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The two newest members of the urocortin (UCN)/corticotropin-releasing hormone (CRH) family of peptides - UCN II and UCN III - bind to the CRH-2 receptor, suppress feeding, and are expressed in the periphery as well as the brain. We used several sensitive techniques to examine their interactions with the blood-brain barrier (BBB). Of the four known peptides in this family, each interacts with the BBB differently. UCN I barely enters the brain from blood unless its latent saturable influx system is activated by leptin or pretreatment with glucose. However, neither leptin nor glucose affected the entry of intact UCN II. UCN II reached brain paranchyma at a moderate rate that was not self-inhibited or cross-inhibited by UCN/CRH peptides. The apparent, but misleading, rapid influx of UCN III (stresscopin) could be explained by degradation at the BBB itself. Influx of CRH into brain was slower than UCN II but faster than UCN I; it was inhibited by excess CRH but not by excess UCN I, II, III, or leptin. CRH is the only member of this family to have a saturable efflux system out of the brain. Determination of hydrogen bonding, newly applied here to ingestive peptides, was not helpful in explaining these differential interactions of the UCN peptides with the BBB., (Copyright 2002 S. Karger AG, Basel)

Galanin-like peptide (GALP) was recently isolated from the hypothalamus, where its expression is influenced by leptin and food deprivation. Since leptin crosses the blood-brain barrier (BBB) by a saturable transport system that is downregulated by fasting, we examined the effect of leptin and fasting on the entry of GALP into mouse brain. Multiple-time regression analysis showed that the basal influx of 125I-GALP from blood was rapid (K(i) = 9.49 +/- 0.72 x 10(-4) ml/g x min). This influx was not affected by leptin but was significantly decreased by food deprivation for 24 or 48 h, accompanied by decreased immunoreactive plasma GALP at 48 h, but not at 24 h. By contrast, pretreatment of mice fasted for 24 h with glucose resulted in a significant increase in the blood-to-brain influx of GALP that was not accompanied by increased immunoreactive plasma GALP. HPLC showed that most of the GALP crossed the BBB in an intact form, and capillary depletion studies showed that more than 93% of the GALP crossing entered the parenchyma of the brain rather than being bound to the endothelial cells of the capillaries composing the BBB or being reversibly associated with the vasculature. Efflux of 125I-GALP occurred at the rate of the normal reabsorption of CSF, and the octanol-buffer partition coefficient showed insufficient lipophilicity to explain the fast rate of influx. When 125I-GALP was perfused in blood-free buffer, the self-inhibition characteristic of a saturable transport system was evident even though capillary gel electrophoresis showed GALP aggregating as a trimer. Capillary zone electrophoresis showed protein binding of GALP in serum, perhaps facilitating its interactions at the BBB. In particular, these studies show for the first time (1) that immunoreactive GALP is present in blood where (2) its concentrations are reduced by food deprivation, and (3) that there is a rapid blood-to-brain influx of intact GALP (4) which is decreased by fasting and (5) increased by pretreatment with glucose., (Copyright 2001 S. Karger AG, Basel)

Since fasting is one of the few factors found to change the rate of entry of leptin into brain, we used multiple-time regression analysis to study the effects of pretreatment with glucose or insulin on leptin transport across the blood-brain barrier (BBB). Two hours after intraperitoneal injection of glucose (3 g/kg), there was a statistically significant increase in the entry rate (K(i)) of leptin in fasted (from 4.91 +/- 0.70 x 10(-4) ml/g x min to 9.03 +/- 1.00 x 10(-4) ml/g x min) but not (p = 0.15) in nonfasted normal (from 4.90 +/- 1.21 x 10(-4) ml/g x min to 6.42 +/- 1.79 x 10(-4) ml/g x min) or fasted streptozotocin (STZ)-treated diabetic mice (from 4.043 +/- 0.959 x 10(-4) ml/g min to 5.395 +/- 1.355 x 10(-4) ml/g min). Insulin (10 U/kg) increased leptin influx in fasted (from 4.77 +/- 0.26 x 10(-4) ml/g x min to 10.6 +/- 0.15 x 10(-4) ml/g x min at 0.5 h) and nonfasted (from 4.64 +/- 0.75 x 10(-4) ml/g x min to 7.46 +/- 1.48 x 10(-4) ml/g x min at 0.5 h) normal mice, but not in STZ-diabetic mice deficient in insulin (and leptin), even though basal concentrations of glucose were similarly increased in the nonfasted normal and STZ-treated mice. Moreover, the basal rate of leptin influx was the same in overnight fasted normal mice, nonfasted normal mice and STZ-diabetic mice. The results indicate that glucose and insulin can increase leptin transport, but they probably are not the principal factors responsible for the regulatory effect of the BBB on leptin entry into the brain., (Copyright 2001 S. Karger AG, Basel)

Insulin-like growth factor-1 (IGF-1) given peripherally has been found effective in clinical trials to slow down neuronal degeneration in some nervous system diseases. This raises the question of whether and how IGF-1 crosses the blood-brain barrier (BBB). In this report, we found that IGF-1 had a half-life of 4.5 min in blood, could remain intact for 20 min, and entered brain and spinal cord linearly. In the brain, IGF-1 had an influx rate of 0.4 microl/g x min after intravenous (iv) bolus injection as determined by multiple-time regression analysis. Intact radiolabeled IGF-1 was present in brain at 20 min after iv injection. Most of the injected IGF-1 entered the brain parenchyma instead of being entrapped in the cerebral vasculature. Addition of nonradiolabeled IGF-1 enhanced the influx of radiolabeled IGF-1 after iv injection, but inhibited the influx of radiolabeled IGF-1 by in-situ brain perfusion, suggesting that protein binding can explain the difference between the iv and perfusion experiments. In the spinal cord, the cervical region had the fastest uptake, followed by lumbar spinal cord. The thoracic spinal cord had the slowest uptake, comparable to that of brain. By contrast, des(1-3)IGF-1, an IGF-1 analogue with little protein binding but similar biological activity, had a shorter half-life in blood, slower influx rate into brain, and no alteration in pharmacokinetics after addition of nonradiolabeled peptide. We conclude that IGF-1 enters the CNS by a saturable transport system at the BBB, which functions in synchrony with IGF binding proteins in the periphery to regulate the availability of IGF-1 to the CNS., (Copyright 2000 S. Karger AG, Basel.)

Corticotropin-releasing hormone (CRH) is produced and acts both within the central nervous system and at several peripheral sites. However, it is not known whether CRH is able to cross the blood-brain barrier (BBB) in either direction, or whether the central and peripheral compartments are independent. We studied the transport across the BBB of both human/rat CRH (hCRH) and ovine CRH (oCRH) using the native peptides labeled with 125I at the histidine residue, thereby avoiding the use of other synthetic modifications. No apparent transport of either hCRH or oCRH into the brain from blood was found, as measured by multiple-time regression analysis after intravenous injection of the labeled peptides. There were no significant differences between the two forms of the CRH peptide. However, both hCRH and oCRH were rapidly transported out of the brain after intracerebroventricular injection, with half-time (t1/2) disappearances of 11.1 (hCRH) and 15.1 min (oCRH); the transport rate was significantly different for the human and ovine forms. The transport of hCRH could be specifically inhibited by 5 nmol of unlabeled hCRH (t1/2 = 17.7 min) but not by the same dose of the synthetic analog alpha CRH12-41. The process could also be inhibited by pretreatment with aluminum chloride (t1/2 = 18.8 min). An indirect influence of the endogenous opiate modulating peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2, 5 nmol) was apparent by a change in the initial distribution within the brain. In conclusion, there is a specific unidirectional brain to blood transport system for CRH. This transport system in the mouse has a greater affinity for the human/rat than for the ovine form of the peptide, is inhibited by hCRH itself, and can be disrupted by pretreatment with aluminum. By facilitating the rapid clearance of central CRH, this transport system could be involved in the regulation of central CRH levels and could allow central CRH to reach the general circulation and act at peripheral sites.

The known occurrence of opiates in the adrenals stimulated us to determine whether Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), an endogenous peptide that can act as an antiopiate, is present in these glands and whether adrenalectomy affects its concentrations in plasma. The presence of Tyr-MIF-1 in the adrenals of the rat was shown by radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). Binding sites for the tetrapeptide also were found in the adrenal. The concentrations and binding of Tyr-MIF-1 were higher in the adrenal medulla than in the adrenal cortex. The adrenals preferentially trapped 125I-Tyr-MIF-1 as compared with 99mTc-albumin after intravenous administration. However, after correction for tissue weight and vascular space, accumulation of 125I-Tyr-MIF-1 by the adrenals was in the general range seen for several other tissues. The concentrations of Tyr-MIF-1-like immunoreactivity determined by RIA in the plasma of adrenalectomized rats were increased in comparison with sham operated or unoperated controls. This approximate doubling was unchanged by the site of sampling: hepatic portal vein, renal vein, inferior vena cava, jugular vein, or truncal blood. Hypophysectomy, removal of the adrenal medulla, or treatment with corticosterone also did not block this increase. Tyr-MIF-1-like immunoreactivity in the plasma of adrenalectomized rats eluted at the same position by HPLC as did the synthetic Tyr-MIF-1 standard. Thus, the adrenal gland contains Tyr-MIF-1 and can affect its concentrations in plasma.

The concentrations in plasma of the biologically active endogenous peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) have not been measured during development or in female rats. By radioimmunoassay, we found that Tyr-MIF-1-like immunoreactivity (Tyr-MIF-1-LI) was first consistently detectable in plasma when the rat was 5 days old, and then gradually increased to adult concentrations by day 15. In male rats, the levels remained relatively constant for the next 21 months. In female rats, plasma concentrations of Tyr-MIF-1-LI at day 15 were about the same as in male rats. At 6 months of age, however, the concentrations in females decreased by half and by 21 months of life were only about a third of the concentrations found at day 15 or in age-matched males. The differences with age were not due to the length of time of storage of the samples, because another group of rats 1 month old was killed on the same day as 5-day-old rats and still showed several times more Tyr-MIF-1-LI in the plasma; again, no differences were found between male and female rats at either 5 days or 1 month. A single injection of estradiol followed by progesterone lowered the concentrations in 1-month-old male rats. In female rats that were either ovariectomized or sham-ovariectomized, the expected similarity in their plasma concentrations of Tyr-MIF-1-LI was found at 1 month of age.(ABSTRACT TRUNCATED AT 250 WORDS)

The transport of 125I-oxytocin from brain to blood was investigated in mice after intraventricular injection of radioactively labeled oxytocin with or without unlabeled candidate inhibitors. Residual radioactivity in the brain detected after decapitation was the principal determinant of transport activity. The half-time disappearance from the central nervous system of labeled oxytocin was 19.1 min. Inhibition by 10 nmol/mouse of oxytocin showed a saturable component to transport. A 10-nmol dose of tyrosine-melanocyte-stimulating hormone release inhibiting factor (Tyr-MIF-1) and pressinamide also significantly inhibited transport of labeled oxytocin (p less than 0.05). There was no inhibition of the system by a 10-nmol dose of tyrosine, iodotyrosine, MIF-1, or arginine vasopressin. Studies performed with 125I-oxytocin injected simultaneously with 131I-Tyr-MIF-1 with or without unlabeled oxytocin or Tyr-MIF-1 were consistent with both peptides being transported by the previously described peptide transport system-1 (PTS-1). Pretreatment with aluminum (100 mg/kg of elemental aluminum given 60-90 min before intraventricular injection), previously shown to inhibit PTS-1 and some other transport systems, inhibited the transport of labeled oxytocin. Radioactivity collected from the blood after intraventricular injection of 125I-oxytocin eluted on HPLC at the same position as the labeled oxytocin standard and differently from tyrosine, Tyr-MIF-1, MIF-1 and tocinamide. It is concluded that a saturable system exists for the transport of intact oxytocin from brain to blood which appears to be the previously described PTS-1.

Complex interactions of the pituitary, hypothalamus, and pineal involving MIF-1 (Pro-Leu-Gly-NH2) were shown many years ago. One of the largest changes consisted of increased amounts of an MIF-1-like material in the plasma of hypophysectomized rats as measured by the skin lightening of darkened frogs. The present study used radioimmunoassay to measure immunoreactive Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in intact, hypophysectomized, pinealectomized, and superior cervical ganglionectomized (SCG) rats. Samples were collected every 4 h for 24 h. Plasma concentrations of Tyr-MIF-1-like immunoreactivity were higher in hypophysectomized rats and lower in pinealectomized rats than in the intact or SCG groups, which were not reliably different from each other. In most groups, the highest plasma concentrations appeared to occur about 04.00 h, a time at which brain concentrations of Tyr-MIF-1-like immunoreactivity were low. However, no diurnal rhythm in plasma was seen in the pinealectomized rats. At none of the times did brain concentrations of immunoreactive peptide differ among the four groups. By high-performance liquid chromatography of plasma, the main peak of immunoreactivity was found to elute at the same position as Tyr-MIF-1, supporting the natural occurrence of this tetrapeptide. Thus, this study demonstrates that the concentration of immunoreactive Tyr-MIF-1 in plasma is increased by hypophysectomy.

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)

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.