Atrial natriuretic factor in human autonomic failure.

To investigate whether excessive circulating levels of atrial natriuretic factor (ANF) are responsible for orthostatic hypotension and nocturnal polyuria in patients with autonomic failure, we determined the circadian variation in the plasma concentration of ANF as well as the response of this peptide to changes in posture and extracellular fluid volume in patients with autonomic failure. We found that the plasma concentration of ANF was significantly lower in patients with autonomic failure than in controls. Patients with autonomic failure had a significantly higher urinary sodium excretion during the night (8 PM to 8 AM) than during the day (8 AM to 8 PM), and the plasma concentration of ANF significantly decreased during the night in these patients, indicating that high circulating levels of ANF were not the cause of the nocturnal natriuresis. Furthermore, circulating levels of ANF responded appropriately to reductions in right atrial pressure induced by head-up tilt and extracellular fluid volume changes induced by mineralocorticoid administration. These results indicate that exaggerated circulating levels of ANF are not responsible for the orthostatic hypotension or nocturnal natriuresis in patients with autonomic failure, and that appropriate regulation of ANF occurs in patients with autonomic failure.

N-arginine dibasic convertase (NRD convertase): a newcomer to the family of processing endopeptidases. An overview.

N-arginine dibasic convertase (NRD convertase) (accession number L27124) is a metalloendopeptidase from rat brain cortex and testis which cleaves peptide substrates on the N-terminus of arginine residues in basic doublets. Its predicted amino acid sequence contains the putative zinc binding motif HXXEH in a region which exhibits 35% and 48% similarity with E coli protease III (pitrilysin E.C 3.4.99.44) and rat or human insulinase (E.C 3.4.99.45) respectively. This feature clearly classifies this endopeptidase as a member of the pitrilysin family of zinc-metalloproteases. However, the NRD convertase sequence contains a distinctive additional feature consisting of a 71 acidic amino acid stretch. Its substrate selectivity and the characteristic motifs of its amino acid sequence allow us to propose this new metalloendopeptidase as the first member of a new class of processing enzymes.

Multiple forms of somatostatin-like immunoreactivity in the hypothalamus and amygdala of the rat: selective localization of somatostatin-28 in the median eminence.

The presence of multiple forms of somatostatin-like immunoreactivity (SSLI) in the rat hypothalamus was confirmed using a sensitive radioimmunoassay in conjunction with gel filtration chromatography and high performance liquid chromatography (HPLC). Gel filtration chromatography of hypothalamic extracts revealed the presence of four forms of SSLI with estimated molecular weights of 1500, 3000, 6000 and 10000. Analysis by HPLC indicated that the 1500 and 3000 mol. wt forms of SSLI corresponded respectively to somatostatin-14 (SS14) and somatostatin-28 (SS28) whereas the 6000 and 10000 mol. wt forms eluted together as a composite peak of high molecular weight somatostatin (HMW-SS). The proportions of SS14 (63%), SS28 (12%) and HMW-SS (25%) present in the hypothalamus were similar to those in the amygdala (59, 9 and 32% respectively). In contrast, the median eminence contained a greater proportion of SS28 than the other tissues: SS14, SS28 and HMW-SS were present in the proportions 40: 24: 26%. These results show that the rat median eminence differs from the hypothalamus as a whole in containing SS14 and SS28 in almost equimolar concentrations. The localized abundance of SS28 in the nerve terminals of the median eminence suggests a specific role for this peptide in the hypothalamic regulation of growth hormone secretion.

Endopeptidase-24.15 in rat hypothalamic/pituitary/gonadal axis.

Endopeptidase-24.15 (E.C. 3.4.24.15; EP-24.15) cleaves several substrates found in the hypothalamic/pituitary/gonadal axis, including gonadotropin-releasing hormone (GnRH) and the opioid peptides of the dynorphin family. We have examined the activity of EP-24.15 in these tissues as a function of maturation, of the estrous cycle, and in response to ovariectomy and estrogen replacement. A developmental regulation of EP-24.15-specific activity is apparent in anterior pituitary, in hypothalamus, and in the gonads. EP-24.15 is increased in the preoptic area and is decreased in the anterior pituitary in both male and female rats prior to puberty. The specific activity of EP-24.15 was increased following ovariectomy in the anterior pituitary and within medial and lateral preoptic nuclei. Testicular specific activity of EP-24.15 increased with age in a linear fashion, while ovarian EP-24.15 activity increased immediately prior to puberty, but returned to prepubertal levels by 65 days of age. The relevance of EP-24.15 to the metabolism of specific peptides is discussed.

Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules.

An aminopeptidase of the B-type, with an apparent M(r) 72,000 and pI = 4.9, was isolated from rat testes and characterized. The enzyme was able to remove only Arg and/or Lys residues from L-amino acid beta-naphthylamide derivatives and from the N-terminus of several peptides. No cleavage occurred in the case of Arg-Pro bonds as found in bradykinin and substance P. The enzyme was sensitive to cysteinyl reagents and to aminopeptidase inhibitors, such as bestatin, amastatin and arphamenines A and B. The aminopeptidase activity, tested with L-Arg beta-naphthylamide and with Arg0-Met-enkephalin as substrates, was inhibited by o-phenanthroline, and restored by Zn2+ suggesting its metallopeptidase character. The partial characterization of an aminopeptidase-B activity in rat brain cortex identified a protein which is biochemically and immunologically related to the testis enzyme. By immunohistochemistry, the aminopeptidase-B was found to be particularly abundant in the seminiferous tubules at late stages of spermatogenesis and was clearly detected in a restricted area of elongated spermatids. Remarkably, the enzyme was observed to concentrate massively in the residual bodies. Since this aminopeptidase-B was able in vitro to trim out N-terminal Arg and/or Lys residues from peptides mimicking processing intermediates, it is proposed that this enzyme may be involved in propeptide and proprotein processing mechanisms in the course of spermatid differentiation.

Endopeptidase EC 3.4.24.15 presence in the rat median eminence and hypophysial portal blood and its modulation of the luteinizing hormone surge.

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically-active peptides, the enzyme degrades gonadotropin-releasing hormone (GnRH) by cleaving the central Tyr5-Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo-hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats. The present results suggest that secretory and post-secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.

Expression of the thimet oligopeptidase gene is regulated by positively and negatively acting elements.

Thimet oligopeptidase (TOP) is a thiol-dependent metallopeptidase, which can cleave and thereby modulate the activity of many neuropeptides. The enzyme is active in many endocrine tissues, including testis, brain, and pituitary. In rat, the richest source of TOP is the testes, with a specific activity fivefold that of brain. The mechanism whereby rat TOP expression is regulated at the transcriptional level has been examined by reporter gene assay and electromobility shift assays after isolation of 1020 bp of upstream sequence. Computer analysis predicts a number of potential transcription factor-binding sites, which were examined by deletion analysis and DNA-binding studies. The promoter or its deletion fragments were fused to luciferase reporter gene vectors and introduced into GH3 pituitary, COS-1 kidney, MAT-Lu prostate, and GC-2spd(ts) spermatid cells. Two regions of the promoter have been identified: a positively acting region (-901/-219) and a strong negatively acting region (-219/-102). Concomitantly, potential transcription factors interacting with the cis-acting elements of the promoter were studied by gel electromobility shift assays. This work has identified a number of transcription factor-binding sites. However, no differences in the binding behavior in the various cell lines was observed.

Multiple forms of somatostatin-like immunoreactivity are not influenced by cholinergic denervation of rat hippocampus.

Hippocampal choline acetyltransferase was reduced by 89% in rats two weeks after electrolytic lesion of the septum. The hippocampal concentrations of somatostatin (SOM)-14, SOM-28 and high-molecular-weight SOM were unaltered, suggesting that the activity of hippocampal SOM neurones is not influenced by cholinergic afferents. The relevance of this finding to Alzheimer-type dementia and Down's syndrome is discussed.

High-molecular-weight forms of somatostatin are reduced in Alzheimer's disease and Down's syndrome.

Four molecular forms of somatostatin-like immunoreactivity (SOM-LI) are present in the human temporal cortex: SOM-14, SOM-28 and high-molecular-weight forms (HMW-SOM) of 7500 and 12,000 daltons. SOM-14 and HMW-SOM are depleted in cortical tissue from cases of pre-senile Alzheimer-type dementia (ATD), but there is a disproportionate reduction in HMW-SOM. In cases of Down's syndrome (DS) with the neuropathological and neurochemical changes of ATD, the total concentration of SOM-LI was similar to that in control cases and the proportions of molecular forms present were comparable. However, there was a significant reduction in the concentration of HMW-SOM. These results show that ATD and DS may share a common abnormality in the biosynthesis and/or post-translational processing of cortical SOM.

Different patterns of molecular forms of somatostatin are released by the rat median eminence and hypothalamus.

The molecular forms of somatostatin (SOM) released from hypothalamic slices and from the isolated median eminence (ME) in vitro were compared by high-performance liquid chromatography and radioimmunoassay. SOM-14 was the predominant form of the peptide released from hypothalamic slices, although small amounts of SOM-28 were detected. Perifusates of ME tissue contained a larger proportion of SOM-28 and higher molecular weight peptides were present; depolarization increased the rates of release of all molecular forms of SOM. These results suggest that the capacity to release SOM-28 and high-molecular-weight forms of SOM may be a specialized function of nerve terminals in the ME.

Gene expression of the dibasic-pair cleaving enzyme NRD convertase (N-arginine dibasic convertase) is differentially regulated in the GH3 pituitary and Mat-Lu prostate cell lines.

NRD convertase (N-arginine dibasic convertase, NRD-C) is a dibasic selective metalloprotease which cleaves on the N-terminal side of an arginine residue in a dibasic pair. Abundant in endocrine tissues, the highest levels are found in testis. The mechanism whereby NRD-C expression is regulated at the transcriptional level has been examined by reporter-gene assay and electrophoretic-mobility-shift assays. Analysis of the rat and human promoters show that they are highly conserved, containing a number of motifs which may correspond to transcription-factor binding sites. The rat promoter has been cloned into a luciferase reporter vector and analysed in a number of cell lines. Full functionality of the promoter is observed with 5' deletions to 411 bp upstream of the transcriptional start site in spermatid, prostate and pituitary cell lines. Further deletion to 101 bp causes a complete loss of activity in spermatid and prostate lines. By contrast, GH3 pituitary cells display no reduction in promoter activity with deletion to 101 bp of upstream sequence. A number of transcription-factor binding sites have been identified by electrophoretic-mobility-shift assays in the region 411-101; however, no differences in binding between the cell lines were observed.

Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides.

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.

Modulation of POMC expression in human neuroectodermal cells.

Neuroblastoma cell lines have been reported to contain two proopiomelanocortin (POMC) mRNA transcripts. We have now shown by immunocytochemistry and radioimmunoassay (RIA) that a number of neuroectodermally derived cell lines contain immunoreactive beta-endorphin although cell concentrations were not characteristic of any tumour type. To explore further the functional significance of beta-endorphin expression, we analysed neuroblastoma cell lines having intermediate (I), substrate adherent (S) and neuronal (N) phenotypes. No differences in cell beta-endorphin content were detected. However, the expression of POMC mRNA and of immunoreactive beta-endorphin was reduced within a few hours of treatment of these cell lines with retinoic acid. Culture of the cell lines in the presence of beta-endorphin resulted in small but significant increases in growth. Although the POMC gene is in the same chromosomal segment as N-myc, which is normally amplified in neuroblastoma, no corresponding amplification of POMC could be demonstrated. The data suggest that POMC gene products may contribute to the autocrine/paracrine growth of neuroectodermal tumours.

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.

Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues.

A metalloendopeptidase that selectively cleaves doublets of basic amino acids on the amino-terminal side of arginine residues was purified to homogeneity from rat testes and analyzed further. Two catalytically active forms with apparent relative molecular masses of 110,000 and 140,000 Da, respectively, were present in the purified preparation of the enzyme. Antibodies raised against the purified testis endopeptidase revealed by immunoblot both the 110- and 140-kDa forms in both rat testis and brain cortex extracts. The isolated enzyme was inhibited by metal chelators and divalent cations. Its activity, lost after preincubation with EDTA, was restored by low concentrations of Zn2+ and Mn2+, thus demonstrating the metallopeptidase nature of the enzyme. This endopeptidase also exhibited a high sensitivity to amastatin (100% inhibition at 20 microM), an aminopeptidase inhibitor. A substrate specificity study using physiologically important or synthetic peptides containing a processing dibasic site indicated that cleavage occurred selectively at the amino-terminal side of an arginine residue, independent of the nature of the basic doublet. The enzyme produced such a cleavage at the Arg-Lys doublet of somatostatin 28 (Km = 43 microM), at the Arg-Arg doublet of dynorphin A (Km = 6.45 microM) and atrial natriuretic factor (Km = 6.25 microM), and at the Lys-Arg doublet of preproneurotensin-(154-170) (Km = 17.3 microM). Moreover, cleavage efficiency was found to be higher for the larger substrates. The distinctive properties of this endopeptidase imply that this protein is a member of a novel class of proteolytic enzymes that may be involved in the endoproteolytic maturation of hormonal precursors.

Distribution of thimet oligopeptidase (E.C. 3.4.24.15) in human and rat testes.

Thimet oligopeptidase (TOP:E.C. 3.4.24.15) is a thiol sensitive metalloendopeptidase which is widely distributed and active in most tissues including testis, brain and pituitary. In the median eminence it is postulated to play a role in the degradation of GnRH released from the hypothalamus and thus to modulate LH levels. In the rat and human, the testis is the richest source of TOP activity with levels 3- to 5-fold higher than that of the brain. In order to define the exact localisation of this enzyme within the rat and human testis, the distribution of TOP in the developing and adult gonad was examined in situ and in isolated cells by immunohistochemistry, western blotting and northern blotting analysis. Ontogeny studies have demonstrated that TOP is detectable by western blotting from 9 days with levels of expression increasing with the age of the animal. Immunolocalisation of the protein in the interstitium was positive from 9 days onwards but was negative within the seminiferous tubules before 35 days of age, whereas TOP mRNA was not detected within the testis until 35 days of age with subsequent stable expression levels up to 90 days. In the adult rat testis, a strong TOP immunoreactivity was observed within seminiferous tubules, in elongating and elongated spermatids and residual bodies. In the interstitial compartment, immunoreactivity was also observed in Leydig cells and throughout the interstitial space. Western blot analyses confirmed the distribution of expression observed using immunochemistry, however Leydig cells display a lower signal than expected from the immunohistochemical data. Northern hybridization showed that the transcript is present in pachytene spermatocytes, early spermatids, and residual bodies, whereas its presence was not observed in Leydig cells probably due to very low levels of expression of the message. Analyses of various human tissue extracts showed that the testis displays the highest levels of TOP mRNA, with immunohistochemical experiments revealing that, as in the rat, the protein is principally expressed in elongated spermatids/residual bodies, and in Leydig cells. It is concluded that in the human and rat testes, TOP is highly expressed, in particular in post-meiotic germ cells and Leydig cells. The possible involvement of TOP in proteolytic events associated with the process of spermiogenesis and Leydig cell function is currently under investigation.

N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes.

N-Arg dibasic convertase is a metalloendopeptidase from rat brain cortex and testis that cleaves peptide substrates on the N terminus of Arg residues in dibasic stretches. By using both an oligonucleotide and antibodies to screen a rat testis cDNA library, a full-length cDNA was isolated. The sequence contains an open reading frame of 1161 codons corresponding to a protein of 133 kDa that exhibits 35% and 48% similarity with Escherichia coli protease III (pitrilysin, EC 3.4.99.44) and rat or human insulinase (EC 3.4.99.45), respectively. Moreover, the presence of the HXXEH amino acid signature (XX = FL) clearly classifies N-Arg dibasic convertase as a member of the pitrilysin family of zinc-metalloendopeptidases. In addition, a Cys residue that may be responsible for the thiol sensitivity of the insulinase and N-Arg dibasic convertase was proposed. The protein sequence contains a distinctive additional feature consisting of a stretch of 71 acidic amino acids. We hypothesize that this metalloendopeptidase may be a member of a distinct class of processing enzymes.