Effects of cholecystokinin and hydrocortisone on DNA and protein synthesis in immature rat pancreas

To define the developmental pattern of the trophic effects of cholecystokinin octapeptide (CCK-8) and hydrocortisone on immature rat pancreas, we injected newborn rats, rats aged 4, 7, 11, 18, and 25 days and 3 months, and adult rats with CCK (5 and 10 micrograms/kg) in gelatin and hydrocortisone (10 mg/kg) for 3 days. Animals were killed, the pancreata were removed, and the concentrations of DNA and protein were measured and DNA and protein synthesis rates determined by incorporation of [3H]thymidine and [14C]leucine, respectively. These values were compared with those of saline-injected controls. DNA concentration was significantly increased over control at ages 2 days to adult by hydrocortisone and by CCK (10 micrograms/kg) in the adult. Protein concentration was increased on days 3-14 by hydrocortisone. DNA synthesis was increased by CCK and decreased by hydrocortisone at 3 months and adult. Protein synthesis was decreased by hydrocortisone at ages 3-14 days. Thus, each agent has its own developmental pattern with age on the rat pancreas.     

Mitochondrial DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rat liver during cold exposure

We have examined in isolated liver mitochondria the effect of cold exposure on DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rats. In normal rats DNA polymerase activity increased from the first day of cold exposure remaining high up to the fifteenth day. RNA polymerase and protein synthesis were stimulated from the fifth day of cold exposure, maintaining a high level up to the fifteenth day. These activities were related to serum triiodothyronine (T3) levels. Indeed propylthiouracil (PTU) administration to cold-exposed rats drastically depressed the above activities, whereas T3 administration to PTU-treated cold-exposed rats restored them to about the values prevalent in normal cold-exposed rats. The translation products analyzed by gel electrophoresis showed that different effects may be exerted by T3 depending on whether its circulating levels are physiologically or pharmacologically modified. These findings suggest that T3 may be involved in the regulation of the acclimation process by acting, presumably with a permissive role, on those activities which determine a modification of the mitochondrial morphometric features and an increase in mitochondria number and turnover.     

Effect of bethanechol chloride on protein synthesis in rat pancreas

It is well established that acetylcholine and related cholinergic drugs stimulate secretion of pancreatic digestive enzymes. However, effects of such agents on pancreatic protein synthesis have been unclear. For these experiments rats were given either saline or bethanechol chloride in doses of 0.1, 1.0, 2.0, 4.0, and 12.0 mg/kg body wt and killed after 10, 20, 40, 60, 90, 120, and 180 minutes. Pancreatic protein synthesis was measured by determiningin vitro incorporation ofl-phenylalanine-¹⁴C into whole tissue and microsomal protein fractions as well as purified amylase. Effects of bethanechol chloride on secretion were estimated by examining residual amylase content. Doses of 1.0 mg/kg resulted in 23% increases in incorporation of label into tissue proteins and 39% increases in incorporation into microsomal proteins at 20 minutes. A dose of 2.0 mg/kg resulted in 18 and 31% increases in incorporation into tissue proteins at 40 and 60 minutes as well as 34 and 14% increases in incorporation into microsomal proteins at 60 and 90 minutes. Doses of 4.0 mg/kg resulted in decreased incorporation into tissue proteins; that is, ?36, ?19, and ?15% at 20, 40, and 60 minutes and ?36, ?22, and ?14% decreases into microsomal proteins. A dose of 12 mg/kg was associated with ?36, ?38, and ?28% decreases in incorporation into microsomal proteins at 60, 120, and 180 minutes. These studies show that small doses of bethanechol chloride were associated with induction of pancreatic protein synthesis and secretion. Large doses, on the other hand, were associated with decreased amino acid incorporation. These studies point out the importance of careful attention to dose and time of study.     

EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro

Epidermal growth factor (EGF) superfamily of peptide growth factors (EGF-GFs) plays a role in male germ cell development, but the precise function is yet to be defined. The present study shows that EGF-GFs stimulate spermatogonial proliferation in vitro. The EGF-GF ligands, EGF, transforming growth factor-alpha and betacellulin all stimulated DNA synthesis in microdissected stage I segments of rat testis seminiferous tubules in vitro, as revealed by 3H-thymidine incorporation and 5-bromo-2'-deoxyuridine (BrdU) labeling. A fourfold increase over control of BrdU labeled cells, identified as spermatogonia, was seen after treatment with EGF. RT-PCR analysis revealed that the EGF receptors erbB1, erbB2, erbB3 and erbB4 were expressed at all stages of the spermatogenic wave, whereas differential expression was found in isolated Leydig, Sertoli and peritubular cells. The results show that EGF-GFs is spermatogonial growth factor(s) in vitro, although we have not discriminated between a direct action and an indirect effect via somatic cells. We suggest that EGF-GFs is involved in the paracrine control of spermatogenesis in vivo.     

Effects of a cholecystokinin-like peptide on DNA and polyamine synthesis in the rat pancreas

The trophic effect of one or multiple subcutaneous injections of two different doses of a cholecystokinin-like peptide (CCK-LP) on the rat pancreas was evaluated by determination of ornithine decarboxylase (ODC) activity, the concentrations of the polyamines putrescine, spermidine, and spermine, and the activities of DNA polymerase and thymidine kinase, in addition to the contents of DNA, RNA, and protein. ODC activity was increased 10- to 20-fold already 2 h after a single injection of CCK-LP. The activity thereafter decreased and approached the control level after 6 to 8 h. The concentration of putrescine also showed a marked increase after a single injection, approaching maximum at 8 h. A slight increase was found for spermidine as well. DNA polymerase and thymidine kinase increased after 2 days of treatment. The DNA content was still normal at that time. The study suggests that the trophic effect of CCK is initiated very early. It shows that ODC activity and putrescine concentrations are early and sensitive determinants of the effect of CCK on the pancreas.     

RNA, protein and DNA synthesis stimulated by testosterone, insulin and prolactin in the rat ventral prostate cultured in chemically defined medium.

In an organ type tissue culture of the rat ventral prostate in a chemically defined medium insulin (0.08 IU/ml) stimulated the synthesis of RNA within 6-12 h, the synthesis of protein within 6-12 h and the synthesis of DNA within 2-4 days. Testosterone (10(-8)m) stimulated these synthetic processes somewhat more slowly: the synthesis of RNA within 12-24 h, protein within 12-24 h and DNA at 4 days. Rather high concentrations of insulin were needed while testosterone was effective at a physiological concentration. Prolactin (1000 ng/ml) stimulated the synthesis of RNA and protein, but not DNA, when added together with either testosterone or insulin, but was completely ineffective when added alone. The response times resembled those of insulin. The lower concentrations of prolactin were ineffective. Growth hormone, luteinizing hormone and follicle stimulating hormone did not stimulate the synthesis of RNA, protein or DNA even when added with testosterone. The results confirm the findings of the numerous in vivo experiments that the hypophyseal hormone prolactin has a direct effect on the ventral prostate.     

Comparative studies of the age-related changes in protein synthesis in the rat pancreas and parotid gland

This study was undertaken to determine whether changes occur in protein synthesis with age in the pancreas of the rat and to compare the pattern of changes with that observed in parotid salivary glands. The rate of incorporation of ³H-leucine into acid-insoluble proteins declines with age in both glands. In the pancreas, the rate of incorporation reaches the highest level at 12 months and declines by 21, 54 and 64% of this level at 18, 24 and 30 months, respectively. In parotid glands, the highest level of the amino acid incorporation occurs at 2 months and the level declines by 21, 29, 49 and 58% of the 2-month level at 12, 18, 24 and 30 months, respectively. There is no age-related difference in the rate of incorporation of ³H-leucine into acid-soluble fractions of the two glands. The cellular level of α-amylase is also reduced in the pancreas and parotid gland of old (24- and 30-month-old) rats. The differences in the cellular level of α-amylase activity among the age groups correlate with the differences in the number of secretory granules present in the acinar cells of the pancreas, indicating that the level of amylase reflects the cellular content of secretory proteins. There is no detectable morphological change that parallels the over 50% reduction in protein synthesis in these glands of young and old rats.     

Glucocorticoid regulation of rat thymus RNA polymerase activity: the role of RNA and protein synthesis.

Treatment of rat thymus cells with the glucocorticoids cortisol and dexamethasone resulted in the stimulation of RNA polymerase B activity within 10 min of steroid addition. This early effect was followed by the inhibition of both RNA polymerase A and B activities. These effects were glucocorticoid-specific and were inhibited by the antiglucocorticoid cortexolone. The inhibitory effect of dexamethasone on RNA polymerase A activity was abolished by prior treatment of the cells with alpha-amanitin, cordycepin or cycloheximide, but cycloheximide was only capable of inhibiting the steroid effect measured at 3 h if added within 10--20 min after steroid addition. Cycloheximide had no effect on the steroid-mediated inhibition of RNA polymerase B activity. Control RNA polymerase A activities were unaffected by the presence of inhibitors of RNA and protein synthesis. It is concluded that the inhibition of ribosomal RNA synthesis by glucocorticoids is dependent on protein synthesis, but that basal RNA polymerase A activity in rat thymus cells is not stringently coupled to protein synthesis.     

An additional secretory protein in the rat pancreas

An additional protein in rat pancreatic juice has been observed, which is present in healthy rats after pancreatic duct cannulation or in rats in which experimental pancreatitis has been induced by either cerulein or taurocholate. The protein appears 1/2-1 day after surgery or onset of pancreatitis, is present for the following 3-4 days and disappears afterwards. It is found in pancreatic homogenate or in zymogen granules from rats with pancreatitis, but not in normal rats. It does not seem to be related to the recently described 'pancreatic stone protein'. We would like to refer to this protein as 'pancreatitis-associated protein'.     

Regulation of the initiation of pancreatic digestive enzyme protein synthesis by cholecystokinin in rat pancreas in vivo

BACKGROUND & AIMS: Cholecystokinin (CCK) is known to stimulate the synthesis of digestive enzymes in the pancreas at the translational level. We investigated in vivo the biochemical regulation of initiation factors important for the stimulation of translation of digestive enzyme protein in rat pancreas by CCK. METHODS: Intraperitoneal injection of CCK or intragastric administration of a trypsin inhibitor to elicit endogenous CCK release was followed by removal and preparation of pancreas for protein evaluation. Isoelectric focusing was used to evaluate the phosphorylation of the initiation factor eIF4E, and Western blotting and immunoprecipitation followed by Western blotting were used to study the phosphorylation state and amount of other interacting factors. RESULTS: CCK treatment induced a time- and dose-dependent phosphorylation of pancreatic eIF4E and its binding protein (PHAS-I). Because the release of eIF4E from its binding protein as a result of phosphorylation is followed by formation of a messenger RNA cap-binding complex that includes the initiation factor eIF4G, we evaluated the association of eIF4G with released eIF4E and showed that it was increased by CCK. These events occurred over a range of CCK doses from 0.2 to 5 microg/kg. We also evaluated the effect of endogenous CCK by administering a synthetic trypsin inhibitor, camostat (100 mg/kg). Camostat treatment markedly increased the phosphorylation of both PHAS-I and eIF4E and the formation of eIF4E-eIF4G complex. Thus, both exogenous and endogenous CCK activate translational initiation factors in vivo. CONCLUSIONS: Activation of translational machinery necessary for initiation of protein synthesis likely contributes to the normal postprandial synthesis of pancreatic digestive enzymes.     

Effects of ethanol,dl-Ethionine,and protein deficiency on rat pancreas

Histochemical methods were used to investigate the role of ethanol in causing metabolic derangement of the pancreas. Rats fed ethanol developed a marked increase in activity of pancreatic leucine aminopeptidase. An increase in the activity of this enzyme was also found in rats givendl-ethionine, and in rats on protein-deficient diets. Although the mechanism of these changes is uncertain, it would appear that an increased activity of leucine aminopeptidase activity is a feature of various forms of pancreatic injury.Supported by the National Health and Medical Research Council of Australia.The authors acknowledge the valuable technical assistance of Miss P. Walker.     

Adrenal/parathyroid regulation of DNA, collagen and protein synthesis in rat epiphysial cartilage and bone

Young, growing rats which had been chronically (2 weeks) adrenalectomized or parathyroidectomized were used to define the roles of the adrenal and parathyroid glands on the maintenance of normal circadian rhythms of DNA, collagen and non-collagen protein synthesis in the skeleton. The animals were conditioned to food being available ad libitum and to 12 h light: 12 h darkness (lights on from 08.00 to 20.00 h). The pace of DNA, collagen and non-collagen protein synthesis in different regions of the tibia (tibial growth cartilage, metaphysial bone and diaphysial bone) was measured by the in-vivo incorporation of tritiated thymidine (1 h) and radioactive proline (48 h). In intact rats there were no regional differences in the phasing of the circadian profiles; peak DNA and non-collagen protein synthesis occurred at the onset of the dark period while peak collagen synthesis occurred during the middle of the period of light. Adrenalectomy selectively abolished the regional DNA synthesis rhythms without altering the phases of the serum Ca and phosphorus (P) rhythms, which peak at mid-day and at the onset of darkness respectively. Parathyroidectomy abolished the regional rhythms for collagen and non-collagen protein synthesis and serum Ca rhythms, without altering the phase of the serum P and corticosterone rhythms. Dietary Ca-lactate supplements, which raised serum Ca levels towards normal in parathyroidectomized rats, were able to correct serum corticosterone values but did not normalize bone collagen and non-collagen protein synthesis values. These data indicate that the adrenal rhythm governs the proliferative activities of bone and cartilage cells, and that parathyroid hormone is essential to maintain normal collagen and non-collagen protein synthesis rhythms.