared to be some exchange between heat-stable and heat-labile cytosolic protein fractions. Cadmium accumulation from solution was strongly affected by ambient calcium concentrations, suggesting competition between Cd and Ca for uptake sites. Finally, we demonstrate the ability to manipulate the whole body calcium content of L. variegatus as a potential tool for examining calcium influences on dietary Cd dynamics. The potential for this species to be an important conduit of Cd to higher trophic levels is discussed, along with its potential as a standardized food source in metal trophic transfer studies.

Effects of ambient calcium concentration on the deposition of calcium oxalate crystals in Antithamnion (Ceramiales, Rhodophyta)

C.M. Pueschel and J.A. West. 2007. Effects of ambient calcium concentration on the deposition of calcium oxalate crystals in Antithamnion (Ceramiales, Rhodophyta). Phycologia 46: 371–379. DOI: 10.2216/06-74.1A survey of 18 species of the Ceramiales grown in culture revealed calcium oxalate crystals in Antithamnion antillanum Børgesen, A. callocladum Itono, and A. sparsum Tokida. The needle-shaped crystals were present within the cytoplasm of cells of the indeterminate axes but not in cells of the determinate lateral branches. No such crystals were present in A. nipponicum Yamada & Inagaki. The four species of Antithamnion and three additional members of the Ceramiales that do not normally deposit calcium oxalate were grown in natural seawater culture medium in which calcium concentration was elevated by addition of 5–20 mM calcium chloride. Elevated calcium supply did not induce the deposition of calcium oxalate crystals in species that did not previously exhibit them, and it did not change the crystal size range or the cellular or subcellular localization patterns in species that normally formed crystals. At high calcium concentrations, all species ceased growth whether or not they were able to sequester calcium as an oxalate salt. In a second experiment, the four species of Antithamnion were grown in artificial seawater with reduced calcium concentrations. All appeared healthy and grew well in artificial seawater containing 2.5–10 mM CaCl2. In 2.5 mM CaCl2, A. antillanum released tetraspores that grew into thalli having abundant calcium oxalate crystals. Even in 1.0 mM CaCl2, A. callocladum and A. sparsum continued to grow and deposit calcium oxalate crystals of the size range typical for the species. Thalli of all four species died within 12 days if artificial seawater containing only 0.1 mM CaCl2 was provided. Calcium oxalate crystals were present in many cells of the dead thalli, indicating that the crystals did not provide a calcium reserve that could be readily mobilized. Culturing the thalli in complete darkness for 14 weeks also did not stimulate mobilization of the crystals. The presence/absence of calcium oxalate crystals over a broad range of calcium concentrations and light intensities suggests that deposition of this mineral is a constitutive feature and thus could serve as a taxonomic character for these species. It also suggests that calcium oxalate deposition in Antithamnion does not have a role in calcium regulation.

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.

Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals

To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation. Sodium dodecyl sulphate gel electrophoresis and Western blotting were used to analyse demineralized extracts of COM and/or COD crystals deposited from the same centrifuged and filtered urine (which contains abundant urinary proteins) by adjusting the calcium concentration to 2 and 7 mmol/L, respectively. Similar analyses were performed on COM and COD crystals deposited from ultrafiltered urine (which contains only proteins of < 10 kDa) and then incubated in centrifuged and filtered urine, as well as crystals generated in the presence of increasing concentrations of proteins derived from the organic matrix of urinary calcium oxalate crystals. Field-emission scanning electron microscopy was used to assess effects of proteinase K and cathepsin D on internal and superficial crystal structure. Osteopontin was undetectable in COM extracts, but clearly visible in COD. Prothrombin fragment 1 was abundant in COM, but present in COD in lesser amounts than osteopontin. The selectivity was also the same with crystals from ultrafiltered urine that were incubated in centrifuged and filtered urine: prothrombin fragment 1 binding was favoured by low calcium concentration, while osteopontin bound at higher levels. Scanning electron microscopy of COM and COD digested with proteinase K and cathepsin D revealed superficial and internal texture, as wells as surface erosion, in crystals from centrifuged and filtered urine, thus confirming the presence of intracrystalline proteins. Such features were absent from crystals precipitated from ultrafiltered urine. Binding of osteopontin and prothrombin fragment 1 to calcium oxalate is dictated primarily by ambient calcium concentration. Each protein may inhibit urolithiasis by inhibiting crystallization of its preferred crystal habit, and by facilitating the intracellular disintegration and dissolution of crystals attached to and internalized by renal epithelial cells.

Calcium limitation in Daphnia magna

The role of ambient calcium concentrations on survival, moulting, growth and egg production was assessed in the cladoceran Daphnia magna. A threshold for survival was found in the range 0.1–0.5 mg Ca l–1, even when ionic strength of the medium was kept constant. Accumulated length and length specific dry weight was retarded at low Ca (0.5–1.0 mg Ca l–1) at food concentrations above incipient limiting level. For lower food levels, the effect of Ca on growth was less clear. The effect of low Ca on growth rate was most manifest during the first days after hatching, reflecting the higher Ca demands of the early juveniles. Age-specific egg production was strongly reduced at Ca concentrations <10 mg Ca l–1. This was partly an indirect effect of reduced growth rates, but could also be an effect of higher energetic costs associated with Ca uptake in a Ca-poor medium. The high Ca demands in D.magna may not be representative of other Daphnia species. Nevertheless, high specific Ca content seems to be a common property of Daphnia spp. and Ca deficiency could be a major determinant of species success and community structure among crustacean zooplankton; it also puts constraints on carbon sequestration in the pelagic food web of softwater lakes.

Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons.

1. Single L-type calcium channels in chick ciliary ganglion neurons were studied at high current resolution in cell-attached patch recordings using quartz-glass micropipettes. 2. A single open-channel current amplitude was observed when Ba2+ was the charge carrier with a conductance of 26 pS at 110 mM barium. However, with 110 mM calcium two current fluctuation amplitudes were observed. These were termed low and high fluctuation amplitudes, CaL and CaH, and had conductances of 8.8 and 12 pS, respectively. These two levels probably reflect two different channel species. CaL was identified as an L-type calcium channel on the basis of resistance to inactivation, conductance, and dihydropyridine sensitivity. 3. Single-channel current fluctuations could be detected with calcium concentrations as low as 1.0 mM. Although the unitary conductance (gamma) was much greater with barium than calcium at every concentration tested, the concentration dependence of conductance was similar for gamma Ba, gamma CaH and gamma CaL. Fitting the concentration dependencies of these conductances with a Langmuir isotherm gave KD estimates of 4.7, 5.6 and 5.0 mM for barium, CaL and CaH, respectively 4. The single-channel conductance of the L-type channel (gamma L) can be described by the relation: conductance (in pS) = 9.2/(1 + 5.6/[Ca]) where [Ca] is the external calcium concentration in the 1.0-110 mM range. Thus, at a physiological external calcium concentration of 2 mM the conductance is 2.4 pS. 5. Ca2+ transport through the L-type calcium channel is particularly sensitive to changes in external calcium concentration in the physiological range but approaches saturation at about 10 mM. this characteristic may optimize the responsiveness of the cell to small changes in ambient calcium concentrations while limiting excess entry in the presence of abnormally high calcium levels.

Differences in PTH (1-84) release in response to ambient calcium concentrations of parathyroid adenoma fragments and dispersed parathyroid adenoma cells in culture.

In a previous study we observed that during perfusion of normal human parathyroid tissue, the release of PTH (1-84) was modulated by ambient extracellular calcium (Ca++) and lithium (Li+) concentrations in the media and preliminary studies indicated that this stimulus-response coupling was absent in human parathyroid adenoma fragments. The present study compares the responsiveness of parathyroid adenoma fragments and isolated parathyroid adenoma cells from the same adenoma and their response to Ca++ changes and Li+ presence in culture media. The data indicate that parathyroid adenoma tissue fragments fail to respond to ambient changes in Ca++ and Li+. In contrast, dispersed parathyroid cells preparations responded with a significant increase of PTH (1-84) release (50%) under the influence of low ambient calcium concentrations. Six of the dispersed cell preparations also responded with a 45% decrease in PTH release under the influence of a high Ca concentration in the medium. Isolated parathyroid cells obtained from the same adenoma's did not respond to the presence of Li++ in the medium. These data suggest tat human parathyroid adenoma tissue functions autonomously and is not sensitive to calcium regulation in the tissue configuration as opposed to the isolated cell suspensions. The nature of this difference remains elusive.

Molecular cloning and expression of the acidic fibroblast growth factor receptors in a rat parathyroid cell line (PT-r). Parathyroid cell-specific calcium-dependent change of ligand accessibility and covalent attachment of heparan sulfate glycosaminoglycan to the receptors.

We have previously identified two fibroblast growth factor (FGF) receptors with higher affinity for acidic FGF rather than basic FGF in a rat parathyroid cell line (PT-r). Carbohydrate analyses of the receptors suggested the presence of three different types of FGF receptors, a 150-kDa glycoprotein receptor, a approximately 150-kDa heparan sulfate-proteoglycan receptor, and a 130-kDa glycoprotein receptor (Sakaguchi, K., Yanagishita, M., Takeuchi, Y., and Aurbach, G.D. (1991) J. Biol. Chem. 266, 7270-7278). Here, we have cloned two isoforms of the FGF receptors from PT-r cells; one with two immunoglobulin (Ig)-like domains (clone a), and the other with an additional Ig-like domain and an acidic box (clone b). They showed highest homology to the mouse and human keratinocyte growth factor receptors among the FGF receptors reported. Clones a and b had one and three possible glycosaminoglycan attachment sites, respectively. Heparitinase treatment of PT-r cells transfected with clone a suggested that the protein for the 130-kDa glycoprotein receptor was encoded by clone a, and that the same protein also served as a core protein for the approximately 150-kDa heparan sulfate-proteoglycan receptor. Heparan sulfate glycosaminoglycan attachment to the 150-kDa receptor encoded by clone b was not detectable by the same enzyme treatment. Site-directed mutagenesis (from Ser to Ala) studies of the consensus sequence for the attachment of glycosaminoglycans further supported the presence of covalently attached heparan sulfate glycosaminoglycan in the approximately 150-kDa heparan sulfate-proteoglycan receptor. These receptors overexpressed in PT-r cells changed ligand accessibility or apparently translocated after changing extracellular calcium concentrations in a manner similar to the native receptors in PT-r cells (Sakaguchi, K. (1992) J. Biol. Chem. 267, 24554-24562), whereas those expressed in CHO-K1 or NIH/3T3 cells did not. These findings strongly suggest that the two FGF receptor isoforms cloned here represent the acidic FGF receptors that we reported earlier. A subpopulation of the receptors carries heparan sulfate glycosaminoglycan covalently attached to the core protein, and the change in ligand accessibility in response to the shift in ambient calcium concentration is specific to the parathyroid cells.