Distribution of the delta sleep-inducing peptide in the brain of rabbits: study by immunofluorescence

Using the indirect immunofluorescence method, the distribution of the Delta Sleep Inducing Peptide (DSIP)-containing neurons was studied in the rabbit brain. DSIP antisera were raised in rat by multiple injections of synthetic DSIP conjugated to thyroglobulin. Some DSIP immunoreactive cell bodies were detected in the diagonal band of Broca and anterior part of the hypothalamus. Large populations of immunofluorescent fibers and terminals were visualized mainly through the organum vasculosum of the lamina terminalis, the preoptic areas, the subfornical organ, the thalamus, the ventromedial hypothalamus and infundibulum. Further, most of the cells of the intermediate lobe of the hypophysis displayed DSIP-immunoreactivity. The predominant localization of DSIP-immunoreactive fibers and terminals in certain circumventricular organs suggests that DSIP could play a specific role in the neurohumoral regulation.     

Immunoreactive delta sleep-inducing peptide in the rat hypothalamus, pituitary and adrenal gland: effects of adrenalectomy

Immunoreactive (IR) delta sleep-inducing peptide (DSIP) was examined by immunocytochemistry in the rat pituitary and adrenal gland and found to be colocalized with IR thyroid-stimulating hormone in the pituitary and with noradrenaline in the adrenal medulla. IR-DSIP was also detectable in nerve fibers in the posterior pituitary. By radioimmunoassay, IR-DSIP was quantified in plasma and tissue extracts after uni- or bilateral adrenalectomy. Significantly elevated plasma levels of IR-DSIP were measured 5 days after bilateral adrenalectomy (p less than 0.001). IR-DSIP was increased (p less than 0.05) in pituitary extracts from bilaterally adrenalectomized rats after 5 days, but not after 14 or 28 days. Sham- and unoperated animals did not significantly differ in plasma or tissue concentration of IR-DSIP. High-performance liquid chromatography of C18 SEP-PAK purified hypothalamus extracts revealed a single peak of IR-DSIP material of lower hydrophobicity than synthetic DSIP. The elevated concentration of IR-DSIP in the rat pituitary and plasma after bilateral adrenalectomy is consistent with the previously suggested role of DSIP to influence the activity of the hypothalamic-pituitary-adrenal axis.     

Structuro-functional organization of delta sleep-inducing peptide

Theoretical conformational analysis was carried out for a nonapeptide hormone (delta sleep-inducing peptide). Possible structure of the neuropeptide under physiological conditions may be described by a set of low-energy conformations belonging to nine different forms of the backbone. A solution of the "reverse conformational problem" for delta sleep inducing peptide enables one to predict modified amino acid sequences (D-Ala3-, Pro4-, Pro6-, Pro7, and Tyr7-analogs), which may assume one of the low-energy states of the native hormone. The influence of the solute was not taken into account in our calculations.     

Enzymatic synthesis of delta sleep-inducing peptide

The delta sleep-inducing peptide was assembled enzymatically from three tripeptide fragments. All the peptide bonds were prepared by either papain- or alpha-chymotrypsin-mediated synthesis. Secondary hydrolysis was suppressed by introducing N alpha-protected amino acid or peptide esters as carboxyl components and using an alkaline pH. The protected nonapeptide was oxidized with ferric chloride to deprotect the C-terminal phenylhydrazide and then hydrogenated. The homogeneous peptide was obtained by reversed phase high-performance liquid chromatography. Comparison of enzymatic and chemical preparations showed no obvious differences.     

Parasympathetic regulation of cardiac rhythm in delta sleep-inducing peptide deficiency

The effect of delta-sleep peptide (DSP) deficiency on the parasympathetic regulation of the heart rate was studied on 35 rabbits. It was established that the injection of an-serum (titer-1:2000-1:3000) leads to the attenuation of parasympathetic influences: heart rate increase in freely behaving animals and a decrease in negative chronotropic effect with direct vagus irritation. Antiserum, like DSP, administration causes practically no damage of the myocardial ultrastructure.     

Analysis of the mechanism of the stress-protective action of delta sleep-inducing peptide

By RIA there were studied the contents of corticosterone, ACTH, beta-endorphin and insulin in the blood plasma, met- and leu-enkephalin in different regions of the rat brain and in the adrenal glands after a 6-hour immobilization. The stress increased the content of corticosterone, ACTH, beta-endorphin, but not insulin in the blood plasma, and the levels of met-enkephalin in the adrenal glands, but decreased the met-enkephalin contents in the striatum. The injection of DSIP (0.1 mg/kg, i/p) blocked partly the elevation of corticosterone only. The authors propose, that stress-protective action of DSIP is realized with the involvements of the hypothalamo-pituitary-adrenal gland system.     

The effect of delta sleep-inducing peptide on the convulsive activity in corasol kindling

The influence of intraperitoneal delta-sleep inducing peptide (DSIP) injection (100 micrograms/kg) on the epileptic activity was investigated in the experiments on Wistar rats and (CBA X C57B1/6)F1 mice. The model of chronically developing epileptic activity--the model of pharmacological kindling--was created by daily repeated corasole injections in subconvulsive doses (30 mg/kg). It has been shown that DSIP injection delayed the manifestation of generalized seizures during kindling, led to the suppression of seizure activity and reduced the mortality rate of animals that developed kindled seizures. The antiepileptic effect of DSIP was observed throughout the period of 5 minutes to 24 hours after the injection. Naloxone (2.5 mg/kg) did not change the antiepileptic effect of DSIP.     

Effects of delta sleep-inducing peptide on the intercentral integration during experimental epilepsy

In free behavior experiments on cats it has been shown, that the intraperitoneal injection of delta-sleep-inducing peptide (100 mg/kg) may change organization of the pathology integration-epileptic discharges did not spread all the structures simultaneously. The slow-waves were registered in central medium of the thalamus and nucl. caudati. The epileptic discharges were registered first in visual and auditory cortex, hippocampus. After that they were observed in the motor cortex, nucl. caudati and centrum medianum of the thalamus.     

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).     

The anticonvulsive action of the intranigral administration of the delta sleep-inducing peptide]

It is shown that injection of delta sleep-inducing peptide (DSIP) into the substantia nigra reticular part (SNrp) suppresses generalized convulsive activity induced in rats by picrotoxin and corazol injection but exerts no influence on the strichnine-induced seizures. The analogous DSIP injection causes the antiepileptic action in rats kindled through picrotoxin injections. The DSIP intranigral anticonvulsant action is blocked by naloxon and enhanced by haloperidol and yohimbin. It can be concluded that DSIP anticonvulsant action may be realized due to activation of SNrp-dependent opioid mechanisms and suppression of dopaminergic ones.     

The delta sleep-inducing peptide as a factor enhancing the content of substance P in the hypothalamus and the resistance of rats to emotional stress]

The influence of the delta-sleep inducing peptide (DSIP, 60 and 120 nmol/kg, intraperitoneally) on the content of substance P (SP) in rats hypothalamus was studied on males of August line. DSIP administration significantly increased the mean SP content in the hypothalamus and also its content in animals, stable and predisposed to emotional stress. Daily DSIP administration before putting the rats in conditions of stress increased the SP content in the hypothalamus decreased at the emotional stress. Preliminary single DSIP administration to the animals subjected to stress also increased the SP content. Single DSIP administration in a dose of 60 nmol/kg sharply reduced classical stress manifestations, such as hypertrophy of adrenals and thymus involution.     

c-fos Gene expression during emotional stress in rats: blocking by delta sleep-inducing peptide

An emotional stress induces an obvious immediate early gene c-fos expression in the brain limbic structures in the rats predisposed to emotional stress. Administration of the delta-sleep-inducing peptide (DSIP) was shown to inhibit the c-fos expression. It led to an obvious inhibition of the c-fos expression in paraventricular nuclei of the hypothalamus, medial and lateral parts of the septum of rats predisposed to emotional stress. This mechanism seems to play an important role in the DSIP anti-stress effects.     

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.     

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several 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.     

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.     

Delta sleep-inducing peptide in the blood and hypothalamus of rats with various resistance to emotional stress

Enzyme immunoassay was used to study delta-sleep peptide content in blood and hypothalamus in rats of Wistar lines under acute emotional stress. It was found that the content of delta-sleep peptide in blood and hypothalamus of stable rats was higher as compared with rats predisposed to emotional stress. After 1.5-hour emotional stress the content of delta-sleep peptide increased in blood and hypothalamus both in stable rats and predisposed ones. After 3-hour stress there was an increase in delta-sleep peptide content in hypothalamus, and contrary to its decrease in blood in both stable and predisposed animals. It is supposed that delta-sleep peptide along with other oligopeptides is one of the factors determining individual animal resistance to emotional stress, which is supported by significant delta-sleep peptide increase in hypothalamus in stable rats.     

Mechanism of the action of the delta sleep-inducing peptide against a background of L-DOPA administration

A reciprocal nature of the shifts in activity of type A and B monoamine oxidases has been observed under the effect of DSIP against the background of L-DOPA administration (50 micrograms/kg) in the subfractions from the rabbit sensorimotor cortex. The results suggest that the activation of type A monoamine oxidase and serotoninergic system is the basis of the adaptive behavior of animals.     

The effect of the delta sleep-inducing peptide on the development of toxic brain edema-swelling]

Antiedematic effects of the drugs are connected with their action on the mediator systems. DSIP has a wide range of modulatory effects on the brain mediator systems. DSIP antiedematic effect was studied on the toxic brain edema-swelling (BES) model. Physical characteristics of the nervous tissue such as thickness and wetness were used as evaluation criteria. According to the findings, the doses of 75-100 micrograms/kg DSIP were optimal. It is suggested that DSIP effect on BES is multicomponent and rather complicated. Inhibition of serotonin, noradrenaline and histamine systems and activation of GABA-ergic system by DSIP act as a possible antiedematic mechanism.     

Effect of delta sleep-inducing peptide, anticonvulsant preparations and nicotinamide on generalized seizure activity

The influence of delta-sleep inducing peptide (DSIP) upon seizures induced by corazol, bicuculline, picrotoxin, strychnine, thiosemicarbazide were investigated in experiments on F1(CBA X C57 BL/6) mice. It was shown that DSIP increased the latency of first seizure manifestation which were induced by corazol, bicuculline and picrotoxin and also resulted in a suppression of seizure severity of corazol and bicuculline induced seizures. Anticonvulsant action of DSIP was evident under the condition of the mild severity seizures development. The effect of DSIP was mostly pronounced in range of its doses from 10 to 100 mcg/kg. DSIP when combined with phenobarbital, carbamazepine, diphenylhydantoin or nicotinamide enhanced the antiepileptic effects of these anticonvulsant drugs.