DSIP Research & Studies

The research aims to elucidate the sleep-promoting mechanism of DSIP-CBBBP, exploring its impact on neurotransmitter levels and sleep regulation, and to analyze its composition and structure.

A structural analogue of the DSIP, peptide KND, previously showed higher detoxification efficacy upon administration of the cytotoxic drug cisplatin, compared to DSIP. DSIP and KND were investigated using the model of acute myocardial infarction in male SD rats and the model of acute focal stroke in C57Bl/6 mice. A significant decrease in the myocardial infarction area was registered in KND-treated animals relative to saline-treated control animals (19.1 ± 7.3% versus 42.1 ± 9.2%). The brain infarction volume was significantly lower in animals intranasally treated with KND compared to the control saline-treated animals (7.4 ± 3.5% versus 12.2 ± 5.6%). Injection of KND in the first minute of reperfusion in the models of myocardial infarction and cerebral stroke reduced infarction of these organs, indicating a pronounced cardioprotective and neuroprotective effect of KND and potentiality for the treatment of ischemia-reperfusion injuries after transient ischemic attacks on the heart and brain, when administered during the reperfusion period. A preliminary pilot study using the model of myocardial infarction with the administration of DSIP during occlusion, and the model of cerebral stroke with the administration of KND during occlusion, resulted in 100% mortality in animals. Thus, in the case of ischemia-reperfusion injuries of the myocardium and the brain, use of these peptides is only possible during reperfusion.

16 DSIP analogues with substitutions of 1-2 amino acid residues were synthesized in order to investigate their potential use in medicine. Antioxidative properties of these peptides were studied in vitro and their detoxifying activity was examined in vivo on a model of toxicosis that was induced by the cisplatin cytostatic, which has been widely used in the cancer treatment. Practically all the studied DSIP analogues were shown to exhibit considerable direct antioxidative activity (AOA), and that of the ID-6 analogue was higher than AOA of DSIP and comparable with AOA of vitamin C and β-carotine. This analogue also demonstrated the most pronounced detoxifying effect towards cisplatin action, resulting in a decrease in the animal death from the acute cisplatin toxicity to 17% (in comparison with 50-67% for the control animals) and restoration of a number of cisplatin-sensitive biochemical blood parameters: decrease in the activity of aspartate aminotransferase and alanine aminotransferase and downregulation of the concentration of the final products of nitrogen exchange (creatinine and urea). Thus, the DSIP-relative peptides could be promising agents for the decrease in the toxic effects of cytostatics that are used in oncology.

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.

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.

The effects of the ACTH (4-10) analogue, ACTH (4-7)-Pro-Gly-Pro, and delta-sleep inducing peptide (DSIP) on the induction of Fos immunoreactivity in the hypothalamic parvocellular paraventricular nucleus (pPVN) and limbic brain regions were studied in Wistar rats with high (resistant) or low (predisposed) resistance to emotional stress, predicted from differences in their open-field behaviour. Fos-immunoreactive (Fos-IR) cells were counted in brain sections automatically with a computer-based image analyser. Under basal conditions, Fos-IR cell numbers were greater in the pPVN in the predisposed rats, but were lower than in the resistant rats in the basolateral amygdala and medial and lateral septum. Intraperitoneal DSIP injection (30 μg/kg) increased basal Fos-IR cell number in the pPVN and lateral septum in resistant rats, with no effects in predisposed rats. ACTH (4-10) analogue (50 μg/kg)increased Fos expression in the pPVN in both resistant and predisposed rats, with essentially no effects in the basolateral amygdala or medial and lateral septum. Emotional stress (60 min restraint and intermittent subcutaneous electrical shocks) increased Fos expression in the pPVN and medial and lateral septum similarly in predisposed and resistant rats, but in the basolateral amygdala in only the predisposed rats. Intraperitoneal DSIP injection reduced the increases in Fos-IR cell number after emotional stress, particularly in predisposed rats. In predisposed rats DSIP decreased the number of Fos-IR cells in the pPVN and the medial and lateral septum, with no change in the basolateral amygdala. In resistant rats, DSIP decreased Fos expression only in the lateral septum. ACTH (4-10) analogue injection inhibited stress-induced Fos expression in the pPVN and the medial septum, but only in predisposed rats. The experiments indicate that DSIP and ACTH (4-10) analogue reduce pPVN and limbic neurone responses to emotional stress in the rats predisposed to emotional stress; the effects on Fos expression may play a role in the biological activities of these peptides.

Up to the beginning of modern sleep research, theories about sleep have dominated the scene. With their philosophic background, they must be considered as erroneous by present standards. The history of sleep-inducing drugs has followed its own pathways, which were independent from sleep research. Up to the 19th century, opium and alcohol were used predominantly as hypnotics, later, empirically found chemical compounds were added. Today, new drugs are tested by methods of modern sleep research before they reach the market. Electrophysiology, whose origins are described, formed the basis of electroencephalography (EEG). The history of EEG is an important part of the present exposé. The discovery of rapid eye movements (REM) during sleep has been one of the most important achievements in modern sleep research. It led to a new stage classification - which is still used today - as well as to the discovery of sleep cycles. Subsequently, polysomnography has been increasingly used. Additional methods are actometry and the spectral analysis of the sleep EEG. Research of endogenous sleep substances such as "Delta Sleep Inducing Peptide (DSIP) has been actively pursued in the last 25 years. It is unlikely that one particular endogenous substance underlies sleep regulation. Rather a complex system involving different neurotransmitters must be postulated. Sleep disorders medicine is a new medical discipline which has undergone a rapid development. In the USA more than 1000 "sleep disorders centers" have arisen in the past few years. A description of the new 1990 classification of sleep disorders is provided, and narcolepsy, sleep apnea syndrome and some disturbances of the sleep-wake cycle are briefly characterized.

In the present study, we examined whether delta sleep-inducing peptide (DSIP) was (1) secreted from neurons on depolarization and (2) degraded by membrane-associated peptidases. Incubation of DSIP with rat brain membrane resulted in the degradation of DSIP with liberation of tryptophan, an N-terminal amino acid of DSIP. Bestatin and puromycin, aminopeptidase inhibitors, significantly inhibited the degradation of DSIP and release of tryptophan. The releases of immunoreactive DSIP-like substance (irDSIP) from rat brain slices and synaptosomes were significantly stimulated by high K(+)-evoked depolarization. The released irDSIP was coeluted with native DSIP on gel filtration chromatography. High K(+)-evoked release of irDSIP did not show extracellular Ca(2+)-dependency. This Ca(2+)-independency suggests that the secretory pathway of DSIP may be different from that of other neurotransmitters. These results demonstrate that DSIP is released from nerve endings on depolarization and inactivated by membrane-associated puromycin-sensitive aminopeptidase. Therefore, DSIP may serve as a neuropeptide-like material in the central nervous systems.

Monoclonal antibodies were produced following immunization of rats with delta sleep-including peptide (DSIP). The spleen cells of the rats were fused with the myeloma cell line SP2/0. The supernatants of hybridomas were screened on a solid-phase immunoassay using dot-immunobinding of DSIP and some DSIP fragments. The supernatants of six stable producer clones were found to react with DSIP. From this procedure it was also deduced that all these monoclonal antibodies recognized epitope(s) of the penta carboxy-terminal region of DSIP (DSIP5-9). Application of these monoclonal antibodies to rat median eminence sections gave a strong immunolabelling of a large population of fibres and terminal-like structures, mainly localized through the lateral areas. Elution-restaining experiments using a monoclonal antibody to DSIP and a polyclonal antiserum to luteinizing hormone-releasing hormone (LHRH) showed that the patterns of immunoreactivity respectively visualized overlap almost completely. Although numerous LHRH-immunoreactive neuronal elements were also easily demonstrated in the median eminence of the mouse, the hamster and the gerbil species, incubation of sections with monoclonal antibodies to DSIP failed to give any immunoreaction. Taken together these data argue for the independence of the DSIP/LHRH immunolabelling systems. Furthermore, it was demonstrated that DSIP5-9-related epitopes detected in the rat median eminence have no counterpart in the three other rodent species investigated. These species differences may reflect the fact that the carboxy-terminal sequence of the nonapeptide DSIP originally discovered in the rabbit is not conserved in all rodent species.

Delta sleep-inducing-peptide (DSIP) has been reported to increase sleep in subjects with insomnia. The authors studied cerebrospinal fluid (CSF) DSIP-like immunoreactivity (DSIP-LI) in 15 drug-free male subjects with a DSM-IIIR diagnosis of schizophrenia. The subjects underwent a lumbar puncture and three nights of polysomnography. CSF DSIP-LI was significantly correlated with polysomnography the night before the LP: with stage 3 sleep (p = 0.05), stage 3 and delta (stages 3 + 4) sleep during the first nonrapid eye movement NREM period (p = 0.02 and p = 0.05, respectively) and the ratio of the first and second NREM period (p < 0.05), and negatively with stage 2% sleep (p < 0.05). Whether this first report of a potential relationship between CSF DSIP-LI and slow-wave sleep in man might be generalized to sleep in nonpsychiatric subjects awaits further study.

Delta sleep-inducing peptide (DSIP) and P-DSIP, phosphorylated analogue, were found to have enhancing effects on hypothermia induced by i.p. injection of apomorphine (2 mg/kg), a dopamine agonist. Further, the action of P-DSIP appeared and diminished more quickly than that of DSIP. A minimal effective dose of these peptides was 10 ng and the dose-response relationship exhibited an inverted bell-shape with a maximal effective dose of 1 microgram. By the pretreatment of anti-DSIP the enhancing effect of DSIP but not P-DSIP, was totally abolished and the action of both peptides was antagonized by haloperidol. These findings suggest that DSIP and P-DSIP have a close relation to the dopaminergic system on the thermoregulatory mechanisms.

The nonapeptide delta-sleep-inducing peptide (DSIP) has been isolated from venous blood of rabbits induced to sleep. Numerous reports have described sleep as well as extra-sleep effects. Radiochemical and immunochemical data suggest a relationship of DSIP with the pineal gland supported by interactions of this peptide with pineal functions such as the serotonin N-acetyltransferase activity. In order to demonstrate the natural occurrence of DSIP-like material associated with high Mr proteins in the ovine pineal, organs were water-extracted and fractionated by ultrafiltration and gel filtration. Radioimmunoassay (RIA) for DSIP-like fragments of the fractions revealed considerable amounts of pineal DSIP-like immunoreactivity (DSIP-LI) apparently existing in small as well as large molecular forms. Acidification of large DSIP-LI forms resulted in the elution from Sephadex G-50 of Mr less than or equal to 1,000 DSIP-like material. This free DSIP-LI form coeluted with the synthetic DSIP nonapeptide from microBondapak C18 on high-performance liquid chromatography. The results, therefore, appear to indicate the presence of a (biospecific) noncovalent intermolecular interaction of DSIP (1-9) with proteins (Mr greater than or equal to 10,000) of the ovine pineal gland.

DSIP and CLIP [ACTH(18-39)] immunoreactive (IR) neurons and fibers were examined in the human hypophysis and pituitary stalk using immunmohistofluorescence and peroxidase-antiperoxidase methods. Double-stained and adjacent stained sections demonstrate that DSIP is colocalized in about 75% of CLIP-IR-like cells in the anterior pituitary and in residual intermediate lobe cells. Only few CLIP-IR-like fibers are observed in the posterior lobe. On the contrary, a high density of DSIP-IR fibers is visualized in the stalk. It is suggested that DSIP acts as a sleep promoting factor (one of many other actions) and that CLIP increases the paradoxical sleep, so that these two peptides could play a role in the regulating system of the sleep-waking cycle.

The biostability of DSIP (delta sleep-inducing peptide) and two analogs in blood was investigated in order to determine if rates of inactivation contribute to variable effects in vivo. Incubation of DSIP in human or rat blood led to release of products having retention times on a gel filtration column equivalent to Trp. Formation of products was dependent on temperature, time, and species. Incubation of 125I-N-Tyr-DSIP and 125I-N-Tyr-P-DSIP, a phosphorylated analog, revealed slower degradation and, in contrast to DSIP, produced complex formation. An excess of unlabeled material did not displace the radioactivity supporting the assumption of non-specific binding/aggregation. It was concluded that the rapid disappearance of injected DSIP in blood was due to degradation, whereas complex formation together with slower degradation resulted in longer persistence of apparently intact analogs. Whether this could explain the sometimes stronger and more consistent effects of DSIP-analogs remains to be examined.

The effect of a single injection of synthetic delta-sleep-inducing peptide (DSIP, 7 nmol/kg) into the lateral ventricle of 10 cats was investigated by monitoring the sleep-wake cycle during an 8 h period. A significant decrease in sleep latency and a significant increase in total sleep and in total slow wave sleep (SWS) was found following DSIP administration. The increase in sleep resulted exclusively from a significant increase in deep slow wave sleep (S2), while light slow wave sleep (S1) was significantly decreased. Neither the total amount of REM sleep, nor hourly values of REM sleep were affected by DSIP application. Additional measures of REM sleep, like REM sleep latency, mean episode number and mean episode length were not different from those found in control conditions. DSIP was immediately effective since the amount of S2 increased to more than 50% in the first postinjection hour and the difference from the control value was highly significant. The increase in S2 was maintained over 7 h, and disappeared by the eighth hour. The increase in S2 was caused by a prolongation of S2 episodes and not by their more frequent occurrence. The results obtained suggest a sleep-facilitating property of DSIP.

The concentrations of delta sleep-inducing peptide (DSIP)-like (DSIP-LI) and P-DSIP-like (phosphorylated, Ser7) immunoreactivity (P-DSIP-LI) were measured by specific radioimmunoassay in the cerebrospinal fluid (CSF) of patients with senile dementia of the Alzheimer type [SDAT, subdivided into early (S1), middle (S2) and late dementia (S3)], multi-infarct dementia (MD), Parkinson's disease (PD), vascular disease (VD) and communicating hydrocephalus (H), as well as in control patients (C1, C2). Mean DSIP-LI and P-DSIP-LI concentrations were found to be significantly higher in the elderly control group (C1, mean age 83 +/- 5 years) than in the middle-aged control group (C2, mean age 40 +/- 16 years). DSIP-LI and P-DSIP-LI were positively correlated with age in both control groups. Significant decreases of DSIP-LI compared with age-matched controls (C1) were observed for S2, S3, MD, PD, VD and H. In contrast, no significant differences corresponding to pathology were found for P-DSIP-LI.

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 nonpeptide 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.

The effects of ICV injections of DSIP and omega-amino-caprilyl-DSIP (C-DSIP) on the sleep-wake activity and brain temperature (Tbr were studied in rats. The substances (7 nmol/kg) were injected at dark onset, and the sleep-wake activity and Tbr were recorded for 24 hr (dark and light periods, 12 hr each). Relative to the control recordings obtained after artificial CSF injection, the duration of sleep did not increase after either DSIP or C-DSIP. The only significant reaction was an increase of W 6 to 9 hr after the injection of either peptide. The course of Tbr after DSIP and C-DSIP was also identical to that recorded after the injection of artificial CSF. It seems that DSIP administered in a single ICV injection at dark onset does not promote sleep. The increase in W might be attributed to an indirect effect of DSIP or to a degradation product of the peptide.

From 1963 to 1970 the possibility of humoral transmission of delta (SWS)-EEG sleep in rabbits by, i.c.v. infusion of extracorporal dialysate from blood of the sinus confluens of donors kept asleep by electrical stimulation of the ventromedian intralaminar thalamus, has been established. From 1970 to 1977 we isolated, characterized and synthesized a nonapeptide called delta-sleep-inducing peptide (DSIP) responsible for this effect. Subsequently, intravenous administration of DSIP was shown to produce, in different animals, sleep lasting for hours. Analogs with exchanged amino acids in the sequence or shortening the peptide by one or two amino acids decreased or abolished the effect, as did breakdown products, suggesting a close structure-specificity. In contrast sleep-induction per se was found to be species specific, i.e. in cats REM-sleep was predominantly produced. I.c.v., i.v. and s.c. administration yielded, in contrast to pharmaka, a parabolic dose-response curve with different effective optima. Additionally to sleep-induction, DSIP acts upon the circadian rhythmicity of the locomotor activity and transmitter concentrations in the brain and on that of plasma proteins and cortisol levels. We then synthesized a manyfold more powerful derivative by phosphorylation of the serine in position 7 (DSIP-P). Both forms, DSIP and DSIP-P occur in human CSF. Immunoreactive DSIP-like material was found in plasma of several mammals and humans, in human urine, CSF and milk. The penetration of the blood-brain barrier by the peptide has been proven and it was shown that unweaned rats are able to take up DSIP by the intestinal tract. The half-life time for proteolytic split-off of tryptophan by brain slices and homogenates is 15 min. Endogenous immunoreactive DSIP-like material in plasma, urine and CSF was found to be bound to a larger protein (carrier ?) and thus protected from proteolysis. Immunohistochemically DSIP was shown to occur in different regions of the rat brain. The multivariate activity of the peptide was then suggested by its interaction with acute and chronic stress and with drug-effects such as morphine, d-amphetamine and barbiturates. An induction of MAO-A and RNA synthesis in the brain was observed and the brain concentration of DSIP increased during progressed hibernation. Finally, alcohol addictism produced a substantial decrease of the DSIP-concentration in the rat brain and a specific electrophysiological effect on isolated neurons of rats and rabbits was established.(ABSTRACT TRUNCATED AT 400 WORDS)

A method for the enzyme immunoassay (EIA) of delta sleep-inducing peptide (DSIP) has been developed and applied to determine the metabolic clearance rate and biological half-life of DSIP administered to dogs. Antisera to DSIP conjugated to bovine serum albumin were raised in rabbits and proved to be specific for the C-terminus of the peptide. DSIP conjugated to horseradish peroxidase served as the labeled antigen in the EIA and enzyme activity was determined by fluorophotometry. The assay sensitivity was approximately 30 pg/ml. 1 or 2 mg of DSIP was injected intravenously into 4 anesthetized dogs and blood was taken at 5-min intervals. Unextracted plasma was subjected to the EIA directly and showed parallel displacement curves to the standards. DSIP was found to have a rapid disappearance with a mean metabolic clearance rate of 30.7 +/- 2.5 ml/kg . min and a mean half-life of 4.0 +/- 0.7 min in the dogs. Additional measurement of the metabolic parameters in a monkey and 3 rats treated similarly revealed a rapid in vivo clearance of DSIP from plasma with a half-life of 2.9 and 2.0 +/- 0.54 min, respectively.

This paper summarizes different investigations into effects of delta sleep-inducing peptide (DSIP) injections on insomnia. Two different studies showed improvement of sleep following single injections of 25 nmol/kg b.w. before sleep. Repeated administrations indicated a buildup with normalization of sleep structure after four administrations. Repeated injections in the morning - besides increasing daytime activity - still had a strong positive effect on night sleep, but not so two doses daily. A case of insomnia in organic brain disease responded well to higher doses. The results are discussed as to the mode of action of DSIP and its possible therapeutic use in insomnia.