Immunocytochemical localization of a novel pituitary polypeptide “7B2” in the gastro-intestinal tract of the rat

Summary Immunoreactivity to the polypeptide designated 7B2 recently isolated from human and porcine pituitary glands, appears to be consistently confined to neuroendocrine and endocrine cells in various tissues. In rat gut, immunoreactive 7B2 was found in endocrine-paracrine cells. Highly labeled cells were found in the antrum of the stomach and, cells with lower concentrations, in the fundus, duodenum, jejunum and ileum. Except for a few cells which were simultaneously positive for 5-hydroxytryptamine, and a few others showing Grimelius's reaction, 7B2 cells do not exhibit argentaffin and/or argyrophil character. The 7B2 polypeptide seems to be distributed amongst several different types of endocrine cells in the gut.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

Homologous IRCM-Serine Protease 1 from pituitary,heart atrium and ventricle: A common pro-hormone maturation enzyme?

IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem. 261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem. 261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones.     

The Somatic Function of the Germ Nucleus in Pseudourostyla cris tat a: Asexual Reproduction and Stomatogenesis

The micronuclei of the ciliated protozoan Pseudourostyla cristata were eliminated by amputation shortly before binary fusion. The amicronucleate cell lines derived from regenerants were maintained for more than a year. They exhibited a lower viability and reduced vigor in asexual propagation. There was some improvement in the growth of the cell lines 1 mo after operation, but the growth rate remained subnormal even up to 1 yr of culture. The exact cause of the poor growth and survival in the first 3 wk after operation, whether the loss of the micronucleus or operational damage, remains to be determined. It is nevertheless clear that the micronucleus is important for subsequent asexual propagation. The amicronucleate cell lines were permanently crippled in morphogenesis, unlike the situation in Paramecium amicronucleates in which stomatogenesis returned to near-normal during asexual propagation. They always included some cells with a characteristically defective adoral zone of membranelles, reduced number of frontal-ventral-transverse cirri, and reduced body length. They were also reluctant to encyst. It is evident that the micronucleus is important for maintaining normality of the oral apparatus. It is postulated that the permanent stomatogenic crippling of amicronucleates might be related to genomic reduction in the developing macronucleus in sexual reproduction, as exhibited by other hypotrichs. The morphological defects associated with the adoral zone of membranelles may be rationalized as arising from the spreading of a zone of degeneration in the cortex affecting the left edge of the membranelles.     

Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1.

Proprotein and prohormone processing at pairs of basic residues is generally thought to be both tissue- and precursor-specific and to be developmentally regulated. Furin, PC1 (also called PC3), and PC2 represent three recently discovered subtilisin-like proteinases which cleave a number of precursors at the same pairs of basic residues normally processed in vivo. Using human prorenin as a model, we show that PC1 can process it to active renin in cells containing secretory granules, such as the somatomammotroph cell line GH4, but not in cells which lack granules, such as the Chinese hamster ovary or African green monkey kidney epithelial (BSC-40) cell lines. In contrast, in both cell types, human prorenin is not activated by either PC2 or furin. Using the vaccinia virus expression system, biosynthetic labeling experiments demonstrated that PC1 and PC2 are themselves cleaved intracellularly at pairs of basic residues and that these two proenzymes are processed to different extents independent of whether the cell line contains dense core secretory granules. Furthermore, we also show that the cells mostly secrete the cleaved forms of PC1 and PC2, and that intracellularly the pro- form of PC2 predominates. Our data demonstrate that propeptide removal from these enzymes, possibly leading to their activation, is not the only criterion which governs precursor processing.     

Anomalous Cases of Conjugation and Spontaneous Autogamy in Stylonychia mytilus: A Note on the Asexuality of Early Conjugants

Four types of anomalous conjugation were documented in Stylonychia mytilus. Type I pairs were formed between mates of different sizes. These pairs exhibited an abnormal site of fusion in at least one of the mates, and the mates might face each other ventrally throughout conjugation instead of the normal side-by-side position. Type I pairs underwent sexual nuclear development and proceeded with the first cortical reorganization as in normal conjugants. Type II involved pairing at the anterior ends of mates with ventral surfaces facing the same direction. These pairs also underwent sexual nuclear development. Hence, aberrant orientation of the mates, and also ectopic sites of cytoplasmic fusion, if extensive, would permit sexual development. Type III pairs were united ventral-to-ventral with their anterior-left sides at the adoral zone of membranelles, and remained as such throughout conjugation. In these pairs, nuclear and cortical events were typical of the asexual development of physiological reorganization. In Type IV pairs, one mate of the pair possessed a fission furrow and developed two sets of ciliature typical of binary fission, while the other mate might undergo physiological reorganization or binary fission. Type III and Type IV pairs thus reveal the asexual state of early conjugants, which can pursue either one of the two modes of asexual cortical reorganization; these cases reinforce the notion of overlap of asexual and sexual cycles during conjugation of hypotrichs. Spontaneous autogamy was documented for the first time for this genus. The autogamonts proceeded with nuclear development and with the first cortical reorganization. Some probably underwent second and third reorganizations, as in conjugants, but accompanied by abnormalities, particularly in the stages beyond fertilization. Post-autogamous clones were nonviable except for one dubious case.     

Chromosomal assignments of the genes for neuroendocrine convertase PC1 (NEC1) to human 5q15-21, neuroendocrine convertase PC2 (NEC2) to human 20p11.1-11.2, and furin (mouse 7[D1-E2] region).

The chromosomal localization of the genes coding for the pro-protein and pro-hormone convertases PC1, PC2, and Furin has been achieved by in situ hybridization. The genes for PC1 and PC2 were located on human chromosomes 5q15-21 and 20p11.1-11.2, respectively. The gene for Furin was assigned to the mouse chromosome 7D1-7E2 region. These data complete the chromosomal localization of these three convertases in both human and mouse. The results confirm the regional correspondence of the human chromosomes 15 and mouse chromosomes 7, as well as between human chromosome 20 and mouse chromosome 2. Furthermore, the identification of the NEC1 locus on human chromosome 5 and mouse chromosome 13 suggests a conservation of synthenic regions between these regions of the human and mouse genomes.     

Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary.

PC1 and PC2 are enzymes involved in the activation of prohormones via the cleavage of pairs of basic amino acids. The expression levels of each of these enzymes were evaluated in the rat anterior and neurointermediate pituitary lobes by in situ hybridization and Northern gel analysis and after various pharmacological manipulations. All intermediate lobe melanotrophs expressed high levels of PC2 mRNA and lower levels of PC1 mRNA. PC1 mRNA was highly expressed throughout the anterior lobe; however, appreciable PC2 mRNA levels were also found. Based on colocalization studies, anterior lobe corticotrophs were found to express PC1 mRNA, but very little PC2 mRNA. Neurointermediate lobe levels of PC1, PC2, and POMC mRNA increased 2- to 6-fold in rats treated with haloperidol, while they decreased to 10-25% of their control values after bromocriptine treatment. These results indicate that in the intermediate lobe, dopamine is involved in the regulation of PC1 and PC2. In the anterior lobe, haloperidol had a strong effect on PC2 mRNA, increasing its levels by 8- to 12-fold compared to the control value, while PC1 mRNA was unaffected. Both PC1 and PC2 mRNA levels were increased 5- to 9-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Adrenalectomy had no significant effect on anterior lobe PC1 mRNA levels. However, both PC1 and PC2 mRNA levels were responsive to dexamethasone treatment in the AtT-20 cell lines. Our results indicate that dopamine, thyroid hormones, and corticosteroids are involved in PC1 and/or PC2 gene expression. These data are also consistent with the role of PC1 and PC2 as prohormone-processing enzymes.     

Presence of vasopressin, oxytocin and neurophysin in the retina of mammals, effect of light and darkness, comparison with the neuropeptide content of the neurohypophysis and the pineal gland

Arginine vasopressin (AVP) and oxytocin (OT) as well as their CNS carrier neurophysins (Np) have been found in the pineal gland. In view of the analogy between the pineal gland and the retina, the contents of these neuropeptides in rat, human and bovine retinae were determined. AVP, OT and Np were detected by specific radioimmunoassay (RIA) and their presence confirmed by RIA measurements (1) in rat and human retinae on HPLC fractions and (2) by the detection of the C-terminal portion of the precursor to AVP and its associated Np = propressophysin (CPP). The AVP and OT content in the retina of the rat was modified by light: AVP and OT content was smaller at 2 a.m. than at 2 p.m., but was increased by a 7 day constant exposure to darkness. In contrast, pituitary content was decreased after 7 days of constant darkness. If one optic nerve was cut we observed a decrease in retinal AVP content compared to the contralateral side and a decrease in pituitary AVP content. Our data clearly demonstrated the presence of AVP, OT and Np in the retina and their variation induced by light. It is probable that these peptides are of central origin.