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.     

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.     

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.     

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.     

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.     

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 Modulates the Stimulation of Rat Pineal N-Acetyltransferase Activity by Involving the α1 -Adrenergic Receptor

Delta sleep-inducing peptide (DSIP) has been isolated and characterized by its capacity to enhance delta sleep in rabbits. Up to now, sleep was the main target of DSIP research, but different extra-sleep effects of the peptide have been reported as well. Several mechanisms of action have been proposed, though no convincing evidence for any of them has been obtained so far. We recently detected that DSIP reduced the nocturnal increase of N-acetyltransferase (NAT) activity in rat pineal in a dose-dependent manner. The activity of this enzyme is known to be induced by adrenergic agonists and several studies have suggested that stimulation of alpha 1-adrenergic receptors potentiates the "basic" effect of beta-receptors. DSIP in the range between 20 and 300 nM significantly enhanced NAT activity induced by 10(-6) M norepinephrine in vitro, and a similar effect was observed with 2nMP-DSIP, a phosphorylated analog. Incubation with prazosin eliminated the enhancement, whereas propranolol reduced norepinephrine stimulation that was still increased by P-DSIP and probably DSIP. It was concluded that the sleep-peptide and its analog modulate the alpha 1-adrenergic receptor of rat pineal in its response to adrenergic agonists. The same mechanism may also be responsible for other biological activities of DSIP such as sleep-induction and stress-tolerance.     

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.     

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.     

Delta sleep-inducing peptide (DSIP)-like material exists in peripheral organs of rats in large dissociable forms

The presence of delta sleep-inducing peptide (DSIP) in brain has been shown by radioimmunoassay (RIA) and by immunocytochemistry. We now describe the occurrence of DSIP-like material in the peripheral organs of the rat as measured by RIA. Tissue from 12 areas was extracted with water, and the amounts of immunoreactive material found to be between 86 pg/mg tissue (muscle) and 849 pg/mg (stomach). Recoveries of about 80% of added DSIP were achieved at tissue concentrations of 1 mg/ml or less. This percentage was reduced in liver at higher concentrations. The percentage of small peptide adsorbed by charcoal was greatly increased at lower tissue concentrations in all organs. This effect was significant and linear. Chromatography on columns of Sephadex G-15 and G-25 showed immunoreactive material mostly larger than DSIP. Digestion with trypsin, however, produced small immunoreactive peptides with only a minimal reduction in total immunoreactivity. Thus, DSIP-like material is widespread in peripheral tissues and appears to exist mainly in a large form, probably bound to protein, that can be reduced in size by tryptic digestion and can be dissociated at lower concentrations of tissue to yield small immunoreactive peptides.     

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).     

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.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

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.     

Interaction of delta sleep-inducing peptide and its analogues with cellular membranes: a structure-function analysis

The possibility of a correlation between the membrane properties of the delta sleep-inducing peptide (DSIP) and its analogues and their biological activity in vivo was examined by a comparative study of the membrane effects of these peptides. The peptides exhibiting biological activity in vivo were shown to cause a statistically reliable disordering of lipids in thrombocyte plasma membranes similar to the effect of DSIP. The membrane effect of the D-Val2, D-Tyr2, and Tyr1, Pro2 analogues of DSIP had the same bimodal dose dependence characteristic of natural DSIP. Only a slight nonspecific lipid disordering was registered for Trp-Asp-Ala-Ser-Gly-Glu, a biologically inactive hexapeptide analogue. These results indicate a correlation between the biological activity of the peptides during in vivo tests and their membrane properties in vitro. The structure-function relationship was studied within the group of DSIP analogues examined in vitro. The DSIP modeling effect, especially pronounced under the action of stress factors, was suggested to be directly associated with the ability of DSIP to change the dynamic structure of biological membranes.     

Sleep-Inducing Properties of DSIP Analogs: Structural and Functional Relationships

The sleep-inducing activity of Delta Sleep-Inducing Peptide (DSIP) and its 13 synthetic analogs has been studied on rabbits with preliminary implanted electrodes. The peptides were injected into the lateral ventricle of cerebrum. Polygraphic computer monitoring of sleep–wake states was carried out at daytime for 7–12 h. DSIP and most analogs had no statistically significant effect on sleep compared to the control administration of saline to the same animals. [NMeAla²]DSIP and [Pro²]DSIP had a pronounced sleep-inducing effect and reliably increased the proportion of slow-wave sleep by 10–15% on average compared to the control. Several other analogs had a week sleep-inducing effect, increasing the proportion of slow-wave sleep during specific recording time only. [-Ala²]DSIP significantly suppressed sleep. In addition, this analog, as well as parent DSIP and four proline-containing nonapeptides, slightly increased the body temperature. The revealed differences may be due to both conformation properties and proteolytic resistance of the studied molecules, and it may reflect their indirect involvement in the control sleep–wake hormonal processes.     

Interaction of delta sleep-inducing peptide and its analogues with cellular membranes: A structure-function analysis

The possibility of a correlation between the membrane properties of the delta sleep-inducing peptide (DSIP) and its analogues and their biological activity in vivo was examined by a comparative study of the membrane effects of these peptides. The peptides exhibiting biological activity in vivo were shown to cause a statistically reliable disordering of lipids in thrombocyte plasma membranes similar to the effect of DSIP. The membrane effect of the D-Val²-, D-Tyr²-, and Tyr¹, Pro² analogues of DSIP had the same bimodal dose dependence characteristic of natural DSIP. Only a slight nonspecific lipid disordering was registered for Trp-Asp-Ala-Ser-Gly-Glu, a biologically inactive hexapeptide analogue. These results indicate a correlation between the biological activity of the peptides during in vivo tests and their membrane properties in vitro. The structure-function relationship was studied within the group of DSIP analogues examined in vitro. The DSIP modeling effect, especially pronounced under the action of stress factors, was suggested to be directly associated with the ability of DSIP to change the dynamic structure of biological membranes.     

Amphetamine-induced locomotor behavior of mice is influenced by DSIP

The delta sleep-inducing peptide (DSIP) has been shown to induce effects other than only delta sleep. One of these effects was the paradoxical thermoregulatory and locomotor response of rats to amphetamine after DSIP administration. In the present investigation we found similar effects of DSIP on the locomotor activity in mice. However, two different doses of DSIP (30 and 120 nmol/kg) and 3 doses of amphetamine (4, 10, and 15 mg/kg) produced a complex pattern of effects in mice tested at 22 degrees C. In general, DSIP-treated mice showed lower locomotor activity after amphetamine than controls, but under two conditions, both using 15 mg/kg amphetamine, DSIP produced higher scores; this occurred in the first two hours after amphetamine for the 30 nmol/kg DSIP group and in the third hour for mice given 120 nmol/kg DSIP. The results indicate that the effects of DSIP on locomotor behavior were dependent on the dosage of the peptide and the time of measurement as well as the level of amphetamine stimulation.     

Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia

Neuromodulatory delta sleep inducing peptide (DSIP) seems to be implicated in the attenuation of stress-induced pathological metabolic disturbances in various animal species and human beings. Mitochondria, as cell organelles, are considered especially sensitive to stress conditions. In this work, the influence of DSIP and Deltaran((R))-a recently developed product based upon DSIP-on processes of oxidative phosphorylation and ATP production in rat brain mitochondria and rat brain homogenates was studied. A polarographic measurement of oxygen consumption was applied to evaluate the impact of DSIP on maximal rates of mitochondrial respiration and coupling of respiration to ATP production. We provide evidence that DSIP affected the efficiency of oxidative phosphorylation on isolated rat brain mitochondria. This peptide significantly increased the rate of phosphorylated respiration V3, while the rate of uncoupled respiration V(DNP) remaining unchanged. It enhanced the respiratory control ratio RCR and the rate of ADP phosphorylation. DSIP and Deltaran exhibited the same action in rat brain homogenates. We also examined the influence of DSIP under hypoxia when mitochondrial respiratory activity is altered. In rats subjected to hypoxia, we detected a significant stress-mediated reduction of V3 and ADP/t values. Pretreatment of rats with DSIP at the dose of 120 microgram/kg (i.p.) prior to their subjection to hypoxia completely inhibited hypoxia-induced reduction of mitochondrial respiratory activity. The revealed capacity of DSIP to enhance the efficiency of oxidative phosphorylation found in vitro experiments could contribute to understanding pronounced stress protective and antioxidant action of this peptide in vivo.