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

Bicarbonate treatment reduces acinar cell vacuole formation but not blebbing.

<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 stained with hematoxylin and examined microscopically. Control (A), CER (B), CARB (C), Bicarbonate (D), CER+Bicarbonate (E) and CARB+Bicarbonate (F). Each is a representative photograph (40x magnification). Vacuoles are indicated by arrowheads.</p

Inhibition of PKA enhances secretagogue stimulated zymogen activation and amylase secretion.

<p>Acini were treated with or without the PKA inhibitor PKI [1 µM] or H89 [10 µM] for 60 min prior to the addition of either CER [100 nM] or CARB [1 mM]. Cells were assayed after one hour of secretagogue treatment for trypsinogen activation (A,D), chymotrypsinogen activation (B,E) and amylase secretion (C,F). *p<0.05 vs. corresponding CER or CARB treatment alone (n≥5).</p

The effect of bicarbonate on secretagogue stimulated zymogen activation decreased by PKA inhibition using myristolated PKI.

<p>Acini were treated with or without the PKA inhibitor PKI [1 µM] for 60 min prior to changing media and replacing it with either 25 mM bicarbonate and a constant flow of air/CO₂ (95/5%) or no added bicarbonate under room air conditions. 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

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

Bicarbonate treatment has no effect on secretagogue stimulated LDH release.

<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 2 hours. Cells were collected and a cell free fraction obtained. Both the cell-free and cell-containing fractions were assayed for LDH and the results expressed as the percentage of LDH released into the media. (n≥3).</p

Inhibition of sAC, but not tmAC, enhances cerulein (CER)-stimulated zymogen activation and amylase secretion, but not CARB stimulated responses.

<p>Acini were treated with or without the sAC inhibitor KH7 (25–50 µM) for 2 hours or the tmAC inhibitor DDA [25 µM] for 1 hour prior to the addition of either CER [100 nM] or CARB [1 mM]. Cells were assayed after one hour of secretagogue treatment for trypsinogen activation (A,D), chymotrypsinogen activation (B,E) and amylase secretion (C,F). *p<0.05 vs. corresponding CER or CARB treatment alone (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

sAC is localized to the apical pole of acinar cell and undergoes translocation after cerulein (CER) stimulation.

<p>Tissue was removed from control and CER treated rats and was probed for sAC and f-actin. (A) Pancreatic tissue was probed with antibodies to sAC (R21). sAC immunoreactivity was concentrated in the apical regions of the acinar cell (arrow heads), but labeling was also observed in the cytoplasm in control tissue (CTL). In CER hyperstimulation (CER) there was a loss of apical staining and the appearance of intense cytoplasmic puncta. 3 representative pictures from different experiments are shown. Representative nuclei are marked with and “N”. B) Control tissue probed for both sAC (green) and also f-actin (rhodaminne-phalloidin, red). In the merged co-localization image of sAC with f-actin is yellow.</p