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.

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.

scholarly journals Caerulein and carbamoylcholine stimulate pancreatic amylase release at resting cytosolic free Ca2+

1986 ◽  
Vol 235 (1) ◽  
pp. 139-143 ◽  
Author(s):  
R Bruzzone ◽  
T Pozzan ◽  
C B Wollheim
Keyword(s):  
Fold Increase ◽  
Free Calcium ◽  
Amylase Secretion ◽  
Rapid Phase ◽  
Secretory Rate ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Significant Stimulation ◽  
Phase 0 ◽  
Stimulation Of

Cytosolic free calcium concentrations ([Ca2+]i) and amylase secretion were measured in isolated rat pancreatic acini loaded with the intracellularly trapped fluorescent indicator quin2. Both caerulein and carbamoylcholine caused a rapid increase in [Ca2+]i, with a maximal 3-fold increase at 10(-9) M-caerulein and 10(-4) M-carbamoylcholine. However, caerulein (10(-12) M and 10(-11) M) as well as carbamoylcholine (10(-7) M) caused a significant stimulation of amylase release, while not inducing any detectable rise in [Ca2+]i. Changes in [Ca2+]i after addition of either secretagogue were transient and did not last more than 2-3 min. By contrast, when amylase secretion was monitored as a function of time, two distinct secretory phases could be observed upon addition of either carbamoylcholine (10(-5) M) or caerulein (10(-10) M). An initial, rapid phase (0-5 min) which caused a 6-7-fold increase above basal, followed by a sustained (5-30 min), but less marked, secretory rate (2-3-fold above basal). Addition of atropine (10(-4) M) 5 min after carbamoylcholine (10(-5) M) (i.e. after termination of the rise in [Ca2+]i and of the first secretory phase) did not cause any significant change in [Ca2+]i, while significantly inhibiting amylase secretion from 5 to 30 min to the same rate observed in the absence of the secretagogue. These results show that caerulein and carbamoylcholine, two agents thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, are capable of eliciting amylase secretion independently of a concomitant rise in [Ca2+]i. Furthermore, with both secretagogues the rise in [Ca2+]i, when observed, was only transient, while the stimulation of amylase release was sustained.

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.

Actions of peptides isolated from amphibian skin on amylase release from dispersed pancreatic acini.

1979 ◽  
Vol 236 (5) ◽  
pp. E571
Author(s):  
E R Uhlemann ◽  
A J Rottman ◽  
J D Gardner
Keyword(s):  
Salivary Gland ◽  
Guinea Pig ◽  
Vasoactive Intestinal Peptide ◽  
Amylase Secretion ◽  
Amphibian Skin ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Stimulation Of

In dispersed acini prepared from guinea pig pancreas, peptides isolated from amphibian skin (caerulein, bombesin, litorin, and physalaemin) as well as eledoisin, a peptide isolated from the posterior salivary gland of a Mediterranean octopod, caused a significant increase in amylase release. Each amphibian peptide potentiated the stimulation of amylase release caused by vasoactive intestinal peptide or secretin in that the increase in amylase release caused by an amphibian peptide plus vasoactive intestinal peptide or secretin was significantly greater than the sum of the increase caused by each secretagogue acting alone. Potentiation of amylase secretion did not occur with an amphibian peptide plus cholecystokinin or carbachol.

Visualization of amylase secretion from individual pancreatic acini

1988 ◽  
Vol 254 (5) ◽  
pp. G664-G670 ◽  
Author(s):  
D. Bosco ◽  
M. Chanson ◽  
R. Bruzzone ◽  
P. Meda
Keyword(s):  
Protein A ◽  
Plaque Assay ◽  
Wide Distribution ◽  
Amylase Secretion ◽  
Functional Heterogeneity ◽  
Active Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Dose Dependent ◽  
Stimulation Of

To assess the secretion of individual rat pancreatic acini, we developed a reverse hemolytic plaque assay that allows for a direct visualization of amylase release. This release was detected around secreting cells by the presence of hemolytic plaques that resulted from the complement-mediated lysis of red blood cells bearing amylase-antiamylase complexes bound to protein A. Controls showed that these plaques reflected specifically the active secretion of amylase. Quantitation of hemolytic plaques showed that after a 30-min incubation approximately 50% of the acini secreted under basal conditions. Stimulation of amylase release by increasing concentrations of carbamylcholine resulted in a dose-dependent recruitment of secreting acini as well as in a time-dependent enhancement in the response of individual acini. Under all conditions, the wide distribution of hemolytic plaque sizes indicated large differences in the secretory output of individual acini. Thus, using a new method to directly visualize and quantitate amylase secretion, we have provided evidence for a functional heterogeneity of pancreatic acini.

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.

Glutathione depletion inhibits amylase release in guinea pig pancreatic acini

1983 ◽  
Vol 244 (3) ◽  
pp. G273-G277
Author(s):  
W. F. Stenson ◽  
E. Lobos ◽  
H. J. Wedner
Keyword(s):  
Guinea Pig ◽  
Reduced Glutathione ◽  
Glutathione Depletion ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Stimulus Secretion Coupling ◽  
Dose Dependent ◽  
Higher Doses

Isolated guinea pig pancreatic acini were specifically depleted of glutathione by treatment with 2-cyclohexene-1-one (2-CHX-1). Untreated acini contained 4.3 +/- 0.6 micrograms of glutathione per milligram protein. Incubation with 1 mM 2-CHX-1 for 5 min at 37 degrees C depleted glutathione to 17% of control values; 5 mM 2-CHX-1 depleted glutathione to less than 4% of control values. Incubation with 2-CHX-1 also impaired the ability of the isolated acini to secrete amylase in response to stimulation with carbachol and the ionophore A23187. The depletion of glutathione and the inhibition of amylase secretion by 2-CHX-1 were both dose dependent and time dependent. Incubation of acini with 2 mM 2-CHX-1 for 15 min at 37 degrees C reduced glutathione levels to 6.6% of control and reduced carbachol-stimulated amylase release to 63% of control. Higher doses of 2-CHX-1 or longer incubations resulted in greater depletion of glutathione and greater inhibition of carbachol-induced amylase release. These data indicate that specific depletion of glutathione impairs the ability of isolated acini to secrete amylase in response to physiological and pharmacologic stimuli and suggest that glutathione has a role in stimulus-secretion coupling in the exocrine pancreas.

Amylase secretion by isolated pancreatic acini after acute cholecystokinin treatment in vivo

1984 ◽  
Vol 246 (4) ◽  
pp. G419-G425 ◽  
Author(s):  
M. Otsuki ◽  
Y. Okabayashi ◽  
A. Ohki ◽  
S. R. Hootman ◽  
S. Baba ◽  
...  
Keyword(s):  
Binding Sites ◽  
Amylase Secretion ◽  
Response Curves ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Cholecystokinin Receptor ◽  
Isolated Pancreatic Acini ◽  
And Control ◽  
Quinuclidinyl Benzilate

A single dose of synthetic cholecystokinin octapeptide (CCK8, 5 micrograms/kg) in a depot carrier was injected subcutaneously into rats 2 and 14 h before the removal of the pancreas and the preparation of isolated pancreatic acini. CCK8 treatment induced no significant change in body weight or total amount of pancreatic DNA, but pancreatic weight, total pancreatic protein and amylase, and the concentration of amylase and total protein relative to DNA were significantly decreased. In acini prepared from CCK8-pretreated rats, responsiveness to maximal and supramaximal concentrations of CCK8 was significantly increased, irrespective of whether the amount of amylase released was expressed relative to DNA or calculated as a percentage of the acinar content. The dose-response curves for CCK8 were similarly shaped in both CCK8-pretreated and control rats but shifted threefold toward higher concentrations of CCK8 2 or 14 h after CCK8 treatment. Specific 125I-CCK binding was significantly increased only for high-affinity binding sites. Although these observations suggest that alterations in pancreatic amylase release could be due to changes at the cholecystokinin receptor, the secretory responsiveness to maximal and supramaximal concentrations of carbachol was also increased without any change in the sensitivity. Moreover, in contrast to the cholecystokinin receptor, there was no change in the number of muscarinic receptors or in their affinity for either agonists or antagonists measured with [3H]quinuclidinyl benzilate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of inhibitors of cyclic nucleotide phosphodiesterase on actions of cholecystokinin, bombesin, and carbachol on pancreatic acini

1983 ◽  
Vol 245 (5) ◽  
pp. G676-G680
Author(s):  
J. D. Gardner ◽  
V. E. Sutliff ◽  
M. D. Walker ◽  
R. T. Jensen
Keyword(s):  
Dose Response ◽  
Cyclic Nucleotide ◽  
Response Curve ◽  
Dose Response Curve ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Enzyme Secretion ◽  
Amylase Secretion ◽  
Pancreatic Acini ◽  
Rightward Shift ◽  
Stimulation Of

In dispersed acini from guinea pig pancreas two inhibitors of cyclic nucleotide phosphodiesterase, Ro 20-1724 and 3-isobutyl-1-methylxanthine (IBMX), augmented the increase in amylase secretion caused by supramaximal concentrations of cholecystokinin but did not alter the stimulation of enzyme secretion caused by bombesin. The augmentations of the action of cholecystokinin caused by Ro 20-1724 or IBMX could be reproduced by 8-bromo-cAMP. When tested alone or with theophylline, cholecystokinin did not alter cAMP in pancreatic acini; however, with Ro 20-1724 or IBMX, concentrations of cholecystokinin that were supramaximal for stimulating amylase secretion caused a significant increase in cellular cAMP. These findings indicate that Ro 20-1724 and IBMX augment the action of cholecystokinin on enzyme secretion by inhibiting cyclic nucleotide phosphodiesterase and allowing a significant cholecystokinin-induced increase in cellular cAMP. IBMX but not Ro 20-1724 caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion caused by carbachol. IBMX also caused a parallel rightward shift in the dose-response curve for the stimulation of outflux of 45Ca caused by carbachol. These results indicate that IBMX, but not Ro 20-1724, can function as a muscarinic cholinergic antagonist.

Calcium fluxes in isolated pancreatic acini: effects of secretagogues

1981 ◽  
Vol 240 (1) ◽  
pp. G38-G49 ◽  
Author(s):  
R. L. Dormer ◽  
J. H. Poulsen ◽  
V. Licko ◽  
J. A. Williams
Keyword(s):  
Time Course ◽  
Calcium Content ◽  
Total Calcium ◽  
Good Correspondence ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinetic Component ◽  
Rapid Drop ◽  
Single Exponential ◽  
Stimulation Of

45Ca2+ exchange and total calcium content were measured in isolated mouse pancreatic acini. 45Ca2+ uptake could be described as the sum of a constant and a single exponential kinetic component; about 60% of total acinar calcium was exchangeable. Stimulation by bethanechol increased 45Ca2+ uptake, but the time course of uptake could be fit only by the addition of a more rapid kinetic component without any change in the total exchangeable Ca2+. 45Ca2+ washout after 1-h loading could be fit as the sum of two exponential components. Stimulation increased the rate of 45Ca2+ washout with the appearance of a third and more rapid kinetic component. There was not, however, a good correspondence between the exponential constants measured in uptake and washout protocols in unstimulated acini. Exponential constants were also affected by the concentration of calcium in the medium, further indicating the presence of nonlinearities in 45Ca2+ exchange. The dose-response relationships were similar for bethanechol stimulation of 45Ca2+ uptake and amylase release, whereas stimulation of 45Ca2+ washout reached a maximum at a higher concentration of bethanechol. Both 45Ca2+ uptake and analytical measurement of total Ca2+ showed a rapid drop in acinar Ca2+ content followed by a gradual reuptake on stimulation by bethanechol. It is concluded that the initial primary effect of secretagogues is to increase Ca2+ efflux, which is interpreted to be the result of release of sequestered calcium into the cytosol.

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