Constant light and dark affect the circadian rhythm of the hypothalamic-pituitary-adrenal axis.

The effect of constant light and constant dark on the circadian rhythm of the concentrations of hypothalamic corticotropin-releasing-factor-like immunoreactivity (CRF-LI), plasma ACTH, and corticosterone was investigated. Groups of rats were maintained under normal light-dark, constant light, or constant dark conditions for 10 days. Rats were then killed over a 24-hour time period and hypothalamic CRF-LI, plasma ACTH, and corticosterone concentrations were determined by radioimmunoassay. Under normal light-dark conditions, troughs in hypothalamic CRF-LI concentrations coincided with peaks in plasma ACTH and corticosterone concentrations. In rats housed under constant dark conditions for 10 days, higher hypothalamic CRF-LI concentrations were detected at 20.00, 24.00 and 04.00 h than at 08.00, 12.00 and 16.00 h. These relatively high hypothalamic CRF-LI concentrations coincided with relatively low plasma ACTH concentrations. The amplitude of plasma ACTH concentrations was markedly attenuated compared to levels of rats housed under normal light-dark conditions. The rats exposed to constant dark continued to demonstrate higher plasma corticosterone concentrations post meridiem than ante meridiem. The peak in plasma corticosterone coincided with the peak in plasma ACTH concentrations; however, the amplitude was normal. In rats maintained in constant light for 10 days, a decrease in hypothalamic CRF-LI concentrations at 20.00 h coincided with a peak in plasma ACTH. The peak in plasma ACTH concentrations was not associated with a peak in plasma corticosterone concentrations. The rhythm of plasma corticosterone concentrations was dramatically attenuated and phase-shifted. Together, these findings indicate that alterations of normal light-dark conditions result in changes in the circadian variation in hypothalamic CRF-LI, plasma ACTH, and corticosterone concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of DSIP-like material from ovine pineal glands: possible peptide-protein interaction.

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.

Degradation and aggregation of delta sleep-inducing peptide (DSIP) and two analogs in plasma and serum.

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.

Delta sleep-inducing peptide modulates the stimulation of rat pineal N-acetyltransferase activity by involving the alpha 1-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.

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 micrograms/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.'

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 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.

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 in spontaneously hypertensive rats.

Delta sleep-inducing peptide has been shown to exert extra-sleep effects as well as effects on sleep. In this study, the concentrations of DSIP-like immunoreactivity were measured by radioimmunoassay in the plasma of spontaneously hypertensive rats (SHR). They were found to be about 25% higher in SHR plasma than in the plasma of the normotensive Wistar-Kyoto (WK) controls. DSIP was then infused for 10 days by osmotic minipump (200 micrograms/kg/day) into SHR. This resulted in in maintenance of BP at a level of about 200 mm Hg as compared with the significant increase to about 220 mm Hg after 10 days in the SHR controls infused with 0.9% NaCl. After daily SC injection of a single dose of 200 micrograms/kg DSIP for each of 5 days in SHR, findings were similar. The results raise the possibility of an involvement of DSIP in the regulation of BP in SHR.

Delta-sleep-inducing peptide reduces CRF-induced corticosterone release.

It has been reported that delta-sleep-inducing peptide (DSIP) can affect several activities other than sleep, including reduction of stress. We studied the effects of this nonapeptide on corticotropin releasing factor (CRF)-stimulated release of corticosterone in rats treated with chlorpromazine-morphine-pentobarbital. Significant reduction of corticosterone levels were observed after intravenous injection of 5-30 micrograms/kg DSIP. No effect of DSIP was found on the corticosterone release elicited by injection of adrenocorticotropic hormone. The results suggest that DSIP attenuates the effects of CRF at the level of the pituitary.

Interaction of arginine vasopressin and corticotropin releasing factor demonstrated with an improved bioassay.

We developed an improved in vivo bioassay for corticotropin releasing factor (CRF) by modifying the injection schedule in the standard chlorpromazine-morphine-pentobarbital assay procedure. A combined injection of chlorpromazine and morphine followed 75 min later by injection of pentobarbital produced low basal levels of corticosterone and rendered the animals highly sensitive to synthetic CRF but insensitive to the stress of ether or histamine. The lowest dose of CRF that significantly elevated plasma corticosterone levels was 0.01 micrograms/kg. Using this assay, we studied CRF-arginine vasopressin (AVP) interactions at doses that were expected to raise systemic peptide concentrations to levels measured in hypophysial portal blood. The threshold for a significant corticosterone response was found to be at least 250-fold lower for CRF-41 than for AVP. The order in which CRF and AVP are injected was also found to be important, potentiation being greater if CRF was given first. In addition, rats deprived of water for 24 hr were more sensitive to CRF than normally hydrated animals.

Delta-sleep-inducing peptide and two of its analogs reduce nocturnal increase of N-acetyltransferase activity in rat pineal gland.

Serotonin N-acetyltransferase, an enzyme of the pineal gland, converts serotonin to N-acetylserotonin. The activity of this enzyme is induced by norepinephrine in the evening to reach high levels during the dark phase. Delta-sleep-inducing peptide, a humoral sleep factor, also seems to affect circadian rhythms. Intravenous injection of this peptide or either of two of its analogs in the evening significantly reduced the increase of N-acetyltransferase 4 h later. The dose-response relationship of the peptides showed an inverted U-shaped pattern with the active dose about 30 nmol/kg. The effect appears to be dependent on the time of day of administration, as injections in the morning did not change the enzymatic activity. These findings indicate that delta-sleep-inducing peptide (and two of its analogs) can affect enzymatic activities and that these influences probably vary throughout a time period of 24 h.

Evidence for peptide aggregation.

Evidence is presented that peptides may occur in aggregated form. Addition of 125I-Tyr-DSIP to serum resulted in four peaks after gel filtration chromatography on a column of Sephadex G-25. One of the peaks (C) eluted at the same position as the labeled peptide standard. Two Peaks (A and B) eluted before the standard and one (peak D) afterwards. The first peak (A) eluted at void volume, a position expected for labeled peptide bound to protein. The other two peaks (B and D), corresponding to smaller molecular size material, were greatly reduced by addition of glacial acetic acid or the chelating agent 1,10-phenanthroline before or even after mixing of the peptide with serum. Iron was one of the ions found to interact with 125I-Tyr-DSIP, and chromatography of a mixture of ferric chloride and peptide without serum resulted in the additional formation of peak B. A substantial portion of peaks A, B, and C (but not D) reacted with a specific DSIP antibody, indicating the presence of intact peptide. The results are consistent with the concept that peptides may occur in multiple forms.

Tyr-MIF-1 and MIF-1 are active in the water wheel test for antidepressant drugs.

MIF-1 [Pro-Leu-Gly-NH2] and Tyr-MIF-1 [Tyr-Pro-Leu-Gly-NH2] were tested in a system in which antidepressant drugs are known to result in increased wheel turning as mice attempt to escape from a small tank of water. One hr after injection, both peptides were found to cause a significant increase of the number of rotations of the wheel at doses as low as 0.01 mg/kg IP, the dose-response pattern for MIF-1 resembling an inverted-U. DSIP and morphine, by contrast, decreased the number of rotations. Under the conditions tested, neither MIF-1 nor Tyr-MIF-1 reversed the effect of morphine. The results demonstrate that MIF-1 and Tyr-MIF-1 are active in another test for antidepressants.

Circadian variation of DSIP-like material in rat plasma.

Levels of delta-sleep-inducing peptide-like immunoreactivity (DSIP-LI) in rat plasma were measured by radioimmunoassay and found to exhibit a circadian rhythm that parallelled the normal rhythm for corticosterone. The maximal plasma levels of both substances were observed to occur at about 1700h. The lowest concentrations of DSIP-LI and corticosterone were detected at 2400h and 1000h, respectively. Exposure to constant levels of illumination abolished the rhythm of DSIP-LI. It is possible that the temporal parallelism between the levels of DSIP-LI and corticosterone may represent a functional relationship between both compounds.

DSIP occurs in free form in mammalian plasma, human CSF and urine.

Although delta-sleep inducing peptide was isolated and characterized several years ago, no definitive evidence has been presented for the natural existence of the free peptide. Several attempts at the partial characterization of DSIP-like immunoreactivity (DSIP-LI) have indicated that a small part of the total immunoreactivity is probably present as the free nonapeptide. Using gel chromatography (Sephadex G-100) and subsequent high performance liquid chromatography on rabbit, human, rat and dog plasma, we now show a distinct peak of DSIP-LI that has the same elution position as synthetic DSIP. Free DSIP was also found in human CSF, whereas in human urine most of the small molecular weight DSIP-LI eluted at a position corresponding to DSIP-P, the phosphorylated analog of DSIP. A newly developed antibody recognizing primarily small molecular weight DSIP-LI was used in a modified, rapid assay to facilitate demonstration of the natural occurrence of free DSIP.

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.

HPLC shadowing: artifacts in peptide characterization monitored by RIA.

High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with 125I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.

DSIP reduces amphetamine-induced hyperthermia in mice.

The effects of delta sleep-inducing peptide (DSIP) have not been fully determined. Besides sleep-inducing activities, effects on locomotor behavior, stress-reduction, and temperature-regulation have been published. It was reported that DSIP reversed the increase in temperature of rats injected at room temperature with 15 mg d-amphetamine per kg body weight. We examined this effect in mice with 9 different concentrations of DSIP in addition to D-Ala4-DSIP and an analog, DSIP-P, phosphorylated at the serine in position 7. A reduction of the increased temperature was observed in mice but not in rats. This effect was only significant after pretreatment with 0.1 and 150 nmol DSIP/kg, but not the other doses. D-Ala4-DSIP decreased the rise in temperature between 50-150 nmol/kg, but DSIP-P showed no such effect nor was DSIP able to significantly reduce the increase of temperature induced by the larger dose of 30 mg/kg d-amphetamine. A bell-shaped dose-response curve was found for D-Ala4-DSIP; for DSIP two active dose ranges were observed. Thus, complex dose-effect relationships seem to exist for DSIP (and perhaps its analogs) in thermoregulation.

Presence of delta-sleep-inducing peptide-like material in human milk.

Delta-sleep-inducing peptide (DSIP)-like material was detected in human breast milk of two women by RIA with a recovery of about 90%. The high concentration of DSIP-like immunoreactivity (DSIP-LI) in colostrum (30 ng/ml) decreased to about 10 ng/ml in milk. The concentration continued to decrease over the next 2 months in one women. In the same woman, a significant circadian rhythm of the amount of breast milk DSIP was found with the peak in the afternoon and the trough in the morning. A significant effect of the sampling procedure was detected in the other woman examined; lower amounts of DSIP-LI were found when the milk was collected before and higher concentrations after nursing. Gel chromatography revealed that most of the immunoreactive DSIP-LI in milk and colostrum occurred in a form larger than the nonapeptide. The presence of DSIP itself, however, was demonstrated by high pressure liquid chromatography, which also showed additional peptides reacting with the antibody. Digestion of the large immunoreactive DSIP-LI by trypsin produced a peak on Sephadex G-10 that coeluted with DSIP. This peak contained three immunoreactive fractions with retention times on high pressure liquid chromatography similar to DSIP, phosphorylated DSIP, and N-tyrosine-DSIP. Plasma samples taken during pregnancy were assayed for DSIP but no difference from normal values was found. Slightly higher amounts were found in placenta than in blood, which might be due to interfering substances. No Tyr-MIF-1 or corticotropin-releasing hormone was detected by RIA in human breast milk. Peptides and proteins of milk can be absorbed from the gastrointestinal tract of babies, but it is not known if the DSIP-LI in human milk is involved in the induction of a sleep-wake cycle in neonates.

Delta-sleep-inducing peptide (DSIP): a review.

Since the turn of the century, it has been postulated that humoral factors induce sleep. Many compounds were proposed as sleep-factors, but only two of the sleep-peptides have been purified to homogeneity and characterized, so far. One of them, DSIP, was shown to be a nonapeptide of MW 849 and to induce mainly delta-sleep in rabbits, rats, mice, and humans, whereas in cats, the effect on REM sleep was more pronounced. A U-shaped activity curve was determined for the dose as well as for the time of infusion. DSIP-like material was found by RIA and immunohistochemistry in brain and by RIA in peripheral organs of the rat as well as in plasma of several mammals. In addition to sleep, the peptide also has been observed to affect electrophysiological activity, neurotransmitter levels in the brain, circadian and locomotor patterns, hormonal levels, psychological performance, and the activity of neuropharmacological drugs including their withdrawal.