Melvin Praissman

Immunoreactive cholecystokinin in human and rat plasma: correlation of pancreatic secretion in response to CCK

Immunoreactive cholecystokinin (CCK) levels in human and rat plasma are described using a radioimmunoassay specific for the biologically active sulfated end of CCK. This assay detected significant changes in plasma cholecystokinin levels during intrajejunal administration of amino acids and intravenous infusions of CCK-8 which were followed by increased pancreatic secretion. In humans, the concentration (pg/ml) of plasma cholecystokinin increased from 10.8 to 18.9 following intrajejunal amino acid instillation and from 15.4 to 31.1 during CCK infusion, while pancreatic trypsin secretion increased more than 15 fold. Ingestion of a test meal also caused a rapid and significant elevation (P less than 0.05) in both plasma CCK (14.5-21.7 pg/ml) and gastrin (50-160 pg/ml) levels. In the rat, an injection of 46 ng of CCK-8 produced a 300% increase in immunoreactive plasma CCK levels (2 min) and caused peak pancreatic protein secretion within 5 min; 4 fold lower doses (11.5 ng) elevated plasma CCK by 38% and pancreatic protein secretion to a small but significant extent. The ability of this assay to detect various forms of sulfated CCK in human plasma was also determined. Following gel chromatography on Sephadex G-50, at least three different immunoreactive peaks were found in plasma from fasted subjects and after intrajejunal amino acid stimulation. While the lower molecular weight CCK peptides (CCK-8 and CCK-12) were detected in plasma from both fasted and stimulated subjects, the larger form (CCK-33) was only present in measurable concentrations after amino acid infusion. The simultaneous measurement of increased plasma CCK levels and pancreatic secretion and the changes in the distribution of CCK peptides following amino acid infusion provides strong support that this assay detects physiologically relevant changes in biologically active CCK peptides.

Effect of sulfated and non-sulfated gastrin and octapeptide-cholecystokinin on cat gall bladder in vitro

This study demonstrates that for the isolated cat gall bladder a smaller molar dose of the sulfated form of OP-CCK and gastrin is required to produce contraction as compared to the respective non-sulfated forms. For OP the D50 for the sulfated form versus the non-sulfated form was 1.94. For gastrin it was 1.10.

Modification of the C-terminal octapeptide of cholecystokinin with a high-specific-activity lodinated imidoester: Preparation, characterization, and binding to isolated pancreatic acinar cells

Abstract Both radiotrace-labeled and high-specific-activity 125I-labeled derivatives of the biologically active C-terminal octapeptide of cholecystokinin (CCK-8) were prepared by reaction with the iodinated form of the imidoester (IIE), methyl p-hydroxybenzimidate. Following gel and ion-exchange chromatography the purified IIE-CCK-8 derivative displayed a new peak in the ultraviolet at 320 nm, and was equally potent as CCK-8 in stimulating cyclic GMP production in dispersed pancreatic acinar cells from guinea pig. The high-specific-activity derivative (125IIE-CCK-8) was prepared with carrier-free iodine-125 and had a specific activity exceeding 2000 μCi/nmol. A sensitive radioimmunoassay using this derivative and a specific antisera was capable of detecting CCK-8, caerulein, and CCK-33, but not gastrin and desulfated CCK-8. Finally, 125IIE-CCK-8 bound specifically to dispersed acinar cells, and was competitively inhibited by CCK-8 but not by other nonrelated peptide hormones.

The binding characteristics of 125I-gastrin and 125I-CCK8 to guinea pig fundic gastric glands differ: is there more than one binding site for peptides of the CCK-gastrin family?

The binding of biologically active 125I-labeled derivatives of the C-terminal octapeptide of cholecystokinin (125I-CCK8) and gastrin (125I-G) to dispersed guinea pig fundic glands were compared at 24 degrees C. Although both peptides share the same C-terminal pentapeptide sequence, differences were found in the amount of each radioligand bound to fundic glands, their dissociation behavior, and their Scatchard plots. However, each peptide was able to displace the other radioligand from the glands at nM concentrations which indicated that both peptides bound to the same site. The different binding characteristics observed for 125I-G and 125I-CCK8 most likely resulted from the different dissociation rates of each peptide.

Preparation of an N-acetyl-octapeptide of cholecystokinin: The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5--4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentration-dependent isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.

Role of cholecystokinin in intestinal phase of human pancreatic secretion

In this study we have utilized a sensitive and specific radioimmunoassay for cholecystokinin (CCK) to determine the effects of a jejunal infusion (5 cc/min) of amino acids (44 g/liter), saline, and amino acids with intravenous atropine (20 μg·kg−1·hr) on pancreatic exocrine secretion. Amino acids were found to stimulate pancreatic output of trypsin and release CCK, while a saline infusion at the same rate and osmolality (320 mosm/liter) failed to do so. In the presence of atropine, the amino acid infusion did not stimulate the pancreatic output of trypsin, despite an augmented CCK release. The total CCK released above baseline was greatest with the infusion of amino acids with atropine, while the total trypsin output above baseline was greatest with the infusion of amino acids. These results indicate that CCK release is not under cholinergic control and that cholinergic blockade inhibits pancreatic secretion by interrupting stimulating cholinergic fibers to the pancreas.

Ion-mediated water flow: I. Electroosmosis

SummaryThe electroosmotic flows of solution produced by the chloride salts of H, Na, K, tetramethylammonium (TMA) and tetraethylammonium (TEA) through three membranes of net negative charge and high water content (40 to 60%) have been obtained. The amount of solution transported, (EOs), increased in the order: HCl, KCl, NaCl, TMACl and TEACl in a membrane of 43% hydration. In membranes 60% hydrated the order became HCl, KCl, NaCl, TEACl and TMACl. (EOs) for a salt increased as membrane hydration became larger. The permselectivity of the three membranes for cations declined in the order: HCl, KCl=NaCl, TMACl and TEACl. Cation permselectivity also declined with increases in membrane hydration. The (EOs) is a net solution flow and is the difference between the cation-induced water flow and the chloride-induced water flow in the opposite direction. In membranes of moderate to high H2O content, co-ion transport is significant and the water-flow associated with co-ion movement must be determined if the contribution of the counter-ion ([EO]cation) to the (EOs) is to be found. Cl-ion induced water flow was determined by assuming an identity of K and Cl ions. [EO]cation increased as the hydrated radii of the cations increased and for any particular cation [EO]cation was at least 100% greater in the 60% hydrated membrane than in the 43% hydrated membrane. The current-induced water flow was found to be composed of both an electroosmotic and an osmotic component. The latter represented between 10 and 40% of the total water flow.

Role of cholecystokinin in the regulation of the interdigestive phase of pancreatic secretion

The rate of pancreatic secretion during the interdigestive state varies with the phase of interdigestive motility. During phases I1 and 111 of interdigestive motility, pancreatic secretion is greatest, and minimal during phases I and IV. Pancreatic polypeptide and motilin have been reported to be increased during phases I1 and 111 but do not appear to be responsible for the stimulation of pancreatic secretion. We have investigated the role of cholecystokinin (CCK) in regulating pancreatic secretion during the interdigestive state. Eight volunteers underwent a study of interdigestive duodenal motility with a catheter that collected pancreatic secretions at the ligament of Treitz. The phase of motility was correlated with the output of trypsin and the plasma CCK levels. The output of trypsin during phases I1 and 111 was 0.9 × 0.2 and 1.0 × 0.2 mg/kg/h, respectively, and decreased to 0.3 × 0.1 mg/kg/h during phase IV-I (p <0.05). To determine if the output of trypsin during phases I1 and 111 was responsible for the increases in plasma CCK, the effect of intra-duodenal trypsin, 3 mg/kg/h, in five volunteers was determined. The infusion significantly increased the output of trypsin to a mean of 3.1 × 1.9 mg/kg/h (p <0.05). The plasma CCK concentration increased with intraduodenal trypsin from 20.4 t 5 to 26.4 2 3.7 pg/ml (p <0.05). The infusion study was repeated in two volunteers with heat-inactivated trypsin. The mean CCK level rose from 19.6 × 4 to 23.8 pg/ml. The results indicate that the variation in interdigestive pancreatic secretion is accompanied by increases in plasma CCK during the phases of maximum output of trypsin, phases I1 and 111. However, the presence of trypsin in the duodenum may result in release of CCK. The stimulation of CCK release by intraduodenal trypsin appears to be independent of tryptic activity and may be the result of the protein content.

Ion-mediated water flow. II. Anomalous osmosis.

SummaryAnomalous osmotic water flows may be the basis of the hypotonicity of gastric juice sampled at low rates of secretion. The anomalous osmotic flows of water produced by the exchange of hydrogen and a series of cations across the three membranes used in the electroosmotic studies (Paper I) have been obtained. The solvent flow results in part from the momentum imparted by the moving ions to the water contained in the membrane matrix. The physical parameters that regulate the rate of bi-ionic exchange and the accompanying anomalous osmotic solvent flows are: the hydration states of the membranes; the molarities of the membranes and the mobilities of the exchanging ions which are a function of ion size. Each ion in the exchange produces a flow of liquid. Assuming that the ions do not interact with one another in the membrane, the anomalous osmotic flux was assumed to be the sum of the water flows produced by each permeant ion. The anomalous osmotic flux produced by a bi-ionic exchange was calculated from electroosmotic coefficients ([EO]cation and [EO]Cl) and the ion-exchange rates. The calculated values were all 10 to 60% less than the observed values. Part of these differences may have resulted from concentration gradients in unstirred boundary layers adjacent to the membrane which caused an osmotic flow of water in the direction of net water movement. As in the stomach, the sodium-hydrogen exchange produced a hypotonic solution.

Size and charge heterogeneity of immunoreactive gastrin in extracts of porcine antra

Abstract Two components immunoreactive to porcine gastrin antisera have been found in porcine antral extracts by starch gel electrophoresis and gel filtration. One corresponds to the heptadecapeptide hormone gastrin. The other displays the same size and charge characteristics reported by Yalow and Berson for BG in human plasma and human tissue extracts. Dialysis was of considerable value in separating the two.