[Perioperative stroke]. / Perioperativer Schlaganfall.

Perioperative stroke is defined as an ischemic cerebrovascular event that occurs during or within 30 days after surgery and is associated with an increased perioperative risk of morbidity and mortality. Depending on the type of surgery stroke is diagnosed in up to 11% of all patients in the perioperative period. Patients with a history of ischemic stroke or transitory ischemic attack have an increased risk for perioperative stroke. Therefore, a critical assessment of indications and the timing of surgery are crucial to prevent recurring stroke in this patient population. Importantly, individualized blood pressure management is essential for optimization of cerebral perfusion during the perioperative period.This article provides a summary of the epidemiology, risk factors, and etiology of perioperative stroke. Moreover, possible preventive strategies relevant for the anesthesiologist are reviewed.

Generation and phenotypic characterization of a galanin overexpressing mouse.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin.

The neuropeptide galanin has been shown to suppress epileptic seizures. In cortical and hippocampal areas, galanin is normally mainly expressed in noradrenergic afferents. We have generated a mouse overexpressing galanin in neurons under the platelet-derived growth factor B promoter. RIA and HPLC analysis revealed up to 8-fold higher levels of galanin in transgenic as compared with wild-type mice. Ectopic galanin overexpression was detected especially in dentate granule cells and hippocampal and cortical pyramidal neurons. Galanin-overexpressing mice showed retardation of seizure generalization during hippocampal kindling, a model for human complex partial epilepsy. The high levels of galanin in mossy fibers found in the transgenic mice were further increased after seizures. Frequency facilitation of field excitatory postsynaptic potentials, a form of short-term synaptic plasticity assessed in hippocampal slices, was reduced in mossy fiber-CA3 cell synapses of galanin-overexpressing mice, indicating suppressed glutamate release. This effect was reversed by application of the putative galanin receptor antagonist M35. These data provide evidence that ectopically overexpressed galanin can be released and dampen the development of epilepsy by means of receptor-mediated action, at least partly by reducing glutamate release from mossy fibers.

Characterisation of a new chimeric ligand for galanin receptors: galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide.

In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR1<1 nM and at hGalR2<10 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.

Mice over-expressing galanin have elevated heat nociceptive threshold.

The neuropeptide galanin may have a role in modulation of nociceptive input at spinal level. Here we report that mice over-expressing galanin exhibit significant elevation of nociceptive threshold to thermal stimulation in comparison to wild-type mice as assessed by the tail flick and paw heat irradiation tests. No change in response to mechanical or cold stimulation was seen. The elevated heat nociceptive threshold in the galanin over-expressing mice was reversed by intrathecal application of the putative galanin receptor antagonist M-35, galanin-(1-12)-pro-bradykinin-(2-9). The results thus support that galanin has an inhibitory function in rodent spinal cord.

Galanin: neurobiologic mechanisms and therapeutic potential for Alzheimer's disease.

The neuropeptide galanin (GAL) is widely distributed in the mammalian CNS. Several lines of evidence suggest that GAL may play a critical role in cognitive processes such as memory and attention through an inhibitory modulation of cholinergic basal forebrain activity. Furthermore, GAL fibers hyperinnervate remaining cholinergic basal forebrain neurons in Alzheimer's disease (AD). This suggests that GAL activity impacts cholinergic dysfunction in advanced AD. Pharmacological and in vitro autoradiographic studies indicate the presence of heterogeneous populations of GAL receptor (GALR) sites in the basal forebrain which bind GAL with both high and low affinity. Interestingly, we have recently observed that GALR binding sites increase in the anterior basal forebrain in late-stage AD. Three G protein-coupled GALRs have been identified to date that signal through a diverse array of effector pathways in vitro, including adenylyl cyclase inhibition and phospholipase C activation. The repertoire and distribution of GALR expression in the basal forebrain remains unknown, as does the nature of GAL and GALR plasticity in the AD basal forebrain. Recently, GAL knockout and overexpressing transgenic mice have been generated to facilitate our understanding of GAL activity in basal forebrain function. GAL knockout mice result in fewer cholinergic basal forebrain neurons and memory deficits. On the other hand, mice overexpressing GAL display hyperinnervation of basal forebrain and memory deficits. These data highlight the need to explore further the putative mechanisms by which GAL signaling might be beneficial or deleterious for cholinergic cell survival and activity within basal forebrain. This information will be critical to understanding whether pharmacological manipulation of GALRs would be effective for the amelioration of cognitive deficits in AD.

Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons.

The neuropeptide galanin colocalizes with choline acetyltransferase, the synthetic enzyme for acetylcholine, in a subset of cholinergic neurons in the basal forebrain of rodents. Chronic intracerebroventricular infusion of nerve growth factor induces a 3- to 4-fold increase in galanin gene expression in these neurons. Here we report the loss of a third of cholinergic neurons in the medial septum and vertical limb diagonal band of the basal forebrain of adult mice carrying a targeted loss-of-function mutation in the galanin gene. These deficits are associated with a 2-fold increase in the number of apoptotic cells in the forebrain at postnatal day seven. This loss is associated with marked age-dependent deficits in stimulated acetylcholine release, performance in the Morris water maze, and induction of long-term potentiation in the CA1 region of the hippocampus. These data provide unexpected evidence that galanin plays a trophic role to regulate the development and function of a subset of septohippocampal cholinergic neurons.

The cloned guinea pig pancreatic polypeptide receptor Y4 resembles more the human Y4 than does the rat Y4.

Pancreatic polypeptide (PP) is involved in gastrointestinal functions and forms, together with neuropeptide Y (NPY) and peptide YY (PYY), the PP-fold family of peptides. The PP-binding receptor subtype Y4 has so far been cloned in human, rat, and mouse, and displays extensive species differences regarding sequence, pharmacology, and distribution. To explore this variability further, we have cloned the Y4 receptor in the guinea pig, which is evolutionarily equally distantly related to both humans and rodents. The guinea pig Y4 receptor is 84% identical to the human Y4 receptor, but only 74-75% identical to the rat and mouse receptors. The two latter are 75-76% identical to human Y4. The guinea pig Y4 receptor bound 125I-hPP with a dissociation constant (Kd) of 29+/-3 pM. The pharmacological profile of guinea pig Y4 has the following rank order of potencies: PP > NPY approximately = PYY approximately = LP-NPY approximately = LP-PYY > NPY2-36 >> [D-Trp32]NPY. Thus, the guinea pig receptor is more similar to the human Y4 than to the rat Y4 both in sequence and pharmacology. This agrees with the greater identity between guinea pig and human PP compared to rat PP. These comparisons suggest that the rodent PPs and Y4 receptors have an accelerated replacement rate.

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.

Mutagenesis study on human galanin receptor GalR1 reveals domains involved in ligand binding.

Many receptor mutants were generated and several NH2-terminally modified galanin analogs synthesized to define the regions of hGalR1 involved in galanin binding. Ligand binding properties and functionality of mutant receptors were evaluated. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remained functional albeit at least 20-fold less efficient than the wild-type receptor in the inhibition of stimulated cAMP production. Hence, His264 and Phe282 of hGalR1 are directly involved in galanin binding. NH2-terminal carboxylic acid analogs of galanin (1-16) have a very low affinity for the wild-type receptor, but substantially increased affinity for the Glu271Lys-hGalR1, suggesting that the NH2-terminus of galanin binds to the receptor near the transmembrane (TM) VI. Based on these findings and computer-aided molecular modeling, we propose a binding site model for the hGalR1 receptor (possibly also for other galanin receptor subtypes): galanin binds with its NH2-terminus to the pocket between TM III and TM VI, Trp2 of galanin interacts with His264 of the receptor, and Tyr9 is involved in an aromatic-aromatic type of interaction with Phe282 of ECIII of GalR1.

Galanin expression within the basal forebrain in Alzheimer's disease. Comments on therapeutic potential.

The inhibitory neuropeptide galanin has widespread distribution throughout the central nervous system. Studies indicate that galanin modulates cognition by regulating cholinergic basal forebrain (CBF) neuron function. The chemoanatomic organization of galanin within the mammalian CBF differs across species. In monkeys, all CBF neurons coexpress galanin, whereas in apes and humans galanin is found within a separate population of interneurons that are in close apposition to the CBF perikarya. Pharmacologic investigations revealed a low and high affinity galanin receptor within the basal forebrain in humans. In vitro autoradiographic investigations of the primate brain indicate that galanin receptors are concentrated within the anterior subfields of the CBF as well as bed nucleus of the stria terminalis, amygdala, and entorhinal cortex. Galaninergic fibers hyperinnervate remaining CBF neurons in Alzheimer's disease. Because galanin inhibits the release of acetylcholine in the hippocampus, it has been suggested that the overexpression of galanin in Alzheimer's disease may downregulate the production of acetylcholine within CBF perikarya, further exacerbating cholinergic cellular dysfunction in this disorder. These observations suggest that the development of a potent galanin antagonist would be a useful step towards the successful pharmacologic treatment of Alzheimer's disease.

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.

Ligand binding and functional effects of systematic double D-amino acid residue substituted neuropeptide Y analogs on Y1 and Y2 receptor types.

In order to identify the signal epitopes of the neuropeptide Y (NPY) molecule, the conformation of the NPY molecule was pertubated by a systematic double D-amino acid replacement of neighbouring residues. These NPY-analogs were examined for receptor affinity and on biological activity. The rat cerebral cortex and hippocampus were used for binding characteristics on Y1 and Y2 binding sites, respectively, while the isolated guinea pig caval vein and rat vas deferens were used in functional characterization of Y1 and Y2 receptors, respectively. The NPY analogs were examined as ligands at [3H]NPY binding sites in homogenates of the rat brain. Pairwise D-substitutions of either of the first 6 amino acid residues in the N-terminal part of the molecule resulted in a 20-100-fold loss of affinity for Y1 binding sites compared with the native peptide. In comparison, the same analogs displayed affinities, which were about 8-40 times lower than NPY itself at Y2 binding sites. Especially [D-Ser3,D-Lys4]NPY had a low affinity to Y1 and Y2 binding sites. For many of the pairwise D-amino acid substituted NPY analogs, there were similar affinities for Y1 and Y2 binding sites in the cerebral cortex and hippocampus, respectively. D-Amino acid residue substitutions in positions 7 and 8 did essentially not affect the affinity to either type of binding site, while such replacements in positions 19 and 20 resulted in a drastic loss of affinity to both types of NPY binding site. In contrast, [D-Tyr21,D-Ser22]NPY was only slightly less potent than NPY itself on either type of binding site. Pairwise D-amino acid substitutions in the C-terminal (positions 27 to 36) decreased the affinity to Y1 and Y2 binding sites by 2 to 3 orders of magnitude. In the guinea pig vena cava the D-amino acid substituted NPY analogs evoked a concentration-dependent contraction with an rank order of potency similar to that of the respective analog at Y1 binding sites in the cerebral cortex. Similarly, in the rat vas deferens the D-amino acid substituted NPY analogs evoked a concentration-dependent inhibition of the electrically-stimulated twitches with a rank order of potency similar to that of the respective analog at Y2 binding sites in the hippocampus. However, D-amino acid replacements in positions 25 and 26 resulted in an analog which was virtually inactive in the vas deferens, but almost equipotent with NPY in the vena cava. In conclusion, the present study has shown that N-terminal double D-amino acid substitutions in the NPY molecule reduced the binding affinity to and activation more of the Y1 receptor, than of the Y2 receptor, while both receptors were quite sensitive to double D-amino acid changes in positions 19 and 20 and in the C-terminal end of the NPY molecule.

Potential-dependent block of human delayed rectifier K+ channels by internal Na+.

The delayed rectifier K+ currents in differentiated human SH-SY5Y neuroblastoma cells were characterized with tight-seal recording techniques. Activation and inactivation parameters were measured. At high positive potentials, the current showed a marked rectification, causing a region of negative slope conductance in the current vs. potential curve. The rectification depended markedly on the pipette Na+ concentration. Without Na+, no rectification was observed, whereas with high Na+ (20-60 mM), a marked rectification was always observed. Tail current measurements showed a fast ( < 400 microseconds) block of K+ currents in the presence of internal Na+. With 60 mM Na+ in the pipette 8% of the K+ current was blocked at 0 mV, 27% at +20 mV, and 82% at +100 mV. Similar degrees of block were often seen with 30 mM Na+ in the pipette. The submembrane Na+ concentration in intact cells was estimated, on the basis of the reversal of Na+ current, to be approximately 15 mM. Single-channel K+ currents, in the cell-attached configuration, showed a conductance of approximately 20 pS at 40-60 mV above rest but showed rectification at high potentials.

Delineation of the peptide binding site of the human galanin receptor.

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th

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.

Functional effects and ligand binding of chimeric galanin-neuropeptide Y (NPY) peptides on NPY and galanin receptor types.

1. The effects and binding characteristics of a series of chimeric galanin-neuropeptide Y (NPY) peptides were examined in various preparations known to contain a predominant population of either Y1 or Y2 receptors for NPY or galanin receptors. 2. NPY suppressed the electrically stimulated twitches of the rat vas deferens (Y2 receptors), while galanin enhanced the electrically stimulated twitches. The galanin-NPY peptides M 32 (galanin(1-13)-NPY(25-36)), M69A (galanin(1-13)-Lys-[epsilon NH-Gly-NPY(4-1)]NPY(25-36)) and M88 (galanin(1-12)-Ala-NPY(25-36)) evoked a concentration-dependent suppression of the electrically stimulated twitches. These chimeric peptides were about equipotent with NPY, while NPY (13-36) was about five times less potent than NPY itself. Also a stochiometric combination of the N- and C-terminal fragments NPY (1-24)NH2 and NPY (25-36) (each at 1 microM) was inactive in vas deferens. M120 (galanin (1-13)-NPY(14-36) (1 microM) did not affect the NPY-mediated suppression of the stimulated twitches. 3. NPY evoked a concentration-dependent contraction in the guinea-pig isolated caval vein (Y1 receptors), while galanin (< or = 1 microM) was inactive. M32, M69A and M88 induced a slight contraction at very high concentrations only (> or = 0.3 M), while M120 was inactive at 1 microM. None of the four chimeric peptides affected the contraction evoked by NPY. 4. Since the number of NPY receptors in the rat vas deferens and guinea-pig caval vein were too low,the affinities of the galanin-NPY peptides for [3H]-NPY binding sites were examined in membranes from rat brain areas known to contain predominant populations of Y1 receptors (cerebral cortex) and Y2 receptors (hippocampus), respectively. The chimeric peptides M32, M69A, M88, M120 and NPY (13-36)all had higher affinities for hippocampal binding sites than for cerebral cortical binding sites. These peptides were 90-440 times less potent than NPY at cerebral cortical binding sites and 15-125 times less potent than NPY at hippocampal binding sites. The most selective chimeric peptide was M32, which had a 20 fold higher affinity for hippocampal than for cerebral cortical binding sites.5. At hypothalamic [125I]-galanin binding sites M32, M88 and M69A were equipotent with galanin,while M120 was about 10 times less potent than galanin. M32, M88 and M69A, like galanin contracted the rat isolated jejunum.6. The N-terminal portion (1-12) of galanin seems to permit a steric conformation of the attached NPY (25-36) part of the chimeric galanin-NPY peptides, which results in a facilitated Y2 but not Y1.receptor recognition and activation. None of the galanin-NPY peptides appeared to act as antagonists at either type of NPY receptor, probably due to their low affinity. Instead, they displayed a very high affinity for hypothalamic galanin receptors and probably act as galanin agonists in the rat jejunum.

Inter- and intraspecific variation of the nucleotide sequence of the cytochrome b gene in Cory's (Calonectris diomedea), Manx Shearwater (Puffinus puffinus) and the Fulmar (Fulmarus glacialis).

The cytochrome b gene of three European taxa of the family of Procellariidae was amplified from total DNA and sequenced. The sequence comparison shows that the Fulmar (Fulmarus glacialis) is significantly distinct from shearwaters, whereas Cory's (Calonectris diomedea) and Manx Shearwater (Puffinus puffinus) are closely related. Although the populations of C. diomedea can be distinguished morphologically, the sequences of cyt b differ only slightly between the Atlantic and Mediterranean subspecies (i.e. C. d. borealis versus C. d. diomedea) and do not reveal other population differences within subspecies.