Supramaximal CCK and CCh concentrations abolish VIP potentiation by inhibiting adenylyl cyclase activity

1998 ◽  
Vol 275 (5) ◽  
pp. G1202-G1208 ◽  
Author(s):  
Toshiharu Akiyama ◽  
Yoshihide Hirohata ◽  
Yoshinori Okabayashi ◽  
Issei Imoto ◽  
Makoto Otsuki
Keyword(s):  
Adenylyl Cyclase ◽  
Cyclase Activity ◽  
Intracellular Camp ◽  
Amylase Secretion ◽  
Adenylyl Cyclase Activity ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Camp Pathway ◽  
Kinase C ◽  
Intracellular Camp Accumulation

Exocrine pancreatic secretion stimulated by vasoactive intestinal polypeptide (VIP), which acts through the adenylyl cyclase-cAMP pathway, is potentiated by stimulation with other secretagogues such as CCK and carbachol (CCh). However, the potentiating effect is abolished by the same secretagogues at supramaximal concentrations. In the present study, we examined the mechanisms by which supramaximal concentrations of CCK octapeptide (CCK-8) or CCh reduce the VIP-induced potentiation of amylase secretion from isolated rat pancreatic acini. VIP-stimulated amylase secretion was potentiated by submaximal stimulatory concentrations of CCK-8 and CCh but was reduced by the same reagents at higher concentrations. Supramaximal concentrations of CCK-8 or CCh also reduced forskolin-induced potentiation of amylase release but did not reduce that induced by 8-bromo-cAMP. Moreover, supramaximal concentrations of CCK-8 or CCh inhibited VIP-stimulated intracellular cAMP production as well as adenylyl cyclase activity. 12- O-tetradecanoylphorbol 13-acetate (TPA) also reduced the magnitude of the potentiation of amylase release caused by VIP plus CCK-8 or CCh, although TPA itself decreased neither VIP-stimulated adenylyl cyclase activity nor intracellular cAMP accumulation. These results indicate that supramaximal concentrations of CCK-8 and CCh reduce the potentiating effect of VIP and forskolin on amylase secretion by inhibiting the adenylyl cyclase activity. In addition, protein kinase C is suggested to be partly implicated in this inhibitory mechanism. The mechanisms that lead to such inhibition may be interlinked but distinct from each other.

scholarly journals Cyclic AMP Levels, Adenylyl Cyclase Activity, and Their Stimulation by Serotonin Quantif ied in Intact Neurons

1997 ◽  
Vol 110 (3) ◽  
pp. 243-255 ◽  
Author(s):  
Leland C. Sudlow ◽  
Rhanor Gillette
Keyword(s):  
Adenylyl Cyclase ◽  
Dissociation Constants ◽  
Fold Increase ◽  
Nucleotide Metabolism ◽  
Cyclase Activity ◽  
Applied Theory ◽  
Intracellular Camp ◽  
Adenylyl Cyclase Activity ◽  
Direct Binding ◽  
The Many

In molluscan central neurons that express cAMP-gated Na+ current (INa,cAMP), estimates of the cAMP binding affinity of the channels have suggested that effective native intracellular cAMP concentrations should be much higher than characteristic of most cells. Using neurons of the marine opisthobranch snail Pleurobranchaea californica, we applied theory and conventional voltage clamp techniques to use INa,cAMP to report basal levels of endogenous cAMP and adenylyl cyclase, and their stimulation by serotonin. Measurements were calibrated to iontophoretic cAMP injection currents to enable expression of the data in molar terms. In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity. Serotonin stimulation of adenylyl cyclase and [cAMP] was inversely proportional to cells' resting adenylyl cyclase activity. Average cAMP concentration at the membrane rose from 3.6 to 27.6 μM, levels consistent with the expected cAMP dissociation constants of the INa,cAMP channels. These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP. Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

Effects of staurosporine on protein kinase C and amylase secretion from pancreatic acini

1989 ◽  
Vol 257 (4) ◽  
pp. G548-G553 ◽  
Author(s):  
T. B. Verme ◽  
R. T. Velarde ◽  
R. M. Cunningham ◽  
S. R. Hootman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Digestive Enzyme ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Functional Protein ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity

The effects of staurosporine, a recently isolated microbial alkaloid, on amylase secretion and protein kinase C activity of guinea pig pancreatic acini were investigated. Staurosporine at a concentration of 1 microM completely inhibited both acinar protein kinase C activity (IC50 = 5.5 +/- 1.4 nM) and amylase secretion induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (IC50 = 4.1 +/- 0.4 nM). At this concentration, staurosporine reduced amylase secretion elicited by maximally effective concentrations of carbachol and cholecystokinin by approximately 50% but did not appreciably alter the potencies of the two secretagogues. In the presence of staurosporine, amylase secretion induced by carbachol was linear for at least 60 min. Staurosporine had no effect on amylase release elicited by the Ca2+ ionophore A23187. It did, however, inhibit secretion induced by vasoactive intestinal peptide, although with a reduced potency relative to its effects on amylase release stimulated by TPA, carbachol, and cholecystokinin (IC50 = 34 +/- 17 nM). These results indicate that staurosporine is a potent inhibitor of protein kinase C activity in pancreatic acini and that protein kinase C has an important role as an intracellular mediator of digestive enzyme secretion induced by cholecystokinin and carbachol in the acinar cell. In addition, a separate staurosporine-insensitive coupling pathway, most likely involving Ca2+, appears to be equally important and can maintain long-term secretion in the absence of functional protein kinase C activity.

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.

Caerulein causes translocation of protein kinase C in rat acini without increasing cytosolic free Ca2+

1988 ◽  
Vol 255 (1) ◽  
pp. G33-G39 ◽  
Author(s):  
R. Bruzzone ◽  
R. Regazzi ◽  
C. B. Wollheim
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Microsomal Fraction ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Enhanced Secretion

We investigated the relationships between changes in cytosolic free Ca2+ ([Ca2+]i) and amylase secretion in dispersed rat pancreatic acini. Although 10 pM caerulein did not raise [Ca2+]i, higher concentrations (1 nM) of the peptide elicited a prompt, marked, but transient (2-3 min) elevation of [Ca2+]i. Both concentrations of caerulein caused an almost identical release of amylase over a 30-min period. To investigate the mechanism(s) underlying Ca2+-independent secretion, we measured the effect of the secretagogue on protein kinase C activity and found that both caerulein concentrations caused a significant translocation of protein kinase C from the cytosolic to the microsomal fraction. Because 1 nM caerulein induced a greater enzyme secretion than 10 pM caerulein during the first 2-5 min of stimulation, we explored further the role of [Ca2+]i transients during the first minutes of secretion. Addition of ionomycin in the presence of 10 pM caerulein resulted in a rise in [Ca2+]i and enhanced secretion as a result of caerulein in a near additive fashion during the first 2 min of stimulation. Second, we pretreated acini for 5 min with 1 microM 12-O-tetradecanoylphorbol-13-acetate. This maneuver inhibited both caerulein-induced inositol trisphosphate formation and [Ca2+]i elevation. These findings were paralleled by a similar inhibition of caerulein-stimulated amylase release only during the first 5 min of secretion. These results indicate that 1) caerulein can stimulate amylase secretion independently of a concomitant [Ca2+]i rise, possibly by activation of protein kinase C, and 2) an elevation of [Ca2+]i serves as a trigger to enhance amylase release only during the initial phase of secretion.

scholarly journals Gonadotropin-Releasing Hormone Inhibits Pituitary Adenylyl Cyclase-Activating Polypeptide Coupling to 3′,5′-Cyclic Adenosine-5′-Monophosphate Pathway in LβT2 Gonadotrope Cells through Novel Protein Kinase C Isoforms and Phosphorylation of Pituitary Adenylyl Cyclase-Activating Polypeptide Type I Receptor

Endocrinology ◽  
2008 ◽  
Vol 149 (12) ◽  
pp. 6389-6398 ◽  
Author(s):  
Sigolène Larivière ◽  
Ghislaine Garrel-Lazayres ◽  
Violaine Simon ◽  
Norihito Shintani ◽  
Akemichi Baba ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Inhibitory Effect ◽  
Intracellular Camp ◽  
Type I ◽  
Camp Pathway ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Underlying Mechanisms

Gonadotrope cells are primarily regulated by GnRH but are also targets of the pituitary adenylyl cyclase-activating polypeptide (PACAP). Although it has been reported that reciprocal interactions between both neuropeptides contribute to regulation of gonadotrope function, the underlying mechanisms remain poorly understood. In this study, we reevaluated PACAP coupling to the cAMP pathway in LβT2 gonadotrope cells and analyzed GnRH effect on PACAP signaling. We established that PACAP38 markedly increases intracellular cAMP levels (EC50 of 4.7 ± 1.3 nm) through the PACAP type 1 receptor (PAC1-R), as evidenced by pharmacological and RT-PCR studies. Interestingly, although GnRH couples to cAMP pathway in LβT2 cells, the effects of both neuropeptides were not synergistic. Instead, the GnRH agonist (GnRHa) triptorelin rapidly and strongly inhibited (70% inhibition as early as 5 min) PACAP38-induced cAMP production. Inhibition was calcium independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindoylmaleimide, indicating that GnRHa inhibitory action relies on PKC. Selective down-regulation of both conventional and novel PKC prevented a GnRHa effect, whereas pharmacological inhibition of conventional PKC only was ineffective, strongly suggesting the involvement of novel PKC isoforms. GnRHa did not inhibit forskolin- or cholera toxin-stimulated cAMP accumulation, suggesting that PAC1-R is the predominant target of GnRH. Accordingly, we demonstrated for the first time that GnRH increases PAC1-R phosphorylation through PKC, providing a potential molecular mechanism which may account for GnRH inhibitory effect.

Amylase release from streptolysin O-permeabilized pancreatic acini

1990 ◽  
Vol 259 (2) ◽  
pp. G157-G164 ◽  
Author(s):  
M. Kitagawa ◽  
J. A. Williams ◽  
R. C. De Lisle
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein S ◽  
Bacterial Toxin ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Streptolysin O ◽  
Gamma S

Intracellular mediators of exocytosis were investigated using isolated mouse pancreatic acini permeabilized with the bacterial toxin streptolysin O (SLO). Permeabilization was demonstrated by fluorescent staining with ethidium bromide and fluorescein diacetate and release of cytoplasmic lactate dehydrogenase. When SLO-permeabilized acini were incubated at 37 degrees C in Ca2(+)-EGTA buffers containing MgATP, amylase secretion was Ca2+ dependent with an EC50 of 0.40 microM Ca2+ and a maximally effective Ca2+ concentration of 1 microM. Maximal amylase secretion was 330% of that in Ca2(+)-free buffer (basal). The nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 30 microM) increased the maximal secretion to 451% of basal in the presence of 1 microM Ca2+ and decreased the EC50 to 0.14 microM Ca2+. Removal of ATP plus addition of antimycin A and 2-deoxy-D-glucose inhibited Ca2(+)-dependent, GTP gamma S-enhanced amylase secretion by 56%. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM) also enhanced maximal secretion to 450% of basal and decreased the EC50 to 0.18 microM Ca2+. Enhancement of amylase secretion by submaximal concentrations of GTP gamma S or TPA was inhibited by the protein kinase C inhibitor staurosporine. These results suggest that Ca2+ stimulation of amylase secretion is potentiated by activation of protein kinase C. However, the enhancement of secretion by GTP gamma S and TPA was additive at their maximally effective concentrations, suggesting that another G protein(s) maybe involved in the terminal steps of exocytosis.

Calcium Entry Attenuates Adenylyl Cyclase Activity

CHEST Journal ◽  
1995 ◽  
Vol 107 (5) ◽  
pp. 1420-1425 ◽  
Author(s):  
William B. Abernethy ◽  
John F. Butterworth ◽  
Richard C. Prielipp ◽  
Jian P. Leith ◽  
Gary P. Zaloga
Keyword(s):  
Adenylyl Cyclase ◽  
Calcium Entry ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity
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