Activation of soluble adenylyl cyclase protects against secretagogue stimulated zymogen activation in rat pancreaic acinar cells.

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis

Zymogen activation in a reconstituted pancreatic acinar cell system

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Bicarbonate treatment reduces acinar cell cytosolic vacuoles induced by hyperstimulation.

<p>Acini were treated with or without bicarbonate 25 mM either alone or in the presence of either CER 100 nM or CARB 1 mM for 1 hour. Cells were collected and fixed in PLP fixative followed by embedding in EPON. Sections were cut and examined via EM. Control (A), CER (B), CARB (C), Bicarbonate (D), CER+Bicarbonate (E) and CARB+Bicarbonate (F). Each is a representative photograph. Vacuoles are indicated by arrowheads.</p

sAC is associated with specific subcellular fractions.

<p>(A) Tissue was removed from control and CER treated rats and separated by differential centrifugation into various membrane fractions or cytosol followed by Immunoblot analysis of the 34 kD band of sAC. The top panel shows representative immunoblots and below is quantitation of all experiments (n = 3). (B) Immunoblot for sAC using Ab (R21) in cytoplasmic and nuclear protein extract from pancreatic acini (n>3).</p

The effect of bicarbonate on secretagogue stimulated zymogen activation is decreased by PKA inhibition using H89.

<p>Acini were treated with or without the PKA inhibitor H89 [10 µM] for 30 min prior to changing media and replacing it with either bicarbonate [25 mM] at a constant flow of air/CO₂ (95/5%) or without the addition of bicarbonate under room air conditions. H89 was added back to the appropriate wells. Secretagogue (CER 100 nM or CARB 1 mM) was added immediately after media change to the appropriate wells and acini incubated for 1 hour. Acini were collected and assayed for trypsinogen activation (A,D), chymotrypsinogen activation (B,E) and amylase secretion (C,F). #p<0.05 vs. corresponding secretagogue alone, *p<0.05 vs. corresponding secretagogue/bicarbonate treatment (n≥5).</p