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Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were previously isolated from bovine brain. Both peptides showed the greatest selectivity and affinity for the mu opiate receptor of any endogenous substance found to date and may serve as natural ligands for the mu-opiate receptor. We have purified them from the fronto-parietal cortex of human brain tissue by solid phase extraction and high performance liquid chromatography. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against endomorphin-1. The isolated endomorphins showed full biological activity. The tetrapeptides were found in human brain in much higher amounts than in bovine frontal cortex.

Insulin derived from the peripheral circulation has been shown to exert various effects on the brain due to its ability to cross the blood-brain barrier (BBB). The relation between diabetes mellitus and insulin has been extensively studied for peripheral tissues but not for central nervous system tissues. We examined the effects that streptozotocin- or alloxan-induced diabetes have on the transport of insulin across the murine BBB. We used multiple-time regression analysis to measure the unidirectional influx rate constant (Ki) and vascular association (Vi) of intravenously injected, radioactively labeled human insulin (I-Ins). Treatment with streptozotocin induced an enhancement of both the Ki and Vi of I-Ins that correlated with the onset of diabetes. Brain perfusion showed that the enhanced uptake was not due to altered vascular space or levels of insulin in the serum. Alloxan enhanced Ki and Vi after 5 days but the early phase of diabetes was associated with a decreased Ki. Hyperglycemia induced by the intraperitoneal injection of glucose elevated the Vi but abolished the Ki. Furthermore, altered I-Ins uptake by brain was not associated with changes in brain or body weight. These results show that there is an increased uptake of I-Ins by the brain in the diabetic state that is not due to acute changes in the serum levels of glucose or insulin, altered vascular space, or catabolic events. Chronic changes in levels of glucose, insulin or other hormone or neuroendocrine agents are likely to underlie the altered rate of transport of insulin across the BBB of diabetic mice.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are endogenous ligands that have greater affinity and selectivity for the mu-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin-biotin-peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing mu receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.

beta-amyloid (A beta), the major component of the amyloid deposited in the brains of patients with Alzheimer's disease, is found in blood and can cross the blood-brain barrier (BBB). This suggests that the circulation could be a source of A beta in brain. The passage of unbound A beta across the BBB is slow. Most of the A beta in blood, however, is likely to be bound to apolipoprotein J (ApoJ). ApoJ and A beta bound to ApoJ have been shown to rapidly cross the BBB of the guinea pig when studied in situ with a blood-free brain perfusion model. ApoJ in blood, however, is found in a concentration 28 times higher than that needed to saturate the ApoJ transporter in situ. This suggests that the putative ApoJ transporter may not be functional in vivo. We found here that when measured in a murine in situ brain perfusion model, 125I-ApoJ crossed the BBB with a unidirectional influx rate constant (Ki) of 3.75 x 10(-3) ml/g-min, which is similar to that found in the guinea pig. After intravenous injection, however, no penetration of ApoJ across the BBB was measured in either the mouse or guinea pig. These results suggest that ApoJ is unlikely to provide a significant route for the transport of A beta across the BBB in vivo.

Blood-borne insulin is known to cross the blood-brain barrier (BBB) where it can act as a satiety peptide. We examined in mice the pharmacokinetics and characteristics of such passage by multiple-time regression analysis. The unidirectional influx constant (Ki) of human insulin radioactively labeled with iodine (I-Ins) ranged from 0.87 to 1.7 microliters/g-min. The transport of I-Ins was inhibited almost 50% by 0.1 micrograms/mouse of unlabeled human insulin, a dose that had no effect on serum glucose. Similar results were found with rat insulin. The results with self-inhibition suggest that any hemoencephalic signal transmitted by the blood to brain transport of insulin is independent of the effects of insulin on glucose. The transport of I-Ins was altered by aluminum but not by administration of tyrosine, verapamil, or leptin, indicating independence from amino acid transport, the p-glycoprotein system, a slow calcium channel, or leptin transport. By contrast with insulin, enzyme degradation limited the uptake and accumulation by brain of intravenously injected, radioactively labeled glucagon and glucagon-like peptide. In conclusion, these results are consistent with the view that insulin can affect satiety and related behaviors independently of its peripheral effects by crossing the BBB to act within the brain.

Endomorphin 1 and 2 are recently discovered endogenous ligands for the mu-opioid receptor. In the present study, responses to intravenous administration of endomorphin 1 and 2 were investigated in the systemic vascular bed of the rat. Endomorphin 1 and 2 induced dose-related decreases in systemic arterial pressure when injected in doses of 10-100 nmol/kg i.v.. The decreases in systemic arterial pressure in response to endomorphin 1 and 2 were associated with significant decreases in heart rate, cardiac output, and total peripheral resistance. The endogenous ligand for the ORL1 receptor, nociceptin/OFQ had similar effects on systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance in the rat. Injections of isoproterenol (1 microgram/kg i.v.) and calcitonin gene-related peptide (CGRP; 0.3 nmol/kg i.v.), decreased systemic arterial pressure and total peripheral resistance. However these decreases in arterial pressure were associated with increases in heart rate and cardiac output. The results of the present study demonstrate that the endomorphin peptides have significant vasodilator activity in the systemic vascular bed of the rat and show that this response is associated with a decrease in heart rate and cardiac output.

Previous results have shown that treatment of rats with morphine during the neonatal period can influence development of peptide transport system-1 (PTS-1), the blood-brain barrier transport system for Tyr-MIF-1 and methionine enkephalin. Previous work has suggested that the activity level of PTS-1 correlates with the concentration of methionine enkephalin in the brain. We show here that rats treated peripherally with morphine sulfate (MS) in both the prenatal and neonatal periods have enhanced activity of PTS-1. The degree of enhancement increases with age to reach a 66% increase in comparison with controls at age 9 weeks. The mu agonist MS was more powerful than the kappa agonist ethylketocyclazocine (EKC) or the delta agonist [D-Pen2.5,pCl-Phe4]enkephalin (pCl-DPDPE) in producing this effect. Opiate antagonists had complex effects with methylnaltrexone blocking the action of MS on PTS-1. These results show that the level of PTS-1 activity in adult rats can be modified by perinatal events that affect opiate tone during development.

Both amyloid beta protein (A beta) and aluminum (A1) have been implicated in Alzheimer's disease. Recently, A beta has been found to be produced by peripheral tissues as well as by the CNS and to cross and accumulate in the vascular bed of the brain, which comprises the blood-brain barrier (BBB). This raises the possibility that blood-borne A beta may be a source of A beta within the CNS. Al has been shown to alter the structure and function of A beta, to inhibit the class of enzymes (metalloproteases) associated with the processing and degradation of A beta, and to alter the permeability of the BBB to peptides of similar size to A beta. Therefore, Al could alter the access of blood-borne A beta to the CNS either by changing the permeability of the BBB or by affecting enzymatic degradation. We examined the effect of Al on both of these parameters and found that Al did not alter the permeability of the BBB to A beta radioactively labeled with 125I (I-A beta) ever after correction for in vivo degradation. However, Al did enhance clearance and degradation of I-A beta in the circulation but not in the brain. Alterations in clearance can indirectly affect the CNS accumulation of circulating substances by modifying their presentation to the brain. In vitro studies of intracellular enzymatic activity of lysates of mouse and human erythrocytes (RBC) showed that Al could inhibit degradation of I-A beta through a mechanism antagonized by calcium and dependent on the concentrations of RBC lysate and Al. Analysis by high performance liquid chromatography showed that Al acted primarily by inhibiting the initial degradation of I-A beta to a peptide intermediate without inducing the aggregation of I-A beta under the conditions of these studies. No difference was found in sensitivity to Al between RBCs from patients with Alzheimer's disease and age- and sex-matched controls. The ability of Al to alter the degradation of A beta suggests a way in which these two potentially neurotoxic substances might interact in conditions such as Alzheimer's disease.

Impairment of the blood-brain barrier (BBB) in experimental autoimmune encephalomyelitis (EAE) has been frequently attributed to disruption, without much consideration of saturable transport processes. In mice with EAE, we studied the permeability of the BBB to radioactively labeled albumin and sucrose, markers of BBB disruption, and tumor necrosis factor-alpha (TNF-alpha), a cytokine transported across the BBB by a saturable system and thought to play a role in the pathogenesis of EAE. Permeation of the BBB was increased to all three substances during the acutely ill stage, was greatest in the lumbar spine, and returned to normal with recovery. The change in BBB permeability to sucrose was greater than to the larger albumin and is consistent with a partial disruption of the BBB. The enhanced permeability to TNF-alpha was comparable to that for sucrose, even though TNF-alpha is similar in size to albumin. This paradoxically high uptake of TNF-alpha could be explained by an enhancement of its endogenous saturable transport system. Thus the changes in BBB function during EAE extend beyond disruption to include changes in the saturable transport systems for substances involved in the disease process.

Insulin found in the CNS may be a key regulator in the balance of energy in the body. Since the peripheral circulation is the principal source of insulin in the CNS, insulin must cross the blood-brain barrier. We examined the retention of radioactively labeled insulin in the brain and its transport from the brain after injection icv in mice. The results were compared with those found in mice after fasting, starvation, refeeding, and the addition of aluminum (previously shown to affect the transport of peptides from the CNS) as well as tumor necrosis factor-alpha (TNF-alpha) (known to interact with peripheral insulin). There was no obvious saturable transport system for insulin from the brain, but the retention of insulin was regulated by both aluminum and starvation. Although TNF-alpha was neither required nor involved chronically in the retention of insulin in the brain, acute ip administration of TNF-alpha produced an early increase in the retention of insulin similar to that found after starvation.

Two endogenous brain peptides (Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2)), a cyclized analog and two fragments of Tyr-W-MIF-1, and hemorphin (Tyr-Pro-Trp-Thr) were tested for binding to mu 1 and mu 2 opiate receptor. All these peptides bound to both mu 1 and mu 2 sites in assays optimized to discriminate these subtypes of the mu opiate receptor in membranes from bovine thalamus. The cyclized analog of Tyr-W-MIF-1, previously shown to have potency near that of Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) and morphine in producing analgesia after intracerebroventricular (i.c.v.) injection, bound to mu 1 and mu 2 sites with affinities similar to those of DAMGO. Tyr-W-MIF-1, previously shown to induce analgesia after i.c.v. injection but with much higher potency after intrathecal (i.t.) injection, also bound to both mu 1 and mu 2 sites with an affinity between that of morphiceptin and hemorphin. Although the highest ratios of Ki's for mu 2/mu 1 were shown by hemorphin, Tyr-W-MIF-1, and Tyr-W-MIF-1, none of the compounds were significantly different in selectivity. The results indicate that the relatively lower potency of Tyr-W-MIF-1 after i.c.v., compared with i.t. injection, is not due to a lack of binding to mu 1 sites. They suggest that it has relatively high efficacy at mu 2, but low efficacy at mu 1 sites, a possibility that might explain some of the novel properties of these peptides.

A series of highly lipophilic comploffs 59Fe(RsalH2)3tach (R = H, NO2, OMe) and 68Ga(NO2salH2)3tach have been evaluated in vivo as potentially clinically useful agents.

Blood-borne cytokines enter the brain by transport across the blood-brain barrier (BBB) or by leakage through extracellular pathways at sites, such as circumventricular organs (CVOs), without a BBB. We used radioactively labeled albumin (T-Alb) to differentiate the relative contribution of transport and extracellular pathways to the passage of interleukin-1 alpha ([125I]IL-1 alpha) across the BBB. The major mechanism of entry for [125I]IL-1 alpha after intravenous (i.v.) injection was a saturable transport system with extracellular pathways accounting for only a small fraction of entry into brain. CVOs concentrated blood-borne [125I]IL-1 alpha in a saturable manner to a much greater extent than did the cerebral cortex and cerebellum, but accounted for less that 5% of total brain uptake. After intracerebroventricular (i.c.v.) injection, [125I]IL-1 alpha and T-Alb were concentrated in the CVOs, especially the median eminence, although CVOs contained less that 1% of the substances injected. Distribution after i.c.v. injection was largely due to diffusion and leakage through extracellular pathways. We conclude that after i.c.v. injection, leakage across extracellular pathways accounts for the small but concentrated amount of [125I]IL-1 alpha found in CVOs. After i.v. injection, transport across the BBB accounts for the majority of [125I]IL-1 alpha in brain.

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.

Spinal administration of morphine or [D-Ala2,MePhe4,Gly(ol)5)]enkephalin (DAMGO) produces potent, naloxone-reversible analgesia that is modulated by alpha 2-adrenoceptors. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) is a naturally occurring, amidated tetrapeptide that is structurally related to the melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) family of endogenous peptides. Tyr-W-MIF-1 displays high selectivity for the mu-opioid receptor. We investigated the effect of spinal administration of Tyr-W-MIF-1 on analgesia using the mouse tail-flick assay. Intrathecal (i.t.) administration of Tyr-W-MIF-1 produced a dose-dependent analgesic response, with an ED50 of 0.41 microgram, that was reversed by naloxone. Pretreatment with the mu-opioid receptor-selective antagonist beta-funaltrexamine blocked the effect of i.t. Tyr-W-MIF-1. However, pretreatment with the mu1-opioid receptor-selective antagonist naloxonazine did not antagonize the analgesia, indicating the effect was mediated through spinal mu2-opioid receptors. Pretreatment with desipramine, an inhibitor of norepinephrine reuptake, potentiated the analgesic effect of i.t. Tyr-W-MIF-1, producing a 3.1-fold leftward shift in the dose-response curve. Spinal administration of yohimbine, an alpha 2-adrenoceptor-selective antagonist, significantly attenuated the analgesic effect of Tyr-W-MIF-1. Thus, the potent analgesic effect of i.t. Tyr-W-MIF-1 is mediated through spinal mu2-receptors, and is modulated by norepinephrine and alpha 2-adrenoceptors.

Macrophage inflammatory proteins (MIP)-1 alpha and -1 beta have been postulated to exert their pyrogenic effects by acting directly at sites within the brain. Such activity would require circulating MIP-1s to cross the blood-brain barrier (BBB). We examined the ability of the monomer and polymer of MIP-1 alpha and the polymer of MIP-1 beta radioactively labeled with 125iodine (I-MIP-1) to cross the BBB. These I-MIP-1s behaved very similarly to each other but in a manner not previously seen for other cytokines. The I-MIP-1s immediately associated to a high degree and in a reversible manner with the vascular space of the brain. This association did not increase over time nor was it self-inhibitable. These results make it unlikely that the MIP-1s are transported into the brain by saturable transport systems in the manner found for some of the other cytokines. Other mechanisms, such as interactions with brain endothelia, leakage into brain through extracellular pathways, and binding at circumventricular organs, are more likely to provide the mechanisms through which blood-borne MIP-1s affect the central nervous system.

The penetration of four radioiodinated materials-Tyr-MIF-1, DAMGO, tyrosine, and albumin-into the periventricular tissue after ICV injection was studied in rats by film autoradiography. Rates of disappearance from the CNS for the injected compounds were also determined by computer-assisted image analysis of the autoradiographic images. The four materials showed distinct patterns of dispersion from the ventricular system, with Tyr-MIF-1 moving farthest into the parenchyma of the brain and albumin primarily restricted to the ventricular space. The other two compounds, tyrosine and DAMGO, had intermediate values. Tyr-MIF-1 also displayed the fastest rate of removal from the brain, which may represent the ability of the peptide to gain access to sites of saturable transport. By contrast, the exit from the brain of DAMGO was minimal, whereas the efflux of albumin and tyrosine was intermediate. These results show the utility of these methods in the simultaneous measurement of both the patterns of distribution within the CNS and the rates of removal from the CNS of compounds injected into the brain.

Leptin, or OB protein, is produced by fat cells and may regulate body weight by acting on the brain. To reach the brain, circulating leptin must cross the blood-brain barrier (BBB). Intravenously injected radioiodinated leptin (125I-leptin) had an influx constant (Ki) into brain of (5.87)10(-4) ml/g-min, a rate 20 times greater than that of labeled albumin. Unlabeled leptin inhibited the influx of 125I-leptin in a dose-dependent manner whereas unlabeled tyrosine and insulin, which have saturable transport systems, were without effect. HPLC and acid precipitation showed that the radioactivity in brain and serum represented intact 125I-leptin. About 75% of the extravascular 125I-leptin in brain completely crossed the BBB to reach brain parenchyma. Autoradiography detected uptake at the choroid plexus, arcuate nuclei of the hypothalamus, and the median eminence. Saturable transport did not occur out of the brain. The results show that leptin is transported intact from blood to brain by a saturable system.

Blood-borne peptides are capable of affecting the central nervous system (CNS) despite being separated from the CNS by the blood-brain barrier (BBB), a monolayer comprised of brain endothelial and ependymal cells. Blood-borne peptides can directly affect the CNS after they cross the BBB by nonsaturable and saturable transport mechanisms. The ability of peptides to cross the BBB to a meaningful degree suggests that the BBB may act as a modulatory pathway in the exchange of informational molecules between the brain and the peripheral circulation. The permeability of the BBB to peptides is a regulatory process affected by developmental, physiological, and pathological events. This regulation sets the stage for the relation between peptides and the BBB to be involved in pathophysiological events. For example, some of the classic actions of melanocortins on the CNS are explained by their abilities to cross the BBB, whereas aspects of feeding and alcohol-related behaviors are associated with the passage of other specific peptides across the BBB. The BBB should no longer be considered a static barrier but should be recognized as a regulatory interface controlling the exchange of informational molecules, such as peptides, between the blood and CNS.

This article is the eighteenth installment of our annual review of research concerning the opiate system. It includes articles published during 1995 reporting the behavioral effects of the opiate peptides and antagonists, excluding the purely analgesic effects. The specific topics covered this year include stress: tolerance and dependence; eating; drinking; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; sex, pregnancy, and development; immunological responses; and other behaviors.

Film autoradiography was used to demonstrate the transport and sites of accumulation of blood-borne radioiodinated interleukin-1 alpha (II-1 alpha) and other cytokines into the brain after intravenous administration. [125 I]Il-1 alpha, [125I]Il-1 beta, [125I]interleukin-1 receptor antagonist (II-1ra), and [125I]tumor necrosis factor-alpha (TNF alpha) labeled the choroid plexus and the capillary network 30 min after injection into the blood, suggesting that these areas may serve as sites of blood-to-brain transport. [125I]Il-1alpha, but not [125I]Il-1beta, [125I]Il-1ra, [125I]TNF alpha, or [125I]interleukin-2 (Il-2), was also found localized to the caudal region of the septal nuclei. Only unlabeled II-1 alpha was able to inhibit this accumulation. These findings provide further evidence for the passage of select cytokines across the blood-brain barrier (BBB) and are the first to identify a target site within the central nervous system (CNS) for a transported cytokine.

Neurotensin (NT) has been suggested to be a neuropeptide with therapeutic potential. We used multiple-time regression analysis to measure the unidirectional influx constant (Ki) of a tritiated analog of NT8-13, NT1, with improved metabolic stability. The Ki of [3H]NT1 across the blood-brain barrier (BBB) was 5.12(10(-4)) ml/g-min and was decreased 66% by unlabeled NT1 system. The amount of NT1 crossing the BBB, 0.087% of the injected dose per gram of brain, is consistent with its exerting central effects after peripheral administration. The stable [3H]NT1 crossed the BBB in intact form as assessed by HPLC and completely crossed the endothelial cells that comprise the BBB as assessed by the capillary depletion method. The presence of a transport system could be important for the development of NT analogs.

One mechanism by which blood-borne cytokines might affect the function of the central nervous system (CNS) is by crossing the blood-brain barrier (BBB) for direct interaction with CNS tissue. Saturable transport systems from blood to the CNS have been described for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist (IL-1ra), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Blood-borne cytokines have been shown to cross the BBB to enter cerebrospinal fluid and interstitial fluid spaces of the brain and spinal cord. IL-2 does not cross the BBB by a saturable transport system. The blood-to-brain uptakes of IL-1 alpha, IL-beta, and IL-1ra are interrelated for most brain sites, but the posterior division of the septum shows selective uptake of blood-borne IL-1 alpha. The saturable transport systems for IL-6 and TNF-alpha are distinguishable from each other and from the IL-1 systems. The amount of blood-borne cytokines entering the brain is modest but comparable to that of other water-soluble compounds, such as morphine, known to cross the BBB in sufficient amounts to affect brain function. CNS to blood efflux of cytokines has also been shown to occur, but the mechanism of passage is unclear. Taken together, the evidence shows that passage of cytokines across the BBB occurs, providing a route by which blood-borne cytokines could potentially affect brain function.

Blood-borne pancreatic polypeptide (PP) affects pancreatic secretion indirectly by acting through the central nervous system (CNS). PP, which is apparently not synthesized by brain, must cross the blood-brain barrier (BBB) to reach areas such as the cerebellum, an area rich in PP receptors, and to account for the PP found in cerebrospinal fluid (CSF). We used multiple-time regression analysis to measure the unidirectional influx constant (Ki) into brain of intravenously injected radioiodinated PP (I-PP). The Ki was 1.15 (10(-3)) ml/g.min and was inhibited by unlabeled PP but not by tyrosine. HPLC showed that radioactivity in the brain was mostly intact I-PP. Up to 0.065% of the injected dose entered each gram of brain with preferential entry into the cerebellum and the pons-medulla. Capillary depletion confirmed that intact I-PP penetrated the BBB. I-PP exited the brain by a nonsaturable process. These results show that I-PP crosses the BBB by a saturable system to reach its receptors behind the BBB.

Soluble receptors for cytokines can be important regulators of cytokine function. By binding to their cytokine ligands, they act as antagonists and carrier proteins. We investigated whether the blood-to-brain saturable transport of human tumor necrosis factor-alpha (TNF) and human interleukin-1 alpha (IL-1) radioactively labeled with 125I could be blocked by preincubation with their soluble receptors. At ratios of 100:1 and 1,000:1 of receptor to cytokine, the soluble p75 human receptor to TNF (rhuTNFR:Fc) totally blocked the entry of human or murine TNF into the brain. However, the soluble murine receptor to IL-1 (muIL-1R) only partially blocked IL-1 entry. Radioactively labeled rhuTNFR:Fc and muIL-1R were not transported across the blood-brain barrier (BBB) and were no more able to penetrate the BBB than the vascular marker serum albumin. This indicates that the transporter at the BBB for IL-1, but not the one for TNF, can strip the cytokine from its soluble receptor. These findings might be useful in determining which, if any, of the actions exerted on the brain by blood-borne cytokines are due to penetration of the BBB.

Two samples of the peptide tyrosine-melanocyte-stimulating hormone release-inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH2) were tritiated on different amino acids (Tyr or Pro) and incubated together at 37 degrees C with fractions of rat brain. The amount of intact tetrapeptide remaining was determined by HPLC. By 3 min, most of the Tyr-MIF-1 was degraded. Because similar amounts of [3H]Pro and [3H]Tyr appeared after incubation of the Tyr-MIF-1 peptides in brain homogenate, even as early as 30 s, examination of only this crude preparation would misleadingly indicate that Tyr-MIF-1 is not a precursor of melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1; Pro-Leu-Gly-NH2) in brain tissue. However, incubation of the mitochondrial fractions of brain under the same conditions resulted in more than three times as much [3H]Tyr being formed as [3H]Pro, with accompanying accumulation of MIF-1. Addition of excess MIF-1 to the mitochondrial fraction completely suppressed the formation of MIF-1 and more than doubled the amount of Tyr-MIF-1 remaining intact. When Tyr-MIF-1 tritiated only on the Tyr was added to the mitochondrial fraction, the main peaks of radioactivity appeared only at the positions of Tyr and Tyr-MIF-1, not at the position of Tyr-Pro. The results indicate that Tyr-MIF-1 can serve as a precursor of MIF-1 in brain mitochondria, an effect not evident when crude brain homogenate is used.

Adrenocorticotropin (ACTH)-related compounds, termed melanocortins, produce a large number of effects on the central nervous system (CNS) after their peripheral administration. Some of the CNS effects of ACTH are mediated through the release of glucocorticoids from the adrenal gland, but there are fragments and analogues of ACTH that do not act on the adrenals. This raises the possibility that some blood-borne melanocortins may be acting directly on the brain, which would necessitate their crossing the blood-brain barrier (BBB). We review here the literature showing that melanocortins can affect the BBB in several ways, including an alteration of the permeability of the BBB to other substances.

Tyr-MIF-1-like immunoreactivity (Tyr-MIF-1-LI) was measured in the plasma of hypophysectomized rats. The concentrations were increased in every rat examined, regardless of sex or time after hypophysectomy, and ranged from 2.5 to 10 times greater than the concentrations in intact rats. The elevated concentrations were not decreased by replacement by thyroxine, estrogen and progesterone, or acute injection of corticosterone. Chronic administration of physiological doses of corticosterone, however, did reduce the concentrations to normal. High performance liquid chromatography of tritiated Tyr-MIF-1 incubated at 37 degrees C for 3 min in plasma obtained by hypophysectomized rats eliminated the possibility that reduced degradation of Tyr-MIF-1 explained the increased concentrations. Thus, the increase in Tyr-MIF-1-LI in the plasma of hypophysectomized rats is robust and reversible by chronic replacement of corticosterone.

This article is the 17th installment of our annual review of research concerning the opiate system. It includes papers published during 1994 involving the behavioral, nonanalgesic, effects of the endogenous opiate peptides. The specific topics covered this year include stress; tolerance and dependence; eating; drinking; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; sex, pregnancy, and development; immunological responses; and other behaviors.

Tyr-MIF-1 previously was isolated from tissue obtained after death. A possible role of autolysis could not be excluded. We report the isolation of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in increased amounts from fresh human brain cortex. This confirms the natural existence of this brain peptide.

Amylin is co-secreted with insulin from the pancreas of patients with non-insulin dependent diabetes mellitus, and its deposition may contribute to the central nervous system (CNS) manifestations of this disease. Amylin, but not its mRNA, is found in brain, suggesting that CNS amylin is derived from the circulation. This would require amylin to cross the blood-brain barrier (BBB). We used multiple-time regression analysis to determine the unidirectional influx constant (Ki) of blood-borne, radioactively labeled amylin (I-Amy) into the brain of mice. The Ki was 8.99(10(-4)) ml/g-min and was not inhibited with doses up to 100 micrograms/kg, but it was inhibited by aluminum (Al). About 0.11 to 0.13 percent of the injected dose of I-Amy entered each gram of brain. Radioactivity recovered from brain and analyzed by HPLC showed that the majority of radioactivity taken up by the brain represented intact I-Amy. Capillary depletion confirmed that blood-borne I-Amy completely crossed the BBB to enter the parenchymal/interstitial fluid space of the cerebral cortex. Taken together, these results show that blood-borne amylin has access to brain tissue and may be involved in some of the CNS manifestations of diabetes mellitus.

Recent work has shown that interleukin-1 alpha (IL-1 alpha) and IL-1 beta are transported from blood to brain across the blood-brain barrier by a saturable system. Here, we show that the endogenous IL-1 receptor antagonist (IL-1ra) radioactively labeled with either 125I or 35S is also transported across the blood-brain barrier by a saturable transport system. Between 0.33 and 0.65% of an intravenous dose of labeled IL-1ra entered each gram of brain. The three cytokines inhibited each other's transport in a way suggesting that their elevated blood levels would tend to favor the entry of IL-1 beta at the expense of IL-1 alpha. High performance liquid chromatography confirmed that radioactivity entering the brain represented intact cytokine. Recovery of radioactivity from cerebrospinal fluid, an area without blood vessels, and from the parenchymal fraction of the cortex, and area without circumventricular organs, after capillary depletion confirmed that blood-borne IL-1ra gained entry into the brain. The transport system for IL-1ra appeared to be linked to that for IL-1 alpha and IL-1 beta, but was not affected by IL-2, IL-6, TNF alpha, or MIP-1 alpha. The results show that IL-1ra circulating in the blood can cross the blood-brain barrier to enter the central nervous system.

Brain-to-blood transport, or efflux, systems play important roles in brain functions and can affect the CNS uptake and activity of endogenous and exogenous blood-borne substances. Several efflux systems have been described for peptides. These efflux systems may play important roles in communication between the CNS and peripheral tissues and may be important in conditions such as alcoholism.

Interleukin-6 (IL-6) can alter brain function after peripheral administration, suggesting that it, like IL-1 alpha, IL-1 beta and TNF-alpha, might be able to cross the blood-brain barrier (BBB). We used multiple-time regression analysis to measure the unidirectional influx constant (Ki) into brain of radioactively labeled murine and human IL-6 given i.v. Ki values ranged from 3.05 to 4.54 (10(-4)) ml/g/min and were inhibited by unlabeled IL-6 but not IL-1 alpha or TNF-alpha, showing that the transport system for IL-6 is distinct from those for IL-1 alpha and TNF-alpha. Approximately 0.2% of the dose injected i.v. entered each gram of brain. The capillary depletion method showed that most of the IL-6 taken up by brain entered the parenchyma. However, only approximately 16% of the radioactivity recovered eluted as intact I-IL-6 in brain and approximately 50% in CSF after chromatographic separation by HPLC/Sephadex. The efflux rate for IL-6 injected into the lateral ventricle of the brain suggests that it enters the blood with the reabsorption of CSF. These results suggest that blood-borne IL-6 can reach sites behind the BBB, but that susceptibility to enzymatic degradation may limit contact time within the CNS.

Mu and delta opiate receptor regulation by opiate agonists and antagonists was studied in the human neuroblastoma cell line SH-SY5Y. Morphine down-regulated both mu and delta receptors, but its effects on each subtype could be dissociated by use of specific antagonists. The selective mu antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) blocked the down-regulation of mu, but not delta receptors. Conversely, the delta antagonist (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH([N,N-diallyl-Tyr1, Aib2,3]Leu- enkephalin)] ICI 174,864 blocked morphine-induced down-regulation of delta but not mu receptors. These selective antagonists also were studied alone for their effects on both receptors. CTAP alone at doses of 0.1 microM and higher up-regulated mu receptors. CTAP did not affect delta receptors at 0.3 microM or less, but it down-regulated them at doses of 1 microM or more, apparently due to its delta agonist activity at higher doses, which was reversed by ICI 174,864. ICI 174,864 alone also showed complex effects on the two subtypes, up-regulating both mu and delta sites. Its effects were most selective at a low dose (0.1 microM), which upregulated delta sites with minimal effects on mu sites. The nonselective antagonist naloxone provided a more robust upregulation (> 40%) of both mu and delta receptors than either selective antagonist alone or in combination. The mu-to-delta ratio (1.4 to 1) was not altered by differentiation of the cells with retinoic acid, which up-regulated both mu and delta receptors. Differentiation with the phorbol agent 12-O-tetradecanoyl-phorbol-13-acetate, however, up-regulated mu, but not delta receptors. The selective mu agonist Tyr-Pro-MePhe-D-Pro-NH2 (PL017) down-regulated mu receptors with a half-maximal effect at 180 nM, but was without effect on delta receptors at concentrations up to 10 microM. Conversely, the selective delta agonist Tyr-D-Pen-Gly-Phe-D-Pen([D-Pen2,5]-enkephalin) (DPDPE) potently down-regulated delta receptors, producing half-maximal decreases at 0.5 nM. At doses above those that reduced the maximum binding of [3H]pCl-DPDPE binding to the delta site, DPDPE also induced an apparent loss of affinity (increased Kd) at the delta site. It was without effect on mu receptors, however, at doses up to 10 microM. Thus, down-regulation of mu and delta receptors was homologous, because selective agonists down-regulated their respective receptors without effect on the heterologous opiate receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Previous work has shown that one mechanism by which blood-borne interleukin-1 alpha (IL-1) may be able to affect the central nervous system (CNS) is by direct transport into the brain across the blood-brain barrier (BBB). The BBB of the brain consists of endothelial (between blood and interstitial fluid) and ependymal (between blood and cerebrospinal fluid) barriers. Which of these barriers IL-1 can cross has not previously been investigated. At the spinal cord, which could be the site of action for some of the effects of IL-1 such as analgesia, the BBB consists only of the endothelial barrier. 2. We show here that IL-1 labelled with 125I (I-IL) is transported across the BBB of the spinal cord by a saturable system similar to the one previously described for the brain. High performance liquid chromatography (HPLC) showed that most of the material entering the spinal cord represented intact I-IL. The BBB of the spinal cord was no more leaky to radioactively labelled albumin than the BBB of the brain and was not disrupted by 50 micrograms kg-1 of IL-1. 3. Capillary depletion showed that most of the I-IL entered the parenchymal-interstitial fluid space of the spinal cord with only a modest amount being sequestered by the endothelial cells of its BBB. 4. I-IL entered the cervical, thoracic and lumbar regions of the spinal cord equally well. I-IL entering at the brain and diffusing caudally was estimated only to account for about 1% of the total radioactivity found in the spinal cord after i.v. injection.(ABSTRACT TRUNCATED AT 250 WORDS)

In this double-blind pilot study, 20 significantly depressed patients who all met the DSM-III R criteria for major depression were given a single subcutaneous injection of either 10 mg MIF-1 (Pro-Leu-Gly-NH2) or placebo on each of 5 consecutive days. Treatments were reversed for a second week of 5 consecutive daily injections. At the end of the first week, the group receiving MIF-1 was significantly improved on all rating scales as compared with the group receiving placebo. Eight out of 9 patients receiving MIF-1 showed marked improvement (score < or = 7 on the Hamilton Scale) as compared with only 2 of 11 patients receiving saline (P < 0.01). Administration of MIF-1 during the second week to the patients who had received placebo during the first week resulted in substantial improvement so that by the end of the second week the two groups were indistinguishable.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), an endogenous brain peptide, was incubated at 37 degrees C in cerebrospinal fluid (CSF) from rats and the amount remaining in intact from determined by high performance liquid chromatography (HPLC). By 3 min, a time at which Tyr-MIF-1 had been considerably degraded by rat plasma, essentially no degradation of the tetrapeptide had occurred in CSF. Tyr-MIF-1 persisted in mainly intact form for the 8 days tested, with a calculated half-time degradation of 11.4 days. The simultaneous addition to CSF of equal amounts of Tyr-MIF-1 tritiated on the Tyr and Tyr-MIF-1 tritiated on the Pro facilitated determination of whether Tyr-MIF-1 serves as a precursor of MIF-1 (Pro-Leu-Gly-NH2) in CSF. This possibility was excluded by the findings that the primary metabolite was Tyr-Pro, with minimal and equal amounts of the free amino acids Pro and Tyr being formed, but no MIF-1. The results show the extreme stability of Tyr-MIF-1 in CSF and are consistent with a role for this peptide in the CNS.

The neurotrophins are a family of polypeptides that promote differentiation and survival of select peripheral and central neurons. Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and neurotrophin-5 are included in this group. In recent years, tremendous advances have been made in the study of these factors. This has stimulated our review of the field, characterizing the neurotrophins from initial isolation to molecular analysis. The review also discusses their synthesis, localization, and responsive tissues, in both the periphery and CNS. The complex receptor interactions of the neurotrophins are also analyzed, as are putative signal transduction mechanisms. Discussion of the observed and postulated involvement in neuropathological disorders leads to the conclusion that the neurotrophins are involved in the function and dysfunction of the nervous system.

The metabolism of the endogenous brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and MIF-1 (Pro-Leu-Gly-NH2) was determined by HPLC after incubation of the tritiated peptides in human and rat plasma. Degradation of Tyr-MIF-1 was rapid in the plasma from both species, in contrast to the slightly delayed degradation of MIF-1 in rat plasma and the extremely prolonged persistence of MIF-1 in human plasma. In rat plasma, more than half of the intact Tyr-MIF-1 and MIF-1 was degraded within 5 min, in contrast to the 5 days required for 50% degradation of MIF-1 in human plasma at 37 degrees. To slow the rapid rate of metabolism, studies were then performed at 0 degree. Incubation of Tyr-MIF-1 in human plasma at 0 degree for 2 hr resulted in HPLC identification of more Tyr-Pro than Tyr at all times. At 0 degree in rat plasma, however, more Tyr than Tyr-Pro was formed after the first 5 min of incubation of the Tyr-MIF-1 that was labeled on the Tyr. This raised the possibility that the tetrapeptide Tyr-MIF-1 might be serving as a precursor of the tripeptide MIF-1. Incubation of Tyr-MIF-1 tritiated at the Pro under the same conditions with and without Tyr-MIF-1 tritiated at the Tyr showed that Tyr-Pro, not MIF-1, was the predominant degradation product of Tyr-MIF-1. In addition to the metabolism of Tyr-MIF-1 being slower at lower temperatures, it was also slowed by some enzyme inhibitors. After 10 min of incubation at 37 degrees, EDTA appeared to be more effective than bestatin, p-chloromercuribenzoic acid (PCMB), pepstatin, or aprotinin, but after 30 min, bestatin was more effective. Intravenous injection of the tritiated peptides into rats showed short half-time disappearances; again, MIF-1 persisted in blood longer than Tyr-MIF-1. Thus, the results show the rapid metabolism of Tyr-MIF-1 in human and rat plasma, the slightly slower metabolism of MIF-1 in rat plasma, the predominant formation of Tyr-Pro rather than MIF-1 from Tyr-MIF-1, and the markedly delayed metabolism of MIF-1 in human plasma.

Tyr-W-MIF-1 is a tetrapeptide recently isolated from brain that has opiate modulating activity. In this study, we used immunocytochemical (ICC) detection of FOS proto-oncogene protein to map brain areas activated by an ICV injection of Tyr-W-MIF-1 (200 micrograms). The analgesic effect of the peptide, which lasted 1 h, was confirmed in each rat with the tail flick test. FOS was activated in several limbic structures, including the cingulate and infralimbic cortex, nucleus accumbens, and central nucleus of the amygdala. FOS activation also occurred in several diencephalic nuclei, including the supraoptic, paraventricular, and periventricular nuclei of the hypothalamus, and the paraventricular nucleus of the thalamus. Several activated areas contained mu-opiate receptors. However, despite the known selectivity of Tyr-W-MIF-1 for mu receptors, FOS immunoreactivity was also induced in nuclei of the amygdala, hypothalamus, and thalamus, where concentrations of kappa receptors were high but those of mu and delta receptors were not detected. The results show that Tyr-W-MIF-1 induces FOS activation in several brain areas, including but not limited to, areas associated with nociception and stress-induced analgesia.

The centrally mediated effects of peptides as published in the journal Peptides from 1986 to 1993 are tabulated in two ways. In one table, the peptides are listed alphabetically. In another table, the effects are arranged alphabetically. Most of the effects observed after administration of peptides are grouped, wherever possible, into categories such as cardiovascular and gastrointestinal. The species used in most cases has been rats; where other animals were used, the species is noted. The route of administration of peptides and source of information also are included in the tables, with a complete listing provided at the end. Many peptides have been shown to exert a large number of centrally mediated effects.

The relative binding to mu, delta, and kappa opiate receptors was characterized for the brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), and two fragments of Tyr-W-MIF-1 (Tyr-Pro-Trp and Tyr-Pro-Trp-Gly) previously shown to have antagonist as well as agonist activity in the guinea pig ileum. Tyr-MIF-1 had relatively low affinity (Ki = 1 microM at the mu site) but high selectivity (400- and 700-fold greater affinity for mu over delta and mu over kappa binding). Tyr-W-MIF-1 (Ki = 71 nM at the mu site) showed higher affinity binding to all three sites than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa receptors. The affinity of the fragments of Tyr-W-MIF-1 was lower for mu but higher for delta receptors. We also tested two cyclized analogs of Tyr-W-MIF-1 that were about 200-fold more active than the parent compound in producing analgesia. These analogs showed higher affinity binding to all three opiate receptors. One of the analogs showed binding affinity to mu sites (Ki = 1.3 nM) that was within 3-fold of that of the potent analog of enkephalin, DAMGO. Thus, brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opiates, can provide high selectivity for mu opiate receptors. Analogs based on one of them, Tyr-Pro-Trp-Gly-NH2, show high affinity as well as potent analgesic activity.

Peptides have been shown to be transported in the direction of both blood to brain and brain to blood. Although blood to brain transport is known to occur at both the choroid plexus and the capillary bed of the brain, comprising the two major components of the blood-brain barrier, the location of efflux systems for peptides remains largely unstudied. We adapted established methodologies to study this question for two peptides known to be transported out of the brain after injection into the cerebrospinal fluid (CSF): Tyr-MIF-1, transported by peptide transport system (PTS)-1 and RC-160, a somatostatin analog transported by PTS-5. Radioactive iodide, known to be transported out of the brain primarily by the capillaries, also was studied. We found that after injection into brain tissue, RC-160 and iodide were rapidly transported out of the brain by saturable mechanisms. By contrast, efflux of Tyr-MIF-1 was slow and nonsaturable after injection into brain tissue, but rapid and saturable after injection into the lateral ventricle of the brain. Autoradiography confirmed that peptide injected into brain tissue did not diffuse far from the site of injection during the study period. The results indicate that the efflux system for RC-160 is located at least partly at the capillaries and suggest that the major location for the efflux system of Tyr-MIF-1 is at the choroid plexus.

Blood-borne interleukin-2 (IL-2), like other cytokines, is known to affect the central nervous system (CNS). One mechanism by which circulating substances can alter brain function is to directly cross the blood-brain barrier (BBB). We investigated the ability of IL-2 to cross the BBB, the interface between the periphery and the CNS. IL-2 labeled with 125I (I-IL-2) was injected into mice intravenously and its rate of entry into the brain determined by multiple-time regression analysis. I-IL-2 was found to enter the brain about 10 times faster than albumin. Neither morphine nor antibodies to IL-2, IL-1 alpha, or the IL-1 receptor affected the entry of I-IL-2. High performance liquid chromatography (HPLC) confirmed that the radioactivity entering the brain represented intact cytokine. However, excess unlabeled IL-2 was unable to impede the entry of I-IL-2, indicating that this transport is nonsaturable. This contrasts with saturable transport systems found for the cytokines IL-1 alpha and TNF-alpha, but still may explain how IL-2 can exert central effects.

This paper is the sixteenth installment of our annual review of research concerning the opiate system. It is restricted to papers published during 1993 that concern the behavioral effects of the endogenous opiate peptides, and does not include papers dealing only with their analgesic properties. The specific topics this year include stress; tolerance and dependence; eating; drinking; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; development; immunological responses; and other behaviors.