Biomedical subjects

M C Beinfeld

Publications and source records attributed to M C Beinfeld.

At least 37 records · Page 2Linked to original sources

PubMed1997

CCK biosynthesis and processing: recent progress and future challenges.

The peptide cholecystokinin (CCK), like other peptides which pass through the regulated secretory pathway, undergoes a number of post-translational modifications during its biosynthesis including tyrosine sulfation, endoproteolytic cleavage, and trimming by carboxypeptidases. This minireview summarizes what is known about this process in endocrine cells and in the Cpe(fat)/Cpe(fat) mouse and points out what challenges remain for future research.

Animals↗

PubMed1997

Expression of rat pro cholecystokinin (CCK) in bacteria and in insect cells infected with recombinant baculovirus.

Neuropeptide prohormones are generally not abundant in nature as they exits to be processed and contain sites which could be cleaved by a number of cellular proteases. In order to study the processing of prohormones in vitro it is necessary to produce them in quantity. Finding an expression system which produces intact prohormone has been a matter of trial and error. We report that intact rat pro CCK was produced with an amino-terminal His-Tag in e. coli, and it was secreted from sf9 and other insect cells infected with a recombinant baculovirus vector. The bacteria contained about 0.1 micrograms pro CCK/ml of cells. The High 5 insect cells produced 4.3 micrograms/ml medium (as determined by RIA), 10 times as much as sf9 or sf21. Using a combination of ion exchange, gel filtration and HPLC, the insect cell protein was purified about 150 fold with a recovery of about 16%. The secreted insect cell pro CCK is tyrosine sulfated like its mammalian equivalent. Using these expression systems it is possible to produce significant (microgram to mg) quantities of pro CCK for immunologic, enzymatic and structural studies.

Animals↗

PubMed1997

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.

Cholecystokinin↗

PubMed1997

Cholecystokinin (CCK) levels are greatly reduced in the brains but not the duodenums of Cpe(fat)/Cpe(fat) mice: a regional difference in the involvement of carboxypeptidase E (Cpe) in pro-CCK processing.

In order to assess the possible role of carboxypeptidase E (Cpe) in pro-CCK processing, tissues from the Cpe(fat)/Cpe(fat) mice were analyzed for CCK content and molecular forms using specific RIAs directed against different portions of the prohormone. Levels of amidated CCK were decreased by about 74% in whole brain of Cpe(fat)/Cpe(fat) mice in comparison to control mice, while levels of amidated CCK in intestine were only reduced by about 36%. In contrast, using an antiserum specific for CCK Gly Arg Arg, Cpe(fat)/Cpe(fat) mice brain had about 13-fold higher levels of this peptide relative to controls, while levels were identical in mutant and control duodenal tissue. This study demonstrates a regional difference in the involvement of Cpe in pro-CCK processing. The accumulation of CCK Gly Arg Arg in Cpe(fat)/Cpe(fat) brains provides definitive proof that the dibasic cleavage of the carboxyl terminus of pro CCK occurs on the carboxyl terminal of the dibasic, between the Arg and Ser as well as confirming that amidated CCK 8 in brain originates from CCK 8 Gly Arg Arg rather than from larger amidated peptides like CCK 22 or CCK 33. The Cpe(fat)/Cpe(fat) mouse phenotype obviously involves multiple endocrine defects, however, it is tempting to speculate that this severe CNS deficiency in CCK 8 may be related to the adult-onset obesity seen in this mutant mouse.

Animals↗

PubMed1996

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.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3.

The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively.

Adrenocorticotropic Hormone↗

PubMed1995

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1993

Pharmacological characterization of the release of neuropeptide Y-like immunoreactivity from the rat hypothalamus.

Radioimmunoassay for NPY and detection by HPLC-EC were used to detect the concurrent release of norepinephrine and NPY from slices of the rat hypothalamus and to examine the modulation by adrenoceptors of the release of this neuropeptide. Basal and potassium-evoked (56 mM K+) release of both compounds were easily measured, with evoked release occurring in a calcium-dependent manner. The effect of the alpha 2-adrenoceptor agonist clonidine, the antagonists prazosin (alpha 1 selective) and yohimbine (alpha 2 selective) and the beta-adrenoceptor antagonist propranolol were all shown to modulate the evoked release of NPY. The alpha 2 agonist clonidine decreased evoked release of NPY, while the alpha 2 antagonist yohimbine increased the potassium-evoked release. Prazosin decreased both the basal and potassium-evoked release of NPY. Propranolol had the most profound effect on release of NPY, causing a significant decrease in basal release and a large decrease in the potassium-evoked release of NPY from slices of hypothalamus.

Animals↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

CSF cholecystokinin octapeptide in patients with bulimia nervosa and in normal comparison subjects.

Cholecystokinin octapeptide (CCK-8) appears to modulate appetitive behavior, and in rodents, anxiety-related behavior. The authors studied CCK-8 in patients with bulimia nervosa. CSF concentrations of CCK-8 were measured in 11 drug-free female patients with DSM-III-R-defined bulimia nervosa and in 16 normal subjects. The bulimic patients had significantly lower levels of CCK-8 than the comparison subjects. CCK-8 concentrations were inversely correlated with scores on the anger-hostility, anxiety, and interpersonal sensitivity subscales of the SCL-90-R. They were not significantly correlated with age, percentage of standardized average body weight, or mean weekly frequency of binge eating or vomiting. The results indicate that central CCK-8 abnormalities may play a role in the pathophysiology of bulimia nervosa.

Adult↗

PubMed1992

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.

Animals↗

PubMed1992

Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.

Amino Acid Sequence↗

PubMed1992

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.

1-Methyl-3-isobutylxanthine↗

PubMed1992

CSF cholecystokinin concentrations in patients with panic disorder and in normal comparison subjects.

Cholecystokinin concentrations in the CSF of 25 patients with panic disorder and 16 normal comparison subjects were ascertained by radioimmunoassay. The patients with panic disorder had significantly lower CSF concentrations of cholecystokinin, which may reflect increased CNS cholecystokinin receptor sensitivity, reduced numbers of receptors, or a compensatory reduction in cholecystokinin octapeptide secondary to theoretically increased central cholecystokinin tetrapeptide activity.

Adolescent↗

PubMed1992

Cholecystokinin release from the rat caudate-putamen, cortex and hippocampus is increased by activation of the D1 dopamine receptor.

Studies were undertaken to examine the role of dopamine in the regulation of cholecystokinin (CCK) release in slices of the rat caudate-putamen (CP), a region where both neuroactive substances are abundant yet not extensively colocalized. It was found that dopamine, acting through a D1 dopamine receptor, is a major factor regulating CCK release in this tissue. Haloperidol and the selective D1 antagonist, SKF 83566, but not the D2 antagonist, sulpiride, inhibited potassium-evoked release from CP slices. Apomorphine enhanced both basal and potassium-evoked release, an effect which was reversed by (+/-)SKF 83566, but not by sulpiride. The D1 agonist, (+)SKF 38393, displayed agonist and antagonist activity in this system, whereas the D2 agonist, quinpirole, had no effect. Depletion of endogenous dopamine by pretreatment of animals with alpha-methyl-para-tyrosine decreased potassium-induced release by 45%, and in these animals, haloperidol no longer inhibited release. These dopaminergic agents altered CCK release in cortex and hippocampus in a similar fashion, although those tissues were less sensitive to their effects. These results suggest that dopamine, acting through D1 receptors, increases CCK release in CP, cortex and hippocampus. The results of previous studies showing that phorbol esters increase CCK release and that occupation of D1 receptors increases phosphoinositol turnover make it possible to hypothesize that dopamine, acting through D1 receptors, is increasing CCK release by increasing phosphoinositol turnover. Further studies will be required to verify this hypothesis and to determine whether dopamine is acting directly or indirectly to modulate CCK release.

Amphetamine↗

PubMed1991

Concentration of cholecystokinin in cerebrospinal fluid is decreased in psychosis: relationship to symptoms and drug response.

1. Cholecystokinin (CCK) is a neuropeptide which is co-localized within some mesolimbic and mesocortical dopamine neurons. 2. CCK resembles an antipsychotic drug in some pharmacological and behavioral tests. 3. Levels of CCK in the cerebrospinal fluid (CSF) are reduced in eleven drug-free schizophrenics in comparison with six controls. 4. Schizophrenic males have lower CSF CCK levels than females. 5. Rapidity of antipsychotic response to haloperidol appeared to be inversely related to drug-free baseline CSF CCK levels.

Adult↗

PubMed1990

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.

Animals↗