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R Gruener

Publications and source records attributed to R Gruener.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1991

Electrophysiological properties and cholinergic responses in guinea-pig celiac ganglion neurons in primary culture.

Prevertebral neurons enzymatically dissociated from celiac ganglia of adult guinea-pigs were maintained in long-term primary culture. Cells were plated at a density of 95 +/- 15 cm-2, and intracellular electrical activity was measured between 2 and 7 weeks after dissociation. Neurite outgrowth began within 24 h of enzymatic dissociation. Cell survival dropped below 50% after more than two weeks in culture. The resting potential (-53 mV +/- 0.8), time constant (12 ms +/- 1.3), input resistance (47 M omega +/- 8.6), rheobase (0.33 nA +/- 0.02), degree of accommodation, spike amplitude (70 mV +/- 3.0), after hyperpolarization amplitude (-9.5 mV +/- 0.55), and after hyperpolarization duration (88 ms +/- 7.6) in these cells were not different from those recorded from neurons in intact celiac ganglia. A larger proportion (greater than 90%) of cells exhibited fast accommodation (phasic) in response to depolarizing current pulses. Unevoked (spontaneous) depolarizations and action potentials were observed. The cells responded to pressure ejected acetylcholine. Two types of responses consisted of an early rapid depolarization which was attenuated by hexamethonium and a later slow depolarization which was attenuated by atropine. We conclude that prevertebral neurons from guinea-pigs can be maintained in long-term primary culture, that they retain electrophysiological properties similar to intact ganglia and exhibit complex responsivity to acetylcholine.

Acetylcholine↗
PubMed1991

Vector-averaged gravity alters myocyte and neuron properties in cell culture.

To investigate whether changes in the gravitational field of developing neurons and myocytes affect cellular development, we rotated cultures of embryonic spinal neurons and myocytes in a horizontal clinostat. Rotation in the clinostat produces, from the cells' perspective, a "vector-free" gravity environment by continuous averaging of the vector. In this way, rotation in the clinostat simulates the microgravity of space where the gravity vector is substantially reduced. At rotation rates of 1-50 rpm, cellular and nuclear areas of myocytes were significantly enlarged and the number of presumptive nucleoli increased. In neurons, frequent and large swellings appeared along neuritic shafts. Some of these changes were reversible after cessation of rotation. Since our data are generally consistent with findings from other cell types subjected to spaceflight, we suggest that the vector-free gravity environment of the clinostat appears to simulate, at least in part, the microgravity of space. Our data further show that cellular processes are sensitive to altered gravity and suggest that cell development in the microgravity of space may be significantly altered.

Animals↗
PubMed1990

Vector-free gravity disrupts synapse formation in cell culture.

Terrestrial organisms evolved under and are subjected to the constancy of gravity. The organisms having adapted to this environmental factor, it is possible that embryonic development may be modified by exposure to altered gravity. To test the effects of gravity on embryonic development, we monitored the formation of nerve-associated acetylcholine receptor patches (NARPs) as an index of synaptogenesis. Embryonic spinal neuron and myotomal myocyte cocultures were placed in a horizontally rotating clinostat. From the cell's perspective, this results in the cancellation of the gravitational vector because of continuous averaging, thus mimicking the reduced gravitational force encountered in space. NARPs from cultures in which nerve-muscle contact was established before the onset of rotation were unaffected. In contrast, cultures in which nerve contact took place during rotation showed a marked inhibition of NARPs. Moreover, in the myocytes which did exhibit NARPs, the area of the patch was significantly reduced compared with control sister cultures. Several paradigms were used to ascertain that these findings did not result simply from loss of contact between neurites and myocytes, accelerated diffusion of a putative aggregating factor secreted by neurites, or from turbulence in the medium. Our data suggest that the process of synapse formation is sensitive to the gravitational vector. Embryonic development of the nervous system, in space, may therefore be markedly different from that normally occurring on earth.

Animals↗
PubMed1987

Vasopressin promotes neurite growth in cultured embryonic neurons.

Vasopressin (AVP) has been identified as a neural peptide which may influence memory function. Because of this action, we investigated the effect of AVP on neurons growing in culture. Vasopressin was found to markedly increase neurite outgrowth from cultured embryonic neurons and to also accelerate the rate of neuritic growth. Maximal stimulation of neurite production occurred after 24-hour incubation in the presence of 1 microM AVP. In AVP-treated cultures the profuse neuritic arborization was characterized by numerous microspikes along the neuritic shafts and at the perimeters of growth cones. These data provide strong evidence for a neurotrophic effect of AVP which, we suggest, may be relevant to neuronal development as well as to morphological changes which occur in the mature nervous system, possibly during memory formation.

Animals↗
PubMed1986

In vitro inhibition of vasopressin release in brain by behaviorally relevant ethanol concentrations.

We have investigated the effect of ethanol upon vasopressin (AVP) content in brain and upon in-vitro release of AVP from the rat median eminence. In-vitro ethanol concentrations (5-25 mM), comparable to behaviorally relevant blood ethanol levels, induce a substantial inhibition of AVP release from the median eminence, whereas higher ethanol concentrations (greater than 50 mM) potentiate release. In vivo, ethanol, at a behaviorally relevant blood ethanol concentration (126 mg%), does not produce a significant difference in AVP content in brain although there is a consistent trend towards an increase in the hypothalamus and neurohypophysis. The results are considered in relation to the effects of ethanol on biogenic amine release and to memory impairments induced by low doses of acute ethanol exposure.

Animals↗
PubMed1986

Halothane-induced changes in acetylcholine receptor channel kinetics are attenuated by cholesterol.

The single-channel recording technique was used to investigate the role of membrane lipids in the action of general anesthetics on ion channels. We examined the effects of halothane on acetylcholine receptor channels in Xenopus laevis myocytes in which the plasma membrane cholesterol level had been changed by pretreatment with cholesterol-rich or cholesterol-free liposomes. We found that the alteration in acetylcholine receptor channel kinetics, elicited in the presence of clinically-relevant concentrations of halothane, is attenuated when membrane cholesterol is increased and enhanced when membrane cholesterol concentration is decreased. These findings support the idea that general anesthetics interact with synaptic receptor channels indirectly through the lipid domains in which these synaptic proteins are embedded.

Animals↗
PubMed1984

Halothane shortens acetylcholine receptor channel kinetics without affecting conductance.

The extracellular patch-clamp technique was used to examine how halothane, a general anesthetic, affects the properties of single nicotinic acetylcholine receptor channels of embryonic Xenopus skeletal muscle cells grown in culture. Under control conditions, single-channel events showed a bimodal distribution on the basis of current amplitudes. This distribution was maintained during exposure to halothane and its washout. In addition, the mean current value of the low-and high-amplitude channels was unaffected by the presence of the anesthetic at clinically relevant concentrations. In contrast, halothane shortened the burst durations of both channel types in a concentration-dependent manner. This shortening of burst durations may be an expression of the more rapid relaxation of the channel protein to the nonconducting state, possibly due to the disordering effect of the anesthetic on membrane lipids in which the receptor protein is embedded. This functional change, in the behavior of the synaptic receptor, provides further direct information on the mode of action of general anesthetics.

Animals↗
PubMed1983

Association of post-anaesthetic hyperthermia with abnormal muscle characteristics: a case report.

A previously healthy 18-year-old male, following appendectomy developed post-anaesthetic hyperthermia (42.1 degrees C) with an elevation of serum creatine kinase and activated partial thromboplastin time. Repeated arterial blood gases were normal. Cooling and anti-pyretic medication did not control the fever. In contrast, sodium dantrolene appeared effective in lowering the patient's temperature and normalizing the vital signs, both acutely and over the following three days. Subsequent muscle biopsy revealed a normal contracture response to caffeine alone or in the presence of halothane. However, the muscle had a larger than normal potentiation of evoked twitch tension in the presence of caffeine and halothane. Electrophoresis of the muscle revealed a marked increase of an unidentified low molecular weight protein. The patient's clinical course, and the results of the muscle studies, suggest that an abnormality of skeletal muscle.

Adolescent↗
PubMed1981

Calcium uptake by isolated sarcoplasmic reticulum: examination of halothane inhibition, pH dependence, and Ca2+ dependence of normal and malignant hyperthermic human muscle.

Ca2+ uptake and release in muscle homogenates and fragmented sarcoplasmic reticulum were examined in biopsy specimens from nonsusceptible and malignant hyperthermia (MH) susceptible patients. Ca2+ flux was examined by the filter binding assay technique using 45Ca. It was found that Ca2+ uptake and release were the same in both normal and MH muscle homogenates. Halothane inhibited the uptake of Ca2+ by the sarcoplasmic reticulum. The halothane inhibition of Ca2+ uptake in normal and MH sarcoplasmic reticulum was fitted to a single line with a correlation coefficient (r) of -0.958. The pH and Ca2+ dependence of Ca2+ uptake were the same for both normal and MH sarcoplasmic reticulum. The pK for Ca2+ uptake is approximately 5.9. It is concluded that the Ca2+ uptake function of the muscle from the five patients with MH examined is not abnormal and might not be the locus for the initiation of MH.

Calcium↗