Net Calcium Flux Research Articles

TRPV6 and Cav1.3 Mediate Distal Small Intestine Calcium Absorption Before Weaning.

Background & AimsIntestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults after peak bone mass is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption during this critical period.MethodsExpression of small intestinal and renal calcium transport genes was assessed by using quantitative polymerase chain reaction. Net calcium flux across small intestinal segments was measured in Ussing chambers, including after pharmacologic inhibition or genetic manipulation of TRPV6 or Cav1.3 calcium channels. Femurs were analyzed by using micro–computed tomography and histology.ResultsNet TRPV6-mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca2+ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Cav1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Cav1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca2+ absorption in the ileum, and increased expression of renal Ca2+ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Cav1.3 expression.ConclusionsWe have detailed novel pathways contributing to transcellular Ca2+ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance early in life.

An in silico method to predict net calcium transfer during hemodialysis.

International guidelines for chronic hemodialysis patients suggest a dialysate calcium concentration between 1.25 and 1.5 mmol/L. However, it is not certain if these dialysate calcium levels result in net calcium transfer into the patient. With ubiquitous prevalence of vascular calcification in hemodialysis patients, it is pertinent to model the mass balance of calcium during dialysis. To this end, we developed a two compartmental patient model and spatiotemporal representation of dialyzer model to investigate and quantify the calcium mass balance during dialysis. The model accounts for calcium-albumin binding and varying protein concentration; the latter accounts for the Gibbs-Donnan effect. The model simulations suggest that despite a lower dialysate calcium concentration of 1.25 mmol/L, some of our patients may be loaded with calcium during dialysis. This net calcium flux from dialysate to blood side may be a potential contributor to vascular calcification, a primary cause of cardiovascular mortality in hemodialysis patients.

Novel Calcium Transport Pathways Mediate Intestinal Calcium Absorption Pre‐Weaning

Calcium is essential to many vital physiological functions including bone mineralization. Peak bone deposition rate occurs in infancy and maximal bone mineral content is achieved early in adulthood, thereafter bone mass is lost. Maintenance of a positive calcium balance during development is thus essential to achieving optimal bone mass. Calcium homeostasis is mediated by interactions between the intestine, kidneys, and bones. Intestinal absorption occurs by either an active transcellular or passive paracellular pathway. Currently, based on studies in adults the duodenum is thought to be a site of largely transcellular absorption whereas the jejunum and ileum are proposed to mediate exclusively paracellular calcium absorption. There are limited studies on young animals. We therefore set out to describe calcium absorption pathways pre and post weaning. Wildtype FVB/N mice at 7 ages from 1 day to 6 months old were examined. Levels of Trpv6 and CalbD9k mRNA, mediators of transcellular calcium transport, increased six‐fold between two weeks and one month of age in the duodenum with a corresponding increase in CalbindinD9k protein. In the jejunum and ileum, mediators of transcellular transport – Trpv6 (jejunum only), Cav1.3, and CalbD9k – are highly expressed prior to weaning, suggesting novel pathways of calcium absorption during development. Consistent with this, net calcium flux, measured in Ussing chambers, was not different from zero across ileum post weaning. However, we identified significant net absorption in the absence of an electrochemical gradient across ileum prior to weaning. Further, this net absorption was abolished by nifedipine, an L‐type calcium channel blocker. Abundance of Cldn‐2 and ‐15 mRNA, mediators of paracellular absorption, peak at 7 days in the duodenum. In the ileum, Cldn‐2 mRNA peaks at 14 days and decreases 10‐fold by 1 month, whereas Cldn‐15 expression increases 3‐fold from day 14 to 1 month of age. In conclusion, these observations suggest that weaning induces a significant shift from paracellular to transcellular calcium absorption in the duodenum and that there is significant, previously unreported, transcellular calcium absorption from the jejunum and ileum pre weaning mediated by Cav1.3.Support or Funding InformationThis research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Stollery Children's Hospital Foundation through the Women and Children's Health Research Institute

Coulometric calcium pump for thin layer sample titrations.

A selective electrochemical calcium pump based on a fast diffusive calcium ionophore-based membrane is reported. An initially nonpolarized ionophore-based membrane allows one to establish a net calcium flux by applying a potential step function (i.e., 250 mV for 30 s). The resulting calcium flux is released into a microliter scale thin layer reservoir, and the resulting ion perturbation is monitored by either a potentiometric or a coulometric readout. This chemical perturbation in the thin layer thus acts as a titration agent that is precisely controlled by coulometry. A linear correlation between released and detected calcium is confirmed by the two different readout modes. Having demonstrated the efficiency of the calcium pump in background electrolyte solutions, a complexometric titration with known concentrations of EDTA in the thin layer sample was performed. With the potentiometric readout, titrations in the range of 0.25-0.75 mM gave a precision of 3%, whereas the coulometric readout gave a range of 0.02-0.12 mM and a precision of 2%. Improved precision is expected by better control of the thin layer geometry by microfabrication. The significance of this work is that the coupling of a selective calcium pump with a thin layer element can give rise to rapid and complete sample concentration changes and result in a promising platform for titrations either on the laboratory bench or for in situ measurements in environmental or diagnostic settings.

Significant contribution of authigenic carbonate to marine carbon burial

Carbon is removed from the Earth’s surface through the formation and burial of carbon-bearing rocks and minerals. An analysis of pore water profiles collected from marine sediments around the globe suggests that the precipitation of authigenic calcium carbonate accounts for around 10% of the carbonate that accumulates in marine sediments globally. Carbon is removed from the Earth’s surface through the formation and burial of carbon-bearing rocks and minerals1,2. The formation of calcium carbonate and its burial in marine sediments accounts for around 80% of the total carbon removed from the Earth’s surface. However, the fraction of calcium carbonate that precipitates in the oceans, versus that which precipitates authigenically in marine sediments, is unclear. Here, we compile measurements of the calcium concentration of pore fluids collected at 672 seafloor sites around the globe to calculate the global flux of calcium within marine sediments. We use these data, combined with alkalinity measurements of pore fluids, to quantify authigenic calcium carbonate precipitation. We estimate that the net calcium flux into marine sediments that can be ascribed to authigenic carbonate precipitation amounts to around 1×1012 mol yr−1. As such, we estimate that authigenic carbonate precipitation accounts for at least 10% of global carbonate accumulation. We show that much of the precipitation occurs along the eastern margins of ocean basins, where organic matter delivery to the sea floor is likely to be high. We suggest that authigenic calcium carbonate precipitation represents a non-negligible component of the global carbon cycle.

Mechanisms of calcium absorption by anterior and posterior segments of the intestinal tract of juvenile lake sturgeon

Rapid growth in juvenile fish increases calcium demand, and the intestine may play a role in calcium homeostasis at this life stage, in addition to branchial and renal transport. This study examined calcium flux in the gastrointestinal tract (GIT) of freshwater juvenile lake sturgeon acclimated to 0.14, 0.34, and 2.26mmolL−1 environmental calcium. Net Ca2+ flux did not differ due to environmental [Ca2+] in either the anterior or posterior intestine. Blocking the apical epithelial calcium channel (ECaC) with ruthenium red (RR, 8.5μmolL−1) significantly decreased Ca2+ influx in the anterior intestine, but exposure to the plasma membrane Ca2+-ATP-ase (PMCA) inhibitor trifluoperazine (TFP, 10mmolL−1) had no effect at any environmental [Ca2+], nor did inhibition of the Na+–Ca2+ exchanger (NCX) with KB-R7943 (10μmolL−1). Neither RR nor TFP affected Ca2+ uptake by the posterior intestine in any of the treatment groups, but KB-R7943 reduced net calcium flux in the posterior intestine at all environmental [Ca2+]. Thus, basolateral Ca2+ influx in the posterior GIT of lake sturgeon relies more heavily on NCX than PMCA. Furthermore, the differing pharmacological effects in the anterior and posterior intestine suggest that the dominant transporters responsible for calcium uptake vary over the length of the GIT in lake sturgeon.

The influence of environmental calcium concentrations on calcium flux, compensatory drinking and epithelial calcium channel expression in a freshwater cartilaginous fish

Calcium metabolism and mRNA levels of the epithelial calcium channel (ECaC) were examined in a freshwater cartilaginous fish, the lake sturgeon Acipenser fulvescens. Lake sturgeon were acclimated for ≥2 weeks to 0.1 (low), 0.4 (normal) or 3.3 (high) mmol l(-1) environmental calcium. Whole-body calcium flux was examined using (45)Ca as a radioactive marker. Net calcium flux was inward in all treatment groups; however, calcium influx was greatest in the low calcium environment and lowest in the high calcium environment, whereas efflux had the opposite relationship. A significant difference in the concentration of (45)Ca in the gastrointestinal tract (GIT) of fish in the low calcium environment led to the examination of drinking rate and calcium flux across the anterior-middle (mid) intestine. Drinking rate was not different between treatments; however, calcium influx across the mid-intestine in the low calcium treatment was significantly greater than that in both the normal and high calcium treatments. The lake sturgeon ECaC was 2831 bp in length, with a predicted protein sequence of 683 amino acids that shared a 66% identity with the closest sequenced ECaCs from the vertebrate phyla. ECaC mRNA levels were examined in the gills, kidney, pyloric caeca, mid-intestine and spiral intestine. Expression levels were highest in the gills, then the kidneys, and were orders of magnitude lower in the GIT. Contrary to existing models for calcium uptake in the teleost gill, ECaC expression was greatest in high calcium conditions and kidney ECaC expression was lowest in low calcium conditions, suggesting that cellular transport mechanisms for calcium may be distinctly different in these freshwater cartilaginous fishes.

In vivo and in vitro effects of a pulsed electromagnetic field on net calcium flux in rat calvarial bone.

Although PEMF's have been found to promote fracture healing and to modulate the activity of bone cells in vitro, effects on bone metabolism are largely unexplored. A bioassay using neonatal rat calvarial bone was used to determine the early effects of a pulsing electromagnetic field (PEMF) exposure in vivo and in vitro on bone metabolic calcium exchange. Bone discs taken from whole body exposed animals (0-4 hours) show a log exposure time-dependent average increase in net Ca uptake in the 0-50% range (r2 = 0.83). This increase could be detected immediately after exposure and also after 24 hours, but not 48 hours later. Animals given whole body PEMF exposure also showed a decrease in serum calcium and did not elevate serum Ca after administration of exogenous parathyroid hormone (PTH). Bone discs from untreated rats, exposed to PEMF for 15 minutes in vitro and then assayed, showed net Ca uptake increases of a similar magnitude and also were refractory to the Ca-releasing effect of PTH. Unexposed discs responded normally to PTH by decreasing net Ca uptake. Treatment of calvarial discs with calcitonin or acetazolamide, both of which inactivate osteoclasts, made the bone refractory to further increases in Ca uptake by PEMF. These results suggest that PEMF exposure produces PTH-refractory osteoclastics and has a relatively rapid effect on increasing net bone Ca uptake, putatively due to a decrease in PTH/paracrine-mediated bone resorption.

Glucose-induced inhibition of in vitro bone mineralization

Patients with diabetes tend to have an increased incidence of osteopenia that may be related to hyperglycemia. However, little is known about how glucose may alter bone formation and osteoblast maturation. To determine whether glucose affects osteoblastic calcium deposition, MC3T3-E1 cells were incubated in media containing either a normal (5.5 mmol/L) or high glucose concentration (15 mmol/L) or mannitol (15 mmol/L), and bone nodule formation was examined. Net calcium flux was measured thrice weekly and cumulative calcium uptake was determined. Compared with control incubations, glucose significantly inhibited daily and cumulative calcium uptake into the nodules. At the time of matrix maturation, cultures undergo a rapid phase of increased calcium deposition; this was significantly inhibited by the presence of glucose. Total calcium uptake, determined by acid digestion, was also significantly inhibited by glucose. Area and number of nodules were quantitated at the end of the incubation period (day 30) by staining with Alizarin Red S calcium stain. Compared with both control and mannitol-treated cultures, the number of nodules was increased by incubation with glucose. Furthermore, both the average total nodular area and calcified nodular area of large nodules were increased by glucose. Cellular proliferation as well as the release of markers of osteoblast activity (osteocalcin and alkaline phosphatase) were determined at the end of the experimental period (day 30). Cellular proliferation and alkaline phosphatase activity was significantly increased in the presence of glucose, however, the release of osteocalcin into culture media was similar in all three groups. In conclusion, the present study shows that elevated glucose concentration present throughout the development of murine osteoblasts stimulates cellular proliferation while inhibiting calcium uptake. The result of glucose inhibition of calcium uptake suggests that bone could be structurally altered in diabetes.

Prostaglandins regulate acid-induced cell-mediated bone resorption.

Metabolic acidosis induces bone calcium efflux initially by physicochemical dissolution and subsequently by cell-mediated mechanisms involving inhibition of osteoblasts and stimulation of osteoclasts. In rat kidney, acidosis increases endogenous prostaglandin synthesis, and in bone, prostaglandins are important mediators of resorption. To test the hypothesis that acid-induced bone resorption is mediated by prostaglandins, we cultured neonatal mouse calvariae in neutral or physiologically acidic medium with or without 0.56 microM indomethacin to inhibit prostaglandin synthesis. We measured net calcium efflux and medium PGE(2) levels. Compared with neutral pH medium, acid medium led to an increase in net calcium flux and PGE(2) levels after both 48 h and 51 h, a time at which acid-induced net calcium flux is predominantly cell mediated. Indomethacin inhibited the acid-induced increase in both net calcium flux and PGE(2). Net calcium flux was correlated directly with medium PGE(2) (r = 0.879, n = 29, P < 0.001). Exogenous PGE(2), at a level similar to that found after acid incubation, induced net calcium flux in bones cultured in neutral medium. Acid medium also stimulated an increase in PGE(2) levels in isolated bone cells (principally osteoblasts), which was again inhibited by indomethacin. Thus acid-induced stimulation of cell-mediated bone resorption appears to be mediated by endogenous osteoblastic PGE(2) synthesis.

Major trauma enhances store-operated calcium influx in human neutrophils.

Chemotaxins from inflammatory sites prime or activate neutrophils (PMN) by using cytosolic calcium ([Ca2+]i) fluxes as second messengers. [Ca2+]i can be mobilized rapidly by receptor-mediated entry or store-release, or more slowly by store-operated calcium influx (SOCI). We studied [Ca2+]i mobilization by chemotaxins and how trauma impacts the calcium entry mechanisms used by chemotaxins. [Ca2+]i flux was studied by spectrofluorometry. The contributions of early and late [Ca2+]i currents to net calcium flux were compared after stimulation by more potent (fMLP, C5a, PAF) or less potent (IL-8, GRO-alpha, and LTB4) agonists. Store operated [Ca2+]i mobilization was reflected by the ratio of area under the [Ca2+]i efflux curve to peak [Ca2+]i (efflux curve). PMN from trauma patients (ISS > 25) and pair-matched volunteer (n = 7 pairs) were then primed and stimulated with thapsigargin to compare cell calcium stores and SOCI. Late [Ca2+]i mobilization made more important contributions to fMLP, PAF, and C5a signals than to IL-8, GRO-alpha, or LTB4 (p < 0.01 all comparisons). Calcium stores and store release were only marginally lower after injury (p = not significant), but trauma PMN showed far higher [Ca2+]i influx after thapsigargin (p = 0.007), and greater net SOCI (p = 0.034). SOCI may play an important role in PMN activation, and trauma increases PMN SOCI. Prolonged elevations of [Ca2+]i due to enhanced SOCI may alter stimulus-response coupling to chemotaxins and contribute to PMN dysfunction after injury.

A coculture model of synoviocytes and bone for the evaluation of potential arthritis therapies

Objective: To evaluate the symbiotic relationship between musculoskeletal cells in the intact joint utilizing a coculture system and to determine if the model can be utilized to evaluate potential treatments for articular diseases. Methods: Two neonatal mouse calvariae were placed on steel supports on a monolayer of rabbit synovial fibroblasts, and net calcium flux, bone cell activity, and undecalcified histology were determined at 6, 24, and 48 h. To determine if the model was predictive of response to known therapies for articular disease, the coculture was incubated in the presence and absence of indomethacin or doxycycline, and the net calcium flux was measured. Results: The coincubation of calvariae with synoviocytes led to a fivefold increase in net calcium efflux compared to calvariae alone. The concentration in the media of the osteoblastic enzyme alkaline phosphatase increased at 6 h but decreased thereafter, whereas the concentration of osteoclastic enzyme β-glucuronidase increased with time. Undecalcified bone histology revealed progressive demineralization and an increase in the number of osteoclasts in calvariae incubated with synoviocytes compared to calvariae alone. Both indomethacin and doxycycline inhibited calcium flux from cocultures but the predominant effect of doxycycline was on the synoviocyte whereas the predominant effect of indomethacin was on bone. Conclusion: The coincubation of synoviocytes with calvariae led to an increase in bone mineral dissolution with time. This effect could be partially inhibited by known treatments for rheumatoid arthritis. Thus, the coculture model may simulate certain aspects of the in vivo processes relevant to rheumatoid arthritis. This model should prove useful for the study of potential therapies for inflammatory arthritis and distinguish between effects of these therapies on different cellular components of the joint.

Immediate and steady state extracellular ionic fluxes of growing Arabidopsis thaliana root hairs under hyperosmotic and hypoosmotic conditions

Osmotic‐induced shifts in extracellular ionic fluxes at the apex of growing Arabidopsis thaliana L. root hairs were examined using the extracellular self‐referencing (vibrating) ion‐selective probe technique. With either APW 7 (artificial pond water, pH 7) or APW 7 plus 200 mM mannitol/sorbitol, a steady state was reached in which growth rates were the same (0.75 and 0.82 µm min−1) as were the net fluxes of calcium (inward), potassium (outward), and chloride (inward). The outward flux of protons was about 20‐fold larger in APW 7 plus 200 mM mannitol/sorbitol compared to APW 7 (P= 0.028). Significant changes in ionic fluxes within 5 min of osmotic changes were observed for hyperosmotic treatment: a 7.5‐fold increase in the inward calcium flux (P= 0.041) and a 1.7‐fold increase in the outward potassium flux (P= 0.007). Although the change in the calcium flux was consistent with a process of osmotic adjustment (and not a consequence of binding/release from the cell wall), the magnitude of the flux was considerably less than the potassium efflux. Unlike the situation in other organisms where volume regulation explicitly relies upon modulation of ionic fluxes (especially sodium, potassium and chloride), the root hairs may rely on other mechanisms of osmotic regulation, possibly coupled to differences in proton efflux under steady state growth conditions. The root hairs do exhibit an osmotic‐induced electrical signal (R. R. Lew. 1996. Plant Physiol. 112: 1089–1100) which may be a component of initial signal transduction controlling osmotic regulation.

Acute effects of prolactin on passive calcium absorption in the small intestine by in vivo perfusion technique

The acute effect of intraperitoneally administered prolactin (0.2, 0.4, and 0.6 mg/kg body weight) on passive calcium transport in duodenum, proximal jejunum, and ileum of sexually mature female Wistar rats was investigated by using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 microCi 45CaCl2 (1 Ci = 37 GBq) was perfused through the 10-cm intestinal loop for 60 min. Results showed that 0.4 mg prolactin/kg body weight significantly increased duodenal net Ca absorption (net Ca) from 23.81 +/- 1.84 to 30.56 +/- 1.57 mmol/g dry weight (p < 0.05) by stimulating the lumen to plasma calcium flux (CaL-P). The jejunum responded to 0.2, 0.4, and 0.6 mg prolactin/kg body weight by reversing from net Ca absorption of 18.60 +/- 1.70 mmol/g dry weight to net secretion of -3.30 +/- 1.56, -10.39 +/- 2.21, and -11.79 +/- 2.04 mmol/g dry weight (p < 0.01), respectively, as a result of a dose-dependent increase in plasma to lumen calcium flux (CaP-L). Calcium fluxes in the ileum on the other hand did not respond to prolactin. There was a close correlation between net water flux and net calcium flux in all three intestinal segments under basal condition regardless of the luminal sodium concentration. However, this correlation was lost after prolactin administration, which while having no effect on net water flux, altered the duodenal and jejunal calcium fluxes. By varying the luminal concentration of sodium, it was found that the stimulatory effect of 0.4 mg prolactin/kg body weight on the duodenal CaL-P was reduced when compared with control, i.e., 17.84 +/- 0.91 vs. 26.64 +/- 1.05 mmol/g dry weight at a sodium concentration of 180 mM, and 14.48 +/- 0.99 vs. 20.12 +/- 1.34 mmol/g dry weight at a sodium concentration of 140 mM. At a sodium concentration of 80 mM, the prolactin effect was absent. Since duodenal Na(+)-K+ ATPase activity was increased by prolactin from 3.77 +/- 0.16 to 4.95 +/- 0.30 mumol Pi.mg-1 protein.h-1 (p < 0.05), sodium dependency of the prolactin-enhanced lumen to plasma calcium flux may be related to both sodium-induced water flow and calcium-sodium exchange across the basolateral membrane. Thus, it was postulated that under basal condition, net calcium transport in the small intestine occurred with the sodium-induced water transport along the paracellular pathway. However, after prolactin administration, this association was lost. Prolactin-enhanced lumen to plasma calcium flux in the duodenum was sodium dependent and involved the Na(+)-K+ ATPase activity. In the proximal jejunum, prolactin stimulated plasma to lumen calcium flux, but the mechanism was not known.

Acute effects of prolactin on passive calcium absorption in the small intestine by in vivo perfusion technique

The acute effect of intraperitoneally administered prolactin (0.2, 0.4, and 0.6 mg/kg body weight) on passive calcium transport in duodenum, proximal jejunum, and ileum of sexually mature female Wistar rats was investigated by using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 microCi 45CaCl2 (1 Ci = 37 GBq) was perfused through the 10-cm intestinal loop for 60 min. Results showed that 0.4 mg prolactin/kg body weight significantly increased duodenal net Ca absorption (net Ca) from 23.81 +/- 1.84 to 30.56 +/- 1.57 mmol/g dry weight (p < 0.05) by stimulating the lumen to plasma calcium flux (CaL-P). The jejunum responded to 0.2, 0.4, and 0.6 mg prolactin/kg body weight by reversing from net Ca absorption of 18.60 +/- 1.70 mmol/g dry weight to net secretion of -3.30 +/- 1.56, -10.39 +/- 2.21, and -11.79 +/- 2.04 mmol/g dry weight (p < 0.01), respectively, as a result of a dose-dependent increase in plasma to lumen calcium flux (CaP-L). Calcium fluxes in the ileum on the other hand did not respond to prolactin. There was a close correlation between net water flux and net calcium flux in all three intestinal segments under basal condition regardless of the luminal sodium concentration. However, this correlation was lost after prolactin administration, which while having no effect on net water flux, altered the duodenal and jejunal calcium fluxes. By varying the luminal concentration of sodium, it was found that the stimulatory effect of 0.4 mg prolactin/kg body weight on the duodenal CaL-P was reduced when compared with control, i.e., 17.84 +/- 0.91 vs. 26.64 +/- 1.05 mmol/g dry weight at a sodium concentration of 180 mM, and 14.48 +/- 0.99 vs. 20.12 +/- 1.34 mmol/g dry weight at a sodium concentration of 140 mM. At a sodium concentration of 80 mM, the prolactin effect was absent. Since duodenal Na(+)-K+ ATPase activity was increased by prolactin from 3.77 +/- 0.16 to 4.95 +/- 0.30 mumol Pi.mg-1 protein.h-1 (p < 0.05), sodium dependency of the prolactin-enhanced lumen to plasma calcium flux may be related to both sodium-induced water flow and calcium-sodium exchange across the basolateral membrane. Thus, it was postulated that under basal condition, net calcium transport in the small intestine occurred with the sodium-induced water transport along the paracellular pathway. However, after prolactin administration, this association was lost. Prolactin-enhanced lumen to plasma calcium flux in the duodenum was sodium dependent and involved the Na(+)-K+ ATPase activity. In the proximal jejunum, prolactin stimulated plasma to lumen calcium flux, but the mechanism was not known.

Sensing and refilling calcium stores in an excitable cell

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.

The body ion loss biomarker. 2. Field validation in coal mine-polluted streams

We performed a field validation of the body ion loss biomarker by developing a predictive model of the level of impact of coal mining on receiving streams. Hatchery brook charr (Salvelinus fontinalis ; wet mass = 1.25 to 32.2 g) were exposed in four mine-impacted streams that exhibited differing levels of acid and trace metals and in two unimpacted control streams. Net whole-body ion (Na, Ca) fluxes over a 4-h exposure were determined. Stepwise regression was used to develop predictive models of body ion flux over 4 h from measured stream water chemistry variables. The predictive power of the best model was assessed using cross validation. The best model predicting net sodium flux explained 48% of the variability in the model with six chemical variables (Mn, Zn, Mg, Al, Fe, and Na). The model of calcium flux explained 42% of the variation with eight variables (H + , Ca, Na, Mn, Al, Ni, Mg, and Zn). A multiple regression model derived from principal components analysis did not significantly improve the predictive power of these models. We conclude that whole-body net sodium flux and to a lesser extent whole-body net calcium flux are acute indicators of the level of impact of acid and trace metals in streams polluted by coal mine discharge and may be useful as biomarkers under field conditions.

Combined magnetic fields increased net calcium flux in bone cells

Low energy electromagnetic fields (EMF) exhibit a large number of biological effects. A major issue to be determined is "What is the lowest threshold of detection in which cells can respond to an EMF?" In these studies we demonstrate that a low-amplitude combined magnetic field (CMF) which induces a maximum potential gradient of 10(-5) V/m is capable of increasing net calcium flux in human osteoblast-like cells. The increase in net calcium flux was frequency dependent, with a peak in the 15.3-16.3 Hz range with an apparent bandwidth of approximately 1 Hz. A model that characterizes the thermal noise limit indicates that non-spherical cell shape, resonant type dynamics, and signal averaging may all play a role in the transduction of low-amplitude EMF effects in biological systems.

Effects of parathyroid hormone and parathyroid hormone‐related protein on the rates of absorption of magnesium, calcium, sodium, potassium and phosphate ions from the reticulo‐rumen of sheep

Not only parathyroid hormone (PTH) but also parathyroid hormone-related protein (PTHrP) may play a role in calcium and phosphate homeostasis in ruminants. In five trained sheep, each with a large rumen cannula, the isolated rumen wash technique was used to measure electrolyte absorption rates from the rumen. After two control periods of measurement, 20 micrograms PTH(1-34) (n = 4), or PTHrP(1-34) (n = 5), was injected intravenously as a loading dose, followed by an infusion of 0.67 micrograms min-1 over 2 h. Both PTH(1-34) and PTHrP(1-34) significantly increased the absorption rates of calcium and phosphate from the reticulo-rumen. The increases in the absorption rates of magnesium, sodium and potassium observed were not significant. The same technique was also used to demonstrate that increasing the intraruminal calcium concentration from 1 to 4 mmol l(-1) caused corresponding increases in the net rate of absorption of both calcium and inorganic phosphate from the reticulo-rumen. Rumen epithelium was taken from four sheep and mounted in Ussing chambers so that fluxes of calcium could be measured in both directions using 45Ca. It was found that the addition of 100 ng ml-1 PTH(1-34) or PTHrP(1-34) to the serosal side increased the net calcium flux rates across the ruminal epithelium. It is concluded that both PTH and PTHrP can influence calcium and phosphate homeostasis in sheep not only by their recognized actions on bone and kidney but also on the absorption of these ions from the forestomachs.

Decreased bone carbonate content in response to metabolic, but not respiratory, acidosis

In vitro cultured neonatal mouse calvariae release calcium and buffer the medium proton concentration in response to a decrease in the medium pH caused by a reduction in bicarbonate concentration ([HCO3-]), a model of metabolic acidosis, but not to an equivalent decrease in pH caused by an increase in the partial pressure of carbon dioxide (PCO2), a model of respiratory acidosis. We have postulated that the medium is in equilibrium with the carbonated apatite in bone. To determine whether bone carbonate is depleted during models of acidosis, we cultured calvariae in control medium (pH approximately 7.4, PCO2 approximately 43, [HCO3-] approximately 26) or in medium in which the pH was equivalently reduced by either a decrease in [HCO3-] (metabolic acidosis, pH approximately 7.1, [HCO3-] approximately 13) or an increase in PCO2 (respiratory acidosis, pH approximately 7.1, PCO2 approximately 86) and determined net calcium flux (JCa) and bone carbonate content. We found that compared with control, after 3, 24, and 48 h there was a decrease in bone carbonate content during metabolic but not during respiratory acidosis. Compared with control, at 3 h JCa increased with both respiratory and metabolic acidosis; however, at 24 and 48 h JCa increased only with metabolic acidosis. JCa was correlated inversely with percent bone carbonate content in control and metabolic acidosis at all time periods studied (r = -0.809, n = 23, P < 0.001). Thus a model of metabolic acidosis appears to increase JCa from bone, perhaps due to the low [HCO3-] inducing bone carbonate dissolution.(ABSTRACT TRUNCATED AT 250 WORDS)