In vivo and in vitro analyses of amygdalar function reveal a role for copper.

Mice with a single copy of the peptide amidating monooxygenase (Pam) gene (PAM(+/-)) are impaired in contextual and cued fear conditioning. These abnormalities coincide with deficient long-term potentiation (LTP) at excitatory thalamic afferent synapses onto pyramidal neurons in the lateral amygdala. Slice recordings from PAM(+/-) mice identified an increase in GABAergic tone (Gaier ED, Rodriguiz RM, Ma XM, Sivaramakrishnan S, Bousquet-Moore D, Wetsel WC, Eipper BA, Mains RE. J Neurosci 30: 13656-13669, 2010). Biochemical data indicate a tissue-specific deficit in Cu content in the amygdala; amygdalar expression of Atox-1 and Atp7a, essential for transport of Cu into the secretory pathway, is reduced in PAM(+/-) mice. When PAM(+/-) mice were fed a diet supplemented with Cu, the impairments in fear conditioning were reversed, and LTP was normalized in amygdala slice recordings. A role for endogenous Cu in amygdalar LTP was established by the inhibitory effect of a brief incubation of wild-type slices with bathocuproine disulfonate, a highly selective, cell-impermeant Cu chelator. Interestingly, bath-applied CuSO4 had no effect on excitatory currents but reversibly potentiated the disynaptic inhibitory current. Bath-applied CuSO4 was sufficient to potentiate wild-type amygdala afferent synapses. The ability of dietary Cu to affect signaling in pathways that govern fear-based behaviors supports an essential physiological role for Cu in amygdalar function at both the synaptic and behavioral levels. This work is relevant to neurological and psychiatric disorders in which disturbed Cu homeostasis could contribute to altered synaptic transmission, including Wilson's, Menkes, Alzheimer's, and prion-related diseases.

Reversal of physiological deficits caused by diminished levels of peptidylglycine alpha-amidating monooxygenase by dietary copper.

Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine alpha-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

Breeding stock-specific variation in peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

Location of neurons that express regulated endocrine-specific protein-18 in the rat diencephalon.

In situ hybridization for regulated endocrine-specific protein-18 messenger RNA showed a distinct and limited pattern of expression in the hypothalamus, midline thalamus, amygdala and hippocampus of the rat. High levels of regulated endocrine-specific protein-18 messenger RNA were found in the magnocellular neurons of the hypothalamic paraventricular, supraoptic and accessory nuclei, in the neurons of the periventricular, medial tuberal, arcuate, lateral and perifornical nuclei, infundibular stalk, and in the ventrolateral division of the ventromedial nucleus and compact division of the dorsomedial nucleus. Lower levels of regulated endocrine-specific protein-18 messenger RNA were found in the parvocellular divisions of the paraventricular nucleus as well as in the bed nucleus of the stria terminalis, median preoptic nucleus, medial preoptic nucleus, medial and lateral preoptic areas, subfornical organ, suprachiasmatic nucleus, anterior hypothalamic area, zona incerta, ventromedial nucleus, dorsomedial nucleus and tuber cinereum. Regulated endocrine-specific protein-18 messenger RNA was also found in thalamic structures including the paraventricular, central medial, intermediodorsal, anterodorsal, rhomboid and reticular nuclei. Signal was also identified in the medial and lateral habenula, in the central, medial, basomedial and anterior cortical nuclei of the amygdala, and in the CA1-CA3 and dentate gyrus of the hippocampus. Dopamine may regulate regulated endocrine-specific protein-18 expression in the CNS because (i) regulated endocrine-specific protein-18 was originally identified in melanotropes based on its regulation by dopaminergic agents and (ii) many of the nuclei that contain regulated endocrine-specific protein-18 also receive dopaminergic input. The localization of regulated endocrine-specific protein-18 in the diencephalon suggests that regulated endocrine-specific protein-18 is involved in regulation of limbic and autonomic function, neuroendocrine control of salt and water balance, reproductive function and feeding behavior.

RESP18, a novel endocrine secretory protein transcript, and four other transcripts are regulated in parallel with pro-opiomelanocortin in melanotropes.

The homogeneous nature of the rat intermediate pituitary makes it a powerful model system in which to study peptide hormone secretion. Adult male rats were treated with bromocriptine, a dopamine agonist, or haloperidol, a dopamine antagonist, for 3 weeks. In cDNA libraries prepared from the neurointermediate pituitaries of these rats, pro-opiomelanocortin (POMC) expression exhibited the expected decrease in response to bromocriptine, and increase in response to haloperidol. We report the identification of six transcripts that are coregulated with POMC in the intermediate pituitary by these dopaminergic agents. In addition to demonstrating parallel dopamine-regulated expression of carboxy-peptidase E, chromogranin B, binding protein/glucose-regulated protein, and tenascin, two novel regulated transcripts are described. The expression of one of these novel transcripts, RESP18, is limited to neural and endocrine tissue. The RESP18 transcript is approximately 800 nucleotides in length; its cognate translation product is 20 +/- 1 kDa, contains a putative signal sequence, and has many characteristics of a secreted protein. Cell-free translation experiments in the presence of microsomal membranes demonstrate that the 20 +/- 1-kDa RESP18 protein is cleaved to an 18 +/- 1-kDa protein and sequestered within the lumen of the rough endoplasmic reticulum. Tissue in situ hybridization analysis shows that RESP18 mRNA is highly expressed in both the intermediate and anterior pituitary, as well as in the paraventricular and supraoptic nuclei of the hypothalamus.

Neurosecretory vesicles contain soluble and membrane-associated monofunctional and bifunctional peptidylglycine alpha-amidating monooxygenase proteins.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of neuropeptides in a reaction requiring the sequential action of two enzymes contained within this bifunctional protein. The CNS contains primarily transcripts encoding rPAM-1 and rPAM-2, integral membrane proteins differing by the presence or absence of a noncatalytic domain separating the two enzymes. Subcellular fractionation of adult rat hypothalamus and hippocampus demonstrated the localization of both enzymatic activities to fractions enriched in neurosecretory vesicles. Upon separation of the soluble contents from the membranes of neurosecretory vesicles, 30-40% of both enzymatic activities was recovered in the soluble fraction. Over 40% of both enzymatic activities remained membrane-associated following removal of peripheral membrane proteins. Antisera specific to different regions of PAM were used to identify intact rPAM-1 and rPAM-2, a monofunctional integral membrane peptidyl-alpha-hydroxy-glycine alpha-amidating lyase protein generated from rPAM-1, and a noncatalytic COOH-terminal fragment as the major PAM proteins in carbonate-washed membranes. Endoproteolytic processing generated large amounts of soluble, monofunctional forms of both enzymes from rPAM-1 and smaller amounts of a soluble, bifunctional PAM protein from rPAM-2. A significant amount of both monofunctional enzymes lacking the transmembrane domain was tightly associated with membranes. Whereas soluble mono- and bifunctional enzymes may be released upon exocytosis of neurosecretory vesicles, membrane-associated PAM proteins may remain on the cell surface or be internalized.

Stable expression of full-length and truncated bovine peptidylglycine alpha-amidating monooxygenase complementary DNAs in cultured cells.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the production of alpha-amidated peptides from their glycine-extended precursors, a posttranslational modification often required for full biological activity. We have previously cloned cDNAs encoding a 108-kDa bovine PAM precursor. To confirm that this cDNA encodes a functional alpha-amidating enzyme and to begin to examine the structural requirements for the biosynthesis of an active PAM enzyme, we constructed expression vectors that placed the cDNA for either the full-sized enzyme or a form truncated at the carboxyl-terminal (and thus lacking the transmembrane domain) under the control of the mouse metallothionein-1 promoter. We used the resultant plasmids to transfect AtT-20 mouse anterior pituitary corticotrope cells and selected stable lines that expressed increased levels of PAM activity. Transfected cells in which expression from the metallothionein promoter had been induced had up to 15-fold higher levels of PAM mRNA and up to 7.5-fold higher levels of PAM activity than wild-type cells. The PAM activity in the transfected cells shared many enzymatic characteristics with PAM-B, a 38-kDa soluble form of PAM purified from bovine neurointermediate pituitary. These included copper- and ascorbate-dependent activity, an alkaline pH optimum for the peptide substrate D-Tyr-Val-Gly, similar affinities for several other synthetic substrates, and comparable apparent size during gel filtration. Compared to extracts of wild-type cells, extracts from transfected cells showed increased production of five different amino acid alpha-amides. These data indicate that a single enzyme can act on a variety of peptide substrates, and that the full structure of the PAM precursor is not necessary during biosynthesis for expression of active PAM enzyme.

Characterization of pro-ACTH/endorphin-derived peptides in rat hypothalamus.

The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of beta-endorphin, gamma-lipotropin (gamma-LPH), corticotropin-like intermediate lobe peptide (CLIP), alpha-melanotropin (gamma-MSH), joining peptide, and glycosylated gamma 3-MSH all represent predominant end products. Equimolar amounts of beta-endorphin-, alpha-MSH-, CLIP-, gamma-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (approximately 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of beta-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to beta-endorphin(1-31), beta-endorphin(1-27), and beta-endorphin(1-26). The alpha-N-acetylated forms of endorphin represent less than 10% of the total beta-endorphin immunoreactivity. Analyses of hypothalamic alpha-MSH-sized molecules with acetyl- and amide-directed alpha-MSH antisera suggest that hypothalamic alpha-MSH is fully amidated, but largely not alpha-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that greater than 85% of the alpha-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to alpha-MSH [alpha-N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic gamma-LPH-sized immunoreactive material is indistinguishable from gamma-LPH synthesized by pituitary melanotropes. The minor extent of alpha-N-acetylation of alpha-MSH and beta-endorphin, the limited carboxyl-terminal proteolysis of beta-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia.

Peptidyl-glycine alpha-amidation activity in tissues and serum of the adult rat.

Bioactive peptides frequently terminate in a carboxyl-terminal alpha-amide. The tissue distribution of enzymatic activity capable of converting [125I]D-Tyr-Val-Gly into [125I]D-Tyr-Val-NH2 has been determined. Assay conditions have been established so that enzyme activity can be measured in crude homogenates. In adult male rats, the highest concentrations of activity are found in the anterior and neurointermediate lobes of the pituitary. Lower concentrations of activity are found in the hypothalamus, submandibular glands, and the rest of the brain. Enzyme activity is also easily assayed in serum. Taking into account the mass of each tissue, the submandibular glands and the brain are the major tissue sources of enzymatic activity; serum contains more enzymatic activity than is found in the pituitary gland. In all tissues and in serum, enzyme activity is stimulated by the addition of copper sulfate and ascorbate and is dependent on molecular oxygen. This activity is, therefore, referred to as peptidyl glycine alpha-amidating monooxygenase (PAM) activity. The pH optima for PAM activity in the pituitary and submandibular glands are near neutrality, whereas the pH optima for PAM activity in serum and hypothalamus are broad, with substantial activity at alkaline pH.

Hormonal, drug, and dietary factors affecting peptidyl glycine alpha-amidating monooxygenase activity in various tissues of the adult male rat.

The factors controlling levels of peptidyl glycine alpha-amidating monooxygenase (PAM) activity in its major tissue sources in the adult male rat were investigated by carrying out a variety of endocrine, pharmacological, and dietary manipulations. Levels of PAM activity and alpha MSH immunoactivity in the neurointermediate lobe of the pituitary gland rose and fell in parallel in rats treated with the dopamine antagonist haloperidol or the dopamine agonist bromocriptine, respectively. PAM activity in the anterior pituitary lobe was increased after adrenalectomy or castration and decreased after thyroidectomy or treatment with haloperidol. PAM activity in the submandibular gland was increased after treatment with the alpha-adrenergic antagonist phenoxybenzamine and decreased after treatment with the alpha-adrenergic agonist phenylephrine. Serum levels of PAM activity were unaltered after hypophysectomy, adrenalectomy, sialectomy, or castration, but rose after thyroidectomy and declined after treatment with the ganglionic blocker chlorisondamine or phenoxybenzamine. Chronic dietary copper deficiency in rats resulted in increased PAM activity in homogenates of anterior pituitary lobe and submandibular gland assayed under optimized conditions; chronic dietary ascorbate deficiency in guinea pigs did not produce consistent changes in PAM activity in the tissues examined.