Physostigmine and atropine potentiate and inhibit neuronal alpha 4 beta 4 nicotinic receptors.
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Acetylcholine↗
Biomedical subjects
R Zwart
Publications and source records attributed to R Zwart.
Cloning of the mouse and human solute carrier 22a3 (Slc22a3/SLC22A3) identifies a conserved cluster of three organic cation transporters on mouse chromosome 17 and human 6q26-q27.
Here we report the isolation of the mouse and human solute carrier genes Slc22a3/SLC22A3. Slc22a3 is specifically expressed in placenta, but the levels of expression decline toward the end of gestation. A BAC contig spanning the mouse Slc22a3 gene was constructed, and Slc22a3 was mapped between the Igf2r and Plg genes in close association with two additional members of the Slc22a gene family, mouse Slc22a1 and Slc22a2. A partial cDNA sequence of the human SLC22A3 gene was reconstituted from sequenced EST clones. SLC22A3 is expressed in first-trimester and term placenta, but also in skeletal muscle, prostate, aorta, liver, fetal lung, salivary gland, and adrenal gland. Using a somatic cell hybrid panel and a human YAC clone, SLC22A3 was mapped to the syntenic region on human chromosome 6q26-q27, between the IGF2R and APO(a)-like genes. SLC22A1 and SLC22A2 localized to the same locus, demonstrating the conservation of the close physical linkage of these three organic cation transporter genes in mouse and human.
Amino Acid Sequence↗
Physostigmine and acetylcholine differentially activate nicotinic receptor subpopulations in Locusta migratoria neurons.
The effects of acetylcholine (ACh) and physostigmine (PHY) on thoracic ganglion neurons of Locusta migratoria were investigated using whole-cell and cell-attached voltage clamp. ACh activated whole-cell currents with variable amplitudes, time course and ion channel block between cells, suggesting differential expression of nicotinic acetylcholine receptor (nAChR) subtypes. This was supported by selective block of the peak of the currents by the alpha7-specific alpha-conotoxin ImI. PHY at 100 microM evoked smaller whole-cell currents with variable amplitudes and marginal desensitization. The PHY/ACh amplitude ratio varied between cells, and was positively related to the time constant of decay of the ACh response. EC50 values for the peak amplitude of the ACh- and PHY-induced currents were 50 microM and 3 microM, respectively. Both agonists activated nAChR, indicated by equal voltage-dependence and reversal potentials and the same pharmacological properties of ACh and PHY responses. In addition, PHY and ACh induced ion channel block. Co-application and cross-desensitization experiments showed that ACh and PHY activate the same nAChR subpopulations. Both agonists activated nicotinic single channels with three conductance levels, which were equal for ACh and PHY, indicating activation of the same nAChR subtypes by both agonists. However, for all levels PHY displayed a lower open probability than ACh. Taken together, different whole-cell responses appear to originate from differential activation, desensitization and ion channel block by ACh and PHY of distinct nAChR populations.
Acetylcholine↗
Familial mitochondrial DNA depletion in liver: haplotype analysis of candidate genes.
Two sons and one daughter of healthy consanguineous parents presented with fatal hepatic failure in association with severe depletion of mitochondrial (mt)DNA in liver; a third son is healthy. Other published cases of mtDNA depletion concern single members of a family, which excludes the use of haplotype analysis. In the family presented here, the inheritance of the genes for mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF-1), mitochondrial single-stranded DNA-binding protein (mtSSBP), and endonuclease G (EndoG) was studied using microsatellite markers linked to these genes. The inheritance of the gene for mtDNA polymerase (pol gamma) was studied using a polymorphic CAG repeat present within the coding region of the gene. EndoG and mtSSBP were excluded, but mtTFA remains a candidate. Pol gamma or NRF-1 involvement would be compatible only with autosomal dominant inheritance. Coding sequence analysis of NRF-1 and mtTFA revealed no novel mutations in affected individuals.
Chromosomes, Human↗
Four pharmacologically distinct subtypes of alpha4beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.
Nicotinic receptors generally are presumed to consist of two alpha and three non-alpha subunits. We varied the relative levels of expression of the neuronal nicotinic alpha4 and beta2 receptor subunits in Xenopus laevis oocytes by nuclear injection of cDNAs coding for these subunits in alpha:beta ratios of 9:1, 1:1, and 1:9. The sensitivities of the receptors to acetylcholine and d-tubocurarine were investigated in voltage-clamp experiments. For receptors expressed at the 9:1 and 1:1 alpha:beta ratios, the EC50 value of acetylcholine is approximately 60 microM. For the majority of the receptors expressed at the 1:9 alpha:beta ratio, the sensitivity to acetylcholine is enhanced 30-fold. No evidence for more than one type of acetylcholine binding site in a single receptor is obtained. The sensitivity to d-tubocurarine decreases with decreasing alpha:beta ratio. IC50 values of d-tubocurarine are 0.2, 0.5, and 2 microM for the 9:1, 1:1, and 1:9 alpha:beta ratios, respectively. At the 1:9 alpha:beta ratio, additional receptors with an IC50 value of 163 microM d-tubocurarine are expressed. At least two components with distinct sensitivities to d-tubocurarine are required to account for the shift in IC50. The combined agonist and antagonist effects reveal four distinct subtypes of alpha4beta2 nicotinic receptors. The results imply that the subunit stoichiometry of heteromeric alpha4beta2 acetylcholine receptors is not restricted to 2alpha:3beta.
Acetylcholine↗
Preferential amplification and phenotypic selection in a population of deleted and wild-type mitochondrial DNA in cultured cells.
In order to study the still poorly understood dynamics of mitochondrial gene segregation, we attempted to alter the percentage of deleted mtDNA (del-mtDNA) over wild-type mtDNA in cell-culture by manipulating respiratory chain capacity. For this purpose, we used a cell-line harbouring a 6-kb mtDNA-deletion which normally was present in 70% of the molecules. The results show that in the presence of low concentrations of doxycycline (DC), an inhibitor of mitochondrial protein synthesis, the average percentage of del-mtDNA in culture steadily declined. After short-term DC treatment most cells still contained del-mtDNA and removal of DC led to a rapid increase in the proportion of del-mtDNA. In contrast, long-term DC treatment rendered del-mtDNA undetectable by Southern analysis, reflecting the complete absence of del-mtDNA in most cells. In this case, del-mtDNA in culture remained at a constant low level after removal of the drug. The results clearly show the importance of phenotypic selection in the segregation of a deleterious mtDNA mutation.
Anti-Bacterial Agents↗
Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects.
Atropine, the classic muscarinic receptor antagonist, inhibits ion currents mediated by neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. At the holding potential of -80 mV, 1 microM atropine inhibits 1 mM acetylcholine-induced inward currents mediated by rat alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nicotinic receptors by 12-56%. Inward currents induced with a low agonist concentration are equally inhibited (alpha3beta2, alpha3beta4), less inhibited (alpha2beta4, alpha7), or potentiated (alpha4beta2, alpha4beta4) by 1 microM atropine. Effects on the more sensitive alpha4beta4 nicotinic receptors were investigated in detail by systematic variation of acetylcholine and atropine concentrations and of membrane potential. At high agonist concentration, atropine inhibits alpha4beta4 nicotinic receptor-mediated ion current in a noncompetitive, voltage-dependent way with IC50 values of 655 nM at -80 mV and of 4.5 microM at -40 mV. At low agonist concentration, 1 microM atropine potentiates alpha4beta4 nicotinic receptor-mediated ion current. This potentiating effect is surmounted by high concentrations of acetylcholine, indicating a competitive interaction of atropine with the nicotinic receptor, and potentiation is also reversed at high atropine concentrations. Steady state effects of acetylcholine and atropine are accounted for by a model for combined receptor occupation and channel block, in which atropine acts on two distinct sites. The first site is associated with noncompetitive ion channel block. The second site is associated with competitive potentiation, which appears to occur when the agonist recognition sites of the receptor are occupied by acetylcholine and atropine. The apparent affinity of atropine for the agonist recognition sites of the alpha4beta4 nicotinic acetylcholine receptor is estimated to be 29.9 microM.
Acetylcholine↗
Cellular aspects of persistent neurotoxicants: effects of Pb2+ on neuronal nicotinic acetylcholine receptors.
Among persistent neurotoxicants inorganic lead (Pb2+) is known to cause a variety of cellular effects associated with alterations in neuronal performance. Here we describe differential effects of Pb2+ on distinct subtypes of neuronal nicotinic acetylcholine receptors (nAChR) in Xenopus oocytes expressing various combinations of alpha and beta receptor subunits. Transient inward currents evoked by ACh in voltage clamped oocytes expressing alpha4beta2 and alpha3beta4 nAChR are inhibited by external Pb2+ at concentrations in the range of 1 nM-200 microM. The sensitivities of oocytes expressing alpha4beta2 and alpha3beta4 nAChR vary greatly. Additional experiments suggest that this variation may be due to receptor heterogeneity, which is demonstrated for alpha4beta2 nAChR in cell-attached patch clamp experiments. Conversely, the inward current evoked by ACh in oocytes expressing alpha3beta2 nAChR is potentiated by Pb2+ at concentrations in the range of 1-250 microM. The potentiating effect shows little variation between oocytes and is similar for inward currents evoked by low and high concentrations of ACh. The distinct effects on alpha3beta2 nAChR as compared to alpha4beta2 and alpha3beta4 nAChR suggest that Pb2+ interacts with both alpha and beta subunits of the neuronal nAChR. The effects of Pb2+ on defined subtypes of nAChR in oocytes are compared to those on ligand-gated ion channels in intact cells.
Animals↗
Oct-6 (SCIP/Tst-1) is expressed in Schwann cell precursors, embryonic Schwann cells, and postnatal myelinating Schwann cells: comparison with Oct-1, Krox-20, and Pax-3.
The POU domain transcription factor Oct-6 (SCIP/Tst-1) is likely to control important stages of Schwann cell development, including the initiation of myelination around birth. Here, we use immunocytochemical and reverse transcriptase-polymerase chain reaction techniques to examine Oct-6 earlier in nerve development, to test the idea that Oct-6 has an additional role in Schwann cell precursors or early embryonic Schwann cells, a possibility raised by previous studies on transgenic mice. Consistent with this, we find low but unambiguous levels of Oct-6 mRNA and protein in Schwann cell precursors of mouse and rat (nerves from 12- and 14-day-old embryos, respectively), with expression levels gradually increasing during early Schwann cell development and towards birth. Unexpectedly, Oct-6 immunoreactivity is clearly present in nuclei of most myelinating cells at least as late as postnatal day 12. Furthermore, many nonmyelinating Schwann cells express Oct-6 in adult life. A comparison of Oct-6 mRNA with other Schwann cell transcription factors-namely, Oct-1, Krox-20, and Pax-3-reveals that each factor exhibits strong developmental regulation and a unique expression pattern in embryonic nerves. Therefore, they are likely to play distinct regulatory roles in early development of the Schwann cell lineage.
Animals↗
Histamine slows the onset of desensitization of rat cortical NMDA receptors.
The effects of histamine on N-methyl-d-aspartate (NMDA) receptor-mediated ion current in cultured rat cortical neurones have been investigated by superfusion of voltage-clamped (-80 mV) cells with 100 mM NMDA and with various concentrations of histamine. In a subpopulation of the neurones tested (30/53), histamine (10-100 mM) enhanced the peak amplitude, and slowed down the onset of desensitization of the NMDA-induced ion currents, both in a concentration-dependent manner. No effects of histamine on the kinetics of recovery from desensitization were detected. In other neurones (21/53), histamine had no effect on the NMDA-induced currents and in two of the neurones tested histamine potentiated NMDA-induced currents without a detectable effect on desensitization kinetics. The results indicate that in a subpopulation of rat cortical neurones histamine potentiates NMDA-induced ion currents by slowing the kinetics of onset of desensitization.
Animals↗
The POU factor Oct-6 and Schwann cell differentiation.
The POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6(-/-) mice at different stages of postnatal development reveals a delay in Schwann cell differentiation, with a transient arrest at the promyelination stage. Thus, Oct-6 appears to be required for the transition of promyelin cells to myelinating cells. Once these cells progress past this point, Oct-6 is no longer required, and myelination occurs normally.
Animals↗
Serotonin 5-HT2 receptor activation potentiates N-methyl-D-aspartate receptor-mediated ion currents by a protein kinase C-dependent mechanism.
Modulation of N-methyl-D-aspartate (NMDA) receptor-mediated ion currents by serotonin was investigated with a two-electrode voltage clamp technique in Xenopus oocytes injected with rat brain RNA. After a 1-min application of 200 nM serotonin a transient potentiation of the NMDA receptor-mediated ion currents was observed. The serotonin-induced enhancement was mimicked by the protein kinase C activators 1-oleoyl-2-acetyl-sn-glycerol (100 microM) and phorbol 12-myristate 13-acetate (10 nM), whereas the inactive phorbol ester 4-alpha-phorbol 12-myristate 13-acetate (10 nM) had no effect. From these observations it was concluded that protein kinase C was involved in the enhancement of NMDA-induced currents. In agreement with this conclusion, it was found that the serotonin effect was inhibited by the protein kinase C inhibitors sphingosine (1 microM) or staurosporine (1 microM) added 20 min before NMDA application and by oocyte injection of protein kinase C (PKC)-inhibitor peptide (500 ng/oocyte) 1 hr prior to recordings. The serotonin receptor involved was identified as a 5-HT2 receptor subtype by the finding that 200 nM of the selective 5-HT2 receptor agonist alpha-methyl-5-hydroxytryptamine mimicked the potentiation of NMDA-induced ion currents by serotonin. Furthermore, the observed potentiation was significantly reduced by co-application of serotonin with 100 microM of the selective 5-HT2 receptor antagonist ketanserin. These results indicate that 5-HT2 receptors enhance NMDA receptor function via phosphoinositol hydrolysis and subsequent stimulation of PKC.
Animals↗
Fatal neonatal liver failure and depletion of mitochondrial DNA in three children of one family.
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Acidosis↗
The restricted expression pattern of the POU factor Oct-6 during early development of the mouse nervous system.
Oct-6 is a POU transcription factor that is thought to play a role in the differentiation of cells of neuroectodermal origin. To investigate whether the Oct-6 protein could play a role in the establishment of neuroectoderm in vivo we studied the expression of the Oct-6 protein during early mouse development. Expression is first observed in the primitive ectoderm of the egg cylinder stage embryo. In gastrulating embryos, Oct-6 protein is found in the extra-embryonic ectoderm of the chorion and the anterior ectoderm of the embryo proper. As development proceeds, Oct-6 expression becomes more restricted to the anterior medial part of the embryo until Oct-6 positive cells are observed only in the neural groove of the headfold stage embryo. In the late headfold stage embryo, Oct-6 expression is detected in the neuroepithelium of the entire brain and later is restricted to a more ventral and anterior position. As the anterior neuropore closes, Oct-6 protein is detected in a segment-like pattern in the mid-and forebrain. Thus, the expression pattern of the Oct-6 gene agrees with a role for the Oct-6 protein in the establishment and regional specification of the neuroectoderm in vivo. The two waves of widespread induction of the Oct-6 gene, one in the primitive ectoderm and another in the primitive brain, both followed by a progressive restriction in the expression patterns suggest a mechanism for the regulation of the gene.
Animals↗
Potentiation and inhibition of subtypes of neuronal nicotinic acetylcholine receptors by Pb2+.
Effects of inorganic lead (Pb2+) on defined subtypes of neuronal nicotinic acetylcholine receptors have been investigated. Voltage clamp experiments have been performed on Xenopus oocytes expressing alpha 3 beta 2, alpha 3 beta 4 and alpha 4 beta 2 neuronal nicotinic acetylcholine receptor subunit combinations. In oocytes expressing the alpha 3 beta 2 subunit combination Pb2+ enhances the peak amplitude of nicotinic acetylcholine receptor-mediated inward currents evoked by superfusion with 100 microM acetylcholine. At concentrations of 1-250 microM, Pb2+ potentiates alpha 3 beta 2 receptor-mediated inward current concentration dependently by a factor of 1.1-11.0. Inward currents evoked by low (3 microM) and high (1 mM) concentrations of acetylcholine are potentiated to a similar extent. Conversely, in oocytes expressing the alpha 3 beta 4 subunit combination Pb2+ inhibits the nicotinic receptor-mediated inward currents evoked with 100 microM acetylcholine. Inhibitory effects are observed in the concentration range of 1 nM-100 microM Pb2+ but the degree of inhibition varies between oocytes. A similar inhibition of the alpha 4 beta 2 nicotinic receptor-mediated inward current by Pb2+ indicates that alpha as well as beta subunits are involved in the potentiating and inhibitory effects. Possible reasons for the variation in the inhibitory effects of Pb2+ on alpha 3 beta 4 and alpha 4 beta 2 nicotinic receptor-mediated inward currents have been investigated and are discussed. The divalent cations Ca2+ and Mg2+ potentiate both alpha 3 beta 2 and alpha 3 beta 4 nicotinic receptor-mediated inward currents. The distinct modulation of receptor function by Pb2+ and by Ca2+ and Mg2+ and the dependence of the modulatory effect of Pb2+ on subunit composition suggest that Pb2+ interacts with multiple sites on the alpha and beta subunits of neuronal nicotinic acetylcholine receptors.
Animals↗
Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy.
Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.
Cells, Cultured↗
Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein.
Murine leukemia viruses (MuLV) have been adapted for use as gene transfer vectors for experimental and human gene therapy applications. Their utility for these purposes has been circumscribed by the limited host range and relatively low titer of available producer clones. Pseudotyping of MuLV particles with the vesicular stomatitis virus envelope protein (VSV-G), expressed transiently in cells producing MuLV Gag and Pol proteins, has yielded vector preparations with a broader host range that can be concentrated by ultracentrifugation. We have explored the use of steroid-inducible and tetracycline-modulated promoter systems (necessary because the VSV-G protein is toxic to cells when constitutively expressed) to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MuLV particles. A packaging cell line and producer clones capable of expressing a chimeric transcription factor, composed of the tetracycline repressor (tetR) and the VP16 trans-activating sequences of herpes simplex virus VP16 gene and containing the VSV-G coding sequences linked to a minimal promoter having seven tandem copies of the tetracycline responsive operator (tetO), exhibited high levels of VSV-G protein expression when cultured in the absence of tetracycline. Vector particles, produced at titers of 10(5)-10(6) infectious colony forming units per ml (cfu/ml), could be concentrated effectively by ultracentrifugation yielding vector preparations having a titer of 10(9) cfu/ml. These cell lines grew normally when VSV-G protein expression was repressed by tetracycline. Such producer clones hold promise for future human gene therapy applications.
Animals↗
Subunit-dependent action of lead on neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.
1. Pb2+ affects neuronal nicotinic acetylcholine receptors (nAChR) in N1E-115 neuroblastoma cells in a dual manner. At nanomolar concentrations a blockade is observed, while at submillimolar concentrations this blocking effect is reversed. 2. The Xenopus oocyte expression system was used to examine whether the dual effect of Pb2+ is related to a differential action on nAChR subtypes. Effects of Pb2+ were investigated in oocytes expressing nAChR after co-injection of alpha 3 and beta 2 or alpha 3 and beta 4 cDNA. 3. At 1-250 mumol/L, Pb2+ causes a 10-1000% increase of the response mediated by the alpha 3 beta 2 nAChR. 4. Pb2+ blocks ACh-induced inward currents mediated by alpha 3 beta 4 nAChR. The inhibitory potency of Pb2+ greatly varies between cells. In 50% of the cells concentrations < or = 1 mumol/L Pb2+ blocked the nicotinic response by 31-93%. In the other cells even at higher concentrations Pb2+ caused only 0-65% inhibition. 5. These results show that Pb2+ may both potentiate and block nAChR, depending on the type of nAChR subunit expressed.
Animals↗