Beta-aminoethyl Ether Research Articles

Comparison of removal of endodontic smear layer using ethylene glycol bis (beta-amino ethyl ether)-N, N, N', N'-tetraacetic acid and citric acid in primary teeth: A scanning electron microscopic study.

Background:Root canal irrigants are considered momentous in their tissue dissolving property, eliminating microorganisms, and removing smear layer. The present study was aimed to compare the removal of endodontic smear layer using ethylene glycol bis (beta-amino ethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) and citric acid solutions with saline as a control in primary anterior teeth.Materials and Methods:Thirty primary anterior teeth were chosen for the study. The teeth were distributed into three groups having ten teeth each. Following instrumentation, root canals of the first group were treated with 17% EGTA and the second group with 6% citric acid. Only saline was used as an irrigant for the control group. Then, the teeth were subjected to scanning electron microscopy (SEM) study. The scale given by Rome et al. for the smear layer removal was used in the present study.Results:The pictures from the SEM showed that among the tested irrigants, 17% EGTA + 5% sodium hypochlorite (NaOCl) group showed the best results when compared to other groups.Conclusion:The results advocate that the sequential irrigation of the pulp canal walls with 17% EGTA followed by 5% NaOCl produced efficacious and smear-free root canal walls.

Calcium Variously Mediates the Effect of Cytokinin on Chlorophyll and LHCPII Accumulation During Greening in Barley Leaves and Cucumber Cotyledons

Abstract During greening, excised etiolated barley leaves and cucumber cotyledons that were depleted of exogenous Ca2+ by a chelating agent (ethylene glycol-bis (beta aminoethyl ether)-N,N,N`N`-tetraacetic acid, EGTA) showed ∼50% reduced chlorophyll (Chl) accumulation and ∼30% accumulation of apoprotein of the light-harvesting chlorophyll a/b-binding protein complex of photosystem II (LHCPII). The Ca2+ channel blocker lanthanum chloride (LaCl3) applied to cucumber cotyledons reduced LHCPII accumulation more than EGTA did. In both plant mate-rials, cytokinins enhanced chlorophyll accumulation by 50-60% and this effect was completely canceled by EGTA application. Hormones significantly increased LHCPII accumulation but EGTA application reduced that effect in barley leaves by ∼30% and in cucumber cotyledons by ∼80%. A similar effect was observed in LaCl3-treated cotyledons. CaCl2 application boosted chlorophyll accumulation in both plant materials. CaCl2 applied together with cytokinin reduced the hormonal effect on chlorophyll accumulation by ∼38% in barley leaves and 23% in cucumber cotyledons, but almost totally inhibited cytokinin-stimulated LHCPII accumulation. Our results indicate that calcium variously mediates the effect of cytokinin on chlorophyll and LHCPII accumulation. Cytokinin-induced enhancement of chlorophyll accumulation seems totally dependent on the exogenous pool of Ca2+, while Ca2+-dependent and Ca2+-independent pathways are involved in the hormonal effect on LHCPII accumulation. The effect of cytokinin on the increase of light-induced LHCPII accumulation appears to be sensitive to exogenously applied Ca2+, which almost totally blocked the hormonal effect. Our results give indirect evidence that the responses to cytokinin and light act on different events leading to Chl and LHCPII accumulation.

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

Sensing of EGTA Mediated Barrier Tissue Disruption with an Organic Transistor

Barrier tissue protects the body against external factors by restricting the passage of molecules. The gastrointestinal epithelium is an example of barrier tissue with the primary purpose of allowing the passage of ions and nutrients, while restricting the passage of pathogens and toxins. It is well known that the loss of barrier function can be instigated by a decrease in extracellular calcium levels, leading to changes in protein conformation and an increase in paracellular transport. In this study, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), a calcium chelator, was used to disrupt the gastrointestinal epithelial barrier. The effect of EGTA on barrier tissue was monitored by a novel label-free method based on an organic electrochemical transistor (OECT) integrated with living cells and validated against conventional methods for measuring barrier tissue integrity. We demonstrate that the OECT can detect breaches in barrier tissue upon exposure to EGTA with the same sensitivity as existing methods but with increased temporal resolution. Due to the potential of low cost processing techniques and the flexibility in design associated with organic electronics, the OECT has great potential for high-throughput, disposable sensing and diagnostics.

An Oxidant- and Organic Solvent-Resistant Alkaline Metalloprotease from Streptomyces olivochromogenes

Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the enzyme was estimated to be 51 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gelatin zymography. The activity was optimum at pH 7.5 and 50 degrees C and stable between pH 7.0 and 10.0. SOMP was stable below 45 degrees C and Ca(2+) increased its thermostability. Ca(2+) enhanced while Co(2+), Cu(2+), Zn(2+), Mn(2+), and Fe(2+) inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity, suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents, and oxidizing agents, and the activity is enhanced by H(2)O(2). The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological applications especially for organic solvent-based enzymatic synthesis.

Calcium Signaling in Neuronal Cells Exposed to the Munitions Compound Cyclotrimethylenetrinitramine (RDX)

Cyclotrimethylenetrinitramine (RDX) has been used extensively as an explosive in military munitions. Mechanisms for seizure production, seen in past animal studies, have not been described. Increased calcium levels contribute to excitotoxicity, so in this study neuroblastoma cells are loaded with calcium-indicating dye before application of 1.5 microM to 7.5 mM RDX, with fluorescence recorded for 30 cycles of 11 seconds each. The lowest concentration of RDX increases calcium fluorescence significantly above baseline for cycles 2 to 8; millimolar concentrations increase calcium fluorescence significantly above baseline for cycles 2 to 30. Increases in calcium, like those of 200 nM carbachol, are prevented with 10 mM of calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N tetra-acetic acid (EGTA, tetrasodium salt). Calcium channel blocker verapamil (20 microM), Ca(2+)-ATPase inhibitor thapsigargin (5 microM), and general membrane stabilizer lidocaine (10 mM) partially attenuate carbachol- and RDX-induced increases in calcium, suggesting that RDX transiently increases intracellular calcium by multiple mechanisms.

Roles of purines in synaptic modulation evoked by hypercapnia in isolated spinal cord of neonatal rat in vitro

The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord. Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods. Hypercapnia (20% CO2) depressed the reflex potentials, which were partially reversed by an adenosine A1 receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A1 receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X7 receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia. These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A1 receptors.

Epidermal Growth Factor-Induced Proliferation of Chicken Primordial Germ Cells: Involvement of Calcium/Protein Kinase C and NFKB11

Epidermal growth factor (EGF) has been shown to stimulate survival in diverse cells in vitro. In the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGCs) were investigated. Results showed that EGF (10-100 ng/ml) increased the number and area of PGC colonies in a time- and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA; an intracellular Ca(2+) chelator). In addition, the degradation of NFKBIA and NFKB1 (p65) translocation was observed after EGF treatment, which was significantly blocked by pretreatment with AG1478, EGTA, H(7), or SN50 (NFKB1-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H(7), and SN50, respectively. On the other hand, inhibition of EGFR, Ca(2+)/PKC, or NFKB1 abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2, and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase 3/9 activity, indicating that EGFR, PKC, and NFKB1 signaling cascades were involved in EGF-stimulated DNA synthesis and antiapoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca(2+)/PKC involving NFKB1 signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.

OOCYTE ACTIVATION BY STRONTIUM IN THE PRESENCE OF CALCIUM SUPPORTS FULL TERM DEVELOPMENT OF SOMATIC CELL CLONED EMBRYOS

Without using sperm, artificial oocyte activation is essential for the current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has been widely used as an activator of oocytes especially in mouse, by which efficient oocyte activation requires calcium-free medium but not culture media since they normally contain calcium. In this study, we considered whether strontium with chelated calcium in culture media is enough to efficiently activate oocytes. Ethylene glycol-bis (beta-aminoethyl ether) - N, N, N', N'-tetraacetic acid (EGTA) were added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because they contain EGTA which preferentially binds calcium ion rather than strontium ion. After optimizing EGTA at 2–5 mM along with 5mM strontium in either CZB, M16 or KSOM, more than 80% of available oocytes were parthenogenetically activated, which was as efficient as standard conditions of strontium in a Ca-free medium. Further, it was demonstrated that this activation method can support full-term development of cloned embryos using trichostatin A with comparable success rates. Thus, addition of EGTA into culture media can easily make activation media without additional preparation of Ca-free medium. (poster)

Permissive role of calcium on regulatory volume decrease in freshly isolated mouse cholangiocytes

Calcium (Ca2+) pathways are important in cell volume regulation in many cells, but its role in volume regulatory processes in cholangiocytes is unclear. Thus, we have investigated the role of Ca2+ in regulatory volume decrease (RVD) in cholangiocytes using freshly isolated bile duct cell clusters (BDCCs) from normal mouse. No significant increase in [Ca2+]i was observed during RVD, while ionomycin and ATP showed significant increases. Confocal imaging also showed no significant changes in the levels or distributions of intracellular Ca2+ during RVD. Cell volume study by quantitative videomicroscopy indicated that removal and chelation of extracellular Ca2+ by ethylene glycol-bis (beta-aminoethyl ether)-N,N,N-tetraacetic acid (EGTA) or administration of nifedipine did not affect RVD but verapamil significantly inhibited the RVD. Moreover, Ca2+ agonists or inhibitors of Ca2+ release from intracellular stores had no significant effect on RVD. However, 1,2-bis (2-aminophenoxy) ethane-N,N,N'N'-tetraacetic acid-AM (BAPTA-AM) showed significant decreases in [Ca2+]i and significantly inhibited RVD, which was reversed with coadministration of valinomycin, suggesting that BAPTA-AM-induced inhibition is due to potassium conductance or other cellular processes requiring permissive [Ca2+](i. These findings indicate that an increase in [Ca2+]i or extracellular Ca2+ is not required for RVD but Ca2+ has a permissive role in RVD of mouse cholangiocytes.

Purification and Characterization of a Hyperthermostable and High Maltogenic α-Amylase of an Extreme Thermophile Geobacillus thermoleovorans

The purified alpha-amylase of Geobacillus thermoleovorans had a molecular mass of 26 kDa with a pI of 5.4, and it was optimally active at 100 degrees C and pH 8.0. The T 1/2 of alpha-amylase at 100 degrees C increased from 3.6 to 5.6 h in the presence of cholic acid. The activation energy and temperature quotient (Q 10) of the enzyme were 84.10 kJ/mol and 1.31, respectively. The activity of the enzyme was enhanced strongly by Co2+ and Fe2+; enhanced slightly by Ba2+, Mn2+, Ni2+, and Mg2+; inhibited strongly by Sn2+, Hg2+, and Pb2+, and inhibited slightly by EDTA, phenyl methyl sulfonyl fluoride, N-ethylmaleimide, and dithiothreitol. The enzyme activity was not affected by Ca2+ and ethylene glycol-bis (beta-amino ethyl ether)-N,N,N,N-tetra acetic acid. Among different additives and detergents, polyethylene glycol 8000 and Tween 20, 40, and 80 stabilized the enzyme activity, whereas Triton X-100, glycerol, glycine, dextrin, and sodium dodecyl sulfate inhibited to a varied extent. alpha-Amylase exhibited activity on several starch substrates and their derivatives. The K m and K cat values (soluble starch) were 1.10 mg/ml and 5.9 x 10(3)/min, respectively. The enzyme hydrolyzed raw starch of pearl millet (Pennisetum typhoides) efficiently.

Regulation ofVrXTH1 expression in mungbean

Xyloglucan endotransglucosylase/hydrolases (XTHs) play roles in plant development by rearranging xyloglucan cross-links. Previously, we isolated a cDNA of XTH,VrXTH1, from mungbean that is thought to be associated with auxin-related growth. Here we report that several factors regulate the expression ofVrXTH1. This gene is predominantly expressed in the elongating regions of seedlings. Environmental stimuli, such as light and temperature, also affect its transcript levels. Because calcium acts as a second messenger in many signal transduction pathways, we examined its involvement in the regulation ofVrXTH1 expression. Interestingly, the application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; a specific calcium chelator) repressed transcription, regardless of the presence of auxin. Furthermore, treatment with either A23187 (a calcium ionophore) or calcium increased gene expression, suggesting that changes in the amount of intracellular Ca2+ is important to the modification of transcript levels. Taken together, our results demonstrate that the expression ofVrXTH1 is closely related to plant growth and may be modulated by the concentration of cytosolic calcium.

Effects of cooling on histamine-induced contractions of human umbilical artery: The role of ion channels

The effects of cooling (to 28 degrees C) on histamine (10(-9) - 3 x 10(-4) M)-induced contractions and the role of calcium (Ca(2+)), potassium (K(Ca) (2+)) and sodium (Na(+)) channel blockers in the cooling-induced responses were investigated in the endothelium-denuded human umbilical artery. Concentration-response curves to histamine were isometrically recorded at 37 and 28 degrees C (control). The same procedure was repeated at 28 degrees C in the presence of tetraethylammonium (TEA, 10(-3) M), pilsicainide (10(-6) M), ouabain (10(-6) M), caffeine (3 x 10(-4) M), verapamil (10(-6) M) and also in Ca(2+)-free medium with ethylene glycol bis-(beta-aminoethyl ether) N,N,N(1),N(1)-tetraacetic acid (EGTA). During cooling, the sensitivity, but not the maximal response, was significantly higher than 37 degrees C. Cooling to 28 degrees C after treatment with verapamil or pilsicainide decreased the sensitivity, whereas treatment with TEA and ouabain significantly increased sensitivity. Treatment with caffeine did not modify the effect of cooling. Furthermore, cooling to 28 degrees C after incubation in Ca(2+)-free solution with EGTA decreased the sensitivity to histamine. The results of this study suggest the role of Ca(2+), K(Ca) (2+) and Na(+)-ion channels in the cooling-induced changes of human umbilical arteries treated with histamine.

Acetylcholine release induced by the volatile anesthetic sevoflurane in rat brain cortical slices.

1. We have investigated the effect of the volatile anesthetic sevoflurane on acetylcholine (ACh) release from rat brain cortical slices. 2. The release of [3H]-ACh into the incubation fluid was studied after labeling the tissue ACh with [methyl-3H]-choline chloride. 3. We observed that sevoflurane induced an increase on the release of ACh that was dependent on incubation time and anesthetic concentration. The sevoflurane-induced ACh release was not blocked by tetrodotoxin (TTX) and therefore was independent of sodium channels. In addition, the sevoflurane effect was not blocked by ethylene glycol-bis(beta-aminoethyl ether (EGTA) or cadmium (Cd2+), thus independent of extracellular calcium. 4. The sevoflurane-induced ACh release was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM), suggesting the involvement of intracellular calcium-sensitive stores in the process. Dantrolene, an inhibitor of ryanodine receptors, had no effect but 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inhibitor of inositol 1,4,5-triphosphate receptor inhibited the sevoflurane-induced release of ACh. 5. It is concluded that sevoflurane-induced release of ACh in brain cortical slices involves the mobilization of calcium from IP3-sensitive calcium stores.

The calpain inhibitor VI prevents apoptosis of adult motor neurons

Motor neuron cell death was studied in organotypic cultures of adult spinal cord slices from the mouse. Six hours after excision, many motor neuron nuclei displayed apoptotic features including nuclear and chromatin condensation. At this time point, many motor neurons also exhibited immunoreactivity to calpain II. Both the calpain inhibitor VI and ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the appearance of apoptotic nuclei whereas the pan caspase inhibitor Z-VAD.fmk had no effect. The results suggest that calpain is required for apoptosis of motor neurons and that this type of apoptosis is independent of caspase activation.

Modulation of phosphoenolpyruvate carboxylase in vivo by Ca 2+ in Amaranthus hypochondriacus, a NAD-ME type C 4 plant: Possible involvement of Ca 2+ in up-regulation of PEPC-protein kinase in vivo

The properties of phosphoenolpyruvate carboxylase (PEPC) were studied, with respect to calcium (Ca2+), in leaves of Amaranthus hypochondriacus, a C4 plant. Experiments were conducted in vitro (by adding Ca2+ during enzyme assay) or in vivo (by feeding Ca2+ to intact leaves through petiole). Inclusion of 10 microM Ca2+ during assay marginally increased (<30%) malate sensitivity of PEPC in extracts from dark-adapted leaves. The effect of Ca2+ was marginal on PEPC in extracts from illuminated leaves. Upon applying a low concentration of Ca2+ to leaves, the PEPC activity in leaves increased by 1.5-fold, while inhibition by malate decreased markedly. The light activation of PEPC in Ca2+-fed leaves was slightly higher than in the absence of Ca2+-ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra acetic acid (EGTA). To assess further the role of Ca2+, 5 mM EGTA (Ca2+ chelator) was either added during the enzyme assay or fed to leaves through petiole. EGTA had no effect on PEPC, when added during enzyme assay. Upon feeding EGTA, the PEPC activity in the dark-adapted leaf extracts increased by 30%, and the effect on malate sensitivity was marginal. However, there was a decrease in PEPC activity in illuminated extracts, resulting in a marked decrease in the extent of light activation of PEPC. The extent of phosphorylation of PEPC was much higher in Ca2+ or Ca2+-EGTA-fed leaves than in the control, but EGTA decreased the light-induced phosphorylation. Our results suggest that optimal alone concentration of Ca2+ is essential for PEPC in leaves of A. hypochondriacus, particularly in vivo. We suggest that Ca2+ regulates PEPC, at an upstream level, such as transcription, by modulating PEPC-protein kinase, thus facilitating the light activation of PEPC.

Warming and response to contractile agents in calf cardiac vein: Role of the Ca2+, KCa2+ and Na+ ion channel blockers

The effects of warming (to 41 degrees C) on the serotonin (5-HT, 10(-8)-3 x 10(-3) M)- and carbachol (10(-)9-3 x 10(-4) M)-induced contractions and the role of calcium (Ca2+), potassium (K+), and sodium (Na+) channel blockers, in the warming-induced responses were investigated in the calf cardiac vein. Concentration-response curves to 5-HT and carbachol were isometrically recorded at 37 and 41 degrees C (control). The same procedure was repeated at 41 degrees C in the presence of verapamil (10(-6) M), caffeine (3 x 10(-4) M), tetraethylammonium (TEA, 10(-3)M), flecainide (10(-6) M), and also in the Ca2+-free medium with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). During warming, the sensitivity, but not the maximal response, was significantly higher. Warming to 41 degrees C after treatment with verapamil or flecainide decreased the sensitivity, whereas treatment with caffeine increased the sensitivity significantly. Treatment with TEA did not modify the effect of warming. Furthermore, warming to 41 degrees C after incubation in Ca2+-free solutions with EGTA decreased the sensitivity to 5-HT and carbachol. The results of this study suggest the role for Ca2+ and Na+ ions in the warming-induced changes of cardiac vein treated with 5-HT and carbachol.

Matrix metalloproteinase-specific inhibition of Ca 2+ entry mechanisms of vascular contraction

Abdominal aortic aneurysm (AAA) is a common disease with as yet unclear cause. Increased matrix metalloproteinase (MMP) levels in the plasma and aorta are a consistent finding in AAA. Although the role of MMPs in AAA has largely been attributed to degradation of the extracellular matrix proteins, the effects of MMPs on the mechanisms of aortic contraction are unclear. The purpose of this study was to test the hypothesis that MMPs promote aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats treated or not treated with MMP-2 and MMP-9. In normal Krebs solution (2.5 mmol/L Ca2+ ) phenylephrine (10-5 mol/L) caused contraction of the aortic strips, which was significantly inhibited (P < .05) by MMP-2 (maximum, 48.9% +/- 5.0%) and to a greater extent by MMP-9 (maximum, 69.8% +/- 6.2%). The MMP-induced inhibition of phenylephrine contraction depended on concentration and time. The inhibitory effects of MMPs on phenylephrine contraction were reversible. In Ca2+ -free (2 mmol/L ethylene glycol bis[beta-aminoethyl ether]-N,N,N',N'-tetraacetic acid) Krebs solution phenylephrine caused a small contraction that was not inhibited by MMP-2 or MMP-9, which suggests that MMPs do not inhibit Ca2+ release from the intracellular stores. Membrane depolarization with 96 mmol/L of potassium chloride, which stimulates Ca2+ entry from the extracellular space, caused a time-dependent and reversible contraction, which was inhibited by MMP-2 and MMP-9. Histologic studies of MMP-treated tissues stained with hematoxylin-eosin or Verhoeff stain for elastin confirmed the absence of degradation of the extracellular matrix. MMP-2 and MMP-9 also caused significant inhibition of 45Ca2+ influx induced by phenylephrine and potassium chloride. These data suggest that MMP-2 and MMP-9 promote aortic dilation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction. Abdominal aortic aneurysm (AAA) is a slow and progressive disease. The late stages of AAA are characterized by degenerative changes in the extracellular matrix and smooth muscle components of the aortic wall. The present study describes novel inhibitory effects of matrix metalloproteinase (MMP) on the Ca2+ entry mechanisms of aortic smooth muscle contraction, even in the absence of extracellular matrix degradation. The MMP-induced inhibition of aortic contraction may further explain the role of increased MMP activity particularly during the early development of AAA. Chronic exposure to MMPs may lead to protracted inhibition of aortic contraction, progressive aortic dilation, and aneurysm formation. MMP-9 is a more potent inhibitor of aortic contraction than MMP-2, consistent with a more dominant role in AAA. Restoration and preservation of smooth muscle contractile function by specific inhibitors of MMPs may represent a new strategy in preventing the progression of small AAA.

Calcium Is Involved in Photomovement of Cyanobacterium Synechocystis sp. PCC 6803¶

The unicellular cyanobacterium Synechocystis sp. PCC 6803 (Syn6803) exhibits photomovement through gliding motility. For a better understanding of photomovement in Syn6803, we examined the effects of Ca2+ on photoorientation and motility using a computer-assisted videomicroscope motion analysis system. When calcium ion was chelated from the basic motility medium by adding 0.5 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the photoorientation was completely inhibited, whereas the gliding motility remained approximately 70% of the control. Photoorientation impaired by EGTA was nearly recovered within 30 min upon addition of 1 mM Ca2+. The recovery of photoorientation by Ca2+ was mimicked by either Mn2+ or Mg2+ but not by Ba2+ or Sr2+. Lanthanum ion at 10 microM completely inhibited both phototactic orientation and gliding motility of Syn6803. Furthermore, pimozide (voltage-gated L-type calcium channel inhibitor), orthovanadate (calcium efflux blocker) and A23187 (calcium ionophore) partially inhibited phototactic orientation and gliding motility. Interestingly, photoorientation was prevented with increasing concentrations of calmodulin antagonist such as trifluoperazine (TFP) and chlorpromazine, but gliding motility was inhibited in proportion to the concentration of TFP. The results we present strongly indicate that Ca2+ plays a significant role in regulating the photomovement of Syn6803.