Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines.

The underlying neurochemical changes contributing to prion-induced neurodegeneration remain largely unknown. This study shows that scrapie infection induced a 2-fold increase of insulin receptor (IR) protein and aberrantly processed IR beta-chain in scrapie-infected N2a neuroblastoma cells (ScN2a) as measured by Western blot of immunoprecipitated IR, in the absence of increased IR mRNA. Elevated IR protein level was further confirmed in an independently scrapie-infected neuroblastoma cell line N1E-115 (ScN1E-115). Proliferation studies showed that the increased IR level in ScN2a did not result in an increased insulin-mediated cell growth compared to normal N2a cells. Binding studies indicated that this apparent paradox was due to a 65% decrease in specific [(125)I]insulin binding sites in ScN2a when compared to the amount of immunoreactive IR, although the IR binding affinity was unchanged. Analysis of insulin stimulated IR tyrosine phosphorylation showed a slight but not significant reduction in ScN2a, when related to the increased level of immunoreactive IR. However, comparing the IR tyrosine phosphorylation to the loss of binding sites in ScN2a, we demonstrated an increased IR tyrosine phosphorylation of the remaining functional IR. In addition to these differences in IR properties, the basal extracellular signal regulated kinase-2 (ERK2) phosphorylation detected by Western blot, was significantly elevated and the insulin stimulated ERK2 phosphorylation was subsequently decreased in ScN2a. Together, these data show that scrapie infection affects the level and processing of the IR and signal transduction mediated by the IR in neuroblastoma cells, as well as induces an elevated basal ERK2 phosphorylation. Aberrant regulation of neuroprotective receptors may contribute to neurodegeneration in prion diseases.

Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells.

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80% decrease in specific (125)I-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1Rbeta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases.

Functional trans-inactivation of insulin receptor kinase by growth-inhibitory angiotensin II AT2 receptor.

The present study demonstrates negative intracellular cross-talk between angiotensin II type 2 (AT2) and insulin receptors. AT2 receptor stimulation leads to inhibition of insulin-induced extracellular signal-regulated protein kinase (ERK2) activity and cell proliferation in transfected Chinese hamster ovary (CHO-hAT2) cells. We show that AT2 receptor interferes at the initial step of insulin signaling cascade, by impairing tyrosine phosphorylation of the insulin receptor (IR) beta-chain. AT2-mediated inhibition of IR phosphorylation is insensitive to pertussis toxin and is also detected in neuroblastoma N1E-115 and pancreatic acinar AR42J cells that express endogenous receptors. We present evidence that AT2 receptor inhibits the autophosphorylating tyrosine kinase activity of IR, with no significant effect on insulin binding properties. AT2-mediated inactivation of IR does not mainly involve tyrosine dephosphorylation by vanadate-sensitive tyrosine phosphatases nor serine/threonine phosphorylation by protein kinase C. As a consequence of IR inactivation, AT2 receptor inhibits tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and signal-regulatory protein (SIRPalpha1) and prevents subsequent association of both IRS-1 and SIRPalpha1 with Src homology 2 (SH2)-containing tyrosine phosphatase SHP-2. Our results thus demonstrate functional trans-inactivation of IR kinase by G protein-coupled AT2 receptor, illustrating a novel mode of negative communication between two families of membrane receptors.

sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1.

We have previously reported in Chinese hamster ovary (CHO) cells expressing sst2 that activation of the sst2 somatostatin receptor inhibits insulin-induced cell proliferation by a mechanism involving stimulation of a tyrosine phosphatase activity. Here we show that the tyrosine phosphatase SHP-1 was associated with the insulin receptor (IR) at the basal level. Activation of IR by insulin resulted in a rapid and transient increase of tyrosine phosphorylation of IR, its substrates IRS-1 and Shc, and also of SHP-1. This was then followed by a rapid dephosphorylation of these molecules, which was related to the insulin-induced increase of SHP-1 association to IR and of SHP-1 activity. On the other hand, addition to insulin of the somatostatin analogue, RC160, resulted in a higher and faster increase of SHP-1 association to IR directly correlated with an inhibition of phosphorylation of IR and its substrates, IRS-1 and Shc. RC160 also induced a higher and more sustained increase in SHP-1 activity. Furthermore, RC160 completely suppressed the effect of insulin on SHP-1 phosphorylation. Finally, in CHO cells coexpressing sst2 and a catalytically inactive mutant SHP-1, insulin as well as RC160 could no longer stimulate SHP-1 activity. Overexpression of the SHP-1 mutant prevented the insulin-induced signaling to be terminated by dephosphorylation of IR, suppressed the inhibitory effect of RC160 on insulin-induced IR phosphorylation, and abolished the cell proliferation modulation by insulin and RC160. Our results suggest that SHP-1 plays a role in negatively modulating insulin signaling by association with IR. Furthermore, somatostatin inhibits the insulin-induced mitogenic signal by accelerating and amplifying the effect of SHP-1 on the termination of the insulin signaling pathway.

Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase.

Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.

Expression of peptides, nitric oxide synthase and NPY receptor in trigeminal and nodose ganglia after nerve lesions.

Using immunohistochemistry and in situ hybridization, the expression of galanin (GAL)/galanin message associated peptide (GMAP)-, neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI)- and nitric oxide synthase (NOS)-like immunoreactivities and mRNAs, and NPY receptor mRNA was studied in normal trigeminal and nodose ganglia and 14 and 42 days after peripheral axotomy. In normal trigeminal ganglia about 11% of the counted neuron profiles contained GAL mRNA, 4% NOS mRNA, 5% NPY mRNA, 7% VIP mRNA, and 19% NPY receptor mRNA. Peptide mRNA- and NPY receptor mRNA-positive neuron profiles were small in size. Fourteen days after axotomy a marked increase in the number of GAL mRNA-(34% of counted neuron profiles), NPY mRNA-(54%) and VIP mRNA-(31%) positive neuron profiles, and a moderate increase in the number of NOS mRNA-(22%) positive neuron profiles were observed in the ipsilateral trigeminal ganglia. The GAL/GMAP, VIP- and NOS-positive profiles were mainly small, the NPY-positive ones mostly large. NPY receptor mRNA was expressed in some large neurons. In normal nodose ganglia, about 3% of the counted neuron profiles contained GAL mRNA, 3% NPY mRNA, 17% NOS mRNA and less than 1% VIP mRNA. Fourteen days after peripheral axotomy, a marked increase in the number of GAL mRNA-(78% of counted neuron profiles), NOS mRNA-(37%) and VIP-(46%) mRNA-positive neuron profiles was seen in the ipsilateral nodose ganglia. The number of NPY-positive (23%) neurons was moderately increased, mainly in small neuron profiles. There were no NPY receptor mRNA-positive neurons, either in normal nodose ganglia or in nodose ganglia ipsilateral to the axotomy. In contralateral nodose ganglia the number of GAL- and NPY-positive neuron profiles was slightly increased, and VIP cells showed a moderate increase. Immunohistochemical analysis revealed parallel changes in expression of peptides and NOS in both trigeminal and nodose ganglia, demonstrating that the changes in mRNA levels are translated into protein. Finally, although not quantified, similar upregulations of peptide and NOS mRNA levels were observed in both ganglia 42 days after nerve injury provided that regeneration was not allowed, suggesting that the changes are long lasting. The present results show that the effect of axotomy on peptide and NOS expression in the trigeminal and nodose ganglia is similar to that previously shown for lumbar dorsal root ganglia. However, no mRNA for the NPY Y1 receptor could be detected in the vagal system. In general the mechanism(s) for and the purpose(s) of the messenger regulation in response to axotomy may be similar in these different sensory systems (dorsal root, trigeminal and nodose ganglia).

cDNA sequence, ligand biding, and regulation of galanin/GMAP in mouse brain.

The cDNA encoding the 29 amino acid-long neuropeptide galanin and its flanking peptide galanin message associated peptide (GMAP), has been cloned and sequenced from mouse hypothalamic cDNA. The primary sequence of mouse galanin is GWTLNSAGYLLGPHAIDNHRSFSDKHGLT, followed by an amidation signal GKR. There are now 12 galanin sequences known: human, porcine, dog, rat, bovine, chicken, sheep, alligator, bowfin, dogfish, trout and mouse. The N-terminal 14 amino acids are identical in all of these species and the whole primary sequence of mouse galanin is identical to that of rate galanin. The mouse C-terminal flanking peptide, the GMAP, which is encoded on the same mRNA as galanin, shows a high degree of homology with all other known GMAP sequences but is not identical to any of them and it is more charged than the other GMAP sequences. Synthetic mouse galanin was found to displace [125I]mono-iodo-Tyr26 galanin (porcine) from receptors in the mouse hypothalamic membranes with high affinity (KD = 0.9 nM). Estrogen treatment of mice (0.1 mg/kg i.p.) for 6 h, which elevates the rat pituitary galanin mRNA levels, does not affect the galanin mRNA levels in mouse hypothalamus and pituitary. Neither does a subchronic glucocorticoid treatment (dexamethasone, 0.5 mg/kg i.p. for 7 days) affect these mRNA levels.

New high affinity peptide antagonists to the spinal galanin receptor.

1. The role of endogenous galanin in somatosensory processing has been studied with galanin receptor antagonists. The new galanin receptor ligands C7, M32, M38 and M40 bind with high affinity (Kd in nanomolar range) to spinal cord galanin receptors and possess oxidative stability as compared to earlier generations of peptide ligands. These peptides have been examined in the spinal flexor reflex model where exogenous galanin exhibited biphasic excitatory and inhibitory effects. 2. Intrathecal administration of C7 [galanin(1-13)-spantide] and M32 [galanin (1-13)-neuropeptide Y(25-36) amide] blocked facilitation of the nociceptive flexor reflex induced by 30 pmol intrathecal galanin in decerebrate, spinalized rats in a dose-dependent manner, thus behaving as antagonists of the galanin receptor. In contrast, M38[galanin(1-13)-(Ala-Leu)3-Ala amide] and M40 [galanin(1-13)-Pro-Pro-(Ala-Leu)2-Ala amide], exhibited only weak antagonism at high doses in this model. Moreover, lower doses of M40 potentiated galanin-induced reflex facilitation. C7 was neurotoxic at high doses in the rat spinal cord. 3. M32 and C7 were potent antagonists of galanin receptors in rat spinal cord, in correlation with their in vitro binding characteristics. In contrast, M38 and M40, despite their high in vitro affinity, exhibited only very weak antagonism. Moreover, M40 may also behave as a partial agonist. 4. Previous studies have shown that the galanin receptor may be heterogeneous. The discrepancy between in vitro binding and in vivo antagonistic potency of M38 and M40 may also suggest the presence of different galanin receptor subtypes within the rat spinal cord. However, other explanations for the discrepancy, such as differences in metabolic stability, diffusion rates and penetration to the site of action are also possible.

Metabolism of galanin and galanin (1-16) in isolated cerebrospinal fluid and spinal cord membranes from rat.

The occurrence of galanin (GAL) in the spinal cord and reports suggesting that it acts as an endogenous inhibitory spinal modulator in sensory/noxious transmission, have focused interest on its metabolism in the spinal cord. The metabolic half-lives and degradation patterns of GAL(1-29) and the high affinity N-terminal fragment GAL(1-16), were determined in isolated cerebrospinal fluid (CSF) from rats, and analysed by reverse phase HPLC. The half-lives for GAL(1-29) and GAL(1-16) in isolated rat CSF at 37 degrees C were 120 min and 60 min, respectively. The first degradation products which we could isolate and identify of GAL(1-16) were: GAL(3-16) and GAL(3-12) and for GAL(1-29): GAL(1-5) and GAL(1-4), all without affinity to spinal galanin receptors. Degradation studies of GAL(1-29) and GAL(1-16) in a spinal cord membrane preparation, in absence or presence of different protease inhibitors: E-64, pepstatin A, 3,4-DCI, bestatin, phosphoramidon, kelatorphan and thiorphan, or metal chelators: EDTA, EGTA and o-phenanthrolin, suggest that a phosphoramidon sensitive zinc-metalloprotease is mainly responsible for the degradation of GAL(1-29) and GAL(1-16), since both o-phenanthrolin (0.3 mM) and phosphoramidon (920 microM) substantially prolong their half-lives.

Peripheral axotomy increases the expression of galanin message-associated peptide (GMAP) in dorsal root ganglion cells and alters the effects of intrathecal GMAP on the flexor reflex in the rat.

We have previously reported that galanin message-associated peptide (GMAP), a fragment of galanin precursor protein, occurs in a limited number of dorsal root ganglion (DRG) cells in rats with intact sciatic nerves. In the present study, the localization of GMAP in dorsal root ganglia, dorsal roots and dorsal horn was analyzed immunohistochemically and compared between rats with intact and sectioned sciatic nerves. Furthermore, the effects of intrathecal (i.t.) GMAP on the flexor reflex in rats with intact and sectioned nerves were examined. In rats with intact sciatic nerves, i.t. GMAP elicited a moderate facilitation of the flexor reflex. The facilitation of the flexor reflex induced by conditioning stimulation (CS) of cutaneous C-fibers was strongly blocked by GMAP. GMAP also selectively antagonized the reflex facilitatory effect of i.t. substance P (SP), but not i.t. vasoactive intestinal peptide (VIP). Unilateral sciatic nerve section induced an upregulation of GMAP in the ipsilateral dorsal root ganglia 2 weeks after axotomy. The effect of GMAP on the baseline reflex was similar in normal and axotomized rats, but the blocking effect of GMAP on C-fiber CS-induced facilitation was significantly reduced after axotomy. GMAP did not antagonize the reflex facilitatory effect of SP after axotomy, whereas an antagonism on VIP-induced facilitation was observed. The possible role of GMAP in spinal transmission and comparison with the effects of galanin are discussed.

Galanin--10 years with a neuroendocrine peptide.

Galanin is a 29/30 amino acids long neuropeptide which does not belong to any known peptide family. The N-terminal first 16 amino acids of the molecule are both necessary and sufficient for receptor recognition and receptor activation. The main pharmacophores of galanin in its central and pancreatic actions are Gly1, Trp2, Asn5 and Tyr9, respectively. The neuropeptide galanin has multiple effects in both the central and peripheral nervous systems. Centrally, galanin potently stimulates fat intake and impairs cognitive performance. Anoxic glutamate release in the hippocampus is inhibited by galanin and the noradrenergic tonus in the brain is influenced by a hyperpolarizing action of galanin in the locus coeruleus. In the spinal cord galanin inhibits spinal excitability and potentiates the analgesic effect of morphine. In the neuroendocrine system galanin acts in a stimulatory manner on the release of growth hormone and prolactin, and peripherally galanin inhibits glucose induced insulin release. Galanin also causes contraction of the jejunum. The galanin receptor is a Gi-protein-coupled, membrane-bound glycoprotein with an estimated molecular mass of 53 kDa. Several putative tissue specific galanin receptor subtypes have been proposed on a pharmacological basis. The distribution of galanin receptors and of galanin like immunoreactivity are overlapping in the CNS, both being high in areas such as the locus coeruleus, raphe nucleus and hypothalamus. Galanin receptor activation leads to a reduced intracellular Ca(2+)-concentration, either by direct action on voltage sensitive Ca(2+)-channels or indirectly via opening of K(+)-channels or via inhibition of adenylyl cyclase activity. The lowered intracellular Ca2+ level subsequently leads to a reduced PLC activity. Galanin also inhibits cGMP synthesis induced by depolarization. A number of synthetic high affinity galanin receptor antagonists of the peptide type were developed recently, which have enabled the elucidation of functional roles of endogenous galanin in several systems. Furthermore, putative subtypes of galanin receptors can be distinguished by the use of these new galanin receptor ligands.

Galanin antisense oligonucleotides reduce galanin levels in dorsal root ganglia and induce autotomy in rats after axotomy.

Antisense (AS) oligonucleotides (ONs) to galanin (GAL) were applied to the proximal end of a transected sciatic nerve, allowing their cellular uptake and transport into injured axons. GAL expression in dorsal root ganglia and self-mutilation behavior (autotomy) were then studied. AS-ONs with phosphorothioate or allyl modifications significantly suppressed the axotomy-induced increase in GAL levels, as demonstrated by immunohistochemistry and exaggerated autotomy behavior, whereas no significant effect on GAL mRNA levels could be demonstrated with in situ hybridization. Allyl-ONs were more effective than phosphorothioate-ONs. An AS-ON with three base mismatches did not induce any of the above effects. These results support the view that the inhibition of axotomy-induced GAL up-regulation is related to autotomy.

Immunohistochemical demonstration of galanin-, and galanin message-associated peptide-like immunoreactivities in sympathetic ganglia and adrenal gland of the guinea pig.

Using the indirect immunofluorescence method, the distribution of galanin (GAL)- and galanin message-associated peptide (GMAP)-like immunoreactivities (LI) were studied in sympathetic ganglia and the adrenal gland of the guinea pig. A rather dense network of GAL-immunoreactive nerve fibers was found in the inferior mesenteric ganglion (IMG) and in the superior mesenteric pole of the celiac-superior mesenteric ganglion complex (C-SMG). The celiac pole of the C-SMG, the stellate ganglion, and the superior cervical ganglion contained fewer, mostly scattered fibers. SIF-cells in prevertebral and paravertebral ganglia contained GAL-LI, as did the adrenal medullary cells. The GAL fibers in the IMG surrounded mainly principal ganglion cells containing somatostatin-immunoreactivity (SOM-IR), whereas fewer fibers were seen around neuropeptide Y (NPY) cells and cells in which SOM and NPY coexisted. Application of colchicine or vinblastine onto the IMG did not result in the appearance of GAL-IR in the principal ganglion cells. In denervation experiments it was revealed that most of the GAL fibers reach the IMG via the lumbar splanchnic nerves. GAL-IR appears to be colocalized with substance P (SP) in fibers of the IMG, indicating an origin of the GAL-containing fibers in dorsal root ganglia (DRG). This conclusion was supported by the finding in lumbar DRGs of GAL-positive cell bodies that contained SP. The role of GAL in prevertebral ganglia is unclear. It may be suggested that GAL modulates the slow, long-lasting membrane depolarization of the principal ganglion cells caused by SP in the primary afferents related to the IMG. GMAP-LI was detected in SIF cells and adrenal medullary cells in which GMAP-LI parallels the immunoreactivity of GAL. GMAP-LI was not observed in neuronal cell bodies or nerve fibers of the ganglia.

Upregulation of nitric oxide synthase and galanin message-associated peptide in hypothalamic magnocellular neurons after hypophysectomy. Immunohistochemical and in situ hybridization studies.

The expression of several bioactive molecules in magnocellular hypothalamic neurons is modified when the axons of these cells are transected. In this study we have evaluated by means of immunocytochemistry and in situ hybridization the effect of hypophysectomy on the expression of nitric oxide synthase (NOS)- and of galanin message-associated peptide (GMAP)-like immunoreactivities (-LIs) as well as on their respective mRNAs in hypothalamic magnocellular neurosecretory neurons. The results show a transient increase in NOS- and GMAP-LIs in magnocellular neurons of both the paraventricular and supraoptic nuclei when compared to normal animals. The maximal increase in staining was observed between 5 and 7 days, and by 14 days NOS-LI was back to normal levels, whereas strong GMAP-LI could still be detected in a few cells. A similar picture was observed for the NOS and GMAP mRNAs. The functional significance of the present findings is unclear, but they indicate a possible role of nitric oxide and GMAP in neurosecretory neurons after injury.

Biological activities of two endogenously occurring N-terminally extended forms of galanin in the rat spinal cord.

The occurrence of two N-terminally extended forms of galanin in the porcine adrenal medulla was reported earlier by Bersani et al. (1991). We have synthesized and examined the ability of these two extended forms of galanin, galanin-(-7-29) and galanin-(-9-29), to bind to galanin receptors in the rat dorsal spinal cord. The effect of intrathecal (i.t.) injection of these peptides on spinal flexor reflex excitability in decerebrate, spinalized, unanesthetized rats was also studied. Both galanin-(-7-29) and galanin-(-9-29) fully displaced specific 125I-monoido-[Tyr26]porcine galanin (125I-galanin) binding to membranes prepared from rat dorsal spinal cord, with IC50 values 0.13 and 0.14 microM, respectively. The metabolic half-lives in spinal cord membranes for galanin-(1-29), galanin-(-7-29) and galanin-(-9-29) were 117 +/- 17, 271 +/- 23 and 185 +/- 19 min, respectively. I.t. injection of galanin-(-7-29) and galanin-(-9-29) mimicked the biphasic facilitatory and inhibitory effect of i.t. galanin-(1-29) on flexor reflex excitability and antagonized C-fiber conditioning stimulus-induced spinal cord hyperexcitability, but with reduced potencies compared to galanin-(1-29). We suggest that the N-terminally extended forms of galanin act as endogenous ligands with low agonist activity.

Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes.

The galanin-receptor ligand M40 [galanin-(1-12)-Pro3-(Ala-Leu)2-Ala amide] binds with high affinity to [mono[125I]iodo-Tyr26]galanin-binding sites in hippocampal, hypothalamic, and spinal cord membranes and in membranes from Rin m5F rat insulinoma cells (IC50 = 3-15 nM). Receptor autoradiographic studies show that M40 (1 microM) displaces [mono[125I]iodo-Tyr26]galanin from binding sites in the hippocampus, hypothalamus, and spinal cord. In the brain, M40 acts as a potent galanin-receptor antagonist: M40, in doses comparable to that of galanin, antagonizes the stimulatory effects of galanin on feeding, and it blocks the galaninergic inhibition of the scopolamine-induced acetylcholine release in the ventral hippocampus in vivo. In contrast, M40 completely fails to antagonize both the galanin-mediated inhibition of the glucose-induced insulin release in isolated mouse pancreatic islets and the inhibitory effects of galanin on the forskolin-stimulated accumulation of 3',5'-cAMP in Rin m5F cells; instead M40 is a weak agonist at the galanin receptors in these two systems. M40 acts as a weak antagonist of galanin in the spinal flexor reflex model. These results suggest that at least two subtypes of the galanin receptor may exist. Hypothalamic and hippocampal galanin receptors represent a putative central galanin-receptor subtype (GL-1-receptor) that is blocked by M40. The pancreatic galanin receptor may represent another subtype (GL-2-receptor) that recognizes M40, but as a weak agonist. The galanin receptors in the spinal cord occupy an intermediate position between these two putative subtypes.

Galanin message-associated peptide (GMAP)- and galanin-like immunoreactivities: overlapping and differential distributions in the rat.

Using the indirect immunofluorescence method the distribution of galanin message associated peptide (GMAP)- and galanin-like immunoreactivities (LI) was compared in brain, intestine and some endocrine tissues of rat. In general, neurons in the peripheral and central nervous system contained both immunoreactivities. However, in retina the cones were GMAP-positive but galanin-negative. A strong GMAP-LI was observed in the prolactin cells in the anterior lobe of the pituitary and in the insulin cells in the islets of Langerhans in the pancreas, whereas incubation with galanin antiserum resulted in staining of fewer cells (anterior pituitary) or a very weak fluorescence (pancreas). The results show that most neurons express both GMAP- and galanin-LI, but raise the possibility that in some systems there is a tissue specific, posttranslational differential processing of preprogalanin.

Galanin-mediated control of pain: enhanced role after nerve injury.

The endogenous inhibitory role of the neuropeptide galanin in pain transmission and spinal cord excitability was demonstrated by the use of a high-affinity galanin receptor antagonist, M-35 [galanin-(1-13)-bradykinin-(2-9)-amide]. M-35, which displaced 125I-labeled galanin from membranes of rat dorsal spinal cord with an IC50 of 0.3 nM, dose-dependently antagonized the effect of intrathecal galanin on the flexor reflex. M-35 potentiated the facilitation of the flexor reflex by conditioning stimulation of cutaneous unmyelinated afferents in rats with intact nerves and the potentiating effect of M-35 on the conditioning-stimulation-induced reflex facilitation of the cutaneous unmyelinated afferents was strongly enhanced after axotomy. These results demonstrate that endogenous galanin plays a tonic inhibitory role in the mediation of spinal cord excitability, and it is particularly noteworthy that this function of galanin is remarkably enhanced after peripheral nerve section.

Galanin receptors and their second messengers in the lumbar dorsal spinal cord.

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr26 galanin as ligand. The equilibrium binding of [125I]galanin showed the presence of a single population of high-affinity binding sites with KD = 0.6 +/- 0.2 nM in a concentration of 55 +/- 15 fmol mg-1 protein (Bmax). The N-terminal fragments galanin (1-16) and galanin (1-12) fully displaced specific [125I]galanin binding from membranes with IC50 values 6 nM and 4 microM, respectively. The C-terminal fragment galanin (17-29) did not displace [125I]galanin when applied in the concentration range 10(-11)-10(-4) M. GTP inhibited the specific binding of [125I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10 microM) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 microM) did not inhibit the noradrenalin (100 microM) activated cAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on Bmax or KD of the galanin receptor was found.

M-15: high-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus, locus coeruleus, and spinal cord.

The 20-amino acid peptide M-15 binds with high affinity (IC50 approximately 0.1 nM) to 125I-labeled galanin (125I-GAL) binding sites in membranes from the ventral hippocampus, midbrain, and rat spinal cord. Receptor autoradiographic studies show that M-15 can displace 125I-GAL from all labeled sites. M-15 acts as a reversible high-affinity antagonist in blocking the inhibitory effects of GAL on the evoked release of acetylcholine in vivo in the hippocampus and on the GAL-induced hyperpolarization of locus coeruleus neurons in slices. M-15 also blocks the facilitatory effects of GAL on the spinal flexor reflex. Thus, the chimeric peptide M-15 [GAL-(1-13)-substance P-(5-11)amide] represents the first antagonist to the neuronal actions of GAL.