Regulatory Influence of Galanin and GALR1/GALR2 Receptors on Inflamed Uterus Contractility in Pigs.

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10-7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10-7 M). GALR1/GALR2 antagonist and GAL (10-7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.

A promising biomarker of elevated galanin level in hypothalamus for osteoporosis risk in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) and osteoporosis are two major healthcare problems worldwide. T2DM is considered to be a risk factor for osteoporosis. Interestingly, several epidemiological studies suggest that bone abnormalities associated with diabetes may differ, at least in part, from those associated with senile or post-menopausal osteoporosis. The growing prevalence that patients with T2DM simultaneously suffer from osteoporosis, puts forward the importance to discuss the relationship between both diseases, as well as to investigate correlative agents to treat them. Emerging evidences demonstrate that neuropeptide galanin is involved in the pathogenesis of T2DM and osteoporosis. Galanin via activation of central GALR2 increases insulin sensitivity as well as bone density and mass in animal models. The disorder of galanin function plays major role in development of both diseases. Importantly, galanin signaling is indispensable for ΔFosB, an AP1 antagonist, to play the bone mass-accruing effects in the ventral hypothalamic neurons of diabetic models. This review summarizes our and other recent studies to provide a new insight into the multivariate relationship among galanin, T2DM and osteoporosis, highlighting the beneficial effect of galanin on the comorbid state of both diseases. These may help us better understanding the pathogenesis of osteoporosis and T2DM and provide useful clues for further inquiry if elevated galanin level may be taken as a biomarker for both conjoint diseases, and GALR2 agonist may be taken as a novel therapeutic strategy to treat both diseases concurrently.

Central administration of galanin N-terminal fragment 1-15 decreases the voluntary alcohol intake in rats.

Alcohol consumption is considered a major risk factor for disease and mortality worldwide. In the absence of effective treatments in alcohol use disorders, it is important to find new biological targets that could modulate alcohol consumption. We tested the role of the N-terminal galanin fragment (1-15) [GAL(1-15)] in voluntary ethanol consumption in rats using the two-bottle choice paradigm as well as compare the effects of GAL(1-15) with the whole molecule of GAL. We describe for the first time that GAL(1-15), via central mechanisms, induces a strong reduction in preference and ethanol consumption in rats. These effects were significantly different than GAL. GAL receptor (GALR) 2 was involved in these effects, because the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions in preference and ethanol intake. Importantly, the mechanism of this action involves changes in GALR expression and also in immediate-early gene C-Fos and receptors-internalization-related gene Rab5 in the striatum. The relevance of the striatum as a target for GAL(1-15) was supported by the effect of GAL(1-15) on the locomotor activity of rats after ethanol administration. These results may give the basis for the development of novel therapeutics strategies using GAL(1-15) analogues for the treatment of alcohol use disorders in humans.

Activation of galanin receptor 1 inhibits locus coeruleus neurons via GIRK channels.

The noradrenergic neurons of the locus coeruleus (LC) are associated with various brain functions and psychiatric disorders, such as addiction and depression. It has been shown that neuropeptide galanin (GAL) inhibits neuronal excitability in LC, but the mechanisms remain unclear. In the present study, we investigated the ionic and signal transduction mechanisms underlying inhibitory effect of GAL on LC neurons using whole-cell patch clamp recording in rat brain slices. Bath application of GAL decreased the spontaneous firings and induced a dose-dependent hyperpolarization of LC neurons and this effect was attenuated by knockdown of Galr1, but not Galr2, confirming that mainly GALR1 mediates the inhibition effect of GAL. The inhibitory effect of GAL was also blocked by treatments of pertussis toxin (PTX), GTP-γ-s or GDP-ß-s, respectively, indicating that the functions of PTX sensitive Gi/o protein are required for GAL-induced hyperpolarization. Moreover, the blockers of GIRK (tertiapin-Q or SCH2 3390 hydrochloride) attenuated the GAL response while blocker of BK/SK/KATP channels or TASK-1/3 channels did not affect it significantly, suggesting that GIRK channels play an important role in GAL-induced hyperpolarization in LC neurons. Taken together, the inhibitory effect of GAL on LC neurons is mediated by GALR1 via PTX-sensitive Gi/o proteins, which activate GIRK channels.

Elevated galanin receptor type 2 primarily contributes to mechanical hypersensitivity after median nerve injury.

In this study, we investigated temporal changes in galanin receptor type 2 (GalR2) expression in NF200-, galanin-, neuropeptide Y (NPY)-, and neuronal nitric oxide synthase (nNOS)-like immunoreactive (LI) dorsal root ganglion (DRG) neurons after median nerve chronic constriction injury (CCI), and the effects of GalR2 on c-Fos expression in the cuneate nucleus (CN). Double immunofluorescence labeling methods were used to appraise changes in GalR2 expression in NF200-LI, galanin-LI, NPY-LI, and nNOS-LI DRG neurons after CCI. The von Frey assay was used to assess the efficiency of intraplantar administration of saline, M871 (a GalR2 antagonist), or AR-M1896 (a GalR2 agonist) on neuropathic signs of rats with CCI. The effects of alterations in c-Fos expression were assessed in all treatments. The percentage of GalR2-LI neurons in lesioned DRGs increased and peaked at 1 week after CCI. We further detected that percentages of GalR2-LI neurons labeled for NF200, galanin, NPY, and nNOS significantly increased following CCI. Furthermore, M871 remarkably attenuated tactile allodynia, but the sensation was slightly aggravated by AR-M1896 after CCI. Consequentially, after electrical stimulation of the CCI-treated median nerve, the number of c-Fos-LI neurons in the cuneate nucleus (CN) was significantly reduced in the M871 group, whereas it increased in the AR-M1896 group. These results suggest that activation of GalR2, probably through NPY or nitric oxide, induces c-Fos expression in the CN and transmits mechanical allodynia sensations to the thalamus.

The antidepressant-like effect of galanin in the dorsal raphe nucleus of rats involves GAL2 receptors.

Galanin is a neuropeptide distributed in human and rat brain regions that are involved with emotional regulation, such as the dorsal raphe nucleus (DRN). Galanin effects in the DRN are mediated by GAL1 and GAL2 receptors. Intracerebral infusion of a GAL2 (AR-M1896) or a GAL1 (M617) agonist induced either antidepressant or depressive-like effect, respectively, in rats exposed to the forced swimming test (FST). However, it is not clear if GAL1 and/or GAL2 receptors present in the DRN would be involved in such effects. Therefore, we investigated the effects induced by intra-DRN infusion of galanin (0.3 nmol), AR-M1896 (1 nmol, GAL2 agonist), or M617 (GAL1 agonist) in rats exposed to the FST. Galanin and AR-M1896 intra-DRN administration induced antidepressant-like effect in the FST. However, M617 did not induce any change in the FST. Neither M617 nor AR-M1896 changed the locomotor activity of rats in the open field test. Intra-DRN pre-treatment with M871 (1 nmol), a selective GAL2 antagonist, counteracted the antidepressant-like effect induced by galanin. These results suggest that galanin signaling through GAL2 receptors in the DRN produces triggers antidepressant-like effect.

Involvement of galanin and galanin receptor 2 in nociceptive modulation in anterior cingulate cortex of normal rats and rats with mononeuropathy.

The present study was performed to explore the role of galanin and galanin receptor 2 in nociceptive modulation in anterior cingulate cortex (ACC) of normal rats and rats with mononeuropathy. Intra-ACC injection of galanin induced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations in both normal rats and rats with mononeuropathy, the increased HWLs were attenuated significantly by intra-ACC injection of galanin receptor 2 antagonist M871, indicating an involvement of galanin receptor 2 in nociceptive modulation in ACC. Interestingly, the galanin-induced HWL was significant higher in rats with mononeuropathy than that in normal rats tested by Randall Selitto test. Furthermore, both the galanin mRNA expression and galanin content increased significantly in ACC in rats with mononeuropathy than that in normal rats. Moreover, both the mRNA levels of galanin receptor 2 and the content of galanin receptor 2 in ACC increased significantly in rats with mononeuropathy than that in normal rats. These results found that galanin induced antinociception in ACC in both normal rats and rats with mononeuropathy. And there may be plastic changes in the expression of galanin and galanin receptor 2 in rats with mononeuropathy, as well as in the galanin-induced antinociception.

Preclinical evaluation of intravenous NAX 810-2, a novel GalR2-preferring analog, for anticonvulsant efficacy and pharmacokinetics.

OBJECTIVE: Potential clinical utility of galanin or peptidic analogs has been hindered by poor metabolic stability, lack of brain penetration, and hyperglycemia due to galanin receptor subtype 1 (GalR1) activation. NAX 810-2, a galanin receptor subtype 2 (GalR2)-preferring galanin analog, possesses 15-fold greater affinity for GalR2 over GalR1 and protects against seizures in the mouse 6 Hz, corneal kindling, and Frings audiogenic seizure models. The purpose of these studies was to further evaluate the preclinical efficacy and pharmacokinetics of NAX 810-2 in mice. METHODS: NAX 810-2 was administered by intravenous (i.v.; tail vein, bolus) injection to fully kindled (corneal kindling assay) or naive CF-1 mice (6 Hz assay and pharmacokinetic studies). Plasma NAX 810-2 levels were determined from trunk blood samples. NAX 810-2 was also added to human plasma at various concentrations for determination of plasma protein binding. RESULTS: In the mouse corneal kindling model, NAX 810-2 dose-dependently blocked seizures following intravenous administration (median effective dose [ED50 ], 0.5 mg/kg). In the mouse 6 Hz (32 mA) seizure model, it was demonstrated that NAX 810-2 dose-dependently blocked seizures following bolus administration (0.375-1.5 mg/kg, i.v.; ED50 , 0.7 mg/kg), with a time-to-peak effect of 0.5 h posttreatment. Motor impairment was observed at 1.5 mg/kg, i.v., whereas one-half of this dose, 0.75 mg/kg, i.v., was maximally effective in the 6 Hz test. Plasma levels of NAX 810-2 show linear pharmacokinetics following intravenous administration and a half-life of 1.2 h. Functional agonist activity studies demonstrate that NAX 810-2 effectively activates GalR2 at therapeutic concentrations. SIGNIFICANCE: These studies further suggest the potential utility of NAX 810-2 as a novel therapy for epilepsy.

Antinociception induced by galanin in anterior cingulate cortex in rats with acute inflammation.

The present study was performed to explore the role of galanin in nociceptive modulation in anterior cingulate cortex (ACC) of rats with acute inflammation, and the changes in galanin and galanin receptor 2 (Gal R2) expressions in rats with acute inflammation. Intra-ACC injection of galanin induced antinociception in rats with acute inflammation, the antinociceptive effects induced by galanin were attenuated significantly by intra-ACC injection of the Gal R2 antagonist M871, indicating an involvement of Gal R2 in nociceptive modulation in ACC in rats with acute inflammation. Furthermore, we found that both the galanin mRNA expression and galanin content increased significantly in ACC in rats with acute inflammation than that in normal rats. Moreover, both the mRNA levels of Gal R2 and the content of Gal R2 in ACC increased significantly in rats with acute inflammation than that in normal rats. These results demonstrated that galanin induced antinociception in ACC in rats with acute inflammation. And there were changes in the expression of galanin and Gal R2 in rats with acute inflammation.

Galanin (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT: involvement of the raphe-hippocampal 5-HT neuron system.

Galanin N-terminal fragment (1-15) [GAL(1-15)] is associated with depression-related and anxiogenic-like effects in rats. In this study, we analyzed the ability of GAL(1-15) to modulate 5-HT1A receptors (5-HT1AR), a key receptor in depression. GAL(1-15) enhanced the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test. These effects were stronger than the ones induced by Galanin (GAL). This action involved interactions at receptor level since GAL(1-15) affected the binding characteristics and the mRNA levels of 5-HT1AR in the dorsal hippocampus and dorsal raphe. The involvement of the GALR2 was demonstrated with the GALR2 antagonist M871. Proximity ligation assay experiments indicated that 5-HT1AR are in close proximity with GALR1 and GALR2 in both regions and in raphe RN33B cells. The current results indicate that GAL(1-15) enhances the antidepressant effects induced by 8-OH-DPAT acting on 5-HT1AR operating as postjunctional or as autoreceptors. These results may give the basis for the development of drugs targeting potential GALR1-GALR2-5-HT1AR heteroreceptor complexes linked to the raphe-hippocampal 5-HT neurons for the treatment of depression.

Involvement of galanin receptor 2 and CaMKII in galanin-induced antinociception in periaqueductal grey of rats.

The present study was performed to explore the effect of the galanin receptor 2 (GalR2) antagonist M871 on the galanin-induced antinociception in periaqueductal grey (PAG), and an involvement of Ca(2+)/calmodulin-dependent kinase II (CaMKII) in the galanin-induced antinociception. Intra-PAG injection of galanin induced marked increases in HWLs to noxious thermal and mechanical stimulation. The increased HWLs to thermal and mechanical stimulation decreased significantly after intra-PAG administration of the GalR2 antagonist M871, indicating an involvement of GalR2 in the galanin-induced antinociception in PAG of rats. Furthermore, rats received intra-PAG injection of galanin, followed 5min later by intra-PAG administration of the CaMKII inhibitor MAP. The galanin-induced increases in HWLs to thermal and mechanical stimulation decreased significantly after intra-PAG administration of MAP, indicating that there is an involvement of CaMKII in the galanin-induced antinociception in PAG, blockade the activity of CaMKII by MAP inhibits the galanin-induced antinociception in PAG of rats. Our results strongly indicate that the galanin-induced antinociception is mediated by GalR2 in the PAG, and CaMKII may be involved in the galanin-induced antinociception in PAG of rats.

Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice.

A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca(2+)] removal and L-type voltage-dependent Ca(2+) channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca(2+)]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders.

The role of galanin receptors in anticonvulsant effects of low-frequency stimulation in perforant path-kindled rats.

Low-frequency stimulation (LFS) has antiepileptogenic effects on kindled seizures. In the present study, the role of galanin receptors in the inhibitory effect of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by perforant path stimulation in a rapid kindling manner (six stimulations per day). LFS (0.1 ms pulses at 1 Hz, 600 pulses, and 80-150 microA) was applied immediately after termination of each kindling stimulation. M35 (0.5 and 1.0 nM per site), a nonselective galanin receptor antagonist and M871 (1.0 microM per site), a selective galanin receptor type 2 (GalR2) antagonist, were daily microinjected into the dentate gyrus before starting the stimulation protocol. The expression of GalR2 in the dentate gyrus was also investigated using semi-quantitative RT-PCR. Application of LFS significantly retarded the kindling acquisition and delayed the expression of different kindled seizure stages. In addition, LFS significantly reduced the increment of daily afterdischarge duration during kindling development. Intra-dentate gyrus microinjection of both M35 and M871 significantly prevented the inhibitory effects of LFS on kindling parameters. During the focal kindled seizure stages (1-3) M871 had no significant effect. However, during generalized seizure stages (4 and 5), M871 had the same effect as M35. Semi-quantitative RT-PCR also showed that after kindling acquisition, the GalR2 mRNA level decreased in the dentate gyrus but application of LFS prevented this decrease. Obtained results show that activation of galanin receptors by endogenous galanin has a role in mediating the inhibitory effect of LFS on perforant path-kindled seizures. This role is exerted through GalR1 during focal- and through GalR2 during generalized-kindled seizures.