Enhanced Calcium Uptake Research Articles

Local calcium chloride infusion after pulsed field ablation enhances acute efficacy of cardiac electroporation.

Pulsed field ablation (PFA) has emerged as an innovative therapy for cardiac arrhythmias. Drawing parallels with PFA's application in solid tumors, calcium chloride (CaCl2) as an adjuvant therapy, known as calcium electroporation, may amplify PFA's apoptotic effects. We propose that PFA in the atrium could enhance calcium uptake through PFA-created pores, thereby increasing ablation efficacy even at reduced power levels by exploiting PFA's permeabilization effects. We conducted in vivo ablations on the atria of seven pigs using low PFA power (250 V, 20 μs for 50 pulses at 200 ms intervals). Post-PFA, we randomly administered an infusion of either 200 mg/2 ml CaCl2 (calcium group) or saline (control) directly to the ablation site via the catheter tip. We evaluated reduction in electrogram voltage amplitude, electrocardiography (ECG) parameters, ablation lesion parameters, and histology after PFA. Nineteen lesions from control and calcium groups were examined. Control lesions showed no voltage decrease post-PFA, whereas calcium-treated lesions exhibited a significant voltage reduction. Gross pathology indicated marked differences in maximum lesion surface diameter, depth, and volume between the lesion groups. Histologically, calcium group lesions were characterized by a more severe acute PFA response with contraction band necrosis, myocytolysis and nuclear pyknosis in adjacent myocardium, in addition to microhemorrhages. Infusing calcium chloride locally after PFA markedly improves the immediate efficacy of electroporation in porcine atria. This study suggests that calcium electroporation could bolster PFA outcomes without higher energy levels, potentially diminishing associated risks. These preliminary findings warrant further research into the long-term efficacy and potential clinical application of calcium electroporation in PFA.

Casein hydrolysates produced via sequential enzymatic hydrolysis: characterisation and their effect on calcium uptake by Caco‐2 cell line

SummaryWhile dietary components such as phytate reduce calcium absorption, bioactive peptides could potentially enhance calcium absorption. This study aimed to characterise the peptides derived from casein and assess their effect on calcium uptake by Caco‐2 cells. Casein was subjected to hydrolysis by Alcalase and Flavourzyme to produce 4‐h and 16‐h casein hydrolysates. High‐performance liquid chromatography analysis showed that hydrolysis caused significant increases and decreases in the content of some amino acids. Casein hydrolysates achieved 40.36% and 52.64% degree of hydrolysis, as determined by the o‐phthaldialdehyde method. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and dynamic light scattering analyses revealed that the peptides had molecular weight less than 15 kDa and diameter between 100 and 1000 nm. The 4‐h casein hydrolysate enhanced calcium uptake by Caco‐2 cells monolayer at low calcium intake, possibly via transcellular absorption. These results provide insight into the nutraceutical potential of casein hydrolysates as enhancer of calcium absorption.

Impact of Calcium Influx on Endoplasmic Reticulum in Excitotoxic Neurons: Role of Chemical Chaperone 4-PBA.

Excessive activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propoinic acid (AMPA) receptors instigates excitotoxicity via enhanced calcium influx in the neurons thus inciting deleterious consequences. Additionally, Endoplasmic Reticulum (ER) is pivotal in maintaining the intracellular calcium balance. Considering this, studying the aftermath of enhanced calcium uptake by neurons and its effect on ER environment can assist in delineating the pathophysiological events incurred by excitotoxicty. The current study was premeditated to decipher the role of ER pertaining to calcium homeostasis in AMPA-induced excitotoxicity. The findings showed, increased intracellular calcium levels (measured by flowcytometry and spectroflourimeter using Fura 2AM) in AMPA excitotoxic animals (male Sprague dawely rats) (intra-hippocampal injection of 10mM AMPA). Further, ER resident proteins like calnexin, PDI and ERp72 were found to be upregulated, which further modulated the functioning of ER membrane calcium channels viz. IP3R, RyR, and SERCA pump. Altered calcium homeostasis further led to ER stress and deranged the protein folding capacity of ER post AMPA toxicity, which was ascertained by unfolded protein response (UPR) pathway markers such as IRE1α, eIF2α, and ATF6α. Chemical chaperone, 4-phenybutric acid (4-PBA), ameliorated the protein folding capacity and subsequent UPR markers. In addition, modulation of calcium channels and calcium regulating machinery of ER post 4-PBA administration restored the calcium homeostasis. Therefore the study reinforces the significance of ER stress, a debilitating outcome of impaired calcium homeostasis, under AMPA-induced excitotoxicity. Also, employing chaperone-based therapeutic approach to curb ER stress can restore the calcium imbalance in the neuropathological diseases.

Discovery of calcium-binding peptides derived from defatted lemon basil seeds with enhanced calcium uptake in human intestinal epithelial cells, Caco-2

It is anticipated that calcium-chelating peptides may serve to enhance the absorption of calcium. This research examined defatted lemon basil seeds (DLBS) which had been treated with Alcalase under optimized parameters for the degree of hydrolysis for proteolysis, discovering that the activity for calcium-binding in a competitive condition with phosphate ion was 60.39 ± 1.545%. The purification of the hydrolysates was performed via ultrafiltration along with reversed-phase high performance liquid chromatography (RP-HPLC). Determination of the purified peptide amino acid sequence was confirmed for both peptides and reported as Ala-Phe-Asn-Arg-Ala-Lys-Ser-Lys-Ala-Leu-Asn-Glu-Asn (AFNRAKSKALNEN; Basil-1), and Tyr-Asp-Ser-Ser-Gly-Gly-Pro-Thr-Pro-Trp-Leu-Ser-Pro-Tyr (YDSSGGPTPWLSPY; Basil-2). The respective activities for calcium-binding were 38.62 ± 1.33%, and 42.19 ± 2.27%. Fluorescence spectroscopy, and fourier transform infrared spectroscopy were employed in order to assess the chelating mechanism between calcium and the peptides. It was found that the calcium ions took place through the activity of the amino nitrogen atoms and the oxygen atoms on the carboxyl group. Moreover, both of these peptides served to improve calcium transport and absorption in Caco-2 cell monolayers, depending on the concentration involved. It was revealed that the peptide-calcium complexes offered an increased calcium absorption percentage when compared to free calcium at similar concentrations. It might be concluded that the peptide within the peptide-calcium complex can promote calcium absorption through both active and passive transport pathways by increasing calcium concentration and promoting cell membrane interaction. Accordingly, DLBS protein can be considered a strong potential source of protein which can be used to produce calcium-binding peptides and might therefore play a role in the production of nutraceutical foods as a bioactive ingredient.

KFP-1, a Novel Calcium-Binding Peptide Isolated from Kefir, Promotes Calcium Influx Through TRPV6 Channels.

Kefir is an acidic and alcoholic fermented milk product with multiple health-promoting benefits. A previous study demonstrated that kefir enhanced calcium absorption in intestinal Caco-2 cells. In this study, kefir-fermented peptide-1 (KFP-1) is isolated from the kefir peptide fraction, and its function as a calcium-binding peptide is characterized. KFP-1 was identified as a 17-residue peptide with a sequence identical to that of κ-casein (residues 138-154) in milk protein. KFP-1 is demonstrated to promote calcium influx in Caco-2 and IEC-6 small intestinal cells in a concentration-dependent manner. TRPV6, but not L-type voltage-gated calcium channels, is associated with the calcium influx induced by KFP-1. An in vitro calcium binding assay indicates that the full-length KFP-1 peptide has a higher calcium-binding capacity than the two truncated KFP-1 peptides, KFP-1∆C5 and KFP-1C5. Alexa Fluor 594 labeling shows that KFP-1 is taken up by Caco-2 cells and interacts with calcium ions and TRPV6 protein. Moreover, KFP-1 is found moderately resistant to pepsin and pancreatin digestions and enhanced calcium uptake by intestinal enterocytes in vivo. These data suggest that KFP-1, a novel calcium-binding peptide, binds extracellular calcium ions and enters Caco-2 and IEC-6 cells, and promotes calcium uptake through TRPV6 calcium channels. The present study is of great importance for developing kefir-derived metal ion-binding peptides as functional nutraceutical additives.

Abstract 437: Mitochondrial Cardiomyopathies Feature Enhanced Mitochondrial Calcium Signaling

Mitochondrial diseases often feature early onset of dilated cardiomyopathy, due to the large energetic demand placed by the heart. Among regulators of mitochondrial respiration, we focus on calcium, which potently stimulates ATP synthesis. We assessed the hypothesis that cells boost mitochondrial calcium to stimulate respiration as this declines in mitochondrial cardiomyopathies. To this end, we studied mice that develop a neonatal, severe cardiomyopathy caused by cardiac-specific knockout (KO) of mitochondrial transcription factor A ( Tfam ). Such deletion impairs transcription of mitochondrial DNA, which encodes multiple subunits of the electron transport chain (ETC). Tfam KO mice exhibited a dilated cardiomyopathy, with decreased fractional shortening and increased LV diameters, and had marked inhibition of multiple ETC complexes. We determined that Tfam KO mitochondria took up calcium twice as fast as mitochondria from littermate controls, while calcium efflux was approximately 70% slower. Whole-mitoplast voltage clamp revealed that the enhanced calcium uptake was due to an increase in the current carried by the uniporter. Furthermore, the larger uniporter current reflected increased amounts of uniporter subunit proteins. This occurred despite a reduction in the transcripts of genes encoding these subunits, suggesting a post-translational mechanism for the enhanced uniporter stability. Finally, we found that the rate of ADP-stimulated oxygen consumption in calcium-free solution was 50% less in the Tfam KO mitochondria compared to controls, but increased substantially more, nearly to control levels, when calcium was present (2.5-fold increase for KO versus 1.7-fold for control). In conclusion, enhanced mitochondrial calcium signaling in a mitochondrial cardiomyopathy model may serve to compensate for energetic failure.

A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

Bioactive Peptides Isolated from Casein Phosphopeptides Enhance Calcium and Magnesium Uptake in Caco-2 Cell Monolayers.

The ability of casein phosphopeptides (CPPs) to bind and transport minerals has been previously studied. However, the single bioactive peptides responsible for the effects of CPPs have not been identified. This study was to purify calcium-binding peptides from CPPs and to determine their effects on calcium and magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1 to P5 were isolated and the amino acid sequences were determined using LC-MS/MS. Compared with the CPP-free control, all five monomeric peptides exhibited significant enhancing effects on the uptake of calcium and magnesium (P < 0.05). Interestingly, when calcium and magnesium were presented simultaneously with P5, magnesium was taken up with priority over calcium in the Caco-2 cell monolayers. For example, at 180 min, the amount of transferred magnesium and calcium was 78.4 ± 0.95 μg/well and 2.56 ± 0.64 μg/well, respectively, showing a more than 30-fold difference in the amount of transport caused by P5. These results provide novel insight into the mineral transport activity of phosphopeptides obtained from casein.

Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells.

Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives.

Effects of Calcium-Binding Peptide from Tilapia Scale Protein Hydrolysates on Calcium Absorption in Caco-2 Cells

ABSTRACTFish scales are usually discarded as an industrial solid waste. In this study the fish scale protein was recovered and employed to prepare protein hydrolysates using commercial proteases. The hydrolysates were further separated into four peptide fractions (F1, F2, F3, and F4) by Sephadex G-15. Among the fractions, the F3 fraction, with the highest calcium-binding activity, was further investigated to enhance calcium uptake in Caco-2 cells. The results showed calcium uptake from F3-Ca by Caco-2 cells after 2 h of incubation was significantly higher than those of CaCl2 and tilapia scale protein hydrolysates (TSPH), respectively, and was similar to that of casein phosphopeptide (CPP). Therefore, the F3 fraction improved Ca uptake versus the CPP samples and can be employed as an alternative to CPP in the manufacture of functional foods.

Growth and Longevity of the „Living fossil”Congeria kusceri(Bivalvia: Dreissenidae) from the Subterranean Dinaric Karst of Croatia

The present study analyzed growth and longevity of the endemic cave dwelling bivalve Congeria kusceri obtained from the pit Jama u Predolcu, Croatia. A water temperature of 13–14°C constituted the lower limit for shell growth of C. kusceri. Obtained data also indicated that enhanced calcium uptake by C. kusceri during its summer growth and reproductive phases coincided with a decrease in alkalinity of the pit water. An analysis of shell growth rings in C. kusceri has identified an impressive longevity of 53 years. Calculations of growth rate suggested maximum theoretical shell lengths (L∞) of 16.7 mm for males and 18.7 mm for females and a growth constant of 0.04 year-1 for both sexes. Congeria kusceri can, however, grow to a shell length of > 24 mm, suggesting that longevity of this species could be greater than that identified in this study. The obtained results add to our understanding of how colonisation and continued and successful occupation of a subterranean freshwater habitat has been achieved.

Early osteoblast responses to orthopedic implants: Synergy of surface roughness and chemistry of bioactive ceramic coating.

Pro-osteogenic stimulation of bone cells by bioactive ceramic-coated orthopedic implants is influenced by both surface roughness and material chemistry; however, their concomitant impact on osteoblast behavior is not well understood. The aim of this study is to investigate the effects of nano-scale roughness and chemistry of bioactive silica-calcium phosphate nanocomposite (SCPC50) coated Ti-6Al-4V on modulating early bone cell responses. Cell attachment was higher on SCPC50-coated substrates compared to the uncoated controls; however, cells on the uncoated substrate exhibited greater spreading and superior quality of F-actin filaments than cells on the SCPC50-coated substrates. The poor F-actin filament organization on SCPC50-coated substrates is thought to be due to the enhanced calcium uptake by the ceramic surface. Dissolution analyses showed that an increase in surface roughness was accompanied by increased calcium uptake, and increased phosphorous and silicon release, all of which appear to interfere with F-actin assembly and osteoblast morphology. Moreover, cell attachment onto the SCPC50-coated substrates correlated with the known adsorption of fibronectin, and was independent of surface roughness. High-throughput genome sequencing showed enhanced expression of extracellular matrix and cell differentiation related genes. These results demonstrate a synergistic relationship between bioactive ceramic coating roughness and material chemistry resulting in a phenotype that leads to early osteoblast differentiation.

Fluorescence Technologies for Evaluating Male Gamete (Dys)Function

For many years, andrologists have sought ways of assessing sperm fertility, especially of new sires entering the breeding chain. As knowledge of the complex processes that enable sperm to fertilize eggs has increased, it has become clearer that quantitative estimation of the fertilizing potential of a sire or an ejaculate is actually unlikely ever to be fully realized. Here, we propose that a better approach is to identify substandard males and semen samples. During the past decades, the use of fluorescence technologies in biomedical science has burgeoned, with the development of very powerful instrumentation such as confocal microscopy and flow cytometers of ever-increasing capabilities together with a vast range of fluorochromes and fluorochrome conjugates. This technology has been applied to andrology but thus far in only a relatively simple way. In this review, we offer strategies for assessing a large range of sperm functions thought to be related to fertilizing ability over a temporal window rather than at a single time point. From such an assessment profile, sperm samples that over-respond or do not respond sufficiently could be identified, termed dysfunctional and rejected. We outline the rationales behind such tests, present information on new potentially useful fluorochromes and current flow cytometer models that would be suitable for the multicolour multifunctional tests we propose, and we offer suggestions as to how andrologists might design such multicolour tests for themselves.

Intestinal Cell Calcium Uptake and the Targeted Knockout of the 1,25D3-MARRS (Membrane-associated, Rapid Response Steroid-binding) Receptor/PDIA3/Erp57

We have crossed ERp57(flx/flx) mice with commercially available mice expressing villin-driven cre-recombinase. Lysates of intestinal epithelial cells were prepared from knock-out (KO) mice and littermates (LM) and used in Western blot analyses with Ab099 against the N terminus of the 1,25D(3)-MARRS (membrane-associated, rapid response steroid-binding) receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [(3)H]1,25D(3) revealed negligible binding in preparations from either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid-stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D(3) elicited enhanced calcium uptake in females and males within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell surface vitamin D receptors. However, antibodies to the vitamin D receptor failed to block 1,25D(3)-stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake and for LM females reached 250% of controls within 5 min, and 150% of controls in cells prepared from LM males. Enterocytes from female or male KO mice failed to exhibit steroid hormone-stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D(3)-MARRS receptor is of central importance to steroid hormone-stimulated calcium uptake in mammalian intestinal cells.

Intestinal Cell Calcium Uptake and the Targeted Knockout Of the 1,25D3‐MARRS Receptor/PDIA3/Erp57

We have crossed ERp57lfx/flx mice with mice expressing villin‐driven cre‐recombinase. Lysates of enterocytes from knockout (KO) mice and littermates (LM) were used in Western analyses with Ab099 against the N‐terminus of the 1,25D3‐MARRS receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [3H]1,25D3 revealed negligible binding in either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D3 elicited enhanced within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell‐surface VDR. However, antibodies to the VDR failed to block 1,25D3‐ stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake. Enterocytes from female or male KO mice failed to exhibit 1,25D3 stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D3‐MARRS receptor is of central importance to steroid hormone stimulated calcium uptake in mammalian intestinal cells.

Calmodulin antagonist W7 increases inositol phosphates in insulin secreting RINm5F cells.

W7, a calmodulin antagonist, has been reported to increase cytosolic free calcium concentration [Ca2+]i in non stimulated rat insulinoma cells (RINm5F). And this effect was not due to enhanced calcium uptake. In the present study the effect of calmodulin antagonist W7 on the inositol phosphate turnover of RINm5F cells was studied. Inositol phosphates were separated using a new modified technique of anion-exchange high performance liquid chromatography (HPLC). It was observed that W7 significantly increased inositol trisphosphate and inositol bisphosphate within 5 and 15 sec, respectively. No changes of inositol phosphates were detected employing W5, a chlorine-deficient analogue of W7 without calmodulin antagonistic action. Our data are in favour of the view that (I) calmodulin may be involved in inositol phosphate metabolism of RINm5F cells and that (II) the increase of [Ca2+]i in response to W7 as reported previously may be due to elevation of inositol trisphosphate.

Mitochondria at the Immunological Synapse

Quintana et al . noted that beads coated with antibodies to CD3, a constituent of the T cell receptor (TCR) complex, produced a larger and more sustained calcium signal in the Jurkat T cell line and primary human T cells than did the same antibodies in solution. This difference correlated with the antibody-coated beads’ ability to stimulate the formation of the immune synapse, a macromolecular structure that forms at the plasma membrane at the site of TCR activation and that is involved in T cell activation. Calcium influx across the plasma membrane in response to TCR activation involves the activation of calcium release-activated calcium (CRAC) channels, the Orai1 channels, in response to depletion of calcium from intracellular stores. Quintana et al . found that when mitochondrial calcium uptake was pharmacologically inhibited, calcium signaling was decreased in response to the antibody-coated beads. Indeed, TCR activation triggered the relocation of mitochondria to within 200 nm of the plasma membrane at the site of the immune synapse (based on total internal reflection microscopy), and these mitochondria that were close to the immune synapse exhibited enhanced calcium uptake compared with that of mitochondria distant from the immune synapse. The authors suggest that the relocation of the mitochondria to the immune synapse and their uptake of calcium prevent calcium-induced inactivation of the Orai1 channels, thereby allowing sustained calcium influx in response to stimuli that induce the formation of the immune synapse. A. Quintana, C. Schwindling, A. S. Wenning, U. Becherer, J. Rettig, E. C. Schwarz, M. Hoth, T cell activation requires mitochondrial translocation to the immunological synapse. Proc. Natl. Acad. Sci. U.S.A. 104 , 14418-14423 (2007). [Abstract] [Full Text]

Effects of a new titanium fluoride derivative on enamel de‐ and remineralization

The potential of a new titanium fluoride (TiF) derivative for caries prevention was tested in a pH-cycling model. Daily treatments with various concentrations (100, 250, and 500 p.p.m.) of TiF were compared with similar sodium fluoride (NaF) treatments given at the same pH. Bovine enamel lesions were subjected to 3 wk of pH cycling. The effects were assessed by analyzing calcium uptake and loss in the re- and demineralizing solutions, respectively, and by post pH cycling microradiographic analysis of the lesions. Treatments with NaF reduced calcium loss, enhanced calcium uptake, and induced overall lesion remineralization. Treatments with TiF derivative gradually caused almost complete inhibition of calcium loss and uptake (lesion 'arrestment'), irrespective of the concentration of the TiF derivative. To test the permanence of protection, sound enamel was pretreated with either TiF derivative or NaF, and demineralized for 14 d at pH 4.4 and 4.6. Calcium loss data show that up to 80% inhibition of demineralization could be achieved for the TiF derivative, which was not possible for NaF treatments. This inhibition was obtained through a combination of concentration and number of treatments. The TiF derivative is a promising agent for the prevention of dental caries, especially when aimed at preventing the onset of caries.

Nucleus isthmi enhances calcium influx into optic nerve fiber terminals in Rana pipiens

We examined the role of nucleus isthmi in enhancing intracellular calcium concentrations in retinotectal fibers in the frog optic tectum in vitro. The intracellular calcium levels were measured using the fluorescent calcium-sensitive dye, Calcium Green-1 3000 mw dextran conjugate (CG-1), which was injected into one optic nerve. Electrical stimulation of the labeled optic nerve alone increased tectal CG-1 fluorescence whereas electrical stimulation of nucleus isthmi alone had no effect on CG-1 fluorescence. Electrical stimulation of the nucleus isthmi ipsilateral to the labeled tectum, followed by electrical stimulation to the optic nerve can enhance calcium uptake more than a double pulse stimulation of the optic nerve alone. Maximum enhancement of the calcium signal by nucleus isthmi occurs when optic nerve stimulation follows the ipsilateral nucleus isthmi stimulation by 10 ms. These results suggest that nucleus isthmi input can facilitate retinotectal neurotransmission, and the mechanism could be used to allow the frog to attend to a single prey stimulus in an environment of several prey stimuli.

Atrial natriuretic peptide induces rat peritoneal mast cell activation by cGMP-independent and calcium uptake-dependent mechanism.

Atrial natriuretic peptide (ANP), a 28 amino acid basic polypeptide, is known to induce histamine release from human and rat mast cells in vitro and cause a wheel formation in rat skin. However, cellular events associated with histamine release are not clearly understood. In this study, we have examined the calcium flux and cGMP formation associated with histamine release in the ANP-treated mast cells. ANP, in vitro, induced mast cell degranulation and histamine release in a dose-dependent manner. ANP also induced an enhanced calcium uptake into cells and increased the cellular level of cGMP in mast cells. A high level of calcium in the media caused an inhibition of ANP-dependent histamine release but enhanced the level of intracellular cGMP of mast cells. ANP inducing a dose-dependent increase in vascular permeability of rat skin was confirmed by the extravasation of the circulating Evans blue. The results indicate ANP induced the histamine release and an increase in vascular permeability through mast cell degranulation in cGMP-independent and calcium uptake-dependent manner.