Stability of the mRNA encoding some pancreatic hydrolases is modulated by dietary protein intake in the rat.

Wistar rats fed on either a high-protein or a protein-free diet were examined to determine their pancreatic hydrolase mRNA stabilities in comparison with those of control animals receiving a standard diet. Actinomycin D was used to inhibit transcription and, after isolating the pancreatic RNA, the specific messengers were quantified by performing dot-blot hybridization with cDNA probes. In the rats fed on a high-protein diet, only the half-lives of anionic trypsinogen I and elastase I (EC 3.4.21.36) were affected. Interestingly, when rats were fed on the protein-free diet, most of the hydrolase mRNA half-lives showed changes, except that corresponding to lipase. In these rats, the half-life values of the mRNA coding for anionic trypsinogen I, chymotrypsinogen and procarboxypeptidase B increased, in sharp contrast with those of the amylase and elastase I mRNA, which decreased. These results strongly suggest that the mechanism whereby the biosynthesis of pancreatic hydrolases is regulated, depending on the presence or absence of proteins in the diet, is not unique and provide evidence that the stability of mRNA encoding most, if not all, the hydrolases in pancreatic cells is modulated by the dietary protein content.

Dietary modulation of the mRNA stability of trypsin isozymes and the two forms of secretory trypsin inhibitor in the rat pancreas.

The stability of the mRNAs encoding pancreatic trypsin isozymes, namely the cationic form and the two anionic forms I and II, as well as that of the secretory trypsin inhibitors I and II, were studied in rats fed on either a high-protein diet, or a protein-free diet compared with a standard diet for a 10-day period. Either immediately or 3 h and 6 h after injecting the transcription inhibitor, actinomycin D, the mRNA levels were quantified by performing dot-blot hybridization with specific oligonucleotide probes. Under high-protein dietary conditions, the stability of the mRNAs coding for anionic trypsin II and cationic trypsin showed no change, whereas that of anionic trypsin I and the two forms of secretory trypsin inhibitor were affected. The mRNA half-life of anionic trypsin I and trypsin inhibitor II increased, in sharp contrast with that of trypsin inhibitor I, which decreased. When rats were fed on a protein-free diet, the stabilities of both anionic trypsin forms and trypsin inhibitor I increased, whereas that of trypsin inhibitor II decreased and that of cationic trypsin remained unchanged. The present results show the existence of differences in the mechanisms whereby gene expression of trypsin isozymes and secretory trypsin inhibitors is regulated, although they are synthesized in parallel in the pancreatic acinar cell and stored in zymogen granules before being secreted into the intestinal lumen.

Antigen specificity and cross-reactivity of fifteen monoclonal antibodies against porcine pancreatic alpha-amylase II and its AB and C domains.

Highly productive hybridoma secreting mabs specific for porcine alpha-pancreatic amylase II were established. Fifteen clones were selected. The mabs produced (KD = 1.68-11.2 nM) were checked for cross-reactivity with six heterologous antigens, namely porcine pancreatic alpha-amylase I, barley amylase, human pancreatic alpha-amylase, Taka amylase and triose phosphate isomerase, using direct ELISA assay; mabs were classified within seven groups: in a few groups mabs cross-reacted with a single heterologous antigen either porcine pancreatic amylase I (6 mabs) or barley amylase (2 mabs) or human pancreatic amylase (3 mabs). Two other groups cross-reacted with two heterologous antigen either porcine I and human or porcine I and barley. Only one mab out of fifteen cross-reacted in direct ELISA binding to all amylases and triose phosphate isomerase. Using sandwich ELISA test only three mabs were found to bind porcine amylase II present at high concentration. Results consistent with direct porcine amylase binding were obtained from binding inhibition assays. Analysis by the additivity test allowed to find that 3 mabs, B10.10, B1.11, C6.4 recognize distinct epitopes while the epitopes for the other pairs tested are either overlapping or at least close to each other. Finally mabs binding specifically either to the AB or to the C domain fragment or to both fragments have been obtained.

Serotonin release from the superior cervical ganglion of the cat.

Serotonin of the superior cervical ganglion is contained in a distinct and separate population of small intensely fluorescent (SIF) cells. We provide evidence, in in vitro experiments, that newly synthesized serotonin can be released in the cat superior cervical ganglion from the serotonin-containing SIF cells. Resting steady state in the release of [(3)H]serotonin was observed 30 min after the beginning of the superfusion with l-[(3)H]tryptophan. A marked increase was seen in the serotonin release either in the presence of fluoxetine, a potent reuptake blocker of serotonin, or during depolarization with potassium chloride or veratridine. Calcium-free medium led to a decrease of spontaneous and potassium-evoked release. The veratridine-stimulating response was abolished by tetrodotoxin. Paradoxically, a slight increase in the spontaneous release of serotonin was observed in the presence of tetrodotoxin. Serotonin released from serotonin-containing SIF cells could be involved in the modulation of ganglionic transmission.

Release of serotonin from perikarya in cat nodose ganglia.

Newly synthesized serotonin (5-HT) can be released in the nodose ganglion from the nerve cell bodies of vago-aortic serotoninergic neurones. Free-calcium led to a decrease of spontaneous and potassium-evoked release. The veratridine-stimulating response was abolished by TTX. The concept that 5-HT released from perikarya in the extracellular space could be involved in the self-regulation of the activity of the vago-aortic pathway is discussed.