Norepinephrine Drives Persistent Activity in Prefrontal Cortex via Synergistic α1 and α2 Adrenoceptors

Optimal norepinephrine levels in the prefrontal cortex (PFC) increase delay-related firing and enhance working memory, whereas stress-related or pathologically high levels of norepinephrine are believed to inhibit working memory via α1 adrenoceptors. However, it has been shown that activation of Gq-coupled and phospholipase C-linked receptors can induce persistent firing, a cellular correlate of working memory, in cortical pyramidal neurons. Therefore, despite its importance in stress and cognition, the exact role of norepinephrine in modulating PFC activity remains elusive. Using electrophysiology and optogenetics, we report here that norepinephrine induces persistent firing in pyramidal neurons of the PFC independent of recurrent fast synaptic excitation. This persistent excitatory effect involves presynaptic α1 adrenoceptors facilitating glutamate release and subsequent activation of postsynaptic mGluR5 receptors, and is enhanced by postsynaptic α2 adrenoceptors inhibiting HCN channel activity. Activation of α2 adrenoceptors or inhibition of HCN channels also enhances cholinergic persistent responses in pyramidal neurons, providing a mechanism of crosstalk between noradrenergic and cholinergic inputs. The present study describes a novel cellular basis for the noradrenergic control of cortical information processing and supports a synergistic combination of intrinsic and network mechanisms for the expression of mnemonic properties in pyramidal neurons.     

Transport of α-Ketoisocaproate in Neuroblastoma NB-2a Cells

Transport of α-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free ketoacid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and β-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, α-cyano-4-hydroxycinnamate, 4,4′-diisothiocyano-2,2′-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na⁺].     

Rat α2u-globulin mRNA expression in the preputial gland

Rat _2u-globulin and the mouse major urinary proteins (MUP) are encoded by homologous multigene families whose members exhibit diverse tissue-specific, developmental, and hormonal controls of expression. Although their patterns of expression and hormonal control appear to be very similar in many respects, we have found high levels of _2u-globulin mRNA in rat preputial glands, whereas MUP mRNA could not be detected in the male mouse preputial gland. Male and female rat preputial have similar concentrations of _2u-globulin mRNA, suggesting an absence of endocrine regulation as occurs in the liver and lachrymal glands. Two-dimensional polyacrylamide gel electrophoresis of proteins encoded by hybrid-selected _2u-globulin mRNA indicates that the liver and lachrymal translation products have different mobilities. However, many of the preputial gland products comigrate with most or all of the liver and lachrymal products. Among the possibilities suggested by these results is that _2u-globulin genes expressed in liver and lachrymal glands under endocrine control are also expressed constitutively in the preputial gland.This work was supported by Public Health Service Research Grant GM25023 from the National Institutes of Health.     

Autoradiographic demonstration of α2 adrenoceptors in the bovine neurohypophysis

Summary Autoradiography of sections from neurohypophyses treated with tritiated clonidine has shown specific binding to ₂-adrenoceptor sites in the neurohypophysis but not in the intermediate lobe. Other studies have shown that ₂ agonists such as clonidine can cause a fall in circulating antidiuretic hormone; it is therefore possible to speculate that this action could be a direct one on the neurohypophysis since the appropriate binding sites have been shown to exist.     

Immunohistochemical localization of human α2macroglobulin in connective tissue

Summary The localization of ₂ macroglobulin (₂M) has been examined by an indirect immunofluorescent technique in frozen sections of various human tissues. The results indicate that ₂M is present only in connective tissues and blood. The outer medulla of the kidney and the submucosa of the gut showed the strongest reaction. Epithelia or endothelial cells were unreactive. In liver, only the Kupffer cells were stained. These results were confirmed with the immunoperoxidase technique and by the study of tissue extracts in crossed immunoelectrophoresis (CIE). As a positive control a polyspecific antiserum prepared against whole human fibroblasts as well as anti-albumin were used. Our findings are interpreted in the light of the observations that ₂M is synthesized and selectively ingested by fibroblasts.Abbreviation used in this Paper ₂M ₂macroglobulin This work was supported by a Grant from the Belgian Cancer Fund (Algemene Spaar en Lijfrente Kas), by Grant 3.0043.79 (Fonds voor Geneeskundig Wetenschappelijk Onderzoek) and by Research Fund OT/VII/30 (K.U. Leuven)F. Van Leuven is a Post Doctoral Research Fellow of the American Cystic Fibrosis Foundation     

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.     

Changes in temperature have opposing effects on current amplitude in α7 and α4β2 nicotinic acetylcholine receptors

We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (nAChR) subtypes: the rapidly desensitizing homomeric α7 nAChR, the more slowly desensitizing heteromeric α4β2 nAChR and on α7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in Xenopus oocytes at room temperature (RT; 21°C) were compared to those recorded at either physiological temperature (37°C) or at lower temperature (4°C). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with α4β2 nAChRs was increased at high temperature (125±9%, n = 6, P<0.01) and reduced at low temperature (47±5%, n = 6, P<0.01), whereas the amplitude of α7 responses was reduced at high temperature (27±7%, n = 11, P<0.001) and increased at low temperatures (224±16%, n = 10, P<0.001). In contrast to the effects of temperature on α4β2 and wild type α7 nAChRs, the amplitude of α7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when α7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonist-evoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of receptor desensitization on all subtypes examined. In all cases, higher temperature resulted in increased rates of desensitization. Thus, it appears that the differing effects of temperature on the amplitudes of whole-cell responses cannot be explained by temperature-induced changes in receptor desensitization rates.     

Genetic polymorphism of αs1- and αs2-caseins in Hungarian Milking Goats

The aim of this study was to perform an initial characterization of milk quality and to determine genetic polymorphism at the CSN1S1 and CSN1S2 locus in two herds of local dairy goats (Hungarian Milking). The fat, protein and lactose level of milk samples in Hungarian Milking Goats were compared to other local goat breeds worldwide and it was concluded that the mean milk production of the Hungarian goats should be improved. The presence of the A, B, C + D, E, F and O alleles of CSN1S1 locus and A + B + C + E, D, F and O alleles of CSN1S2 locus were genotyped for by PCR-AS and PCR-RFLP methods in 103 goats. The strong B allele of CSN1S1 is more frequent in the local Hungarian Milking than in the imported Alpine and Saanen goats. The relatively high incidence of the O allele of CSN1S2 gene is also characteristic for the Hungarian Milking Goats and those special allele distribution patterns could be used to develop selection strategies to breed specialised lines of Hungarian local breeds.     

Relationship between α-L-fucosidase deficiency in plasma and α-L-fucosidase activity in leukocytes

Summary After testing a population sample of 185 hospitalized Italian children for the plasma -L-fucosidase deficiency and establishing an approximate threshold value between heterozygotes and wild-type homozygotes, we analyzed by two statistical methods the distribution of the two genotypes. The results obtained by probit analysis agree with threshold and average values expected on the basis of the Hardy-Weinberg equilibrium.In addition, the level of -fucosidase in leukocytes of 12 individuals with deficiency of -fucosidase in plasma was found to be significantly lower than that of 61 controls (P<0.005). These results indicate that the mutation(s) causing a deficiency of -fucosidase in plasma is (are) also expressed in leukocytes.     

Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

PLK2 Modulates α-Synuclein Aggregation in Yeast and Mammalian Cells

Phosphorylation of α-synuclein (aSyn) on serine 129 is one of the major post-translation modifications found in Lewy bodies, the typical pathological hallmark of Parkinson’s disease. Here, we found that both PLK2 and PLK3 phosphorylate aSyn on serine 129 in yeast. However, only PLK2 increased aSyn cytotoxicity and the percentage of cells presenting cytoplasmic foci. Consistently, in mammalian cells, PLK2 induced aSyn phosphorylation on serine 129 and induced an increase in the size of the inclusions. Our study supports a role for PLK2 in the generation of aSyn inclusions by a mechanism that does not depend directly on serine 129 phosphorylation.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

Both α1- and α2-adrenoceptors in the Insular Cortex Are Involved in the Cardiovascular Responses to Acute Restraint Stress in Rats

The insular cortex (IC) is a limbic structure involved in cardiovascular responses observed during aversive threats. However, the specific neurotransmitter mediating IC control of cardiovascular adjustments to stress is yet unknown. Therefore, in the present study we investigated the role of local IC adrenoceptors in the cardiovascular responses elicited by acute restraint stress in rats. Bilateral microinjection of different doses (0.3, 5, 10 and 15 nmol/100 nl) of the selective α₁-adrenoceptor antagonist WB4101 into the IC reduced both the arterial pressure and heart rate increases elicited by restraint stress. However, local IC treatment with different doses (0.3, 5, 10 and 15 nmol/100 nl) of the selective α₂-adrenoceptor antagonist RX821002 reduced restraint-evoked tachycardia without affecting the pressor response. The present findings are the first direct evidence showing the involvement of IC adrenoceptors in cardiovascular adjustments observed during aversive threats. Our findings indicate that IC noradrenergic neurotransmission acting through activation of both α₁- and α₂-adrenoceptors has a facilitatory influence on pressor response to acute restraint stress. Moreover, IC α₁-adrenoceptors also play a facilitatory role on restraint-evoked tachycardiac response.     

Lrrk2 interaction with α-synuclein in diffuse Lewy body disease

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson’s disease (PD) and its more severe variant diffuse Lewy body disease (DLB). Pathological mutations in Lrrk2 are autosomal dominant, suggesting a gain of function. Mutations in α-synuclein also produce autosomal dominant disease. Here we report an interaction between Lrrk2 and α-synuclein in a series of diffuse Lewy body (DLB) cases and in an oxidative stress cell based assay. All five cases of DLB, but none of five controls, showed co-immunoprecipitation of Lrrk2 and α-synuclein in soluble brain extracts. Colocalization was also found in pathological deposits in DLB postmortem brains by double immunostaining. In HEK cells transfected simultaneously with plasmids expressing Lrrk2 and α-synuclein, co-immunoprecipitation of Lrrk2 and α-synuclein was detected when they were exposed to oxidative stress by H₂O₂. Taken together, these results suggest the possibility that in PD and related synucleinopathies, oxidative stress upregulates α-syn and Lrrk2 expression, paving the way for pathological interactions. New therapeutic approaches to PD and the synucleinopathies may result from limiting the interaction between Lrrk2 and α-synuclein.     

Seed Storage Glutelin α-2 Subunit Lacking Mutants in Rice

Nine rice Oryza sativa L.） mutant lines lacking the seed storage glutelin α-2 subunit were obtained from the progenies of fertilized egg cells treated with N-methy-N-nitrosourea (MNU). The mutants could be classified into three types: the α-1 subunit increased type (α-1H/α-2L), decreased the β-2 subunit decreased type (β-2L/α-2L) and the α-3 subunit increased type (α-3H/α-2L) according to their SDS-PAGE profiles. Two-dimensional electrophoresis analysis revealed that all of the mutants lacked a polypeptide of pI 6.71/α-2, while new polypeptides of pI 6.50/α-1 and pI 6.90/α-3 formed in α-1H/α-2L and α-3H/α-2L mutants respectively. Although the β-2L/α-2L mutants did not form new polypeptide, their pI 8.74/β-2 polypeptide was also decreased, suggesting that the two polypeptides decreased in β-2L/α-2L mutants might derive from the same glutelin precursor. These mutant lines are very useful in studying genetic characterisation,the mechanism of genetic regulation on biosynthesis, gene function and proteomics of rice seed storage glutelin.     

Human α2macroglobulin

Summary Human ₂macroglobulin combines two unique features: the non-active site directed inhibition of virtually all endoproteases and the selective clearance of ₂M-endoprotease complexes by receptor-mediated endocytosis. To study the molecular details of the mechanisms involved, primary amines were found to be worthwhile probes at three specific levels: the inactivation of native ₂M, the derivatization by factor XIII and the cellular process of receptor-recycling. In this paper published data are supplemented with recently obtained evidence to discuss and speculate on the possible action or involvement of transglutaminase activities, indicated by the effects of the primary amines.     

Interaction of Insulin-Like Growth Factor-Binding Protein 2 with α2-Macroglobulin in the Circulation

Insulin-like growth factors (IGFs) play active role in mitogenic and metabolic processes. In the peripheral circulation, they are mostly bound to specific IGF-binding proteins (IGFBPs). Proteolysis of IGFBPs releases free, active IGFs. IGFBP-2 is the second most abundant of the six binding proteins and its concentration increases in catabolic states. The possible interaction between IGFBP-2 and other proteins in the circulation was investigated in this study. Our results showed that IGFBP-2 associates with α2-macroglobulin (α2M), a protease inhibitor. Formation of IGFBP-2/α2M complexes most likely contributes to the regulation of IGFBP-2 proteolysis and, thus, the activity of IGFs.     

Actions of terazosin and its enantiomers at subtypes of α- and α2-Adrenoceptors in vitro

Abstract

Terazosin and its enantiomers, antagonists of α1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of α1- and α2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of α1-adrenoceptors with K values in the low nanomolar range, and showed potent antagonism of α1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each α1-adrenoceptor subtype. At α2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial α2B receptors. These agents were not significantly different in their potencies at α2a or α2A sites. Since the high affinity for α2B sites of quinazoline-type α-adrenoceptor antagonists has been used to differentiate α2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at α1 -adrenoceptor subtypes, the lower potency of R(+) terazosin at α2B receptors indicates a somewhat greater selectivity for α1- compared to α2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.     

Hydroxylysine in the N-terminal regions of the α1- and α2-chains of various collagens

The degree of hydroxylation of the lysine residue located in both alpha(1)- and alpha(2)-chains of collagen in the N-terminal, non-helical telopeptide region of the molecule has been determined in collagen from various sources after isolation of the peptides (alpha(1)- and alpha(2)-CB1) that contain the lysine residue in question and are obtained by cyanogen bromide cleavage of collagen alpha(1)- and alpha(2)-chains respectively. As with collagen from chick tibia, bone collagens from rat tibia and femur and embryonic chick frontal bone, have a high degree of hydroxylation (approx. 50% or more) of the lysine residue in both alpha(1)- and alpha(2)-CB1 peptides. This is in contrast with the lack of hydroxylation of this residue in both alpha(1)- and alpha(2)-chains of all skin collagens so far examined. The presence of hydroxylysine in alpha(1)- and alpha(2)-CB1 peptides from tendon collagen is also indicated. In rat tail tendon collagen the amount of hydroxylation is only slight but in the much less soluble tendon collagen from embryonic chick leg tendons, approximately one-third of the lysine is hydroxylated.     

Identification of α-dystroglycan binding sequences in the laminin α2 chain LG4–5 module

The biological activities of the laminin α2 chain LG4–5 module result from interactions with cell surface receptors, such as heparan sulfate proteoglycans and α-dystroglycan. In this study, heparin and α-dystroglycan binding sequences were identified using 42 overlapping synthetic peptides from the LG4–5 module and using recombinant LG4–5 protein (rec-α2LG4–5). Physiological activities of the active peptides were also examined in explants of submandibular glands. Heparin binding screens showed that the A2G78 peptide (GLLFYMARINHA) bound to heparin and prevented its binding to rec-α2LG4–5. Furthermore, alanine substitution of the arginine residue in the A2G78 site on rec-α2LG4–5 decreased heparin binding activity. When α-dystroglycan binding of the peptides was screened, two peptides, A2G78 and A2G80 (VQLRNGFPYFSY), bound α-dystroglycan. A2G78 and A2G80 also inhibited α-dystroglycan binding of rec-α2LG4–5. A2G78 and A2G80 specifically inhibited end bud formation of submandibular glands in culture. These results suggest that the A2G78 and A2G80 sites play functional roles as heparan sulfate- and α-dystroglycan-binding sites in the module. These peptides are useful for elucidating molecular mechanisms of heparan sulfate- and/or α-dystroglycan-mediated biological functions of the laminin α2 chain.