Richard S. Izzo

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.

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.

Binding and internalization of VIP in rat intestinal epithelial cells

We have prepared villous cells from the jejunum of the rat small intestine and studied the effects of divalent cations and bacitracin on the binding and internalization of VIP. Villous epithelial cells (4 x 10(6) cells/ml) were suspended in a Hepes-NaCl buffer with 1.0% BSA, (pH 7.4) and the cells were incubated for varying periods of time with 125I-VIP at 24 degrees C. Specific binding of radiolabeled VIP was maximal within 10 min (10%) and slowly declined to 9.0 percent after 30 min. In the presence of 1.0 mg/ml bacitracin, however, maximal specific binding of VIP was only 2.7 percent (P less than or equal to 0.001). The addition of CA2+ or Mg2+ to the buffer significantly decreased binding of VIP in a concentration dependent manner. At 8.0, 4.0, 2.0 and 1.0 mM Ca2+, binding of 125I-VIP decreased by 70, 60, 40 and 25 percent, whereas in the presence of the same concentrations of Mg2+ binding was decreased to 50, 38, 25 and 10 percent (P less than or equal to 0.01). To determine if epithelial cells internalize VIP, we bound 125I-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150 micrograms/ml). Surface bound radioligand was the same at both 24 and 4 degrees C (5.3%), while internalized 125I-VIP was 4.0% at 24 degrees C compared to only 1.0% at 4 degrees C (P less than or equal to 0.001). At 24 and 4 degrees C, both Ca2+ (4.0 mM) and Mg2+ (8.0 mM) decreased surface bound radioligand by 60 percent (P less than or equal to 0.01) and lowered internalized radioactivity. These data demonstrate that (1) bacitracin decreases the binding of VIP to small intestinal epithelial cells, (2) both Ca2+ and Mg2+ affect the binding of VIP to its surface receptor and (3) VIP is internalized into epithelial cells.

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.

Dysplasia expresses altered nuclear matrix protein composition in human ulcerative colitis

We compared nuclear matrix protein compositions (NMPS) in colonic tissue from three different patient groups: 1) normal controls; 2) ulcerative colitis (UC) and 3) ulcerative colitis with dysplasia (UC‐dysplasia). NMPS were separated by two‐dimensional electrophoresis using isoelectrofocusing and SDS‐PAGE. Six specific NMPS (59,500 Kd, pI 6.6 and 6.3; 49,250 Kd, pI 7.4; 33,750 Kd, pI 7.5; 20,000 Kd, pI 7.5; and 19,000 Kd, pI 6.3) were identified in UC‐dysplasia that were not found in normal controls nor in UC colonic tissue. These observations suggest that NMPS may serve as important biochemical markers of dysplasia in UC.

Chloroquine alters the processing of secretin in isolated rat pancreatic acinar cells

In this study we considered the effect of chloroquine on the processing and intracellular distribution of internalized secretin radioligand in acinar cells. Chloroquine (100 microM) had no effect on the total amount of 125I-secretin bound but had marked effects on the processing of this radioligand in acinar cells. After an initial 60 min of radioligand binding in the presence and absence of chloroquine, cells were washed free of unbound radioligand, resuspended and then processed for different times at 37 degrees C. During 60, 120 and 180 min of processing, the amount of internalized radioligand in the presence of 100 microM chloroquine was increased by 116, 194 and 273%, respectively, compared to untreated control samples. Chloroquine also increased the amount of intact 125I-secretin radioligand within the cell as measured by rebinding to pancreatic plasma membranes. After 120 and 180 min of processing, intact peptide within the acinar cell was 25 and 66% greater in the presence of this agent than in control samples (P less than or equal to 0.01). To determine if chloroquine affected intracellular localization of the secretin radioligand, we measured the amount of radioactivity in soluble and particulate fractions of cell homogenates. Chloroquine decreased radioactivity entering particulate fractions of the cell by greater than 35% after 120 and 180 min of processing (P less than or equal to 0.01). This study demonstrates that (1) chloroquine inhibits the intracellular degradation of secretin in acinar cells and (2) chloroquine alters intracellular localization of this peptide during processing.

Peptide purity: lack of quality control in preparation of CCK-8.

To The Editor: The recent availability of large quantities of synthetically prepared peptide hormones of interest to scientists studying gastrointestinal function has led to a rapid and welcome increase in biochemical and physiological studies using these materials. Most vendors supplying these peptides certify their composition either explicitly or implicitly. We have recently found that such claims are not necessarily true. In two separate studies using the C-terminal octapeptide of cholecystokinin (CCK-8) obtained from Bachem Fine Chemicals (Torrance, California) (lot No. R4642), we were unable to reproduce data obtained previously using CCK-8 supplied by the Squibb Institute for Medical Research. As we suspected a concentration problem, we then weighed out the same amount of CCK-8 from both Bachem and Squibb, dissolved them in the same volume of buffer, and examined them spectrally. Although both gave identical ultraviolet spectral patterns, the material from Bachem displayed only one third the maximum absorption at 280 nm as that found with the Squibb material. Using a molar extinction coefficient of 5500, the Squibb material was at least 90% peptide while the material from Bachem was only 33% by weight. This was confirmed by amino acid analysis which showed the composition expected for CCK-8. However, the absolute recovery of amino acids indicated that the sample was only 29% by weight peptide based on a molecular weight of 1158. We have not determined the composition of the remaining material in the Bachem sample but HPLC analysis showed only a single peptide component. This type of problem might be avoided if all vendors supplying peptides provided data sheets specifying the exact amount of peptide contained in the vial and the amount and nature of any counterions. Unfortunately, the CCK-8 just described from Bachem Fine Chemicals was sent without this type of information. We certainly hope that the oft quoted expression caveat emptor, will not become a necessary consideration in the purchase of these important peptides. M E L P R A I S S M A N