The brain decade in debate: VIII. Peptide hormones and behavior: cholecystokinin and prolactin

This article is a transcription of an electronic symposium held on November 28, 2000 in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the peptide field with particular focus on central actions of prolactin and cholecystokinin. The comments in this symposium reflect the diversity of prolactin and cholecystokinin research and demonstrate how the field has matured. Since both peptides play a role in reproductive behaviors, particularly mother-infant interactions, this was the starting point of the discussion. Recent findings on the role of the receptor subtypes as well as interaction with other peptides in this context were also discussed. Another issue discussed was the possible role of these peptides in dopamine-mediated rewarding systems. Both prolactin and cholecystokinin are involved in mechanisms controlling food intake and somatic pain thresholds. The role of peripheral inputs through vagal afferents modulating behavior was stressed. The advent of knockout animals as potential generators of new knowledge in this field was also addressed. Finally, interactions with other neuropeptides and investigation of the role of these peptides in other fields such as immunology were mentioned. Knowledge about the central functions of prolactin and cholecystokinin has shown important advances. The role of these peptides in neurological and psychiatric syndromes such as anorexia, drug abuse and physiological disturbances that lead to a compromised maternal behavior seems relevant

An introduction to neuronal cholecystokinin.

This issue of Peptides was inspired by a gathering of CCK researchers at the first Neuronal Cholecsytokinin Gordon Conference. The papers in this issue reflect the diversity of CCK research and demonstrate how the field has matured. Reviews describe the regulation of CCK gene expression and CCK release, the nature of the hormone binding site of the CCK A receptor, interaction of CCK, dopamine and GABA, the role of CCK in thermoregulation, sexual behavior and satiety in rodents and humans. The research articles document features of cardiovascular regulation, reduced cocaine sensitization and decreased satiety in rats that lack the CCK A receptor. Pro CCK processing in neuroblastoma cells and the elevation of CCK levels in CSF in a model of chronic pain are detailed in other articles. Three articles using different behavioral paradigms in rat and sheep examine CCK in learning and memory. Two articles that examine CCK in different behaviors that have a dopaminergic component are included. Other articles describe the interaction between a 5HT(3) antagonist and CCK-induced satiety and c-fos activation and document secretion of oxytocin and vasopressin in female patients and controls in response to CCK 4 administration. There is good reason to believe that the future is bright for research on CCK. With the organization of national and international meetings, CCK researchers have a forum for communication. Opportunities for cooperation and collaboration have never been better. The easy integration of academic basic and clinical science with industrial science bodes very well for the advancement of our understanding of the multiple roles that CCK plays in the brain and for the future development of CCK-based therapies.

Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22.

Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1-7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.

Role of tyrosine sulfation and serine phosphorylation in the processing of procholecystokinin to amidated cholecystokinin and its secretion in transfected AtT-20 cells.

Mammalian procholecystokinin (pro-CCK) is known to have three sulfated tyrosine residues, one of which is present in the CCK 8 moiety and two additional residues present in the carboxyl-terminal extension. In the present study, inhibition of tyrosine sulfation by sodium chlorate decreased the secretion of processed CCK 8 in CCK-expressing endocrine cells in culture. It was then demonstrated that when each of these tyrosines individually, as well as all three together, was mutated to phenylalanine and expressed in endocrine cells, CCK was still processed and secreted. However, the amount of CCK secreted varied with the type of mutation. Substitution of Phe to Tyr in CCK 8 reduced the quantity of secreted CCK 8 by 50%, and when all the sulfated Tyr were mutated to Phe the quantity of secreted CCK was reduced by about 70%, similar to what is observed with chlorate treatment. Changing of the putative phosphorylation site serine to alanine does not affect the processing. Serine phosphorylation at this site may play a functional role in regulatory events. Our results demonstrate that tyrosine sulfation alters the amount of secretion but is not an absolute requirement for the processing and secretion of CCK in this cell line. Tyrosine sulfation of CCK may still be important for its solubility, stabilization, and/or functional interaction.

Methodologies to study the induction of rat hepatic and intestinal cytochrome P450 3A at the mRNA, protein, and catalytic activity level.

Studies were conducted to characterize assays for the isolation and quantitation of rat cytochrome P450 (CYP) 3A isoforms from hepatic and intestinal tissues. Isolated intestinal microsomes were analyzed for their alkaline phosphatase activity and CYP 3A immunoreactivity. The involvement of CYP 3A in the in vitro hydroxylation of midazolam (MDZ) was also evaluated using isoform specific chemical and antibody inhibitors. The effect of glycerol (a common constituent of the microsomal reconstitution buffer) concentration on in vitro MDZ hydroxylation was also investigated. Additionally, to verify that the intestinal preparation was adequate for use in studies investigating the induction of CYP3A at the MRNA, protein, and catalytic activity within a single animal, a separate induction study was carried out with the CYP 3A inducer dexamethasone (DEX). A reverse transcription-polymerase chain reaction (RT-PCR) assay and a quantitative Western blotting method were used to reliably detect differences in CYP 3A mRNA and immunoreactivity between DEX- and vehicle (VH)-treated tissues. The in vitro hydroxylation of MDZ evaluated CYP 3A catalytic activity and identified increases in CYP 3A activity caused by DEX in comparison to VH. Collectively, these described techniques provide an experimental model to study xenobiotic induction of rat hepatic and intestinal CYP 3A from the molecular to the catalytic level in individual rats without the need for pooling of tissue.

Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture.

Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 microM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 microM CCK 8 secretion was inhibited by two thirds and at 100 microM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 microM lovastatin. Our results clearly demonstrate that lovastatin at 1 microM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.

Identification of glycine-extended CCK peptides in endocrine cells and modulation of CCK amide and CCK Gly content and secretion from endocrine tumor cells by an inhibitor of amidation.

Immunoreactive glycine-extended CCK peptides are found in normal mouse cerebral cortex and are very abundant in some CCK expressing endocrine tumor cells in culture. The glycine-extended forms in mouse cortex and in cell lines mirror their respective amidated forms. Mouse cerebral cortex, mouse AtT20 and rat WE cells produce mainly CCK 8 amide and CCK 8 Gly. In contrast, mouse intestinal STC-1 cells produce CCK 22 and CCK 8 amide along with forms of CCK Gly which are slightly larger than their respective amidated forms. The CCK 8 Gly-like peptide from AtT20 cells, after desulfation, co-eluted on HPLC with unsulfated CCK 8 Gly. Addition of copper and ascorbate to culture medium of WE cells caused a small increase in secretion of amidated CCK, without changing cellular levels of this peptide. Treatment with the amidation inhibitor diethyldithiocarbamate greatly decreased cellular content and secretion of CCK amide while it increased cellular content and secretion of CCK Gly. These results provide further evidence that glycine-extended CCK peptides are the immediate precursors of amidated CCK peptides.

Prohormone convertase 2 is necessary for the formation of cholecystokinin-22, but not cholecystokinin-8, in RIN5F and STC-1 cells.

Two endocrine tumor cell lines from pancreas (RIN5F) and intestine (STC-1) express cholecystokinin (CCK) messenger RNA and are able to posttranslationally process pro-CCK to CCK-22 and CCK-8 amide. Both of these forms are also secreted by these cells. Because they make and secrete forms of amidated CCK larger than CCK-8, they represent a model of pro-CCK processing in the gut and allow investigation of possible mechanisms for tissue differences in prohormone processing. Both of these cells express two endoproteases convertase-1 (PC1) also known as PC3 and prohormone convertase-2 (PC2), which may be involved in pro-CCK processing. We have previously shown than inhibition of PC1 expression in these cells using stable expression of antisense messenger RNA caused a significant reduction in cellular content of amidated CCK and caused a selective depletion of CCK-8 with a comparative sparing of CCK-22. We demonstrate here that inhibition of PC2 expression in these cells also caused a large initial decrease in CCK content and produced a selective depletion of CCK-22 and a comparative sparing of CCK-8. These results support both a role for both PC1 and PC2 in pro-CCK processing in these cells and the hypothesis that tissue-specific processing of pro-CCK may be explained by differences in expression or activity of PC1 and PC2.

Prohormone convertase 1 is necessary for the formation of cholecystokinin 8 in Rin5F and STC-1 cells.

Several immortalized cell lines serve as models for procholecystokinin (pro-CCK) processing. Rin5F cells, derived from a rat insulinoma, and STC-1 cells, derived from a murine intestinal tumor, process pro-CCK mainly to amidated CCK 8. Both also make significant quantities of amidated CCK 22, a slightly larger form found in the gut. Many modifications are necessary during pro-CCK processing including cleavages performed by endoproteases, the identities of which are unknown. A candidate endoprotease is prohormone convertase 1 (PC1) also known as PC3, a Ca2+-dependent serine endoprotease of the subtilisin family. Constitutive expression of antisense PC1 message in stably transfected Rin5F cells resulted in a significant reduction of the cellular content of CCK 8 as measured by radioimmunoassay. Several affected cell lines displayed about 80% reduction in CCK content in early passages after transfection. Expression of antisense PC1 message in these cell lines resulted in a selective depletion of CCK 8 and a comparative sparing of CCK 22. The induction of antisense PC1 message within a single subclone of Rin5F cells using the Lac Switch system also resulted in a significant inhibition of CCK content. Expression of antisense PC1 message in a stably transfected STC-1 cell line also resulted in a decrease in CCK content and in PC1 protein expression, and the specific depletion of CCK 8 with comparative sparing of CCK 22. These observations support the hypothesis that PC1 is necessary for pro-CCK processing in Rin5F and STC-1 cells and suggests a role for PC1 endoprotease in the biosynthesis of CCK 8 in vivo.

Cholecystokinin (CCK) release from rat brain slices in vitro is enhanced by agents which elevate intracellular cAMP.

Cholecystokinin (CCK) release from rat brain slices in vitro is enhanced by agents which elevate intracellular cAMP. Treatment of several CCK expressing tumor cells in culture with agents which increase intracellular cAMP increased secretion of CCK. The possibility that elevation of cAMP also alters potassium-evoked release of CCK from rat brain slices incubated in vitro was also investigated. Treatment of slices of rat prefrontal cortex and caudate-putamen with 5 microM forskolin plus 0.5 mM IBMX (3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor) caused a significant elevation of potassium-evoked CCK release. That cAMP can increase CCK release in intact brains slices as well as endocrine tumor cells from thyroid, pancreas, pituitary and intestine suggests that cAMP is a common intracellular mediator for the regulation of CCK release.

Expression of antisense PC1 in stably transfected RIN5F cells significantly reduces CCK 8 biosynthesis.

The subtilisin-like endoprotease PC1 (PC3) has been implicated in the processing of a number of prohormones. To evaluate whether PC1 may be important for the processing of pro CCK to CCK 8, stable cell lines expressing a portion of the PC1 cDNA in the antisense orientation were established from RIN5F cells. These cells express CCK mRNA, produce and display regulated secretion of CCK 8. One of the clones, R1E8, expresses antisense PC1 mRNA as determined by reverse transcriptase-PCR (RT-PCR) and contains a significantly reduced level of PC1 protein. As compared to both RIN5F and RIN5F control cells (transfected with the expression plasmid containing no antisense message), R1E8 contains only about 30% cell content of CCK 8. These results suggest that PC1 may be important for the processing of CCK 8 from pro CCK.

Inhibition of pro-cholecystokinin (CCK) sulfation by treatment with sodium chlorate alters its processing and decreases cellular content and secretion of CCK 8.

Pro-cholecystokinin (CCK) has three sulfated tyrosine residues. Sulfation of the tyrosine residue in CCK 8 is known to be important for its activity at CCK A receptors. The role of these sulfated tyrosines in the sorting and processing of pro-CCK was examined by treatment of CCK-secreting rat thyroid medullary carcinoma cells with 10 nM sodium chlorate (a non-toxic inhibitor of tyrosine sulfation). This treatment caused a 50% decrease in the cellular content of immunoreactive CCK and an 80% decrease in its secretion. Sephadex G-50 chromatography of cellular extracts and culture media showed a selective depletion of CCK 8. There was a comparative sparing of CCK 33 and larger molecular forms in cellular extracts which was not observed in the media. These results suggest that the sulfation of the tyrosines of pro-CCK is clearly important for the correct sorting and/or processing of pro-CCK. The pattern of immunoreactive CCK peptides seen with chlorate treatment is consistent with the substrate specificity of a recently identified putative CCK cleaving enzyme and suggests that unsulfated pro-CCK is not efficiently processed to CCK 8 in vivo. The large decrease in CCK content and secretion observed with sodium chlorate may also be due to inefficient sorting of unsulfated pro-CCK into secretory vesicles.

Processing, release and metabolism of cholecystokinin in SK-N-MCIXC cells.

The human cholinergic neuroepithelioma cell line SK-N-MCIXC, which expresses high levels of cholecystokinin (CCK) mRNA and secretes intact CCK into the media, was used to examine CCK processing and metabolism. Our data provide evidence for the existence of specific candidate processing enzymes in SK-N-MCIXC cells which may be involved in processing proCCK in the brain and indicate that SK-N-MCIXC cells provide a model system for studying the regulation of these enzymes. mRNAs for the intracellular processing enzymes, prohormone convertase 1 (PC1), PC2 and furin were present in SK-N-MCIXC cells. PC1 and/or PC2 and/or furin may cleave at the dibasic amino acid pairs Arg-Arg at the C-terminal part of proCCK, and Arg-X-X-Arg at the N-terminal of the CCK-58 sequence in proCCK. The SK-N-MCIXC cell line demonstrated spontaneous and regulated release of CCK and large amounts of CCK-precursors, as measured with region specific radioimmunoassays coupled to high performance liquid chromatography. Storage granules containing glycine-extended CCK were shown in SK-N-MCIXC cells using indirect immunofluorescence. The extracellularly localized CCK-metabolizing enzyme, neutral endopeptidase 24.11 (EC 3.4.24.11), was present in membranes from both SK-N-MCIXC cells and in intact slices of rat cerebral cortex. The rat cerebral cortex is a brain region known to be rich in CCK. The SK-N-MCIXC cell line provides an in vitro model to study the regulation of CCK synthesis and metabolism in neuronal systems since it contains the storage granules, mRNA, intact peptide, and complement of enzymes necessary for biosynthesis and metabolism of CCK.

Cholecystokinin concentrations and peptide immunoreactivity in the intact and deafferented medullary dorsal horn of the rat.

To further address the hypothesis that cholecystokinin (CCK) in the medullary dorsal horn (MDH) arises from intrinsic or higher-order neurons, CCK-8-specific radioimmunoassay (RIA) and immunohistochemical (IHC) experiments were carried out in adult rats after trigeminal tractotomy. RIA of punches from deafferented superficial layers of the MDH revealed no significant change in CCK levels vs. the control right side. In this same area, IHC revealed modest reductions in CCK, gastrin, and substance P staining. Calcitonin gene-related peptide (CGRP) staining was reduced substantially. Gastrin immunoreactive cell bodies, present normally in inner lamina II, were reduced in number. RIA and IHC methods were also used to assess MDH CCK concentrations in adult rats subjected to left infraorbital nerve section at birth. The left medulla contained significantly higher levels of CCK than the control right medulla (1.27 +/- 0.19 vs. 0.97 +/- 0.11 ng/mg protein). IHC revealed a dense band of CCK-like staining in laminae I and II ipsi- and contralateral to the lesion. Thus, neonatal deafferentation elevates medullary CCK. To determine if the neonatal lesion-induced increase in medullary CCK is due to primary afferent or higher-order reorganization, RIA and IHC experiments were run after infraorbital nerve section at birth and trigeminal tractotomy in adulthood. RIA revealed no significant change in CCK levels caudal to the tractotomy, although they were higher than control levels in 9 of 12 cases. IHC revealed modest reductions in CCK, substance P, and gastrin staining that resembled the reductions observed in tractotomy-alone cases. These data suggest that 1) most MDH CCK is of non-primary afferent origin, 2) gastrin immunoreactivity in layer II probably originates in CCK-containing cells intrinsic to layer II, the expression of which is dependent upon trigeminal primary afferent input, 3) neonatal V deafferentation induces increased CCK in the superficial MDH, reflecting reorganized intrinsic or higher-order inputs, and 4) higher-order substance P in the MDH is robust.

Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells in culture.

The release of peptides immunoreactive for a synthetic peptide (V-9-M) contained in the amino-terminal of pro-CCK was examined. The potassium-evoked release of V-9-M immunoreactive peptides from rat cerebral cortical slices in vitro was calcium dependent. Cholecystokinin-secreting rat medullary thyroid carcinoma cells also secreted significant quantities of these peptides. Sephadex column chromatography of the release media from slices and cells showed two V-9-M immunoreactive peptides, one larger and one smaller than V-9-M itself. Previous behavioral studies have suggested that V-9-M has a distinct neuropharmacological profile. These results demonstrate that V-9-M-like peptides are released along with CCK-8 and are consistent with the hypothesis that V-9-M-like peptides may be neurotransmitters or neuromodulators or may be involved in the sorting or transport of CCK-8.

CCK mRNA expression, pro-CCK processing, and regulated secretion of immunoreactive CCK peptides by rat insulinoma (RIN 5F) and mouse pituitary tumor (AtT-20) cells in culture.

The rat insulinoma RIN 5F and the mouse pituitary AtT-20 cell line, which are known to express several biologically active peptides, were found to express CCK mRNA, to correctly process, and to release immunoreactive cholecystokinin (CCK) peptides. They expressed low levels of these peptides (about 0.4 and 0.2 ng/mg protein, respectively) and both cell lines processed pro-CCK to a form which co-eluted with CCK 8 sulfate on Sephadex gel filtration chromatography and HPLC. The major CCK 8 immunoreactive peptide which they secreted co-eluted with CCK 8 on Sephadex G-50 chromatography. The secretion of CCK from both cell lines was significantly enhanced by treatment for 24 h with forskolin + IBMX (3-isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor). This treatment also doubled the CCK content of the AtT-20 cells. It appears that the ability of different endocrine tumor cells to express and process CCK is not as uncommon as previously thought. These cells should be useful for future studies of CCK expression, processing, and regulation of secretion.

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.

Neuropeptide content and connectivity of the rat claustrum.

The rat claustrum has a homogeneous distribution of the neuropeptides somatostatin (SOM), cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) along its rostrocaudal axis. In general, neuropeptide levels are comparable to those of overlying pyriform cortex. Visualization of mRNA encoding SOM, CCK and VIP in cell bodies of the claustrum by in situ hybridization histochemistry demonstrates that all 3 neuropeptides are contained in intrinsic claustral neurons. Mid-coronal section of the claustrum itself, or interruption of potential rostral, caudal or medial connections between the claustrum and the rest of the brain did not significantly alter levels of VIP, SOM or CCK in claustrum, cerebral cortex, or basal ganglia. Isolation of the claustrum from the cerebral cortex immediately dorsal to it along its rostrocaudal aspect caused no change in peptide levels in claustrum indicating that VIP, SOM and CCK projections to claustrum do not arrive from dorsal cortical areas. Transection of the external capsule above the claustrum caused a 50-100% elevation of all 3 peptides on the contralateral side of the lesion, suggesting that VIP, SOM and CCK synthesis and/or release within the claustrum may be regulated by projections from the contralateral side. VIP, SOM and CCK are candidates for neurotransmitters contained in neurons whose cell bodies are within the claustrum and possibly also immediately overlying lateral neocortex, and have their terminals mainly within the claustrum itself.

Role of peptide histidine isoleucine in relaxation of cat lower esophageal sphincter.

Vasoactive intestinal peptide (VIP) is a candidate as an inhibitory neurotransmitter mediating relaxation of the lower esophageal sphincter (LES) because VIP antiserum reduces LES relaxation in response to neural stimulation. Vasoactive intestinal peptide antiserum, however, does not completely block LES relaxation. Thus it is possible that other neurotransmitters may be involved. Peptide histidine isoleucine has structural homologies with VIP, is synthesized with VIP from a common precursor protein, coexists in some nerve cells, and is coproduced with VIP in some tumors. In numerous organ systems VIP and peptide histidine isoleucine (PHI) produce similar effects, with PHI being less potent than VIP by approximately one log number. In the LES both VIP and PHI produce tetrodotoxin-resistant dose-dependent relaxation, with PHI being almost equipotent with VIP. We therefore tested the hypothesis that PHI may be a second neurotransmitter, partly responsible for relaxation of the cat LES, by using a highly specific rabbit PHI antiserum that exhibits minimal cross-binding with VIP, secretin, and glucagon. In 3 animals, LES and brain tissue were extracted in 0.1 N HCl and assayed with a PHI radioimmunoassay. The antiserum cross-reacted with cat brain and LES showing PHI concentrations greater than 100 ng/g, with the LES containing equal or greater concentrations of PHI than brain tissue. In other animals consecutive LES circular muscle strips were cut, mounted in 1-ml muscle chambers, and stimulated with 6-s square-wave trains of 0.1-, 0.2-, 0.4-, and 0.8-ms pulses at 1, 2, and 5 Hz. These parameters produced relaxation that was completely blocked by tetrodotoxin, and reduced by VIP antiserum, but not affected by adrenergic or cholinergic receptor antagonists. Some strips were incubated in 5% or 10% PHI antiserum, whereas others were incubated in the same concentration of preimmunization serum from the same animal. Incubation in normal serum did not significantly affect relaxation, whereas in the antiserum-treated strips, LES relaxation was reduced by a significant amount (20%-30%) at all parameters of stimulation tested. Incubation in antiserum however had no effect on relaxation induced by VIP (10(-8)-10(-6) M). These data suggest that PHI may play a role in LES relaxation induced by electrical stimulation.

Establishment of a cholecystokinin-producing rat medullary thyroid carcinoma cell line.

A rat medullary thyroid carcinoma, which was previously shown to produce high levels of immunoreactive cholecystokinin (CCK), was used to establish a stable cell line. Transplantable tumors were subjected to four series of alternate in vitro and in vivo passages. Cells were prepared from the fourth series of tumors under serum-free medium conditions that prevent fibroblast growth. Subcloning of these cells yielded several propagatable clonal cell lines. One cell line with immunoreactive CCK-8 production was selected for further studies. This high CCK cell line, WE4/2, produces and secretes a CCK-immunoreactive product that coelutes with synthetic CCK-8 sulfate during Sephadex chromatography and HPLC. Northern analysis with a rat CCK cDNA revealed that the cultured cells produce a CCK RNA the same size and with the same 5' end as that previously reported for brain and intestines. In addition, a recombinant plasmid containing about 800 basepairs of 5' flanking sequence of the rat CCK gene linked to the coding sequence of the bacterial chloramphenicol acetyltransferase gene elicited a high level of chloramphenicol acetyltransferase activity when transfected into the WE4/2 cell line. Therefore, the WE4/2 cell line provides a model system for studying CCK gene expression and biosynthesis.