A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini

2005 ◽  
Vol 289 (1) ◽  
pp. C22-C32 ◽  
Author(s):  
Yan Bi ◽  
John A. Williams
Keyword(s):  
Actin Cytoskeleton ◽  
Late Phase ◽  
Inhibitory Effect ◽  
Pancreatic Acinar Cell ◽  
Dominant Negative ◽  
Amylase Secretion ◽  
Apical Region ◽  
Filamentous Actin ◽  
Amylase Release ◽  
Pancreatic Acini

The actin cytoskeleton has long been implicated in protein secretion. We investigated whether Rho and Rac, known regulators of the cytoskeleton, are involved in amylase secretion by mouse pancreatic acini. Secretagogues, including cholecystokinin (CCK) and the acetylcholine analog carbachol, increased the amount of GTP-bound RhoA and Rac1 and induced translocation from cytosol to a membrane fraction. Immunocytochemistry revealed the translocation of Rho and Rac within the apical region of the cell. Expression by means of adenoviral vectors of dominant-negative Rho (RhoN19), dominant-negative Rac (RacN17), and Clostridium Botulinum C3 exotoxin, which ADP ribosylates and inactivates Rho, significantly inhibited amylase secretion by CCK and carbachol; inhibiting both Rho and Rac resulted in a greater reduction. This inhibitory effect of RhoN19 on CCK-induced amylase secretion was apparent in both the early and late phases of secretion, whereas RacN17 was more potent on the late phase of secretion. None of these three affected the basal Ca2+ or the peak intracellular Ca2+ concentration stimulated by CCK. Latrunculin, a marine toxin that sequesters actin monomers, time-dependently decreased the total amount of filamentous actin (F-actin) and dose-dependently decreased secretion by secretagogues without affecting Ca2+ signaling. These data suggest that Rho and Rac are both involved in CCK-induced amylase release in pancreatic acinar cell possibly through an effect on the actin cytoskeleton.

Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

2005 ◽  
Vol 289 (3) ◽  
pp. G561-G570 ◽  
Author(s):  
Yan Bi ◽  
Sophie Le Page ◽  
John A. Williams
Keyword(s):  
Actin Polymerization ◽  
Morphological Changes ◽  
Dominant Negative ◽  
Amylase Secretion ◽  
Filamentous Actin ◽  
Pancreatic Acini ◽  
Actin Reorganization ◽  
Supramaximal Stimulation ◽  
Rho Family ◽  
High Concentrations

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.

Effects of synthetic cyclic AMP analogs on amylase exocytosis from rat pancreatic acini

1994 ◽  
Vol 72 (10) ◽  
pp. 1138-1147 ◽  
Author(s):  
Claus Schäfer ◽  
Hanna Steffen ◽  
Hartmut Printz ◽  
Burkhard Göke
Keyword(s):  
Protein Kinases ◽  
Vasoactive Intestinal Polypeptide ◽  
Inhibitory Effect ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Dependent Protein ◽  
Maximal Induction ◽  
Intestinal Polypeptide

Diastereomers of adenosine 3′,5′-phosphorothioate activate cAMP-dependent protein kinases (cAMP-PK) in vitro. We found that these compounds are highly selective tools to monitor cAMP-dependent PKA activation and its effect on amylase exocytosis from pancreatic acini. In permeabilized rat acinar cells, (Sp)-cAMPS dose-dependently stimulated amylase secretion, while (Rp)-cAMPS inhibited (Sp)-cAMPS-induced amylase release. In intact rat acini, 8-Br-(Sp)-cAMPS reduced the secretory responses to secretin, vasoactive intestinal polypeptide (VIP), 8-Br-cAMP, and 8-Br-(Sp)-cAMPS, but not to cerulein. Another derivative, dibutyryl-(Rp)-cAMPS, induced a small inhibitory effect against 8-Br-(Sp)-cAMPS and VIP, which was overlapped by an unspecific stimulatory effect on amylase exocytosis induced by the degradation product butyrate. Furthermore, (Sp)-5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3′,5′-monophosphorothioate ((Sp)-5,6-DCl-cBIMPS), a specific cAMP-PK activator, induced a maximal induction of cAMP-PK activity, but its stimulation of amylase secretion was less than that by secretin. (Sp)-5,6-DCl-cBIMPS regulated the phosphorylation of several proteins, which were also affected by secretin. However, secretin had additional effects. Its action was most likely mediated by a dual effect on the cAMP and the calcium pathway. Our results indicate that the cAMP-dependent pathway is involved in amylase exocytosis from rat pancreatic acini.Key words: secretin, vasoactive intestinal polypeptide, (Rp)-cAMPS, (Sp)-cAMPS, 8-Br-(Rp)-cAMPS, 8-Br-(Sp)-cAMPS, dibutyryl-(Rp)-cAMPS, (Sp)-5,6-DCl-cBIMPS, cAMP-dependent protein kinases, pancreatic acini, amylase secretion.

EGF inhibits secretagogue-induced cAMP production and amylase secretion by Gi proteins in pancreatic acini

1995 ◽  
Vol 269 (5) ◽  
pp. G676-G682 ◽  
Author(s):  
D. Stryjek-Kaminska ◽  
A. Piiper ◽  
S. Zeuzem
Keyword(s):  
Adenylyl Cyclase ◽  
G Proteins ◽  
Egf Receptor ◽  
Inhibitory Effect ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Camp Accumulation ◽  
Camp Production ◽  
Amylase Release ◽  
Pancreatic Acini

In pancreatic acinar cells, the epidermal growth factor (EGF) receptor interacts with both cholera toxin- and pertussis toxin (PTX)-sensitive G proteins. In the present study, isolated rat pancreatic acini were used to investigate the effect of EGF on basal and secretagogue-induced adenosine 3',5'-cyclic monophosphate (cAMP) production and amylase release. EGF increased cAMP production and amylase release in pancreatic acini. However, cAMP accumulation and amylase release elicited by either vasoactive intestinal peptide (VIP) or forskolin were inhibited by EGF (17 nM). EGF inhibited the VIP-induced cAMP production and amylase release with a half-maximal effective concentration of 3 and 2 nM, respectively. EGF had no effect on the N6,2'-O-dibutyryladenosine-3',5'-monophosphate-stimulated amylase release, suggesting that the inhibitory effect of EGF on the VIP- and forskolin-induced cAMP production is due to inhibition of adenylyl cyclase. PTX pretreatment of the acini led to an increase of the basal, EGF-, and VIP-stimulated cAMP accumulation and amylase release, indicating that PTX-sensitive G proteins exert tonic inhibition of adenylyl cyclase even in the absence of agonist. In PTX-pretreated acini, the inhibitory effect of EGF on the VIP-induced cAMP production and amylase release was abolished. In conclusion, these results suggest that EGF inhibits secretagogue-induced cAMP production via activation of PTX-sensitive G proteins in rat pancreatic acini, whereas EGF-induced cAMP production and amylase release occurs via a PTX-insensitive pathway.

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.

scholarly journals Inhibitory effect of high leucine concentration on α-amylase secretion by pancreatic acinar cells: possible key factor of proteasome

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Long Guo ◽  
Baolong Liu ◽  
Chen Zheng ◽  
Hanxun Bai ◽  
Hao Ren ◽  
...  
Keyword(s):  
Amylase Activity ◽  
Acinar Cells ◽  
Inhibitory Effect ◽  
Proteasome Activity ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
High Concentration ◽  
Amylase Release ◽  
Cholecystokinin Receptor ◽  
Key Factor

The present study aimed to investigate whether leucine affects the pancreatic exocrine by controlling the antisecretory factor (AF) and cholecystokinin receptor (CCKR) expression as well as the proteasome activity in pancreatic acinar cells of dairy calves. The pancreatic acinar cells were isolated from newborn Holstein bull calves and cultured using the Dulbecco’s modified Eagle’s medium/nutrient mixture F12 Ham’s liquid (DMEM/F12). There were six treatments of leucine dosage including 0 (control), 0.23, 0.45, 1.35, 4.05, and 12.15 mM, respectively. After culture for 3 h, the samples were collected for subsequent analysis. As the leucine concentration increased from 0 to 1.35 mM, the α-amylase activity in media decreased significantly (P<0.05), while further increase in leucine concentration did not show any decrease in α-amylase activity. Addition of leucine inhibited (P<0.05) the expression of AF and CCKR, and decreased the activity of proteasome (P<0.05) by 76%, 63%, 24%, 7%, and 9%, respectively. Correlation analysis results showed α-amylase secretion was negatively correlated with leucine concentration (P<0.01), and positively correlated with proteasome activity (P<0.01) and the expression of CCK1R (P<0.01) and AF (P<0.05). The biggest regression coefficient was showed between α-amylase activity and proteasome (0.7699, P<0.001). After inhibition of proteasome by MG-132, low dosage leucine decreased (P<0.05) the activity of proteasome and α-amylase, as well as the expression of CCK1R. In conclusion, we demonstrated that the high-concentration leucine induced decrease in α-amylase release was mainly by decreasing proteasome activity.

Regulation of CCK-induced amylase release by PKC-δ in rat pancreatic acinar cells

2004 ◽  
Vol 287 (4) ◽  
pp. G764-G771 ◽  
Author(s):  
Chenwei Li ◽  
Xuequn Chen ◽  
John A. Williams
Keyword(s):  
Acinar Cells ◽  
Adenoviral Vectors ◽  
Dominant Negative ◽  
Pancreatic Acinar Cells ◽  
Amylase Secretion ◽  
Wild Type ◽  
Amylase Release ◽  
Pkc Isoforms ◽  
Chemical Inhibitors ◽  
Dominant Negative Variant

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, α, δ, ε, and ζ, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-α, -δ, and -ε, but not -ζ from soluble to membrane fraction. CCK-induced amylase release was inhibited ∼30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-δ inhibitor, but not by Gö6976, a PKC-α inhibitor, at concentrations from 1 to 5 μM. Neither overexpression of wild-type or dominant-negative PKC-α affected CCK-induced amylase release. Overexpression of PKC-δ and -ε enhanced amylase release, whereas only dominant-negative PKC-δ inhibited amylase release by 25%. PKC-δ overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-δ only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-δ and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-δ but had no further effect in the presence of dominant-negative PKC-δ. These results indicate that PKC-δ is the PKC isoform involved with amylase secretion.

A new biological contribution of cyclo(His-Pro) to the peripheral inhibition of pancreatic secretion

1997 ◽  
Vol 273 (6) ◽  
pp. E1127-E1132 ◽  
Author(s):  
Pascal Fragner ◽  
Olivier Presset ◽  
Nicole Bernad ◽  
Jean Martinez ◽  
Claude Roze ◽  
...  
Keyword(s):  
Pancreatic Secretion ◽  
Biological Activities ◽  
Systemic Circulation ◽  
Inhibitory Effect ◽  
Pancreatic Acinar Cells ◽  
Exocrine Pancreatic Secretion ◽  
Amylase Release ◽  
Pancreatic Acini

The tripeptide pyro-Glu-His-Pro-NH2[thyrotropin-releasing hormone (TRH)] was isolated from the hypothalamus as a thyrotropin-releasing factor. It has a broad spectrum of central nervous system-mediated actions, including the stimulation of exocrine pancreatic secretion. TRH is also synthesized in the endocrine pancreas and found in the systemic circulation. Enzymatic degradation of TRH in vivo produces other bioactive peptides such as cyclo(His-Pro). Because of the short half-life of TRH and the stability of cyclo(His-Pro) in vivo, we postulated that at least part of the peripheral TRH effects on the exocrine pancreatic secretion may be attributed to cyclo(His-Pro), which has been shown to have other biological activities. This study determines in parallel the peripheral effects of TRH and cyclo(His-Pro) as well as the putative contribution of other TRH-related peptides on exocrine pancreatic secretion in rats. TRH and its metabolite cyclo(His-Pro) dose dependently inhibited 2-deoxy-d-glucose (2-DG)-stimulated pancreatic secretion. TRH and all the related peptides tested had no effect on the basal and cholecystokinin-stimulated amylase release from pancreatic acinar cells in vitro. These data indicate that cyclo(His-Pro) mimics the peripheral inhibitory effect of TRH on 2-DG-stimulated exocrine pancreatic secretion. This effect is not detected on isolated pancreatic acini. Our findings provide a new biological contribution for cyclo(His-Pro) with potential experimental and clinical applications.

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)

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.

A new CCK analogue differentiates two functionally distinct CCK receptors in rat and mouse pancreatic acini

1989 ◽  
Vol 257 (4) ◽  
pp. G594-G600 ◽  
Author(s):  
T. Matozaki ◽  
J. Martinez ◽  
J. A. Williams
Keyword(s):  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Leucine Incorporation ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
Partial Agonists ◽  
Low Concentrations ◽  
Phenylalanine Residue ◽  
Stimulation Of

Analysis of the competitive inhibition of 125I-labeled cholecystokinin octapeptide (CCK-8) binding to isolated rat or mouse pancreatic acini showed that in both species CCK-8 interacts with two different affinity sites. A newly synthesized CCK analogue modified at the COOH-terminal phenylalanine residue totally inhibited 125I-CCK binding. This interaction occurred with sites of a single affinity in rat acini but with two different affinity sites in mouse acini. When acini were incubated with increasing concentrations of CCK-8, a biphasic stimulation of amylase release was observed. By use of rat acini, the analogs stimulated amylase release but caused no inhibition at supramaximal concentrations. By contrast, in mouse pancreatic acini, analogues showed a biphasic stimulation of amylase release similar to CCK-8. Both CCK-8 and the analogue stimulated [3H]leucine incorporation into protein at low concentrations in rat pancreatic acini. Higher concentrations of CCK-8 profoundly inhibited [3H]leucine incorporation, whereas the analogue had no inhibitory effect. Moreover, the analogue at higher concentrations blocked the inhibition of [3H]leucine incorporation caused by CCK-8 but did not affect carbamylcholine-induced inhibition. In mouse acini, however, the CCK analogue inhibited [3H]leucine incorporation similar to the effect of CCK-8. The results support the concept that occupancy of distinct affinity sites or states of the CCK receptor is associated with specific biological actions. A model of the CCK receptor is proposed in which two interchangeable affinity states exist. By occupying all the receptors in only one state, the new CCK analogues serve as partial agonists of some and antagonists of other actions of CCK.

Sign in / Sign up

Export Citation Format

Share Document