Cholecystokinin-58 is the major circulating form of cholecystokinin in canine blood

Cholecystokinin-58 (CCK-58) is the largest and most abundant, biologically active form of cholecystokinin in canine intestinal mucosa. Despite the high amounts in mucosa, CCK-58 has not been detected in significant amounts in the circulation. The release of CCK-58 into the peripheral blood in response to an intraduodenal perfusion of sodium oleate (9.0 mmol h-1) was studied in seven conscious dogs. Plasma (50 ml) was obtained before and after endogenous stimulation by a newly developed method that prevents in vitro degradation of large cholecystokinins. The relative abundance of immunoreactive forms of CCK was studied by high pressure liquid chromatography (HPLC) which separated the gastrin and CCK forms. Column eluates were measured with an antibody which recognizes the intact carboxyl terminus of both gastrin and CCK. Cholecystokinin immunoreactivity increased over basal in plasma by 7 fmol/ml after intraduodenal perfusion with sodium oleate. The most abundant form of stimulated cholecystokinin immunoreactivity eluted on HPLC in the position of CCK-58 (63% of total immunoreactivity found). Since CCK-58 is biologically active and is the most abundant circulating form, it should play an important role in the physiology of cholecystokinin.     

Lipids, proteins and carbohydrates stimulate the secretion of intestinal cholecystokinin in the pig

Food intake enhances the release of intestinal cholecystokinin (CCK) in the pig but the contribution of individual nutrients to the CCK response has not yet been established in this species. Six hogs (mean weight 50 kg) were fitted with a duodenal fistula for instillation of nutrients and with portal (PV) and carotid (CA) catheters for blood sampling. After a 24-h fast, the animals received 1,000 ml of isotonic solution containing 440 kcal of carbohydrate (starch hydrolysate), or of protein (casein hydrolysate) or fat (Intralipid) or a control saline solution by 60-min intraduodenal perfusion after a 60-min control period during which the animals received saline. Portal and peripheral blood samples were collected at 15-min intervals for CCK radioimmunoassay. Intraduodenal perfusion of fat provoked a sharp increase in CCK-Like immunoreactivity (CCK-LI) in PV (peak 76.6 +/- 12.2 pM from basal 10.8 +/- 1.2 pM) and in peripheral blood (peak 46.7 +/- 8.4 pM from basal 9.1 +/- 1.0 pM). The protein hydrolysate induced a transient increase in plasma CCK-LI during the first 30 min of intestinal perfusion (PV: peak 40.1 +/- 5.0 pM from basal 11.9 +/- 1.4 pM; CA: 31.8 +/- 4.0 pM from basal 8.5 +/- 0.8 pM). The transient effect of proteins on CCK release might reflect the consequence of somatostatin release from intestinal stores. Starch hydrolysate promptly raised plasma CCK-LI level to a plateau value (PV: 52.5 +/- 13.1 pM from basal 11.9 +/- 1.4 pM; CA: 35.4 +/- 8.0 from basal 8.5 +/- 0.8 pM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cholecystokinin octapeptide on the pancreatic exocrine secretion in the pig

In conscious pigs, intravenous infusion of serial doses of cholecystokinin octapeptide (CCK8; 2.9-232.3 pmol.kg-1.min-1) upon a background of secretin resulted in a linear increase of plasma CCK-like immunoreactivity (CCK-LI) concentration and evoked a dose-related increase of pancreatic volume and bicarbonate and protein outputs. The threshold plasma CCK-LI concentration for significant pancreatic response was 103.8 +/- 10.2 pM using a CCK8 dose of 8.8 pmol.kg-1.min-1. The maximum pancreatic response was observed for a plasma CCK-LI level of 498.0 +/- 15.3 pM using 77.2 pmol CCK8.kg-1.min-1. In anesthetized pigs, the threshold plasma CCK-LI concentration for pancreatic response was 1500 pM (actual CCK8 dose of 60.3 pmol.kg-1.min-1). The physiological relevance of this finding was assessed by comparing the food-induced increase of pancreatic secretion with that of plasma CCK-LI. Food ingestion was followed by a sharp pancreatic response and by a progressive increase of plasma CCK-LI to a peak increment of about 15 pM. Gel chromatography of portal and peripheral plasma from fed animals revealed three major peaks in the volumes of CCK33/39 and CCK8, and in a volume intermediate between CCK33/39 and CCK8. An additional minor component eluted ahead of CCK33/39. CCK8, which is one of the CCK components released after food intake, appears to be a fairly weak pancreatic stimulant in pigs.     

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.     

Endocrine cells with gastrin/cholecystokinin-like immunoreactivity in the pancreas of the spiny dogfish,Squalus acanthias

Endocrine cells containing gastrin/cholecystokinin (CCK)-like immunoreactivity were localized to the islet tissue in the pancreas of the spiny dogfish. Most of these cells were located in the 'intestinal' lobe of the pancreas; only occasional cells were observed in the 'splenic' lobe. The gastrin/CCK-like immunoreactive cells were often co-localized with the 'classical' pancreas hormones (insulin, glucagon and somatostatin). Radioimmunoassay of water extracts with a C-terminally directed antiserum revealed high levels of immunoreactive material in the intestinal part (48.6 +/- 19.9 pmol/g) and lower levels (4.5 +/- 0.6 pmol/g) in the splenic part. Acetic acid extracts of the intestinal lobe contained low levels (6.8 +/- 3.3 pmol/g) of gastrin/CCK-like immunoreactivity, whereas corresponding extracts of the splenic part showed no immunoreactivity. When the extracts were subjected to DEAE ion-exchange chromatography the gastrin/CCK-like peptides eluted as a major peak. After Sephadex gel filtration, pooled immunoreactive material from the main DEAE chromatographic peak eluted at a position close to that of CCK4. Further characterization by ion-exchange and reversed-phase HPLC showed that, in general, the immunoreactive material behaved like the shorter forms of the gastrin/CCK family (CCK4/G5 and CCK8/Cae 3-10).     

Biologically active gastrin/CCK-related peptides in the stomach of a reptile, Crocodylus niloticus; Identified and characterized by immunochemical methods

We have used immunochemical, chromatographic, and bioassay techniques to characterize peptides related to gastrin and CCK, from the stomach of the reptile Crocodylus niloticus. By immunocytochemistry gastrin/CCK-like peptides were localized in specific mucosal cells of the pylorus and in the duodenum. Boiling water extracts of pyloric antrum cross reacted with four antisera specific for the C-terminal region of gastrin or CCK, but estimates of concentration varied between antisera. Antisera specific for the N-terminus of heptadecapeptide gastrin (G17), intact G17, or the amphibian CCK-like peptide caerulein did not cross react with the crocodile extracts. Gel filtration of the extracts on Sephadex G50 resolved one major peak eluting significantly before G17 or CCK8, suggesting larger molecular size, whereas ion exchange on DE52 cellulose resolved two major immunoreactive peaks, both eluting before G17, indicating that they are less acidic. The more acidic of the two peptides stimulated gastric acid secretion in the rat, but had no CCK-like actions on the rat pancreas. Thus crocodile antrum contains gastrin-like peptides, which are however clearly distinguishable from any of the known mammalian forms of gastrin and CCK.     

Pharmacokinetics and organ catabolism of cholecystokinin octapeptide in pigs

The pharmacokinetics and organ catabolism of cholecystokinin octapeptide (CCK8) were studied in pigs. In conscious animals, intravenous infusion of increasing doses of CCK8 (2.9-232.3 pmol.kg-1.min-1) resulted in a linear increase of plasma CCK-like immunoreactivity (CCK-LI). At the cessation of infusion of the largest dose of CCK8, plasma CCK-LI promptly returned to near basal values. The half-disappearance time (t1/2), metabolic clearance rate (MCR) and distribution volume (DV) were estimated to be 0.55 +/- 0.03 min, 134.8 +/- 10.8 ml.kg-1.min-1 and 107.9 +/- 13.0 ml.kg-1, respectively. In another group of anesthetized animals, infusion of CCK8 at similar doses produced higher plateau plasma CCK levels and the t1/2, MCR and DV were calculated to be 0.68 +/- 0.06 min, 32.5 +/- 3.9 ml.kg-1.min-1 and 45.2 +/- 5.6 ml.kg-1, respectively. Estimates of first-pass immunological degradation through various vascular beds were in the range 27-66%, with in decreasing order the kidney, liver, hindleg, followed by the brain and gut. These results indicate that immunoreactive CCK8 is rapidly cleared from plasma during passage through several vascular beds. The peptide is only partly inactivated during hepatic transit and so may exert hormonal effects upon its release from intestinal stores.     

Molecular forms of cholecystokinin in human plasma during infusion of bombesin

Bombesin is a tetradecapeptide with stimulatory actions on several gastrointestinal functions. Infusion of bombesin (60 pmol/kg. 20 min) into 7 normal subjects induced significant increases in plasma cholecystokinin (CCK) as measured with 2 sequence-specific radioimmunoassays. Employing antibody 1703, specific for carboxyl-terminal CCK-peptides containing at least 14 amino acid residues, plasma CCK concentrations rose from 0.8 +/- 0.2 pmol/l to 9.9 +/- 1.7 pmol/l (p less than 0.005), while using antibody T204, specific for the sulfated tyrosine region of CCK, plasma CCK levels increased from 2.9 +/- 0.5 pmol/l to 12.4 +/- 1.3 pmol/l (p less than 0.005). Plasma samples obtained from 3 subjects during bombesin infusion were fractionated by Sephadex column chromatography. Fractionation revealed 4 molecular forms of CCK: peak I eluted in the void volume and comprised 0-7% of CCK-like immunoreactivity, peak II eluted at 35% and comprised 8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised 44-61% of CCK-like immunoreactivity, and peak IV eluted at 75% and comprised 15-27% of CCK-like immunoreactivity. Radioimmunoassay with a carboxyl-terminal CCK-antibody fully cross-reacting with gastrin did not reveal additional molecular forms of CCK. Since both the carboxyl-terminus and the sulfated tyrosine region are required for biological activity of CCK, it is likely that all these molecular forms of CCK possess biological activity.     

Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis

Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.     

Effect of infusion of salmon calcitonin on the secretion of somatostatin and gastrin in man

In vitro and animal studies have pointed out complex interrelations between gastrointestinal hormones and calcitonin. To analyse the acute effects of calcitonin in more detail, patients undergoing surgery were infused intravenously with synthetic salmon calcitonin, a potent analog of the human hormone. Samples were taken after 0, 30 and 60 minutes from the hepatic, portal and a peripheral vein. Somatostatin and gastrin were determined by radioimmunoassay. The mean basal levels of somatostatin in peripheral and hepatic venous plasma (14.2 and 15.6 pg/ml) were significantly lower than in portal plasma (45.6 pg/ml), indicating effective removal by the liver. After infusion of calcitonin there was a general rise in somatostatin levels and an increase in the gradient between hepatic and portal blood. Basal gastrin levels were highest in the portal vein when compared intraindividually. The differences disappeared after calcitonin infusion with a concomitant systemic reduction of gastrin levels. Thus, calcitonin is able to stimulate the secretion of somatostatin from the gastrointestinal tract and does reduce gastrin secretion, possibly via the stimulation of somatostatin secretion.     

Effect of nicotine on basal and bombesin stimulated canine plasma levels of gastrin, cholecystokinin and pancreatic polypeptide

The influence of nicotine on the basal and bombesin (BBS) stimulated plasma levels of gastrin, cholecystokinin (CCK) and pancreatic polypeptide (PP) was investigated in conscious dogs. Plasma levels of nicotine and gastrointestinal (GI) hormones were measured by employing gas liquid chromatography and specific radioimmunoassay (RIA). The basal levels of gastrin, CCK and PP were found to be in pg/ml (pmol/l) (mean +/- S.E.), 28 +/- 5 (13 +/- 3), 252 +/- 32 (66 +/- 8) and 347 +/- 136 (83 +/- 32), respectively and these values remained unchanged with nicotine. Significant increases in levels of gastrin, CCK and PP were, however, found with infusions of BBS alone or with BBS in combination with nicotine. Gastrin levels were higher whereas CCK and PP levels were lower with BBS alone than with BBS plus nicotine. The peak values for CCK and PP, but not gastrin, were less during second BBS infusion. These results indicate that nicotine, in presence of bombesin, has an inhibitory effect on the release of gastrin and a stimulatory effect on the release of PP and CCK.     

Release of Cholecystokinin-Like Immunoreactivity in the Frontal Cortex of Conscious Rats as Assessed by Transcerebral Microdialysis: Effects of Different Depolarizing Stimuli

Abstract: The release of cholecystokinin-like immunoreactivity (CCK-LI) from the frontal cortex of freely moving rats has been studied using a transcerebral microdialysis technique coupled to a radioimmunoassay procedure. Basal levels of CCK-LI in the dialysate were above detection limits (2.4 ± 0.7 pg/20 min; n = 8). High-K⁺ media evoked CCK-LI overflow in a concentration-dependent manner. The threshold concentration was 50 mM KCI. The peak overflow evoked by 100 mM K⁺ amounted to 42.7 ± 2.8 pg/20 min (n = 6); it was totally Ca²⁺ dependent but insensitive to 1 μM tetrodotoxin. Infusion of 4-aminopyridine (1 mM ; 20 min) evoked an overflow of CCK-LI (32 ± 2.3 pg/ 20 min; n = 4), wnich was totally Ca²⁺ dependent and tetrodotoxin sensitive. Depolarization with 100 μg/ml of veratrine (20 min) provoked a CCK-LI overflow (62.2 ± 10 pg/20 min; n = 6), which was also blocked by tetrodotoxin or by the absence of Ca²⁺ ions. The CCK-LI material collected under basal conditions or during veratrine infusion consisted essentially of CCK octapeptide sulfate. The veratrine-induced CCK-LI overflow did not change significantly when the infusion time was prolonged to 100 min. A second 20-min stimulus with 100 μg/ml of veratrine applied 200 min after a first 20-min stimulus evoked a barely significant CCK-LI overflow. These data suggest that one single 20-min stimulus with 100 μg/ml of veratrine may be sufficient to deplete the CCK-LI releasable stores and that >200 min are required to replenish the depleted CCK-containing vesicles. Taken together the data allow us to conclude that the physiology and the pharmacology of CCK release can be adequately studied in vivo by brain microdialysis.     

Gastrin/cholecystokinin-like immunoreactive peptides in the Dungeness crab, Cancer magister (Dana): immunochemical and biological characterization

The purpose of this investigation was to characterize a gastrin/cholecystokinin-like immunoreactant (G/CCK-LI) extractable from the crab, Cancer magister. G/CCK-LI was extracted best in boiling water and was found mainly in the stomach, hemolymph and carapace. A relatively large immunoreactive peptide in the stomach and apparently smaller forms in the hemolymph and carapace were separated by Sephadex G-50 fractionation. Anion-exchange chromatography further fractionated the stomach form into three major peaks. The crab material cross-reacted with three antisera specific for the common C-terminus of gastrin/CCK, but cross-reacted much less with three antisera directed against other portions of the gastrin molecule. Partially purified crab stomach G/CCK-LI inhibited the binding of labeled CCK to mouse brain G/CCK receptors but not to rat pancreatic CCK receptors. The crab peptide did not stimulate rat gastric acid or rat pancreatic amylase secretion. These results indicate that the crab peptides are structurally similar to, but distinguishable from, the bioactive C-terminal amino acid sequence common to gastrins and CCKs.     

Morphine-induced in vivo release of spinal cholecystokinin is mediated by δ-opioid receptors – effect of peripheral axotomy

Morphine and other opioid agonists induce spinal in vivo release of cholecystokinin (CCK), a neuropeptide with anti-opioid properties. However, so far the opioid receptor subtype responsible for this effect has not been determined. In the present in vivo microdialysis study, the morphine-induced release of cholecystokinin-like immunoreactivity (CCK-LI) in the dorsal horn was completely blocked by the delta-opioid antagonist naltrindole (10 microM in the perfusion fluid). Neither the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP; 10 microM in the perfusion fluid), nor the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI); 10 microM in the perfusion fluid) had any significant effect in this respect. In addition, systemic administration of the delta-opioid receptor agonist BW373U86 (1 mg/kg, s.c.) and spinal administration of the delta(2)-opioid receptor agonist, Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide ([D-Ala(2)] deltorphin II) (1 microM in the perfusion fluid) induced a significant increase of the CCK-LI level. The effect of BW373U86 on spinal CCK-LI release was completely blocked by spinal administration of naltrindole. The mu-opioid receptor agonist [D-ala(2)-N-Me-Phe(4)-Gly(5)-ol]-enkephalin (DAMGO) (1 microM in the perfusion fluid or 1 mg/kg, s.c.) failed to alter the CCK-LI level. Peripheral nerve lesions have previously been shown to down-regulate mu- and delta-opioid receptors in the dorsal horn, to increase the gene-expression of CCK and CCK-receptor mRNA in dorsal root ganglion neurons and to alter the potassium-induced spinal CCK-LI release. After complete sciatic nerve transection, administration of the two selective delta-opioid receptor agonists induced a significant release of CCK-LI, which was comparable to controls. In contrast, neither systemic nor spinal administration of morphine and DAMGO altered the spinal CCK-LI release in axotomized animals. The present data indicate that the delta-opioid receptor mediates morphine-induced CCK-LI release in the spinal cord.     

The molecular nature of vascularly released cholecystokinin from the isolated perfused porcine duodenum

Using sequence-specific radioimmunoassays, the quantities and molecular nature of cholecystokinin (CCK) have been determined in extracts of porcine duodenal mucosa and in the vascular perfusate from the isolated porcine duodenum. The basal concentration of CCK in the perfusate was 84 pM equiv. CCK-8 (mean; range: 32–173 pM, n = 5). After intraluminal stimulation with amino acids, acidified fat emulsions and hydrochloric acid, the concentrations increased 2–5-fold. Both in the basal and stimulated state the concentrations of the related hormone, gastrin, were below 5 pM equiv. gastrin-17. CCK in the perfusate was concentrated by affinitychromatography using antibodies directed against the bioactive C-terminus. Subsequent gel chromatography revealed a form with a size like or slightly larger than the C-terminal dodecapeptide (CCK-12), a predominant form resembling the C-terminal octapeptide (CCK-8), and a form resembling the C-terminal tetrapeptide (CCK-4). The duodenal mucosa contained in addition CCK-33, -39 and CCK-peptides with further N-terminal extensions. The results suggest that small CCK peptides are the principal circulating forms, while CCK-33 and larger forms are biosynthetic precursors.     

Gastrin/CCK-like peptides in the spiny dogfish, Squalus acanthias; concentrations and actions in the gut

1. The presence of gastrin/CCK-like immunoreactive material in both muscle and mucosal layers of the whole gut, except the cardiac stomach, in Squalus acanthias has been confirmed by radioimmunoassay. The highest levels were measured in rectum and spiral intestine respectively. 2. Fractionation of the spiral intestine mucosal extract on DEAE 52 columns indicated the presence of multiple forms of gastrin/CCK in the elasmobranch gut. 3. Synthetic mammalian gastrin- and CCK-like peptides, when effective, increased the rhythmic activity and sometimes the basal tonus in smooth muscle preparations from the intestine or rectum. 4. The irregular effects of mammalian peptides on the motility indicated an essential difference from the mammalian counterpart in the structure of the elasmobranch peptide present in the gastrointestinal nerves.     

Pharmacological Characterization of Morphine-Induced In Vivo Release of Cholecystokinin in Rat Dorsal Horn

Previous studies indicate that an increased release of cholecystokinin (CCK) in response to morphine administration may counteract opioid-induced analgesia at the spinal level. In the present study we used in vivo microdialysis to demonstrate that systemic administration of antinociceptive doses of morphine (1-5 mg/kg, s.c.) induces a dose-dependent and naloxone-reversible release of CCK-like immunoreactivity (CCK-LI) in the dorsal horn of the spinal cord. A similar response could also be observed following perfusion of the dialysis probe for 60 min with 100 microM but not with 1 microM morphine. The CCK-LI release induced by morphine (5 mg/kg, s.c.) was found to be calcium-dependent and tetrodotoxin-sensitive (1 microM in the perfusion medium). Topical application of either the L-type calcium channel blocker verapamil (50 microg) or the N-type calcium channel blocker omega-conotoxin GVIA (0.4 microg) onto the dorsal spinal cord completely prevented the CCK-LI release induced by morphine (5 mg/kg, s.c.). Our data indicate that activation of L- and N-type calcium channels is of importance for morphine-induced CCK release, even though the precise site of action of morphine in the dorsal horn remains unclear. The present findings also suggest a mechanism for the potentiation of opioid analgesia by L- and N-type calcium channel blocking agents.     

Isolation from chicken antrum, and primary amino acid sequence of a novel 36-residue peptide of the gastrin/CCK family

A peptide that cross-reacted with C-terminal gastrin/CCK antisera was isolated from chicken antral extracts by a combination of gel filtration and reversed-phase HPLC. The sequence was: Phe-Leu-Pro-His- Val-Phe-Ala-Glu-Leu-Ser-Asp-Arg-Lys-Gly-Phe-Val-Gln-Gly-Asn-Gly-Ala- Val-Glu-Ala-Leu-His-Asp-His-Phe-Tyr-Pro-Asp-Trp-Met-Asp-Phe(NH2). Aside from the C-terminal tetrapeptide and the Tyr residue, the molecule does not resemble other known forms of gastrin or CCK. The peptide was a potent stimulus of avian gastric acid but not pancreatic secretion. The results have important implications for the structure-activity and evolutionary relationships of the gastrin/CCK family.     

paH gradients in isoelectric focusing experiments at subzero temperatures

We have developed a simple enzymatic procedure for evaluation of antisera reactivity against the large molecular forms of gastrin and cholecystokinin (CCK). The procedure can be used for radioimmunochemical quantitation of the precursor molecules. The different molecular forms of gastrin or CCK in tissue extracts or plasma were separated by gel chromatography. The concentration of each form was then measured with 17 different antisera before and after tryptic cleavage. The ratio between the molar concentrations before and after tryptic cleavage varied from 0.32 to 1.00. Such variation can explain the variable hormone concentrations in serum and tissue measured with different radioimmunoassays. The present procedure can be performed with any biological fluid containing the precursor forms. It does not require the large molecular forms in pure state. In principle the procedure can be used for quantitation of all peptide precursors.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)