RT-PCR of cDNA from testis and brain from wild type (WT) and Sacy^tm1Lex/Sacy^tm1Lex mice (KO).

<p>(A) PCR across exons 15-16. (B) PCR across exons 5-11. (C) PCR for β-actin loading control. (D) PCR for LacZ/Neo. (−) is a no template control. The number in the lower right corner of each panel is the number of cycles used in each experiment.</p

Somatic sAC activity in brain is lower than activity in testis, but it is not diminished in brains from Sacy^tm1Lex/Sacy^tm1Lex mice.

<p>(A) Adenylyl cyclase activity (in pmol cAMP formed per ml) in mouse IgG or R37 IP from detergent extracts from a single mouse brain or mouse testis from wild type mice. Activity from testis may be under-represented; we did not confirm antibody was in excess. MM is adenylyl cyclase reaction conditions alone (no IP added). Control IgG activity is derived from pooled brain and testis detergent extracts. Values represent averages of duplicate determinations. (B) Adenylyl cyclase activity in R37 IPs from wild type (WT) or Sacy^tm1Lex/Sacy^tm1Lex (KO) mice. MM is adenylyl cyclase reaction conditions alone (no IP added). Extracts were precleared through mouse IgG prior to immunoprecipitation. Values represent quadruplicate determinations from two wild type and two knockout brains with error bars indicating S.E.M.</p

PCR from Exons 1 through 5 from WT and knockout mouse tissues.

<p>(A) and (C) PCR across exons 1-5. (A) Testis first strand from WT (+/+), Sacy^tm1Lex/+ heterozygote (+/−), and Sacy^tm1Lex/Sacy^tm1Lex homozygous knockout (−/−) mice. (C) First strand cDNA from brain (B), heart (H), kidney (K), or liver (Li) from Sacy^tm1Lex/Sacy^tm1Lex homozygous knockout (−/−) mice; (−) indicates no template control. (B) and (D) β-actin controls. The number in the lower right corner of each panel is the number of cycles used in each experiment.</p

Sequence of 5′ RACE product defining new mRNA start site from mouse brain.

<p>Sequence in bold is the newly defined 5′UTR which corresponds to the region previously assigned to be the intron between Exons 4 and 5.</p

Somatic sAC isoforms unaffected in Sacy^tm1Lex locus.

<p>Immunoprecipitations (IP) using mAb R37 or IgG control antibody from detergent solubilized whole cell extracts (lysates) of brains (A,B) or kidney (C) from wild type or Sacy^tm1Lex/Sacy^tm1Lex mice were subjected to Western analysis using (A,C) biotinylated R21 mAb (R21B) or (B) biotinylated R37 mAb (R37B). White circles denote nonspecific bands detected with streptavidin (no primary antibody) alone. The smear at ∼50 kDa in the R37 IP from brain resolves to at least two bands when less of the IP is loaded for Western blotting (inset).</p

Schematic organization of (A) previously identified, testicular sAC transcripts and (B) the newly identified somatic sAC transcript.

<p>Boxes denote exons. C1 and C2 refer to the two catalytic domains. Red exons contain stop codons. (A) sAC_fl is encoded by all known coding exons (32), and sAC_t is generated by skipping exon 12. Yellow exons (2-4) are removed in the Sacy^tm1Lex allele. Arrows indicate approximate locations of epitopes for the indicated monoclonal antibodies (R40, R21, and R37). (B) Somatic sAC transcripts derive from a unique start site upstream of exon 5 and continue through at least exon 16 to an unknown stop.</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

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

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

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