Interaction of boar spermatozoa with porcine oocytes: Increase in proteins with high affinity for the zona pellucida during epididymal transit

Methods for the investigation of cell-associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using ⁴⁵Ca²⁺ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de-esterify the intracellular fluorescent calcium indicator Quin-2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage-sensitive calcium channels.     

Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.     

Role of calcium in secretion of chorionic gonadotropin by first trimester human placenta.

The role of calcium in regulation of secretion of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro has been investigated. Depletion of calcium in the medium by addition of EGTA resulted in a drastic decrease in the levels of immunoreactive hCG in the medium with consequent of accumulation of hCG in the tissue. Addition of A 23187 which is a calcium ionophore resulted in a dose dose dependent increase in the hCG in the medium and this stimulatory response could not be observed in the absence of calcium. Use of lanthanum (a calcium antagonist) in place of calcium in the medium used resulted in a significant decrease in the levels of hCG in the medium. Addition of veratridine (a sodium channel activator) stimulated hCG secretion in a dose dependent manner. These results suggest that calcium is essential for normal secretion of hCG by human placenta.     

A soluble extract from human spermatozoa activates ascidian oocytes

A soluble extract from human spermatozoa induced calcium oscillations and extrusion of the first polar body when injected into oocytes of the ascidian Ciona intestinalis . The properties of calcium oscillations and time of polar body extrusion precisely mimic oocyte activation induced by C. intestinalis sperm or sperm extracts. The data suggest that human sperm extracts can activate oocytes of different phyla by the same mechanism as homologous spermatozoa. Injection of inositol 1,4,5-trisphosphate (IP₃) into C. intestinalis oocytes mimicked to some extent the initial stages of oocyte activation, but the results demonstrate that ascidian oocyte activation by human sperm extract cannot be explained solely in terms of IP₃-induced calcium release. Injection of other calcium releasing second messengers, cyclic adenosine diphosphate ribose, or calcium ions, does not lead to oocyte activation or release intracellular calcium in ascidian oocytes. It was concluded that human spermatozoa contain one or more molecules that can trigger intracellular calcium release in oocytes from different phyla.     

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line

Background  

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.     

Effect of pH on the speciation and solubility of divalent metals in human and bovine milks

Computer models estimated the ligand speciation and solubility of calcium, magnesium, zinc, and copper over a pH range for low molecular weight fractions characteristic of either human or bovine milks. Above pH 4 calcium is the only metal predicted to precipitate. Most of the remaining soluble calcium, magnesium, and zinc should be complexed with citrate. The solubility of calcium, magnesium, and zinc in human and bovine milks was measured experimentally from pH 2 to 7. The solubility of all three metals decreased as the pH increased. Calcium and zinc were soluble over a narrower pH range in bovine milk than in human milk. Increasing the levels of either calcium or inorganic phosphate alone in decaseinated human milk did not affect the solubility of zinc, but when both calcium and inorganic phosphate were added at levels comparable to bovine milk the solubility of zinc decreased at the higher pH's. The decreased solubility of zinc in skimmed milks in pH's characteristic of the small intestine is likely due to coprecipitation of zinc with calcium phosphate--a reaction not predicted for milk systems from known chemical solubility product data.     

The binding of calcium ions by erythrocytes and `ghost''-cell membranes

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400mug-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140mug-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca(2+) concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte ;ghost' cells, the membranes of which appear to be permeable to Ca(2+) ions, can bind about 1000mug-atoms of calcium per ;ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.     

Calcium transport and regulation in human primary and metastatic melanoma.

Thioredoxin reductase (TR) activity on primary melanomas and in surrounding skin is regulated by calcium and, therefore, TR activity can be used to measure the flux of calcium between primary tumors and their surrounding epidermis. Calcium uptake in human melanotic melanoma cell lines SKmel-23 (metastatic) and BC-PT-1 (primary) is related to the density of beta-2-adrenoceptors. The non-pigmented cell line HT-144 (metastatic), did not express beta-2-adrenoceptors, yielding a slow rate of calcium uptake compared to SKmel-23 and BC-PT-1. Cell extracts from melanotic and amelanotic melanoma tissues did not contain a phenylethanolamine-N-methyltransferase (PNMT) for the biosynthesis of epinephrine from norepinephrine and S-adenosylmethionine. However, human full-thickness skin, epidermis and cell cultures of human keratinocytes contained significant PNMT activities. Taken together, these results indicate that (a), TR can be used to monitor calcium flux between primary melanomas and their surrounding skin and vice versa and (b), calcium uptake may be regulated by stimulation of beta-2-adrenoceptors on melanotic melanomas by epinephrine synthesized in the surrounding skin.     

UVB irradiation-induced dysregulation of plasma membrane calcium ATPase1 and intracellular calcium homeostasis in human lens epithelial cells

Ultraviolet B (UVB) could lead to the apoptosis of human lens epithelial cell and be hypothesized to be one of the important factors of cataractogenesis. In the human lens, Ca²⁺-ATPase is a major determinant of calcium homeostasis. Plasma membrane calcium ATPase1 (PMCA1) is a putative “housekeeping” isoform and is widely expressed in all tissues and cells, which plays an important role in calcium homeostasis. However, the effects of UVB-irradiation on the expression of PMCA1 and the cellular calcium homeostasis are still unclear. In the present study, we cultured human lens epithelial cells (HLE B-3) in vitro and investigated the effects of UVB irradiation on the expression of PMCA1 and the intracellular calcium homeostasis using real-time cell electronic sensing system, flow cytometry, fluo-3/AM probes, real-time quantitative PCR, and enzyme-linked immunosorbent assay techniques. We found that UVB irradiation could induce human lens epithelial cell death, cause intracellular calcium ion (Ca²⁺) elevation, inhibit Ca²⁺-ATPase activity and decrease the expression of PMCA1 at gene and protein levels, suggesting that the downregulation of PMCA1 and the disruption of calcium homeostasis may play important roles in UVB-induced HLE B-3 cell apoptosis.     

Investigation on the surface hydrophobicity and aggregation kinetics of human calprotectin in the presence of calcium

Calcium and zinc binding protein, calprotectin is a multifunctional protein with broad spectrum antimicrobial and antitumoural activity. It was purified from human neutrophil, using a two-step ion exchange chromatography. Since surface hydrophobicity of calprotectin may be important in membrane anchoring, membrane penetration, subunits oligomerization and some biological roles of protein, in this study attempted to explore the effect of calcium in physiological range on the calprotectin lipophilicity. Incubation of human calprotectin (50 microg/ml) with different calcium concentrations showed that 1-anilino-8-naphthalene sulfonic acid (ANS) fluorescence intensity of the protein significantly elevates with calcium in a dose dependent manner, suggesting an increase in calprotectin surface hydrophobicity upon calcium binding. Our study also indicates that calcium at higher concentrations (6, 8 and 10 mM) induces aggregation of human calprotectin. Our finding demonstrates that the starting time and the rate constant of calprotectin aggregation depend on the calcium concentration.     

Bioavailability of zinc and its binding to casein in milks and formulas

Differences in zinc bioavailability among milk and formulas may be attributed to binding of zinc to various ligands. We determined the distribution of zinc and protein at different pHs and zinc and calcium concentrations. We used radiolabelled cow's milk, human milk, whey-predominant (WPF) and casein-predominant (CPF) infant formula. Lowering the pH changed zinc and protein distribution: zinc shifted from pellet (casein) to whey in cow's milk, from fat to whey in human milk and from fat and pellet to whey in formulas. Protein shifted from whey to pellet in human milk and from whey and pellet to fat in formulas. Increasing zinc and calcium concentrations shifted protein and zinc from pellet to whey for cow's milk and from whey and pellet to fat for the formulas. Protein distribution was not affected by calcium or zinc addition in human milk or CPF, while zinc shifted from whey to fat in human milk and from fat and pellet to whey in CPF. Zinc and calcium binding to isolated bovine or human casein increased with pH. At 500 mg/L of zinc, bovine casein bound 32.0 +/- 1.8 and human casein 10.0 +/- 0.9 mg zinc/g protein. At 500 mg/L of calcium, calcium was preferentially bound over zinc. Adding calcium and zinc resulted in 32.0 +/- 1.8 mg zinc/g bound to bovine casein and 17.0 +/- 0.8 mg zinc/g to human casein, while calcium binding was low. Suckling rat pups dosed with 65Zn labelled infant diets were killed and individual tissues were gamma counted. Lower zinc bioavailability was found for bovine milk at pH = 4.0 (%65Zn in liver = 18.7+1.4) when compared to WPF (22.8 +/- 1.6) or human milk (26.9 +/- 0.8). Lowering the pH further decreased zinc bioavailability from human milk, but not from cow's milk or WPF. Knowledge of the compounds binding minerals and trace elements in infant formulas is essential for optimizing zinc bioavailability.     

Lack of a central role for calcium in the induction and release of human interleukin-1

This study examines the potential role of calcium in the activation of human monocytes by bacterial lipopolysaccharide (LPS) to produce interleukin-1 (IL-1). Monocytes cannot be triggered to produce IL-1 through addition of calcium ionophores. Triggering of IL-1 production by LPS cannot be blocked by depletion of extracellular calcium, blockade of calcium channels, or addition of agents which antagonize the effects of intracellular calcium. Finally, the addition of LPS does not induce the mobilization of intracellular free calcium as measured by quin-2 fluorescence.     

Extracellular calcium increases free cytoplasmic calcium and DNA synthesis in human osteoblasts.

A high concentration of extracellular calcium (8 mM) induced an increase in free cytoplasmic calcium, a lower cyclic AMP level and increased DNA synthesis in primary cultures of human osteoblast-like cells. Inhibition of protein kinase C with bisindolylmaleimide I inhibited the stimulatory effect of extracellular calcium on DNA synthesis in human osteoblast-like cells, whereas inhibition of protein kinase A with Rp-cAMPs had no effect on DNA synthesis. This indicates that protein kinase C, possibly via increased free cytoplasmic calcium, mediates the effect of extracellular calcium on DNA synthesis in osteoblast-like cells rather than a relative decrease in cyclic AMP and protein kinase A activity. Furthermore, a low concentration (0.5 mM) of extracellular calcium decreased DNA synthesis. In conclusion, these data suggest that a high extracellular calcium level may be a coupling factor that recruits osteoblasts in the bone remodeling process, and that a low level of extracellular calcium may also regulate osteoblast function.     

Metal binding characteristics of human salivary and porcine pancreatic amylase

With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.     

Identification of a novel human subfamily of mitochondrial carriers with calcium-binding domains

Aralar1 and citrin were identified as calcium binding aspartate/glutamate carriers (AGC) in mitochondria. The presence of calcium binding motifs facing the extramitochondrial space allows the regulation of the transport activity of these carriers by cytosolic calcium and provides a new mechanism to transduce calcium signals in mitochondria without the requirement of calcium entry in the organelle. We now report the complete characterization of a second subfamily of human calcium binding mitochondrial carriers named SCaMC (short calcium-binding mitochondrial carriers). We have identified three SCaMC genes in the human genome. All code for highly conserved proteins (about 70-80% identity), of about 500 amino acids with a characteristic mitochondrial carrier domain at the C terminus, and an N-terminal extension harboring four EF-hand binding motifs with high similarity to calmodulin. All SCaMC proteins were found to be located exclusively in mitochondria, and their N-terminal extensions were dispensable for the correct mitochondrial targeting of the polypeptides. SCaMC-1 is the human orthologue of the rabbit Efinal protein, which was reported to be located in peroxisomes, and SCaMC-2 is the human orthologue of the rat MCSC protein, described as up-regulated by dexamethasone in AR42J cells. One of the SCaMC genes, SCaMC-2, has four variants generated by alternative splicing, resulting in proteins with a common C terminus but with variations in their N-terminal halves, including the loss of one to three EF-hand motifs. These results make SCaMC one of most complex subfamilies of mitochondrial carriers and suggest that the large number of isoforms and splice variants may confer different calcium sensitivity to the transport activity of these carriers.     

Substance P immunoreactive nerve fibres are related to gastric cancer differentiation status and could promote proliferation and migration of gastric cancer cells

Tachykinins such as SP (substance P) may be involved in the progression of gastric adenocarcinoma through binding to NK-1 receptor. However, the existence and relationship between SP and gastric cancer progression and differentiation remained unknown. We have studied the NK-1 receptor in human gastric cancer tissue and MKN45 cell line and found SP-containing nerve fibres in human gastric cancer and found that the amounts of SP-positive nerves were related to gastric cancer differentiation. SP could promote proliferation, adhesion, migration and invasion of MKN45 cells in vitro. In addition, the intracellular calcium level of MKN45 cells was elevated after SP stimulation, and administration of CRACs (calcium release-activated calcium channels) inhibitor SKF-96365 could partially abolish these effects induced by SP. These results demonstrated that NK-1 receptor and SP-containing nerves existed in human gastric cancer; SP positive nerves may play an important role in human gastric cancer progression, and calcium is critically significant among SP-induced biological effects.     

Modulation of the intestinal Ca2+ uptake by a cheese whey protein digest

We have previously constructed a system which enables the search for factors that could modulate the intestinal calcium transporter, CaT1 (TRPV6; Takano et al., Cytotechnology, 43, 113 (2003)). This system evaluates the CaT1-mediated calcium uptake by using CHO cells stably expressing human CaT1 (CHO-hCaT1 cells). We found that a cheese whey protein digest (CWP-D) increased the calcium uptake by the CHO-hCaT1 cells. CWP-D also enhanced the calcium uptake in human intestinal Caco-2 cells. The in vivo effects of CWP-D were then measured by using rats with enteral feeding. Although enteral feeding decreased the portal calcium concentration, CWP-D partially suppressed the decrease, suggesting that CWP-D could be used for food to enhance calcium absorption.     

Human T lymphocyte mitogenesis in response to the B oligomer of pertussis toxin is associated with an early elevation in cytosolic calcium concentrations

Pertussis toxin was found to serve as a mitogen in the human T lymphocyte, an effect which could be mimicked by its resolved binding component, the B oligomer. The mechanism of action of this component appeared to involve a rapid and sustained elevation of cytosolic calcium levels, as monitored by fura-2 fluorescence. The source of mobilized calcium was predominantly extracellular, suggesting that the binding of the B oligomer to the T cell plasma membrane in some way elicited calcium channel activation. Notably, the influx of calcium was not observed with cholera toxin, an AB toxin lacking mitogenic effects on the human T lymphocyte.     

Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.     

Effect of verapamil on the contractions produced by high potassium and by noradrenaline in human isolated saphenous vein

The effect of a calcium channel blocker, e.g. verapamil, on the contractions produced by high potassium (K+) and noradrenalne (NA), was studied in the isolated saphenous vein in man. The aim of the present experiments was to see which of the two types of contractions was more sensitive to blockade by a calcium channel blocker, e.g. verapamil, and if verapamil had a differential effect on KCl and NA, whether this could be interpreted in terms of the presence of two calcium activation mechanisms in human saphenous vein. The results of the present investigation showed that KCl and NA contracted whereas verapamil relaxed the human saphenous vein. NA produced larger contraction (3.4 g tension) than did KCl (1.3 g tension). Lowering the calcium concentration in the external medium, from 2.5 mM to 1 mM, resulted in a reduced contraction in both NA and KCl responses, indicating dependence on influx of calcium. However, verapamil (1 microM) produced greater reduction in the KCl than NA-induced contraction, indicating that the NA contraction may involve additional mechanism, i.e. dependence on the release of calcium from intracellular Ca2+ stores. These results are in favour of the suggestion that the KCl-induced contraction was due to depolarization and voltage-dependent activation of calcium channels, whereas the NA-induced contraction was due to both depolarization and receptor-activation of the calcium channels, the latter being less sensitive to calcium channel blockers, e.g. verapamil. Thus, the KCl and NA-induced contractions in human saphenous vein may be due to two different calcium activation mechanisms; one is more sensitive (KCl) than the other (NA) to the presence of the calcium antagonist, verapamil.