Structural requirements for galanin action in the guinea-pig ileum.

Galanin fragments and galanin analogues were tested on neurally evoked muscle contractions in guinea-pig ileum in vitro. Galanin fragments inhibited the neurally evoked circular muscle contractions with the following order of potency: Galanin(1-29), galanin(2-29), galanin(1-15). In contrast, galanin(3-29), galanin(10-29), galanin(21-29), [D-Trp2]galanin, [Phe2]galanin and [Tyr2]galanin were ineffective. Galanin(1-29), galanin(2-29) and galanin(1-15) did not affect the neurally evoked longitudinal muscle contractions. These results indicate that (1) the two N-terminal amino acid residues of the galanin molecule are essential for the inhibitory action of galanin on neurally-evoked circular muscle contraction and (2) for the full potency also the C-terminal end is required.     

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.     

Motility regulating effects of galanin on smooth muscle of porcine ileum.

We studied the effect of synthetic porcine galanin on circular and longitudinal oriented strips of pig ileal muscle. Galanin 10(-11)-10(-6) M had no effect on resting tension in the two layers. In circular muscle precontracted with carbachol 10(-6) M, galanin dose-dependently inhibited the amplitude of contractions to a maximum of 33 +/- 8% at 10(-6) M. In longitudinal muscle the amplitude of contractions induced by carbachol 10(-7) M or transmural field stimulation increased after addition of galanin 10(-9)-10(-7) M to a maximum of 21 +/- 6%, while at higher concentrations inhibition occurred. Maximal inhibition was 36 +/- 14% at galanin 10(-6) M. Tetrodotoxin did not influence the effects of galanin in the preparations. The results indicate that in the homologous species galanin inhibits the circular muscle layer, possibly by a direct action on the smooth muscle. In the longitudinal muscle the effect of galanin is apparently excitatory. The inhibition observed with high concentration of galanin could be due to tachyphylaxis and desensitization. Alternatively, an additional population of low affinity, inhibitory receptors may exist.     

Galanin receptor and its ligands in the rat hippocampus

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 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 +/- 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 +/- 0.1 M-1 min-1 at 37 degrees C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I--galanin-binding site is a trypsin-sensitive membrane protein, which is heat-denaturated at 60 degrees C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP-ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 microM.     

Galanin: an inhibitory neural peptide of the canine small intestine

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.     

Functional and immunocytochemical evidence that galanin is a physiological regulator of human jejunal motility.

The neuropeptide galanin has species-dependent effects on intestinal motility. It has a contractile effect on rat jejunal muscle while it relaxes guinea-pig ileum by inhibiting cholinergic transmission. Its effect on human gut motility has been unknown. Extensive work led to the discovery of selective galanin analogues such as M15 [galanin(1-12)-Pro-substance-P(5-11)], M35 [galanin(1-12)-Pro-bradykinin(2-9)-amide] that competitively inhibit various actions of galanin in the central nervous system. The present study was designed to examine the effect of galanin, M15 and M35 on longitudinal jejunal smooth muscle strips isolated from humans and rats, and to localize galanin-immunoreactivity in human jejunum. Galanin and ACh were equally effective in stimulating contractions of the isolated jejunal muscle: sigmoid curve fitting showed that maximal contractile response to galanin and ACh were 25.7+/-11.1 mN and 23.7+/-9.7 in humans, while 8.0+/-0.6 and 8.1+/-0.3 mN in rats, respectively. These effects of galanin were not inhibited by either atropine (5 x 10(-6) M) or tetrodotoxin (3 x 10(-6) M). The potency of galanin inducing the contractile actions were similar in humans and rats. Interestingly, neither M15 nor M35 (up to 10(-7) M) were able to inhibit the responses of the smooth muscle to galanin. However, both putative galanin receptor antagonists showed agonist effects in our experimental models. In accordance with the functional studies, both the longitudinal and the circular muscle layers were abundant in nerve fibers and varicosities showing galanin immunoreactivity. Our data suggest that galanin is a potent physiological regulator of jejunal contractions in humans. Its action on the jejunum, however, is mediated by galanin receptors that are different from those located in the central nervous system.     

Characterization of porcine gastric galanin.

The presence of a peptide capable of producing powerful contractions of rat small intestinal smooth muscle was detected in chromatographic fractions derived from porcine gastric corpus extracts. The pharmacological characteristics of this entity suggested that it might be galanin and on its purification to homogeneity, amino acid composition and sequence analysis demonstrated the identify of the gastric and intestinal forms of galanin. The presence of galanin in the gastric corpus tissue and its ability to affect gastric smooth muscle activity, gastrin release, and gastric acid secretion suggest potential important physiological roles for galanin in the stomach.     

The effect of vasoactive intestinal polypeptide (VIP) on the canine gallbladder motility.

1. VIP at doses of 10(-9) to 10(-8) M was ineffective and at doses of 5 x 10(-8) to 10(-7) M exerted a slight inhibitory effect on the tone of the canine gallbladder muscle strip. However, VIP (0.1-1 micrograms/kg) injected intravenously (i.v.) in conscious dogs dose-dependently decreased the gallbladder pressure. 2. VIP did not influence significantly the acetylcholine (ACh)- or carbachol- induced contractions of canine gallbladder under in vitro or in vivo conditions, but it decreased the electrically-induced, atropine-sensitive contractions of gallbladder muscle strips. 3. VIP (5 x 10(-9) to 5 x 10(-8) M) did not influence significantly the dose-response curve for cholecystokinin octapeptide (CCK OP) of canine and guinea-pig gallbladder muscle strips. VIP injected i.v. (0.1-0.5 micrograms/kg) in conscious dogs greatly decreased the CCK OP-induced gallbladder pressure.     

Effects of galanin on smooth muscle and mucosa of porcine jejunum.

The enteric neuropeptide galanin (GAL) increased the amplitude of spontaneous contractions in longitudinally oriented muscle strips and inhibited short-circuit current (Isc) elevations induced by transmural electrical stimulation (ES) of mucosal sheets from porcine jejunum in vitro. GAL-induced contractions (GAL EC50 = 9 nmol/l) were maximally 25% of those elicited by 10 mumol/l carbamylcholine and remained unaffected by atropine, tetrodotoxin, or tachyphylaxis to substance P. The presynaptic Ca2+ channel blocker, omega-conotoxin (0.1 mumol/l), inhibited GAL-induced contractions by 66%. GAL attenuated mucosal Isc elevations induced by ES with an IC50 = 13 nmol/l and at 0.1 mumol/l produced rapid decreases in basal Isc averaging 8 +/- 2 microA cm-1 in 77% of tissues examined. The alpha-adrenoceptor blocker phentolamine or the opiate antagonist naloxone did not alter tissue Isc responses to GAL. These results suggest that GAL modulates neuronal activity linked to secretomotor function in the porcine small intestine.     

Effect of galanin and galanin antagonists on peristalsis in esophageal smooth muscle in the opossum

Galanin, a neuropeptide that is widely distributed in the esophageal nerves, is known to exert a neuromodulatory action in the gut. These studies examined the effect of galanin and galanin antagonists on esophageal peristalsis in anesthetized opossums in vivo. Intraluminal esophageal pressures were recorded at 1, 3, 5, 7, and 9 cm above the lower esophageal sphincter. Esophageal peristaltic contractions were induced by swallow and short- (1-s) and long-train (10-s) vagal stimulation (VS). Galanin (1 nmol/kg) inhibited the amplitude of swallow-induced peristaltic contractions and increased peristaltic velocity by enlarging the latency periods in the upper part of the esophagus and reducing them in the lower part. Galinin nearly abolished esophageal contractions caused by short-train VS at 5 Hz and inhibited the contractions at 10 Hz. Galanin increased latency periods induced by short-train VS with little change in the velocity of peristalsis and reduced the amplitude of both A (cholinergic) and B (noncholinergic) contractions due to long-train VS. However, the decrease in amplitude of B contractions was more marked. Galantide (3 nmol/kg) antagonized the inhibitory action of exogenous galanin on esophageal contractions elicited by short-train VS, but by itself galantide had no significant effect on esophageal contractions. In conclusion, exogenous galanin inhibits the amplitude of swallow-induced peristaltic contractions and converts them into nonperistaltic contractions by inhibiting both the cholinergic and noncholinergic components.     

Galanin: structural requirements for binding and signal transduction in RINm5F insulinoma cells.

Receptors for galanin, a neuropeptide inhibiting insulin release, have been described on RINm5F insulinoma cells. To characterize structural requirements for binding and biological activity of galanin, we studied binding and inhibition of hormone stimulated intracellular cAMP-production of N-terminal galanin fragments and -analogues in RINm5F cells. Half-maximal binding and potency were the same for all peptides used. Active peptides had the following rank of potency: galanin = galanin(1-22(23)Cys) greater than galanin(1-29(4)NLe) greater than galanin(1-18) greater than galanin(1-29(7)DAla) greater than galanin(1-29(2)DTrp4NLe7DAla) greater than galanin(1-29(2)DTrp). Galanin(3-29) was inactive. Therefore the first two amino acids of the galanin molecule with the indole side chain of the tryptophane residue in the right steric position are crucial for receptor binding.     

Structural requirements for galanin interaction with receptors from pancreatic beta cells and from brain tissue of the rat

The binding activity of several galanin fragments and analogs was measured on specific receptors present in rat brain and the rat pancreatic beta cell line Rin m 5F. In both tissues it was observed that: 1) galanin(3-29), galanin(10-29) and [Ile2]-galanin were ineffective for inhibiting [125I] galanin binding and 2) active peptides had the following rank order of potency: galanin(1-29) greater than [Ac-Trp2]-galanin(2-29) greater than galanin(2-29) greater than galanin(1-15) greater than [Phe2]-galanin greater than [Tyr2]-galanin. It was concluded that the N-terminal portion of galanin is very important for interaction with central or peripheral receptors. The aromatic amino acid in position 2 (Trp in native galanin) plays a crucial role.     

Galanin: evidence for a hypothalamic site of action to release growth hormone

Galanin is a 29 amino acid peptide that was isolated and characterized from porcine intestinal extracts. The presence of galanin-like immunoreactivity in neuronal elements in the hypothalamus and median eminence suggested a role for it in the hypothalamic control of anterior pituitary function. A hypothalamic site of action of galanin to stimulate growth hormone (GH) release is suggested by our observation that doses as low as 50 picomoles when infused into the third cerebroventricle of conscious, unrestrained ovariectomized rats resulted in significantly elevated plasma levels of GH. This effect was specific for GH and was dose-related. The failure of galanin to alter GH release from dispersed, cultured anterior pituitary cells in vitro further suggests that endogenous galanin plays a neuromodulatory role at the level of the median eminence, possibly affecting the release of known GH-releasing and/or inhibiting factors.     

Presence of galanin in human pancreatic nerves and inhibition of insulin secretion from isolated human islets

Summary In several animal species, galanin occurs in pancreatic nerves and inhibits insulin secretion. However, the presence and action of galanin in the human pancreas have not been established. Therefore, we examined the presence and nature of human pancreatic galanin-like immunoreactive material (GLIR) and the effects of galanin on glucose-stimulated insulin secretion from isolated human islets. Immunofluorescent staining of human pancreas revealed GLIR in fine varicose fibers in both islets and exocrine parenchyma. Furthermore, acid extracts of pancreas (n=3) and isolated islets (n=3) contained 0.17±0.06 and 0.23±0.11 pmol GLIR/mg protein. Human pancreatic GLIR coeluted with synthetic porcine galanin from Sephadex G-50. Moreover, synthetic porcine galanin inhibited glucose-stimulated insulin secretion from collagenase-isolated human islets at dose rates >10^-8 M. Thus, (1) human pancreas is innervated by galanin-containing nerves, (2) human pancreatic GLIR is of similar size as synthetic porcine galanin, and (3) porcine galanin inhibits glucose-stimulated insulin secretion from human islets. Therefore, galanin could be an important local regulator of insulin secretion in man.     

Comparison of the biological activity of canine and porcine motilin in rabbit

The biological activity of porcine and canine motilin was studied in rabbits by establishing dose-response curves of both peptides using two different methods. The dissociation constant, obtained from the displacement of iodinated porcine motilin by canine motilin was 0.6 +/- 0.3 nM, versus 1.2 +/- 0.4 nM for porcine motilin. For the 13-norleucine and 13-leucine analogues of porcine motilin a value of 0.8 +/- 0.3 nM was obtained. Both motilins were almost equipotent in stimulating the in vitro contractile response of longitudinal smooth muscle strips: half-maximal effect was achieved at a concentration of 1.0 +/- 0.1 nM for canine versus 1.3 +/- 0.2 nM for the 13-norleucine analogue of porcine motilin. We conclude that porcine and canine motilin have a comparable bioactivity in the rabbit, although canine motilin is slightly more effective. The motilin receptor is probably specific for the N-terminal portion which is identical in both molecules.     

Effects of 17β-estradiol on galanin(1-29)- and galanin(1-16)-like immunoreactivities

There are reasons to believe that the galanin neuropeptide family could include more than the two hitherto known members (galanin(1-29) and galanin-like peptide), such as the existence of at least three galanin receptors and the fact that synthetic short-chain homologues have effects and binding sites that are distinct from those of galanin(1-29). The current study uses a radioimmunoassay based on a polyclonal rabbit antiserum raised against galanin(1-16) to study the concentrations of galanin(1-16) like immunoreactivity (LI) in the various parts of the brain and gut of ovariectomized female rats, and investigates the effects of different concentrations of estradiol on these concentrations in relation to galanin(1-29)-LI. Galanin(1-29) concentrations were increased by 17β-estradiol administration in almost all examined tissues whereas galanin(1-16)-LI was increased by 17β-estradiol treatment in most of the gut, but only in the pituitary of the brain. Furthermore, the relation between galanin(1-29)-LI and galanin(1-16)-LI varied substantially from tissue to tissue. The main hypothesis, that galanin(1-16)-LI would be affected by 17β-estradiol in brain and/or gut, was confirmed in addition to the secondary hypothesis, stating that the pattern of galanin(1-16)-LI changes would differ from that of galanin(1-29). The study indicates that galanin(1-16)-LI is estrogen-responsive but that its concentrations are regulated differently from that of galanin(1-29). This is strongly indicative of a biological relevance of this potentially new member of the galanin neuropeptide family.     

Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor.

The neuropeptide galanin (1-29) binds with high affinity to hypothalamic receptors (KD approximately 0.9 nM) and regulates feeding behavior. The N-terminal fragments (1-16), (1-16)NH2 are high affinity (KD approximately 6 nM) full agonists in vivo and in vitro. L-Ala substitutions show that amino acid residues Gly1, Trp2, Asn5, Tyr9, and Gly12 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 (1-15), (1-14), and (1-13) have submicromolar KD values and galanin (1-12) has KD approximately 3 microM. Cyclic analogs of galanin (1-12) of different ring size were synthesized by condensing Gly1 and Gly12 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.     

Autoradiographic localization of 125I-galanin binding sites in the blowfly brain.

The localization of porcine galanin (pGAL) binding sites in the brain of the blowfly Phormia terraenovae was investigated by autoradiography using the following radioiodinated ligands: pGAL 1-29 (two isoforms), pGAL 15-29 and rat (r) GAL 1-29. The different porcine radioligands bound specifically with the following intensity: 125I-[Tyr26]-pGAL15-29 > > 125I-[Tyr26]-pGAL1-29 > > 125I-[Tyr9]-pGAL1-29. With rat galanin 125I-[Tyr9]-rGAL1-29 no specific binding could be shown. In addition, displacement of 125I-[Tyr26]-pGAL1-29 was tested with pGAL 1-29, pGAL 1-22 and pGAL 15-29 (at 0.1 nM-1 microM). A gradual displacement was achieved with increasing concentrations of pGAL 1-29 and pGAL15-29, whereas no displacement with pGAL 1-22 was detected. The results indicate that the C-terminal portion of pGAL is important for binding in the blowfly. The pGAL binding sites were localized in synaptic neuropils of the central body, the antennal lobes, the optic lobes, the pars intercerebralis and the subesophageal ganglion, all of which contain GAL-like immunoreactive neural processes.     

Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.     

The effect of galanin, CGRP and ANP on spontaneous smooth muscle activity of rat uterus

In the present study the effect of the newly isolated peptides galanin, calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) was examined on spontaneous uterine smooth muscle activity on the rat in vitro. Galanin showed a slight and insignificant stimulatory effect on the amplitude, while both CGRP and ANP were found to be potent inhibitors of the uterine smooth muscle contractions. In connection with the recent demonstration of galanin and CGRP nerves in the genital tract, these pharmacological findings suggest that the peptides may participate in the control of nervous control of uterine muscular activity, although the exact physiological roles remain to be clarified.