Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis

Birch, Nigel P.; Rodríguez, Carlos Aníbal; Dixon, Jack E.; Mezey, Éva

doi:10.1016/0169-328x(90)90073-m

Cited by 30 publications

(10 citation statements)

References 15 publications

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“…There was also no cross-reactivity between the BDP-and RPC2-specific probes. Coronal sections (12 ^m) were cut from fresh-frozen adult rat brain and pituitary and processed for in situ hybridization, as described previously (34). Sections were hybridized with either 1 x 10 6 cpm/section BDP A or RPC2 A probe (for brain) or 1 x 10 6 cpm/section of both BDP A and B or RPC2 A and B (for pituitary).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Isolation of Two Complementary Deoxyribonucleic Acid Clones from a Rat Insulinoma Cell Line Based on Similarities to Kex2 and Furin Sequences and the Specific Localization of Each Transcript to Endocrine and Neuroendocrine Tissues in Rats

et al. 1991

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We have identified two rat insulinoma cDNAs that code for proteins homologous to the Kex2 dibasic protease of yeast and the mammalian furin gene product. A 5.0-kilobase (kb) cDNA, termed BDP, coding for a 752-amino acid protein and a 2.5-kb cDNA coding for a 636-amino acid protein, which was found to be the rat equivalent of the human insulinoma PC2 protein, were isolated. The proteins encoded by these clones contain a specific N-terminal signal sequence, indicating that both enter the secretory pathway. Neither protein contains a C-terminal transmembrane domain as is found in kex2 and furin, suggesting that the proteins may be soluble. Both proteins contain regions surrounding the active site residues which show amino acid identities to both kex2 (43% for BDP and 41% for RPC2) and furin (57% for BDP and 53% for RPC2). Probes specific for the mRNAs of each protein were used to localize the expression of each protein in endocrine and neuroendocrine tissues.

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Section: In Situ Hybridizationmentioning

confidence: 99%

Isolation of Two Complementary Deoxyribonucleic Acid Clones from a Rat Insulinoma Cell Line Based on Similarities to Kex2 and Furin Sequences and the Specific Localization of Each Transcript to Endocrine and Neuroendocrine Tissues in Rats

et al. 1991

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“…Control experiments included the use of sense strand riboprobes, as well as pretreatment of tissue with RNase; labeling was never encountered in these experiments. [19,21], CPE and PAM mRNA were widely distributed in brain and generally detected at high levels; CPE mRNA was detected in all brain areas, while regional differences were apparent for PAM. The cellular localization of CPE and PAM mRNA transcripts was primarily neu ronal, although the ventricular ependymal cells also labeled positively for both, and in the case of CPE, the signal was detected in the choroid plexus.…”

Section: Experiments 1: In Situ Hybridizationmentioning

confidence: 99%

Neuroanatomical and Functional Studies of Peptide Precursor-Processing Enyzmes

Cullinan

Day

Schäfer

et al. 1991

Enzyme

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An overview of in situ hybridization mapping studies comparing the brain distributions of mRNA transcripts encoding the proprotein convertases Furin, PCI and PC2 in relation to transcripts encoding carboxypeptidase FI (CPE) and peptidylglycine a-amidating monooxygenase (PAM) is presented. Furin mRNA was detected in both neurons and nonneuronal cells throughout all brain areas. The cellular localization of PCI and PC2 was primarily neuronal, with PC2 generally more widely distributed, although many regional variations were detected. The detection of specific combinations of the convertases, CPE and PAM in peptide-rich brain regions suggests that specific enzymatic pathways are involved in neuropeptide processing. Results are also described from a series of functional studies on the processing of pro-opiomelanocortin (POMC) in a heterologous neuronal cell line, Neuro-2A, which expresses low levels of PC2 mRNA but no detectable PCI mRNA. Two contrasting POMC-processing patterns were observed; one where the precursor was processed at a number of cleavage sites to produce several peptides, and another where POMC was processed at a single cleavage site to produce βE only. If PC2 is responsible for POMC processing in transfected cells, this enzyme may have favored cleavage of the amino terminal-processing site above other sites in the latter type of cell line.

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“…This is likely due to more rapid and complete dissociation of the zinc cofactor from the active site and replacement by cobalt at low pH because of the ionization of the zinc-binding histidine residues. Indeed, recent immunohistochemical studies, using specific antiserum to carboxypeptidase H (Lynch et al, 1990) or in situ hybridization of carboxypeptidase H mRNA in rat brain (Birch et al, 1990;MacCumber et al, 1990), revealed some discrepancies with earlier studies using [3H]GEMSA binding Strittmatter et al, 1984). Thus, previous studies using these techniques to assess carboxypeptidase H activity in brain could have included CPM as well.…”

Section: Discussionmentioning

confidence: 94%

“…In addition, the ICs0 for inhibition of CPM by GEMSA, which was thought to be a more specific inhibitor for carboxypeptidase H Strittmatter et al, 1984;Fricker, 1988), is -100-fold lower for the membrane-bound CPM at pH 5.5 than when the solubilized, purified enzyme is used at pH 7.5 (Deddish et al, 1989). Certain brain regions that were positive for carboxypeptidase H by inhibitor binding were less reactive when the more specific techniques of immunohistochemistry or in situ hybridization were used (Lynch et al, , 1990Strittmatter et al, 1984;Birch et al, 1990;MacCumber et al, 1990). Indeed, recent immunohistochemical studies, using specific antiserum to carboxypeptidase H (Lynch et al, 1990) or in situ hybridization of carboxypeptidase H mRNA in rat brain (Birch et al, 1990;MacCumber et al, 1990), revealed some discrepancies with earlier studies using [3H]GEMSA binding Strittmatter et al, 1984).…”

Section: Discussionmentioning

confidence: 99%

Carboxypeptidase M in Brain and Peripheral Nerves

Nagae

Deddish

Becker

et al. 1992

Journal of Neurochemistry

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Carboxypeptidase M (CPM), a plasma membrane‐bound enzyme, cleaves C‐terminal basic amino acids with a neutral pH optimum. We studied its distribution in human, baboon, and dog brain and in dog peripheral nerves. Areas were dissected, homogenized, centrifuged, and assayed for activity with dansyl‐Ala‐Arg. The corpus callosum and the pyramidal and optic tract were especially rich in CPM, whereas basal ganglia and cortex had low activity. The identity of the basic carboxypeptidase activity with CPM was shown by similarities in subcellular localization, membrane attachment, substrate hydrolysis, inhibition by a specific basic carboxypeptidase inhibitor, and cross‐reaction with anti‐human CPM antiserum. This antiserum immunoprecipitated an average of 85% of the activity in human and baboon brain and ∼66% in dog brain. CPM copurified with myelin extracted from the brain. Consistent with results obtained in placenta and cultured kidney cells, CPM in the brain appears to be membrane‐bound via a phosphatidylinositol glycan anchor. In the peripheral nerves, the specific activity in dog sciatic nerve and in vagus was high (98 and 149 nmol/h/mg of protein, respectively). In immunohistochemical studies, glia in the brain, which appear to be oligodendrocytes or astrocytes, and the outer aspects of myelin sheaths and Schwann cells in sciatic and vagus nerves were stained. We conclude that in some areas of the CNS and the PNS, CPM is closely associated with myelin and myelin‐forming cells. Northern blot analysis revealed the presence of mRNA coding for CPM in the brain, showing that the enzyme is indeed synthesized there.

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Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis

Cited by 30 publications

References 15 publications

Isolation of Two Complementary Deoxyribonucleic Acid Clones from a Rat Insulinoma Cell Line Based on Similarities to Kex2 and Furin Sequences and the Specific Localization of Each Transcript to Endocrine and Neuroendocrine Tissues in Rats

Isolation of Two Complementary Deoxyribonucleic Acid Clones from a Rat Insulinoma Cell Line Based on Similarities to Kex2 and Furin Sequences and the Specific Localization of Each Transcript to Endocrine and Neuroendocrine Tissues in Rats

Neuroanatomical and Functional Studies of Peptide Precursor-Processing Enyzmes

Carboxypeptidase M in Brain and Peripheral Nerves

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