Peptidylglycine Α-amidating Monooxygenase Expression Research Articles

Multiple roles for peptidylglycine α-amidating monooxygenase in the response to hypoxia.

The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (Km = 99 ± 19 μM) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2[Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (Pam Myh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam 0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by Pam Myh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by Pam Myh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

PERK-mediated expression of peptidylglycine \u03b1-amidating monooxygenase supports angiogenesis in glioblastoma

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

PAM haploinsufficiency does not accelerate the development of diet- and human IAPP-induced diabetes in mice.

Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes. PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam+/- and Pam+/+ mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1β, IFN-γ and TFN-α. Despite Pam+/- only having 50% PAM expression and enzyme activity as compared with Pam+/+ mice, glucose tolerance and body mass composition were comparable in the two models. After 24weeks of HFD, both Pam+/- and Pam+/+ mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam+/- and Pam+/+ mice with beta cell expression of hIAPP. Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

25-OR: Investigating a Candidate Causal Allele in Type 2 Diabetes Susceptibility Gene PAM as a Cause of Neonatal Diabetes Mellitus

Peptidylglycine α-amidating monooxygenase (PAM) catalyses the amidation of glycine-extended peptides, thereby maximising their biological potency. We recently showed that coding alleles in PAM mediate type 2 diabetes (T2D) risk through alterations to PAM protein activity and expression in the β cell. Identifying alleles along a continuum of functional severity informs on the relationship between protein perturbation and clinical effect. We hypothesised that rare, loss of function PAM alleles could cause severe β cell dysfunction presenting as neonatal diabetes. Sequencing of 15 probands presenting with diabetes at ≤ 6 months of age and their unaffected immediate family, identified a novel de novo nonsynonymous coding variant (p.R36S) in PAM in a single proband. We investigated the impact of the variant allele on amidating activity, protein stability and localisation as a measure of PAM function and the consequences on β cell viability. Recombinant R36S-PAM exhibited normal amidating activity in vitro which was consistent with PAM activity measured in patient serum (WT: 391pmol/μl/hr vs. R36S: 305pmol/μl/hr). In human β cells (EndoC-βh1) the secreted luminal R36S-PAM isoform displayed reduced protein expression (-78%, p=0.004) compared to WT-PAM, which could be rescued by inhibiting the proteosomal pathway (+120% of WT); whilst the non-secreted membrane-integral isoform was retained in the endoplasmic reticulum (ER). We observed that overexpression of R36S-PAM elevated levels of ER stress (p-IRE1: +196%, p=0.02; CHOP: +114%, p=0.051) and pro-apoptotic markers (ASK1: +145%, caspase-3: +154%) compared with WT-PAM. We have identified a novel, de novo coding variant, in a gene with a proven role in T2D risk, in a single patient with neonatal diabetes. Functional studies provide evidence for protein mislocalisation and instability and support a role for this allele in ER stress and β cell dysfunction consistent with neonatal diabetes pathogenesis. Disclosure S. Sengupta: None. L.L. Bonnycastle: None. B. Hastoy: None. A. Grotz: None. M.M. Umapathysivam: None. A. Raimondo: None. A. Swift: None. P.S. Chines: None. A. Clark: None. H. Huopio: None. F.S. Collins: None. M. Laakso: None. A.L. Gloyn: Consultant; Spouse/Partner; Eli Lilly and Company, Merck & Co., Inc. Consultant; Self; Merck & Co., Inc. Consultant; Spouse/Partner; Novo Nordisk A/S, Pfizer Inc. Research Support; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Other Relationship; Self; Diabetes UK, European Foundation for the Study of Diabetes. Funding UK Wellcome Trust

Changes in Corticotrope Gene Expression Upon Increased Expression of Peptidylglycine α-Amidating Monooxygenase.

Throughout evolution, secretion has played an essential role in the ability of organisms and single cells to survive in the face of a changing environment. Peptidylglycine α-amidating monooxygenase (PAM) is an integral membrane monooxygenase, first identified for its role in the biosynthesis of neuroendocrine peptides released by the regulated secretory pathway. PAM was subsequently identified in Chlamydomonas reinhardtii, a unicellular green alga, where it plays an essential role in constitutive secretion and in ciliogenesis. Reduced expression of C. reinhardtii PAM resulted in significant changes in secretion and ciliogenesis. Hence, a screen was performed for transcripts and proteins whose expression responded to changes in PAM levels in a mammalian corticotrope tumor cell line. The goal was to identify genes not previously known to play a role in secretion. The screen identified transcription factors, peptidyl prolyl isomerases, endosomal/lysosomal proteins, and proteins involved in tissue-specific responses to glucose and amino acid availability that had not previously been recognized as relevant to the secretory pathway. Perhaps reflecting the dependence of PAM on molecular oxygen, many PAM-responsive genes are known to be hypoxia responsive. The data highlight the extent to which the performance of the secretory pathway may be integrated into a wide diversity of signaling pathways.

Expression of Peptidyl-Glycine α-amidating Mono-Oxygenase (PAM) Enzymes in Morphological Abnormalities Adjecent to Pulmonary Tumors

This is an investigation of an endocrine cell type in the midgut of the migratory locust Locusta migratoria. This cell type is found in the posterior region of the midgut and is especially common in the ampullae through which Malpighian tubules drain into the gut at the midgut-hindgut junction. Strong Locusta diuretic hormonelike immunoreactivity in these cells was colocalized with FMRFamide- and substance P-like immunorectivities. At the uitrastuctural level, immunoreactivities for Locusta diuretic hormone was found in spherical granules (mean diameter of 450 nm), the contents of which showed variable electron density. Fractionation of a methanolic extract of the ampullae by reversed-phase high performance liquid chromatography revealed the presence of two peaks of Locusta diuretic hormone-like immunoreactive material, both of which stimulate cyclic AMP production by isolated Malpighian tubules. The more hydrophobic material is most likely Locusta diuretic hormone, which has the same retention time when chromatographed under identical conditions.

Peptidylglycine α‐amidating monooxygenase heterozygosity alters brain copper handling with region specificity

Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in post-synaptic vesicular fractions. Cu followed a similar pattern, with ~ 20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) was selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not in the hippocampus in PAM+/− mice, GABAB receptor mRNA levels were similarly affected. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not in the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. Atp7a, a Cu-transporting P-type ATPase, is localized to the trans-Golgi network and to vesicles distributed throughout the dendritic arbor. Tissue-specific alterations in Atp7a expression were found in mice heterozygous for peptidylglycine α-amidating monooxygenase (PAM), an essential neuropeptide-synthesizing cuproenzyme. Atp7a and PAM are highly expressed in amygdalar interneurons. Reduced amygdalar expression of Atox-1 and Atp7a in PAM heterozygous mice may lead to reduced synaptic Cu levels, contributing to the behavioral and neurochemical alterations seen in these mice.

Haploinsufficiency in Peptidylglycine α-Amidating Monooxygenase Leads to Altered Synaptic Transmission in the Amygdala and Impaired Emotional Responses

The mammalian amygdala expresses various neuropeptides whose signaling has been implicated in emotionality. Many neuropeptides require amidation for full activation by peptidylglycine α-amidating monooxygenase (PAM), a transmembrane vesicular cuproenzyme and regulator of the secretory pathway. Mice heterozygous for the Pam gene (PAM(+/-)) exhibit physiological and behavioral abnormalities related to specific peptidergic pathways. In the present study, we evaluated emotionality and examined molecular and cellular responses that characterize neurophysiological differences in the PAM(+/-) amygdala. PAM(+/-) mice presented with anxiety-like behaviors in the zero maze that were alleviated by diazepam. PAM(+/-) animals were deficient in short- and long-term contextual and cued fear conditioning and required higher shock intensities to establish fear-potentiated startle than their wild-type littermates. Immunohistochemical analysis of the amygdala revealed PAM expression in pyramidal neurons and local interneurons that synthesize GABA. We performed whole-cell recordings of pyramidal neurons in the PAM(+/-) amygdala to elucidate neurophysiological correlates of the fear behavioral phenotypes. Consistent with these observations, thalamic afferent synapses in the PAM(+/-) lateral nucleus were deficient in long-term potentiation. This deficit was apparent in the absence and presence of the GABA(A) receptor antagonist picrotoxin and was abolished when both GABA(A) and GABA(B) receptors were blocked. Both evoked and spontaneous excitatory signals were enhanced in the PAM(+/-) lateral nucleus. Phasic GABAergic signaling was also augmented in the PAM(+/-) amygdala, and this difference comprised activity-independent and -dependent components. These physiological findings represent perturbations in the PAM(+/-) amygdala that may underlie the aberrant emotional responses in the intact animal.

The influence of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and its metabolite—dichlorodiphenyldichloroethylene (DDE) on mRNA expression for NP-I/OT and PGA, involved in oxytocin synthesis in bovine granulosa and luteal cells

The effect of polychlorinated biphenyls (PCBs) congeners (PCB 77, PCB 126, PCB 153) and their technical mixture—Aroclor (Ar) 1248, as well as dichlorodiphenyltrichloroethane (DDT) and its metabolite—dichlorodiphenyldichloroethylene (DDE; two individual isomers p,p′- and o,p′- or their mixture, 95% and 5%, respectively) at the dose of 10 ng/ml each, on the gene expression of (a) oxytocin (OT) precursor—neurophysin–oxytocin (NP-I/OT) and (b) peptidyl glycine-α-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of OT synthesis, was studied. Granulosa cells from follicles >1 cm in diameter, collected on days 19–21 of estrous cycle, and luteal cells from corpora lutea (CL) collected on days 8–12 of the estrous cycle were used. The cells were incubated (6 h) with these xenobiotics and the expression of NP-I/OT and PGA genes was determined. All PCBs increased ( P < 0.05) NP-I/OT gene expression in granulosa cells. Similarly, all PCBs but PCB 126 increased ( P < 0.05) PGA gene expression in these cells. DDT and DDE increased ( P < 0.05) gene expression of NP-I/OT in granulosa cells, while gene expression of PGA in these cells was stimulated ( P < 0.05) by DDE only. The mRNA expression for NP-I/OT and PGA in luteal cells was increased ( P < 0.05) by PCB 77 and PCB 153. Both DDE isomers and mixture also stimulated ( P < 0.05) of NP-I/OT mRNA expression, while increase ( P < 0.05) of PGA mRNA expression was elicited by incubation of these cells with DDE mixture and Ar 1248. Obtained data suggest that PCBs, DDT and DDE can affect the mRNA expression for NP-I/OT and PGA in bovine granulosa and luteal cells.

Substrate specificity of transthyretin: identification of natural substrates in the nervous system

Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTR(prot-) (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine alpha-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTR(prot-), was measured. Cells grown with TTR(prot-) displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.

Neuropeptide amidation: cloning of a bifunctional α-amidating enzyme from Aplysia

One of the most common mechanisms of posttranslational modifications to generate biologically active (neuro)peptides is the process of peptide α-amidation. The only enzyme known to catalyze this important modification is peptidylglycine α-amidating monooxygenase (PAM): a (bifunctional) zymogen, giving rise to a monooxygenase (PHM) and a lyase (PAL). The highly peptidergic central nervous system and endocrine system of the marine mollusk Aplysia has homologs of various mammalian peptide processing enzymes, including furin, Afurin2, prohormone convertase 1 (PC1), PC2, carboxypeptidase E (CPE) and CPD. Previously, it has been shown that the abdominal ganglion of Aplysia, which contains ∼800 peptidergic bag cell neurons, contains the highest specific α-amidating activity. We have identified and cloned multiple overlapping central nervous system and bag cell cDNAs that encode a predicted 748-residue protein that is a member of the PAM family. The protein sequence contains the contiguous sequence of the catalytic domains of PHM and PAL, clearly demonstrating the existence of bifunctional Aplysia PAM, the first invertebrate PAM zymogen with an organization similar to that in vertebrates. None of the characterized clones encoded the so-called exon A domain between the PHM and PAL domains. Furthermore, in a specific search by reverse transcription-polymerase chain reaction of RNA from multiple tissues we could only detect exon A-less transcripts. PAM expression was detected in the central nervous system, and in several endocrine and exocrine organs. Aplysia PAM is a candidate prohormone processing enzyme that plays an important role in the processing of Aplysia prohormones in the secretory pathway.

Characterization and regulation of peptidylglycine alpha-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts.

Peptidylglycine alpha-amidating monooxygenase (PAM), which catalazyes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, has been found in H9c2 myoblasts. The expression of PAM has been evaluated in H9c2 cells. Northern blot analysis and amplification of fragments derived from rat PAM by the reverse transcription/polymerase chain reaction method has demonstrated the presence of rPAM-1, -2, -3, -3a and -3b mRNA transcripts. These forms of PAM mRNA may be generated by alternative splicing. PAM mRNA levels are increased to 160 +/- 12% of control values by treatment with dexamethasone but are unchanged during triiodothyronine incubation of the cells. PAM activity is very low, which is not comparable to the high levels found in adult atrium tissue. Western blot analysis has demonstrated 86-, 76-, and 46-kDa PAM proteins in the particulate fraction. The soluble fraction contains major PAM proteins of 110, 86, and 46 kDa. In situ hybridization studies with 35S-labeled full length RNA antisense transcripts of rat PAM-1 cDNA have localized autoradiographic grains around the nucleus. Our data clearly demonstrate PAM expression in H9c2 rat heart cells, suggesting the ability of these cardiac cells to make bioactive alpha-amidated hormones and/or neuropeptides.

Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.

Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.

Evidence of high expression of peptidylglycine alpha-amidating monooxygenase in the rat uterus: estrogen regulation.

In the present study, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17beta-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

Embryonic and Uterine Expression Patterns of Peptidylglycine α-Amidating Monooxygenase Transcripts Suggest a Widespread Role for Amidated Peptides in Development

Posttranslational processing of peptide precursors frequently includes COOH-terminal amidation by the bifunctional enzyme peptidylglycine α-amidating monooxygenase (PAM). We examined the ontogeny of PAM gene expression usingin situhybridization and detected expression in the cardiogenic region beginning at embryonic day 9 (e9) and in decidualizing uterine endometrium and myometrial smooth muscle at even earlier postimplantation stages. PAM expression in the CNS at e10 was highest in the dorsal spinal cord and floor plate and exhibited complex patterning in several CNS regions, including the ventricular zone, over the next several days with PAM expression first detected in neurons at e13. High levels of PAM expression characterized several nonneural cell populations as well, including limb mesoderm and the mesenchyme immediately adjacent to nasal, maxillary, palatal, and dental epithelia during tissue fusion and remodeling. Since alternative splicing generates PAM transcripts encoding proproteins that are differentially localized and processed, we used probes that distinguish major subsets of PAM transcripts to determine that transcripts encoding integral membrane PAM isoforms predominate in most, if not all, PAM-expressing cell types throughout development. Further, transcripts that encode soluble and cleavable PAM isoforms are essentially absent from two CNS areas that are rich in transcripts encoding integral membrane, bifunctional PAM: the ependymal region of the spinal cord and the ventricular zone of the hippocampus. These results provide evidence for widespread expression and cell-type-specific alternative splicing of PAM during development and raise the possibility that region-specific amidation of PAM substrates contributes significantly to several developmental processes.

Expression of peptidyl-glycine α-amidating mono-oxygenase (PAM) enzymes in morphological abnormalities adjacent to pulmonary tumors: Martinez A, Treston AM, Saldise L, Montuenga LM, Linnoila RI. BPRB, DCS, National Cancer Institute, 9610 Medical Center Drive, Rockville, MD 20850-3300. Am J Pathol 1996;149:707–716

Expression of peptidyl-glycine α-amidating mono-oxygenase (PAM) enzymes in morphological abnormalities adjacent to pulmonary tumors: Martinez A, Treston AM, Saldise L, Montuenga LM, Linnoila RI. BPRB, DCS, National Cancer Institute, 9610 Medical Center Drive, Rockville, MD 20850-3300. Am J Pathol 1996;149:707–716

Tissue-Specific Regulation of Peptidyl-Glycine α-Amidating Monooxygenase Expression*

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is present in a variety of tissues, where it plays a vital role in the processing of numerous peptide substrates, often conferring bioactivity. PAM is present in high amounts in heart atrial myocytes and the pituitary gland, where activity is present in both soluble and membrane forms. We used AtT-20 cells, a mouse corticotrope tumor cell line, and primary heart atrial cultures to establish the occurrence of tissue-specific regulation of PAM expression. In the AtT-20 cells, PAM expression is regulated in parallel with the source of its peptide substrate, pro-ACTH/endorphin. PAM mRNA levels are increased to 132 +/- 5% of control values by treatment with (Bu)2-cAMP and decreased to 55 +/- 7% of control values by treatment with dexamethasone. Treatment with (Bu)2cAMP decreases PAM specific activity in the AtT-20 cells to 68 +/- 4% of the control value, presumably due to secretion of enzyme from the cells; dexamethasone treatment decreases PAM specific activity to 57 +/- 1% of the control value. In contrast, in heart atrial cultures, dexamethasone stimulates PAM expression. In atrial cultures exposed to dexamethasone for 48 h, PAM mRNA and PAM specific activity are elevated to 230 +/- 50% and 220 +/- 20% of control values, respectively; secretion of PAM activity is increased to 230% of the control value. As for AtT-20 cells, treatment of atrial cultures with (Bu)2cAMP increases PAM mRNA levels. Thus, PAM expression is regulated in a tissue-specific manner by dexamethasone in the two tissues examined. In AtT-20 cells, time-course studies and studies with cycloheximide indicate that dexamethasone exerts its effects on PAM mRNA levels by an indirect mechanism involving protein synthesis.

Developmental regulation of peptidylglycine α-amidating monooxygenase (PAM) in rat heart atrium and ventricle: Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.