Localization and inhibitory actions of galanin at the feline lower esophageal sphincter

Intrinsic reflexes of the lower esophageal sphincter (LES) are mediated by specific arrangements of excitatory and inhibitory nerves. We have previously described an excitatory reflex at the feline LES mediated by a bombesin-like peptide (BN) which causes release of substance P (SP) to directly contract the LES. Galanin is a neurotransmitter in the enteric nervous system which colocalizes in neurons containing vasoactive intestinal peptide (VIP). The aims of this study were to determine: (1) the distribution of galanin at the feline LES; (2) the effect of galanin on basal LES tone; (3) the effect of galanin on agonist-induced LES contractions by BN, SP and bethanechol; and (4) the effect of galanin on LES relaxation induced by esophageal distension and exogenous VIP. Galanin-like immunoreactivity (galanin-LI) was localized in neurons that were widely distributed throughout the LES and adjacent organs. Galanin-LI was most abundant in the circular muscle, muscularis mucosa and myenteric plexus of the LES. In anesthetized cats, intra-arterial galanin had no effect on basal LES pressure in a dose range of 10⁻¹¹ to 10⁻⁶ g/kg. Galanin (5 10⁻⁷ g/kg) reduced the LES contractile response to SP by 65 ± 8% (P = 0.0001). This galanin-mediated inhibition of SP was not blocked by tetrodotoxin. Galanin similarly decreased the LES contractile response to BN (63 ± 7%, P = 0.005) and bethanechol (55 ± 17%, P = 0.012). Galanin had no effect on the LES relaxation induced by esophageal distension or exogenous VIP. We conclude: (1) galanin-LI is present in neurons at the feline LES; (2) galanin has no effect on basal sphincter tone, but inhibits contractions of the LES by both direct and indirect agonists; and (3) galanin does not effect the LES relaxation induced by esophageal distension or VIP.     

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.     

Inhibitory effects of galanin on evoked [Ca2+]i responses in cultured myenteric neurons

Galanin modulates gastrointestinal motility by inhibiting the release of ACh from enteric neurons. It is, however, not known whether galanin also inhibits neuronal cholinergic transmission postsynaptically and whether galanin also reduces the action of other excitatory neurotransmitters. The aim of the present study was thus to investigate the effect of galanin on the evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in myenteric neurons. Cultured myenteric neurons from small intestine of adult guinea pigs were loaded with the Ca(2+) indicator fluo-3 AM, and the [Ca(2+)](i) responses following the application of different stimuli were quantified by confocal microscopy and expressed as a percentage of the response to high-K(+) solution (75 mM). Trains of electrical pulses (2 s, 10 Hz) were applied to stimulate the neuronal fibers before and after a 30-s superfusion with galanin (10(-6) M). Substance P (SP), 5-HT, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and carbachol were used as direct postsynaptic stimuli (10(-5) M, 30 s) and were applied alone or after galanin perfusion. Galanin significantly reduced the responses induced by electrical fiber stimulation (43 +/- 2 to 35 +/- 3%, P = 0.01), SP (15.4 +/- 1 to 8.0 +/- 0.3%, P < 0.01), and 5-HT (26 +/- 2 to 21.4 +/- 1.5%, P < 0.05). On the contrary, galanin did not affect the responses induced by local application of DMPP and carbachol. We conclude that in cultured myenteric neurons, galanin inhibits the excitatory responses induced by electrical stimulation, SP, and 5-HT. Finally, the inhibitory effect of galanin on electrical stimulation, but not on DMPP- and carbachol-induced responses, suggests that, at least for the cholinergic component, galanin acts at the presynaptic level.     

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.     

Effects of cholinergic stimulation on human esophageal peristalsis.

The effects of cholinergic stimulation on esophageal peristalsis were studied in normal subjects. An intraesophageal transducer assembly was used to measure the dynamics of esophageal peristalsis before and after edrophonium chloride, 80 mug/kg intravenously. Following endrophonium, there was a marked increase in amplitude of esophageal persistalsis associated with a significant reduction in velocity of esophageal peristalsis and a significant increase in width and time of appearance of the contraction wave following a swallow. Graded doses of endrophonium all resulted in significant increases in peristaltic amplitude with the maximal response occurring at doses of 80 and 160 mug/kg. Similar results were obtained with a more direct-acting cholinergic agent, bethanechol, 80 mug/kg subcutaneously. The relevance of these results as an indication of the importance of cholinergic innervation in regulating esophageal motility are discussed.     

Relationship between esophageal muscle thickness and intraluminal pressure in patients with esophageal spasm

We previously showed, in normal subjects, a positive correlation between the esophageal contraction amplitude and peak muscle thickness. The goal of this study was to determine the relationship between esophageal muscle thickness and contraction amplitude in patients with high-amplitude peristaltic and simultaneous contractions. Eleven patients with high-amplitude peristaltic contractions, 8 with diffuse esophageal spasm (DES), 7 with nonspecific (NS) motor disorder of the esophagus, and 10 normal subjects were studied using simultaneous pressure and ultrasound imaging. Pressure was recorded by manometry and ultrasound imaging with a high-frequency ultrasound probe catheter. Recordings were performed in the lower esophageal sphincter (LES) and at 2, 4, 6, 8, and 10 cm above the LES during resting state and swallow-induced contractions. Baseline esophageal muscle was thicker in the distal, compared with the proximal esophagus both in normal subjects and patient groups. Patients with DES and nutcracker esophagus (NC) have a higher baseline muscle thickness compared with normal and NS patients. Correlation between the peak pressure and the peak muscle thickness was weaker in patients with NC and DES compared with normal subjects and patients with NS. Whereas normal subjects have good correlation between delta (difference between peak and baseline) muscle thickness and peak pressures, this relationship was absent in patients with NC and DES. Increase in contraction amplitude in patients with NC and DES was associated with an increase in baseline thickness of esophageal muscularis propria. Increase in baseline thickness was specific to patients with spastic motor disorders and was not seen in patients with NS.     

Absence of a deglutitive inhibition equivalent with secondary peristalsis

This study aimed to determine the interactions between closely paired swallow-induced primary peristalsis (PP) and air injection-induced secondary peristalsis (SP). Ten subjects (7 men, 18-42 yr) were studied using a catheter, including two sleeves (upper and lower esophageal sphincters), a midesophageal infusion port, and seven esophageal and two pharyngeal recording sites. Ten iterations of PP and SP were induced by 5-ml water swallows and 20-ml intraesophageal air injections, respectively. Thereafter, the interactions between PP and SP, separated by 1- to 12-s intervals, were studied in all four possible sequences: paired swallows, swallow preceded by air injection, air injection preceded by swallow, and paired air injections. Tracings were analyzed for lower esophageal sphincter relaxation, presence and integrity of peristalsis, and event interaction. Eight subjects with success rates of both >/=90% PP and >/=80% SP were analyzed (PP 97 +/- 2%, SP 90 +/- 3%). During paired PP interactions and SP followed by PP, the first sequence was inhibited by the second with intervals < 4-6 s. However, no inhibition of the first peristaltic sequence was found in either PP followed by SP trials or SP followed by air injection. In contrast to swallowing or proximal esophageal distention, air injection into the lumen of the midesophagus does not inhibit an ongoing peristaltic event. Being that the elicitation of SP in the smooth muscle esophagus is intramurally mediated, this suggests that deglutitive inhibition is a centrally mediated phenomenon rather than an intrinsic property of peristalsis.     

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.     

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.     

Immunocytochemical localization of galanin in the rat male and female genital tracts and motor effects in vitro

Galanin, a recently discovered neuropeptide, was studied in the rat male and female reproductive tracts by immunocytochemistry and in vitro pharmacology. Nerve fibers containing galanin immunoreactivity were most abundant in the female paracervical tissue, where they surrounded non-immunoreactive ganglion cells. Galanin nerves were also found in the uterus and Fallopian tubes, as well as in the vas deferens. When tested in vitro galanin contracted the smooth muscle of both the uterine horn and cervix. Galanin also slightly potentiated the response to electrical field stimulation in preparations from the uterine cervix and vas deferens, but it had no effect on the seminal vesicle. Galanin-(1–10), an N-terminal residue of galanin, also contracted the uterine horn, though higher concentrations were required. The neurally induced contractions were not influenced by galanin-(1–10) in any of the smooth muscle preparations tested. The muscle receptors mediating the direct contractile effects in the uterine horn seem to require the N-terminus of galanin, while the neuromodulatory effects on the electrically induced contractile activity seem to need the C-terminal part or the whole galanin molecule. Galanin may thus function as a neuromediator in the rat male and female genital organs.     

Esophageal wall blood perfusion during contraction and transient lower esophageal sphincter relaxation in humans

We recently reported that esophageal contraction reduces esophageal wall perfusion in an animal study. Our aim was to determine esophageal wall blood perfusion (EWBP) during esophageal contraction and transient lower esophageal sphincter relaxations (TLESRs) in humans. We studied 12 healthy volunteers. A custom-designed laser Doppler probe was anchored to the esophageal wall, 4-6 cm above the LES, by use of the Bravo pH system so that the laser light beam stay directed toward the esophageal mucosa. A high-resolution manometry equipped with impedance electrodes recorded esophageal pressures and reflux events. Synchronized pressure, impedance, pH, and EWBP recordings were obtained during dry and wet swallows and following a meal. Stable recordings of laser Doppler EWBP were only recorded when the laser Doppler probe was firmly anchored to the esophageal wall. Esophageal contractions induced by dry and wet swallows resulted in 46 ± 9% and 60 ± 10% reduction in the EWBP, respectively (compared to baseline). Reduction in EWBP was directly related to the amplitude (curvilinear fit) and duration of esophageal contraction. Atropine reduced the esophageal contraction amplitude and decreased the EWBP reduction associated with esophageal contraction. TLESRs were also associated with reduction in the EWBP, albeit of smaller amplitude (29 ± 3%) but longer duration (19 ± 2 s) compared with swallow-induced esophageal contractions. We report 1) an innovative technique to record EWBP for extended time periods in humans and 2) contraction of circular and longitudinal muscle during peristalsis and selective longitudinal muscle contraction during TLESR causes reduction in the EWBP; 3) using our innovative technique, future studies may determine whether esophageal wall ischemia is the cause of esophageal pain/heartburn.     

Effects of galanin on wide-dynamic range neuron activity in the spinal dorsal horn of rats with sciatic nerve ligation

Galanin is a 29-amino acid peptide with a suggested role in nociception. The effect of galanin on wide-dynamic range neuron discharge frequency in rats with nerve ligation, used as a model of neurogenic pain, was investigated by extracellular recording methods. Seven to 14 days after sciatic nerve ligation, 0.1, 0.5 or 1 nmol of galanin was administered directly on the dorsal surface of the L3-L5 spinal cord of rats with sciatic nerve ligation. It was found that galanin inhibited the activity of wide-dynamic range neurons dose-dependently, an effect was more pronounced in sciatic nerve ligated rats than intact rats. Furthermore, when 1 nmol of galantide, the galanin antagonist, was administered on the dorsal surface of the L3-L5 spinal cord, the wide-dynamic range neuron discharge frequency increased significantly. The results suggest that galanin plays an important role in the modulation of presumed nociception in mononeuropathy.     

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.     

Relationship between esophageal muscle thickness and intraluminal pressure: an ultrasonographic study

A number of studies show a close temporal relationship between the rate of change in muscle thickness as detected by high-frequency intraluminal ultrasonography (HFIUS) and intraluminal pressure measured by manometry. There is a marked variability in esophageal contraction amplitude from one swallow to another at a given level in the esophagus and along the length of the esophagus. Furthermore, peristaltic pressures are higher in the distal compared with the proximal esophagus. The goal of this study was to evaluate the relationship between the baseline and peak muscle thickness and the contraction amplitude during swallow-induced contractions along the length of the esophagus. Fifteen normal subjects were studied using simultaneous esophageal pressures and HFIUS or HFIUS alone. Recordings were made during baseline and standardized swallows in the lower esophageal sphincter (LES) and at 2, 4, 6, 8, and 10 cm above the LES. HFIUS images were digitized, and esophageal muscle thickness and peak contraction amplitudes were measured. In the resting state, muscle thickness is higher in the LES compared with the rest of the esophagus. Baseline muscle thickness is also significantly higher at 2 cm vs. 10 cm above the LES. In a given subject and among different subjects, there is a good relationship between peak muscle thickness and peak peristaltic pressures (r = 0.55) at all sites along the length of the esophagus. The positive correlation between pressure and muscle thickness implies that the mean circumferential wall stress is fairly uniform from one swallow to another, irrespective of the contraction amplitude.     

Galanin and pancreastatin inhibit stimulated insulin secretion in the mouse: comparison of effects

The effects of the two intrapancreatic peptides galanin and pancreastatin on basal and stimulated insulin and glucagon secretion in the mouse were compared. It was found that at 2 min after intravenous injection of galanin or pancreastatin (4.0 nmol/kg), basal plasma glucagon and glucose levels were slightly elevated. Galanin was more potent than pancreastatin to elevate basal plasma glucagon levels: they increased from 60 +/- 15 to 145 +/- 19 pg/ml (p less than 0.01) after galanin compared to from 35 +/- 5 to 55 +/- 8 pg/ml (p less than 0.05) after pancreastatin. Plasma insulin levels were lowered by galanin (p less than 0.05), but not by pancreastatin. CCK-8 (6.3 nmol/kg) or terbutaline (3.6 mumol/kg) markedly increased the plasma insulin levels. Galanin (4.0 nmol/kg) completely abolished the insulin response to CCK-8 (p less than 0.001), but pancreastatin (4.0 nmol/kg) was without effect. Galanin inhibited the insulin response to terbutaline by approximately 60% (p less than 0.01), but pancreastatin inhibited the insulin response to terbutaline by approximately 35% only (p less than 0.05). CCK-8 and terbutaline did both elevate plasma glucagon levels by moderate potencies: neither pancreastatin nor galanin could affect these responses. Thus, in the mouse, galanin and pancreastatin both inhibit basal and stimulated insulin secretion, and stimulate basal glucagon secretion. Galanin is thereby more potent than pancreastatin. The study also showed that galanin potently inhibits insulin secretion stimulated by the octapeptide of cholecystokin and by the beta 2-adrenoceptor agonist terbutaline, and that pancreastatin inhibits terbutaline-induced insulin secretion.     

The canine sympathetic neuropeptide galanin: a neurotransmitter in pancreas, a neuromodulator in liver.

Our laboratory has investigated the role of the neuropeptide galanin in the sympathetic neural control of both the canine endocrine pancreas and liver. Galanin mRNA and peptide were found in the neuronal cell bodies of the celiac ganglion, which projects fibers to both organs. Galanin fibers formed dense networks around the islets. Galanin was released from these nerves and the amount released appeared sufficient to markedly inhibit basal insulin secretion. We therefore propose that galanin is a sympathetic neurotransmitter in canine endocrine pancreas. Galanin was also found in hepatic nerves usually co-localized with tyrosine hydroxylase, a sympathetic marker. Further, intraportal administration of the sympathetic neurotoxin, 6-hydroxydopamine, abolished galanin staining in the hepatic parenchyma. We evaluated the role of galanin in mediating the actions of sympathetic nerves to increase hepatic glucose production and decrease hepatic arterial conductance. Local infusion of synthetic galanin had little effect by itself, but it did potentiate the action of norepinephrine to stimulate hepatic glucose production, demonstrating a neuromodulatory action. In contrast, galanin had no effect on hepatic arterial blood flow. We therefore propose that in the liver galanin functions as a neuromodulator of norepinephrine's metabolic action.     

Binding site prediction of galanin peptide using evolutionary trace method

Galanin is a neuropeptide with aminoacid length ranging from 29 to 31 is widely distributed in central and peripheral nervous system. Galanin controls various psychological processes such as sensation of pain, learning, feeding, and sexual behaviour. The N-terminal region of this neuropeptide has highly conserved 15 amino acids, which is triggered by galanin receptors. We performed evolutionary trace analysis for galanin sequences to gather information about functional residues. The consensus pattern given by the evolutionary trace (ET) analysis is supported by CLUSTALW and WEBLOGO results. Our observations strongly suggest the presence of functional residues in the N-terminal region of galanin for agonist-receptor binding.     

Central galanin and N-terminal galanin fragment induce c-Fos immunoreactivity in the medulla oblongata of the anesthetized rat

This immunohistochemical study analyzed the c-Fos expression (c-Fos-ir) induced by galanin injections. Galanin and N-terminal galanin fragment (1-15) induced a significant increase of c-Fos expression (c-ir) within the medulla oblongata 90 min and 6 h. after intracisternal injections. This expression has been studied mainly in the nucleus of the solitary tract and in the ventrolateral medulla showing different temporal profiles for both peptides. The presence of c-Fos-ir in TH-positive cells was analyzed in all the groups. These results may be relevant to understand the role of galanin in several functions including central cardiovascular control.     

甘丙肽及其受体在中枢神经系统疾病中的作用

中枢神经系统疾病因其发病机制复杂而难以找到药物作用的有效靶点。甘丙肽(galanin, GAL)因其广泛的中枢神经系统分布并与多种神经系统疾病密切相关而进入人们的视线。现已证明,GAL与三种G蛋白偶联受体(GALR1-3)结合后,通过抑制cAMP/PKA(GALR1、GALR3)和激活磷脂酶C(GALR2)等信号通路调节众多生理和病理过程。本文概述了近年来GAL及其受体在中枢神经系统疾病中的作用的研究进展,旨在为理解这些疾病的发病机制以及靶向药物的研发提供新的指导。     

Inhibition of insulin release by galanin and gastrin-releasing peptide in the anaesthetized rat

The present study was designed to determine the effects of intravenously administered galanin or gastrin-releasing peptide (GRP) on glucose- and/or glucose-dependent insulinotropic peptide (GIP)-stimulated insulin release in the anaesthetized rat. Galanin inhibited glucose-stimulated insulin responses in a dose-related manner. Galanin also inhibited insulin release in response to glucose administered with GIP; this effect was due largely to inhibition of the glucose-stimulated component since galanin did not inhibit GIP-stimulated insulin release. Galanin also inhibited insulin responses to ingestion of a mixed meal. GRP inhibited glucose-stimulated insulin responses, and the insulin responses to glucose plus GIP; unlike galanin, GRP inhibited both glucose- and GIP-stimulated insulin release. GRP also inhibited insulin release following ingestion of a mixed meal. The results suggest a possible modulatory role for these neuropeptides in regulation of insulin secretion.