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U Langel

Publications and source records attributed to U Langel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Differential regulation of GTPase activity by mastoparan and galparan.

The chimeric peptide galparan, composed of galanin (1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin (1-13) was found in this tissue. EC50 of 12.1 +/- 2.1 microM and Hill coefficient of 2.1 +/- 0.6 was calculated for galparan from a dose-response curve and Ki of 19.1 +/- 0.3 microM was obtained by fitting the experimental data to the Michaelis-Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose-response curve for galparan inhibition. The rate of GTP gamma S binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel beta-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of alpha-helix. Critical micelle concentration of galparan in buffer solution of 22 microM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.

Animals↗
PubMed1998

Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormone-mastoparan constructs.

Ca2+ efflux, Ca(2+)-ATPase, and membrane permeability measurements were used to investigate the biochemical mechanisms of Ca2+ release induced by mastoparan (MP) and the chimeric hormone-MP constructs incorporating galanin (galparan) or vasopressin antagonist (M375 and M391) moieties. Comparative studies utilised preparations of porcine cerebellar microsomes and rabbit skeletal muscle sarcoplasmic reticulum (SR). MP and chimeric peptides galparan, M375 and M391 induce Ca2+ release over a range of concentrations from 0.3-10 microM. Comparison of MP and three chimeric, N-terminal extended, constructs indicates that N-terminal extension modifies the biological properties of MP, producing changes in efficacy which are enzyme-isoform-specific. Biochemical studies indicate that the chimeric analogues and MP inhibit Ca(2+)-ATPases and directly activate the ryanodine receptor (RyR) to release Ca2+ from both heavy SR (HSR) and microsomes. The same peptides have no effect on the InsP3 receptor (InsP3R). Other actions that include modest changes in membrane permeability may also contribute to the Ca(2+)-mobilising action of MP and chimeric constructs.

Amino Acid Sequence↗
PubMed1998

Galaninergic signalling and adenylate cyclase.

Galanin is a hyperpolarizing, inhibitory neurotransmitter; its recognition by seven transmembrane spanning G-protein-coupled receptors leads to a change in accumulation of cAMP (3'5'-cyclic AMP). Different subtypes of galanin receptor and G-proteins could be manifested in the mode of inhibitory action of galanin receptor on the production of cAMP by adenylate cyclase. Galanin analogues, acting at the adenylate cyclase level as subtype-specific galanin antagonists, can selectively block the inhibitory effect of endogenous galanin and thereby have potential as therapeutic agents for several endocrine, neuroendocrine and neuronal disorders. In this review, the latest results in the field of interplay between galanin-initiated signal transduction and the cAMP pathway are summarized.

Adenylyl Cyclases↗
PubMed1998

Purification of a galanin degrading 70 kDa metallo-peptidase from bovine spinal cord.

Galanin is a 29/30 amino acids long neuroendocrine peptide, acting as an inhibitory modulator in the spinal cord. Several studies show that galanin is involved in control of pain threshold and acts synergistically with morphine, hence, inhibition of galanin degradation may be a pharmaceutical target for treatment of pain. In this study we have designed an 11 amino acids long substrate based on the first 10 N-terminal amino acids of galanin (this part contains the major pharmacophores of galanin), with a N-terminal fluorescent marker, anthranilic acid, and a C-terminal internal fluorescence quencher, 3-nitrotyrosine, coupled to the epsilon-amino group of the linker Lys11. Using this substrate to detect galanin degradation, we have purified a membrane bound galanin inactivating metallo-peptidase from bovine spinal cord. This enzyme, cleaving galanin between Trp2 and Thr3, is a novel 70 kDa, Zn2+ dependent metallo-peptidase.

Animals↗
PubMed1998

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Amino Acid Sequence↗
PubMed1998

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells: studies with chimeric and other peptides.

Structure-activity relationships for the binding of human alpha-calcitonin gene-related peptide 8-37 (halphaCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (halphaCGRP stimulation of adenylate cyclase). On SK-N-MC cells the potency order was halphaCGRP8-37 > halphaCGRP19-37 = AC187 > rat amylin8-37 > halpha[Tyr0]-CGRP28-37 (apparent pKBs of 7.49+/-0.25, 5.89+/-0.20, 6.18+/-0.19, 5.85+/-0.19 and 5.25+/-0.07). The SK-N-MC receptor appeared CGRP1-like. On Col 29 cells, only halphaCGRP8-37 of the above compounds was able to antagonize the actions of halphaCGRP (apparent pKB=6.48+/-0.28). Its receptor appeared CGRP2-like. halpha[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal alpha-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than halphaCGRP8-37. On SK-N-MC cells, halphaCGRP8-18,28-37 (M433) and mastoparan-halphaCGRP28-37 (M432) had apparent pKBs of 6.64+/-0.16 and 6.42+/-0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. M433 was almost as potent as halphaCGRP8-37 on Col 29 cells (apparent pKB=6.17+/-0.20). Other antagonists were inactive.

Adenylyl Cyclases↗
PubMed1998

Cell penetration by transportan.

Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine. Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using N epsilon13-biotinyl-transportan. The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37 degrees, 4 degrees, and 0 degrees C. Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable. The kinetics of internalization was studied with the aid of the 125I-labeled peptide. At 37 degrees C, the maximal intracellular concentration is reached in about 20 min. The internalized transportan is protected from trypsin. The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide. In Bowes' melanoma cells, transportan first localizes in the outer membrane and cytoplasmatic membrane structures. This is followed by a redistribution into the nuclear membrane and uptake into the nuclei where transportan concentrates in distinct substructures, probably the nucleoli.

1-Octanol↗
PubMed1998

Novel galanin receptor ligands.

Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G-protein-coupled central nervous system receptors via its N-terminus. We synthesised several galanin receptor ligands and fragments around C-terminal extensions of galanin(1-13) to yield chimeric peptides with C-terminals corresponding to bioactive peptides like bradykinin(2-9), mastoparan, neuropeptide Y(25-36) or substance P(5-11), respectively. We also synthesised short galanin analogs in which galanin(1-13) was C-terminally elongated with Lys14; different pharmacologically active small molecules were then attached to the epsilon-amino group of Lys14. Several cysteine-substituted linear and ring closed analogs of galanin(1-9) and galanin(1-16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure-activity relationship analysis of peptide-type ligands to galanin receptors.

Amino Acid Sequence↗
PubMed1998

Co-localized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission.

We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABA(B) receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (-ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABA(B) agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABA(B) antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.

Animals↗
PubMed1997

Mutagenesis and ligand modification studies on galanin binding to its GTP-binding-protein-coupled receptor GalR1.

In this study, a large number of receptor mutants were generated and several N-terminally modified galanin analogues synthesized to refine the previously proposed binding site model for galanin to its GTP-binding-protein-coupled receptor GalR1. In addition to ligand-binding studies, the functionality of mutant receptors was evaluated by assessing their ability to mediate galaninergic inhibition of isoproterenol-stimulated adenylyl cyclase activity. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remain functional albeit 20-fold less efficient than the wild-type receptor in mediating inhibition of stimulated cAMP production by galanin. The His267Ala mutant is, apart from being deficient in galanin binding, also severely impaired in functional coupling. While His264 and Phe282 seem to be important in forming the binding pocket for galanin, His267 might play a role in forming or stabilizing the active conformation of the GalR1 receptor rather than directly participating in the formation of the binding pocket for galanin. N-terminal carboxylic acid analogues of galanin have low affinity to wild-type GalR1, but substantially increased affinity to the Glu271Lys receptor mutant. This, together with the finding that an alanine substitution of Phe115 in TM III results in a tenfold decrease in affinity for galanin, suggests that the N-terminus of galanin interacts with Phe115. In contrast to the Phe282Ala mutation in TM VII, a conservative mutation of Phe282 to tyrosine did not alter the affinity for galanin. Thus, the interaction between Tyr9 of galanin and Phe282 is likely to be of an aromatic-aromatic nature.

Adenylyl Cyclases↗
PubMed1997

Galparan induces in vivo acetylcholine release in the frontal cortex.

The chimeric peptide galparan (galanin(1-13)-mastoparan) induced the in vivo release of acetylcholine in the frontal cortex of rats when injected intracerebroventricularly, i.c.v. The ACh-releasing effects of galparan are reversible, dose-dependent, and not exerted at galanin receptors or at sites where mastoparan acts. Pertussis toxin pretreatment (i.c.v.) of the rats for 96 h prior to injection of galparan or of mastoparan completely prevented the ACh-releasing effects of both galparan and mastoparan. It appears that galparan acts at a novel site in the release of ACh in the cerebral cortex in vivo.

Acetylcholine↗
PubMed1997

Conformational analysis of galanin using end to end distance distribution observed by Förster resonance energy transfer.

The structural dynamics of the flexible neuropeptide galanin in solution were studied by Förster resonance energy transfer measurements at different temperatures by time-resolved fluorescence spectroscopy to determine its conformational heterogeneity. Endogenous tryptophan at position 2 acted as the fluorescent donor and the non fluorescent acceptor dinitrophenyl or the fluorescent acceptor dansyl were selectively attached to lysine 25 in porcine galanin. The coexistence of different structures of the neuropeptide galanin in trifluoroethanol solution was revealed by the model independent analysis of the distribution of relaxation times from the time-resolved resonance energy transfer data. Multiple conformational states are reflected by distinct end-to-end distance populations. The conformations differ in mean donor-acceptor distance by about 15, and are consistent with the extended and folded backbone conformations of two alpha-helical regions separated by a flexible hinge. The effect that the labelling of galanin has on binding to the receptor was also evaluated. DNP-galanin showed the same high affinity to galanin receptors as unlabelled galanin, whereas DNS-galanin had significantly reduced affinity.

Chemical Phenomena↗
PubMed1997

Chimeric strategies for the rational design of bioactive analogs of small peptide hormones.

The aim of this study was to evaluate a variety of synthetic strategies pertinent to the development of chimeric analogs of the structurally divergent nonapeptide hormones arginine vasopressin (AVP) and bradykinin (BK). Single-chain peptides combining AVP and BK directly, AVP(1-9)-BK(1-9) or via a flexible aminohexanoic acid (epsilonAhx) linker, AVP(1-9)-epsilonAhx-BK(1-9), bind with relatively high affinity to the bovine kidney medulla B2a bradykinin receptor (B2a BKR). Significantly, amino-terminal extended chimeric analogs of BK, including AVP(1-9)-BK(1-9) and galanin(1-13)-BK(1-9), are functional B2 BKR agonists. These findings illustrate that chimeric peptides can activate G-protein-coupled receptors (GPCRs) in a manner analogous to that of endogenous monomeric agonists. Further development, combining the sequences of receptor subtype-selective antagonists, produced high-affinity chimeric antagonists of the V1a vasopressin receptor (V1a VPR) and the B2a BKR. We also determined the pharmacological characteristics of high-affinity chimeric hormone analogs derivatized with the membrane targeting function of mastoparan. Homodimers of an amino-terminal extended BK analog and a V1a-selective antagonist represent the first examples of new classes of B2 BKR and V1a VPR antagonists, respectively. These findings are discussed in relation to the GPCR binding site for small peptides and the development of novel biological probes and therapeutic agents.

Amino Acid Sequence↗
PubMed1997

L-Ala-substituted rat galanin analogs distinguish between hypothalamic and jejunal galanin receptor subtypes.

In order to explore which amino acids or which blocks of amino acids in the 29 amino acid neuropeptide galanin are important for recognition of the endogenous ligand by galanin receptor subtypes present in the jejunum and in the hypothalamus, respectively, we have carried out L-Ala substitutions of individual amino acids or of blocks of amino acids in the rat galanin sequence and examined the binding of the obtained analogs to the rat hypothalamic and jejunal galanin receptor subtypes. This study reveals that the galanin sequence YLLGPH9-14 is essential for recognition of galanin by both the rat hypothalamic and jejunal galanin receptor subtypes. Substitution of the N-terminal amino acids. GWTL1-4, leads to total loss of affinity of galanin for both hypothalamic and jejunal galanin receptors. The alpha-helical C-terminal amino acid (25-29) part of galanin has no greater influence on the affinity of galanin to the hypothalamic galanin receptor subtype. L-Ala substitution of the C-terminal amino acids of galanin KHGLT25-29 shows, however, that this C-terminal motif is essential for the recognition by the jejunal galanin receptor subtype, whereas amino acids in the middle portion of galanin NSAG5-8 are of importance for binding to the hypothalamic but not to the jejunal receptor. [Ala5-8]Galanin thus has a more than 100-fold higher affinity to jejunal receptor than to the hypothalamic receptor, while [Ala25-29]galanin has a more than 100-fold higher affinity for the hypothalamic than for jejunal galanin receptor subtypes. pH dependence of the galanin binding to these receptor subtypes is also different.

Alanine↗