Secretagogue-induced changes in subcellular Ca2+ distribution in isolated pancreatic acini

1981 ◽  
Vol 240 (2) ◽  
pp. G130-G140
Author(s):  
R. L. Dormer ◽  
J. A. Williams
Keyword(s):  
Atomic Absorption Spectrometry ◽  
High Speed ◽  
Microsomal Fraction ◽  
Absorption Spectrometry ◽  
Subcellular Fractionation ◽  
Differential Centrifugation ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Induced Changes ◽  
Stimulation Of

In a prior study, we demonstrated that pancreatic secretagogues increased both the uptake into and washout of 45Ca2+ from isolated mouse pancreatic acini. The net result of these processes was an initial fall in total acinar cell Ca2+ content. In the present study, we have employed subcellular fractionation of acini under conditions that minimized posthomogenization redistribution of Ca2+ in order to localize those organelles involved in intracellular Ca2+ fluxes. Homogenization and differential centrifugation of acini, preloaded with 45Ca2+ and subjected to a period of washout, showed that carbachol induced an increased loss of 45Ca2+ from all fractions isolated. The high-speed microsomal fraction lost 45Ca2+ to a greater extent than did whole acini; measurement of total Ca2+ by atomic absorption spectrometry showed a net loss of Ca2+ from this fraction. Purification of the lower-speed fractions indicated that carbachol increased 45Ca2+ exchange with both zymogen granules and mitochondria, but net Ca2+ levels in these organelles were unchanged. It was concluded that stimulation of pancreatic acini by carbachol results in the release of calcium from a microsomal compartment leading to a rise in cytoplasmic Ca2+, increased exchange with granule and mitochondrial Ca2+, and increased efflux of Ca2+ from the cell.

K, Ca, Mg, and Zn in platelets, as determined by atomic absorption spectrometry with use of a sealed decomposition bomb and discrete nebulization.

1985 ◽  
Vol 31 (4) ◽  
pp. 609-612 ◽  
Author(s):  
T Makino
Keyword(s):  
Metal Ions ◽  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Accurate Determination ◽  
Absorption Spectrometry ◽  
New Method ◽  
Differential Centrifugation ◽  
Individual Variations

Abstract In this new method for precise and accurate determination of K, Ca, Mg, and Zn in platelets, small amounts of platelets, prepared by differential centrifugation and cell washing, are decomposed in a homemade mini-vessel, a sealed Teflon bomb. The metal ions in the decomposed sample are measured by atomic absorption spectrometry with discrete nebulization. Overall, CVs ranged from 0.8 to 4.8%. We investigated sex-related differences (none were found) and intra-individual variations.

Secretagogue-induced changes in intracellular pH and amylase release in mouse pancreatic acini

1987 ◽  
Vol 253 (5) ◽  
pp. G690-G696 ◽  
Author(s):  
K. J. Carter ◽  
P. L. Rutledge ◽  
M. L. Steer ◽  
W. Silen
Keyword(s):  
Intracellular Ph ◽  
Total Content ◽  
Ethylene Glycol Tetraacetic Acid ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Drug Concentrations ◽  
Wide Range ◽  
Induced Changes ◽  
Stimulation Of

The response of the intracellular pH (pHi) to stimulation of enzyme secretion in pancreatic acini was measured using the fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Acini suspended in pH 7.40 buffer demonstrated cytoplasmic alkalinization of 0.17, 0.14, and 0.15 pH units 2 min after addition of the secretagogues carbachol (10(-5) M), caerulein (10(-10) M), and bromo-A23187 (10(-6) M). Corresponding net stimulated amylase secretion over 30 min was 9.2, 10.3, and 5.6% of total content, respectively. Pretreatment of acini with atropine blocked the pHi rise induced by carbachol; addition of atropine 2 min after the carbachol did not reverse the alkalinization. Acini suspended in Ca2+ free buffer containing 0.1 or 0.2 mM ethylene glycol tetraacetic acid showed 0.21 and 0.10 pH unit alkalinization in response to caerulein (10(-10) M) and carbachol (10(-5) M) but no change in pHi after addition of bromo-A23187. Amylase release in response to increasing concentrations of caerulein was maximal at 10(-10) M, with decreasing rates of amylase release at higher drug concentrations (10(-8), 10(-7) M). Alkalinization in response to stimulation of secretion was maximal at 10(-8) M caerulein (0.30 pH units at 2 min) but was of lesser magnitude at 10(-7) M. Pancreatic acini demonstrated autoregulation of pHi over a range of external pH from 7.4 to 7.1. Net amylase release over 30 min in response to 10(-5) M carbachol was sustained at normal levels in buffers of pH varying between 7.7 and 6.5. In contrast, cytoplasmic alkalinization in response to carbachol occurred only in buffers with pH values between 7.40 and 7.10. These results indicate that amylase release occurs over a wide range of pHi and is not invariably associated with secretagogue-induced alkalinization.

Application of the high-speed self-reversal background corrector to the determination of cadmium by chemical vapor generation atomic absorption spectrometry

2012 ◽  
Vol 90 (10) ◽  
pp. 874-879 ◽  
Author(s):  
Christophe Waterlot ◽  
Aurélie Pelfrêne ◽  
Francis Douay
Keyword(s):  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Flame Atomic Absorption Spectrometry ◽  
High Speed ◽  
Absorption Spectrometry ◽  
Standard Reference Materials ◽  
Vapor Generation ◽  
Flame Atomic Absorption ◽  
Interference Problems

Concentrations of cadmium (Cd) in extracting solutions (neutral salts) from contaminated soils are often too low to be determined by conventional flame atomic absorption spectrometry. For this reason, determination of Cd requires sensitive analytical methods free from interference problems generated by samples. In this context, vapor generation atomic absorption spectrometry (HGAAS) was combined with a high-speed self-reversal background corrector. This new approach was successfully applied after optimization of the analytical parameters to obtain a maximal absorbance signal of the volatile Cd species. The optimum condition was achieved with a 3% (m/v) NaBH4 in 1.5% (m/v) NaOH reducing solution and a solution containing 0.3 mol/L HNO3. The detection limit was 1 ng mL–1 under the previous conditions and the relative standard deviation was up to 5% for 10 replicate analyses of Cd at 0.2 and 1 ng mL–1, reflecting a very highly sensitive and reproducible method. Moreover, the results showed that the proposed combination was an efficient method to overcome the interference problems caused by different coexisting cations, As, Al, Ca, Cu, Fe, Mg, Mn, Ni, Pb, Se and Zn, up to 10 µg mL–1. The method was validated with analyses of two standard reference materials and was used for Cd determination in 0.01 mol/L CaCl2 extracts from contaminated kitchen garden soils. The data were compared with those obtained by two other more conventional methods, electrothermal atomic absorption spectrometry (ETAAS) and flame atomic absorption spectrometry (FAAS). The analytical results obtained by the ETAAS and HGAAS were in a good agreement, suggesting the suitability of the method for Cd determination in 0.01 mol/L CaCl2 extracting solution.

Relation between free cytosolic calcium and amylase release by pancreatic acini

1985 ◽  
Vol 249 (3) ◽  
pp. G389-G398 ◽  
Author(s):  
D. L. Ochs ◽  
J. I. Korenbrot ◽  
J. A. Williams
Keyword(s):  
Acinar Cells ◽  
Cytosolic Calcium ◽  
Pancreatic Acinar Cells ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Intracellular Ca ◽  
Free Media ◽  
Induced Changes ◽  
Stoichiometric Relation ◽  
Stimulation Of

Pancreatic acini were loaded with the Ca-selective fluorescent indicator quin-2 by incubation with its acetyoxymethyl ester. Loading acini with 844 +/- 133 microM quin-2 altered neither their ultrastructure nor their viability. The rate of amylase release from quin-2-loaded acini in response to the secretagogue carbachol, however, was significantly smaller than that of control acini. Studies in which acini were loaded with both quin-2 and a similar Ca-chelating compound, BAPTA, indicated that this reduced amylase release was related to the Ca buffering properties of quin-2. The concentration of free intracellular Ca calculated from the fluorescence of quin-2 was 90 +/- 18 nM. Stimulation by carbachol of acini suspended in media containing 1.25 mM Ca caused a rapid, transient enhancement of this value. After stimulation amylase release, the onset of the rise in free cytosolic Ca levels was observed in 1.1 +/- 0.1 s following the addition of agonist, and peak Ca levels (545 +/- 112 nM) were obtained within 5.3 +/- 0.3 s. For concentrations of carbachol less than or equal to 10(-6) M, a stoichiometric relation was found between stimulated amylase release and the peak concentration of free cytosolic Ca achieved. At higher concentrations of carbachol, however, the peak free cytosolic Ca remained constant while amylase release declined. The latency of the rise in intracellular Ca following stimulation of acini suspended in Ca-free media was not different from that observed for acini suspended in normal media, but the rise time was significantly prolonged. In the presence of extracellular Ca, the intracellular level of Ca remained elevated 2.8-fold above basal levels for at least 15 min following stimulation with 10(-6) M carbachol, whereas it had returned to near resting levels by 15 min when either 3 X 10(-7) or 3 X 10(-5) M carbachol was the stimulus. The Ca ionophore ionomycin (10–6 M) induced changes in the level of free cytosolic Ca similar to those caused by 10(-6) M carbachol. Ionomycin, however, stimulated only approximately one-third as much amylase release. These data suggest that factors in addition to changes in free cytosolic Ca may be important in regulating enzyme secretion by pancreatic acinar cells.

A High Speed Method of Continuous Background Correction in Atomic Absorption Spectrometry

1975 ◽  
Vol 29 (2) ◽  
pp. 149-153 ◽  
Author(s):  
T. H. Donnelly ◽  
A. J. Eccleston ◽  
R. L. Gully
Keyword(s):  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
High Speed ◽  
Pulse Generator ◽  
Absorption Spectrometry ◽  
Background Correction ◽  
Light Sources ◽  
Electronic Circuitry ◽  
Continuous Background ◽  
Speed Method

Background correction in atomic absorption spectrometry using high speed electronic circuitry has been developed. Electrical responses are generated from two light sources: an atomic spectral lamp and a modified Beckman hydrogen arc lamp. Repetition rate of the pulses (2.6 msec) was limited by the increasing distortion of the pulse shape. Instantaneous relationships between the various electronic components was established using a synchronizing pulse generator.

A High Speed Method of Continuous Background Correction in Atomic Absorption Spectrometry

1975 ◽  
Vol 29 (2) ◽  
pp. 154-158 ◽  
Author(s):  
T. H. Donnelly ◽  
A. J. Eccleston
Keyword(s):  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
High Speed ◽  
Broad Band ◽  
Absorption Spectrometry ◽  
Point Of View ◽  
Background Correction ◽  
Continuum Source ◽  
Continuous Background ◽  
Band Absorption

The need for background correction in atomic absorption spectrometry (AAS), particularly when graphite furnaces are used to generate the atomic vapor, is discussed. It is shown that a Beckman hydrogen arc lamp is suitable as a continuum source from the point of view of noise, extent of its useful broad band absorption, and light intensity for background-corrected absorption (BCA) measurements over the wavelength range examined (200 to 460 nm). The standard method of determining tin present in rock samples as cassiterite, by extraction as the volatile tin iodide, was examined by flameless AAS with BCA. The method corrects for the large nonatomic absorption present, it is rapid, and it enables easier examination of solutions containing low concentrations of tin (detection limit for a 1 g starting sample is ∼1 jug tin).

ACTIVATION OF SOLUBILIZED STEROID-TRANSFORMING ENZYMES OF ADRENAL MICROSOMAL ORIGIN BY SERUM PROTEINS

1972 ◽  
Vol 54 (2) ◽  
pp. 297-315 ◽  
Author(s):  
MARGOT A. HAMILTON ◽  
R. W. McCUNE ◽  
SIDNEY ROBERTS
Keyword(s):  
High Speed ◽  
Microsomal Fraction ◽  
Adrenal Glands ◽  
Serum Proteins ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Supernatant Fraction ◽  
Rat Serum ◽  
Microsomal Preparation ◽  
Stimulation Of

SUMMARY The reactions which result in the conversion of pregnenolone to progesterone and of progesterone to deoxycorticosterone in undisrupted microsomal preparations from rat adrenal glands were stimulated by homologous serum. The active materials were shown to be firmly associated with serum proteins. The dialysable fraction of serum was either without effect on these transformations or was inhibitory. The enzyme systems involved were partially solubilized by exposure of the microsomal preparation to prolonged sonic treatment or to 1% Triton N-101. After either treatment, 35–40% of the original specific activity of the steroid 21-hydroxylase system responsible for the conversion of progesterone to deoxycorticosterone was found in the supernatant fraction after high-speed centrifugation. However, this solubilized system did not respond to serum preparations. The same procedures also resulted in a supernatant fluid which showed about 50–60% of the initial specific activity of the multi-enzyme system involved in the conversion of pregnenolone to progesterone. In these instances, the stimulatory effect of serum was retained or accentuated. Acetone powders prepared from the adrenal microsomal fraction were also active in the conversion of pregnenolone to progesterone and responded to serum with enhanced activity. Earlier observations that activation of steroid 21-hydroxylase by homologous rat serum was specific for the β-globulin fraction were confirmed in the present investigations. In contrast, stimulation of the conversion of pregnenolone to progesterone was apparently due principally to the albumin fraction. Albumin preparations from a number of other sources, as well as whole human serum protein, were also effective in this regard. The active protein preparations selectively stimulated 4-ene-3β-hydroxysteroid dehydrogenase activity, but did not activate 5-ene-3-oxosteroid isomerase in the microsomal fraction. This finding suggested that activation of 5-ene-3β-hydroxysteroid dehydrogenase was responsible for stimulation of progesterone synthesis from pregnenolone. The results indicate that the protein-bound factor in rat serum which was capable of stimulating the conversion of progesterone to deoxycorticosterone in microsomal preparations from rat adrenal glands was different from that which activates the conversion of pregnenolone to progesterone. Moreover, these diverse factors appeared to act by different mechanisms.

Nicotine stimulation of protein secretion from isolated rat pancreatic acini

1985 ◽  
Vol 248 (2) ◽  
pp. G158-G163 ◽  
Author(s):  
A. P. Majumdar ◽  
G. A. Davis ◽  
M. A. Dubick ◽  
M. C. Geokas
Keyword(s):  
Protein Synthesis ◽  
Incubation Period ◽  
Protein Secretion ◽  
Time Course ◽  
Zymogen Granules ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Isolated Rat ◽  
Stimulation Of

The secretion of amylase and trypsinogen from isolated rat pancreatic acini was greatly stimulated by 3–25 mM nicotine. In the presence of 12.5 mM nicotine, a concentration used in the study, amylase and trypsinogen release was increased by 95 and 400%, respectively, when compared with the corresponding control and showed further a preferential release of trypsinogen. A 90-min time course release of the enzymes revealed that in the presence of nicotine trypsinogen-to-amylase ratio remained two- to threefold elevated over that of the control throughout the incubation period. The nicotine-induced stimulation of trypsinogen and amylase release from the acini could not be blocked by 2.5 mM cycloheximide, a dose that inhibited overall acinar protein synthesis by about 85%. When isolated acini were incubated with [3H]leucine in the presence of nicotine, the released proteins revealed a fourfold higher radioactivity (dpm/microgram acinar DNA) than the control. Cycloheximide dramatically decreased this increment. The rate of release of 3H-pulse-labeled proteins from the acini was greatly accelerated by nicotine. It is concluded that nicotine stimulates the secretion of preformed zymogen granules and newly synthesized proteins from dispersed rat pancreatic acini in vitro.

Effect of extracellular manganese on amylase release from dispersed pancreatic acini

1981 ◽  
Vol 241 (5) ◽  
pp. G359-G364 ◽  
Author(s):  
S. Abdelmoumene ◽  
J. D. Gardner
Keyword(s):  
Secretory Process ◽  
Amylase Secretion ◽  
Enzyme Release ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Fourfold Increase ◽  
Maximal Stimulation ◽  
Basal Release ◽  
Induced Changes ◽  
Stimulation Of

In dispersed acini from guinea pig pancreas, adding extracellular manganese increased amylase release. A significant effect could be detected with 0.25 mM manganese, and maximal stimulation occurred with 1 mM manganese. When manganese was added, the rate of amylase release did not change during the first 20 min of incubation and then gradually increased to a new steady state by 80 min, which with 1 mM manganese represented a fourfold increase in the rate of enzyme release. Extracellular manganese inhibited the stimulation of amylase release caused by those secretagogues that mobilize cellular calcium but augmented the stimulation caused by those secretagogues whose actions are mediated by cellular cAMP. The mechanism by which manganese altered stimulated amylase secretion differed from the mechanism by which manganese stimulated basal amylase release because the change in stimulated release was maximal within 10 min, whereas the change in basal release did not occur until after 20 min. The actions of manganese on secretagogue-stimulated amylase release were not attributable to manganese-induced changes in secretagogue-stimulated calcium outflux or cAMP and, instead, appear to result from actions of manganese on one of the later steps in the mechanisms for stimulating the secretory process.

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