Rate Of Calcium Transport Research Articles

Responses of primary osteoblasts and osteoclasts from hemizygous \u03b2-globin knockout thalassemic mice with elevated plasma glucose to 1,25-dihydroxyvitamin D3

β-thalassemia is often associated with hyperglycemia, osteoporosis and increased fracture risk. However, the underlying mechanisms of the thalassemia-associated bone loss remain unclear. It might result from abnormal activities of osteoblasts and osteoclasts, and perhaps prolonged exposure to high extracellular glucose. Herein, we determined the rate of duodenal calcium transport in hemizygous β-globin knockout thalassemic (BKO) mice. Their bones were collected for primary osteoblast and osteoclast culture. We found that BKO mice had lower calcium absorption than their wild-type (WT) littermates. Osteoblasts from BKO mice showed aberrant expression of osteoblast-specific genes, e.g., Runx2, alkaline phosphatase and osteocalcin, which could be partially restored by 1,25(OH)2D3 treatment. However, the mRNA expression levels of RANK, calcitonin receptor (Calcr), c-Fos, NFATc1, cathepsin K and DMT1 were similar in both BKO and WT groups. Exposure to high extracellular glucose modestly but significantly affected the expression of osteoclast-specific markers in WT osteoclasts with no significant effect on osteoblast-specific genes in WT osteoblasts. Thus, high glucose alone was unable to convert WT bone cells to BKO-like bone cells. In conclusion, the impaired calcium absorption and mutation-related aberrant bone cell function rather than exposure to high blood glucose were likely to be the principal causes of thalassemic bone loss.

Is the renal kallikrein-kinin system a factor that modulates hypercalciuria?

Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK). Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H) that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%). These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.

¿Es el sistema calicreína/quinina renal un factor modulador de la calciuria?

Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK). Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H) that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%). These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics.

Alteration of Tight Junction Gene Expression by Calcium- and Vitamin D-Deficient Diet in the Duodenum of Calbindin-Null Mice

Calcium absorption is regulated by both active (transcellular) and passive (paracellular) pathways. Although each pathway has been studied, correlations between the two pathways have not been well elucidated. In previous investigations, the critical transcellular proteins, calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), were shown to affect other transcellular pathways by buffering intracellular calcium concentrations. The rate of paracellular calcium transport in the duodenum is generally determined by the expression of tight junction genes. In the present study, the effect of dietary calcium and/or vitamin D supplementation on the expression of tight junction genes (occludin, ZO-1 and claudin 2, 10b, 12 and 15) in the duodenum of CaBP-9k- and/or -28k-deficient mice was examined. With a normal diet, the expression of most tight junction genes in the duodenum was significantly increased in CaBP-9k knockout (KO) mice compared to wild-type (WT) animals. With a calcium- and vitamin D-deficient diet, tight junction gene expression was significantly decreased in the duodenum of the CaBP-9k KO mice. These findings suggest that expression of paracellular tight junction genes is regulated by transcellular CaBP proteins, suggesting that active and passive calcium transport pathways may function cooperatively.

Impaired body calcium metabolism with low bone density and compensatory colonic calcium absorption in cecectomized rats

An earlier study reported that cecal calcium absorption contributes less than 10% of total calcium absorbed by the intestine, although the cecum has the highest calcium transport rate compared with other intestinal segments. Thus, the physiological significance of the cecum pertaining to body calcium metabolism remains elusive. Herein, a 4-wk calcium balance study in cecectomized rats revealed an increase in fecal calcium loss with marked decreases in fractional calcium absorption and urinary calcium excretion only in the early days post-operation, suggesting the presence of a compensatory mechanism to minimize intestinal calcium wasting. Further investigation in cecectomized rats showed that active calcium transport was enhanced in the proximal colon but not in the small intestine, whereas passive calcium transport along the whole intestine was unaltered. Since apical exposure to calcium-sensing receptor (CaSR) agonists similarly increased proximal colonic calcium transport, activation of apical CaSR in colonic epithelial cells could have been involved in this hyperabsorption. Calcium transporter genes, i.e., TRPV6 and calbindin-D(9k), were also upregulated in proximal colonic epithelial cells. Surprisingly, elevated serum parathyroid hormone levels and hyperphosphatemia were evident in cecectomized rats despite normal plasma calcium levels, suggesting that colonic compensation alone might be insufficient to maintain normocalcemia. Thus, massive bone loss occurred in both cortical and trabecular sites, including lumbar vertebrae, femora, and tibiae. The presence of compensatory colonic calcium hyperabsorption with pervasive osteopenia in cecectomized rats therefore corroborates that the cecum is extremely crucial for body calcium homeostasis.

Direct stimulation of the transcellular and paracellular calcium transport in the rat cecum by prolactin

Prolactin (PRL) is reported to stimulate calcium absorption in the rat's small intestine. However, little is known regarding its effects on the cecum, a part of the large intestine with the highest rate of intestinal calcium transport. We demonstrated herein by quantitative real-time polymerase chain reaction and Western blot analysis that the cecum could be a target organ of PRL since cecal epithelial cells strongly expressed PRL receptors. In Ussing chamber experiments, PRL enhanced the transcellular cecal calcium absorption in a biphasic dose-response manner. PRL also increased the paracellular calcium permeability and passive calcium transport in the cecum, which could be explained by the PRL-induced decrease in transepithelial resistance and increase in cation selectivity of the cecal epithelium. PRL actions in the cecum were abolished by inhibitors of phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK), but not inhibitors of gene transcription and protein biosynthesis. In conclusion, PRL directly enhanced the transcellular and paracellular calcium transport in the rat cecum through the nongenomic signaling pathways involving PI3K, PKC, and ROCK.

Effects of NH4Cl-induced systemic metabolic acidosis on kidney mitochondrial coupling and calcium transport in rats

We have previously shown that chronic metabolic acidosis, induced in rats by NH(4)Cl feeding, leads to nephron hypertrophy and to a decreased water-salt reabsorption by the kidneys. Since mitochondria are the main source of metabolic energy that drives ion transport in kidney tubules, we examined energy-linked functions (respiration, electrochemical membrane potential and coupling between respiration and ADP phosphorylation) in mitochondria isolated from rat kidney and liver at 48 h after metabolic acidosis induced by NH(4)Cl. Mitochondria isolated from the kidneys and liver of metabolic acidotic rats, induced by NH(4)Cl, was used to study of the oxygen consumption by Clark-type electrode, mitochondrial electrical transmembrane potential estimated by the safranine O method and the variations in free medium Ca(2+) concentrations examined by absorbance spectrum of Arsenazo III set at the 675-685 nm wavelength pair. Whole kidney and liver mitochondria isolated from 48 h acidotic rats presented higher resting respiration, lower respiratory control and a lower ADP/O ratio than controls. These differences in mitochondrial coupling, between respiration and oxidative phosphorylation (ATP synthesis), were totally corrected when experiments were carried out in the presence of carboxyatractyloside, GDP and BSA, indicating that mitochondrial uncoupling proteins are more active in acidotic rat kidneys. Interestingly, determination of Ca(2+) transport demonstrated a faster rate of initial Ca(2+) uptake by acidotic kidney mitochondria, which resulted in a lower concentration of extra-mitochondrial Ca(2+) under steady-state conditions (Ca(2+) set point) when compared with control mitochondria. In contrast, there were no significant differences in the rates of Na(+) or ruthenium red induced Ca(2+) efflux. We suggest that the mild uncoupling and higher Ca(2+) accumulation represents an adaptation of the mitochondria to cope with conditions of oxidative stress and high cytosolic Ca(2+), which are associated with a decreased efficiency of oxidative phosphorylation that may explain, at least in part, the striking natriuresis observed under chronic acidosis. Finally, there were no changes in Ca(2+) transport or coupling in liver mitochondria isolated from the acidotic rats.

Elevated Ca2+ ATPase (SERCA2) activity in tuna hearts: Comparative aspects of temperature dependence

Tunas have an extraordinary physiology including elevated metabolic rates and high cardiac performance. In some species, retention of metabolic heat warms the slow oxidative swimming muscles and visceral tissues. In all tunas, the heart functions at ambient temperature. Enhanced rates of calcium transport in tuna myocytes are associated with increased expression of proteins involved in the contraction-relaxation cycle. The cardiac SR Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling. Measurements of oxalate-supported Ca2+-uptake in atrial SR vesicles isolated from four species of tunas indicate that bluefin have at least two fold higher Ca2+-uptake than all other tunas examined between 5 and 30 degrees C. The highest atrial Ca2+-uptake was measured in bluefin tuna at 30 degrees C (23.32+/-1.58 nmol Ca2+/mg/min). Differences among tunas in the temperature dependency of Ca2+-uptake were similar for ATP hydrolysis. Western blot analysis revealed a significant increase in SERCA2 content associated with higher Ca2+ uptake rates in the atrial tissues of bluefin tuna and similar RyR expression across species. We propose that the expression of EC coupling proteins in cardiac myocytes, and the higher rates of SERCA2 activity are an important evolutionary step for the maintenance of higher heart rates and endothermy in bluefin tuna.

3-Nitrotyrosine Modification of SERCA2a in the Aging Heart: A Distinct Signature of the Cellular Redox Environment

In the aging heart, decreased rates of calcium transport mediated by the SERCA2a isoform of the sarcoplasmic reticulum (SR) Ca-ATPase are responsible for the slower sequestration of cytosolic calcium and consequent prolonged muscle relaxation times. We report a 60% decrease in Ca-ATPase activity in the senescent Fischer 344 rat heart relative to that of young adult hearts; this functional decrease can be attributed, in part, to the 18% lower abundance of SERCA2a protein. Here, we show that the additional loss of activity is a result of increased 3-nitrotyrosine modification of the Ca-ATPase. Age-dependent increases in nitration of cardiac SERCA2a are identified using multiple analytical methods. In the young (adult) heart 1 molar equivalent of nitrotyrosine is distributed over at least five tyrosines within the Ca-ATPase, identified as Tyr(122), Tyr(130), Tyr(497), Tyr(586), and Tyr(990). In the senescent heart, the stoichiometry of nitration increases by more than two nitrotyrosines per Ca-ATPase, coinciding with the appearance of nitrated Tyr(294), Tyr(295), and Tyr(753). The abundant recovery of native analogues for each of the nitrated peptides indicates partial modification of multiple tyrosines within cardiac SERCA2a. In contrast, within skeletal muscle SERCA2a, a homogeneous pattern of nitration appears, with full site (1 mol/mol) nitration of Tyr(753), in young, with additional nitration of Tyr(294) and Tyr(295), in senescent muscle. The nitration of these latter vicinal sites correlates with diminished transport function in both striated muscle types, suggesting that these sites provide a mechanism for downregulation of ATP utilization by the Ca-ATPase under conditions of nitrative stress.

A Theory of Plasma Membrane Calcium Pump Stimulation and Activity

The ATP-driven Plasma Membrane Calcium pump or Ca(2+)-ATPase (PMCA) is characterized by a high affinity for calcium and a low transport rate compared to other transmembrane calcium transport proteins. It plays a crucial role for calcium extrusion from cells. Calmodulin is an intracellular calcium buffering protein which is capable in its Ca(2+) liganded form of stimulating the PMCA by increasing both the affinity to calcium and the maximum calcium transport rate. We introduce a new model of this stimulation process and derive analytical expressions for experimental observables in order to determine the model parameters on the basis of specific experiments. We furthermore develop a model for the pumping activity. The pumping description resolves the seeming contradiction of the Ca(2+):ATP stoichiometry of 1:1 during a translocation step and the observation that the pump binds two calcium ions at the intracellular site. The combination of the calcium pumping and the stimulation model correctly describes PMCA function. We find that the processes of calmodulin-calcium complex attachment to the pump and of stimulation have to be separated. Other PMCA properties are discussed in the framework of the model. The presented model can serve as a tool for calcium dynamics simulations and provides the possibility to characterize different pump isoforms by different type-specific parameter sets.

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.

Evidence for stanniocalcin and a related receptor in annelids

Stanniocalcin (STC) is a prime example of a hormone whose discovery in fish led to its subsequent discovery in mammals. STC is considered to be first and foremost a vertebrate polypeptide hormone with regulatory effects on ion transport, mitochondrial function and steroid hormone synthesis. The gene is widely expressed in both fishes and mammals, and the hormone can operate via both local and endocrine signaling pathways. In spite of the growing catalogue of vertebrate hormones and receptors with homologues in invertebrates, the notion that there might be an invertebrate STC homolog has received scant attention to date. In the present study, we have provided evidence for STC in annelid worms (freshwater leeches). Western blot analysis revealed the presence of two STC immunoreactive (STCir) proteins in leech tissue extracts of 100 and 193 kDa. These same extracts significantly lowered the rate of gill calcium transport upon injection into fish. Similarly, fish STC increased the rate of whole body calcium uptake when administered to leeches, and STC receptors of high affinity were identified on isolated leech plasma membranes. Two discrete populations of STC-positive cells were also identified in leeches using antibodies to fish STC and fish STC cRNA probes. One of the cell types was confined to the skin. The second cell type was confined to the coelomic cavity and identified as an adipose cell, which in leeches is a major repository of fat. Collectively, the data constitutes compelling evidence for the existence of STC-related proteins and receptors in annelids that share structural and functional similarities with those in vertebrates.

Ordered and Cooperative Binding of Opposing Globular Domains of Calmodulin to the Plasma Membrane Ca-ATPase

We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation.

Lysophosphatidylcholine modulates catalytically important motions of the Ca-ATPase phosphorylation domain.

Catalytically important motions of the Ca-ATPase, modulated by the physical properties of surrounding membrane phospholipids, have been suggested to be rate-limiting under physiological conditions. To identify the nature of the structural coupling between the Ca-ATPase and membrane phospholipids, we have investigated the functional and structural effects resulting from the incorporation of the lysophospholipid 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphocholine (LPC) into native sarcoplasmic reticulum (SR) membranes. Nonsolubilizing concentrations of LPC abolish changes in fluorescence signals associated with either intrinsic or extrinsic chromophores that monitor normal conformational transitions accompanying calcium activation of the Ca-ATPase. There are corresponding decreases in the rates of calcium transport coupled to ATP hydrolysis, suggesting that LPC may increase conformational barriers associated with catalytic function. Fluorescence anisotropy measurements of the lipid analogue 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) partitioned into SR membranes indicate that LPC does not significantly modify lipid acyl chain rotational dynamics, suggesting differences in headgroup conformation between LPC and diacylglycerol phosphatidylcholines. Complementary measurements using phosphorescence anisotropy of erythrosin isothiocyanate at Lys464 on the Ca-ATPase provide a measure of the dynamic structure of the phosphorylation domain, and indicate that LPC restricts the amplitude of rotational motion. These results suggest a structural linkage between the cytosolic phosphorylation domain and the conformation of membrane phospholipid headgroups. Thus, changes in membrane phospholipid composition can modulate membrane surface properties and affect catalytically important motions of the Ca-ATPase in a manner that suggests a role for LPC generated during signal transduction.

ORCHARD FACTORS AFFECTING FRUIT STORAGE QUALITY AND PREDICTION OF HARVEST DATE OF APPLES

Numerous and well-recognised factors may affect the quality of pome fruits at harvest and following storage. Generally, fruit quality is associated with climatic and soil conditions, tree characteristics and cultural practices. The apple fruit is especially sensitive to low calcium concentration and susceptible to a number of physiological disorders during storage. Many environmental and cultural factors influence calcium content of apple fruits at maturity. When dormancy has been broken and bloom period begins, many important factors come into play that can affect ultimate fruit quality. These are related to fertilisation, pollination, seed number and fruit set. Number of seeds may influence the rate of calcium transport to the apples, probably by production and transport of auxins. On the other hand. fruits, which develop from terminal flowers, are richer in calcium than those developing from lateral ones. The location of fruits in the tree crown is also influencing calcium concentration, and incidence of physiological disorders; more fruits are affected in the upper parts of canopy. This is probably due to a poorer calcium input to these fruits. Cropping level of the tree is also a factor. Fruits from trees with light crops show generally a lower calcium concentration and they are more prone to bitter pit and internal breakdown. It seems that fruits compete in some manner with vegetative tissues for calcium transported from the root system. Summer pruning can have a strong positive effect on fruit quality, which at least in part is associated with increased Ca levels. Calcium treatments have also been shown to reduce the occurrence of physiological disorders. Wide variability of individual fruits within a tree as well as of trees within an orchard block, in respect to fruit maturation and subsequent storability was found to be associated with fruit calcium levels and differences in bloom time. Apples low in calcium show an earlier onset of the endogenous ethylene climacteric as fruits mature on the tree. On the other hand, fruits developed from late blooming flowers were considerably smaller than those from early blooming ones. Fruits from late blooming flowers produced less ethylene, exhibited a lower starch index and developed superficial scald during storage. This indicates that apple cultivars with a prolonged bloom period should be harvested at least twice. The date of harvest plays an important role in the quality of stored apple Ethylene is regarded as a more reliable indicator of physiological maturity than starch hydrolysis. In addition to this information, we attempted to predict the onset of the ethylene climacteric. The rapidity of the development of the ethylene climacteric is related to fruit maturity and we used this developmental phenomenon to predict the optimal harvest date.

The effect of oxygen free radicals on myocardial nuclear calcium transport in rat

The 1H and 13C NMR spectra of the chiral NADH models 3 and 4 were studied both in the presence or absence of Mg2+ ions. These two compounds have a chiral carboxamide part derived from (S)-phenylalaninol but contrary to 3, compound 4 has the ability to free-rotate about the C3CO bond. Careful analysis of the spectral results and comparison with some other studies recently published in the literature allowed the following assumptions: 1-the 1,4-dihydropyridine structure is planar both in the absence or presence of magnesium ions. 2-in the case of the rigid model 3, the CO is out of the plane of the dihydropyridine structure. 3-with Mg 2+ the chiral amino alcohol moiety adopts a quasi-cyclic conformation in the case of 4 but not in the case of 3. The observed enantioselectivities are explained by the mean of ternary complexes between the model, Mg2+ and the substrate.

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.

Stimulation by calcitriol of the uptake of calcium and of its transplacental flux in the ovine interplacentomal chorion: Work in progress

Our past results have shown that the placenta of an intact twin lamb fetus transports calcium more rapidly in vivo than that of its twin that had been totally nephrectomized 8 to 19 d earlier. Nephrectomy reduces the calcitriol concentration in the fetal circulation. The addition of calcitriol to the medium used to perfuse the placenta of a previously nephrectomized fetus rapidly increased the rate of calcium transport. We are now focusing more narrowly on potential individual components of this calcium transport process, which is fundamental to successful long bone growth and production of a viable neonate. Initially, we assessed the effects of calcitriol on the entry of calcium ions into the maternal-facing surface of discs of interplacentomal chorion in vitro, and our preliminary results clearly indicate the value of this new methodology in dissecting the basic mechanism of placental calcium transport. The chorion was obtained from fetuses between 120 and 145 d of gestation that had been nephrectomized 6 to 21 d earlier. These fetuses included 5 pairs of twins, 1 member of each pair of which had been nephrectomized. Our results demonstrate that previous nephrectomy, with subsequent reduction in calcitriol and development of fetal hypocalcaemia, was associated with a significant reduction in the rate of uptake of calcium ions into the maternal-facing aspect of the chorion. The addition of 600 pM calcitriol to the fluid bathing this chorionic surface led to a rapid, significant increase in the rate of uptake of calcium ions by the chorionic trophoblast epithelia cells. No such effect was noted with chorion from the placentas of fetuses with intact kidneys. The addition of diltiazem (50μM) to the incubation medium caused a reduction in the uptake of calcium ions by chorionic discs from either intact or nephrectomized fetuses. These results indicate for the first time that the ovine placental trophectodermal barrier is at least partly under calcitriol control and demonstrate the value of separation of a complex process into its constituent parts.

Benzophenone-Sensitized Photooxidation of Sarcoplasmic Reticulum Membranes: Site-Specific Modification of the Ca 2+-ATPase

Benzophenone (BP) was used as a photosensitizer to initiate lipid peroxidation in model and native biological membranes at concentrations of BP that do not perturb bilayer structure, as assessed by stearic acid spin label dynamics. Illumination of BP partitioned into sarcoplasmic reticulum membranes (SR) results in an exponential decay of BP and a linear accumulation of conjugated dienes and other products of lipid peroxidation as observed previously for micelles of linoleic acid [Marcovic and Patterson. Photochem. Photobiol. 58:329–334, 1993]. Lipid peroxidation was substantially inhibited in the presence of membrane-spanning proteins in SR compared to protein-free lipid vesicles, suggesting the competitive reaction of the initiator (triplet BP) and BP-derived radical species with protein groups. Modification of the predominant integral membrane protein, the Ca 2+-ATPase, was demonstrated by changes in Ca 2+-ATPase amino acid composition as well as by its functional inhibition. The rate of calcium transport showed an immediate exponential decay to completion, while calcium-dependent ATPase activity exhibited an initial lag before modest inactivation. These results are consistent with the respective localization of calcium transport sites within membrane-spanning peptides and the ATP-binding site within the cytosolic domain of the Ca 2+-ATPase, further suggesting that photosensitization of BP models oxidative stress inside the hydrophobic interior of the SR membrane.