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A method of estimating the amount of calcium bound to the metallochromic indicator arsenazo III.

As a metallochromic indicator for ionized calcium, arsenazo III is approximately 50 times more sensitive than murexide. However, because of the high binding constant for calcium, the following problems may occur: (a) a considerable amount of calcium is bound to arsenazo III, thereby causing an error in estimating the concentration of ionized calcium; (b) the amount of bound calcium varies with the concentrations of calcium;, arsenazo III, magnesium ion and monovalent cations; (c) the amount also varies with pH, (d) the relationship between the absorbance change and the concentration of ionized calcium is nonlinear; and (e) the binding constant of arsenazo III for calcium cannot be determined by the conventional double reciprocal plot. A new experimental and theoretical method is presented which copes with these problems.

Arsenazo III↗

Ouabain increases aldosterone release from bovine adrenal glomerulosa cells: role of renin-angiotensin system.

To evaluate the potential physiological significance of ouabain or a ouabainlike substance, we investigated the effect of nanomolar concentrations of ouabain on aldosterone release by cultured bovine adrenal glomerulosa cells. Ouabain (10 nM) increased aldosterone release from 0.35 to 0.89 ng.mg-1.4 h-1 in the serum-containing medium. Losartan prevented this increase. When angiotensinogen was added to the nonserum medium, 10 nM ouabain enhanced the aldosterone release. Losartan again blocked the increase. These findings together with a stimulation of renin release by ouabain indicate that angiotensin II generated by the adrenal cell renin-angiotensin system in the presence of exogenous serum or exogenous angiotensinogen is necessary for the ouabain-induced stimulation of aldosterone release. Ouabain (10 nM) enhanced the intracellular calcium concentration increase elicited by 0.1 nM angiotensin II severalfold. Addition of 1 nM ouabain enhanced the aldosterone secretion resulting from the addition of 1 nM angiotensin II. Nanomolar levels of ouabain, therefore, interact with both locally formed and exogenous angiotensin II to stimulate aldosterone production. A suggested mechanism is that ouabain increases calcium stores in the endoplasmic reticulum, thereby increasing the agonist response.

Aldosterone↗

Comparison of the contractile and metabolic effects of muscarinic stimulation with those of KCl.

Urinary bladder emptying is mediated primarily by a coordinated contraction of the bladder body in response to parasympathetic stimulation and muscarinic receptor activation. In previous studies we presented evidence that the contractile response to bethanechol stimulation could be dissociated from the metabolic response through the use of diltiazem (calcium channel blockade). The conclusion from these studies was that muscarinic stimulation resulted in a significant increase in metabolic activity which was not directly associated with contraction. KCl stimulates contraction in isolated strips by directly depolarizing the membrane rather than from binding to specific membrane receptors. The current study directly compares the metabolic and contractile activity of bethanechol (muscarinic stimulation) with KCl (direct membrane depolarization). Isolated strips of rabbit urinary bladder body were monitored in vitro for changes in intracellular-free calcium, NADH/NAD ratio, and contraction. Intracellular-free calcium was monitored by preincubation of isolated bladder smooth muscle strips with FURA-2 AM and continuously measuring the fluorescence with an MB2 surface spectrofluorometer using excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm. The NADH/NAD ratio was monitored with the MB2 surface spectrophotometer using an excitation wavelength of 366 nm and an emission wavelength of 450 nm. Contraction was monitored using a isometric force transducer connected to a Grass model D polygraph. The results can be summarized as follows. (1) Both bethanechol and KCl stimulate a sharp decrease in the NADH/NAD ratio, a rapid increase in intracellular-free calcium, and a slower increase in contractile force.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells.

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.

Adenosine Triphosphatases↗

Effects of muscarinic stimulation on intracellular calcium in the rabbit bladder: comparison with metabolic response.

Urinary bladder emptying is mediated primarily by a co-ordinated contraction of the bladder body in response to parasympathetic stimulation and muscarinic receptor activation. In a previous study using surface spectrophotometry to monitor the nicotinamide adenine dinucleotide (oxidized form)/nicotinamide adenine dinucleotide (reduced form) (NADH/NAD) ratio, we demonstrated that muscarinic stimulation results in a rapid decrease in this ratio which precedes the contractile response, and that the ED50 for the NADH response is significantly lower than the ED50 for contraction. The current study was designed to correlate changes in intracellular free calcium using FURA-2 fluorescence with both the contractile and metabolic response to muscarinic stimulation. Isolated strips of urinary bladder body were monitored in vitro for changes in intracellular free calcium, NADH/NAD ratio, and contraction. Intracellular free calcium was monitored by preincubation with FURA-2 AM and continuously measuring the fluorescence with an MB2 surface spectrofluorometer using excitation wavelengths of 340 and 380 nm, and an emission wavelength of 510 nm. The NADH/NAD ratio was monitored with the MB2 surface spectrophotometer using an excitation wavelength of 366 nm and an emission wavelength of 450 nm. Contraction was monitored using an isometric force transducer connected to a Grass model D polygraph. The results can be summarized as follows: (1) Bethanechol stimulates a sharp decrease in the NADH/NAD ratio, a rapid increase in intracellular free calcium, and a slower increase in contractile force. (2) The ED50 for NADH fluorescence was significantly less than the ED50 for either contraction or calcium fluorescence which were equal to each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Mechanism of modulating effect of histamine on the excitation and contraction of smooth muscles of the ureter].

Experiments were carried out on smooth muscle cells (SMC) of the guinea-pig ureter by the double sucrose gap method with simultaneous recording of electrical and contractile activities. The effect of histamine on SMC was studied in normal Krebs solution and in sodium-free Krebs solution with TEA. In normal Krebs solution, histamine was shown to increase the duration of AP plateau and contraction. In sodium-free Krebs solution with TEA, the APs of ureter SMC also had a plateau that was determined by the increased calcium conduction of the membrane. In these conditions, histamine provoked a decrease in the duration of the AP plateau and contraction. The histamine effect was blocked by phencarol both in normal and sodium-free Krebs solution with TEA. A possible role of sodium, calcium and potassium ions in the modulating action of histamine on excitation of ureter SMC is discussed.

Action Potentials↗

Effect of sodium content on the crystallization behavior of fluoramphibole glass-ceramics.

The purpose of this study was to evaluate the effect of sodium content on the crystallization behavior of glass-ceramics in the system SiO(2)-MgO-CaO-Na(2)O-K(2)O-F. Five glass compositions were prepared with increasing sodium content. The compositions were melted at 1400 degrees C for 2 h and cast into 60 x 12-mm ingots. Differential thermal analyses were performed on the glasses up to 1400 degrees C. Discs (1.5 x 12 mm) were cut from the ingots, nucleated in the temperature range 600-650 degrees C for 1-2 h, and crystallized in the temperature range 900-1000 degrees C for 0.5-4 h. The crystalline phases were analyzed by x-ray diffraction. The microstructure was investigated by scanning electron microscopy. Differential thermal analyses showed that the crystallization exotherm occurred in the temperature range 600-750 degrees C. There was a linear relationship between the amount of sodium and the transformation temperatures. X-ray diffraction revealed the presence of mica and diopside as major crystalline phases in the sodium-free composition. Mica, diopside, and fluorrichterite were present in all other glass-ceramics. The microstructure of the sodium-free glass-ceramic was characterized by the presence of hexagonal mica crystals and prismatic diopside crystals. All other compositions exhibited needle-shaped fluorrichterite crystals (2-5 microm in length) in addition to mica and diopside crystals.

Aluminum Silicates↗

Protection by diphenyliodonium against glutamate neurotoxicity due to blocking of N-methyl-D-aspartate receptors.

The protective effect of diphenyliodonium, known as an inhibitor of flavin enzymes including nitric oxide synthases, was examined against the neurotoxicity of excitatory amino acids on cultured spinal neurons of the rat. Diphenyliodonium reduced the neuronal damage induced by 15-min exposure to glutamate or N-methyl-D-aspartate in a dose-dependent manner; half effective concentrations (EC50) were about 3 microM for both. Protection was only observed when diphenyliodonium was added into the exposure medium. Diphenyliodonium showed no effect on the toxicity induced by 24 h exposure to non-N-methyl-D-aspartate receptor agonists. Using a microfluorometry technique with Fura 2, we observed that diphenyliodonium reversibly inhibited the N-methyl-D-aspartate-evoked intracellular Ca2+ elevation. The amount of 45Ca2+ influx induced by N-methyl-D-aspartate was also inhibited by diphenyliodonium in a dose-dependent manner; EC50 was about 3 microM. Furthermore, we examined the effect of diphenyliodonium on an opening activity of the N-methyl-D-aspartate receptors estimated by binding of dizocilpine maleate to membrane fractions from whole brain of adult rat and from cultured spinal neurons. Diphenyliodonium inhibited the binding of dizocilpine maleate dose-dependently; EC50 was 5-8 microM. These results suggest that diphenyliodonium is a new antagonist to the N-methyl-D-aspartate receptors and that diphenyliodonium protects neurons against glutamate toxicity due to a direct blocking of the Ca2+ influx. This conclusion is supported by the similarity of the stereochemical structures predicted by computer between diphenyliodonium and dizocilpine maleate.

Animals↗

Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+ fluxes and prolactin release in GH3/B6 pituitary tumor cells.

Xenoestrogens (XEs) are widespread in our environment and are known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E2) to disrupt normal estrogenic signaling. We investigated the effects of E2 versus several XEs representing organochlorine pesticides (dieldrin, endosulfan, o',p'-dichlorodiphenylethylene), plastics manufacturing by-products/detergents (nonylphenol, bisphenol A), a phytoestrogen (coumestrol), and a synthetic estrogen (diethylstilbestrol) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-alpha. Picomolar to nanomolar concentrations of both E2 and XEs caused intracellular Ca2+ changes within 30 sec of administration. Each XE produced a unique temporal pattern of Ca2+ elevation. Removing Ca2+ from the extracellular solution abolished both spontaneous and XE-induced intracellular Ca2+ changes, as did 10 microM nifedipine. This suggests that XEs mediate their actions via voltage-dependent L-type Ca2+ channels in the plasma membrane. None of the Ca2+ fluxes came from intracellular Ca2+ stores. E2 and each XE also caused unique time- and concentration-dependent patterns of prolactin (PRL) secretion that were largely complete within 3 min of administration. PRL secretion was also blocked by nifedipine, demonstrating a correlation between Ca2+ influx and PRL secretion. These data indicate that at very low concentrations, XEs mediate membrane-initiated intracellular CCa2+ increases resulting in PRL secretion via a mechanism similar to that for E2, but with distinct patterns and potencies that could explain their abilities to disrupt endocrine functions.

Animals↗

139La nuclear magnetic resonance characterisation of La2O3 and La1-xSrxMO3 where M = Cr, Mn or Co.

139La Nuclear magnetic resonance (NMR) spectra have been used to obtain nuclear quadrupole coupling parameters for La2O3 and a series of perovskites La1-xSrxMO3 (where M = Cr, Mn or Co). Depending on the doping level of SrO2 these materials are either paramagnetic or ferromagnetic at room temperature. Magnetic transferred hyperfine effects are strongly in evidence in the Mn compounds. A 59Co NMR spectrum was observed in LaCoO3. A precision measurement of the nuclear quadrupole coupling constant in La2O3 was made by nuclear quadrupole resonance (NQR) spectroscopy.

Calcium Compounds↗

Calcium imaging of individual erythrocytes: problems and approaches.

Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E(2) and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission. Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable.

Aniline Compounds↗

Evaluation of quicklime mixing for the remediation of petroleum contaminated soils.

Quicklime mixing is an established solidification/stabilization technique to improve mechanical properties and immobilise contaminants in soils. This study examined the effects of quicklime mixing on the concentrations and leachability of petroleum hydrocarbon compounds, in two natural soils and on a number of artificial sand/kaolinite mixtures. Several independent variables, such as clay content, moisture content and quicklime content were considered in the study. After mixing the soils with the quicklime, pH, temperature, moisture content, Atterberg limits and concentrations of petroleum hydrocarbon compounds were determined on soil and leachate samples extracted from the treated soils. Significant decreases in concentrations of petroleum hydrocarbon compounds were measured in soils and leachates upon quicklime mixing, which may be explained by a number of mechanisms such as volatilization, degradation and encapsulation of the hydrocarbon compounds promoted by the quicklime mixing. The increase in temperature due to the exothermic hydration reaction of quicklime when in contact with porewater helps to volatilize the light compounds but may not be entirely responsible for their concentration decreases and for the decrease of heavy aliphatics and aromatics concentrations.

Aluminum Silicates↗

Expression of L-type calcium channels associated with postnatal development of skeletal muscle function in mouse.

Several factors have an influence on the improvement of muscle activity and motor co-ordination of mammals during post-natal development. One of them is voltage sensitive L-type calcium channel function. In striated muscles of adult mammals these channels are located in T-tubule membranes thus linking the on-coming action potential to the molecular process of muscle contraction. The postnatal development of L-type calcium channels is therefore critical not only for contraction but also for all subsequent motor learning. We used high affinity enantiomer of dihydropyridine labelled with a fluorophore in order to show the relative amount of L-type calcium channels by histofluorescence in tissue. We found by qualitative microscopical analysis that the amount of L-type calcium channels increased during the postnatal development in the mouse skeletal muscle (m. rectus femoris and m. gastrocnemius). We also noted variation between different fibre types in the increase of the amount of L-type calcium channels, as judged by the intensity of histofluorescence. We showed by histochemical staining and statistical analysis that the high density of L-type calcium channels in adult muscles is correlated with fast oxidative glycolytic fibre type of striated muscles rather than slow oxidative or fast glycolytic fibres. Based on this finding we propose that the development of L-type calcium channels can be considered as one of the factors determining the different physiological properties of fibre types.

Aging↗

A clinical trial in type 2 diabetic nephropathy.

A prospective, randomized, three-armed, double-blind, placebo-controlled clinical trial has been completed in 210 sites worldwide to determine whether the angiotensin II receptor blocker irbesartan or the calcium channel blocker amlodipine has a renoprotective effect in patients with overt type 2 diabetic nephropathy. A total of 1,715 subjects randomized during a 3-year period were followed a minimum of 2 years. The goal for all treatment groups was to achieve equivalent blood pressure control, with the blinded study drug (irbesartan, amlodipine, or placebo) as primary therapy with additional antihypertensive drugs, excluding angiotensin-converting enzyme inhibitors, calcium antagonists, and angiotensin II receptor antagonists, to achieve seated systolic blood pressure less than 135 mm Hg and diastolic blood pressure less than 85 mm Hg. The primary outcome was the combined endpoint of time to doubling of entry serum creatinine, end-stage renal disease, or death. Secondary outcomes included fatal and nonfatal cardiovascular events. A Clinical Management Committee monitored the conduct of the study. An Outcome Confirmation Committee classified all study outcome events in blinded fashion. An external Data Safety Monitoring Committee monitored unblinded data for interim safety and efficacy analyses of the study. Eligibility criteria included informed consent, age 30 to 70 years, adult-onset diabetes, hypertension, urine protein excretion greater than 900 mg/24 hours, and serum creatinine values of 90 to 265 micromol/L in women and 110 to 265 micromol/L in men. Baseline characteristics were age, 59 +/- 8 years; body mass index, 31 +/- 7 kg/m(2); 67% male; 73% white, 14% black, and 13% other; duration of diabetes, 15 +/- 9 years; retinopathy, 66%; neuropathy, 48%; congestive heart failure, 7.5%; screening seated systolic blood pressure, 156 +/- 18 mm Hg, and diastolic blood pressure, 85 +/- 11 mm Hg; urine protein excretion, 4.0 +/- 3.5 g/24 hours; serum creatinine, 150 +/- 53 micromol/L; serum potassium, 4.6 +/- 0.5 mEq/L; total cholesterol, 229 +/- 58 mg/dL; and hemoglobin A(1c), 8.1 +/- 1.7%. This large-scale international trial should help define the clinical course and standards of care for hypertensive adults with type 2 diabetes mellitus and nephropathy. Results available on May 19, 2001, will help in defining the current controversy of the risks and benefits of blockade of the renin-angiotensin system versus calcium channel blockade versus standard antihypertensive therapy in this large patient population.

Adult↗

A simple histochemical technique for the identification of gunshot residue.

Alizarin red S (ARS) is a commonly used organic dye useful in the histologic identification of calcium deposits. ARS also forms colored reaction products with other metal ions, including barium and lead, which are present in primer residue. In histochemical studies, ARS is shown to identify primer residues from several manufacturers as well as primer residue deposited in tissue, either experimentally or in close-range gunshot wounds. This can be easily accomplished with routine histologic techniques. ARS does not stain gunpowder residue, tattoo pigment, melanin, graphite, india ink, or anthracotic pigment.

Anthraquinones↗

Chemical transformation of some biologically relevant calcium phosphates in aqueous media during a steam sterilization.

The purpose of this study was to investigate the effect of steam sterilization on some biologically relevant calcium phosphates: CaHPO4 . 2H2O (DCPD), calcium deficient apatite (CDA) and biphasic calcium phosphate (BCP). Suspensions of 0.2 g of each calcium phosphate compound with 5.0 ml of deionized water were prepared and steam sterilized in an autoclave (20 min at 121 degrees C). After sterilization the suspensions were filtered and the dried solids characterized with scanning electron microscopy, IR-spectroscopy and X-ray diffraction. The pH and calcium concentrations of the filtrates were determined with ion selective electrodes. Similar measurements were made with the same samples which were not sterilized. The sterilization procedure was found to result in the dehydration of DCPD and hydration of calcium oxide incorporated into the BCP. Solution pH was observed to change from 7.3 to 5.5 for the solutions in equilibrium with DCPD and from 8.5 to 10.6 for those in equilibrium with BCP. Minor changes both with the solid and liquid phases were found to occur during the steam sterilization of CDA. These results indicate that steam sterilization may have different effects on different calcium phosphate suspensions: it can result in dehydration of DCPD, fast hydration for CaO in BCP, but no significant effect on CDA.

Journal Article↗