Biomedical subjects

Lynn Wecker

Publications and source records attributed to Lynn Wecker.

7 recordsLinked to original sources

PubMed2004

The alpha7 nicotinic acetylcholine receptor subunit exists in two isoforms that contribute to functional ligand-gated ion channels.

Fast synaptic transmission in mammalian autonomic ganglia is mediated primarily by nicotinic receptors, and one of the most abundant nicotinic acetylcholine receptor subtypes in these neurons contains the alpha7 subunit (alpha7-nAChRs). Unlike alpha7-nAChRs expressed in other cells, the predominant alpha7-nAChR subtype found in rat intracardiac and superior cervical ganglion neurons exhibits a slow rate of desensitization and is reversibly blocked by alpha-bungarotoxin (alphaBgt). We report here the identification of an alpha7 subunit sequence variant in rat autonomic neurons that incorporates a novel 87-base pair cassette exon in the N terminus of the receptor and preserves the reading frame of the transcript. This alpha7 isoform was detected using reverse transcriptase-polymerase chain reaction techniques in neonatal rat brain and intracardiac and superior cervical ganglion neurons. Immunoblot experiments using a polyclonal antibody directed against the deduced amino acid sequence of the alpha7-2 insert showed a pattern of expression consistent with alpha7-2 subunit mRNA distribution. Moreover, the alpha7-2 subunit could be immunodepleted from protein extracts by solid-phase immunoprecipitation techniques using the anti-alpha7 monoclonal antibody 319. The alpha7-2 subunit was shown to form functional homomeric ion channels that were activated by acetylcholine and blocked by alpha-bungarotoxin when expressed in Xenopus laevis oocytes. This alpha7 isoform exhibited a slow rate of desensitization, and inhibition of these channels by alphaBgt reversed rapidly after washout. Taken together, these data indicate that the alpha7-2 subunit is capable of forming functional alphaBgt-sensitive acetylcholine receptors that resemble the alpha7-nAChRs previously identified in rat autonomic neurons. Furthermore, the distribution of the alpha7-2 isoform is not limited to peripheral neurons.

Acetylcholine↗

PubMed2004

Developmental differences in nicotine place conditioning.

To understand the motivations and implications of the prevalence of smoking, studies have compared the behavioral effects of nicotine, the psychoactive drug in tobacco, in adolescent and adult animals. The present study used a biased three-chambered conditioned-place preference procedure without prior habituation to examine the potential rewarding and anxiolytic effects of nicotine across adolescence and adulthood to assess the presence of age-dependent differences in response to nicotine.

Age Factors↗

PubMed2003

Transdermal selegiline: targeted effects on monoamine oxidases in the brain.

BACKGROUND: The oral administration of monoamine oxidase (MAO) inhibitors has the potential to cause a hypertensive reaction after the ingestion of tyramine-containing compounds. Because transdermal drug administration bypasses gastrointestinal absorption, it is possible that inhibition of MAO-A in brain may be achieved without enzyme inhibition in the gastrointestinal system, thereby eliminating the possibility of this drug interaction. These studies determined whether the transdermal administration of selegiline has differential effects on MAOs in brain versus the gastrointestinal system. METHODS: Rats were exposed to various doses of selegiline via a transdermal patch for up to 30 days, and MAO-A and MAO-B activities were determined in brain regions and gastrointestinal tissue. RESULTS: In all brain regions, transdermal selegiline, at doses that produced maximal MAO-B inhibition, led to a dose- and time-dependent MAO-A inhibition. The inhibition of MAOs in gastrointestinal tissue was less than that in brain, and doses that produced maximal MAO-A inhibition in brain inhibited MAO-A in gastrointestinal tissue by only 30%-40%. CONCLUSIONS: Results suggest that transdermal selegiline preferentially inhibits MAO-A in brain relative to the gastrointestinal system. As a consequence, transdermal selegiline should be devoid of the potential to cause a hypertensive reaction after the ingestion of tyramine-containing compounds.

Administration, Cutaneous↗

PubMed2003

Phosphorylation of the alpha4 subunit of human alpha4beta2 nicotinic receptors: role of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC).

This study determined whether the alpha4 subunit of human alpha4beta2 neuronal nicotinic receptors is phosphorylated in situ by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). To accomplish this, human cloned epithelial cells stably transfected with the human alpha4beta2 nicotinic receptor (SH-EP1-halpha4beta2) were incubated with 32P-orthophosphate to label endogenous ATP stores, and the phosphorylation of alpha4 subunits was determined in the absence or presence of PKA or PKC activation. Autoradiographs and immunoblots indicated that alpha4 subunits immunoprecipitated from a membrane preparation of SH-EP1-halpha4beta2 cells exhibited a single 32P-labeled band corresponding to the alpha4 subunit protein; no signals were associated with untransfected SH-EP1 cells. The alpha4 subunits from SH-EP1-halpha4beta2 cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was decreased in the presence of the PKA inhibitor H-89 (5 microM), but unaltered in the presence of the PKC inhibitor Ro-31-8220 (0.1 microM). Activation of PKA by forskolin (10 microM), dibutyryl-cAMP (1 mM), or Sp-8-Br-cAMP (1 mM) enhanced phosphorylation nearly threefold; the inactive isomer, Rp-8-Br-cAMP (1 mM) had no effect. In addition, the forskolin effect was totally blocked by the PKA inhibitor H-89 (5 microM). Activation of PKC by the phorbol esters PDBu (200 nM) or PMA (200 nM) increased alpha4 subunit phosphorylation approximately twofold, and the PDBu effect was blocked by the selective PKC inhibitor Ro-31-8220 (0.1 microM). These findings indicate that the alpha4 subunit of human alpha4beta2 nicotinic receptors is phosphorylated in situ by PKA and PKC.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2003

Phosphorylation sites within alpha4 subunits of alpha4beta2 neuronal nicotinic receptors: a comparison of substrate specificities for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC).

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human alpha4 subunit of alpha4beta2 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 microM; Kemptide had a Km of 7.7 microM. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 microM and 2896 microM, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 microM; GS 1-8 had a Km of 2.1 microM. VRCRSRSI had a comparative affinity for PKC with a Km of 327 microM. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 microM, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human alpha4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.

Amino Acid Sequence↗

PubMed2002

Identification of three cAMP-dependent protein kinase (PKA) phosphorylation sites within the major intracellular domain of neuronal nicotinic receptor alpha4 subunits.

This study determined whether all protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites on the alpha4 subunit of rat alpha4beta2 neuronal nicotinic receptors could be localized to the M3/M4 cytoplasmic domain of the protein, and investigated specific amino acid substrates for the kinases through two-dimensional phosphopeptide mapping and site-directed mutagenesis. Experiments were conducted using alpha4beta2 receptors expressed in Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(333-594) ). When oocytes expressing alpha4beta2 receptors were incubated with [(32) P]orthophosphate in order to label endogenous ATP stores, phosphorylation of alpha4 subunits was evident. Incubation of either immunoprecipitated receptors or the fusion protein with [(32) P]ATP and either PKA or PKC followed by trypsinization of the samples demonstrated that the kinases phosphorylated alpha4 subunits on multiple phosphopeptides, and that the phosphorylated full-length alpha4 protein and fusion protein produced identical phosphopeptide maps. Site-directed mutagenesis of Ser365, Ser472 and Ser491 to alanines in the fusion protein eliminated phosphopeptides phosphorylated by PKA, but not by PKC. Other mutations investigated, Ser470, Ser493, Ser517 and Ser590, did not alter the phosphopeptide maps. Results indicate that Ser365, Ser472 and Ser491 on neuronal nicotinic receptor alpha4 subunits are phosphorylated by PKA and are likely to represent post-translational regulatory sites on the receptor.

Animals↗

PubMedDate not supplied

The controversy concerning plasma homocysteine in Parkinson disease patients treated with levodopa alone or with entacapone: effects of vitamin status.

Levodopa treatment of Parkinson disease results in hyperhomocysteinemia (HHcy) as a consequence of levodopa methylation by catechol-O-methyltransferase (COMT). Although inhibition of COMT should theoretically prevent or reduce levodopa-induced HHcy, results from several prospective studies are conflicting. Our review of these studies suggests that the ability of COMT inhibition to reduce or prevent levodopa-induced HHcy in Parkinson disease patients may be attributed to differences in the vitamin status of the study participants. In patients with low or low-normal folate levels, levodopa administration is associated with a greater increase in homocysteine and concomitant entacapone administration is associated with a greater reduction in homocysteine.

Catechol O-Methyltransferase↗