Radioimmunoassay of delta sleep-inducing peptide using an iodinated p-hydroxyphenylpropionic acid derivative as tracer

A highly sensitive radioimmunoassay for delta sleep-inducing peptide (DSIP) has been developed. A p-hydroxyphenylpropionic acid conjugate of DSIP was used for radioiodination. Using reversed-phase high performance liquid chromatography the labelled DSIP derivative was isolated in a high yield and with a high specific activity. The assay allows measurement of DSIP-like material in body fluids with a minimum detectable concentration of 0.1 ng/ml standard DSIP (10 pg/tube).     

Immunohistochemical localization of delta sleep-inducing peptide (DSIP) in the brain and pituitary of the cartilaginous fish Scyliorhinus canicula.

The distribution of delta sleep-inducing peptide (DSIP) in the brain and pituitary of the cartilaginous fish Scyliorhinus canicula was investigated using the indirect immunofluorescence technique. Delta sleep-inducing peptide-like immunoreactive cell bodies were mainly observed in the nucleus lateralis tuberis of the hypothalamus. Immunolabeled perikarya were also distributed in the nucleus lobi lateralis hypothalami and in the dorso-lateral wall of the recessus posterioris. Most of these cells, located in the subependymal layers of the infundibulum and lateral lobes, had the typical aspect of cerebrospinal fluid-contacting elements. The DSIP-like immunoreactive fibers were localized in the basal telencephalon, within the regions of the nucleus interstitialis commissurae anterioris and the nucleus entopeduncularis. A dense network of DSIP-positive fibers was seen throughout the midcaudal hypothalamus, the lateral lobes, and the posterior lobe. In the pituitary, numerous DSIP-like immunoreactive cells were detected in the median lobe of the pars distalis. In particular, a high concentration of cells was seen in the dorsal wall of the median lobe, an area which is known to contain melanin-concentrating hormone (MCH)-producing cells. Comparison of the distribution of DSIP- and MCH-like immunoreactive cells revealed that the two neuropeptides are stored in the same cells of the median lobe of the pituitary. These findings provide the first evidence for the presence of a DSIP-related peptide in fish. The distribution of the immunoreactive material supports the view that DSIP may act as a neuromodulator and/or a hypophysiotropic factor. Moreover, the presence of DSIP-like immunoreactive cells in the pars distalis suggests that this peptide may exert autocrine or paracrine effect in the pituitary.     

Delta sleep-inducing peptide (DSIP)-like immunoreactivity in gut: coexistence with known peptide hormones

Delta sleep-inducing peptide-like immunoreactivity (DSIP-LI) has previously been demonstrated in brain neurons and in endocrine cells of the pituitary and the adrenal medulla. By means of three different antisera against synthetic DSIP we now describe the occurrence and distribution of DSIP-LI in several gut endocrine cells. The human gut was the richest source, where DSIP-LI was located in gastrin/CCK, secretin and PYY/glicentin cells. The rat and pig gut harbour a moderate number of immunoreactive cells in the antral mucosa but in the intestines DSIP-LI-containing cells were very few. By radioimmunoassay, the concentration of DSIP-LI was determined in extracts of various gut regions from man, pig and rat. The highest concentrations were found in all human specimens compared with corresponding samples in the pig and rat. In all three species, high-performance liquid chromatography revealed a single peak of DSIP-like material with approximately the same retention time as DSIP 3-9. Taken together, the present results provide evidence for the presence of DSIP-LI in gut endocrine cells in man, pig and rat; the human gut seems to be the richest source of DSIP-like peptides.     

Biosynthesis and processing of delta sleep-inducing peptide-like precursors in primary cultures of mouse anterior pituitary cells

The biosynthesis and processing of material resembling delta sleep-inducing peptide (DSIP) have been studied in mouse anterior pituitary primary cell cultures. Cells were pulse/chase incubated with 3H-labelled amino acids (Gly, Arg or Ala) and cell extracts were immunoprecipitated with DSIP antiserum. Labelled DSIP-related proteins were resolved by SDS/PAGE. Multiple forms of DSIP-immunoprecipitable material were observed, including three precursors of molecular mass 50-60 kDa which were processed to two major groups of intermediates of 35-45 kDa and 9-16.5 kDa. These intermediates appear to be processed to a DSIP-related peptide (molecular mass less than 3 kDa), which co-ran on reversed-phase HPLC with an endogenous form of DSIP in mouse anterior pituitary, but not with rabbit DSIP. This less than 3-kDa peptide incorporated [3H]Gly, but not [3H]Arg or [3H]Ala. In addition, it incorporated [3H]glucosamine, indicating that it was a glycopeptide. Secretion studies showed release of the less than 3-kDa DSIP-like glycopeptide and the 9-16.5-kDa group of intermediates into the medium. The present study demonstrates the biosynthesis of a small DSIP-like glycopeptide in mouse anterior pituitary cells, which is not identical with, but has similarities to, rabbit DSIP.     

Delta sleep-inducing peptide (DSIP): a still unresolved riddle

Delta sleep-inducing peptide (DSIP) was isolated from rabbit cerebral venous blood by Schoenenberger-Monnier group from Basel in 1977 and initially regarded as a candidate sleep-promoting factor. However, the link between DSIP and sleep has never been further characterized, in part because of the lack of isolation of the DSIP gene, protein and possible related receptor. Thus the hypothesis regarding DSIP as a sleep factor is extremely poorly documented and still weak. Although DSIP itself presented a focus of study for a number of researchers, its natural occurrence and biological activity still remains obscure. DSIP structure is different from any other known representative of the various peptide families. In this mini-review we hypothesize the existence of a DSIP-like peptide(s) that is responsible (at least partly) for DSIP-like immunoreactivity and DSIP biological activity. This assumption is based on: (i) a highly specific distribution of DSIP-like immunoreactivity in the neurosecretory hypothalamic nuclei of various vertebrate species that are not particularly relevant for sleep regulation, as revealed by the histochemical studies of the Geneva group (Charnay et al.); (ii) a large spectrum of DSIP biological activity revealed by biochemical and physiological studies in vitro; (iii) significant slow-wave sleep (SWS) promoting activity of certain artificial DSIP structural analogues (but not DSIP itself!) in rabbits and rats revealed by our early studies; and (iv) significant SWS-promoting activity of a naturally occurring dermorphin-decapeptide that is structurally similar to DSIP (in five of the nine positions) and the sleep-suppressing effect of its optical isomer, as revealed in rabbits. Potential future studies are outlined, including natural synthesis and release of this DSIP-like peptide and its role in neuroendocrine regulation.     

Demonstration of delta sleep inducing peptide in a strain of human small cell lung cancer by immunocytology]

The "delta sleep inducing peptide" (DSIP) is a regulatory peptide localized in the brain, the hypophysis and some endocrine cells of the gut. The present immunological study, performed with a monoclonal antibody to DSIP, provides evidence for the presence of DSIP-like immunoreactivity (DSIP-LI) in a strain of small cell carcinoma. The specificity of the immunoreaction was assessed by the tests using heterologous antigen known to be secreted by these cells. The DSIP could play a role in the course of this disease.     

Separation of tryptophan from DSIP, a Trp-nonapeptide, by adsorption to aluminum oxide

Delta sleep-inducing peptide (DSIP) has been found to induce sleep as well as extra-sleep effects. Although the presence of endogenous DSIP-like material has been demonstrated, the metabolic fate of injected DSIP has not been clarified so far. A major obstacle in monitoring degradation of DSIP has been the lack of an easy method to separate DSIP from tryptophan (Trp). Cleavage of the N-terminal Trp apparently represents the first and most important step in the metabolism of the peptide. Adsorption to aluminum oxide has been found to separate the two compounds and optimal conditions for the separation are described. Quantitative determination of the degradation of DSIP in plasma or serum is now rapidly achieved. The method should help to advance metabolic studies of DSIP. Other applications such as extraction of DSIP from solutions are also possible.     

Delta-sleep-inducing peptide (DSIP): An update

The isolation and characterization of delta-sleep-inducing peptide (DSIP) achieved from 1963 to 1977 were reviewed in 1984. The first reports describing sleep as well as extra-sleep effects of DSIP also were included in that work. Only two years later, much additional literature concerning DSIP has accumulated. Besides further sleep-inducing and/or-supporting effects of DSIP in animals, considerable work has been carried out to evaluate the potential use of the peptide for therapeutic purposes such as treatment of insomnia, pain, and withdrawal. Immunohistochemical as well as radioimmunochemical studies provided further insights into the natural occurrence of the nonapeptide and the distribution of DSIP-like material in the body, suggesting possible relations of the peptide to certain diseases. Various physiological functions of DSIP and a possible mechanism of action involving the modulation of adrenergic transmission remain to be established.     

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP

Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.     

Distribution and characterization of immunoreactive porcine C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is a new member of the natriuretic peptide family recently identified in porcine brain (1). We raised an antiserum against porcine CNP and set up a radioimmunoassay (RIA) for CNP. Using this RIA system, distribution of immunoreactive (ir-) CNP in porcine tissue was measured and compared with that of ir-atrial natriuretic peptide (ANP) and ir-brain natriuretic peptide (BNP). Tissue concentration of ir-CNP in brain was the highest of the three natriuretic peptides at about 0.79 pmol/g wet wt. CNP was present in medulla-pons in high concentration, with a significant concentration detected in cerebellum. In contrast, ir-CNP was not detected in peripheral tissue, including heart, in a significant concentration. These data demonstrated sharp contrasts in the distribution of the three natriuretic peptides, suggesting that CNP is a natriuretic peptide functioning in the central nervous system.     

Delta sleep-inducing peptide (DSIP)-like material is absorbed by the gastrointestinal tract of the neonatal rat

Entry of delta sleep-inducing peptide (DSIP) into the circulation from the gastrointestinal (GI) tract was studied in unweaned rat pups. The pups were fed an analog of DSIP (N-Tyr-DSIP) or 125I-N-Tyr-DSIP and blood samples collected. Significant increases in plasma DSIP-like immunoreactivity occurred after the feeding of 100 ωg/animal of N-Tyr-DSIP but not after vehicle (normal saline) or 1 ωg/animal. Column chromatography showed this immunoreactivity to coelute with intact DSIP and des-Trp¹- DSIP. A small but statistically significant increase of immunoreactivity occurred in the plasma of pups whose nursing mothers were injected with N-Tyr-DSIP but not in those whose mothers were injected with saline. Radioactivity appeared in both the brain and blood of 1–2 and 10 day old rat pups fed 125I-N-Tyr-DSIP. Although only a small amount of the radioactivity in plasma co-eluted with intact 1251I-N-Tyr-DSIP on column chromatography, almost all of the radioactivity in brain did, suggesting that the radioactivity in the brain represented crossing of the blood-brain-barrier by the peptide and not just contamination by blood. The results cannot be explained by either regurgitation of intestinal contents, or by stimulation of endogenous peptide. They show that a DSIP peptide administered orally can be absorbed through the GI tract into the systemic circulation.     

Atrial natriuretic factor in human plasma

A reproducible and sensitive radioimmunoassay (RIA) was developed to measure ANF in human plasma. Immunoreactive ANF was extracted from plasma with Sep-Pak cartridges, using 0.2% ammonium acetate (pH 4) with acetonitrile. The sensitivity of the assay was 3.9 pg/ml. The coefficient of variance for inter-assay and intra-assay was 16.8% and 6.8%, respectively. In normal healthy subjects (n = 67), ANF content was 11.9 +/- 1.3 pg/ml (mean +/- SEM). Significantly-higher ANF concentrations were found in proximal coronary sinus blood, being 6 to 37 times greater than in the peripheral circulation. Comparison of the prior extraction method with direct RIA revealed a good correlation (r = 91) in samples containing higher than 100 pg/ml ANF. No correlation was observed with lower values. The elution profiles of reverse-phase HPLC of peripheral and coronary sinus plasma extracts were similar but somewhat complex, with the main immunoreactive peak corresponding to a low-molecular-weight peptide.     

Presence and distribution of immunoreactive and bioactive FMRFamide-like peptides in the nervous system of the horseshoe crab, Limulus polyphemus

FMRFamide immunoreactivity was detected in all regions of the Limulus nervous system, including the brain (6.5 +/- 0.6 pg FMRFamide/mg), cardiac ganglion (2.06 +/- 0.67 pg FMRFamide/mg), and ventral nerve cord (5.8 +/- 0.7 pg FMRFamide/mg). The distribution of immunoreactive FMRFamide (irFMRFamide) was mapped by immunofluorescence and the distribution corresponded to regional RIA data. A good proportion of the CNS and cardiac ganglion neuropile contained irFMRFamide, and fluorescent cell bodies were observed in several areas. High performance liquid chromatography (HPLC) was employed to separate and characterize the FMRFamide-like peptides from extracts of Limulus brains. HPLC fractions were analyzed using coincidental radioimmunoassay and bioassay (the radula protractor muscle of Busycon contrarium). There appear to be at least three FMRFamide-like peptides in the Limulus brain, including one similar to clam FMRFamide. FMRFamide acts on Limulus heart in a biphasic manner at relatively high concentrations (10(-5)M), but has no effect on the activity of the isolated ventral nerve cord. These data suggest that in Limulus FMRFamide-like peptides are acting as neurotransmitters, or neuromodulators.     

Neurohypophyseal hormone-like peptides in the ovine pineal gland using reverse-phase liquid chromatography and radioimmunoassay

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.     

Peptidergic modulation of sleep: a comparative study of analogs of the peptide DSIP

Effects of intracerebroventricular administration of 3 DSIP analogues with higher stability against proteolysis, on subsequent sleep were studied in rabbits and rats previously implanted with electrodes and cannulas. Significant increase of total sleep time (mainly due to slow wave sleep) after administration of the peptides (D-Trp1) DSIP and (D-Tyr1) DSIP as compared with the control injections to the same animals was found during the first 3-5 hours in rabbits and 12 hours in rats. Shortened analogue (D-Trp1) DSIP1-6 had no effect on rabbit sleep and significantly reduced slow wave sleep in rats during the first 3 hours. It is concluded that some DSIP-like peptides which are more stable against aminopeptidases, can modulate rodent sleep.     

Extraction and immunochemical characterization of delta sleep-inducing peptide-like material from the porcine pituitary and adrenal gland

The naturally occurring forms of delta sleep-inducing peptide (DSIP) are not fully identified. In the present study, porcine pituitaries and adrenal glands were extracted in water, saline or acid under various conditions and immunoreactive DSIP (IR-DSIP) quantified by radioimmunoassay. The highest concentrations were measured in anterior pituitary extracts (40.8 +/- 2.6 ng/g tissue weight) recovered using water with aprotinin. However, high performance liquid chromatography (HPLC) indicated degradation of hydrophobic forms of IR-DSIP in water extracts. Extraction in acetic acid including C18 Sep-Pak purification resulted in an elution profile of IR-DSIP in adrenal extracts with a major peak coeluting with synthetic DSIP [DSIP(1-9)], whereas anterior pituitary extract showed material of higher hydrophobicity. Approximately 30% of IR-DSIP in anterior pituitary as well as in adrenal gland extracts seemed to be glucosylated, as based on concanavalin A chromatography. One of the DSIP-immunoreactive components by immunoblotting (molecular mass 25 kDa) was identified in both pituitary and adrenal gland extracts. In conclusion, several chromatographically distinct forms of IR-DSIP are present in the porcine pituitary and adrenal gland. IR material eluting as DSIP(1-9) is present in adrenal gland extract. The procedure and solution used for tissue extraction seem to be essential in order to obtain reliable elution positions on HPLC.     

Delta sleep-inducing peptide (DSIP) stimulates LH release in steroid-primed ovariectomized rats

Delta sleep inducing peptide (DSIP) has been shown to increase sleep in various animals and it is found in various parts of the brain including the hypothalamus. While intraventricular administration of DSIP (2 or 10 micrograms) failed to affect LH release in ovariectomized rats, in two separate experiments DSIP (2 or 10; 15 or 30 micrograms) promptly stimulated LH release in ovariectomized estrogen, progesterone-primed rats. However, DSIP (10(-8) or 10(-6)M) had no effect on either basal or luteinizing hormone-releasing hormone-induced in vitro LH release from the hemipituitaries of ovarian steroid-primed rats. These findings are in accord with the hypothesis that DSIP or DSIP-like peptide(s) may activate the hypothalamic neural circuitry responsible for stimulation of LH release reported to occur during sleep.     

Characterization of endozepine-related peptides in the central nervous system and in peripheral tissues of the rat

Endozepines represent a novel family of regulatory peptides that have been isolated by their ability to displace benzodiazepines from their binding sites. All endozepines derive from an 86 amino acid precursor polypeptide called diazepam binding inhibitor (DBI), which generates, through proteolytic cleavage, several biologically active endozepines. The aim of the present study was to compare the molecular forms of endozepines present in different regions of the rat brain and in various peripheral organs using an antiserum raised against the central (biologically active) region of DBI. Combination of HPLC analysis and RIA detection revealed the existence of two major forms (peaks I and II) of endozepine-immunoreactive peptides. The retention times of the two peaks (36 and 39 min, respectively) were identical in all tissues or organs tested. Western blotting analysis of cerebral cortex extracts confirmed the existence of two immunoreactive species with apparent molecular weights 4000 and 6000 Da, which respectively correspond to peaks I and II. Tryptic digestion of peaks I and II generated a single immunoreactive peptide that coeluted with the synthetic octadecaneuropeptide ODN [DBI(33–50)]. These results show that, in different parts of the brain and in various peripheral organs, DBI is rapidly processed to generate two peptides of apparent molecular weight of 4000 and 6000 Da, which both possess the biologically active determinant of endozepines.     

ProCNP and CNP are expressed primarily in male genital organs

Lack of knowledge about the cellular origin of C-type natriuretic peptides (CNP) in the body has hampered the understanding of their biology. We examined the tissue specific expression of proCNP and CNP in the pig. The concentration of the CNP precursor, proCNP, was measured in extracts of 32 different tissues using a newly developed RIA. In 22 tissue extracts, we also measured CNP using a commercial RIA. In selected tissues, CNP mRNA was quantified by PCR, and the cellular CNP and proCNP localization was visualized by immunocytochemistry. Extracts from selected tissues were examined by gel chromatography. The highest peptide concentrations were found in extracts from the epididymis, seminal vesicles and prostate. CNP mRNA in the seminal vesicles and epididymis was 125-fold higher than in the other tissues examined. Gel chromatography showed that a CNP-53-like peptide is the dominant CNP tissue-form. Immunocytochemistry confirmed the pattern of peptide expression measured by RIA. In conclusion most proCNP-derived peptides are synthesized in epithelial cells in the epididymis, the prostate gland and in the seminal vesicles. The expression in male genital organs suggests a role of CNP in reproduction.     

The hypnogenic effects of delta sleep-inducing peptide (DSIP) analogs: a comparative study in rabbits and rats]

Hypnogenic effects of 3 DSIP analogs with a higher stability against aminopeptidase activity have been studied in rabbits and rats using intraventricular administration (injections and infusions). An analog (D-Ala-2) DSIP augmented slow wave and paradoxical sleep within the 5th, 8th and 11th hours of the recording period. An analog (D-Val-2) DSIP made the same within the 8th and 10th hours, and hexapeptide (D-Ala-2) DSIP (1-6) increased sleep during the 1st, 3rd, and 5th hours. Both nonapeptides augmented sleep in rabbits as well as in rats, though hexapeptide produced this effect in rabbits only, that might be related to some difference in distribution and colocalization of endogenous DSIP-like peptide in the pituitary of two rodent species. It may be suggested that hypnogenic activity of DSIP analogs is determined by the structure of administrated molecule, being mediated by such hormones as GRF and CLIP.