Biomedical subjects

Stuart E Dryer

Publications and source records attributed to Stuart E Dryer.

17 recordsLinked to original sources

PubMed2006

Evidence for two-pore domain potassium channels in rat cerebral arteries.

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.

Actins↗

PubMed2005

Growth factors mobilize multiple pools of KCa channels in developing parasympathetic neurons: role of ADP-ribosylation factors and related proteins.

In developing ciliary ganglion (CG) neurons, movement of functional large-conductance (BK type) Ca(2+)-activated K+ (K(Ca)) channels to the cell surface is stimulated by the endogenous growth factors TGF(beta)1 and beta-neuregulin-1 (NRG1). Here we show that a brief NRG1 treatment (0.5-1.5 h) mobilizes K(Ca) channels in a post-Golgi compartment, but longer treatments (>3.5 h) mobilize K(Ca) channels located in the endoplasmic reticulum or Golgi apparatus. Specifically, the effects of 3.5 h NRG1 treatment were completely blocked by treatments that disrupt Golgi apparatus function. These include inhibition of microtubules, or inhibition of the ADP-ribosylation factor-1 (ARF1) system by brefeldin A, by over-expression of dominant-negative ARF1, or over-expression of an ARF1 GTPase-activating protein that blocks ARF1 cycling between GTP- and GDP-bound states. These treatments had no effect on stimulation of K(Ca) evoked by 1.5 h treatment with NRG1, indicating that short-term responses to NRG1 do not require an intact Golgi apparatus. By contrast, both the acute and sustained effects of NRG1 were inhibited by treatments that block trafficking processes that occur close to the plasma membrane. Thus mobilization of K(Ca) was blocked by treatments than inhibit ADP-ribosylation factor-6 (ARF6) signaling, including overexpression of dominant-negative ARF6, dominant-negative ARNO, or dominant-negative phospholipase D1. TGF(beta)1, the effects of which on K(Ca) are much slower in onset, is unable to selectively mobilize channels in the post-Golgi pool, and its effects on K(Ca) are completely blocked by inhibition of microtubules, Golgi function and also by plasma membrane ARF6 and phospholipase D1 signaling.

ADP-Ribosylation Factor 1↗

PubMed2005

Measuring circadian rhythms in olfaction using electroantennograms.

Circadian clocks control daily rhythms in many behavioral, physiological, and metabolic processes. Despite remarkable advances in our understanding of the circadian timekeeping mechanism and how it responds to environmental cycles, relatively little is known about how the timekeeping mechanism regulates behavior, physiology, and metabolism. One of the most extensively characterized timekeeping mechanisms is that of Drosophila melanogaster. In this species, autonomous circadian clocks are found in many neuronal and nonneuronal tissues, including essentially all sensory structures. We have shown that sensory neurons in the antenna mediate a robust rhythm in electrophysiological responses to the food odorant ethyl acetate. This article describes how rhythms in olfactory responses are measured and provides a perspective on the generality of these rhythms and their regulation by the clock.

Animals↗

PubMed2005

The p38 mitogen-activated protein kinase pathway negatively regulates Ca2+-activated K+ channel trafficking in developing parasympathetic neurons.

The trafficking of large-conductance Ca2+-activated K+ channels (K(Ca)) in chick ciliary ganglion neurons is regulated by growth factors. Here we show that a canonical p38 cascade inhibits K(Ca) trafficking in ciliary ganglion neurons. Two different p38 inhibitors (SB202190 or SB203580) or over-expression of dominant-negative forms of several components of the p38 cascade increased K(Ca) in ciliary neurons. Inhibition of protein synthesis or Golgi processing had no effect on this phenomenon, suggesting that p38 is acting at a distal step of the trafficking pathway. Depolymerization of filamentous actin (F-actin) increased functional expression of K(Ca), whereas stabilization of F-actin inhibited the effect of SB202190 on K(Ca) trafficking. SB202190 also caused an immunochemically detectable increase in K(Ca) on the plasma membrane. Inhibition of p38 decreased the extent of cortical F-actin in ciliary neurons. Macroscopic K(Ca) is suppressed by transforming growth factor (TGF) beta3. Application of TGFbeta3 increased the phosphorylation of p38 in ciliary neurons and increased cortical F-actin. Thus, the p38 signaling cascade endogenously suppresses development of functional K(Ca), in part by stabilizing an F-actin barrier that prevents plasma membrane insertion of functional channel complexes. This cascade also appears to mediate inhibitory effects of TGFbeta3 on the expression of K(Ca).

Actins↗

PubMed2004

Akt activation is necessary for growth factor-induced trafficking of functional K(Ca) channels in developing parasympathetic neurons.

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Circadian and cAMP-dependent modulation of retinal cone cGMP-gated channels does not require protein synthesis or calcium influx through L-type channels.

Circadian oscillators of chicken retinal cone photoreceptors modulate the gating properties of cGMP-gated channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. This effect is driven in part by cAMP, which acts through Erk MAP kinase to initiate a cascade leading to modulation of CGNCs. Here, we show that cAMP effects on the gating properties CNGCs persist when protein synthesis is blocked. The effects is cAMP also persist when calcium influx through L-type channels is blocked by nitrendipine. The mechanisms whereby cAMP modulates CNGCs therefore differ from those previously reported to underline regulation of melatonin synthesis and secretion.

Animals↗

PubMed2004

A-current expression is regulated by activity but not by target tissues in developing lumbar motoneurons of the chick embryo.

The functional expression of A-type K+ channels (IA) was examined in chick lumbar motoneurons (LMNs) at embryonic days 6 and 11 (E6 and E11). We observed a threefold increase in IA density between E6 and E11 in spinal cord slices and acutely dissociated LMNs. There was no change in current density, kinetics, or voltage dependence of IA in E11 homozygous limbless mutants or in E11 embryos in which hindlimbs were surgically removed at E6. Moreover, chronic in ovo administration of D-tubocurarine, which causes an increase in motoneuron branching on the surface of target muscles, had no effect on IA. Electrical activity played an important role in IA regulation in LMNs in vitro and in ovo. Blocking spontaneous electrical activity of LMNs by chronic in ovo application of mecamylamine or muscimol reduced IA by 80%. LMNs cultured in the presence of TTX also failed to express normal densities of IA, even when the cultures also contained target tissues. The portion of IA that remained after in ovo or in vitro blockade of activity inactivated more quickly than the IA of LMNs that were allowed to discharge spikes. The developmental expression of LMN IA increases significantly during development, and this increase is activity dependent but does not require interactions with target tissues. Ongoing activity also seems to regulate the kinetics of IA inactivation.

Animals↗

PubMed2004

Circadian clocks in antennal neurons are necessary and sufficient for olfaction rhythms in Drosophila.

BACKGROUND: The Drosophila circadian clock is controlled by interlocked transcriptional feedback loops that operate in many neuronal and nonneuronal tissues. These clocks are roughly divided into a central clock, which resides in the brain and is known to control rhythms in locomotor activity, and peripheral clocks, which comprise all other clock tissues and are thought to control other rhythmic outputs. We previously showed that peripheral oscillators are required to mediate rhythmic olfactory responses in the antenna, but the identity and relative autonomy of these peripheral oscillators has not been defined. RESULTS: Targeted ablation of lateral neurons by using apoptosis-promoting factors and targeted clock disruption in antennal neurons with newly developed dominant-negative versions of CLOCK and CYCLE show that antennal neurons, but not central clock cells, are necessary for olfactory rhythms. Targeted rescue of antennal neuron oscillators in cyc(01) flies through wild-type CYCLE shows that these neurons are also sufficient for olfaction rhythms. CONCLUSIONS: Antennal neurons are both necessary and sufficient for olfaction rhythms, which demonstrates for the first time that a peripheral tissue can function as an autonomous pacemaker in Drosophila. These results reveal fundamental differences in the function and organization of circadian oscillators in Drosophila and mammals and suggest that components of the olfactory signal transduction cascade could be targets of circadian regulation.

ARNTL Transcription Factors↗

PubMed2004

Circadian regulation of cGMP-gated channels of vertebrate cone photoreceptors: role of cAMP and Ras.

Circadian oscillators in chicken cone photoreceptors regulate the gating properties of cGMP-gated cationic channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. Here we show that cAMP, acting through protein kinase A (PKA), Ras, and Erk, is part of the circadian output pathway controlling CNGCs. Endogenous and exogenous cAMP cause activation of Erk and Ras, which are more active at night in cones, and increase the apparent affinity of CNGCs for cGMP. The Ras farnesyl transferase inhibitor manumycin-A, and a dominant-negative form of Ras (RasN17) block the circadian rhythms in CNGC gating, as well as the effects of cAMP. A dominant-negative form of the MEK kinase B-Raf also blocks circadian and cAMP modulation of CNGCs. The circadian output pathway modulating CNGC channels is comprised in part of cAMP --> PKA --> Ras --> B-Raf --> MEK --> Erk --> --> CNGCs. cAMP activation of Ras and Erk occur within minutes, whereas modulation of CNGCs requires >1 hr. However, cAMP protagonists do not alter rhythms in cPer2 mRNA, and their effects on CNGCs cannot be attributed to clock phase-shifting.

Animals↗

PubMed2003

Ras is a mediator of TGFbeta1 signaling in developing chick ciliary ganglion neurons.

Large-conductance Ca(2+)-activated K(+) channels (K(Ca)) in chick ciliary ganglion neurons are regulated by target-derived TGFbeta1. Here we show that TGFbeta1 stimulation of K(Ca) expression was blocked by the structurally dissimilar Ras protein farnesyl transferase inhibitors manumycin-A and FTI-277. A similar effect was produced in ciliary neurons overexpressing RasN17, a widely used dominant-negative form of Ras. Moreover, TGFbeta1-evoked increases in phosphorylation of SMAD2 were reduced by manumycin-A, suggesting that Ras-dependent transduction cascades activated by TGFbeta1 feed back onto SMAD signaling. Thus, Ras is a mediator of pleiotropic TGFbeta1 signaling in developing neurons.

Alkyl and Aryl Transferases↗

PubMed2003

Circadian phase-dependent modulation of cGMP-gated channels of cone photoreceptors by dopamine and D2 agonist.

The affinity of cGMP-gated ion channels (CNGCs) for cGMP in chick retinal cone photoreceptors is under circadian control. Here we report that dopamine (DA) and D2 receptor agonists evoke phase-dependent shifts in the affinity of CNGCs for activating ligand. Inside-out patch recordings from cultured chick cones were performed at circadian time (CT) 4-7 and CT 16-19 on the second day of constant darkness. Exposing intact cells to DA or the D2 agonist quinpirole for 2 hr before patch excision caused a significant increase in the K(D) for cGMP during the night (CT 16-19) but had no effect during the day (CT 4-7). DA or quinpirole treatment had no effect on the Hill slope or the average number of channels per patch. The effect of DA was blocked by the D2 antagonist eticlopride and was not mimicked by D1 agonists or blocked by D1 antagonists. By contrast, a brief (15 min) exposure to DA or quinpirole caused a decrease in K(D) during the subjective day and had no effect during the subjective night. Thus, the effect of D2 agonists depends on both the duration of agonist exposure and the time of day. Application of DA or quinpirole evoked a transient activation of the MAP kinase Erk (extracellular signal-related kinase) during the day but caused a sustained inhibition during the night. Conversely, D2 agonists caused activation of Ca2+/calmodulin-dependent protein kinase II during the night and inhibited this enzyme during the day. A circadian oscillator in cones appears to regulate the nature of the transduction cascade used by D2 receptors.

Animals↗

PubMed2003

Molecular identification of the Na+-activated K+ channel.

Progress in understanding sodium-activated potassium channels (K(Na)), suggested to function in excitable cells both during physiological conditions and protectively during hypoxia, has been limited by their unknown molecular identity. In this issue of Neuron, Salkoff and coworkers now show that members of the Slo gene family, Slo2.1 and Slo2.2, encode functional K(Na) channels.

Animals↗

PubMed2003

Expression of K(Ca) channels in identified populations of developing vertebrate neurons: role of neurotrophic factors and activity.

Changes in the intrinsic spike discharge properties in one neuronal population can alter the functions and even the formation of an entire neuronal network. Therefore it is important to understand the factors that regulate acquisition of a mature electrophysiological phenotype. Here we focus on large-conductance K(Ca) channels, which shape the pattern of repetitive discharge and which are therefore likely to play a role in the refinement of neural networks during development. In the parasympathetic ciliary ganglion of chick, the developmental expression of K(Ca) channels coincides with stages at which ciliary cells form synapses with target tissues. Moreover, K(Ca) expression requires formation of synapses with target tissues, and with afferent preganglionic inputs. The trophic effect of targets is mediated by TGFbeta1, whereas the effect of the preganglionic input is mediated by an isoform of beta-neuregulin-1. These trophic factors act synergistically, and this appears to be a normal feature of their actions in vivo. The acute effects of TGFbeta1 entail translocation of preexisting K(Ca) channels from intracellular stores to the plasma membrane. This requires activation of the signaling enzymes Ras, Erk MAP kinase and PI3 kinase. TGFbeta1 also causes a more sustained increase in K(Ca) channels (i.e. for up to 2 weeks) that requires synthesis of new channel proteins. Inductive regulation of K(Ca) expression is also observed in CNS cells that form more complex networks. In lumbar motoneurons, the largest changes in K(Ca) expression coincide with the elimination of synapses with hindlimb targets. Interactions with target tissues play a key role in regulation of motoneuron K(Ca) expression, and this trophic effect of target muscle is mediated by GDNF or a closely related factor. In addition, K(Ca) expression in motoneurons is dependent on ongoing electrical activity both in vivo and in vitro. This provides an additional mechanism for use-dependent refinement of neural networks during embryonic development.

Animals↗

PubMed2003

Central and peripheral circadian oscillators in Drosophila.

Drosophila circadian oscillators comprise interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLOCK (CLK) and CYCLE (CYC) bind E-box elements to activate per and tim transcription, and we now show that at the same time CLK-CYC repress Clk by activating the transcriptional repressor vrille (vri), thus accounting for the opposite cycling phases of these transcripts and identifying vri as the negative component of the Clk-feedback-loop. The core oscillator mechanism is assumed to be the same for oscillators in different tissues. However, we have shown that CRYPTOCHROME (CRY) has a light-independent function in the oscillator that controls olfaction rhythms, suggesting that CRY may function within the oscillator mechanism itself as it does in mammals. These olfaction rhythms require the function of 'peripheral' oscillators which are distinct from the 'central' lateral neuron (LN) oscillators that mediate locomotor activity rhythms. Preliminary results show that antennal oscillator cells are sufficient and LNs are not necessary for olfaction rhythms, indicating that unlike the situation in mammals, the central oscillator has little impact on the olfaction rhythm oscillator under these conditions.

Animals↗

PubMed2002

Glial cell line-derived neurotrophic factor and target-dependent regulation of large-conductance KCa channels in developing chick lumbar motoneurons.

The functional expression of large-conductance Ca2+-activated K+ (K(Ca)) channels in lumbar motoneurons (LMNs) of the developing chick embryo is regulated in part by interactions with striated muscle target tissues. Here we show that the functional expression of K(Ca) channels in LMNs developing in vitro can be stimulated by application of a skeletal muscle extract (MEX) or by coculture with hindlimb myotubes. A similar stimulation of K(Ca) channels in vitro can be produced by the trophic factors glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor but not by neurotrophin (NT)-3 or NT-4. The actions of MEX and hindlimb myotubes are blocked by a GDNF-neutralizing antiserum. Moreover, injection of this same antiserum into the embryonic hindlimb reduced the functional expression of K(Ca) channels in vivo to levels seen in LMNs deprived of interactions with the hindlimb. The effects of GDNF on K(Ca) channel expression in LMNs require 24 hr of continuous exposure to reach maximum and are blocked by the translation inhibitor anisomycin, indicating the need for synthesis of new proteins. GDNF actions are also blocked by the farnesyl transferase inhibitor manumycin, suggesting a role for Ras in the actions of GDNF. Finally, the actions of GDNF are inhibited by PP2, an inhibitor of Src family tyrosine kinases, and by LY29003, an inhibitor of phosphatidylinositol 3 kinases, but not by PD98059, an inhibitor of the Erk signaling cascade. None of these treatments alter expression of voltage-activated Ca2+ channels. Thus, the actions of GDNF on LMN K(Ca) channel expression appear to use a transduction pathway similar to that used for regulation of apoptosis.

Animals↗

PubMed2002

Activity- and target-dependent regulation of large-conductance Ca2+-activated K+ channels in developing chick lumbar motoneurons.

The functional expression of large-conductance (BK-type) Ca2+-activated K+ (K(Ca)) channels was examined in developing chick lumbar motoneurons (LMNs) between embryonic day 6 (E6) and E13 using patch-clamp recording techniques. The macroscopic K(Ca) current of E13 LMNs is inhibited by iberiotoxin and resistant to apamin. The average macroscopic K(Ca) density was low before E8 and increased 3.3-fold by E11, with an additional 1.8-fold increase occurring by E13. BK-type K(Ca) channels could not be detected in inside-out patches from E8 LMNs but were readily detected at E11. The density of voltage-activated Ca2+ currents did not change between E8 and E11. Surgical ablation of target tissues at E5 caused a significant reduction in average K(Ca) density in LMNs measured at E11. Conversely, chronic in ovo administration of d-tubocurarine, which causes an increase in motoneuron branching on the surface of the muscle target tissue, evoked a 1.8-fold increase in average LMN K(Ca) density measured at E11. Electrical activity also contributed to developmental regulation of LMN K(Ca) density. A significant reduction in E11 K(Ca) density was found after chronic in ovo treatment with the neuronal nicotinic antagonist mecamylamine or the GABA receptor agonist muscimol, agents that reduce activation of LMNs in ovo. Moreover, 3 d exposure to depolarizing concentrations of external K+ to LMNs cultured at E8 caused an increase in K(Ca) expression. Conversely, tetrodotoxin caused a decrease in K(Ca) expression in cultured E8 LMNs developing for 3 d in the presence of neurotrophic factors that promote neuronal survival in the absence of target tissues.

Animals↗

PubMed2002

Developmental regulation of neuronal K(Ca) channels by TGFbeta1: an essential role for PI3 kinase signaling and membrane insertion.

TGFbeta1 is a target-derived factor responsible for the developmental expression of large-conductance Ca(2+)-activated K(+) (K(Ca)) channels in ciliary neurons of the chick ciliary ganglion. The acute effects of TGFbeta1 on K(Ca) channels are mediated by posttranslational events and require activation of the MAP kinase Erk. Here we show that TGFbeta1 evokes robust phosphorylation of Akt/PKB, a protein kinase dependent on the products of phosphatidylinositol 3-OH kinase (PI3K). TGFbeta1-evoked stimulation of K(Ca) channels is blocked by the PI3K inhibitors wortmannin and LY294002. These drugs also inhibit TGFbeta1 effects on Akt/PKB phosphorylation but have no effect on TGFbeta1-evoked Erk activation. Application of the MEK1 inhibitor PD98059 blocked TGFbeta1 effects on Erk but had no effect on Akt/PKB phosphorylation. These results indicate that PI3K and Erk represent parallel signaling cascades activated by TGFbeta1 in ciliary neurons. The effects of TGFbeta1 on functional expression of K(Ca) are blocked by the microtubule inhibitors colchicine and nocodazole, by botulinum toxins A and E, and by brefeldin-A, an agent that disrupts the Golgi apparatus. These data indicate that translocation of a membrane protein, possibly Slowpoke (SLO), is required for the acute posttranslational effects of TGFbeta1 on K(Ca) channels. Confocal immunofluorescence studies with three different SLO antisera showed robust expression of SLO in multiple intracellular compartments of embryonic day 9-13 ciliary neurons, including the cell nucleus. These data suggest that TGFbeta1 evokes insertion of SLO channels into the plasma membrane as a result of signaling cascades that entail activation of Erk and PI3K.

3-Phosphoinositide-Dependent Protein Kinases↗