Effect of two new cholecystokinin antagonists on gallbladder emptying in opossums.

In this investigation we evaluated the effect of two new cholecystokinin (CCK) antagonists, CR 1409 and L364,718, on gallbladder emptying in the opossum. Gallbladder emptying was elicited by both exogenous and endogenous CCK. The three test challenges were 1) intravenous infusion of CCK octapeptide (OP) (10 ng.kg-1.min-1), 2) feeding, and 3) intraduodenal infusion of Isocal (0.4 ml/min), a fat-containing nutrient. During control conditions each test challenge elicited approximately 60% gallbladder emptying within 30 min and 70% emptying by 60 min. At given doses both CR 1409 and L364,718 substantially antagonized or abolished the gallbladder emptying elicited by each of the test challenges. The antagonism for postprandial gallbladder emptying was diminished between 30 and 50 min compared with that for CCK-OP infusion and intraduodenal infusion of Isocal. Unexpectedly, the gallbladder emptying induced by infusion of motilin (5 micrograms.kg-1.h-1) was antagonized by either CR 1409 or L364,718. In anesthetized animals, gallbladder contraction was induced by a variety of agonists, such as bethanechol, histamine phosphate, 5-hydroxytryptamine, and phenylephrine. In this later model CR 1409 and L364,718 functioned solely as selective antagonists. We conclude that for the opossum gallbladder 1) the CCK antagonists CR 1409 and L364,718 antagonize or abolish gallbladder emptying induced by exogenous or endogenous CCK; 2) the pattern of CCK antagonism after feeding suggests that the early phase of postprandial gallbladder emptying is mediated by a mechanism other than endogenous CCK, whereas late postprandial emptying is mediated by release of endogenous CCK; and 3) CR 1409 and L364,718 are not totally specific antagonists for gallbladder CCK receptors alone but also antagonize gallbladder contraction induced by motilin.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

DNA and protein synthesis in developing rat pancreas.

To define developmental patterns, we determined the rate of protein and DNA synthesis in rat pancreas at birth, 1, 2, 3, 7, 10, 14, 21, 28 days, 2 months, 3 months, and in adults. Fragments of freshly minced pancreas were incubated with [3H]-thymidine and [14C]-leucine and the DNA and protein synthesis rates were measured. We found that DNA content was lowest at birth, rose through day 3, plateaued at about 8 mg/g wet weight through day 14, then slowly decreased to the adult value of about 5 mg/g at 2 months; protein content, although high at birth, decreased rapidly to a value of 80 mg/g at day 3 and slowly rose to the adult value of 160 mg/g; protein synthesis, low at birth, rapidly increased to about five times the adult value by day 3, and remained elevated for the 1st month; DNA synthesis was 15 times the adult rate at birth, increased to 30 times at 3 days of age, then declined slowly in an exponential fashion to the adult value. We conclude that the pancreas at birth is poised biosynthetically to undergo a rapid hyperplastic and hypertrophic response, and this process reaches a maximal rate at about 3 days of age.

Ontogeny of secretory function and cholecystokinin binding capacity in immature rat pancreas.

Isolated acini were prepared from the pancreas of immature rats (age less than 1 hr. - 48 hrs) in order to study the development of the secretory process. The ultrastructural integrity of the acinar cells was maintained after digestion and stimulation with secretagogues. Acini prepared from rats aged 24 - 48 hours responded to both CCK-8 and carbachol with significant increases in amylase release. Although typical biphasic dose response curves were obtained, the curves were shifted to the right by 1 - 2 log units, compared to the responses of adult acini. At ages younger than 24 hours, acini were insensitive to secretagogues but were sensitive to the calcium ionophore A23187. CCK receptors were virtually absent from membranes prepared from newborn pancreases, but binding of CCK, although small, was measurable at 12 hours and slowly increased up to 48 hours. A greater amount of binding was seen at 72 hours, which appeared constant up to 14 days. At 21 days, adult levels of binding were found. These results confirm previous studies that the rat pancreas is insensitive to secretagogues in the first 24 hours of life. After age 24 hours the secretory process is intact but less sensitive to secretory agents than the more mature pancreas. In the case of CCK, this may be due to lesser numbers of CCK receptors and/or affinity of CCK for its receptor.

Development of an assay for H2-receptor antagonists using isolated fat cells.

Fat cells were isolated by collagenase digestion of adipose tissue from male dogs. The cells were shown to be responsive to both histamine and norepinephrine in producing glycerol, a marker for lipolysis. The histamine-stimulated response was shown to be mediated by H2-receptors because it was inhibited by the H2-receptor antagonists metiamide and cimetidine, but not by H1 blockers such as tripelennamine, or by other agents such as propranolol. Conversely, norepinephrine-stimulated lipolysis was inhibited by propranolol but not by metiamide. The KD estimated for cimetidine and metiamide was approximately 10(-6) M, which compares favorably with that observed in other H2-receptor systems. The relative potency of various histamine H2-receptor agonists in this system was comparable to that seen in other H2-receptor systems. This assay has the advantage of utilizing a homogeneous cell preparation and is not complicated by tachyphylaxis. The dogs which are the source of the adipose tissue can potentially be used for other studies after a suitable recovery period. This system appears useful in screening novel compounds for H2-receptor antagonist or agonist activity.

Effects of 5,5'-diphenyl-2-thiohydantoin on respiration and oxidative phosphorylation of rat liver mitochondria.

5,5'-Diphenyl-2-thiohydantoin (DPTH) administered in vitro, inhibited state 3 oxidation, stimulated state 4 oxidation and decreased ADP:O ratio when 3-hydroxybutyrate and succinate were used as substrates. Considerably lower DPTH concentrations were required for the inhibition of 3-hydroxybutyrate oxidation (50% inhibition occurred at approximately 0.17 mumoles DPTH/mg protein) than were needed for inhibition of succinate oxidation (50% inhibition occurred at about 0.62 mumoles DPTH/mg protein). DPTH showed no inhibitory effects when ascorbate plus tetramethylphenylenediamine (TMPD) served as the substrate. The inhibition of state 3 respiration was not reversed by 2,4-dinitrophenol (DNP), although there was a slight increase in the DNP rate:state 3 rate suggesting the presence of a weak DPTH inhibotory site located within the Site I energy transport chain. Uncoupling, in the presence of DPTH, was observed with all substrates. In experiments utilizing sonicated mitochondria, DPTH inhibited NADH-linked oxidation, but did not inhibit succinate or ascorbate plus TMPD oxidation. The effects of DPTH were reversed by dilution and by addition of albumin. DPTH concentrations which produced inhibition of state 3 respiration in vitro were reached, in vivo, in the livers of rats receiving a single oral dose of 40 mg/kg of DPTH.

Species variability in the modification of erythrocyte surface proteins by enzymatic probes.

Bovine and equine erythrocytes have been studied by three different surface modification techniques to investigate the accessibility of the surface components to the external medium. Lactoperoxidase labeling of equine erythrocytes results in a significant labeling of only one membrane component, a 100 000-mol.wt polypeptide corresponding to the membrane-spanning Component III of human erythrocytes. The major sialoglycoprotein of the equine erythrocyte is not labeled. This is in contradistinction to the situation for human and bovine cells, where both components are labeled. The equine membrane sialoglycoprotein is also not markedly affected by pronase, chymotrypsin or trypsin treatment of whole cells under the treatment conditions used, although it can be cleaved by pronase in isolated membranes. Experiments with the isolated glycoprotein show that its cleavage by trypsin is quite selective, whereas cleavage by pronase and chymotrypsin is much more extensive. Labelling of bovine red cells by galactose oxidase treatment followed by reduction with 3H-labeled borohydride yields radioactivity in only one major peak, that corresponding increase in labeling. Equine erythrocytes don not show significant labeling by this technique unless a neuraminidase pretreatment has been performed. Then only the major glycoprotein is labeled. Thus the equine glycoprotein is apparently inaccessible to the cell surface by standard surface modification methods, although it is clearly a surface component. These experiments point out some of the limitations of surface labeling and proteolysis methods in probing the accessibility of membrane components. The results suggest that apparent inaccessibility of the equine glycoprotein is due partially to its structure and partially to its localization in the membrane.