The galanin N-terminal fragment [galanin-(1-16) The 29-amino-acid-long C-terminal amidated peptide galanin (GAL) (1) has been shown to be widely distributed in the central nervous system of mammals (2-6). GAL-like immunoreactivity has been localized, among other brain regions, in the septal area (4-8) where GAL coexists with acetylcholine in rat (7) and monkey (9) in a subpopulation of cholinergic cell bodies projecting to the hippocampus. and equilibrium binding studies with 1251I-labeled GAL (125I-GAL) have shown a high density of putative high-affinity GAL receptors in the ventral part of the hippocampus (13).In the ventral, but not in the dorsal, hippocampus GAL has been shown to inhibit, in a dose-dependent manner, the release of acetylcholine both in vitro and in vivo (12). A possible role for GAL as presynaptic modulator of the cholinergic function in the ventral hippocampus has been suggested by a recent electrophysiological study (14) and a behavioral study (15). Interactions between acetylcholine and GAL in the ventral hippocampus are not restricted to presynaptic sites but also involve actions that are considered postsynaptic (16) such as the GAL-mediated inhibition of the muscarinic agonist-stimulated breakdown of inositol phospholipids in the ventral hippocampus (17) and GAL inhibition of acetylcholine on a t-maze memory task in ventral forebrain lesioned rats (33).Previous studies carried out on intestinal smooth muscle showed the importance of the N-terminal portion of GAL for biological activity (18,19). Furthermore, N-terminal GAL fragments and analogs were able to mimic the effects of GAL on the pancreatic 83-cell line Rin m 5F, inhibiting forskolinstimulated cAMP production and insulin release (20).In this study, we focus our attention on the structureactivity relationship for the N terminus of GAL at receptors in the ventral hippocampus of the rat. Using equilibrium binding and autoradiographic techniques, we compare the synthetic N-terminal fragment GAL-(1-16) with rat GAL-(1-29) for its ability to displace 1251I-GAL from its receptors.GAL-(1-16) is also tested for its ability to inhibit the scopolamine-induced acetylcholine release in vivo and carbacholstimulated inositol phospholipid breakdown in a slice preparation from the rat ventral hippocampus. The importance of [Trp2] in the activity of the N-terminal fragment is examined.

Cyclic peptides with alternating d- and l-amino acid residues containing tert-leucine residues in every second position can form peptide nanotubes only when both enantiomers of the peptide are present in the solution. These results strongly indicate the formation of peptide nanotubes that assemble with one enantiomer in every second position, thereby forming a lamellar structure.

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The neuropeptide galanin (1-29) binds with high affinity to hypothalamic receptors (K, z 0 . 9 1~) and regulates feeding behavior. The N-terminal fragments (l-l6), (I-16)NH2 are high affinity (K, z 6 n~) full agonists in vivo and in vitro. L-Ala substitutions show that amino acid residues Gly', Trp', Asn', Tyr', and Glyl'are important for the high affinity binding of galanin (1-16). Shortening the fragment (1-16) to galanin (1-7) causes a gradual drop of affinity: galanin (I-IS), (l-l4), and (1-13) have submicromolar K, values and galanin (1-12) has K, z 3 p M . Cyclic analogs of galanin (1-12) of different ring size were synthesized by condensing Gly' and Gly" without or with spacer groups. These analogs, independent of ring size. had a lower affinity than the linear galanin (1-12). Derivatization of the N-terminus of galanin (1-29), (1-16). and (1-12) all resulted in a large drop of affinity for the receptors, suggesting again the importance of the free N-terminal Gly. K c j word.?: galanin fragments and analogs; receptor binding; solid phase synthesis; structure-activity relationship Galanin, a 29 amino acid long C-terminally amidated gut and brain peptide (I), possesses several important biological activities; galanin is a potent inhibitor of the glucose induced insulin release in dog (2), it inhibits acetylcholine release in the rat ventral hippocampus (3) and stimulates the release of human growth hormone (4), furthermore, it potentiates the effects of morphine on suppression of the spinal flexor reflex (9, and potently stimulates feeding behavior when injected into the hypothalamus or the lateral ventricles (6).The amino acid sequence of galanin is known from three species: porcine (1). rat (7), and bovine (8). These available amino acid sequences (Fig. I ) show a complete conservation of the first 15 amino acids in the N-terminal portion of galanin, while amino acid substitutions occur (in residues 23, 26, and 29) in the C-terminal portion of the three sequences.Studies with galanin (1-15) (9) and porcine galanin

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of I2'I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 -t 0.40 nM). The concentration of the high-affinity-binding sites was 107 ? 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 f 0.1 M-' min-l at 37°C.The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The '"I g galanin-binding site is a trypsin-sensitive membrane protein, which is heatdenaturated at 60°C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADPribosylation on the binding of 12'I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein).'271-galanin was shown to be a ligand with affinity equal to that of galanin in displacing '251-galanin. The primary sequence of porcine galanin, a 29-aminoacid, C-terminal-amidated peptide, was determined in 1983 by Tatemoto et al. [l]. Within a short period a series of findings were published on the immunohistochemical mapping of the distribution of this peptide in the central [2 -41 and peripheral nervous system [S], using antisera raised against the porcine peptide. Autoradiography of 251-galanin binding indicated the presence of putative galanin receptors in several brain regions [6]. Among its numerous physiological and pharmacological actions it is noteworthy that galanin acts as a potent inhibitor of insulin release [7], of dopamine release from the median eminence [8] and of acetylcholine release from the ventral hippocampus [9]. This latter finding is of great interest, since galanin turned out to be a potent endogenous regulator of acetylcholine release in the hippocampus, a brain region where cholinergic deficit, such as that in Alzheimer's disease, is accompanied by a loss of cognitive function [lo].Interest in the biochemistry and pharmacology of galanin action is thus well motivated. The rat galanin cDNA was recently cloned and sequenced [II, 121 and the rat galanin sequence became available to us towards the end of this study.Meanwhile, the galanin receptor sites on hamster pancreatic 0-cell tumor [I31 and in rat brain membranes [14] wereCorrespondence to