Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini

Abstract

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.

Fig. 1

PKA inhibitors reduce the maximal effects of the cAMP analog 8-Br-cAMP on carbachol-stimulated zymogen activation and amylase secretion. Pancreatic acini were stimulated with either supraphysiological carbachol (1 mM) alone or costimulated with 8-Br-cAMP (100 μM). Samples were pretreated with KT-5720 (1 μM) or PKI (1 μM) to inhibit PKA activity. Trypsin and chymotrypsin activities (A), as well as amylase secretion (B) were assayed. Data are means ± SE from 3 independent experiments, performed in duplicate. *,#,◆P < 0.05 compared with carbachol + 8-Br-cAMP for trypsin and chymotrypsin activity and amylase secretion, respectively.

Fig. 2

The Epac agonist 8-pCPT-2′-O-Me-cAMP enhances supraphysiological (1 mM) carbachol-stimulated zymogen activation and amylase secretion. Acini were either stimulated with supraphysiological carbachol (1 mM) or costimulated with 8-pCPT-2′-O-Me-cAMP (10–1,000 μM). Trypsin and chymotrypsin activity (A) and amylase secretion (B) were assayed. Data are means ± SE from 3 independent experiments, performed in duplicate. *,#P < 0.05 compared with carbachol alone for trypsin and chymotrypsin activity and for amylase secretion, respectively. ■ in B, basal amylase secretion.

Fig. 3

PKA inhibition does not affect Epac-dependent zymogen activation or amylase secretion. Pancreatic acini were stimulated with either supraphysiological carbachol (1 mM) alone or costimulated with 8-pCPT-2′-O-Me-cAMP (100 μM) or 8-pHPT-2′-O-Me-cAMP (100 μM) in the absence or presence of PKI (1 μM). Trypsin and chymotrypsin activities (A), as well as amylase secretion (B) were assayed. Although 8-pCPT-2′-O-Me-cAMP and 8-pHPT-2′-O-Me-cAMP both enhanced carbachol-induced zymogen activation and amylase secretion, PKI did not affect these responses. Data are means ± SE from 3 independent experiments, performed in duplicate. NS, not statistically significant; *P < 0.05 compared with carbachol + 8-pHPT-2′-O-Me-cAMP.

Fig. 4

Carbachol with 8-Br-cAMP, but not an Epac agonist, causes a prominent PKA-dependent increase in cAMP-responsive element binding protein (CREB) phosphorylation. Pancreatic acini were stimulated with either or 8-pCPT-2′-O-Me-cAMP (100 μM) or supraphysiological carbachol (1 mM) alone or costimulated with 8-Br-cAMP (100 μM) or 8-pCPT-2′-O-Me-cAMP (100 μM) in the absence or presence of PKI (1 μM). Only the combination of carbachol and 8-bromo-cAMP caused a significant in crease in CREB phosphorylation, and this increase in phosphorylation was blocked by preincubation with PKI. Data are means ± SE from 3 independent experiments. *P < 0.05 compared with control; #P < 0.05 compared with carbachol + 8-bromo-cAMP.

Fig. 5

The Epac agonist 8-pCPT-2′-O-Me-cAMP enhances physiological secretagogue-stimulated zymogen activation and amylase secretion. Acini were stimulated with either physiological carbachol (1 μM) or costimulated with 8-pCPT-2′-O-Me-cAMP (100 μM). Trypsin and chymotrypsin activity (A) and amylase secretion (B) were assayed. Similar studies were performed using physiological concentrations of caerulein (100 pM; C and D). Data are means + SE from 3 independent experiments, performed in duplicate. Zymogen activity was normalized to a maximum for each secretagogue [carbachol (1 mM) or caerulein (100 nM)]. *P < 0.05 compared with carbachol (1 μM) for trypsin and chymotrypsin activity and for amylase secretion.

Fig. 6

Disruption of subapical F-actin is unaffected by 8-Br-cAMP or Epac. A and D: pancreatic acini were stimulated with either carbachol or caerulein with or without 8-Br-cAMP (100 μM) or the Epac agonist 8-pCPT-2′-O-Me-cAMP (100 μM). Cells were fixed and stained with rhodamine-phalloidin (red) for F-actin and TOPRO-3 (blue) for nuclei. Representative images from 3 independent experiments are shown. B and E: apical intensities were measured for each sample and expressed relative to unstimulated cells. C and F: ratio of fluorescence intensities of the apical to basolateral regions were determined for each condition. Unstim., unstimulated; Carb, carbachol. Seven cells were analyzed per condition and expressed as means ± SE. *,#,◆P < 0.05 for either carbachol (1 mM) alone, with 8-Br-cAMP, or with 8-pCPT-2′-O-Me-cAMP compared with unstimulated, respectively.