Regulation of cholecystokinin secretion from a rat medullary thyroid carcinoma cell line: role of calcium, cyclic nucleotides, glucocorticoids, neurotensin, and calcitonin gene-related peptide.

CCK-secreting WE rat medullary thyroid carcinoma cell line resembles other calcitonin-producing (C-cell) lines in that calcium, cAMP, or agents which raise cAMP, dexamethasone, and beta-adrenergic agents all stimulate peptide secretion. Unlike other C-cell lines, the WE cells respond similarly to IBMX (3-isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor) in the presence and absence of forskolin, implying that these cells secrete substances that raise cAMP levels, whose effect is accentuated by IBMX. Both CGRP and neurotensin, peptides that may be secreted by these cells, caused a small, but significant, increase in CCK secretion. It is possible that these or other secreted substances that activate adenylate cyclase are responsible for the cell's high rate of CCK secretion. Their high rate of CCK synthesis and their regulated secretion suggest that these cells will be a good model for studies of CCK expression, biosynthesis, and processing.

Inhibition of potassium-evoked release of cholecystokinin from rat caudate-putamen, cerebral cortex and hippocampus incubated in vitro by phencyclidine and related compounds.

Potassium-evoked release of cholecystokinin (CCK) from slices of caudate-putamen, hippocampus, and cerebral cortex was inhibited in a dose-related fashion by phencyclidine (PCP). In order to further examine this effect, PCP-like ligands (dexoxadrol, levoxadrol, PCMP and MK-801) as well as compounds known to interact with the sigma receptor ((+)-SKF, DTG, (+)-3-PPP, and pentazocine) were tested. While some of these compounds inhibited CCK release, their rank order potency (Dex = Lev greater than PCP = PCMP greater than DTG = MK-801 = (+)-3-PPP) differs from that of known PCP-N-methyl-D-aspartate linked effects or sigma interactions. These results suggest that the mechanism by which PCP acts to inhibit CCK release may involve a novel type of PCP interaction.

Phorbol esters stimulate the potassium-induced release of cholecystokinin from slices of cerebral cortex, caudato-putamen and hippocampus incubated in vitro.

Incubation of slices of caudato-putamen, cerebral cortex and hippocampus for 5 to 15 minutes with phorbol 12,13-dibutyrate (PDB) or phorbol 12-myristate 13-acetate (PMA) increased potassium evoked cholecystokinin (CCK) release from 139% to 296% of control. The inactive 4 alpha phorbol and 4 alpha PDB did not alter CCK release. None of the active or inactive phorbols tested altered basal CCK release. These results suggest that there may be similarities in the regulation of CCK release in different brain regions. Although the physiological factors which regulate CCK release may differ in these tissues, it is possible that their common action is mediated by the products of inositol phospholipid turnover.

Peptides in rat brain immunoreactive for the amino terminus of cholecystokinin 33: distribution and chromatography.

Antisera directed against the amino-terminus of porcine CCK 33 detects related immunoreactivity in rat brain extracts, the distribution of which follows that of CCK 8. Sephadex chromatography indicates that several immunoreactive peptides are present with a molecular weight range of 2600-3500. These peptides are likely to be CCK 39 or CCK 33 and the amino terminal segments of CCK 39/33 without the CCK 8 sequence. The presence of CCK 39/33 and its amino-terminal fragments without CCK 22 and its amino-terminal fragments confirms the absence of CCK 22 in the rat brain. This cleavage at CCK 22 is one of the major differences between the processing of CCK in rat brain and gut and may reflect differences in their physiological roles.

Lithium preincubation stimulates the potassium-induced release of cholecystokinin from slices of cerebral cortex and caudate-putamen incubated in vitro.

Potassium-evoked cholecystokinin (CCK) release from slices of caudate-putamen and cerebral cortex, but not hippocampus incubated in vitro was increased by 152-175% by preincubation for 40 min with 10 mM lithium. These results and previous studies suggest that although different physiological agents regulate CCK release in these brain regions, these agents may share a common intracellular mediator which may be a product of inositol phospholipid turnover.

Vasoactive intestinal polypeptide (VIP) inhibits potassium-induced release of cholecystokinin (CCK) from rat caudato-putamen but not from cerebral cortex.

CCK release elicited by 40 mM potassium from slices of rat caudato-putamen (cp) was inhibited by VIP. The effect of VIP was maximal at 10(-7) M. VIP does not inhibit CCK release from cerebral cortex at either 10(-7) or 10(-6) M. VIP is known to elevate levels of cAMP in rat brain. VIP inhibition of CCK release appears to be independent of activation of adenylate cyclase because treatment of cp slices with forskolin (2 X 10(-6) to 10(-4) M) does not mimic the inhibitory action of VIP.

Growth inhibition of Escherichia coli by E colicin plasmids.

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.

The subcellular distribution of peptides immunoreactive for the amino-terminal of pro-cholecystokinin in rat brain.

Antiserum 1942 raised against the synthetic peptide V-9-M is specific for the amino-terminus of pro-cholecystokinin (pro-CCK). It detects three major peptides in whole rat brain extracts with molecular weights of about 13 000 (peak 1), 8000 (peak 2) and 2700 (peak 3), of which the major one is peak 3. Rat brain was found to contain large quantities of these V-9-M-like peptides. Subcellular fractionation of whole rat brain was performed to determine what cellular component was enriched in these peptides. The molecular weight of the V-9-M-like and CCK-8-like peptides enriched in various subcellular fractions has been determined by Sephadex G-50 chromatography. Primary subcellular fractionation experiments indicated a significant enrichment of V-9-M-like peptides in the mitochondrial pellet (P2), a lesser amount in the microsomal pellet (P3), and a slight enrichment in the soluble fraction (S3). Further purification of the P2 fraction demonstrated an increase of V-9-M-like immunoreactivity in purified synaptosomes. With the exception of the enrichment in the soluble fraction, V-9-M-like peptides follow a similar distribution to that of CCK-8-like peptides. Sephadex chromatography of P2 and P3 fractions indicates that the major form of V-9-M present is the peak 3 (2700) form. This V-9-M-like peptide may represent an intermediate in the processing of CCK, and its presence in synaptosomes may indicate that the proteolytic cleavage of pro-CCK into CCK 58 and peak 3 takes place in synaptic vesicles.

The subcellular distribution of peptides immunoreactive for the carboxyl-terminal extension of cholecystokinin in rat brain.

An antiserum raised against a synthetic peptide (D-10-Y) comprising the carboxyl-terminal extension of cholecystokinin (CCK) detects several immunoreactive peptides, which were found to be widely distributed in rat brain (1). This article reports the subcellular distribution of these D-10-Y-like immunoreactive peptides in whole rat brain. Primary subcellular fractionation yielded a mitochondrial (P2) and microsomal (P3) fraction, both of which were enriched in D-10-Y and CCK 8 peptide immunoreactivity. Further fractionation of P2 yielded a purified synaptosome fraction (P4) which was further enriched in D-10-Y and CCK 8-like peptides. Whole rat brain contains two major molecular forms of D-10-Y-like immunoreactivity, one similar in size to CCK 33 (peak 1) and one slightly larger than CCK 8 (peak 2). In the P2 and P3 fractions, most of the D-10-Y-like immunoreactivity was similar in size to peak 2. It is likely that this D-10-Y-like immunoreactive peptide is an intermediate in the processing of CCK 8, and its enrichment in nerve endings is consistent with the final cleavage and amidation reactions taking place in the nerve terminals.