Cholecystokinin Levels in Prohormone Convertase 2 Knock-out Mouse Brain Regions Reveal a Complex Phenotype of Region-specific Alterations

Abstract

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA. Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA. Cholecystokinin (CCK) 2The abbreviations used are: CCK, cholecystokinin; CA1-CA4, CA1-CA4 subfields of ammon's horn of the hippocampal formation; HPLC, high pressure liquid chromatography; PFC, prefrontal cortex; PC, prohormone convertase; TPCK, l-(tosylamido-2-phenyl)ethyl chloromethyl ketone; QPCR, quantitative polymerase chain reaction; VTA/SN, ventral tegmental area, substantia nigra; RIA, radioimmunoassay. is one of the most abundant and widely distributed neuropeptides in mammalian brain (1.Beinfeld M.C. Meyer D.K. Eskay R.L. Jensen R.T. Brownstein M.J. Brain Res. 1981; 212: 51-57Crossref PubMed Scopus (483) Google Scholar). Pre-pro-CCK has a molecular weight of 12,826, and the active CCK 8 sequence is flanked by a short C-terminal segment and a long N-terminal segment. During its post-translational processing, pro-CCK is tyrosine-sulfated and cleaved at multiple sites by endoproteases. C-terminal arginines are removed by a carboxypeptidase, and its C-terminal glycine is converted to an amide (2.Beinfeld M.C. Life Sci. 2003; 72: 747-757Crossref PubMed Scopus (43) Google Scholar). The sequence of mouse pro-CCK and its cleavage sites are shown in Fig. 1. The cleavage of pro-CCK occurs exclusively at single or double basic residues. The best candidates for these enzymes are the prohormone convertases (PC). They are responsible for processing a wide variety of peptide hormones, growth factors, neuropeptides, and bacterial and viral virulence factors. Among them, the most likely candidates for CCK processing are PC1 (3.Seidah N.G. Marcinkiewicz M. Benjannet S. Gaspar I. Beaubien G. Mattei M.G. Lazure C. Mbikay M. Chretien M. Mol. Endocrinol. 1991; 5: 111-122Crossref PubMed Scopus (406) Google Scholar), PC2 (4.Smeekens S.P. Steiner D.F. J. Biol. Chem. 1990; 265: 2997-3000Abstract Full Text PDF PubMed Google Scholar), and PC5 (5.Nakagawa T. Hosaka M. Torii S. Watanabe T. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1993; 113: 132-135Crossref PubMed Scopus (183) Google Scholar, 6.Lusson J. Vieau D. Hamelin J. Day R. Chretien M. Seidah N.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6691-6695Crossref PubMed Scopus (234) Google Scholar). Prohormone convertases have their own unique distribution in rat brain (7.Villeneuve P. Lafortune L. Seidah N.G. Kitabgi P. Beaudet A. J. Comp. Neurol. 2000; 424: 461-475Crossref PubMed Scopus (26) Google Scholar, 8.Villeneuve P. Seidah N.G. Beaudet A. Neuroscience. 1999; 92: 641-654Crossref PubMed Scopus (23) Google Scholar, 9.Winsky-Sommerer R. Benjannet S. Rovere C. Barbero P. Seidah N.G. Epelbaum J. Dournaud P. J. Comp. Neurol. 2000; 424: 439-460Crossref PubMed Scopus (49) Google Scholar, 10.Schafer M.K. Day R. Cullinan W.E. Chretien M. Seidah N.G. Watson S.J. J. Neurosci. 1993; 13: 1258-1279Crossref PubMed Google Scholar, 11.Dong W.J. Seidel B. Marcinkiewicz M. Chrétien M. Seidah N.G. Day R. J. Neurosci. 1997; 17: 563-575Crossref PubMed Google Scholar, 12.Dong W. Marcinkiewicz M. Vieau D. Chrétien M. Seidah N.G. Day R. J. Neurosci. 1995; 15: 1778-1796Crossref PubMed Google Scholar) and are often found in the same areas that express CCK. They are widely co-localized with CCK in specific neuronal populations in rat brain (13.Cain B. Connolly K. Blum A. Vishnuvardhan D. Marchand J.E. Beinfeld M.C. J. Comp. Neurol. 2003; 467: 307-325Crossref PubMed Scopus (30) Google Scholar, 14.Cain B.M. Connolly K. Blum A.C. Vishnuvardhan D. Marchand J.E. Zhu X. Steiner D.F. Beinfeld M.C. J. Neurochem. 2004; 89: 307-313Crossref PubMed Scopus (19) Google Scholar). The most dramatic example of co-localization is the pyramidal cell layer of the CA1-CA4 subfield of hippocampus where all three enzymes are present in at least 80% of CCK neurons (13.Cain B. Connolly K. Blum A. Vishnuvardhan D. Marchand J.E. Beinfeld M.C. J. Comp. Neurol. 2003; 467: 307-325Crossref PubMed Scopus (30) Google Scholar). PCs are also found in a number of endocrine and neuronal tumor cell lines, including those which naturally express CCK mRNA and are capable of cleaving pro-CCK into appropriate products that can be further acted on by carboxypeptidase and amidation enzymes to yield amidated CCK8 or CCK22 (15.Beinfeld M.C. Neuropeptides. 1992; 22: 213-217Crossref PubMed Scopus (29) Google Scholar, 16.Haun R.S. Beinfeld M.C. Roos B.A. Dixon J.E. Endocrinology. 1989; 125: 850-856Crossref PubMed Scopus (29) Google Scholar, 17.Yoon J.Y. Beinfeld M.C. Endocrine. 1994; 2: 973-977Google Scholar). A number of studies support the hypothesis that PC1, PC2, and PC5 are all capable of appropriately cleaving pro-CCK (2.Beinfeld M.C. Life Sci. 2003; 72: 747-757Crossref PubMed Scopus (43) Google Scholar). Mice lacking PC1 or PC2 (18.Furuta M. Yano H. Zhou A. Rouillé Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Crossref PubMed Scopus (354) Google Scholar, 19.Zhu X. Zhou A. Dey A. Norrbom C. Carroll R. Zhang C. Laurent V. Lindberg I. Ugleholdt R. Holst J.J. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10293-10298Crossref PubMed Scopus (280) Google Scholar) have defects in the processing of a wide variety of peptide hormones and neuropeptides. In PC1 or PC2 knock-out mice, CCK levels were reduced in specific brain regions relative to wild-type mice (14.Cain B.M. Connolly K. Blum A.C. Vishnuvardhan D. Marchand J.E. Zhu X. Steiner D.F. Beinfeld M.C. J. Neurochem. 2004; 89: 307-313Crossref PubMed Scopus (19) Google Scholar, 20.Vishnuvardhan D. Connolly K. Cain B. Beinfeld M.C. Biochem. Biophys. Res. Commun. 2000; 273: 188-191Crossref PubMed Scopus (27) Google Scholar, 21.Rehfeld J.F. Lindberg I. Friis-Hansen L. J. Neurochem. 2002; 83: 1329-1337Crossref PubMed Scopus (22) Google Scholar). The reduction in CCK levels varied in different brain regions and correlated well with their co-localization with CCK at the mRNA level. In no tissue was CCK undetectable; other enzymes appear to exist that produce some CCK. The current study was performed to better correlate the known distribution of CCK and PC2 with the levels of CCK in specific brain regions of PC2 knock-out and wild-type mice. Tissue was also harvested for RNA to perform quantitative real-time polymerase chain reaction (QPCR) measurements of CCK, PC1, PC2, and PC5 mRNA abundance to better understand the compensatory changes that occur in the absence of PC2. Mice—Animal experiments were approved by the Tufts University Animal Care and Use Committee and conform to National Institutes of Health guidelines. Male and female PC2 knock-out (B6;129-Pcsk2tm1Dfs) and wild-type (B6129SF2/J) mice, ages 9–13 weeks at sacrifice, were obtained from Jackson Laboratories (Bar Harbor, ME). Their genotype was confirmed using tail DNA, as previously described (18.Furuta M. Yano H. Zhou A. Rouillé Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Crossref PubMed Scopus (354) Google Scholar). Mouse Sacrifice and Dissection—Mice were euthanized with 9:1 mixture of ketamine/xylazine (about 0.15 ml, intraperitoneal) and decapitated. The brains were removed quickly and the olfactory bulbs and hypothalamus were free hand-dissected. The cerebellum was removed and discarded, and the pons, medulla, and spinal cord were free hand-dissected. The remaining forebrain was sectioned on wet ice into 1-mm coronal slices using a mouse brain mold and razor blades. The brain regions to be analyzed were free hand-dissected from these slices. The hippocampal tissue for RIA was dissected from the anterior portion of this region, which has the highest density of CCK-positive cells (13.Cain B. Connolly K. Blum A. Vishnuvardhan D. Marchand J.E. Beinfeld M.C. J. Comp. Neurol. 2003; 467: 307-325Crossref PubMed Scopus (30) Google Scholar) and included hippocampal subfields CA1-CA4 and dentate gyrus. For QPCR measurements, the whole hippocampus was removed. For RNA extractions, the mouse brains were rapidly dissected on wet ice under RNase-free conditions. They were free hand-dissected without being sectioned. All tissues were frozen immediately in Eppendorf tubes on dry ice and stored at -80 °C until extraction. Tissue Extraction for RIA—For RIA, tissues were sonicated in 0.1 n HCl, and an aliquot was removed for total protein determination by the BCA method (Pierce). CCK RIA—CCK was measured using an RIA as previously described (1.Beinfeld M.C. Meyer D.K. Eskay R.L. Jensen R.T. Brownstein M.J. Brain Res. 1981; 212: 51-57Crossref PubMed Scopus (483) Google Scholar). The R5 antiserum cross-reacts equally well with sulfated CCK8, CCK12, CCK22, CCK33, and unsulfated gastrin 17. It has about a 10-fold lower cross-reactivity with nonsulfated CCK peptides. The difference in RIA data between groups was determined with an unpaired Student's t test (Prizm graphics), with significance set at p < .05. CCK-Gly RIA—CCK-Gly-like immunoreactive peptides were detected with an antiserum that detects gastrin 13-Gly and CCK8-Gly. These have very low (<0.1%) cross-reactivity with CCK8 Gly-Arg or CCK8 Gly-Arg-Arg and use 125I-gastrin13 Gly as its tracer and CCK8 Gly as its standard (22.Cain B.M. Vishnuvardhan D. Beinfeld M.C. Peptides. 2001; 22: 1271-1277Crossref PubMed Scopus (24) Google Scholar, 23.Beinfeld M.C. Perloff M.D. Venkatakrishnan K. Peptides. 1998; 19: 1393-1398Crossref PubMed Scopus (12) Google Scholar). Trypsin and Carboxypeptidase Treatment—Both enzymes were purchased from Worthington (Lakewood, NJ). Extracts were neutralized with one-fifth volume of 1 m sodium phosphate, pH 7.5, and an equal volume of TPCK trypsin (2 mg/ml) was added. After 30 min at 20 °C, the samples were incubated at 100 °C for 10 min. They were then treated with carboxypeptidase B (5.6 μg per microliter sample) for 30 min at 20 °C and for 10 min at 100 °C prior to the CCK-Gly RIA. RNA Extraction—Frozen tissues were homogenized with an Ultraturrax homogenizer in 1 ml of TRIzol reagent (Invitrogen). Two hundred microliters of chloroform was added, the samples were shaken at room temperature for 15 s and at for 15 min at To the ml of was added, and the RNA was further with The RNA were determined using a The was between and and was using 2 μg of total RNA treated for 10 min at room temperature with in a reaction mixture using the were performed in a reaction mixture volume of with 5 of were performed using sequence and the were using the The expression of CCK, PC1, PC2, and PC5 were with expression of mRNA. Mouse CCK were and Mouse PC1 were and and Mouse PC2 were from The for were and for were °C for 2 °C 10 min and then for between °C for 30 s and °C for 1 min. The of CCK, PC1, PC2, and PC5 was from the of the The of the was set to and the was from the the The difference in expression between and was as as described 2001; PubMed Scopus Google Scholar). from male and female knock-out and wild-type mice were with the volume of knock-out hippocampus with wild type. The were by and from was with a using a The was with a of to in at 1 were ml of was in a in RIA tubes for the CCK RIA. CCK8 was obtained from of amidated CCK were measured in different brain regions from male and female wild-type and PC2 knock-out mice CCK levels were reduced in hippocampus and elevated in prefrontal cortex and cortex in male and female knock-out mice relative to wild type. CCK levels of female knock-out mice were more likely to from female wild than the male from their wild This was in the olfactory bulb, ventral tegmental and In the male were more affected than the in the and levels in brain regions of male and female wild-type and PC2 knock-out wild wild as number of is in as The number of is in p in a data from male and female knock-out mice were and with data from wild-type mice, changes were CCK levels were in hippocampus, thalamus, septum, VTA/SN, and pons of knock-out mice. CCK levels were increased in olfactory bulb, PFC, cerebral cortex, and forebrain in knock-out mice. CCK levels were the same as wild in medulla, and spinal cord in knock-out levels in brain regions of wild-type and PC2 knock-out as number of is in 0.15 as The number of is in p in a To better understand CCK is increased in and in hippocampus, levels of glycine CCK which are the of amidated CCK were was a in these peptides in knock-out PFC, whereas the levels in the hippocampus were the same in knock-out and wild CCK-Gly levels were also measured in these had treated with trypsin and carboxypeptidase B to all CCK into This increased the levels of CCK-Gly peptides in all In the PFC, levels of CCK-Gly were higher in the whereas the levels in the hippocampus of those groups were the same Gly levels in brain regions of wild-type and PC2 knock-out as number of is in the with trypsin by carboxypeptidase B prior to CCK Gly with trypsin by carboxypeptidase B prior to CCK Gly as The number of is in the p with trypsin by carboxypeptidase B prior to CCK Gly RIA in a To better understand peptide levels are to the mRNA expression levels for CCK, PC1, PC2, and QPCR was performed on RNA from cerebral cortex, hippocampus, spinal and olfactory bulbs of mice of and The are in and CCK mRNA levels were in the cerebral cortex of knock-out mice relative to wild type, mRNA expression levels were not different from wild in knock-out hippocampus, or spinal PC1 mRNA levels were also in male and female knock-out cerebral cortex and male knock-out olfactory bulb whereas were in knock-out hippocampus, or spinal PC1 mRNA levels increased in male knock-out PC5 mRNA levels were in male knock-out cerebral cortex, hippocampus, spinal and olfactory bulb relative to wild type. were in male knock-out of CCK, PC1, and PC5 mRNA levels in wild-type and knock-out mouse cerebral cortex, hippocampus, spinal and olfactory as wild of the wild-type is shown in number of is in < < wild < wild wild wild 0.1 < wild wild < wild wild wild wild < wild as The of the wild-type is shown in the number of is in p < in a of the in CCK, PC1, PC2, and PC5 mRNA levels in male and female wild-type and knock-out as wild of the female is in number of is in wild < < not < < wild wild 0.15 < < < wild wild < < wild wild < < < wild wild wild wild < < < as The of the female is in The number of is in p < not detected in a The QPCR data were further analyzed to examine in the expression of CCK, PC1, PC2 and PC5 the in were higher than this was more often with CCK and These were equally distributed between wild and In one CCK was higher in than The molecular of CCK in and hippocampus were by and measured with the CCK RIA. The detected with CCK8 in wild-type and knock-out tissue from tissues The of the than CCK8 in the is not It not with sulfated CCK12, CCK22, or It is to be CCK8, which on the the volume M.C. Jensen R.T. Brownstein M.J. J. Scopus Google Scholar). It appear to be more in the knock-out In the absence of PC2, CCK levels were reduced in hippocampus, VTA/SN, septum, thalamus, and This is with as PC2 is co-localized with CCK in hippocampus, VTA/SN, and (13.Cain B. Connolly K. Blum A. Vishnuvardhan D. Marchand J.E. Beinfeld M.C. J. Comp. Neurol. 2003; 467: 307-325Crossref PubMed Scopus (30) Google Scholar). The a CCK from the hippocampus G. Beinfeld M.C. Peptides. PubMed Scopus (24) Google Scholar), the of CCK in the the in the described in D. Connolly K. Cain B. Beinfeld M.C. Biochem. Biophys. Res. Commun. 2000; 273: 188-191Crossref PubMed Scopus (27) Google Scholar, 21.Rehfeld J.F. Lindberg I. Friis-Hansen L. J. Neurochem. 2002; 83: 1329-1337Crossref PubMed Scopus (22) Google Scholar), the loss of PC2 CCK levels in specific brain regions. The of of CCK levels in the and the decrease in forebrain cerebral cortex and in the study D. Connolly K. Cain B. Beinfeld M.C. Biochem. Biophys. Res. Commun. 2000; 273: 188-191Crossref PubMed Scopus (27) Google Scholar) is with the of the current The decrease in CCK levels in whole mouse brain J.F. Lindberg I. Friis-Hansen L. J. Neurochem. 2002; 83: 1329-1337Crossref PubMed Scopus (22) Google Scholar) and in cerebral cortex D. Connolly K. Cain B. Beinfeld M.C. Biochem. Biophys. Res. Commun. 2000; 273: 188-191Crossref PubMed Scopus (27) Google Scholar) is in of the current in of and tissue were It is that CCK is elevated in some brain regions and that other areas where CCK be reduced are CCK levels actually The in glycine CCK peptides in in the current study was also in whole brain in the study J.F. Lindberg I. Friis-Hansen L. J. Neurochem. 2002; 83: 1329-1337Crossref PubMed Scopus (22) Google Scholar). Female knock-out mice were more affected by the loss of PC2 than male mice. some female mice of had higher levels of CCK, PC1, PC2, and PC5 mRNA than These the of the of the loss of in The loss of PC2 not the of CCK by cerebral cortex or pro-CCK in the absence of PC2 to produce the same It is that PC2 is important for of CCK in some brain most hippocampus, thalamus, septum, VTA/SN, and pons where the loss of PC2 in lower levels of CCK. on the number of areas increased and CCK levels in PC2 knock-out mice as with the PC1 knock-out mice (14.Cain B.M. Connolly K. Blum A.C. Vishnuvardhan D. Marchand J.E. Zhu X. Steiner D.F. Beinfeld M.C. J. Neurochem. 2004; 89: 307-313Crossref PubMed Scopus (19) Google Scholar), the loss of PC2 appears to have for CCK processing. The loss of PC1 (14.Cain B.M. Connolly K. Blum A.C. Vishnuvardhan D. Marchand J.E. Zhu X. Steiner D.F. Beinfeld M.C. J. Neurochem. 2004; 89: 307-313Crossref PubMed Scopus (19) Google Scholar) also reduced CCK levels in the hippocampus, that PC1 and PC2 be responsible for the of CCK processing in this In all tissues in was a loss of CCK, that in the enzymes that PC1 PC5 were by the loss of PC2, but levels of these enzymes be to of some CCK. CCK levels in the olfactory bulb in PC2 knock-out an about the of the responsible for CCK processing in this In a study of rat brain (13.Cain B. Connolly K. Blum A. Vishnuvardhan D. Marchand J.E. Beinfeld M.C. J. Comp. Neurol. 2003; 467: 307-325Crossref PubMed Scopus (30) Google Scholar), PC2 was PC1 was co-localized whereas PC5 co-localization with CCK in the olfactory PC1 knock-out mice had the same levels of CCK as the wild-type mice in their olfactory bulbs (14.Cain B.M. Connolly K. Blum A.C. Vishnuvardhan D. Marchand J.E. Zhu X. Steiner D.F. Beinfeld M.C. J. Neurochem. 2004; 89: 307-313Crossref PubMed Scopus (19) Google Scholar) and PC2 knock-out mice had elevated CCK These that of these enzymes are responsible for the processing of CCK in this Two other of the and are present in the olfactory bulb W. Marcinkiewicz M. Vieau D. Chrétien M. Seidah N.G. Day R. J. Neurosci. 1995; 15: 1778-1796Crossref PubMed Google Scholar, N.G. Hamelin J. M. W. H. Mbikay M. Chretien M. Day R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and be responsible for CCK processing The in CCK levels in olfactory bulb, cerebral cortex and structures CCK from the cerebral cortex is CCK is increased in and cortex, this appears to be a cerebral A study J.F. Lindberg I. Friis-Hansen L. J. Neurochem. 2002; 83: 1329-1337Crossref PubMed Scopus (22) Google Scholar) found increased levels of glycine CCK in whole PC2 knock-out brains relative to whereas levels of amidated CCK The current study found CCK-Gly levels to be elevated in but not in hippocampus whereas CCK levels increased in and in The of this the of brain regions. It is not known the loss of PC2 elevated levels of CCK in the and olfactory levels are elevated in PFC, which the for higher levels of amidated CCK. levels of glycine CCK in the trypsin and carboxypeptidase are with increased The loss of PC2 have the expression or of enzyme that is to CCK and in higher levels of CCK-Gly and CCK The in CCK in the olfactory bulb is not by cerebral as CCK is present in the It is that the same that levels of CCK in the cortex is also in the It is that the decrease in expression of CCK and PC1 mRNA and the increased levels of amidated CCK are actually The CCK J.F. Mol. Endocrinol. 1999; 13: Google Scholar, K. J.F. Biol. 15: PubMed Scopus Google Scholar) and PC1 W.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, W.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (30) Google Scholar) have and is known to CCK from rat brain slices M.C. PubMed Scopus Google Scholar) and endocrine tumor cells J.Y. Beinfeld M.C. Endocrine. 1994; 2: 973-977Google Scholar). It is that the loss of PC2 the processing of peptide or protein that This reduction in decrease expression of CCK and PC1 mRNA and decrease the of CCK in the cerebral This an of CCK in in higher tissue levels of CCK. This not be unique to CCK. mechanisms be responsible for increased levels of neuropeptides in some brain regions in PC2 knock-out mice R. T. Vishnuvardhan D. Beinfeld M. Neuropeptides. 2003; PubMed Scopus Google Scholar).