Brain Peptides As Neurotransmitters (2)

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as normal faults in Fig. 3 are actually complex 26. R. B. Herrnann and J. A. Canas, Bull. Seismol. 30. See, for example, L. R. Sykes, Rev. Geophys. zones with multiple offsets. Some of the faults Soc. Am. 68, 1095 (1978); R. B. Herrmann, J. Space Phys. 16, 621 (1979). can be interpreted as high-angle reverse faults Geophys. Res. 84, 3543 (1979). 31. W. Stauder et al., Cent. Miss. Val. Earthquake (line S-6). 27. R. R. Heinrich, Bull. Seismol. Soc. Am. 31, 187 Bull. 19 (1979). 23. R. G. Stearns, U.S. Nucl. Regul. Comm. Rep. (1941). 32. We thank R. E. Anderson, W. H. Diment, and NUREGICR-0874 (1979). 28. T. G. Hildenbrand, personal communication. 0. W. Nuttli, who reviewed the manuscript and- 24. W. M. Caplan, Arkansas Div. Geol. Bull. 20 29. The research well near line S-10 encountered suggested improvements. Appreciation is ex- (1954). highly fractured and altered Paleozoic rocks. tended to the U.S. Nuclear Regulatory Commis- 25. T. C. Buschbach, U.S. Nucl. Regul. Comm. The mineralogy and geochemistry of the rocks sion for providing the funds for the research Rep. NUREGICR-0450 (1978). suggests a nearby igneous body. well.

addition, analgesia that follows electrical stimulation of the brainstem of rats could be partially reversed by the opiate antagonist naloxone, an indication that a morphine-like substance was being released Brain Peptides as Neurotransmitters (2). To identify the postulated morphine- like factor two approaches were taken. Solomon H. Snyder Hughes (3) showed that brain extracts can mimic morphine's effects on electri- cally induced contractions of smooth muscle in a fashion that is blocked by It is generally agreed that information represent only 10 percent or less of the naloxone. Terenius and Wahlstrom (4) processing in the brain largely involves total, whose number then may exceed and Pasternak et al. (5) identified in brain communication among neurons through 200. extracts a substance that competes for release of neurotransmitters at synapses. There has been much debate as to cri- opiate receptor binding. The specificity In theory, the brain might make do with teria that should be fulfilled before it can of this effect was established by showing one excitatory and one inhibitory trans- be designated a "'neurotransmitter." In that the marked regional variations in mitter. Until the 1960's the amines ace- this article, I regard as transmitters those opiate receptor density are paralleled by tylcholine, norepinephrine, and sero- peptides localized in specific neuronal similar variations in concentrations of the morphine-like substance (1, 3, 5). Naloxone blockade of the morphine-like Summary. Numerous peptides appear to be neurotransmitter candidates in the actions on smooth muscle and a regional brain. Some, such as the opioid peptide enkephalins, neurotensin, and substance P, distribution closely mimicking that of the were first isolated from the brain. Peptides, such as cholecystokinin and vasoactive opiate receptor ensured that the sub- intestinal polypeptide, were known as intestinal hormones and later recognized as stance under study was biologically rele- brain constituents. Certain hypothalamic-releasing hormones, pituitary peptides, and vant to opiate receptors. Such guaran- blood-derived peptides like angiotensin 11 and bradykinin, may also be central neuro- tees of biological relevance are impor- transmitters. The diversity of localization of these peptides throughout the brain im- tant, as attempts to isolate a substance plies a multiplicity of potential roles. solely based on its ability to inhibit the binding of a radioactive drug to membranes run the risk that the substance tonin were the only well-recognized systems and released on depolarization, being isolated may not be physiologically transmitters. Then came an appreciation which produce changes in neuronal ac- meaningful. that amino acids such as y-aminobutyric tivity, even though for the most recently Hughes et al. (6) isolated the mor- acid (GABA), glutamic acid, aspartic identified peptides some of these criteria phine-like substance from pig brain and acid, and glycine, might serve as trans- have not yet been examined. showed that it consists of two pen- mitters. A dramatic explosion in the tapeptides, methionine enkephalin (met- number of possible neurotransmitters enkephalin) and leucine enkephalin (leu- came with increasing recognition in the Opiate Receptor and Enkephalins enkephalin) which differ only in having past decade that various peptides may methionine or leucine at the carboxyl be neurotransmitters. At present there In most instances, neurotransmitters terminal. Using the assay based on com- seem to be about two dozen peptide neu- are identified as endogenous substances petition for receptor binding, Simantov rotransmitter candidates, and the num- and on this basis their receptor effects and Snyder (7) isolated the same two ber is increasing rapidly (Table 1). Most are characterized. In the case of the en- peptides from calf brain, confirming the of the brain peptide transmitters have kephalins, the receptors, which were dis- findings of Hughes et al. (6). Even before been discovered serendipitously with no covered first, provided a means to identi- the amino acid sequence of enkephalin systematic search. It would not be sur- fy and then isolate these opiate-like pep- was established, we showed by radio- prising if the known peptide transmitters tides. Dramatic properties of the opiate receptor assay that enkephalin was local- receptor, such as its discriminating agon- ized in nerve endings, which is consist- The author is Director of the Department of Neu- ists and antagonists and the intimate re- ent with a neurotransmitter role, that its roscience and Distinguished Service Professor of lation between receptor localization and detailed distribution in Neuroscience, Pharmacology and Experimental regional monkey Therapeutics, and Psychiatry and Behavioral Sci- central sites of pain perception (I) sug- brain closely paralleled that of opiate re- ences, Johns Hopkins University School of Medi- cine, 725 North Wolfe Street, Baltimore, Maryland gested that it might interact with a nor- ceptors, and that its phylogenetic distri- 21205. mally occurring opiate-like substance. In bution was the same as that of opiate re-

0036-8075/80/0829-0976$02.00/0 Copyright 1980 AAAS SCIENCE, VOL. 209, 29 AUGUST 1 2 www.sciencemag.org Downloaded from ceptors (8). Enkephalin is released in a Table 1. Peptide neurotransmitter candidates. have been identified, mu receptors, with calcium-dependent fashion with brain Gut-brain peptides distinct preference for morphine, and del- depolarization, further supporting a neu- Vasoactive intestinal polypeptide (VIP) ta receptors with selectivity for certain rotransmitter role (9). Cholecystokinin octapeptide (CCK-8) enkephalin derivatives, such as [D-Ala2- Once the amino acid sequence of en- Substance P D-Leu5Jenkephalin (Ala, alanine). Dif- Neurotensin kephalin was known it became evident Methionine enkephalin ferential autoradiographic localizations that the five amino acids (residues) con- Leucine enkephalin suggest that the two types of receptors stituting met-enkephalin are contained Insulin mediate different functions (20). Thus, within the 91 amino acids of the peptide Glucagon the mu receptors are preferentially local- /3-lipotropin isolated 10 years earlier Hypothalamic-releasing hormones ized to layers 1 and 4 of the cerebral cor- Thyrotropin-releasing hormone (TRH) from the pituitary by Li (/0). Several Luteinizing hormone-releasing tex, which (especially layer 4) are in- groups of investigators showed that a va- hormone (LHRH) volved preferentially in integrating sen- riety of lipotropin fragments, all incorpo- Somatostatin (growth hormone sory perception. The nucleus accumbens rating the met-enkephalin sequence, pos- release-inhibiting factor, SRIF) and olfactory tubercle, parts of the emo- Pituitary peptides sess opiate activity (11). Within the pitui- Adrenocorticotropin (ACTH) tion-regulating limbic system, are selec- tary most opiate-like activity can be ac- f3-Endorphin tively enriched in delta receptors. En- counted for by fl-endorphin, the 31 a-Melanocyte-stimulating hormone kephalin derivatives with preferential ac- amino acid peptide at the carboxyl termi- (a-MSH) tions at mu receptors are more potent an- nal portion of /3-lipotropin, although Others algesics, suggesting that mu receptors Angiotensin II another recently identified pituitary pep- Bradykinin mediate analgesic actions. Perhaps delta tide, dynorphin, also possesses high Vasopressin receptors preferentially regulate emo- opiate receptor activity (12). /8-Lipo- Oxytocin tional behavior. tropin itself derives from a 31,000-dalton Camosine My co-workers and I have proposed Bombesin precursor peptide which also incorporat- that the two types of opiate receptors in- es the sequence of adrenocorticotropic teract, respectively, with met- and leu- hormone (ACTH), and hence is referred enkephalins (20). Accordingly, the two to as 31K ACTH (13). /3-Endorphin oc- of sensory "pain" neurotransmitters types of enkephalin neurons may be in- curs in the brain at levels about 10 per- such as substance P. Enkephalin tracts volved, in part, in different brain func- cent those of enkephalin in specific neu- and opiate receptors in the limbic sys- tions. In support of this notion is the ronal systems (14) other than enkephalin tem, which regulates emotional behav- finding that brain regions with more mu neurons and has been reviewed recently ior, may explain euphoric effects of than delta receptors, such as the hippo- along with other pituitary hormones in opiates. Respiratory depression, which campus and thalamus, also have more the brain (15). accounts for lethal effects of opiates, met-enkephalin neurons than leu-en- may involve receptors in the solitary nu- kephalin neurons (20, 21). Conversely, cleus of the brainstem; which regulates the central amygdala, which has more Localization of Enkephalin visceral reflexes including respiration. delta than mu receptors, has more leu- Neurons and Opiate Receptors The existence of two distinct enkepha- enkephalin than met-enkephalin neurons lin molecules differing only at the car- (20, 21). The substantia gelatinosa of Considerable insight into the functions boxyl terminal raises questions about spinal cord and the caudate, which have of neurotransmitters has been attained their disposition. Does the same neuron similar levels of mu and delta recep- by light microscopic examination of neu- synthesize both met- and leu-enkephalin tors, also have similar numbers of met- ronal systems containing them. His- or are they contained in distinct neuronal enkephalin and leu-enkephalin neurons tochemical techniques mapping norepi- populations? Most antiserums to the in- (20,21). nephrine, dopamine, and serotonin path- dividual enkephalins fail to discriminate ways were crucial for our present appre- them histochemically. A recently devel- ciation of their role in brain function. For oped antiserum to met-enkephalin ad- Enkephalin and Opiate Receptor peptide neurotransmitters, antibodies sorbed with leu-enkephalin shows abso- Interactions have been raised against the peptides lute specificity for met-enkephalin, while themselves usually linked to a carrier oxidative destruction of met-enkephalin The central question in synaptic trans- protein. Opiate receptors were visual- with permanganate permits selective mission is how recognition of a neuro- ized microscopically prior to the isola- staining of leu-enkephalin (18). Using transmitter is translated into an altera- tion of enkephalin by means of the au- these procedures, we showed that met- tion in cell functioning, be it a change in toradiographic techniques developed by enkephalin and leu-enkephalin occur in ion permeability or cyclic nucleotide for- Kuhar and co-workers (16). Maps local- completely separate neurons in brain and mation. The first generation of receptor izing opiate receptors and enkephalin intestine (18). The neuronal patterns of studies dealt with the binding or recogni- neurons coincide fairly closely and in- met- and leu-enkephalin differ consid- tion site. Subsequent work has shed light volve brain structures whose functions erably. For instance, in the globus pal- on how recognition of a transmitter al- are linked to opiate actions. lidus, leu-enkephalin nerve fibers are dis- ters cellular function and how receptors Localizations of opiate receptors and posed as narrow bands surrounding axon differentiate agonists and antagonists. enkephalins can explain many of the bundles while the met-enkephalin fibers Very low concentrations of sodium ion pharmacological actions of opiates (16, are in dense clusters between the leu-en- enhance the binding of opiate antago- 17). For instance, it appears that small kephalin bands. nists and reduce the binding of opiate enkephalin-containing interneurons in Pharmacological and biochemical evi- agonists, thus providing a way to predict the dorsal spinal cord synapse on opiate dence suggests the existence of multiple whether a test drug is a pure agonist, or receptors localized to nerve endings of populations of opiate receptors (/9). In has both agonist and antagonist func- sensory neurons, inhibiting their release binding studies two distinct receptors tions (22).

29 AUGUST 1980 977 2 www.sciencemag.org Downloaded from Table 2. Sites for enzymatic cleavage of enkephalins. All the peptide bonds of the enkephalins cleaving enkephalin from its precursor can be cleaved by one or another known peptidase. Aminopeptidases and carboxypeptidases differ in the two types of neurons. can remove the NH2-terminal and COOH-terminal amino acids, respectively, but they have no known selectivity for enkephalins. Enkephalinase A1, enkephalinase A2, and angiotensin-con- Identification of a specific inactivating verting enzyme display substantial regional variations in mammalian brain which parallel to system for a neurotransmitter facilitates some extent regional variations in opiate receptor and enkephalin distribution. Enkephalinase B an understanding of its function. For in- activity does not vary much regionally. The regional distribution of cathepsin C is not well stance, behavioral effects of drugs that characterized. block the inactivating enzyme provide Met-enkephalin: Tyrosine Glycine Glycine Phenylalanine Methionine clues to transmitter actions and may af- Leu-enkephalin: Tyrosine t Glycine - Glycine Phenylalanine Leucine ford new therapeutic drugs. Could en- Aminopeptidases Enkephalinase B kephalin be inactivated by a specific pep- Cathepsin C tidase analogous to the enzymatic hy- Enkephalinase A1 drolysis of acetylcholinesterase? Like all Enkephalinase A2 peptides, enkephalin can be acted on by Angiotensin-converting enzyme numerous peptidases (Table 2). Recent- Carboxypeptidases ly, an enzyme activity that cleaves enkephalin between glycine and phenylalanine has been described (36, 37) gener- The actions of sodium at the opiate re- ably also linked to the adenylate cyclase ating the Tyr-Gly-Gly fragment (Tyr, ceptor suggested that sodium per- alterations. tyrosine; Gly, glycine), referred to here meability is altered in synaptic actions of The chain of cellular events following as enkephalinase A. This membrane-as- opiates and enkephalins. Indeed, Ziegl- opiate receptor stimulation has recently sociated enzyme was first reported to gansberger and his co-workers showed been traced yet another step. Opiate re- have affinity for enkephalin detectable in that opiates block the excitatory effects ceptor stimulation in neuroblastoma the nanomolar range, to display regional of glutamic acid and acetylcholine on ce- clones reduces synthesis of gangliosides variations like those of opiate receptors, rebral neurons by altering sodium chan- (lipid related molecules on membrane and to double in activity in morphine-ad- nel functioning (23). Peripheral pharma- surfaces), while phospholipid, protein, dicted mice (37). Our subsequent studies cologic actions of morphine are also reg- nucleic acid, and other metabolic con- (38) and those of others (39) showed that ulated by sodium and other ions in paral- stituents are not affected (30). Another the affinity of the enzyme for enkephalin lel with their effects on opiate receptor lipid, cerebroside sulfate, may play a is only 1 percent of that originally report- binding (24). role in opiate recognition at receptors ed, and its activity increases only 20 per- Receptor interactions of opiate ago- (3/). cent in addicted mice. We separated two nists and antagonists are also distin- enkephalinase A activities (A1 and A2) in guishable by their reactivity with guano- solubilized brain membranes and puri- sine triphosphate (GTP). Like sodium, Enkephalin Metabolism fied both to homogeneity. We also de- GTP decreases receptor affinity of opiate tected a second peptidase, designated agonists but not antagonists (25). The Most biological peptides are formed enkephalinase B, which cleaves en- GTP effects on numerous hormonal neu- by cleavage from larger precursor pep- 1kephalin between the two glycines and rotransmitter receptors are associated tides, just as 3-endorphin is formed by then gives rise to the Tyr-Gly fragment with linkage of the receptor to adenylate cleavage from /8-lipotropin. Although (38). This enzyme has a similar affinity as cyclase. Opiates and enkephalins dimin- met-enkephalin is contained in the amino enkephalinase A for enkephalin. In addi- ish the adenylate cyclase activity and re- acid sequence of,-endorphin, it appears tion, we characterized a membrane-asso- duce cyclic adenosine monophosphate unlikely that /8-endorphin could be the ciated aminopeptidase that cleaves tyro- (cyclic AMP) levels in neuroblastoma biological precursor of met-enkephalin, sine from enkephalin. clones, which possess specific opiate re- since they are localized in different brain Whether any of these enzymes repre- ceptors (26). Thus, cyclic AMP also regions. Several attempts have been sents a specific synaptic inactivating seems to be a second messenger for en- made to identify large peptides in the mechanism for enkephalin is unclear. kephalin. Are the sodium and cyclic brain which generate enkephalin. La- Enkephalinase A and B have similar af- AMP effects related or are they inde- beled amino acids are incorporated into finities for enkephalin, whereas amino- pendent second messengers communi- enkephalin in brain slices (32). Large peptidase has somewhat lesser affinity. cating different kinds of information to peptides in the brain can be converted in- Although there are regional variations in the cell? It has been shown that cyclic to small peptides with enkephalin-like activity of all three enzymes, the most AMP reduction elicited by opiates is cru- immunoreactivity and opiate-like activi- marked variations occur for enkepha- cially dependent on both extracellular ty in systems such as the guinea pig in- linase A1 and A2 and angiotensin-con- sodium and guanine nucleotides (27). testine (33). Peptides of 6, 7, and 15 verting enzyme. Initially, it had been Sodium also regulates receptors for amino acids containing enkephalin se- suggested that enkephalinase A is identi- other neurotransmitters such as alpha- quences have been identified in brain and cal with the angiotensin-converting en- adrenergic, histamine Wr and mus- adrenal tissue (34). For example, Kimura zyme that generates the physiologically carinic cholinergic recepiors (28). In ad- et al. (35) identified peptides incorporat- active angiotensin II from its inactive dition, opiate receptors as well as alpha- ing both met-enkephalin and leu-en- precursor angiotensin I (40). Although adrenergic, dopamine, and histamine H1- kephalin sequences in the adrenal gland both enzymes can cleave enkephalin be- receptors are also regulated by divalent of the cow, which contains as much en- tween glycine and phenylalanine, we cations with manganese generally being kephalin as brain. If these putative en- separated by physical means the two en- the most effective and calcium relatively kephalin precursors occur also in the zymes, indicating that they are distinct inactive (28, 29). Receptor regulation by brains of rodents, then our finding of dis- entities (38) with different sensitivities to divalent cations appears associated with tinct neurons that contain met- and leu- metals, reagents, and enzyme inhibitors regulation by GTP and therefore is prob- enkephalin (18) implies that the enzymes (38, 39).

978 SCIENCE, VOL. 209 Neurotensin of pain has been extensively supported. tonin pathway to the spinal cord which in Substance P occurs in 20 percent of dor- turn elicits analgesia (56). Certain raphe Neurotensin was identified as a by- sal root ganglia cells with some process- nuclei contain both substance P and product of substance P isolation (41). Be- es extending to the skin and others enter- serotonin (57), indicating that a single sides lowering blood pressure by dilating ing the spinal cord and giving rise to ter- neuron can contain more than one neuro- blood vessels, neurotensin alters pitui- minals in the substantia gelatinosa (49, transmitter. tary hormone release (41), and, when in- 50). Whereas cutting the dorsal root has More neuronal pathways have been jected in the brain, lowers body temper- no influence on spinal cord enkephalin, a demonstrated for substance P than for ature (42). Highest concentrations of similar lesion causes degeneration of any of the other peptide transmitters. neurotensin occur in the hypothalamus substance P terminals in the spinal cord. The pathway of sensory neurons that and the basal ganglia of the brain; high Removal of the tooth pulp, which con- contain substance P and the striatonigral concentrations are also found in nerve tains only pain-sensitive sensory fibers, pathway have already been discussed. A ending fractions of brain homogenates causes a loss of nerve endings that con- substance P-containing pathway with (41, 43). Neurotensin is released in a tain substance P in the trigeminal nucle- cells in the caudate and terminals in the calcium-dependent fashion when brain us of the brainstem, where sensory fibers globus pallidus is analogous to the appar- slices are depolarized (44). It produces terminate (51). Since the tooth pulp con- ent enkephalin-containing pathway that selective inhibition of neuronal firing in tains only pain-sensitive fibers, the sub- connects these structures. Cells in the areas such as the locus coeruleus, which stance P sensory neurons presumably medial habenular nucleus that contain has a high density of neurotensin neu- mediate pain perception. Moreover, spi- substance P give rise to axons that termi- rons (45). Abundant neurotensin recep- nal cord neurons excited by substance P nate in the interpeduncular nucleus, par- tor binding sites have been demonstrated are the same as those that respond selec- alleling a similar acetylcholine-contain- throughout the brain (46). All of these tively to painful stimuli (52). Substance P ing pathway. Within the amygdala, sub- findings are consistent with a neurotrans- sensory fibers may also regulate axon re- stance P-containing cells in the medial mitter role. flexes in the skin. Axon reflexes are re- nucleus give rise to terminals in the cen- Histochemical mapping of neurotensin sponsible for the local vasodilation medi- tral nucleus, differing from the amygda- reveals dramatic similarities to the dis- ated by sensory nerves that occurs loid disposition of neurotensin and en- position of enkephalin. Indeed, neuro- around an injured area. Sir Henry Dale kephalin, whose cells are in the central tensin localizations bear a closer resem- (53) suggested that the vasodilation was nucleus. Substance P cells in the bed nu- blance to those of enkephalin than other elicited by release from sensory nerves cleus of the stria terminalis project to the peptides do (47). For instance, as with of the same transmitter that the primary medial preoptic area. These pathways enkephalin, neurotensin neurons are afferent terminals release in the spinal within the limbic system may play a role highly localized to the dorsal gray, sub- cord, a proposal consistent with the po- in emotional behavior. stantia gelatinosa region of the spinal tent vasodilatory action of substance P. cord, implying a role in pain perception; Since substance P is a "pain" trans- this implication has been confirmed by mitter, the demonstrated blockade of its Cholecystokinin and Gastrin direct studies revealing neurotensin's release in spinal cord by opiates (54) may potent analgesic effects (48). The neuro- account in part for opiate analgesia. In- Cholecystokinin (CCK) was originally tensin-elicited analgesia is unrelated to teractions between enkephalin and sub- isolated from the duodenum as a sub- the opiate system, since it is not blocked stance P may occur in other parts of the stance that contracted the gall bladder, by opiate antagonists such as naloxone. central nervous system, since neurons hence its name. Independently, a duode- Drugs that mimic neurotensin might containing the two peptides are juxta- nal substance that stimulated pancreatic have analgesic properties but lack typi- posed in such areas as the raphe nuclei, secretion was designated pancreozymin cal opiate side effects. the ventral tegmental area, the septum, (58). Chemical isolation, sequencing, The most dense collection of neuro- and the amygdala (49, 50). In the sub- and synthesis of CCK in classical studies tensin cells is in the central nucleus of stantia nigra of the brainstem, substance by Mutt and colleagues (59) revealed that the amygdala, a distribution similar to P release is regulated by another trans- it is a 33 amino acid peptide and is identi- that of enkephalin. Another similarity to mitter, GABA. Substance P is contained cal to pancreozymin. In radioimmunoas- enkephalin is that a neurotensin neuronal in a long pathway with cell bodies in the say of brain extracts CCK was first mis- pathway has cell bodies in the central nu- corpus striatum and fibers that descend taken for gastrin (60). Dockray (61) then cleus of the amygdala with axons pro- to terminate in the substantia nigra, showed that the immunoreactivity was ceeding through the stria terminalis to which contains the highest concentration due to CCK and not gastrin, the cross- terminate in part in its bed nucleus. But of substance P in the brain (55). GABA, reactivity deriving from the fact that gas- although neurotensin and enkephalin which occurs in a parallel striatonigral trin and CCK share the same COOH- have closely similar localizations, they pathway, inhibits the release of sub- pentapeptide sequence at the COOH-ter- are stored in distinct neurons (47). stance P that follows the depolarization minal. Whereas intestinal CCK largely of substantia nigra slices (55). consists of the 33 amino acid residue Substance P also seems to be closely peptide, the major CCK entity in the Substance P associated with serotonin, perhaps in brain is the COOH-terminal octapeptide regulating pain perception. Areas that (CCK-8) with a lesser amount of the Next to enkephalin, substance P is the are enriched in serotonin as well as sub- COOH-terminal tetrapeptide and very most studied brain peptide. Its local- stance P and enkephalin include the little CCK-33 (62). ization throughout the brain resembles amygdala, periaqueductal gray, raphe True gastrin is contained in the large neurotensin and enkephalin, although nuclei, and substantia nigra. Stimulation cell system of the hypothalamus, which the similarities are not as great as be- of the nucleus raphe magnus of the brain- projects to the posterior pituitary gland tween neurotensin and enkephalin. Sub- stem, many of whose neurons contain (61). Conceivably, gastrin occurs in stance P's role as a sensory transmitter serotonin, activates a descending sero- a hypothalamic-pituitary pathway sim- 29 AUGUST 1980 979 ilar to the one containing enkephalin. activities of CCK is suggested by find- essentialty restricted to the intestine, its Initial histochemical staining for CCK ings that very low doses of CCK-8 inject- associated organs, and the central ner- in rabbit cerebral cortex suggested label- ed intraperitoneally cause satiety in pre- vous system. Bradykinin presents a dif- ing of the majority of cells as well as viously hungry rats (70). The CCK-8 sa- ferent pattern. It was discovered 30 much white matter (63). Quite different tiety is elicited by lower doses when in- years ago as a biologically active factor results were obtained in the rat and guin- jected peripherally than when admin- released from a2-globulin fractions of ea pig (64). Both rat and guinea pig cere- istered directly in the brain (70). Where blood by incubation with trypsin or bral cortex contain many CCK cells and might the peripheral "satiety receptor" snake venom, and called bradykinin be- fibers, coinciding with the high levels of for CCK be located? The vagal nerves cause of the slow "brady" contractions CCK in the cerebral cortex. CCK and of the liver or stomach are reason- it elicited from the guinea pig ileum vasoactive intestinal peptide (VIP) are able candidates. Evidence for a cen- (75). Being generated in the circula- the only brain peptides with cells in the tral influence of CCK in regulating fluid tion, bradykinin acts (as would be cerebral cortex. Because of the consid- intake derives from experiments show- expected) in many parts of the body and erable mass of the cerebral cortex, the ing that as little as 0.01 picomole of has been implicated in the genesis of in- total brain content of CCK, about 1 to 2 CCK-8 per minute infused into the later- flammation, cardiovascular shock, hy- milligrams in the human, is far greater al ventricles of sheep suppress feeding pertension, pain, and even rheumatoid than that of any other peptide. CCK is a behavior (71). arthritis. Bradykinin is probably the rapid and powerful excitant of cerebral most potent pain-producing substance cortical cell firing (65). known. Its release by tissue damage and The most prominent group of CCK Vasoactive Intestinal Polypeptide action at specific receptors on sensory cells in the brain occurs in the peri- neurons has been suggested as a first aqueductal gray area of the brainstem Vasoactive intestinal polypeptide was step in pain perception. Interestingly, (64), where CCK might be involved in identified in extracts of the gut as a sub- when injected near the periaqueductal the pain-integrating functions of this re- stance that causes vasodilation. It was gray of the brainstem, bradykinin has po- gion. Like substance P, CCK occurs in isolated as a 28 amino acid peptide with tent analgesic effects; in the lateral septal sensory fibers with cell bodies in dorsal many similarities in amino acid sequence area, bradykinin injections increase root ganglia and terminals in the dorsal and biological activity to the intestinal blood pressure (76). gray matter of the spinal cord (64). As peptides secretin and glucagon (72). Immunohistochemical studies have re- with substance P, neurotensin, and en- Thus, besides causing vasodilation, VIP vealed the location of bradykinin neu- kephalin, CCK cells are abundant in the stimulates the conversion of glycogen to rons (77). The principal group of bra- hypothalamus, whereas the central nu- glucose, enhances lipolysis and insulin dykinin cells occurs in the hypothalamus cleus of the amygdala has a dense collec- secretion, inhibits the production of gas- with fibers throughout the hypothalamus tion of CCK fibers but no cells (64). tric acid, and stimulates secretion by the as well as in the lateral septal area and Just as substance P coexists with sero- pancreas and small intestine (72). periaqueductal gray. The septal bradyki- tonin in some neurons, CCK appears to Seven years after its isolation from the nin neurons might participate in the regu- coexist with dopamine not in the sub- intestine, VIP was demonstrated in the lation of blood pressure, which would be stantia nigra neurons, which project to brain (73). Like CCK, highest levels of consistent with the hypertensive effects the caudate nucleus, but in brainstem VIP occur in the cerebral cortex, al- of injections there, whereas bradykinin dopamine neurons, which project to the though cortical VIP levels are only one- neurons in the periaqueductal gray might limbic systems (66). This neuronal sys- tenth those of CCK. VIP appears to be have a role in pain perception. tem is implicated in the actions of anti- contained in small neurons within the ce- schizophrenic neuroleptic drugs, sug- rebral cortex since severing connections gesting a role for CCK in schizophrenia. between the cerebral cortex and the rest Angiotensin Recently CCK receptor binding has ofthe brain does not lower cortical levels been identified in the pancreas (67, 68) of VIP. The VIP nerve terminals are also The angiotensin story is probably old- and brain (68). In the pancreas the pep- localized in the central amygdaloid nu- er than that of almost any biologically ac- tide specificity of binding sites corre- cleus and in the medial preoptic and an- tive peptide. In 1898, the kidney was sponds to relative CCK-like biological terior hypothalamic nuclei (74). shown to contain a proteolytic activity, potencies in the pancreas, with penta- In the cerebral cortex, VIP and CCK renin, which converts a large peptide in gastrin, the COOH-terminal pentapep- neurons are bipolar and oriented per- blood plasma, angiotensinogen, to the tide of CCK, having no activity. By con- pendicular to the surface. This pattern decapeptide angiotensin I (78). Angio- trast, at the brain receptors, pentagastrin makes these peptidergic neurons ideally tensin I is almost devoid of biological ac- and CCK-4 are highly potent (68). The suited to activate and synchronize neu- tivity in the periphery and the brain. It is characteristic CCK receptor binding ronal activity within the vertical columns "activated" by angiotensin-converting sites in the brain may therefore not rep- of cerebral cortical cells. Like CCK, VIP enzylne which removes a COOH-termi- resent receptors for the CCK-8 or CCK- is a potent and rapid neuronal excitant in nal histidyl-leucine (His-Leu) to form 33, the major forms in brain, but for the CCK-enriched hippocampus, is angiotensin II. Most of the peripheral ac- CCK-4 (the COOH-terminal tetrapeptide stored in vesicles, and is released with tions of angiotensin II center around the of CCK), which has recently been de- neuronal depolarization. regulation of the circulatory system. scribed in specific brain areas and is Angiotensin II is a potent vasoconstric- quite distinct from the localization of tor and causes renal sodium retention by CCK-8 (69). Bradykinin stimulating aldosterone secretion from Cholecystokinin is a good example of the adrenal cortex. an intestinal hormone subsequently Enkephalin, neurotensin, substance P, Interestingly, the effects of centrally shown to occur in the brain. A possible CCK, and VIP all fit in the group of administered angiotensin II are com- link between the intestinal and central brain-gut hormones with distributions plementary to its peripheral actions. 980 SCIENCE, VOL. 209 Central injections of angiotensin II po- of the trigeminal, facial, and hypoglossal Insulin and Glucagon tently stimulate drinking behavior and nerves, the ventral spinal cord, the nu- raise blood pressure (79). Regional mi- cleus accumbens, the lateral septal nu- While all the peptide transmitters dis- croinjections have localized the dipso- clei, and the bed nucleus of the stria ter- cussed above are relatively small mole- genic effects of angiotensin II to the sub- minalis (86). We identified specific TRH cules, generally with fewer than 20 fornical organ where application of angi- receptor binding in numerous brain re- amino acids, insulin is substantially otensin II activates neuronal firing and gions with receptor specificity essen- larger, a protein comprising 86 amino enhances drinking behavior (80). tially the same as for pituitary receptors acids. Insulin secreted from the pancreas The striking central actions of angio- (87). TRH elicits behavioral excitation does not penetrate into the brain. Recent tensin suggest that endogenous angioten- and anorexia in animals and may cause evidence reveals that the brain possesses sin should exist in the brain. The brain mood enhancement in humans (88). its own insulin (95). Radioimmunoassay does contain renin-like activity as well as Somatostatin is a hypothalamic, cyclic reveals that insulin concentrations in the considerable angiotensin-converting en- peptide consisting of 14 amino acids; it brain are 10 to 100 times higher than in zyme activity. Attempts to isolate en- inhibits the release of growth hormone plasma. The immunoreactive insulin is dogenous angiotensin itself have in- from the pituitary gland (89). In addition, biologically active at receptor binding dicated extremely low levels and these it blocks the release of pituitary thyrotro- sites and in stimulating glucose oxidation amounts may not in fact represent au- pin (TSH) and prolactin. Apart from its in fat cells. Highest brain insulin concen- thentic angiotensin (81). In contrast to role as a hypothalamic release inhibiting tration (80 nanograms per gram) occurs the very low levels of apparent endoge- factor, somatostatin appears to be a typi- in the hypothalamus with similar levels nous angiotensin, the brain possesses cal "gut-brain" peptide, being localized in the olfactory bulb. abundant angiotensin receptor binding both to neurons throughout the brain and What might be the role of insulin in the higher than in any other tissue of the the stomach, intestine, and pancreas (50, brain? Could such a large molecule func- body (82). 90); it inhibits the secretion of glucagon, tion as a neurotransmitter? The possi- Immunohistochemical studies have re- insulin, and gastrin (91). Somatostatin- bility that insulin has some neurotrans- vealed neuronal systems that stain with containing cells occur in the amygdala, mitter or neuromodulator function is antiserum to angiotensin (83). Whether parts of the hypothalamus, the hippo- suggested by the demonstration in brain this material represents authentic chem- campus, and the cerebral cortex with ter- of specific insulin receptor binding sites ical angiotensin is unclear. Some cells minals located in all of these areas as (96). Properties of insulin receptors in stain in the paraventricular and peri- well as the caudate nucleus, nucleus ac- the brain are essentially the same as fornical area of the hypothalamus, while cumbens, and the olfactory tubercle (50, those of peripheral insulin receptors. dense terminal patterns are observed in 90). Somatostatin also occurs in about 20 Glucagon is a pancreatic hormonal the substantia gelatinosa of the spinal percent of primary sensory neurons as peptide of 29 amino acids. A much larger cord, the spinal cord, the spinal nucleus well as dorsal root ganglion cells. Like form (12,000 daltons) of glucagon has re- of the trigeminal nerve, the central substance P, somatostatin in these un- cently been identified in the intestine amygdala, locus coeruleus, and the peri- myelinated sensory neurons may be a (97). Whether or not this is a precursor of ventricular gray. This pattern resembles major transmitter of pain sensation. pancreatic glucagon is unclear. Intestinal that of substance P, neurotensin, and en- Luteinizing hormone-releasing hor- glucagon, also referred to as glicentin, kephalin. Dorsal root lesions reduce the mone (LHRH), a hypothalamic decapep- has been detected in brain tissue (98). In- angiotensin staining in the dorsal spinal tide, stimulates the secretion of both lu- testinal glucagon may represent another cord, suggesting that the angiotensin-like teinizing and follicle-stimulating hor- very large peptide that serves as a neuro- material may be contained in sensory mones from the anterior pituitary (92). transmitter candidate. Support for this neurons. There is less evidence for a widespread possibility comes from histochemical distribution of LHRH than for TRH or studies in our own and other (99) labora- somatostatin outside the hypothalamus tories, demonstrating central glucagon- Hypothalamic-Releasing Factors or brain. LHRH is contained within the containing neurons. We have detected a pre- and suprachiasmatic and arcuate nu- dense plexus of glucagon-containing The hypothalamic-releasing factors clei of the hypothalamus (93). It is not nerve fibers in the hypothalamus, but were identified as agents that are con- clear which of these LHRH systems spe- have not yet localized cell bodies. tained within the median eminence of the cifically regulates pituitary secretion. hypothalamus and pass through the por- Most LHRH nerve fibers outside the hy- tal capillaries to the pituitary gland pothalamus probably originate from hy- Other Peptides where they regulate the synthesis and re- pothalamic cell bodies, although studies lease of pituitary hormones. Immuno- of lesions separating the hypothalamus The posterior pituitary nonapeptides, histochemical procedures have shown from the rest of the brain have been am- vasopressin and oxytocin, were the first that most of these factors are fairly wide- biguous (94). Some LHRH fibers project peptides isolated from the brain. Vaso- ly distributed outside of the hypothala- to the organum vasculosum of the lamina pressin is the pituitary's antiduretic hor- mus. terminalis, a portion of the central ner- mone, and oxytocin plays a role in regu- Thyrotropin-releasing hormone (TRH) vous system outside of the blood brain lating uterine contraction and milk ejec- was the first of these factors to be isolat- barrier, the suprachiasmatic nucleus, the tion. Vasopressin- and oxytocin-contained and has now been identified as the mammillary bodies, and the ventral teg- ing cells, which project to the pituitary, tripeptide pyroglutamylhistidylprolin- mental area. Some LHRH neurons in the are localized in the supraoptic and para- amide (84). In the rat brain, 80 percent of medial septal area, diagonal band, and ventricular nuclei of the hypothalamus. TRH occurs outside the hypothalamus olfactory tubercle seem to contact blood Besides innervating the pituitary, they (85). Immunohistochemical studies re- vessels rather than other neurons. Con- may give rise to small recurrent collater- veal TRH-containing fibers in many ceivably, these neurons regulate blood al neurons that make connections with areas of the brain, including motor nuclei supply. the vasopressin and oxytocin cells of the 29 AUGUST 1980 981 hypothalamus (100). These peptides are bladder, stimulating enzyme secretion 271, 677 (1978); L. L. Iversen, S. D. Iversen, F. E. Bloom, T. Vargo, R. Guillemin, ibid., p. stored along with carrier hormones re- from the exocrine pancreas, and altering 679. ferred to as I, which binds intestinal motility. Radioimmunoassays 10. C. H. Li, Nature (London) 201, 924 (1964). neurophysin 11. R. Guillemin, N. Ling, R. Burgus, C. R. Acad. oxytocin, and neurophysin II, which with antibodies to bombesin have re- Sci., Ser. D 282, 7831 (1976); B. M. Cox, A. Goldstein, C. H. Li, Proc. Natl. Acad. Sci. binds vasopressin. Neurophysin-con- vealed bombesin-like material in the gas- U.S.A. 73, 1821 (1976); A. F. Bradbury, D. G. taining pathways have been traced histo- trointestinal tract, the lung, and brain Smyth, C. R. Snell, N. J. Birdsall, E. C. Hulme, Nature (London) 260, 793 (1976); M. chemically from the hypothalamic nuclei (106). However, in the brain the material Chretien, S. Benjannet, N. Dragon, N. G. Sei- to various autonomic centers in the reacting with the bombesin antibodies is dah, M. Lis, Biochem. Biophys. Res. Com- mun. 72, 472 (1976); N. Ling, R. Burgus, R. brainstem and the spinal cord (101). In chemically distinct from authentic bom- Guillemin, Proc. Natl. Acad. Sci. U.S.A. 73, the brainstem these fibers project to the besin. High affinity binding of bombesin 3942 (1976). 12. A. Goldstein, S. Tachibana, L. I. Lowney, M. locus coeruleus, parabrachial nucleus, to brain membranes has been reported; Hunkapiller, L. Hood, Proc. Natl. Acad. Sci. various the regional distribution of this bombesin U.S.A. 76, 6666 (1979). Edinger-Westphal nucleus, vagal 13. R. E. Mains, B. A. Eipper, N. Ling, ibid. 74, nuclei, the septum, the subfornical or- binding differs considerably from that of 3014 (1977); R. E. Mains and B. A. Eipper, J. Biol. Chem. 253, 651 (1978); J. L. Roberts and gan, the bed nucleus of the stria termi- immunoreactive bombesin (107). When E. Herbert, Proc. Natl. Acad. Sci. U.S.A. 74, nalis, and the medial nucleus of the injected intraventricularly in rats, bomb- 5300 (1977); J. L. Roberts, M. Phillips, P. A. Rosa, E. Herbert, Biochemistry 17, 3609 amygdala. Although they have similar esin quite potently lowers body temper- (1978). pathways, oxytocin and vasopressin ature. Thus, whether bombesin or a re- 14. J. Rossier et al., Proc. Natl. Acad. Sci. U.S.A. 74, 5162 (1977); S. J. Watson, J. D. Barchas, C. neurons are physically distinct. Oxyto- lated peptide has a biological role in the H. Li, ibid., p. 5155; F. Bloom, E. Battenberg, cin and vasopressin synthesis has been brain is as yet unclear. J. Rossier, N. Ling, R. Guillemin, ibid. 75, 1591 (1978); F. Pelletier, L. Desy, J. C. Lis- traced by injecting radioactive precursor In summary, rapidly increasing num- sitszy, F. Labrie, C. H. Li, Life Sci. 22, 1799 and seem to have neu- (1978). amino acids into the supraoptic para- bers of peptides likely 15. D. T. Krieger and A. S. Liotta, Science 205, ventricular nuclei and then by following rotransmitter-related roles in the brain. 366 (1979); A. Goldstein, ibid. 193, 1081 (1976). 16. S. Atweh and M. J. Kuhar, Brain Res. 124, 53 incorporation into the peptides at dif- Detailed investigation over many years (1977); ibid. 129, 1 (1977); ibid. 134, 393 (1977); ferent points in the hypothalamic- of individual neurotransmitters, such as C. B. Pert, M. J. Kuhar, S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 73, 3729 (1976). pituitary pathway (102). norepinephrine and dopamine, has re- 17. R. Elde, T. Hokfelt, 0. Johannson, L. Te- Behavioral studies suggest a role for vealed links to normal and abnormal be- renius, Neuroscience 1, 349 (1976); R. Sim- antov, M. J. Kuhar, G. Uhl, S. H. Snyder, vasopressin in learning and memory. De- havior and facilitated development of Proc. Natl. Acad. Sci. U.S.A. 74, 467 (1977); Wied and Versteeg showed that major psychotropic drugs. The proper- M. Sar, W. E. Stumpf, R. J. Miller, K. J. impaired Chang, P. Cuatrecasas, J. Comp. Neurol. 182, learning behavior in Brattleboro rats, ties of any one of the peptides discussed 17 (1978); G. R. Uhl, R. R. Goodman, M. J. Kuhar, S. R. Childers, S. H. Snyder, Brain which lack vasopressin-synthesizing in this article are as potentially "inter- Res. 166, 75 (1979); S. J. Watson, H. Akil, S. mechanisms, can be reversed by intra- esting" as those of norepinephrine or Sullivan, J. D. Barchas, Life Sci. 21, 733 (1977). ventricular injections of vasopressin dopamine. The characterization of most 18. L.-I. Larsson, S. R. Childers, S. H. Snyder, (103). In normal rats, peripheral vaso- of these peptides is just commencing. Nature (London) 282, 407 (1979). 19. W. R. Martin, C. G. Eades, J. A. Thompson; pressin injections influence learning and Development of drugs with selective ef- R. E. Huppler, D. E. Gilbert, J. Pharmacol. memory, and in clinical trials vasopres- fects on peptide disposition awaits clari- Exp. Ther. 197, 517 (1976); J. A. H. Lord, A. A. Waterfield, J. Hughes, H. W. Kosterlitz, sin may improve memory in brain-dam- fication of the biosynthesis and in- Nature (London) 267, 495 (1977); K.-J. Chang, B. R. Cooper, E. Hazum, P. Cuatrecasas, Mol. aged human subjects. activation of most of the peptides. The Pharmacol. 16, 91 (1979); K.-J. Chang and P. The dipeptide carnosine (8-alanylhisti- ability to identify specific receptor bind- Cuatrecasas, ibid., p. 91; J. Biol. Chem. 254, 2610 (1979); S. H. Snyder and R. R. Goodman, dine) is the smallest of the neurotrans- ing sites for several peptides provides a J. Neurochem. 35, 5 (1980). mitter candidate peptides in the brain. It simple means to screen for drugs that 20. R. R. Goodman, S. H. Snyder, W. S. Young, M. J. Kuhar, Proc. Natl. Acad. Sci. U.S.A., in is highly concentrated in the primary ol- might mimic or block the effects of pep- press. which passes from tides. Thus, in addition to enhancing our 21. L.-I. Larsson, personal communication. factory pathway, the 22. C. B. Pert, G. Pasternak, S. H. Snyder, Sci- nasal epithelium to the olfactory bulb understanding of brain function, it is ence 182, 1359 (1973); C. B. Pert and S. H. Snyder, Mol. Pharmacol. 10, 868 (1974). (104). Destroying the nasal epithelium or likely that further studies of peptide neu- 23. W. Zieglgansberger and H. Bayerl, Brain Res. severing the olfactory nerve dramatically rotransmitters may result in important 115, 111 (1976); W. Zieglgansberger and J. Champagnat, ibid. 160, 95 (1979). decreases carnosine levels and the activ- therapeutic applications. 24. M. A. Enero, Eur. J. Pharmacol. 45, 379 ity of the carnosine-synthesizing enzyme (1977). References and Notes 25. A. J. Blume, Life Sci. 22, 1843 (1978); S. R. in the olfactory bulb. Moreover, stereo- Childers and S. H. Snyder, ibid. 23, 759 (1978); and saturable carnosine binding 1. S. H. Snyder, Nature (London) 257, 185 (1975). A. J. Blume, Proc. Nati. Acad. Sci. U.S.A. 75, specific 2. H. Akil, D. J. Mayer, J. C. Liebeskind, Sci- 1713 (1978); S. R. Childers and S. H. Snyder, has been demonstrated in the olfactory ence 191, 961 (1976); D. Mayer and J. Liebes- J. Neurochem. 24, 583 (1980). kind, Brain Res. 68, 73 (1974). 26. W. A. Klee and M. Nirenberg, Proc. Natl. bulb. Unlike most other brain peptides, 3. J. T. Hughes, Brain Res. 88, 295 (1975). Acad. Sci. U.S.A. 71, 3474 (1974); S. K. which are presumably synthesized as 4. L. Terenius and A. Wahlstrom, Acta Pharma- Sharma, M. Nirenberg, W. A. Klee, ibid. 72, col. Toxicol. Suppl. 1 33, 55 (1974). 590 (1975). large precursors by ribosomal mecha- 5. G. W. Pasternak, R. Goodman, S. H. Snyder, 27. D. Lichtshtein, G. Boone, A. J. Blume, Life nisms, carnosine is formed in a single en- Life Sci. 16, 1765 (1975). Sci. 25, 985 (1979). 6. J. Hughes, T. W. Smith, H. W. Kosterlitz, L. 28. D. A. Greenberg, D. C. U'Prichard, P. Shee- zymatic step. The olfactory bulb appears Fothergill, B. A. Morgan, H. R. Morris, Na- han, S. H. Snyder, Brain Res. 140, 378 (1978); to be the only area of the central nervous ture (London) 258, 577 (1975). L. B. Rosenberger, H. I. Yamamura, W. R. 7. R. Simantov and S. H. Snyder, Proc. Nati. Ruscke, J. Biol. Chem., in press; R. S. L. system that contains high levels of car- Acad. Sci. U.S.A. 73, 2515 (1976). Chang and S. H. Snyder, J. Neurochem. 34, 8. R. Simantov, A. M. Snowman, S. H. Snyder, 916 (1980). nosine (10-3M), which are ten to a hun- Brain Res. 107, 650 (1976); R. Simantov, M. J. 29. G. W. Pasternak, A. M. Snowman, S. H. Sny- dred times higher than levels in any other Kuhar, G. W. Pastemak, S. H. Snyder, ibid. der, Mol. Pharmacol. 11, 735 (1975); R. Sim- 106, 189 (1976); R. Simantov, R. Goodman, D. antov, A. M. Snowman, S. H. Snyder, ibid. 12, part of the brain. Aposhian, S. H. Snyder, ibid. 111, 204 (1976). 977 (1976); I. Creese, T. B. Usdin, S. H. Sny- Bombesin is a 14 amino acid peptide 9. T. W. Smith, J. Hughes, H. W. Kosterlitz, R. der, ibid. 16, 69 (1979); D. C. U'Prichard and P. Sosa, in Opiates and Endogenous Opioid S. H. Snyder, J. Neurochem. 34, 385 (1980). that has been isolated from frog skin Peptides, H. W. Kosterlitz, Ed. (North-Hol- 30. G. Dawson, R. McLawhon, R. J. Miller, Proc. In bombesin influences land, Amsterdam, 1976), p. 57; M. M. Puig, P. Natl. Acad. Sci. U.S.A. 76, 605 (1979). (105). mammals, Gascon, G. L. Craviso, J. M. Musacchio, Sci- 31. T. M. Cho, J. S. Cho, H. H. Loh, Mol. Phar- the gastrointestinal tract, stimulating se- ence 195, 419 (1977); R. Schulz, M. Wuster, R. macol. 16, 393 (1979). Simantov, S. H. Snyder, A. Herz, Eur. J. 32. A. T. McKnight, J. Hughes, H. W. Kosterlitz, cretion of gastrin and gastric acid, mim- Pharmacol. 41, 347 (1977); G. Henderson, J. Proc. R. Soc. London 205, 199 (1979). icking cholecystokinin effects on the gall Hughes, H. W. Kosterlitz, Nature (London) 33. S. R. Childers and S. H. Snyder, in CNS Ef- 982 SCIENCE, VOL. 209 fects of Hypothalamic Hormones and other 61. G. J. Dockray, ibid. 264, 568 (1976). M. Bassiri, R. D. Utiger, Science 185, 267 Peptides, R. Collu, A. Barbeau, J. R. Cich- 62. J. E. Muller, E. Straus, R. S. Yalow, Proc. (1974); A. Winokur, R. Davis, R. D. Utiger, arme, J. C. Rochefort, Eds. (Raven, New Nat!. Acad. Sci. U.S.A. 74, 3035 (1977); J. F. Brain Res. 120, 423 (1977). York, 1979), p. 253; R. S. Lewis, S. Stein, L. Rehfeld, J. Biol. Chem. 253, 4016 (1978); ibid., 86. T. H6kfelt, K. Fuxe, 0. Johansson, S. Jeff- D. Gerber, M. Rubinstein, S. Udenfriend, p. 4022. coate, N. White, Eur. J. Pharmacol. 34, 389 Proc. Natl. Acad. Sci. U.S.A. 75, 4021 (1978); 63. E. Straus, J. E. Muller, H. Choi, F. Paronetto, (1975). H. Y. T. Yang, W. Fratta, J. S. Hong, A. M. R. S. Yalow, Proc. Natl. Acad. Sci. U.S.A. 74, 87. D. R. Burt and S. H. Snyder, Brain Res. 93, Digiulio, E. Costa, Neuropharmacology 17, 3033 (1977). 309 (1975). 433 (1978). 64. R. B. Innis, F. M. A. Correa, G. R. Uhl, B. 88. A. J. Prange, I. C. Wilson, P. P. Lara, L. B. 34. K. Kangawa, H. Matsuo, M. Igarishi, Bio- Schneider, S. H. Snyder, ibid. 76, 521 (1979); Alltop, G. R. Breese, Lancet 1972-II, 999 chem. Biophys. Res. Commun. 68, 153 (1979); L.-I. Larsson and J. F. Rehfeld, Brain Res. (1972); A. J. Prange, C. B. Nemeroff, M. A. W. Y. Huang, R. C. C. Chang, A. J. Kastin, D. 165, 201 (1979); I. Loren, J. Alumets, R. Ha- Lipton, G. R. Breese, I. C. Wilson, in Hand- H. Coy, A. V. Schally, Proc. Natl. Acad. Sci. kanson, F. Sundler, Histochemistry 59, 249 book of Psychopharmacology, L. L. Iversen, U.S.A. 76, 6177 (1979); A. S. Stem, R. V. (1979). S. D. Iversen, S. H. Snyder, Eds. (Plenum, Lewis, S. Kimura, J. Rossier, S. Stein, S. 65. J. Dodd and J. S. Kelly, J. Physiol. (London), New York, 1978), vol. 13, p. 1. Udenfriend, ibid., p. 6680. in press. 89. P. Brazeau et al., Science 179, 77 (1973). 35. S. Kimura, R. V. Lewis, A. S. Stern, J. Ros- 66. T. H6kfelt, J. M. Lundberg, M. Schultzberg, 90. M. P. Dubois, Proc. Natl. Acad. Sci. U.S.A. sier, S. Stein, S. Udenfriend, Proc. Natl. 0. Johansson, A. Ljungdahl, J. Rehfeld, in 72, 1340 (1975); T. Hokfelt, S. Efendic, C. Acad. Sci. U.S.A. 77, 1681 (1980). Regulation and Function of Neural Peptides, Hellerstrom, 0. Johansson, R. Luft, A. Ari- 36. B. Malfroy, J. P. Swerts, A. Guyon, B. P. E. Costa and M. Trabucchi, Eds. (Raven, New mura, Acta Endocrinol. 80 (Suppl. 220), 5 Roques, J. C. Schwartz, Nature (London) 262, York, in press). (1975); T. Hdkfelt, S. Efendic, 0. Johansson, 523 (1978). 67. M. Deschodt-Lanckman, P. Robberecht, J. R. Luft, A. Arimura, Brain Res. 80, 165 (1974). 37. S. Sullivan, H. Akil, J. D. Barchas, Commun. Camus, J. Christophe, Eur. J. Biochem. 91, 21 91. S. R. Bloom et al., Lancet 1974-fl, 1106 (1974). Psychopharmacol. 2, 525 (1978). (1978); H. Sankaran, I. D. Goldfine, C. W. De- 92. A. Amoss, R. Burgus, R. Blackwell, W. Vale, 38. C. Gorenstein and S. H. Snyder, in Endoge- veney, K.-Y. Wong, J. A. Williams, J. Biol. R. Fellows, R. Guillemin, Biochem. Biophys. nous and Exogenous Opiate Agonists and An- Chem. 255, 1849 (1980); R. T. Jensen, G. F. Res. Commun. 44, 205 (1971); A. V. Schally et tagonists, E. L. Way, Ed. (Pergamon, New Lemp, J. D. Gardner, Proc. Natl. Acad. Sci. al., ibid. 43, 393 (1971). York, 1980) p. 345; Life Sci. 25, 2065 (1979); U.S.A. 77, 2079 (1980). 93. J. Barry, M. P. Dubois, P. Poulain, Z. Zell- Philos. Trans. R. Soc. London, in press; Proc. 68. R. B. Innis and S. H. Snyder, Eur. J. Pharma- forsch. Mikrosk. Anat. 146, 351 (1973); J. Bar- Natl. Acad. Sci. U.S.A., in press. col., in press; S. H. Snyder, R. F. Bruns, J. W. ry, Neurosci. Lett. 2, 201 (1976); D. W. Naik, 39. B. Malfroy, J. P. Swerts, C. Llorens, J. C. Daly, R. B. Innis, Fed. Proc. Fed. Am. Soc. Cell Tissue Res. 157, 423 (1975); G. Setalo, S. Schwartz, Neurosci. Lett. 11, 329 (1979); J. P. Exp. Biol., in press; R. B. Innis and S. H. Sny- Vigh, A. V. Schally, A. Arimura, B. Flerko, Swerts, R. Perdrisot, G. Patey, S. de la der, Proc. Natl. Acad. Sci. U.S.A., in press; Brain Res. 103, 597 (1976); A. J. Silverman, Baume, J. C. Schwartz, Eur. J. Pharmacol. 57, A. Saito, H. Sankaran, I. Goldfine, J. Wil- Endocrinology 99, 30 (1976); E. A. Zimmer- 279 (1979). liams, Science 208, 1155 (1980). man, K. G. Ksu, M. Ferin, G. P. Kozlowski, 40. J. P. Swerts, R. Perdrisot, B. Malfroy, J. C. 69. J. F. Rehfeld et al., Nature (London) 284, 33 ibid. 95, 1(1974). Schwartz, Eur. J. Pharmacol. 53, 209 (1979). (1980). 94. M. Brownstein, A. Arimura, A. V. Schally, M. 41. R. E. Carraway and S. E. Leeman, J. Biol. 70. J. Gibbs, R. C. Young, G. P. Smith, ibid. 245, Palkovits, J. S. Kizer, Endocrinology 98, 662 Chem. 248, 6854 (1973); ibid. 250, 1907 and 323 (1973); C. B. Nemeroff, A. J. Osbahr III, (1976); R. I. Weiner, E. Rattou, B. Kerdelhue, 1912 (1975). G. Bissette, G. Jahnke, M. A. Lipton, A. J. C. Kordon, ibid. 97, 1597 (1975). 42. C. Nemeroff, G. Bissette, A. Prange, P. Loos- Prange, Jr., Science 200, 793 (1978). 95. J. Havrankova, D. Schmechel, J. Roth, M. en, T. Barlow, M. Lipton, Brain Res. 128, 485 71. M. A. Della-Fera and C. A. Baile, Science 206, Brownstein, Proc. Natl. Acad. Sci. U.S.A. 75, (1977). 471 (1979). 5737 (1978). 43. G. Uhl and S. H. Snyder, Life Sci. 19, 1827 72. S. I. Said and V. Mutt, ibid. 169, 1217 (1970); 96. J. Havrankova, J. Roth, M. Brownstein, Na- (1976). V. Mutt and S. I. Said, Eur. J. Biochem. 42, ture (London) 272, 827 (1978). 44. L. L. Iversen, S. D. Iversen, F. E. Bloom, C. 581 (1974). 97. H. S. Tager and J. Markese, J. Biol. Chem. Douglas, M. Brown, W. Vale, Nature (Lon- 73. M. G. Bryant, J. M. Polak, I. Modlin, S. R. 254, 229 (1979); F. Sundby, H. Jacobsen, A. J. don) 273, 161 (1978). Bloom, R. H. Albequerque, A. G. E. Pearse, Moody, Horm. Metab. Res. 8, 366 (1976); L.-I. 45. W. S. Young, G. Uhl, M. J. Kuhar, Brain Res. Lancet 1976-I, 991 (1976); L.-I. Larsson et al., Larsson, H. Holst, R. Hakanson, F. Sundler, 150, 431 (1978). Proc. Natl. Acad. Sci. U.S.A. 73, 3197 (1976); Histochemistry 44, 281 (1975). 46. G. Uhl, J. P. Bennett, Jr., S. H. Snyder, ibid. S. I. Said and R. N. Rosenberg, Science 192, 98. J. M. Conlon, W. K. Samson, R. E. Dobbs, L. 130, 299 (1977); P. Kitabgi et al., Proc. Natl. 907 (1976). Orei, R. H. Under, Diabetes 28, 700 (1979), H. Acad. Sci. U.S.A. 74, 1846 (1977); L. Lazarus, 74. K. Fuxe, T. H6kfelt, S. I. Said, V. Mutt, Neu- Tager, personal communication. M. Brown, M. Perrin, Neuropharmacology 16, roscience Lett. 5, 241 (1977); I. Loren et al., 99. I. Loren, J. Alumets, R. Hakanson, F. Sun- 625 (1977). Neuroscience, in press. dler, J. Thorell, Histochemistry 61, 335 (1979); 47. G. R. Uhl, M. J. Kuhar, S. H. Snyder, Proc. 75. M. Rocha e Silva, W. T. Beraldo, G. Rosen- R. B. Innis, H. Tager, S. H. Snyder, in prepa- Natl. Acad. Sci. U.S.A. 74, 4059 (1977); G. R. feld, Am. J. Physiol. 156, 261 (1949). ration. Uhl, R. R. Goodman, S. H. Snyder, Brain Res. 76. S. A. Ribeiro, A. P. Corrado, F. G. Graeff, 100. R. A. Nicoll and J. L. Barker, Brain Res. 35, 167, 77 (1979); G. R. Uhl and S. H. Snyder, Neuropharmacology 10, 725 (1971); F. M. A. 501 (1971). ibid. 161, 522 (1979). Correa and F. G. Graeff, J. Pharmacol. Exp. 101. L. W. Swanson, ibid. 128, 346 (1977); R. De- 48. B. V. Clineschmidt, J. C. M. Guffin, P. B. Bun- Ther. 192, 670 (1975). fendi and E. A. Zimmerman, in The Hypothal- ting, Eur. J. Pharmacol. 54, 129 (1979). 77. F. M. A. Correa, R. B. Innis, G. R. Uhl, S. H. amus, S. Reichlin, R. J. Baldessarini, J. B. 49. T. Hokfelt, A. Ljungdahl, L. Terenius, R. Snyder, Proc. Natl. Acad. Sci. U.S.A. 76, Martin, Eds. (Raven, New York, 1978), p. 137. Elde, G. Nilsson, Proc. Natl. Acad. Sci. 1489 (1979). 102. M. J. Brownstein, J. T. Russell, H. Gainer, U.S.A. 74, 3081 (1977); T. H6kfelt, 0. Johann- 78. R. Tigerstedt and P. 0. Bergman, Skand. Arch. Science 207, 373 (1980). son, A. Ljungdahl, J. M. Lundberg, M. Physiol. 8, 223 (1898). 103. D. DeWied and D. H. G. Versteeg, Fed. Proc. Schultzberg, Nature (London) 284, 515 (1980). 79. J. T. Fitzsimons, Physiol. Rev. 52, 468 (1972); Fed. Am. Soc. Exp. Biol. 38, 2348 (1979). 50. T. H6kfelt, R. Elde, 0. Johansson, R. Luft, G. and J. Kucharczyk, J. Physiol. (Lon- 104. J. D. Hirsch, M. Grillo, F. L. Margolis, Brain Nilsson, A. Arimura, Neuroscience 1, 131 don) 276, 419 (1978); W. B. Severs and A. E. Res. 158, 407 (1978); F. L. Margolis, N. Rob- (1976). Daniels-Severs, Pharmacol. Rev. 25, 415 erts, D. Ferriero, J. Feldman, ibid. 81, 469 51. S. Gobel and J. M. Buck, Brain Res. 132, 347 (1973). (1974); J. Harding and F. Margolis, ibid. 110, (1977). 80. J. B. Simpson and A. Routtenberg, Science 351 (1976). 52. J. L. Henry, ibid. 114, 439 (1976). 181, 1172 (1973); M. I. Phillips and D. Felix, 105. A. Anastasi, V. Erspamer, M. Bucci, Ex- 53. H. M. Dale, Proc. R. Soc. Med. 28, 319(1935). Brain Res. 109, 531 (1976). perientia 27, 166 (1976). 54. T. M. Jessell and L. L. Iversen, Nature (Lon- 81. I. A. Reid, Fed. Proc. Fed. Am. Soc. Exp. 106. J. H. Walsh, H. C. Wong, G. J. Dockray, Fed. don) 268, 549 (1977); A. Mudge, S. E. Leeman, Biol. 38, 2255 (1979); M. I. Phillips, J. Weyhen- Proc. Fed. Ann. Soc. Exp. Biol. 38, 2315 G. D. Fishbach, Proc. Natl. Acad. Sci. U.S.A. meyer, D. Felix, D. Ganten, W. E. Hoffman, (1979); J. Wharton, J. M. Polak, S. R. Bloom, 76, 526 (1979). ibid., p. 2260. A. G. E. Pearse, Nature (London) 273, 769 55. J. S. Hong, H.-Y. T. Yang, G. Racagni, E. 82. J. P. Bennett, Jr., and S. H. Snyder, J. Biol. (1978); T. J. McDonald, G. Nilsson, M. Vagne, Costa, Brain Res. 122, 521 (1977); K. Gale, J.- Chem. 251, 7423 (1976); N. E. Sirett, A. S. S. R. Bloom, M. A. Ghatei, V. Mutt, Scand. J. S. Hong, A. Guidotti, ibid. 136, 371 (1977); T. McLean, J. J. Bray, J. I. Hubbard, Brain Res. Gastroenterol. 13 (Suppl. 49), 119(1978); J. M. M. Jessell, ibid. 151, 469 (1978); J.-S. Hong, E. 122, 229 (1977). Polak et al., ibid., p. 148; J. Villareal, J. Rivier, Costa, H.-Y. Yang, ibid. 118, 523 (1976). 83. D. G. Changaris, L. C. Keil, W. B. Severs, M. Brown, Endocrinology 102, A390 (1978). T. 56. H. L. Fields and A. J. Basbaum, Annu. Rev. Neuroendocrinology 25, 257 (1978); K. Fuxe, W. Moody and C. B. Pert, Biochem. Biophys. Physiol. 40, 217 (1978). D. Ganten, T. H6kfelt, P. Bolme, Neurosci. Res. Commun. Ser. B 90, 79 (1979). 57. T. H6kfelt et al., Neuroscience 3, 517 (1978); Lett. 2, 229 (1967); V. E. Nahmod, S. Finkiel- 107. T. W. Moody, C. B. Pert, J. Rivier, M. R. V. Chan-Palay, G. Jonsson, S. L. Palay, Proc. man, 0. S. de Gorodner, D. J. Goldstein, in Brown, Proc. Natl. Acad. Sci. U.S.A. 75, 5372 Nat!. Acad. Sci. U.S.A. 75, 1582 (1978). Central Actions of Angiotensin and Related (1978). 58. A. C. Ivy, H. M. Kloster, H. C. Leuth, G. E. Hormones, J. P. Buckley and C. M. Ferrario, 108. Supported by PHS grants MH-18501, DA- Drewyer, Am. J. Physiol. 91, 336 (1929), A. A. Eds. (Pergamon, New York, 1977), p. 573. 00266, and DA-01645 and grants of the Harper and H. S. Raper, J. Physiol. (London) 84. R. Burgus, T. F. Dunn, D. Desidero, D. N. McKnight and John A. Hartford Foundations. 102, 115 (1943); V. Mutt, Ark. Kemi 15, 69 Ward, W. Vale, R. Guillemin, Nature (Lon- I thank the following individuals for advice and (1959). don) 276, 321 (1970); R. M. G. Nair, J. F. Bar- for providing unpublished data: C. Gorenstein, 59. V. Mutt and J. E. Jorpes, Eur. J. Biochem. 6, rett, C. Y. Bowers, A. V. Schally, Biochemis- V. Mutt, G. Aghajanian, S. Watson, J. Kelly, 156 (1968); Biochem. Biophys. Res. Commun. try 9, 1103 (1970). A. Blume, T. H6kfelt, and L. L. Iversen. I par- 26, 392 (1967); Biochem. J. 125, 570 (1971). 85. C. Oliver, R. L. Gskay, N. Ben-Jonathan, J. C. ticularly thank P. Emson for advice and access 60. J. J. Vanderhaeghen, J. C. Signeau, W. Gepts, Porter, Endocrinology 95, 540 (1974); M. J. to the manuscript form of his review, in Prog. Nature (London) 257, 604 (1975). Brownstein, M. Palkovits, J. M. Saavedra, R. Neurobiol. 13, 61 (1979).

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