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R Reitstetter

Publications and source records attributed to R Reitstetter.

13 recordsLinked to original sources
PubMed2000

Antisense knockdown of calcium-dependent K+ channels in developing cerebellar Purkinje neurons.

Normal developmental upregulation of K(Ca) channel activity in cultured rat cerebellar Purkinje neurons was selectively inhibited by antisense oligonucleotide sequence (3 microM) targeted against the rslo transcript. The knockdown was specific; delayed rectifier and apamin-sensitive K+ channel abundances in Purkinje neurons were not affected by rslo antisense. Sense oligonucleotides (3 microM), used as a control, had no effect on channel abundance. Quantitative morphometric analyses of anti-calbindin-labeled Purkinje neurons showed no differences between neurons in control, sense and antisense treatment groups, and confirmed that the presence of the added oligonucleotide in the sense and antisense treatment conditions had no discernable toxic effects on neuronal health, for which neurite outgrowth is a sensitive indicator. These results confirm the identification of the developmentally regulated K(Ca) channel as the product of the gene rslo in cerebellar Purkinje neurons.

Animals↗
PubMed1999

Dependence of nicotinic acetylcholine receptor recovery from desensitization on the duration of agonist exposure.

When subjected to prolonged exposure to nicotinic agonists, nicotinic acetylcholine receptors undergo desensitization, resulting in an inactive receptor that does not allow for the passage of ions. The induction of desensitization of diverse nicotinic acetylcholine receptor subtypes in muscle, ganglia, or brain is likely to play important modulatory roles in synaptic transmission. Furthermore, nicotinic receptor desensitization may contribute to behavioral changes in humans or animals subjected to prolonged nicotine exposure pharmacologically or through the use of tobacco products. We investigated the recovery from desensitization of muscle-type nicotinic acetylcholine receptors in TE671/RD cells induced by exposure to acetylcholine or nicotine. Rates of recovery from desensitization are dependent on the length of agonist exposure and on the agonist used to induce desensitization. Increasing the time of exposure results in an increase in the time constant of recovery for both agonists. The recovery from nicotine-induced desensitization is consistently faster than the recovery from acetylcholine-induced desensitization regardless of whether nicotine or acetylcholine is used to assess levels of desensitization. These findings suggest the existence of more than one state of receptor desensitization and that nicotinic agonists vary in their efficiency of inducing receptors to states of differing depths of desensitization.

Acetylcholine↗
PubMed1998

Morphological consequences of altered calcium-dependent transmembrane signaling on the development of cultured cerebellar Purkinje neurons.

Morphometric analyses of cultured rat Purkinje neurons, visualized with anti-calbindin, demonstrated that elevated KCl (10 mM) significantly increased dendritic outgrowth and branching. The response was blocked by NiCl2 (50 microM; R-type Ca2+ channel antagonist). Cells grown in low external Ca2+ (100 nM) showed no loss of responsiveness to elevated potassium. However, thapsigargin (1 microM; Ca(2+)-ATPase blocker) inhibited dendrite outgrowth, suggesting that intracellular calcium stores may be important in governing development.

Animals↗
PubMed1998

Regulation of Ca2+-dependent K+ channel expression in rat cerebellum during postnatal development.

Potassium channels govern duration and frequency of excitable membrane events and may regulate signals that are important in neuronal development. This study assesses the developmental expression of the large conductance Ca2+-dependent K+ channel in vivo and in vitro in rat cerebellum. In vivo, transcript levels for the Ca2+-dependent K+ channel (KCa) were shown by Northern analysis to increase during development, whereas transcript levels for the voltage-gated K+ channel Kv3.1, a delayed rectifier (KD), remained relatively constant. A comparable pattern was demonstrated by expression in Xenopus oocytes of poly(A)-enriched RNA isolated from postnatal rat cerebella. In cerebellar cultures, increased external K+ provided a simple manipulation of cell excitability that influenced KCa transcript levels during development. With low external K+ (5.3 mM), the levels of KCa channel transcript (assessed by semiquantitative PCR) remained constant throughout development. However, in culture medium that supported significant dendritic outgrowth (10 mM extracellular K+), an upregulation of KCa transcript level was observed similar to that seen in vivo. Tetraethylammonium (TEA; 1 mM) similarly enhanced KCa expression, suggesting that depolarizing stimuli increased KCa expression. The stimulatory effects of increased K+ or TEA on KCa expression required extracellular Ca2+ and were abolished in low external calcium (0.1 mM, buffered with EGTA), although morphological development and survival were not impaired. The regulation of KCa channel expression by depolarization and Ca2+ entry provides evidence of a logical feedback mechanism governing Ca2+ signals that may be significant in cerebellar development.

Animals↗
PubMed1995

Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept.

The ubiquity of cholesterol in cell membranes and changes in its concentration during development, aging and in various diseases suggest that it plays an important role in modulating cell function. We examined this possibility by monitoring the effects of cholesterol on the activity of the calcium-activated potassium (BK) channel reconstituted into lipid bilayers from rat brain homogenates. Increasing the cholesterol concentration to 11% of total lipid weight resulted in a 70% reduction in channel mean open time and a reduction of the open probability of the channel by 80%. Channel conductance was reduced by 7%. Cholesterol is known to change the order state and the modulus of compressibility of bilayers. These physico-chemical changes may be translated into an overall increase in the structural stress in the bilayer, and this force may be transmitted to proteins residing therein. By examining the characteristics of the BK channel as a function of temperature, in the presence and absence of cholesterol, we were able to estimate the activation energy based on Arrhenius plots of channel kinetics. Cholesterol reduced the activation energy of the BK channel by 50% for the open to closed transition. This result is consistent with an increased stress energy in the bilayer and favors the channel moving into the closed state. Taken together, these data are consistent with a model in which cholesterol induces structural stress which enhances the transition from the open to the closed state of the channel. We suggest that this is an important mechanism for regulating the activity of membrane-integral proteins and therefore membrane function, and that the concept of structural stress may be relevant to understanding the modulation of ion channel activity in cell membranes.

Animals↗
PubMed1995

Lipid-ion channel interactions: increasing phospholipid headgroup size but not ordering acyl chains alters reconstituted channel behavior.

We have recently shown (Chang et al., 1995) that lipid-channel interactions, exemplified by the effects of cholesterol on the calcium-activated potassium (BK) channel, profoundly affect channel properties. The present study further explores such interactions by monitoring changes in BK channel behavior after reconstitution into bilayers where the size of phospholipid (PL) headgroups is increased and where the freedom of motion (inverse order) of fatty acid chains is incremented. Increasing the PL headgroup cross-sectional area, from that of N-meth-DOPE to that of DOPC (an increase from ca. 60 to 70 A2), is associated with a doubling of the channel mean opentime. Channel conductance, however, was unaffected. Increasing the order of the fatty acid chains, from that of DOPE to POPE and to that of DEPE, had no significant effect on channel properties (at 22 degrees C). We interpret the changes reported here to reflect lipid-protein interactions through the induction of structural stress related to the headgroup structures of phospholipids.

Fatty Acids↗
PubMed1994

Oxotremorine-M activates single nicotinic acetylcholine receptor channels in cultured Xenopus myocytes.

Oxotremorine methiodide (oxotremorine-M) is the quaternary amine derivative of oxotremorine and is known to be a potent and oft-reported pure, muscarinic receptor agonist. We report here, for the first time, that oxotremorine-M also has strong nicotinic actions at the single channel level. Although previous reports have suggested that oxotremorine-M has mixed cholinergic properties, its nicotinic actions have only been reported in systems which contain both muscarinic and nicotinic receptors, or in skeletal neuromuscular systems where the site of action of oxotremorine-M may have been ambiguous. We tested the possibility that oxotremorine-M is a nicotinic receptor agonist by examining the responses of single nicotinic acetylcholine receptors in primary cultures of myocytes from skeletal myotomes of Xenopus larvae. Myotomal myocytes are known to express the nicotinic acetylcholine receptor and no evidence exists that muscarinic receptors are expressed in these progenitors of the skeletal musculature. Furthermore, because we used aneural myocyte cultures, the effects of oxotremorine-M cannot be attributed to action on presynaptic receptors. Using cell-attached patches, we compared the responses of the nicotinic acetylcholine receptors to suberyldicholine and oxotremorine-M. Our results show that (1) both agonists activate the receptor channel in nanomolar concentrations; (2) the mean channel open-time is significantly smaller in oxotremorine-M; and (3) activation of the nicotinic acetylcholine receptor by oxotremorine-M is accompanied by a large percentage of short openings and a high frequency of event flickering. We conclude that oxotremorine-M is a mixed function agonist, showing partial blocking behavior, which effectively activates pure nicotinic acetylcholine receptors.

Animals↗
PubMed1994

Gravity effects on membrane processes.

Application of the Gouy-Chapman-Debye-Hückel (GCDH) theory to a model membrane in contact with electrolytes of various concentrations and composition predict density variations within an interfacial layer. Assuming that on cellular dimensions hydrodynamics can be applied (the objections are briefly discussed) two types of gravity effects can be defined, 1. convection along the surface of vertically oriented membranes and 2. surface potential variations by layer deformations at horizontally oriented membranes. Both effects should affect transport across the layer to the membrane surface and across the membrane. According to the theoretical predictions first experiments with gramicidin channels incorporated into artificial phosphatidylserine bilayer membranes show a significant difference in single channel currents in vertical and horizontal membranes. The complexity of biological membrane functions requires investigation of isolated membrane surface reactions and transport systems to study the gravisensitivity for each process separately.

Cell Membrane↗
PubMed1994

Vector-averaged gravity does not alter acetylcholine receptor single channel properties.

To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

Acetylcholine↗
PubMed1994

Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight.

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.

Acetylcholine↗
PubMed1992

Gravity effects on biological systems.

Theoretical investigations of the membrane-solution interface predict different effects of gravity on vertically and horizontally oriented planar membranes. Single channel events of gramicidin incorporated into phosphatidylserine planar bilayer membranes were measured in 0.1 M KCl solution, pH 7, at room temperature. The potential difference across the membrane was set to +/- 70 mV. The mean channel current was observed to be about 20% higher in horizontally oriented membranes compared to vertical membranes. This is in good agreement with the theoretical considerations and demonstrates that gravity does affect membrane processes by interaction with the membrane-solution interface which is a ubiquitous structure in biological systems.

Animals↗