The neurohormonal peptide somatostatin acts as an important regulator of a variety of functions in the gastrointestinal tract. In the previous 12 years of its funding, this project has focused on three aspects of gastrointestinal somatostatin: 1) mechanisms of somatostatin biosynthesis and post-translational processing, 2) mechanisms of somatostatin secretion from gastric D-cells, and 3) mechanisms of somatostatin's inhibitory actions on gastric acid secretion. The current application proposes to extend ongoing studies in these three areas. To explore the mechanisms of somatostatin processing, somatostatin cDNA will be expressed in heterologous endocrine cells and the expressed products will be analyzed. The substrate specificity of the processing enzymes will be studies by mutating the key processing sites and examining the expression products. The cDNAs encoding PC2 and PC3, two putative mammalian dibasic cleavage enzymes, will be co-expressed in cells that do not process somatostatin efficiently to determine whether they can function as somatostatin """"""""convertases."""""""" An effort will be made to isolate cDNA clones encoding somatostatin processing enzymes by screening canine D-cell and RIN cell cDNA libraries with probes derived by polymerase chain reaction using primers based on homology to PC2 and PC3. In other studies, the mechanism by which carbachol, cholecystokinin (CCK), and gastrin all have different actions in D-cells despite the observation that they all induce D-cell membrane inositol phospholipid (PI) turnover will be explored. For these studies, we will examine the effects of these agonists alone and in combination with each other and with reagents such as 12-0-tetradecanoyl- phorbol-13-acetate and thapsigargin not only on PI turnover but also on [Ca++]i in single D-cells by microspectrofluorometry and on pertussis toxin and cholera toxin sensitive G-proteins by studies of ADP-ribosylation. To explore the mechanisms of somatostatin's inhibitory actions on acid secretion we will pursue our previous observation that somatostatin inhibits parietal cell carbonic anhydrase II (CA II) gene expression in a fashion that is paralleled by inhibition of the expression of the immediate early gene c-fos. We will examine the cis-regulatory elements of the CA II that are sensitive to somatostatin via expressing in somatostatin receptor- containing GH3 cells a construct consisting of the CA II promoter linked to a chloramphenicol acetyl transferase (CAT) reporter gene. To confirm the linkage between the effects of somatostatin on expression of CA II and c- fos, we will attempt to overcome the inhibitory effect of somatostatin on CA II-CAT expression by overexpressing c-fos in the same cells. Furthermore, we will examine the effect of mutating the c-fos sensitive AP- 1 binding sites of the CA II promoter on the inhibitory actions of somatostatin. An effort will be made to identify the somatostatin sensitive elements of the c-fos gene using portions of the c-fos promoter/enhancer segment in constructs with the CAT reporter gene. Through these studies we hope to gain insight into the cellular and molecular mechanisms governing the synthesis, secretion, and actions of somatostatin.
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