Biomedical subjects

A C Charles

Publications and source records attributed to A C Charles.

At least 19 recordsLinked to original sources

PubMed2001

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1).

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.

4-Aminopyridine↗

PubMed2000

Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells.

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.

Adenylyl Cyclases↗

PubMed1999

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells.

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.

Calcium↗

PubMed1999

ATP released from astrocytes mediates glial calcium waves.

Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.

Adenosine Triphosphate↗

PubMed1999

Spontaneous action potentials initiate rhythmic intercellular calcium waves in immortalized hypothalamic (GT1-1) neurons.

GT1-1 cells exhibit spontaneous action potentials and transient increases in intracellular calcium concentration ([Ca2+]i) that occur in individual cells and as spatially propagated intercellular Ca2+ waves. In this study, simultaneous cell-attached patch-clamp recording of action currents (indicative of action potentials) and fluorescence imaging of [Ca2+]i revealed that Ca2+ transients in GT1-1 cells were preceded by a single action current or a burst of action currents. Action currents preceded Ca2+ transients in a similar pattern regardless of whether the Ca2+ transients were limited to the individual cell or occurred as part of an intercellular Ca2+ wave. Both the action currents and Ca2+ transients were abolished by 1 microM tetrodotoxin. Removal of extracellular Ca2+ abolished all spontaneous Ca2+ transients without inhibiting the firing of action currents. Nimodipine, which blocks L-type Ca2+ currents in GT1-1 cells, also abolished all spontaneous Ca2+ signaling. Delivery of small voltage steps to the patch pipette in the cell-attached configuration elicited action currents the latency to firing of which decreased with increasing amplitude of the voltage step. These results indicate that spontaneous intercellular Ca2+ waves are generated by a propagated depolarization, the firing of action potentials in individual cells, and the resulting influx of Ca2+ through L-type Ca2+ channels. These patterns of spontaneous activity may be important in driving the pulsatile release of GnRH from networks of cells.

Action Potentials↗

PubMed1998

A conditionally immortalized glial cell line that expresses mature myelin proteins and functional GABA(A) receptors.

We have isolated and characterized a conditionally immortalized glial cell line that expresses mature myelin proteins, as well as functional GABA(A) receptors. Glial cells were isolated from postnatal day 1 H-2Kb-tsA58 transgenic mice that contain the temperature-sensitive SV40 large T antigen oncogene under the control of an interferon-gamma-inducible promoter. A clonal line was isolated that grew rapidly under permissive conditions (33 degrees C in the presence of interferon-gamma), but not under nonpermissive conditions (37 degrees C in the absence of interferon-gamma). Cells expressed mRNAs of mature myelin proteins (myelin basic proteins and proteolipid protein) when grown under either permissive or nonpermissive conditions, but myelin basic proteins were detected only when cells were maintained at 37 degrees C. GABA induced an increase in intracellular calcium concentration, indicating the presence of functional receptors for this transmitter. Characterization of the GABA-evoked calcium response and whole-cell currents indicated that these responses were mediated by GABA(A) receptors. Reverse transcription-polymerase chain reaction revealed differential expression of mRNAs for specific GABA(A) receptor subunits. Messages for the alpha2, alpha4, beta1, and delta subunits were readily detected by reverse transcription-polymerase chain reaction, whereas messages for the alpha1, alpha3, alpha5, alpha6, beta2, beta3, and gamma1-3 subunits were not. As this cell line can be easily propagated and differentiated, it should provide a valuable tool in studying not only the development of oligodendroglia, but also the function of their transmitter receptors and myelin proteins.

Animals↗

PubMed1998

Spiral intercellular calcium waves in hippocampal slice cultures.

Complex patterns of intercellular calcium signaling occur in the CA1 and CA2 regions of hippocampal slice organotypic cultures from neonatal mice. Spontaneous localized intercellular Ca2+ waves involving 5-15 cells propagate concentrically from multiple foci in the stratum oriens and s. radiatum. In these same regions, extensive Ca2+ waves involving hundreds of cells propagate as curvilinear and spiral wavefronts across broad areas of CA1 and CA2. Ca2+ waves travel at rates of 5-10 mu m/s, are abolished by thapsigargin, and do not require extracellular Ca2+. Staining for astrocytes and neurons indicates that these intercellular waves occur primarily in astrocytes. The frequency and amplitude of Ca2+ waves increase in response to bath application of N-methyl-D-aspartate (NMDA) and decrease in response to removal of extracellular Ca2+ or application of tetrodotoxin. This novel pattern of intercellular Ca2+ signaling is characteristic of the behavior of an excitable medium. Networks of glial cells in the hippocampus may behave as an excitable medium whose spatial and temporal signaling properties are modulated by neuronal activity.

Animals↗

PubMed1996

Intercellular calcium waves in neurons.

Spontaneous intercellular Ca2+ waves were observed in groups of neurons in two different culture preparations: primary mouse cortical neurons and GT1-1 immortalized neurons. Waves of increased intracellular Ca2+ concentration propagated at rates of 100-200 microns/s over as many as 200 cells and were abolished by the removal of extracellular calcium, by nimodipine, by tetrodotoxin, and by the gap junction inhibitor octanol. A sister clone of the GT1 line, GT1-7 neurons, showed no intercellular Ca2+ waves and were found to have a significantly lower level of connexin26 mRNA than the GT1-1 line. Although we cannot definitively rule out a role for synaptic communication, we propose that intercellular Ca2+ waves in cultured neurons are generated by Ca2+ influx caused primarily by the propagation of depolarization via gap junctions. Intercellular Ca2+ signaling via gap junctions may represent an important mechanism for nonsynaptic neuronal signaling.

Animals↗

PubMed1995

Intercellular calcium waves mediated by diffusion of inositol trisphosphate: a two-dimensional model.

In response to mechanical stimulation of a single cell, airway epithelial cells in culture exhibit a wave of increased intracellular free Ca2+ concentration that spreads from cell to cell over a limited distance through the culture. We present a detailed analysis of the intercellular wave in a two-dimensional sheet of cells. The model is based on the hypothesis that the wave is the result of diffusion of inositol trisphosphate (IP3) from the stimulated cell. The two-dimensional model agrees well with experimental data and makes the following quantitative predictions: as the distance from the stimulated cells increases, 1) the intercellular delay increases exponentially, 2) the intracellular wave speed decreases exponentially, and 3) the arrival time increases exponentially. Furthermore, 4) a proportion of the cells at the periphery of the response will exhibit waves of decreased amplitude, 5) the intercellular membrane permeability to IP3 must be approximately 2 microns/s or greater, and 6) the ratio of the maximum concentration of IP3 in the stimulated cell to the Km of the IP3 receptor (with respect to IP3) must be approximately 300 or greater. These predictions constitute a rigorous test of the hypothesis that the intercellular Ca2+ waves are mediated by IP3 diffusion.

Animals↗

PubMed1995

Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1-7) neurons.

1. Individual immortalized gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons in semiconfluent cultures showed spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) as measured by video fluorescence microscopy and fura-2. In parallel experiments, GT1-7 neurons also showed spontaneous bursts of action potentials that were recorded as action currents from intact cells. The bursts of action currents occurred in characteristic patterns, suggesting an underlying rhythmic oscillation in membrane potential. 2. Depolarization with increased extracellular K+ evoked a concentration-dependent increase in the frequency of Ca2+ oscillations or a sustained plateau of increased [Ca2+]i in GT1-7 neurons. Increased extracellular K+ (30 mM) caused an initial increase in the frequency of action currents, after which they were reversibly abolished. 3. The Ca2+ channel blockers Ni2+ and nimodipine abolished Ca2+ oscillations, whereas nifedipine, gadolinium, omega-conotoxin and omega-agatoxin had no effect on Ca2+ oscillations. These results indicate that Ca2+ oscillations are generated by influx of Ca2+ through voltage-gated Ca2+ channels that are not sensitive to nifedipine and are not N-type or P-type channels. 4. Thapsigargin caused a small, transient rise in baseline [Ca2+]i but had no effect on Ca2+ oscillations. Caffeine and ryanodine had no effect on baseline [Ca2+]i or Ca2+ oscillations. These results indicate that the release of Ca2+ from inositol 1,4,5-trisphosphate (IP-3)-sensitive or caffeine sensitive intracellular stores does not play a major role in Ca2+ oscillations in GT1-7 neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1994

Mechanisms and function of intercellular calcium signaling.

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.

Animals↗

PubMed1994

Neurotransmitters and cytokines in CNS pathology.

In summary, we have demonstrated an in vitro model for oligodendrocyte cell death that may be relevant to events in formation of lesions in MS. It involves cell contact to oligodendrocytes with activated, viable microglia (or inflammatory macrophages), surface TNF-alpha, surface adhesion molecules, and production of NO. Precise mechanisms of TNF-alpha and ICAM-1/LFA-1 participation and the nature of the susceptibility of the oligodendrocyte are currently being studied.

AIDS Dementia Complex↗

PubMed1994

A model for the propagation of intercellular calcium waves.

In response to mechanical stimulation of a single cell, intercellular Ca2+ waves propagate through airway epithelial and glial cell cultures, providing a mechanism for intercellular communication. Experiments indicate that intercellular propagation of the Ca2+ wave is mediated by the movement of inositol 1,4,5-trisphosphate (IP3) through gap junctions. To explore the validity of this hypothesis, we have constructed and solved a system of partial differential equations that models the Ca2+ changes induced by the movement of IP3 between cells. The model is in good qualitative agreement with experimental data, including the behavior of the wave in the absence of extracellular Ca2+, the shape of the subsequent asynchronous Ca2+ oscillations, and the passage of a wave through a cell exhibiting Ca2+ oscillations. However, the concentration of IP3 that is required in each cell to propagate the wave may not be achieved by passive diffusion of IP3 through gap junctions from the stimulated cell. We therefore suggest that Ca(2+)-independent regenerative production of IP3 might be necessary for the propagation of intercellular Ca2+ waves.

Animals↗

PubMed1994

Glia-neuron intercellular calcium signaling.

There is increasing evidence for bidirectional communication between glial cells and neurons. In this study, calcium signaling in primary glia/neuron cultures was investigated using video fluorescence imaging and fura-2. Glial cells in culture without neurons showed occasional spontaneous intracellular Ca2+ oscillations but not intercellular Ca2+ waves. By contrast, glial cells in culture with neurons showed frequent spontaneous Ca2+ oscillations as well as propagated intercellular Ca2+ waves. These spontaneous glial intercellular Ca2+ waves often emanated from sites of contact with neurons, but were only occasionally associated with increases in neuronal Ca2+. Mechanical stimulation of a single glial cell induced a glial intercellular Ca2+ wave which was similar in its temporal and spatial characteristics to spontaneous glial Ca2+ waves. Mechanically induced glial Ca2+ waves, but not spontaneous Ca2+ waves, evoked a transient increase in [Ca2+]i or a change in the pattern of spontaneous Ca2+ oscillations in a small percentage (< 10%) of neighboring neurons. Mechanical stimulation of a single neuron consistently evoked an intercellular Ca2+ wave in neighboring glial cells. These results suggest distinct mechanisms for direct glial-neuronal and neuronal-glial communication. These signaling pathways may play important roles in both function and pathology in the central nervous system.

Animals↗

PubMed1994

GABA has excitatory actions on GnRH-secreting immortalized hypothalamic (GT1-7) neurons.

The effects of gamma-aminobutyric acid (GABA) on clonal gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons were investigated using patch-clamp and fura-2 imaging techniques. Local application of GABA (100 microM) to GT1-7 cells voltage-clamped in the whole-cell configuration immediately increased membrane conductance and noise consistent with activation of the GABAA receptor-Cl- channel complex. Depolarization activated transient Na+ currents which were abolished by tetrodotoxin (TTX; 0.5 microM), and more sustained Ca2+ currents. Under constant current conditions, GT1-7 cells fired spontaneous action potentials, and depending on the Cl- equilibrium potential, GABA either depolarized cells, causing a rapid activation of action potentials, or hyperpolarized cells. In order to determine the effect of GABA on intact cells, the cell-attached patch configuration was used to record extracellularly. Under these conditions, application of GABA (100 microM), but not the GABAB receptor agonist baclofen (10 microM), immediately evoked multiple action potentials. Measurement of [Ca2+]i using fluorescence video microscopy and fura-2 revealed spontaneous, transient, repetitive increases in [Ca2+]i which had a periodicity ranging from 1 to 60 s. These Ca2+ oscillations were abolished by TTX (1 microM) and by the removal of extracellular Ca2+. Application of GABA (1 and 10 microM) induced an immediate increase in [Ca2+]i in all cells and increased the frequency of Ca2+ oscillations in a dose-dependent manner. The GABA-induced increase in [Ca2+]i was abolished by bicuculline and by the removal of extracellular Ca2+, and was inhibited by TTX. Baclofen (1 microM) had no effect on [Ca2+]i. These results suggest that activation of GABAA receptors has an excitatory action on GnRH-secreting immortalized hypothalamic neurons caused by a Cl(-)-dependent depolarization. GABA has been reported to increase GnRH secretion; a direct stimulatory action of the neurotransmitter on GABAA receptors of GnRH-secreting hypothalamic neurons may be responsible for this effect.

Animals↗

PubMed1993

bcl-2 inhibits death of central neural cells induced by multiple agents.

The protooncogene bcl-2, which has been implicated in B-cell lymphoma development, inhibits apoptosis due to growth factor withdrawal in some, but not all, hematopoietic cells. Recently we found that bcl-2 also inhibits apoptosis in PC12 pheochromocytoma cells. We now report that bcl-2 inhibits the death of a central neural cell line due to serum and growth factor withdrawal, the calcium ionophore A23187, glucose withdrawal, membrane peroxidation, and, in some cases, free radical-induced damage. This broad range of protective effects of BCL-2 protein suggests that BCL-2 may interact with a central step in neural cell death. Measurements of intracellular free calcium suggest that BCL-2 alters the transduction of neural death signals at a point distal to the rise in intracellular free calcium.

Animals↗

PubMed1993

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin.

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca2+]i) that was communicated to surrounding cells. Following propagation of the Ca2+ wave, many cells showed asynchronous oscillations in [Ca2+]i. Dantrolene sodium (10 microM) inhibited the increase in [Ca2+]i associated with this Ca2+ wave by 60-80%, and prevented subsequent Ca2+ oscillations. Despite the markedly decreased magnitude of the increase in [Ca2+]i, the rate of propagation and the extent of communication of the Ca2+ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 microM) induced an initial increase in [Ca2+]i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca2+]i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of Ca2+ wave from the stimulated cell to adjacent cells. Glutamate (50 microM) induced an initial increase in [Ca2+]i in most cells that was followed by sustained oscillations in [Ca2+]i in some cells. Dantrolene (10 microM) inhibited this initial [Ca2+]i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 micron) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca2+ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca2+ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP3) mediates the propagation of Ca2+ waves whereas Ca(2+)-induced Ca2+ release amplifies Ca2+ waves and generates subsequent Ca2+ oscillations.

Animals↗

PubMed1992

Substance P stimulates IL-1 production by astrocytes via intracellular calcium.

There is increasing evidence that local substance P (SP) exacerbates peripheral inflammations, partly by stimulating production of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). SP may play similar roles in certain central nervous system inflammations. Multiple sclerosis plaques, for example, form around veins which are innervated by unmyelinated SP-containing fibers, and astrocytes in multiple sclerosis plaques stain for SP. We tested whether SP could stimulate IL-1 and TNF alpha production by cultured astrocytes and whether calcium was the second messenger in this process. We found that both SP and the calcium ionophore A23187 raised intracellular calcium ([Ca2+]i) and stimulated IL-1 production in astrocytes. SP also nonsignificantly increased TNF alpha production by astrocytes. Treatment with dibromo BAPTA/AM, an intracellular calcium buffer, blocked SP-induced IL-1 production. These findings indicate that SP induces IL-1 production by astrocytes and uses calcium as a second messenger. Our results indicate local SP may play a role in multiple sclerosis and certain other central nervous system inflammations.

Animals↗