Omeprazole reverses the decrease in phosphorylation of progastrin-derived peptides induced by fasting in rat

40 GENERATION OF HIGH SPECIFIC ACTIVITY cDNA PROBES BY THE POLYMERASE CHAIN REACTION, FOR USE IN GUT HORMONE RESEARCH. R.DIMALINE Physiology Department, University of Liverpool, U.K. The new and powerful Polymerase Chain Reaction (PCR) technique has revolutionised many aspects of molecular biological research. One application is the production of high specific activity cDNA probes without recourse to cloning. Total RNA was extracted from rat antrum and brain, and used to generate double stranded eDNA. Antral eDNA was used as template in PCR with pairs of oligonucleotide primers corresponding to bases -18 to +4 (sense) and 344 to 325 (antisense) of rat gastrin cDNA, bases -66 to -43 (sense) and 387-364 (antisense) of rat somatostatin eDNA, and bases -I12 to -90 (sense) and 388 to 365(antisense)of rat GRP eDNA. The eDNA from brain was used as template with primers corresponding to bases 352 to 375 (sense) and 996 to 973 (antisense) of rat /%actin eDNA. S2p was incorporated by reducing 40fold, dCTP in the PCR and substituting 100#Ci S2p-dCTP. Agarose gel electrophoresis indicated PCR products of 362bp (gastrin), 453 bp(somatostatin), 500 bp(GRP) and 644 bp(/~aetin). The probes were used in Northern Blot analysis to demonstrate changes in gut hormone gene expression in response to changes in the gut luminal environment. Changes in mRNA were compared with the unregulated/%actin message. CONCLUSION: PCR generates high specific activity cDNA probes without cloning, that permit analyses comparable to those performed using conventional cloned eDNA and cRNA probes.

OMEPRAZOLE REVERSES THE DECREASE IN PHOSPHORYLATION OF PROGASTRINDERIVED PEPTIDES INDUCED BY FASTING IN RAT. G.J.DOCKRAY, A. VARRO, R.DIMALINE. Physiology Dept., University of Liverpool, Liverpool, UK The C-terminal region of progastrin contains a serine that occurs in phosphorylated and unphosphorylated forms and is located immediately adjacent to the cleavage site yielding the biologically active gastrins. Phosphorylation at this site is of potential regulatory significance in gastrin biosynthesis. In the rat, withdrawal of food for 48hr decreases plasma and tissue gastrinlevels, gastrin mRNA, and the phosphorylation of this serine. We have examined the effects of omeprazole (400/tmol.kg "1, daily, po), which inhibits acid secretion by suppressing H+/K+ATPase, on the changes in phosphorylation of progastrin-derived peptides (pGDP). The latter were extracted with boiling water, fractionated by ion exchange chromatography and assayed with an antibody specific for the C-termlnal part of progastrin. The decreases in tissue and plasma gastrin concentrations, and in gastrin mRNA seen in fasted compared with fed rats were reversed in omeprazole-treated fasted rats. The proportion of pGDP in the phosphorylated state was 51.6 +_4,4 % in fed ad libitum rats (mean + S.E., n ffi 4) and 33.0 + 3.9o/o (n = 6, p < 0.05) in fasted rats; treatment of fasted rats with omeprazole increased this proportion to 52.2 +_2.9°/o. (n = 5) which was not significantly different from fed ad libitum rats. CONCLUSIONS. 1. The decrease in phosphorylation of pGDP that occurs with fasting is reversed by omeprazole, and so is likely to be controlled by luminal acid. 2. Changes in the phosphorylation of pGDP may be of regulatory significance since they are directly related to rates of biosynthesis.