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Biomedical subjects

S W Halvorsen

Publications and source records attributed to S W Halvorsen.

At least 19 recordsLinked to original sources
PubMed1999

Ciliary neurotrophic factor and phorbol ester each decrease selected STAT3 pools in neuroblastoma cells by proteasome-dependent mechanisms.

Many cytokines and growth factors activate common signal transduction pathways and yet are able to elicit distinct cell-specific responses. We are defining mechanisms regulating signalling molecules in order to understand how cytokines can produce unique responses. It was found that individual members of the signal transducer and activator of transcription (STAT) family are regulated by ciliary neurotrophic factor (CNTF) and by protein kinase C. Treatment of SH-SY5Y human neuroblastoma cells with the phorbol ester, 12- O -tetradecanoylphorbol 13-acetate (TPA), for 4-5 h caused a 60% decline in both STAT2 and STAT3 levels and no decline in levels of STATs 1, 5 or 6, or in Jaks 1 or 2. The decline in STAT3 was inhibited by treatment with MG132, an inhibitor of proteasome-dependent protein degradation. Treatment of cells with CNTF induced a rapid tyrosine phosphorylation of STAT3 followed by a time-dependent decay of this signal. Loss of tyrosine phosphorylated STAT3 was inhibited by MG132 but did not require protein kinase C activity. These results suggest that STAT3 availability can be controlled by proteasome-dependent pathways activated either by protein kinase C or by cytokines.

Ciliary Neurotrophic Factor↗
PubMed1998

Reciprocal regulation of ciliary neurotrophic factor receptors and acetylcholine receptors during synaptogenesis in embryonic chick atria.

Ciliary neurotrophic factor (CNTF) has been implicated in the development, survival, and maintenance of a broad range of neurons and glia in the peripheral nervous system and the CNS. Evidence also suggests that CNTF may affect development of cells outside the nervous system. We have found that functional CNTF and its receptor are expressed in developing embryonic chick heart and may be involved in parasympathetic synapse formation. CNTF and CNTF receptor mRNA levels were highest at embryonic day 11 (E11)-E13, the period of parasympathetic innervation in chick atria. Levels of atrial CNTF receptor mRNA were fourfold greater at E13 than at E6 and at E13 were 2.5-fold higher in atria than in ventricle, corresponding to the higher degree of parasympathetic innervation occurring in atria. Treatment of isolated atria or cultured atrial myocytes with recombinant human or avian CNTF resulted in the tyrosine phosphorylation and nuclear translocation of the signal transducer and activator of transcription STAT3. The developmental increase in atrial CNTF receptor mRNA was enhanced by stimulating muscarinic receptors with carbachol in ovo and was inhibited by blocking muscarinic cholinergic receptors with atropine. Treatment of cultured atrial myocytes with CNTF resulted in a twofold increase in the levels of muscarinic receptors. Thus, CNTF was able to regulate a key component of parasympathetic synapses on atrial myocytes. These results suggest a postsynaptic role for CNTF in the onset of parasympathetic function in the developing heart and provide new clues to molecular mechanisms directing synapse formation at targets of the autonomic nervous system.

Animals↗
PubMed1998

Retinoic acid up-regulates ciliary neurotrophic factor receptors in cultured chick neurons and cardiomyocytes.

Retinoic acid is an important developmental factor in the heart and nervous system and regulates the expression of trophic factor receptors in neural cell lines. Here we show the effects of retinoic acid on cytokine responsiveness in embryonic chick neurons and myocytes. Treatment of cultured cardiomyocytes and retinal and ciliary ganglion neurons with retinoic acid resulted in increased expression of receptors for the neuropoietic cytokine, CNTF. All-trans-retinoic acid induced as much as a 3-fold increase in CNTF receptor alpha subunit mRNA in a time and concentration dependent manner and resulted in an enhanced CNTF-induced tyrosine phosphorylation of the transcription factor, STAT3. These results indicate that neurons and myocytes expressing CNTF receptors are responsive to retinoic acid and suggest that retinoids may regulate cell sensitivity to cytokines during development.

Animals↗
PubMed1997

Tissue-specific regulation of G-protein-coupled inwardly rectifying K+ channel expression by muscarinic receptor activation in ovo.

We investigated the effects of muscarinic acetylcholine receptor stimulation on the expression levels of the G-protein-coupled inwardly rectifying K+ channel (GIRK) subunits using solution hybridization and immunoblot analyses. We report here that treatment of chick embryos in ovo with muscarinic agonist causes decreases in mRNA levels encoding GIRK1 and GIRK4 in atria but does not alter GIRK1 expression in ventricles. In addition, GIRK1 protein levels also demonstrate a decrease in atria upon muscarinic acetylcholine receptor stimulation. Numerous receptors couple to the activation of the GIRK family of inwardly rectifying K+ channels; thus, these decreases represent a novel mechanism for regulating physiological responses to chronic agonist exposure.

Animals↗
PubMed1997

Ciliary neurotrophic factor stimulates the phosphorylation of two forms of STAT3 in chick ciliary ganglion neurons.

Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that was identified, purified, and cloned based on its neurotrophic activity on cultured chick ciliary ganglion neurons. The molecular mechanisms by which CNTF elicits its effects on these neurons are unknown. We have previously identified functional receptors for CNTF on ciliary ganglion neurons and demonstrated the CNTF-specific tyrosine phosphorylation of an approximately 90-kDa protein. Here we show that CNTF induced the rapid tyrosine phosphorylation and nuclear accumulation of this protein and identify it as an avian form of the transcription factor, STAT3. Identification was confirmed by its recognition with two distinct anti-STAT3 antibodies and the lack of binding to antibodies against STAT1, -2, -4, -5, or -6. The phosphorylation was stable for up to 2 h but required the continued presence of CNTF. CNTF also induced the tyrosine phosphorylation of a similar protein in cultured chick dorsal root ganglion and retinal neurons. In addition, we identify a second, 100-kDa form of STAT3 that appears in response to CNTF. Unlike previous reports, utilizing mammalian cell lines that detected a slower migrating form of STAT3 resulting from H7-sensitive protein phosphorylation, H7 did not prevent the appearance of the 100-kDa form in ciliary neurons. Thus, the 100-kDa avian protein may represent a novel form of CNTF-inducible STAT3.

Animals↗
PubMed1997

Channel activators regulate ATP-sensitive potassium channel (KIR6.1) expression in chick cardiomyocytes.

ATP-sensitive potassium channels (K(ATP)) are widely expressed and yet little is known about the mechanisms regulating their expression. Here we report that expression of chick heart Kir6.1 is regulated by channel activators. Activation of K(ATP) with either ATP depletion or pinacidil, up-regulated Kir6.1 mRNA 1.8- to 2.4-fold in cultured ventricular myocytes as measured by competitive PCR. Pinacidil treatment also increased Kir6.1 protein as detected using an antibody to Kir6.1. Glibenclamide, a K(ATP) inhibitor, completely blocked the pinacidil-induced increase in Kir6.1 levels. It appears that Kir6.1 is up-regulated by an unknown signal transduction pathway initiated by K(ATP) opening.

Adenosine Triphosphate↗
PubMed1997

Opposing regulation of ciliary neurotrophic factor receptors on neuroblastoma cells by distinct differentiating agents.

We have used SH-SY5Y neuroblastoma cells as a model for differentiating neurons to examine the mechanisms that regulate responses to the neuropoietic cytokine ciliary neurotrophic factor (CNTF). Retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) each induced differentiation of SH-SY5Y cells. Cells treated for 24 h with retinoic acid (10 microM) showed a threefold increase in 125I-CNTF binding sites and were up to five times more sensitive to CNTF than untreated cells in stimulating the tyrosine phosphorylation of the transcription factor STAT3. TPA (10 nM) induced a transient 42% decrease in 125I-CNTF binding sites after 4 h of treatment that recovered to near control levels after 7 h of continuous exposure. TPA-treated cells showed a decreased sensitivity to CNTF and a sevenfold decrease in levels of STAT3. The retinoic acid-induced increase in 125I-CNTF binding could be prevented by administration of either cycloheximide or actinomycin D, whereas neither agent altered the TPA-induced decrease in 125I-CNTF binding. In addition, levels of mRNA for both the CNTF receptor alpha and gp130 subunits increased twofold as measured by RNase protection after treatment with retinoic acid for 30 h. The increase in CNTF receptor alpha subunit mRNA was not due to a decrease in its turnover rate, and therefore, was likely due to an increase in gene expression. Thus, retinoic acid and TPA regulate CNTF receptors on neuroblastoma cells differently, and the results demonstrate the importance of transcriptional control of CNTF receptors and also implicate translational and post-translational mechanisms in the regulation of cytokine receptors and responses on neurons.

Antineoplastic Agents↗
PubMed1996

Ciliary neurotrophic factor regulates nicotinic acetylcholine receptors on human neuroblastoma cells.

We have investigated the effects of several neurokine/cytokine family members on the level of alpha-bungarotoxin-binding to neuronal nicotinic acetylcholine receptors. Exposure of human neuroblastoma cells (SH-SY5Y and IMR-32) to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or oncostatin-M resulted in a 30-40% decline in alpha-bungarotoxin receptors on the cells with no decrease seen in either muscarinic acetylcholine receptors or in L-type Ca2+ channels. The level of nicotinic receptor was not affected by the related cytokine, interleukin-6. Treatment of IMR-32 cells with 40 pM CNTF produced a half-maximal decrease of alpha-bungarotoxin binding which compared well with the affinity estimated from binding of 125I-CNTF (Ki approximately 40 pM) and the concentration causing c-fos activation in SH-SY5Y cells, as detected by nuclear run-on assays (60-120 pM). Previous results have indicated that the differentiating agents, phorbol esters and retinoic acid, also decrease nicotinic receptor numbers. Here the effects of CNTF, which did not induce neural differentiation, were enhanced by differentiation with 12-O-tetradecanoylphorbol 13-acetate (10 nM) and prevented by retinoic acid (10 microM). Therefore, the response of neuroblastoma cells to cytokines may be under developmental control. These cells offer a system to examine cytokine responses and signal transduction mechanisms during neural development.

Bungarotoxins↗
PubMed1996

Identification of functional receptors for ciliary neurotrophic factor on chick ciliary ganglion neurons.

Ciliary neurotrophic factor and an avian homolog, growth promoting activity, are members of the cytokine/neurokine family of trophic factors and have been proposed to function as survival and developmental factors for ciliary ganglion neurons in vivo. Here we identify for the first time functional receptors for ciliary neurotrophic factor and growth promoting activity on cultured ciliary ganglion neurons. [(125)I]Rat ciliary neurotrophic factor binding studies indicate that rat ciliary neurotrophic factor and growth promoting activity bind to these receptors with a single affinity, while human ciliary neurotrophic factor recognizes both a high- and low-affinity site. Comparison of the relative potency of human ciliary neurotrophic factor and avian growth promoting activity in biological assays indicates that growth promoting activity is three to five times more active in promoting survival and in regulating acetylcholine receptors. The binding of ciliary neurotrophic factor is specific, sensitive to phosphatidylinositol-specific phospholipase C and partially inhibited by leukemia inhibitory factor, but not inhibited by other members of the human neurokine family, including interleukin-6, interleukin-22 and oncostatin M. Cross-linking of [(125)I]rat ciliary neurotrophic factor to ciliary neurons results in the specific labeling of three proteins with estimated molecular masses of 153,000, 81,000 and 72,000. Only the 81,000 molecular weight component is released from the cells after treatment with phosphatidylinositol-specific phospholipase C, suggesting a membrane attachment via a glycosylphosphatidylinositol linkage. Stimulation with ciliary neurotrophic factor or growth promoting activity, but not by other neurokines, results in the rapid tyrosine phosphorylation of a 90,000 molecular weight protein that is inhibited by pretreatment with phosphatidylinositol-specific phospholipase C. In conclusion, we report here the pharmacological and functional properties of ciliary neurotrophic factor receptors on embryonic ciliary ganglion neurons. These results provide the means for elaborating the molecular mechanisms of ciliary neurotrophic factor action and understanding its physiological role in a defined neuronal population.

Animals↗
PubMed1995

Regulation of nicotinic acetylcholine receptors on human neuroblastoma cells during differentiation.

Neuronal nicotinic acetylcholine receptors are expressed on a variety of cells in the nervous system where they play key roles in synaptic transmission and information transfer. Little is known, however, about the molecular mechanisms that control their expression, distribution, and function during nervous system development. We have investigated the control of expression during differentiation of one class of acetylcholine receptors that bind alpha-bungarotoxin of human neuroblastoma cells. We report that induction of differentiation of SH-SY5Y, SK-n-SH or IMR-32 cells by the phorbol ester 12-O-tetradecanoyl phorbol 13-myristate (10 nM, TPA) or by retinoic acid resulted in as much as a 70% decline in alpha-bungarotoxin receptors on the cells. The response to the phorbol ester was blocked by the protein kinase C inhibitors staurosporine and bisindolylmaleimide. The decrease in receptors induced by 10 microM retinoic acid was not affected by either agent. However, responses to lower (10 nM) concentrations of retinoic acid were blocked by staurosporine but not bisindolylmaleimide, suggesting a dual mechanism of action for retinoic acid in regulating acetylcholine receptors. It appears that acetylcholine receptors on neuroblastoma cells are regulated during differentiation by both protein kinase C-dependent and -independent mechanisms.

Binding Sites↗
PubMed1995

Regulation of glibenclamide receptors in cultured chick cardiomyocytes.

We found that the expression of sulphonylurea receptors which control ATP-dependent potassium channels was up-regulated both by a receptor agonist and by potassium channel activators. Sulphonylurea receptors detected on cell membranes of cultured chick cardiomyocytes by [3H]glibenclamide were increased 2.8-fold following growth of the cells for 1-2 days in the presence of 30 nM glibenclamide. [3H]glibenclamide binding was also increased approximately 2-fold following growth of the cells in the presence of the ATP-dependent potassium channel activators, pinacidil, minoxidil and diazoxide, but was diminished to 75% of control following treatment with depolarizing concentrations of KCl.

Animals↗
PubMed1991

Regulation of acetylcholine receptors on chick ciliary ganglion neurons by components from the synaptic target tissue.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A soluble component of about 50 kDa from embryonic eye tissue, the synaptic target of the ganglion, increases the development of ACh sensitivity by the neurons 10-fold over a 1-week period in culture. The increased sensitivity does not arise from a change in agonist affinity or esterase activity. Both the basal ACh response obtained in the absence of the 50-kDa component and the elevated responses obtained with it can be inhibited by neuronal bungarotoxin (nBgt) but not by alpha-bungarotoxin (alpha Bgt). Increases of less than twofold are observed for the binding of anti-AChR monoclonal antibody 35 (mAb 35), nBgt, and alpha Bgt to the neurons under these conditions. Extract fractions containing the 50-kDa component also enable the neurons to enhance their ACh responses through a cAMP-dependent mechanism. Either the 50-kDa fraction induces the appearance of a new type of AChR regulated by cAMP, or it alters the function of existing AChRs. The 50-kDa fraction produces no change in neuronal growth but can increase GABA responses sixfold, indicating that its effects are not confined to AChRs. It is not clear whether a single molecular species is responsible for the diverse regulatory effects or whether several types of active components are present in the fraction. The component which enhances ACh sensitivity is trypsin-sensitive and heat-labile, as expected for a protein. The component may be widely distributed since the 50-kDa fraction from a number of tissues can increase the ACh response. The fraction from eye tissue, however, has a specific activity 5-10 times greater than that of the liver fraction. A wide distribution would suggest multiple targets and roles for the component during development.

Acetylcholine↗
PubMed1990

Cyclic AMP-dependent phosphorylation of a neuronal acetylcholine receptor alpha-type subunit.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A cAMP-dependent process has previously been shown to enhance the ACh response of the neurons 2- to 3-fold without requiring the synthesis of new receptors. We show here that the receptors can be phosphorylated in situ by a cAMP-dependent process. The phosphorylation occurs predominantly on components of 50 and 58 kDa. Both derive from putative ligand-binding alpha 3 subunits, with the smaller phosphorylated species probably representing a degradation product of the larger. The increase in receptor phosphorylation caused by incubating the neurons with a cAMP analog parallels the increase observed in the ACh response, with respect to both time course and relative extent. The phosphorylation of ciliary ganglion AChRs differs from that reported for electric organ AChRs, which occurs primarily on the non-ligand-binding gamma and delta subunits and increases the rate of agonist-induced receptor desensitization.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Subunit composition of nicotinic acetylcholine receptors from chick ciliary ganglia.

Nicotinic ACh receptors were immunoaffinity-purified from chick ciliary ganglia, radioiodinated, and examined by SDS-PAGE. Components with Mr's of 49, 52, and 60 kDa were obtained. Limited proteolysis produced different peptide maps from the components, confirming the 3 as distinct species. All are glycoproteins since treatment with glycopeptidase F altered their migration during electrophoresis. The 60 kDa component appears to be encoded by the AChR alpha 3 gene since it was selectively immunoprecipitated by an antiserum to a fusion protein containing a putative cytoplasmic region of the predicted alpha 3 gene product. The 49 kDa component selectively cross-reacted on immunoblots with 4 monoclonal antibodies that recognize a component of similar size in AChR preparations from chicken brain. The 52 kDa component is a novel species not previously identified in preparations of brain AChRs. If all 3 components represent integral AChR subunits, they may compose 2 receptor subtypes in the ganglion, e.g., one containing 49 and 60 kDa subunits and another containing 52 and 60 kDa subunits. This is supported by the finding that a receptor preparation can be obtained containing only the 49 and 60 kDa components and is consistent with reports of brain AChRs having only 2 types of subunits. Alternatively, ganglionic AChRs may contain 3 or more types of subunits, with at least one being selectively lost under certain conditions.

Animals↗
PubMed1989

Antisera against an acetylcholine receptor alpha 3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh-binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of alpha 3 ACh-binding subunits expressed in bacteria, we show that ganglionic AChRs contain alpha 3 ACh-binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit-specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.

Amino Acid Sequence↗
PubMed1989

Regulating the number and function of neuronal acetylcholine receptors.

Nicotinic acetylcholine receptors (AChRs) on vertebrate neurons represent a family of receptors distinct from the well-characterized AChR of skeletal muscle. New probes for neuronal AChRs are now being used to examine the regulation of receptor number and function. The results suggest that neuronal AChRs differ from muscle AChRs in regulation by presynaptic input and by at least one second messenger system, and that neuronal AChRs are additionally regulated by retrograde signals from the synaptic target tissue. The forms of regulation provide potential mechanisms by which cell-cell interactions could stabilize synaptic contacts on neurons and modulate synaptic function.

Animals↗
PubMed1989

Specific down-regulation of the alpha-bungarotoxin binding component on chick autonomic neurons by ciliary neuronotrophic factor.

Chick ciliary ganglion neurons have a cholinergic membrane component that binds alpha-bungarotoxin with high affinity but has no known function. The component is different from the nicotinic ACh receptor on the neurons that mediates cholinergic transmission through the ganglion. Ciliary neuronotrophic factor (CNTF) has been shown to enhance the survival of ciliary ganglion neurons in cell culture and has been postulated to act as a target-derived trophic factor for the neurons in vivo. We show here that a factor indistinguishable from CNTF specifically down-regulates alpha-bungarotoxin binding sites on the neurons while increasing cell growth and the number of ACh receptors on the cells. Similar effects, though reduced in magnitude, are seen with chick sympathetic neurons. CNTF has no effect on the number of ACh receptors found on chick myotubes in culture. The down-regulation of alpha-bungarotoxin binding sites on neurons caused by CNTF occurs with a half-time of about 19 hr and is largely reversed within a 4 d period following CNTF removal. It is distinct from the down-regulation caused by cholinergic agonists. Nerve growth factor and fibroblast growth factor have no apparent effect on the number of alpha-bungarotoxin binding sites on the neurons, though fibroblast growth factor does stimulate neuronal growth. The results indicate that the effects of CNTF on the alpha-bungarotoxin binding component are both novel for a growth factor and specific, and they suggest a relationship between the component and the regulation of growth by the target tissue.

Animals↗