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Using adsorption to C-18 reverse phase resin we have developed a simple, fast and efficient procedure for extracting tryptolines from perchloric acid homogenates of brain tissue. Furthermore, using fused silica capillary chromatography coupled to negative chemical ionization mass spectrometry of heptafluorobutyl derivatives, we have developed a sensitive and specific procedure for identifying and quantitating tryptolines in extracts of brain tissue. Of the five members of the tryptoline class which have been studied by us, we have been able to find consistent evidence for the presence of only one, tryptoline. We find tryptoline to be present in extracts of whole rat brain in concentrations of approximately 370 pg/g of tissue. The finding of low concentrations of this compound mandates additional investigation to completely rule out artifactual formation.

Aaron Lerner's work provides a stunning set of examples of substances that help to transmit information in the brain and body. His characterization of alpha-MSH and melatonin and his sparking of interest in the further discovery of previously unknown substances have been of inestimable value for the field of neurobiology. Efforts such as those that Lerner undertook so successfully in the field of investigative dermatology now constitute a major research thrust in the field of behavioral neurochemistry and are directly related to advances in psychiatry and neurology. This review considers aspects of research on the neuropeptides, with particular attention to the endogenous opioid (morphine-like) peptides that are active on neural tissue. Neuropeptide research can be categorized broadly as efforts to discover and characterize new families and classes of active agents, investigations of their genetic and molecular processing, and studies of their relationships to behavior in animals and human beings. This review selectively considers some key research questions and strategies that arise from such research.

Acid acetone extracts of caudate nucleus from bovine brain were found to contain an amidated opioid octapeptide with the following structure: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2. The peptide has been named metorphamide. Bovine metorphamide appears to be derived by proteolytic cleavage from proenkephalin, the common precursor to [Met5]enkephalin and [Leu5]enkephalin. The cleavage within the precursor giving rise to the carboxyl terminus of metorphamide occurs at a single arginine residue and is followed by transformation of a carboxyl-terminal glycine into an amide group. Metorphamide was detected in bovine caudate nucleus extracts by radioimmunoassay, and it was purified to homogeneity by gel filtration and reversed-phase high performance liquid chromatography. Amino acid composition analysis and automated Edman degradation in the gas-phase sequencer confirmed the postulated amino acid sequence. Carboxyl-terminal amidation of bovine metorphamide was shown by stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the carboxyl-terminal amide as part of the recognition site. A synthetic replicate of metorphamide as well as several synthetic analogs were tested for opioid activity in several bioassays and binding assays, and metorphamide was found to have a high mu-binding activity. Metorphamide is the only known naturally occurring opioid peptide that has a high mu-binding activity. The kappa-binding activity is approximately equal to 50% that of the mu-binding activity, but delta-binding activity is negligible.

The relationship between rapid-eye-movement (REM) sleep tonic and phasic activity measures and the pre- and postprobenecid cerebrospinal fluid (CSF) concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was examined in a sample of 14 unmedicated psychiatric patients. The average duration of an eye movement burst and the average number of eye movements within a burst were found to be inversely related to the probenecid corrected accumulation of 5-HIAA in the CSF. These results are interpreted as indicating an association between REM burst eye movement activity and serotonin turnover in psychiatric subjects.

The effect of IV physostigmine administration on the cerebrospinal fluid (CSF) levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in normal subjects was determined. After an adjustment for differing CSF concentrations of probenecid, physostigmine was found to elevate CSF HVA and DOPAC concentrations. The authors discuss these changes in CSF HVA and DOPAC and their possible relationship to the ability of physostigmine to decrease the symptoms of some patients with tardive dyskinesia.

The tissue distribution and function of opioid peptides in humans is incompletely defined. We report here that, unlike that in other species, the human anterior pituitary gland contains high concentrations of methionine-enkephalin (met-enkephalin). The met-enkephalin immunoreactive material was isolated and identified as authentic met-enkephalin by fast atom bombardment-mass spectrometry and Edman degradation sequencing. The met-enkephalin was localized in a large subpopulation of TSH immunoreactive cells (thyrotrophs). No other proenkephalin-derived opioid peptides were found in the pituitary, and there was no overlap between proopiomelanocortin and met-enkephalin immunoreactive cells. These results suggest that the human anterior pituitary gland contains a novel met-enkephalin precursor and a possible role for met-enkephalin in regulating human thyroid function.

In mice, chlordiazepoxide, diazepam and flurazepam reduced 5-HT synthesis and metabolism in the teldiencephalon following an i.v. injection of 3H-tryptophan. Only diazepam produced a significant effect in the brainstem. The steady state level of brain 5-HT or of tryptophan was not altered by any of the drugs. Chlordiazepoxide and diazepam, but not flurazepam, decreased the rate of norepinephrine loss from the brain following inhibition of synthesis with alpha-methyltyrosine.

Immunohistofluorescence studies of the rat central nervous system with antibodies to Phe-Met-Arg-Phe-NH2 (molluskan cardioexcitatory peptide) revealed a widespread neuronal system in the brain, spinal cord, and posterior pituitary. Immunoreactive axons and cell bodies were mainly located in cortical, limbic, and hypothalamic areas. Immunostaining of serial sections of the brain and pituitary showed that the Phe-Met-Arg-Phe-NH2 immunoreactive neurons were different from neurons labeled by antibodies to either Met-enkephalin or the putative Met-enkephalin precursor Tyr-Gly-Gly-Phe-Met-Arg-Phe, which is structurally related to Phe-Met-Arg-Phe-NH2. Control staining by antiserum absorption and radioimmunoassay indicated that the antibodies that caused the specific immunofluorescence recognized peptides with an amidated Arg-Phe sequence at the carboxyl terminus.

Details are presented of an improved selected ion monitoring assay for the major biogenic amine metabolites and probenecid in human lumbar cerebrospinal fluid (CSF). The metabolites and probenecid are simultaneously extracted with ethyl acetate from an acidified aqueous phase, and are simultaneously converted to pentafluoropropionyl esters by reaction with pentafluoropropionic anhydride and pentafluoropropanol. The esters of the metabolites are analyzed following a single injection of the derivatized sample onto the gas chromatographic column, while the ester of probenecid is analyzed following a separate injection onto the gas chromatographic column. Quantitation is achieved using for internal standards dueterated analogues of the metabolites and a chemical analogue of probenecid. Data are presented on the concentration of free and conjugated forms of the metabolites in lumbar CSF taken from healthy volunteers.

Human neuroendocrine tumors are known to demonstrate immunoreactivity to the amphibian peptide bombesin. The recent observation that gastrin-releasing peptide (GRP), a 27 amino acid peptide first isolated from porcine intestine, may be the mammalian analog of bombesin led us to look for this peptide in intestinal carcinoid tumors. Formalin-fixed tissues from 20 of these tumors were examined by the immunoperoxidase technic, using specific antisera to the GRP molecule (1-27) and the GRP fragment (1-16). Intense diffuse cytoplasmic immunoreactivity was observed in carcinoids from the small intestine (7/10), appendix (4/5), and colon (1/5). In each positive case, the pattern of staining for GRP (1-16) and GRP (1-27) was identical. These findings indicate that bombesin-like immunoreactivity in human intestinal carcinoid tumors is attributable to GRP-like molecules and that GRP is a useful marker for neuroendocrine differentiation.

This paper presents a technique for glyoxylic acid-induced monoamine histofluorescence in the central nervous system. Unperfused rat brains are sectioned in a cryostat, immersed in 2% glyoxylic acid solution, warm-air dried and gassed at 100 degrees C. Intense, well-localized catecholamine fluorescence is produced and all known catecholamine-containing structures are demonstrated. The fluorescence obtained by this method was evaluated by a variety of agents and was shown to be catecholaminergic in origin. In contrast to the Vibratome-glyoxylic acid technique, this procedure reliably produces thin, whole-brain sections of even thickness and allows protracted use of the tissue block. Because unperfused tissue is used, the technique can be applied to a broad variety of material, such as post-mortem tissue or invertebrate preparations. Alternate sections can be prepared for a variety of techniques requiring unperfused tissue (e.g., enzymatic localization, chemical assay, anatomical techniques). The reasons for choosing each of the parameters in the technique are discussed.

An enzymatic preparation from human brain converts tryptamine to tryptoline (9H-1,2,3,4-tetrahydropyrido[3,4-b]indole) in the presence of 5-methyltetrahydrofolic acid. Similarly, N-methyltryptamine and 5-hydroxytryptamine yield 1-methyltryptoline and 5-hydroxytryptoline, respectively. Neither in vitro nor in vivo formation of these compounds by human tissues has been described.

Using an antiserum directed at the COOH-terminus of tachykinins, we have examined postmortem tissue from two cases of metastatic ileal carcinoid for the presence of tachykinin-like immunoreactivity. The vast majority of the immunoreactive tachykinin-like material eluted from a Sephadex G-50 column as two peaks at positions corresponding to molecular weights of 1300 and 850. The 1300 dalton peak was resolved by reverse-phase-HPLC into two components which by Edman sequencing, amino acid analysis, and fast atom bombardment (FAB)-mass spectrometry criteria, were identified as substance P and substance K. The 850 dalton peak was also resolved on RP-HPLC into two peaks which were resistant to Edman degradation but from amino acid analysis and FAB-mass spectrometry criteria were identified as pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11. In control experiments substance P 5-11 was converted to pyro-Glu-substance P 5-11 during the extraction procedure. Both tumors also contained a minor immunoreactive peak which eluted from a Sephadex G-50 sizing column at a position corresponding to a molecular weight of 4000 which probably represents neuropeptide K. These results suggest that beta-preprotachykinin is preferentially expressed in carcinoid tumors and that substance K may also play a role in the carcinoid syndrome.

It has been proposed that the etiologies of tardive dyskinesia and Huntington's chorea and of some forms of schizophrenia and the affective disorders involve a cholinergic imbalance with respect to a second neurotransmitter. This relative over- or underactivity of the cholinergic system could result from altered synthesis, storage, release, degradation, or reuptake or from a variety of receptor interactions. Under these hypotheses, clinical symptoms would reflect both the brain region in which the imbalance occurs and the neurotransmitter with which acetylcholine is interacting. Effective treatments could involve the correction of this hypothetical imbalance by changing the relative availability of either one or both of the neurotransmitters. Both precursor loading with choline or dimethylaminoethanol and cholinesterase inhibition may be useful in evaluating the effects of increased cholinergic activity in these disease states; the relative merits of these strategies are discussed.

We looked for opioid peptides and their precursors in 108 tumors of both neuroendocrine and nonneuroendocrine origin, using a monoclonal "pan-opioid" antibody, 3-E7, which recognizes the tetrapeptide Tyr-Gly-Gly-Phe (the sequence responsible for pharmacologic activity in all known opioid peptides), in conjunction with polyclonal antibodies directed against representative peptides of each of the three precursors (alpha-endorphin, [met]enkephalin-Arg-Gly-Leu, and dynorphin B). Using the avidin-biotin immunoperoxidase technique, we observed consistent cytoplasmic immunoreactivity (at least focally) in all of 15 adrenal pheochromocytomas, all of 6 thyroid medullary carcinomas, and all of 5 pituitary adenomas. Opioid staining was also observed in parathyroid adenomas (8 of 9), pancreatic islet-cell tumors (7 of 10), carcinoid tumors from various sites (18 of 26), and paragangliomas (1 of 2). There was no immunoreactivity in pulmonary small-cell carcinomas, Merkel-cell tumors of skin, neuroblastomas, or any of the non-neuroendocrine tumors examined. The expression of alpha-endorphin, [met]enkephalin-Arg-Gly-Leu, and dynorphin B varied from tumor to tumor; however, positive staining with the "pan-opioid" antibody was found in each tumor containing at least one of the three precursors. Opioid peptide immunoreactivity was also detected in non-neoplastic cells of the adrenal medulla, pancreatic islets, pituitary, intestinal and bronchial mucosa, and intestinal myenteric plexuses. We conclude that opioid expression within tumors is most likely due to enhanced expression of a normal cell product and that opioid peptides are useful markers of neuroendocrine differentiation in many tumors.

In this light microscopic immunocytochemical study beta-Endorphin (beta-END), leu-enkephalin and dopamine-beta hydroxylase (DBH) antisera are used to obtain an overview of the interaction of the noradrenergic and opiate peptide systems in brain. Serial brain areas were analyzed for DBH and then for beta-END or leu-enkephalin. Several areas were evaluated for cell and fiber interactions between these systems. The areas of richest possible contact between beta-END and DBH positive systems include the rostral locus coeruleus region, the periaqueductal grey, possibly the dorsal thalamus, the paraventricular hypothalamus and the arcuate nucleus. Enkephalin cells and fibers were seen surrounding the locus coeruleus throughout its length with a few fibers in the nucleus itself.

A nearly full-length cDNA clone isolated from the rat pheochromocytoma cell line, PC12, revealed extensive nucleotide sequence similarity between the rat cDNA and the Drosophila melanogaster hsp70 gene. The rat recombinant clone encodes a 71,000-dalton protein that is 70% identical with the dipteran hsp70 protein. Remarkably, a truncated segment of this cDNA clone was originally isolated by immunoreactivity with antisera raised to catecholamine-synthesizing enzymes, suggesting that this heat shock protein and these catecholamine enzymes shared antigenic determinants. The rat hsp70-related mRNA is responsible for the production of a constitutive hsp70 protein, because it is present in abundant amounts in various tissues at normal growth temperatures and is only minimally induced by hyperthermia. The rat hsp70-related sequence is part of a multigene family that extends across species to mice and humans.

The distribution and endogenous concentrations of norepinephrine, epinephrine, dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid were determined in the brains of adult dogs. Norepinephrine and epinephrine were localized primarily in 'central core' areas in brain stem and hypothalamus. Dopamine (DA) and its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acids (HVA), were localized primarily in basal ganglia with relatively high concentrations also found in amygdala, septum and substantia nigra. HVA was also found in relatively high concentrations in areas where DA concentrations was very low. Serotonin and 5-hydroxyindole-3-acetic acid levels were highest in brain stem, hypothalamus, globus pallidus and nucleus accumbens. Epinephrine levels were higher than in previously studied species, at times as much as 25-30% of norepinephrine and frequently greater than dopamine in brain stem and hypothalamus. Using the ratios 5-hydroxyindole-3-acetic acid/serotonin and homovanillic acid/dopamine as indicators of serotonin and dopamine turnover and utilization, both putative transmitters were found to be generally more highly utilized in areas of lower concentration, especially in brain stem and cortex. Catecholamines were found to be unconjugated in dog brain. DOPAC and HVA were found to exist primarily in the unconjugated form. DOPAC was found to be slightly conjugated in most areas while 10-20% of HVA was present in the conjugated form in most cases.

Rat striatal tyrosine hydroxylase (TH) was assayed 2 hr following treatment with 1 mg/kg haloperidol. TH activity in striata from haloperidol-treated rats (haloperidol TH) was increased significantly relative to control when assayed at pH 7.0, but not at pH 6.0, in the presence of 175 muM tetrahydrobiopterin (BH4). TH was also phosphorylated in vitro, catalyzed by sufficient quantities of the catalytic subunit of bovine heart protein kinase to cause greater than 90% activation after 10 min. TH was activated by phosphorylation at both pH 6.0 and pH 7.0, but the activation was greater at pH 7.0. Haloperidol TH, activated relative to control TH at pH 7.0, was activated by phosphorylation, but there was no difference between haloperidol TH and control TH activity at either pH 6.0 or 7.0 following phosphorylation. Comparison of Lineweaver-Burk plots of nonphosphorylated and phosphorylated TH indicated that activation by phosphorylation was due to a 5-fold change in Km for BH4 and a 2-fold change in Vmax at pH 7.0. Haloperidol TH kinetics were intermediate between those of nonphosphorylated and phosphorylated TH at pH 7.0. Analysis by Lineweaver-Burk, Hanes-Woolf, and Eadie-Scatchard plots suggested that the haloperidol TH kinetic data were the result of a mixture of two forms of the enzyme, with different affinities for cofactor. Theoretical calculations of TH activity of mixtures of nonphosphorylated and phosphorylated TH suggested that the haloperidol data could be explained by postulating a mixture of 25-35% phosphorylated TH molecules with 65-75% nonphosphorylated TH molecules. An hypothesis of the role of TH phosphorylation during conditions of increased neuronal firing rate, such as may occur with haloperidol treatment, is presented.

The probenecid-induced accumulation of the acidic metabolites of dopamine and serotonin is widely used to estimate the CNS turnover of these amines in human subjects. The theoretical basis of the probenecid test is discussed, including the assumptions on which the test is based, and the limitations of the procedure. Suggestions are offered on appropriate methods for analyzing test data. Available evidence suggests that maximal inhibition of the efflux of the acidic metabolites of dopamine and serotonin from the CSF is not achieved with probenecid doses up to 100 mg/kg. Therefore, before a comparison can be made between the accumulation of CSF metabolites from different groups of subjects, a correction for CSF probenecid concentrations is necessary. Moreover, in addition to the measurement of dopamine and serotonin turnover, the probenecid test can be extended to include comparisons of CNS turnover of other monoamines including octopamine, p-tyramine, and tryptamine.

Information which has emerged thus far relates to the overall transmitter mechanisms of sleep. The data, while conflicting, point to the involvement of many neuroregulators at numerous integrative levels of the process. However the long term question still remain: what triggers and maintain sleep, what stops sleep, what occurs to the body and brain during sleep--in essence, why sleep? These questions are now problems for behavioral neurochemists, whereas in a previous era, they were problems for philosophers. Unfortunately, our answers to date, while in another idiom, have hardly been more complete or satisfying. To answer these questions, it will be necessary to understand, in detail, the manner in which neurobiochemical processes relate to the functional physiology of sleep. Although existing studies have given invaluable insight into the neurochemical anatomy of sleep, we have only recently acquired the technical and biochemical expertise necessary to investigate sleep as it occurs normally. Future research must focus on the dynamic changes associated with the regulatory mechanisms of neurotransmitters. Many questions can be asked. With sleep transitions, what changes occur in transmitter content, synthesis, or release? Are there changes in metabolic pathways, reflecting a shift from intra- to interneuronal metabolism? What changes occur in pre- and postsynaptic neurotransmitter receptors to affect sensitivity? What constraints do genetic (245) and environmental (246) factors impose upon these mechanisms? Knowledge of such parameters will allow us to construct more complete models of the neuroregulatory basis of sleep and waking. However, as we acquire this knowledge, we must avoid the temptation of assuming causation when the evidence merely shows correlation. Neuroregulation are involved in the control of number different behaviors; and, at present, we have few, if any, methods of establishing causative links between a specific neuroregulator and a specific behavioral state. Yet, even without an understanding of what "causes" sleep, we may be able to develop pharmacological agents which permits discrete alteration of sleep mechanisms in a more physiological and specific manner. This potential for manipulation of sleep is of obvious importance in illnesses such as insomnia, narcolepsy, and sleep apnea (247, 248). In addition, it may be valuable in the treatment of such conditions as psychosis and depression, where sleep disturbances are an important component of the illness. For example, delirium tremens might be best understood as a psychotic episode which is the result of an aspect of sleep emerging into wakefulness. The range and breadth of both the basic questions and the potential application of sleep research portend an exciting future for this field.

BAM 18 is a derivative of the opioid precursor proenkephalin A. Although it exists in rat and guinea-pig brain in relatively high concentrations, its physiological function is presently unknown. In the present study we have determined the opioid receptor selectivity of this peptide using radioligand binding and peripheral tissue bioassay. When selective binding conditions were used, BAM 18 bound to the mu opioid receptor with an affinity three times that of the kappa opioid receptor and over 10 times that of the delta opioid receptors (Ki = 0.29, 0.75, and 3.2 nM respectively). BAM 18 also displayed mixed receptor selectivity in in vitro bioassay. Ke values for naloxone antagonism of BAM 18 agonist activity in the electrically stimulated guinea-pig ileum and the mouse vas deferens were 4.3 and 9.9 nM, respectively. These data indicate that BAM 18 binds to all three opioid receptor subtypes with a selectivity profile of mu greater than kappa greater than delta.

Dynorphin(1-8) immunoreactivity was visualized by immunohistofluorescence in hypothalamic magnocellular neurons of the rat. No immunoreactive met-enkephalin-Arg6-Gly7-Leu8, a fragment of the adrenal medulla pro-enkephalin molecule, was detected in magnocellular neurons. However, a strong met-enkephalin-Arg6-Gly7-Leu8-like immunostaining was seen in other regions of the brain. These results suggest that in magnocellular neurons dynorphin(1-8) exists independently from pro-enkephalin and therefore the magnocellular neurons represent a third opioid peptide neuronal system in brain. These observations, however, do not rule out a coexistence of proenkephalin and dynorphin-related peptides in other regions of the brain.

Detailed analysis of the dose-response and time-course relationship of ethanol to changes in adenosine 3',5'-cyclic monophosphate (cAMP) content of mouse brain revealed several patterns of response, including both decreases and increases depending on brain area. Whole-brain cAMP content was decreased with ethanol injection at all doses (0.4-3.2 g/kg), and reflected the decreased levels in the cortex. The subcortical and cerebellar levels underwent a significant increase with doses of 1.6 and 3.2 g/kg, respectively. In all brain areas, significant changes in cAMP content occur within 10 min after ethanol injection; elevated levels persist for 30-60 min in the subcortex and cerebellum, but remain significantly depressed in the cortex at 3 h. These dynamic changes in brain cAMP levels after ethanol administration may reflect variations in neurotransmitter activity.

To study the effects of beta-endorphin in chronic schizophrenia, nine male patients participated in a double-blind crossover comparison of a single intravenous 20-mg injection of beta-endorphin and saline. Bolus injection of beta-endorphin from an albumin-coated syringe produced markedly higher plasma concentrations than did slow intravenous infusion from a non-albumin-coated syringe. Beta-endorphin intravenously injected in nine patients produced a statistically significant increase in serum prolactin levels. In one patient, both 10 mg of morphine sulfate and 20 mg of beta-endorphin produced similar increases in the alpha power of the EEG. In eight patients, beta-endorphin administration was associated with a statistically significant but not clinically obvious improvement in schizophrenic symptoms.

Administration of 4 antipsychotic drugs, haloperidol, chlorpromazine, thioridazine and clozapine, for 21 days elicited increased dopamine metabolism in frontal cortex of rat brain. Only clozapine failed to decrease the apparent firing rate of dopamine neurons in the striatum, as indexed by [homovanillic acid]/[dopamine]. These data support the hypotheses that frontal cortex dopamine neurons may be a common site for antipsychotic action while decreased release of dopamine in the striatum may be associated with the development of extrapyramidal side effects.

An inbred colony of narcoleptic doberman pinschers has been analyzed for muscarinic receptor levels in 19 discrete brain regions. In comparison to age-matched controls, receptors were generally elevated in the brainstem and reduced in forebrain areas. No changes in receptor binding affinity were detected. The increased receptor levels found in the brainstem suggest that cholinoceptive neurons in this region are hypersensitive and may be involved in the initiation of cataplexy and other aspects of the narcolepsy syndrome.

Liver tyrosine transaminase activity is low during the day when the rats are mostly asleep and high during the night when they are awake. When wakefulness was imposed for 8 hr during daylight on the day of the experiment and the rats were allowed to sleep for the following 3 hr during darkness, the tyrosine transaminase activity became high during the day and low at night. That this reversal in enzyme activity is not mediated by the pituitary-adrenal axis is demonstrated by the fact that in adrenalectomized rats tyrosine transaminase activity increased during the day in the sleep deprived rats. However, in these rats the enzyme activity did not become low in the sleep-deprived-sleeping condition. Changes in tryptophan pyrrolase activity during sleep deprivation were demonstrated to be mediated by the pituitary-adrenal axis.