Biomedical subjects

Wallace B Thoreson

Publications and source records attributed to Wallace B Thoreson.

9 recordsLinked to original sources

PubMed2003

Reciprocal interactions between calcium and chloride in rod photoreceptors.

This study used imaging and electrophysiological techniques in salamander retinal slices to correlate Ca2+ and Cl- levels in rods and thus test the hypothesis of a feedback interaction between Ca2+- and Ca2+-activated Cl- channels whereby Cl- efflux through Cl- channels can inhibit Ca2+ channels. Increasing [K+]o levels produced a concentration-dependent depolarization of rods accompanied by increases in [Ca2+]i measured with Fura-2. The voltage dependence of increases in [Ca2+]i was compared with the voltage dependence of the calcium current (ICa). [Cl-]i was measured with the dye, MEQ. Depolarization with high K+ to membrane potentials below -20 mV reduced [Cl-]i; larger depolarizations increased [Cl-]i. The Na/K/Cl cotransport inhibitor, bumetanide, shifted the apparent Cl- equilibrium potential (ECl) to more negative potentials, suggesting that this cotransporter helps establish a relatively depolarized ECl. MEQ fluorescence changes evoked by high K+ were inhibited by niflumic acid (0.1 mM), NPPB (2 microM), or replacement of Ca2+ with Ba2+, suggesting that depolarization-evoked Cl- changes result partly from stimulation of Ca2+-activated Cl- channels. Replacing >/=12 mM [Cl-]o with CH3SO4- produced a significant reduction in [Cl-]i. [Ca2+]i increases evoked by 20 or 50 mM K+ were also significantly inhibited by replacing >/=12 mM [Cl-]o with CH3SO4-. Thus modest depolarization can evoke increases in [Ca2+]i that lead to reductions in [Cl-]i, and conversely, reductions in [Cl-]i inhibit depolarization-evoked [Ca2+]i increases. These findings support the hypothesis that feedback interactions between Ca2+- and Ca2+-activated Cl- channels may contribute to the regulation of presynaptic Ca2+ currents involved in synaptic transmission from rod photoreceptors.

Ambystoma↗

PubMed2003

Endogenous adenosine reduces glutamatergic output from rods through activation of A2-like adenosine receptors.

Adenosine is released from retina in darkness; photoreceptors possess A2 adenosine receptors, and A2 agonists inhibit L-type Ca2+ currents (ICa) in rods. We therefore investigated whether A2 agonists inhibit rod inputs into second-order neurons and whether selective antagonists to A1, A2A, or A3 receptors prevent Ca2+ influx through rod ICa. [Ca2+]i changes in rods were assessed with fura-2. ICa in rods and light responses of rods and second-order neurons were recorded using perforated patch-clamp techniques in the aquatic tiger salamander retinal slice preparation. Consistent with earlier results using the A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), the A2A agonist CGS-21680 significantly inhibited ICa and depolarization-evoked [Ca2+]i increases in rods. The A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and A2A antagonist, ZM-241385, but not the A3 antagonist, VUF-5574, inhibited effects of adenosine on Ca2+ influx in rods. DPCPX and ZM-241385 also inhibited effects of CGS-21680, suggesting they both act at A2A receptors. Both A2 agonists, CGS-21680 and DPMA, reduced light-evoked currents in second-order neurons but not light-evoked voltage responses of rods, suggesting that activation of A2 receptors inhibits transmitter release from rods. The inhibitory effects of CGS-21680 on both depolarization-evoked Ca2+ influx and light-evoked currents in second-order neurons were antagonized by ZM-241385. By itself, ZM-241385 enhanced the light-evoked currents in second-order neurons, suggesting that endogenous levels of adenosine inhibit transmitter release from rods. The effects of these drugs suggest that endogenous adenosine activates an A2-like adenosine receptor on rods leading to inhibition of ICa, which in turn inhibits l-glutamate release from rod photoreceptors.

Adenosine↗

PubMed2003

Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay.

PURPOSE: The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS: Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RT-PCR, and electrophysiological analyses. RESULTS: The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogen-exposed retinal culture. CONCLUSIONS: The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.

Animals↗

PubMed2002

Adult corneal limbal epithelium: a model for studying neural potential of non-neural stem cells/progenitors.

Recent studies suggest that tissue-specific stem cells possess much wider potential for differentiation than previously thought and can, in some instances, even cross germ layer boundaries. However, information is lacking regarding the efficiency and the fidelity of their differentiation along heterologous lineages. To address these issues of transdifferentiation, we have analyzed the heterologous potential of stem cells within the same germ layer. We report the neural potential of cells isolated from the limbal epithelium of the adult cornea. Limbal epithelium, which, like the neuroepithelium, is ectodermally derived, participates in the regeneration of cornea throughout life. We have observed that limbal epithelial cells, when removed from their niche and cultured in the presence of mitogens, begin to express neural progenitor markers. Based on the self-renewal property, it is likely that the nestin-positive progenitors are derived from limbal stem cells rather than transit-amplifying (TA) cells that have limited proliferating potential. In differentiation conditions, a subset of these cells acquire neural morphology and express transcripts and proteins specific to neurons and glia, suggesting their differentiation along neural lineage. The acquisition of neural properties is regulated by BMP signaling. Neural differentiation of these cells is also observed upon heterotopic transplantation. Investigation of functional differentiation of cells by electrophysiological analysis reveals properties consistent with the presence of glia that are influenced by extracellular cues. However, similar analyses coupled with Ca(2+) imaging suggest an incomplete differentiation of limbal epithelial-derived neural progenitors into neurons in the condition studied. Our study, therefore, draws attention toward the necessity for rigorous characterization of transdifferentiation and offers a model for characterizing neural potential of heterologous stem cells/progenitors.

Animals↗

PubMed2002

Calcium-dependent inactivation and depletion of synaptic cleft calcium ions combine to regulate rod calcium currents under physiological conditions.

L-type Ca2+ currents (I(Ca)) in rod photoreceptors exhibit Ca2+-dependent inactivation. Perforated-patch whole-cell recordings were obtained from isolated rods of the tiger salamander using 1.8 mm Ca2+ in the bathing medium to determine the extent of Ca2+-dependent inactivation of I(Ca) with physiological [Ca2+] and endogenous buffering. I(Ca) was measured with voltage ramps applied before and after 5-s steps to -40, -30, -20, or -10 mV. Long depolarizing steps in isolated rods produced inactivation of I(Ca) ranging from 15% at -40 mV to > 80% at -10 mV. Because, in addition to Ca2+-dependent inactivation, depletion of synaptic cleft Ca2+ accompanying activation of I(Ca) can reduce presynaptic I(Ca) at calycal synapses, we investigated whether a similar mechanism worked at the invaginating rod synapse. Rods from retinal slices with intact synapses were compared with isolated rods in which synaptic cleft depletion is absent. I(Ca) was more strongly depressed by depolarization of rods in retinal slices, with ICa reduced by 47% following voltage steps to -40 mV. The depression of currents by depolarization was also greater for rods from retinal slices than isolated rods when Ca2+ was replaced with Ba2+ to reduce Ca2+-dependent inactivation. The stronger depolarization-evoked inhibition of I(Ca) in retinal slices compared to isolated rods probably reflects depletion of synaptic cleft Ca2+ arising from sustained Ca2+ influx. Inactivation of I(Ca) exhibited slow onset and recovery. These findings suggest that Ca2+-dependent inactivation and depletion of synaptic cleft Ca2+ may combine to regulate I(Ca) in response to light-evoked changes in rod membrane potential.

Ambystoma↗

PubMed2002

A2 adenosine receptors inhibit calcium influx through L-type calcium channels in rod photoreceptors of the salamander retina.

Presynaptic inhibition is a major mechanism for regulating synaptic transmission in the CNS and adenosine inhibits Ca(2+) currents (I(Ca)) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type Ca(2+) channels that regulate the release of L-glutamate. In the retina, adenosine is released in the dark when L-glutamate release is maximal. We tested whether adenosine inhibits I(Ca) and intracellular Ca(2+) increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both I(Ca) and the [Ca(2+)]i increase evoked by depolarization in a dose-dependent manner with approximately 25% inhibition at 50 microM. An A2-selective agonist, (N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N(6)-(1-methyl-2-phenylethyl)adenosine and N(6)-2-(4-aminophenyl)ethyladenosine, also inhibited I(Ca) and depolarization-induced [Ca(2+)]i increases. An inhibitor of protein kinase A (PKA), Rp-cAMPS, blocked the effects of DPMA on both I(Ca) and the depolarization-evoked [Ca(2+)]i increase in rods. The results suggest that activation of A2 receptors stimulates PKA to inhibit L-type Ca(2+) channels in rods resulting in a decreased Ca(2+) influx that should suppress glutamate release.

Adenosine↗

PubMed2002

Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells.

PURPOSE: Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K(+) currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS: Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS: LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to G(alpha(i)/o/t/z). LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-1, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca(2+)](i) increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca(2+)](i) increases were blocked by PD98059 and by loading cells with antibodies to G(alpha(i)/o/t/z). RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS: LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca(2+) from intracellular stores that appears independent of IP(3) and ryanodine receptor activation.

Animals↗

PubMedDate not supplied

D2-like dopamine receptors promote interactions between calcium and chloride channels that diminish rod synaptic transfer in the salamander retina.

Activation of D2-like dopamine receptors in rods with quinpirole stimulates L-type calcium currents (ICa). This result appears inconsistent with studies showing that D2-like dopamine receptor activation diminishes rod signals in second-order retinal neurons. Since small reductions in [Cl-] can inhibit photoreceptor ICa, we tested the hypothesis that enhancement of ICa with the D2/D4 receptor agonist, quinpirole, increases calcium-activated chloride currents (ICl(Ca)) causing an efflux of Cl- from rods that would provide a negative feedback inhibition of ICa. In agreement with studies from Xenopus, quinpirole reduced rod input to second-order neurons of tiger salamander retina without significantly altering rod voltage responses. Quinpirole also diminished the amplitude of depolarization-evoked increases in [Ca2+]i measured with Fura-2 in rods, a finding consistent with inhibition of synaptic transmission from rods. Electrophysiological and Cl(-)-imaging experiments indicated ECl in rods is approximately -20 mV. Quinpirole enhanced ICl(Ca) and elicited an efflux of Cl- at the resting potential. A similar Cl- efflux was produced by extracellular replacement of 24 mM Cl- with CH3SO4- and this low Cl- solution inhibited Ca2+ responses to a similar degree as quinpirole did. When ICl(Ca) was inhibited with niflumic acid, quinpirole enhanced both ICa and depolarization-evoked increases in [Ca2+]i. Furthermore, with niflumic acid, quinpirole no longer inhibited rod inputs into horizontal and bipolar cells. These results suggest an initial enhancement of ICa by quinpirole is followed by a stimulation of Cl- currents, including ICl(Ca). The net result is a Cl- efflux that inhibits depolarization-evoked increases in [Ca2+]i and synaptic transmission from rods.

Ambystoma↗

PubMedDate not supplied

Contrast encoding in retinal bipolar cells: current vs. voltage.

To investigate the influence of voltage-sensitive conductances in shaping light-evoked responses of retinal bipolar cells, whole-cell recordings were made in the slice preparation of the tiger salamander, Ambystoma tigrinum. To study contrast encoding, the retina was stimulated with 0.5-s steps of negative and positive contrasts of variable magnitude. In the main, responses recorded under voltage- and current-clamp modes were remarkably similar. In general agreement with past results in the intact retina, the contrast/response curves were relatively steep for small contrasts, thus showing high contrast gain; the dynamic range was narrow, and responses tended to saturate at relatively small contrasts. For ON and OFF cells, linear regression analysis showed that the current response accounted for 83-93% of the variance of the voltage response. Analysis of specific parameters of the contrast/response curve showed that contrast gain was marginally higher for voltage than current in three of four cases, while no significant differences were found for half-maximal contrast (C50), dynamic range, or contrast dominance. In sum, the overall similarity between current and voltage responses indicates that voltage-sensitive conductances do not play a major role in determining the shape of the bipolar cell's contrast response in the light-adapted retina. The salient characteristics of the contrast response of bipolars apparently arise between the level of the cone voltage response and the postsynaptic current of bipolar cells, via the transformation between cone voltage and transmitter release and/or via the interaction between the neurotransmitter glutamate and its postsynaptic receptors on bipolar cells.

Animals↗