The neuronal α6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells

We examine some of the biological and physiological properties of the avian α₆ neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α₆ mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as α₃, β₂ and β₄. In contrast, α₆ mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the α₆ subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca²⁺ transients in human BOSC 23 cells transfected with chick α₆ in combination with other chick nAChR neuronal subunits. Heterologously expressed α₆ and β₄ subunits form functional heteromeric nAChRs, which are permeable to Ca²⁺ ions and blocked by the nicotinic antagonist methyllycaconitine (10 μm ). Likewise, ACh elicits measurable currents in cells transfected with α₆ and β₂. Hill analysis of the dose–response curves in cells transfected with α₃, β₄ and α₆ cDNAs, suggests the assembly of functional α₃β₄α₆ receptor, with an apparent affinity for ACh threefold lower than α₃β₄. Our results indicate that α₆-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.     

Changes in the ratio of urinary α1-microglobulin to ulinastatin levels in patients with Alzheimer-type dementia and vascular dementia

Abstract Relationships between urinary levels of α1-microglobulin (α1M) and ulinastatin (UT) in patients with dementia were investigated. There were no significant differences in α1M and UT levels and α1M: UT ratios among three groups: age-matched control subjects, patients with either Alzheimer-type senile dementia (ATD) or vascular dementia (VD). Although a positive correlation was established between α1M and UT levels in these groups, the regression of the demented patients differed significantly from that of controls ( P <0.05). A tendency towards a negative correlation between α1M: UT ratios and the levels of severity or duration of the disease was displayed in the ATD group, whereas a tendency toward a positive correlation between α1M: UT ratios and the levels of severity was observed in the VD group. These results suggest that changes in the relationships between urinary levels of α1M and UT may provide a useful biochemical index for diagnoses of ATD and VD.     

Stimulation of α1-adrenoceptors inhibits memory consolidation in the chick

Investigation of the effects of the different adrenoceptor (AR) subtypes in memory formation may reveal discrete actions of noradrenaline in memory modulation and storage mediated through particular AR subtypes. Noradrenaline injected intracerebrally in the chick produced biphasic effects on memory consolidation with enhancement at low doses and inhibition at high doses. We have previously shown that the enhancement by the lower doses of noradrenaline is attributable to actions at beta2- and beta3-adrenoceptors, whereas the inhibitory effect of higher doses is attributable to alpha1-adrenoceptors. The present studies show that the inhibition of memory by high doses of noradrenaline is mimicked by the alpha1-AR agonist methoxamine, and the dose-response curve is shifted to the right by pretreatment with the alpha1-AR antagonist prazosin. alpha1-ARs may play a critical role in memory formation in highly stressful situations, when noradrenaline levels are high in particular brain regions. It is not known where the alpha1-ARs responsible for the effect on memory are localized. alpha1-ARs are found on neurons and astrocytes and in the cerebral vasculature and therefore the action of high doses of noradrenaline via alpha1-AR agonists could be via an action at any of these sites. Activation of alpha1-adrenoceptors in the intermediate hyperstriatum ventrale in the chick forebrain by the alpha1 adrenoceptor agonist methoxamine inhibits the consolidation of memory. Because the same effect is produced by high levels of noradrenaline, it is likely that stimulation of alpha1-ARs is the mechanism underlying this effect.     

Calcium Signalling in Mouse Bergmann Glial Cells Mediated by α1-adrenoreceptors and H1 Histamine - Receptors

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20–25 days was determined using fura-2 Ca²⁺ microfluorimetry. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i), either individual cells were loaded with the Ca²⁺-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane-permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 μM) and histamine (10-100 μM) triggered a transient increase in [Ca²⁺]_i. The involvement of the α₁-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca²⁺]_i responses were blocked by the selective α¹-adreno-antagonist prazosin and were mimicked by the α¹-adreno-agonist phenylephrine. The monoamine-induced [Ca²⁺]_i signals were not affected by β- and α₂-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β- and α₂-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca²⁺]_i responses demonstrated specific sensitivity to only H₁ histamine receptor modulators. [Ca²⁺]_i responses to monoamines and histamine did not require the presence of extracellular Ca²⁺ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca²⁺]_i increase is due to release from intracellular pools. No [Ca²⁺]_i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with α₁ -adrenoreceptors and H₁ histamine receptors which induce the generation of intracellular [Ca²⁺]_i signals via activation of Ca²⁺ release from inositol-l,4,5-trisphosphate-sensitive intracellular stores.     

Linkage analysis between idiopathic generalized epilepsies and the GABAA receptor α5, β3 and γ3 subunit gene cluster on chromosome 15

Introduction - We tested the hypothesis that genetic variants within the GABA_Aα₅, β₃ and γ₃ subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). Material and methods - Ninety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA_Aα₅, β₃ and γ₃ subunit gene cluster and three different IGE trait models. Results - Evidence against linkage to the GABA_Aα₅, β₃ and γ₃ subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Z_max=1.40 at θ_max=0.00) was obtained for a broad IGE spectrum ("idiopathic" generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (α=0.37; P =0.06) and an autosomal recessive mode of inheritance. Conclusion - The possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA_Aα₅, β₃ and γ₃ subunit gene cluster contributes to the pathogenesis of "idiopathic" generalized seizures and associated EEG abnormalities in a proportion of families.     

Inhibition of stress-stimulated colonic propulsion by α2-adrenoceptor antagonists in rats

Alpha2-adrenoceptor antagonists have been reported to stimulate colonic motor activity, but the effect on colonic motor dysfunction is unclear. We have investigated the effect of alpha 2-adrenoceptor antagonists on wrap-restraint stress-stimulated and normal colonic propulsion in rats. Colonic propulsion was evaluated by the transit of a charcoal marker along the colon. Faecal pellets output was also measured. A 30-min exposure to wrap-restraint stress starting 120 min after infusion of the charcoal marker significantly stimulated colonic transit with a concomitant increase in faecal pellets. Yohimbine and idazoxan, alpha 2-adrenoceptor antagonists, clonidine, an alpha 2-adrenoceptor agonist, and atropine suppressed wrap-restraint stress-stimulated colonic transit and faecal excretion in a dose-dependent manner. Ondansetron and YM060, 5-hydroxytryptamine3 (5-HT3) receptor antagonists, potently inhibited wrap-restraint stress-stimulated colonic transit, but only weakly inhibited faecal excretion. Neither alpha 2-adrenoceptor antagonists nor atropine had any significant effect on normal colonic transit, whereas clonidine and the 5-HT3 receptor antagonists inhibited it. alpha 2-Adrenoceptor antagonists as well as clonidine, atropine and 5-HT3 receptor antagonists inhibit the stress-induced colonic motor dysfunction in rats.     

α2-Adrenoceptors couple to inhibition of R-type calcium currents in myenteric neurons

Alpha2-adrenoceptors inhibit Ca2+ influx through voltage-gated Ca2+ channels throughout the nervous system and Ca2+ channel function is modulated following activation of some G-protein coupled receptors. We studied the specific Ca2+ channel inhibited following alpha2-adrenoceptor activation in guinea-pig small intestinal myenteric neurons. Ca2+ currents (I(Ca2+)) were studied using whole-cell patch-clamp techniques. Changes in intracellular Ca2+ (delta[Ca2+]i) in nerve cell bodies and varicosities were studied using digital imaging where Ca2+ influx was evoked by KCl (60 mmol L(-1)) depolarization. The alpha2-adrenoceptor agonist, UK 14 304 (0.01-1 micromol L(-1)) inhibited I(Ca2+) and delta[Ca2+]i; maximum inhibition of I(Ca2+) was 40%. UK 14 304 did not affect I(Ca2+) in the presence of SNX-482 or NiCl2 (R-type Ca2+ channel antagonists). UK 14 304 inhibited I(Ca2+) in the presence of nifedipine, omega-agatoxin IVA or omega-conotoxin, inhibitors of L-, P/Q- and N-type Ca2+ channels. UK 14 304 induced inhibition of I(Ca2+) was blocked by pertussis toxin pretreatment (1 microg mL(-1) for 2 h). Alpha2-adrenoceptors couple to inhibition of R-type Ca2+ channels via a pertussis toxin-sensitive pathway in myenteric neurons. R-type channels may be a target for the inhibitory actions of noradrenaline released from sympathetic nerves on to myenteric neurons.     

Molecular genetic analysis of the α-synuclein and the parkin gene in Parkinson''s disease in Finland

Two mutations in the alpha-synuclein gene and various mutations in the parkin gene are associated with familial Parkinson's disease (PD). The present study was performed to analyse if mutations in these genes could be detected in Finnish patients with familial PD. The subjects comprised 22 unrelated patients with familial PD. The molecular genetic analysis consisted of sequence analysis of the non-coding and coding exons of the alpha-synuclein gene and screening of eight point mutations in the parkin gene. In addition, a total of 67 controls and 45 patients with sporadic PD were included in the association analysis on polymorphism of the alpha-synuclein gene. Screened point mutations in the parkin gene were not detected. Sequencing of the coding exons 2-6 of the alpha-synuclein gene did not reveal any mutations or polymorphisms. However, three novel alterations in the T10A7 sequence at the 5' end of the non-coding exon 1' of the alpha-synuclein gene were found. The frequencies of the exon 1' polymorphic genotypes or alleles between familial PD patients and control subjects revealed no statistically significant differences. No association for sporadic PD was observed. The results do not support a role for the alpha-synuclein gene or point mutations of the parkin gene in familial PD in our sample.     

Serum levels of β-carotene, α-carotene and vitamin A in patients with Alzheimer''s disease

To elucidate the possible role of carotenoids and vitamin A as risk factors for Alzheimer's disease (AD), we compared serum levels of beta-carotene and alpha-carotene, and vitamin A, measured by isocratic high performance liquid chromatography, of 38 AD patients and 42 controls. The serum levels of alpha-carotene did not differ significantly between AD patients and control groups. However, the serum levels of beta-carotene and vitamin A were significantly lower in the AD-patient group. These values did not correlate to age, age at onset or score on the MiniMental State Examination. Weight and body mass index were significantly lower in AD patients than in controls. These results suggest that low serum beta-carotene concentrations in AD patients could be related to a deficiency in dietary intake of this provitamin, although its possible relationship with risk for AD could not be excluded.     

Suppression of Penicillin-Induced Focal Epileptiform Activity by Locus Ceruleus Stimulation: Mediation by an α1-Adrenoceptor

Application of penicillin to the cerebral cortex of anesthetized rats by pressure ejection from a micropipette resulted in the appearance of focal epileptiform activity with low rates of penicillin release and focal penicillin spikes with higher rates. Electrical stimulation of the locus ceruleus (LC), a major norepinephrine-containing nucleus in the brainstem, or of its axons projecting to the forebrain, the dorsal noradrenergic bundle, suppressed penicillin-induced focal epileptiform activity but was less effective in suppressing focal penicillin spikes. Depletion of monoamines with reserpine blocked the suppressant effect of LC stimulation. Neither the selective depletion of 5-hydroxytryptamine with p-chlorophenylalanine nor administration of methysergide reduced the effectiveness of LC stimulation, suggesting that 5-hydroxytryptamine probably does not mediate the suppression. Pimozide partially antagonized the suppression of focal epileptiform activity induced by LC stimulation, which is consistent with antagonism of alpha-adrenoceptors but not dopamine receptors. beta-Receptor antagonists did not block the suppression of focal epileptiform activity by LC stimulation, suggesting that beta-receptors are not important in the observed suppression. Prazosin, a selective alpha 1-antagonist, at low doses blocked the suppression of focal epileptiform activity by LC stimulation whereas yohimbine, an alpha 2-antagonist enhanced the stimulation-induced suppression. Taken together, the data are consistent with LC and dorsal bundle stimulation releasing norepinephrine, which in turn suppresses focal epileptiform activity by an action mediated by an alpha 1-adrenoceptor.     

Noradrenaline Increases K-conductance and Reduces Glutamatergic Transmission in the Mouse Entorhinal Cortex by Activation of α2-Adrenoreceptors

The entorhinal cortex is a gateway to the hippocampus; it receives inputs from several cortical associative areas as well as subcortical areas. Since there is evidence showing that noradrenaline reduces the epileptic activity generated in the entorhinal cortex, we have examined the action of noradrenaline in the superficial layer of the entorhinal cortex, which is the main source of afferents to the hippocampus. In a previous study we showed that noradrenaline hyperpolarized layer II entorhinal cortex neurons and reduced global synaptic transmission via α₂-adrenoreceptors. Here we present a detailed analysis of the effect of noradrenaline on membrane resistance and on the pharmacologically isolated postsynaptic potentials in layer II entorhinal cortex neurons of mice. Noradrenaline (50 μM) hyperpolarized most layer II entorhinal cortex neurons. This hyperpolarization corresponded to an outward current with a reversal potential following the Nernst equilibrium potential for potassium. The hyperpolarizing effect of noradrenaline was blocked by 10 μM yohimbine. These observations suggest that noradrenaline activates a potassium conductance via an α₂-adrenoreceptor. Noradrenaline (10–50 μM) reversibly reduced the amplitude of the pharmacologically isolated excitatory potentials mediated by both NMDA and α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors, the former being more strongly affected. Again this effect was blocked by 10 μM yohimbine. In contrast, GABA_A-mediated synaptic transmission was virtually unaffected by noradrenaline. Thus, noradrenaline appears to strongly inhibit the glutamate-mediated synaptic transmission in the entorhinal cortex without affecting inhibitory post-synaptic potentials. These observations suggest that α₂-adrenergic receptor agonists may exert a beneficial effect in the control of hyperexcitability in temporal lobe epilepsy.     

Inhibition of N-type Calcium Channels: The Only Mechanism by which Presynaptic α2-Autoreceptors Control Sympathetic Transmitter Release

α₂-Adrenoceptors are known to inhibit voltage-dependent Ca²⁺ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca²⁺ entry, underlie the presynaptic α₂-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [³H]noradrenaline was elicited in the presence of extracellular Ca²⁺ by electrical pulses, 25 mM K⁺ or 10μM nicotine, or by adding Ca²⁺ to otherwise Ca²⁺-free medium when cells had been made permeable by the calcium ionophore A23187 or by α-latrotoxin. Pretreatment of neurons with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA and application of the α₂-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K⁺ or nicotine, but not the overflow caused by Ca²⁺ after permeabilization with α-latrotoxin or A23187. In contrast, the L-type Ca²⁺ channel blocker nitrendipine reduced the overflow due to K⁺ and nicotine, but not the overflow following electrical stimulation or α-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca²⁺ channel agonist BayK 8644, which per se enhanced overflow. In ω-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca²⁺ currents corroborated these results: UK 14304 inhibited Ca²⁺ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α₂-adrenergic effects. Our results indicate that the α₂-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω-conotoxin GVIA-sensitive N-type Ca²⁺ channels. Mechanisms downstream of these channels and voltage-sensitive Ca²⁺ channels other than N-type appear not to be important.     

α2 and β Adrenoceptors in the Mediobasal Hypothalamus and α2 Adrenoceptors in the Preoptic-Anterior Hypothalamus Stimulate Prolactin Secretion in the Conscious Male Rat

Plasma prolactin concentrations were measured in unanaesthetized male rats before and after stereotaxic microinjection of adrenergic agents into the mediobasal and preoptic-anterior hypothalamus. In the mediobasal hypothalamus injection of the α₂ agonist clonidine produced a dose-dependent increase in prolactin secretion over the dose range 0.1 to 10 nmoles, the stimulation due to 1 nmole being blocked by idazoxan (α₂ antagonist). Stimulation of prolactin release was also caused by isoprenaline (β agonist) and was significantly reduced by the β antagonist propranolol. The β₂ agonist salbutamol was also effective in stimulating prolactin secretion. However, the adrenergic agonists, noradrenaline (mixed α and β), phenylephrine (α₁) and tyramine (sympathomimetic) failed to affect prolactin secretion. In the preoptic-anterior hypothalamus clonidine caused a dose-dependent increase in prolactin secretion over the dose range 0.001 to 10 nmoles, the stimulation due to 0.1 nmole being abolished by idazoxan. While prolactin levels were significantly elevated by noradrenaline and tyramine, phenylephrine was ineffective. We conclude that the activation of α₂ and β₂ adrenoceptors in the mediobasal hypothalamus and of α₂ adrenoceptors in the preoptic-anterior hypothalamus, on or near prolactin-regulating neurons, results in increased prolactin secretion. An α₁ inhibitory action in the mediobasal hypothalamus has however not been ruled out. Adrenergic inputs in the preoptic-anterior hypothalamus appear to exert a predominant facilitatory effect on prolactin secretion.     

Serum and urinary manganese levels in patients with Parkinson's disease

To elucidate the possible role of manganese in the risk of developing Parkinson's disease (PD), we compared serum levels of manganese, and 24-h manganese excretion by urine in 29 PD patients and in 27 matched controls. We also measured chromium and cobalt in the same samples. All these values did not differ significantly between the groups, they were not influenced by antiparkinsonian drugs, and they did not correlate with age, age at onset and duration of the PD, scores of the Unified PD Rating Scale or the Hoehn & Yahr staging in the PD group. These results might suggest that serum levels and urinary excretion of manganese are apparently unrelated to the risk of developing PD.     

Amyloid β protein (Aβ) deposition in dementia with Lewy bodies: predominance of Aβ42(43) and paucity of Aβ40 compared with sporadic Alzheimer's disease

Amyloid β protein (Aβ) deposition was investigated by quantitative immunohistochemistry in 13 cases of dementia with Lewy bodies (DLB) and compared with that in a series of age, gender and ApoE genotype matched cases of Alzheimer's disease (AD). In DLB the predominant Aβ peptide species deposited was Aβ₄₂₍₄₃₎ and this was similar in amount to that in AD. By contrast, Aβ₄₀ deposition was sparse in DLB and was lower than that in AD as was the total Aβ (Aβ₄₀+Aβ₄₂₍₄₃₎ ) deposition. These data reinforce the viewpoint that in all disorders in which Aβ deposition is characteristic, the initial and predominant peptide species deposited is the longer form, Aβ₄₂₍₄₃₎ . The density of Lewy bodies (LB) in DLB was unrelated to the extent of Aβ deposition, although those cases possessing one or more copies of the apolipoprotein E E4 allele had a higher LB density than those without an E4 allele. This suggests that the apolipoprotein E E4 isoform might facilitate, though not necessarily trigger, the formation of LB in susceptible individuals.     

Plasma levels of the β-carbolines harman and norharman in Parkinson's disease

Several lines of evidence suggest that endogenous and exogenous toxins may play a major role in the pathogenesis of Parkinson's disease (PD). In vivo aromatic β-carbolines, like harman or norharman, may easily be formed by cyclization of indoleamines with e.g. aldehydes. Because of the structural similarity to MPTP, β-carbolines have been proposed as endogenous toxins. For further elucidation of the role of β-carbolines in neurodegenerative disorders, harman and norharman plasma levels were measured in 36 patients with PD and compared to an age- and sex-matched control group. Plasma levels of norharman in PD were significantly higher compared to the control group. Harman in the plasma of Parkinsonian patients was also elevated compared to controls, but this difference was not significant. Correlation of β-carbolines with plasma levels of L-dopa, oral doses of bromocriptine and selegiline in treated Parkinsonian patients showed no significant results. On the one hand these results may suggest a possible role of β-carbolines in the pathophysiological processes initiating PD, by, e.g., inducing mitochondrial respiratory inhibition like MPP ⁺. One may speculate, however, that elevated levels of norharman and harman are due to an endogenous upregulation caused by unknown metabolic processes.