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W G Wier

Publications and source records attributed to W G Wier.

At least 37 records · Page 2Linked to original sources
PubMed1997

Caffeine-induced release of intracellular Ca2+ from Chinese hamster ovary cells expressing skeletal muscle ryanodine receptor. Effects on full-length and carboxyl-terminal portion of Ca2+ release channels.

The ryanodine receptor (RyR)/Ca2+ release channel is an essential component of excitation-contraction coupling in striated muscle cells. To study the function and regulation of the Ca2+ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca2+]. Unlike the native RyR channels in muscle cells, which display localized Ca2+ release events (i.e., "Ca2+ sparks" in cardiac muscle and "local release events" in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca2+ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca2+ release events observed in muscle cells may involve gating of a group of Ca2+ release channels and/or interaction of RyR with muscle-specific proteins.

Animals↗
PubMed1997

Ca2+ 'sparks' and waves in intact ventricular muscle resolved by confocal imaging.

The [Ca2+]i transient in heart is now thought to involve the recruitment and summation of discrete and independent "units" of Ca2+ release (Ca2+ "sparks") from the sarcoplasmic reticulum, each of which is controlled locally by single coassociated L-type Ca2+ channels ("local control theory of excitation-contraction coupling"). All prior studies on Ca2+ sparks, however, have been performed in single enzymatically dissociated heart cells under nonphysiological conditions. In order to understand the possible significance of Ca2+ sparks to normal working cardiac muscle, we used confocal microscopy to record Ca2+ sparks, spatially averaged [Ca2+]i transients and Ca2+ waves in individual cells of intact rat right ventricular trabeculae (composed of < 15 cells in cross section) microinjected with the Ca2+ indicator fluo 3 under physiological conditions ([Ca2+]o, 1 mmol/L; temperature, 33 +/- 1 degree C). Twitch force was recorded simultaneously. When stretched to optimal length (sarcomere length, 2.2 microns) and stimulated at 0.2 Hz, the trabeculae generated approximately equal to 700 micrograms of force per cell. Spatially averaged [Ca2+]i transients recorded from individual cells within a trabecula were similar to those recorded previously from single cells. The amplitude distribution of the peak ratio of Ca2+ sparks was bimodal, with maxima at ratios of 1.8 +/- 0.3 and 2.7 +/- 0.2 (mean +/- SD), respectively. The amplitude of the peak of Ca2+ sparks was approximately equal to 170 nmol/L. Ca2+ sparks occurred at a frequency of 12.0 +/- 0.8/s (mean +/- SEM) in line scans covering 94 sarcomeres. Ca2+ waves occurred randomly at a frequency of 0.57 +/- 0.08/s and propagated with a velocity of 29.5 +/- 1.7 microns/s. The extent of Ca2+ wave propagation was 3.9 +/- 0.3 sarcomere lengths (sarcomere length, 2.2 microns). Ca2+ sparks could be identified along the leading edge of the waves at intervals of 1.30 +/- 0.11 sarcomere length. Our observations suggest that (1) Ca2+ sparks, similar to those recorded in single cells, occur in trabeculae under physiological conditions and (2) coupling of Ca2+ spark generation between neighboring sites occurs and may lead to (3) the development of Ca2+ waves, which propagate under physiological conditions at a low velocity over limited distances. The results suggest that concepts of excitation-contraction coupling recently derived from isolated myocytes are applicable to intact cardiac trabeculae [corrected].

Aniline Compounds↗
PubMed1996

Ca2+ sparks involving multiple Ca2+ release sites along Z-lines in rat heart cells.

1. High spatial resolution confocal imaging was used to investigate the fundamental nature of "Ca2+ sparks' in rat cardiac myocytes loaded with the fluorescent calcium indicator, fluo-3. 2. The sites at which calcium sparks occurred (Ca2+ release sites) were packed closely and irregularly in transverse planes along Z-lines (mean spacing between sites of 0.76 microns). In contrast, sites were spaced more regularly in the longitudinal direction, at intervals of 1.8 microns (i.e. the sarcomere length). 3. Diffusion of released Ca2+ was slower transversely (apparent diffusion coefficient, D, 7.9 microns 2 s-1) than longitudinally (D, 17.1 microns 2 s-1). 4. Frequently, discrete sites several hundred nanometres apart transversely activated in near synchrony. The probability of transverse synchronous activity fell to low levels (< 20%) at sites separated by more than 1.0 micron. Synchronous activation was not observed between sites on different Z-lines (i.e. separated longitudinally by 1.8 microns). 5. High temporal resolution confocal microscopy (stationary spot) revealed Ca2+ sparks with "stepped' rises, consistent with multiple sites of origin. 6. We conclude that the Ca2+ spark as originally described is usually not an "elementary' event, in the sense of being indivisible, but is often comprised of yet smaller, triggered units of Ca2+ release.

Aniline Compounds↗
PubMed1995

Local Ca2+ transients (Ca2+ sparks) originate at transverse tubules in rat heart cells.

1. The origins of local [Ca2+]i transients (Ca2+ sparks) were studied using dual-channel confocal laser scanning microscopy. Line scan images showing [Ca2+]i (as fluo-3 fluorescence) and the transverse tubule membranes (as Di-8 fluorescence) were obtained simultaneously in single rat cardiac ventricular cells. 2. Line scan images of Di-8 fluorescence showed peaks regularly spaced at intervals of 1.83 +/- 0.30 microns (mean +/- S.D.). These peaks corresponded to the transverse tubules (T-tubules) in cross-section. 3. Line scan images of fluo-3 fluorescence showed local [Ca2+]i transients (LCTs or Ca2+ sparks) evoked by electrical stimulation. 4. Eighty-five per cent (85%) of all Ca2+ sparks evoked by electrical stimulation (n = 138, in 5 cells) occurred within 0.5 micron of a T-tubule. Thirty per cent (30%) occurred within 1 pixel (0.20 micron) of a T-tubule. 5. In some cells studied (3 out of 5), certain T-tubules had a higher probability of being sites of origin of Ca2+ sparks than others. 6. These results support local control theories of excitation-contraction coupling in which Ca2+ release from the sarcoplasmic reticulum (SR) is triggered by a high local [Ca2+]i established between the L-type Ca2+ channels in the T-tubules and associated ryanodine receptor(s) in the junctional SR.

Animals↗
PubMed1995

Local calcium transients triggered by single L-type calcium channel currents in cardiac cells.

Excitation-contraction coupling was studied in mammalian cardiac cells in which the opening probability of L-type calcium (Ca2+) channels was reduced. Confocal microscopy during voltage-clamp depolarization revealed distinct local transients in the concentration of intracellular calcium ions ([Ca2+]i). When voltage was varied, the latency to occurrence and the relative probability of occurrence of local [Ca2+]i transients varied as predicted if Ca2+ release from the sarcoplasmic reticulum (SR) was linked tightly to Ca2+ flux through L-type Ca2+ channels but not to that through the Na-Ca exchanger or to average [Ca2+]i. Voltage had no effect on the amplitude of local [Ca2+]i transients. Thus, the most efficacious "Ca2+ signal" for activating Ca2+ release from the SR may be a transient microdomain of high [Ca2+]i beneath an individual, open L-type Ca2+ channel.

Animals↗
PubMed1995

Calcium signalling in cardiac muscle cells.

In heart cells, several distinct kinds of transient spatial patterns of cytoplasmic calcium ion concentration ([Ca2+]i) can be observed: (1) [Ca2+]i waves, in which regions of spontaneously increased [Ca2+]i propagate at high velocity (100 microns/s) through the cell; (2) Ca2+ 'sparks', which are spontaneous, non-propagating changes in [Ca2+]i that are localized in small (approximately 2 microns) subcellular regions; and (3) evoked [Ca2+]i transients that are elicited by electrical depolarization, in association with normal excitation-contraction (E-C) coupling. In confocal [Ca2+]i images, evoked [Ca2+]i transients appear to be nearly spatially uniform throughout the cell, except during their rising phase or during small depolarizations. In contrast to [Ca2+]i waves and spontaneous Ca2+ sparks, evoked [Ca2+]i transients are triggered by L-type Ca2+ channel current and they are 'controlled', in the sense that stopping the L-type Ca2+ current stops them. Despite their different characteristics, all three types of Ca2+ transient involve Ca(2+)-induced release of Ca2+ from the sarcoplasmic reticulum. Here, we address the question of how the autocatalytic process of Ca(2+)-induced Ca2+ release, which can easily be understood to underlie spontaneous regenerative ('uncontrolled'), propagating [Ca2+]i waves, might be 'harnessed', under other circumstances, to produce controlled changes in [Ca2+]i, as during normal excitation-contraction coupling, or changes in [Ca2+]i that do not propagate. We discuss our observations of Ca2+ waves, Ca2+ sparks and normal Ca2+ transients in heart cells and review our results on the 'gain' of Ca(2+)-induced Ca2+ release. We discuss a model involving Ca2+ microdomains beneath L-type Ca2+ channels, and clusters of Ca(2+)-activated Ca2+ release channels in the sarcoplasmic reticulum which may form the basis of the answer to this question.

Animals↗
PubMed1995

Confocal microscopy reveals local SR calcium release in voltage-clamped cardiac cells.

Confocal microscopy during voltage-clamp depolarization of mammalian heart cells revealed distinct local [Ca2+]i-transients that resulted from Ca2+ released through sarcoplasmic reticulum (SR) release channel(s). When voltage was varied, the latency to occurrence and the relative probability of local [Ca2+]i-transients varied as predicted if SR Ca(2+)-release is linked tightly to Ca2+ flux through co-associated L-type Ca(2+)-channels.

Animals↗
PubMed1995

Mapping miniature synaptic currents to single synapses using calcium imaging reveals heterogeneity in postsynaptic output.

The amplitudes and kinetics of miniature excitatory synaptic currents (MESCs) in mammalian central neurons vary widely. It is unclear whether this variability occurs at each synapse or arises from differences among a heterogeneous population of synapses. Furthermore, it is not known how variability in these currents would affect their associated postsynaptic Ca2+ transients. To address these questions, we conducted simultaneous Ca2+ imaging and patch-clamp recordings from cultured cortical neurons and mapped individual MESCs to identified synapses displaying coincident dendritic miniature synaptic Ca2+ transients (MSCTs). Measurements of MSCTs at dendritic sites that displayed multiple events revealed that MSCT amplitude varied considerably at each site. Simultaneous measurement of MESCs and MSCTs at these sites indicated that variability in coincident synaptic currents contributes to the differences in Ca2+ transient amplitude. The ability of single synapses to exhibit variable output may enable them to engage intracellular signaling pathways at different levels of intracellular Ca2+.

Animals↗
PubMed1995

Simultaneous measurements of Ca2+ and nitric oxide in bradykinin-stimulated vascular endothelial cells.

The production of endothelium-derived relaxing factor (EDRF), known to be nitric oxide (NO), is triggered by a rise in the cytoplasmic calcium concentration ([Ca2+]i) subsequent to receptor binding of vasoactive agonists. In vascular endothelial cells, NO is synthesized from L-arginine by the Ca2+/calmodulin-dependent NO synthase. In this study, we report the first simultaneous measurements of [Ca2+]i and [NO] at the level of single endothelial cells. In cultured bovine aortic endothelial cells, extracellular application of bradykinin (BK, 10 to 20 mumol/L) caused transient (sometimes oscillatory) increase in [Ca2+]i, which was measured with the fluorescent Ca2+ indicator fura 2 and fluorescence imaging microscopy. BK caused an increase in [Ca2+]i, primarily through release from intracellular stores. Under identical experimental conditions, BK caused a transient increase in [NO], which was measured by application of a porphyrinic NO microsensor. [NO] peaked at approximately 0.5 mumol/L. Simultaneous measurements of [Ca2+]i and [NO] in BK-stimulated endothelial cells revealed that a transient increase in [Ca2+]i was rapidly followed by an increase in [NO] that outlasted the [Ca2+]i transient.

Animals↗
PubMed1994

Local, stochastic release of Ca2+ in voltage-clamped rat heart cells: visualization with confocal microscopy.

1. Confocal microscopy and the fluorescent Ca2+ indicator fluo-3 (K+ salt) were used to measure cytosolic free calcium ion concentration ([Ca2+]) during excitation-contraction (E-C) coupling in single, voltage-clamped, rat cardiac ventricular cells. 2. Local [Ca2+]i transients were measured nearly simultaneously in different, separate, subcellular volumes of approximately 2.0 microns 3. During depolarization, local [Ca2+]i transients were distinctly different from each other and from whole-cell [Ca2+]i transients. These differences were particularly apparent during small depolarizations, and were substantially reduced by ryanodine. 3. Components of the local [Ca2+]i transients, particularly those evoked by small depolarizations, were closely similar, in time course and amplitude, to spontaneous local [Ca2+]i transients, or 'sparks' (which have been shown previously to be Ca2+ released from sarcoplasmic reticulum). 4. Analysis of local [Ca2+]i transients in the spatial frequency domain (power spectrum) revealed that high power at spatial frequencies of 0.05-0.2 microns-1 was always associated with spontaneous calcium 'sparks' and with local [Ca2+]i transients evoked by small depolarizing pulses (e.g. to -31 mV). Evoked local [Ca2+]o transients in the presence of ryanodine, and those evoked by depolarization to very positive clamp-pulse potentials (+45 mV), were associated with considerably lower power at this frequency. 5. The results suggest that whole-cell [Ca2+]i transients evoked by voltage-clamp depolarization, and thus by L-type Ca2+ current, are comprised of local [Ca2+]i transients that are similar to the spontaneous calcium 'sparks'. At very positive clamp-pulse potentials, however, the electrically evoked local [Ca2+]i transients may be smaller, perhaps as a result of smaller unitary L-type Ca2+ current.

Aniline Compounds↗
PubMed1994

Processes that remove calcium from the cytoplasm during excitation-contraction coupling in intact rat heart cells.

1. The processes that remove Ca2+ rapidly from the cytoplasm were studied in isolated rat ventricular myocytes subjected to whole-cell voltage clamp and internal perfusion with the Ca2+ indicator, indo-1. Na(+)-Ca2+ exchange was eliminated in most experiments by removing Na+ both internally and externally. 2. When the Ca(2+)-pumping ATPase of the sarcoplasmic reticulum (SR) was inhibited with cyclopiazonic acid and ryanodine interfered with the release of Ca2+ from the SR, [Ca2+]i transients rose slowly and declined extremely slowly. We concluded that transport of Ca2+ by mitochondria and the surface membrane Ca(2+)-pumping ATPase would be negligible over the time course of a single [Ca2+]i transient. 3. The influence of cytoplasmic Ca2+ ligands was characterized by internal perfusion with high concentrations of diffusible Ca2+ ligands (indo-1) or by superfusion with the membrane-permeant Ca2+ ligand, BAPTA AM. As the concentration of indo-1 in the cell increased from < 0.1 mM to at least 0.5 mM, the time constant of the decline of [Ca2+]i increased from about 0.15 s to nearly 3 s. 4. Calcium bound to endogenous Ca2+ ligands during depolarizing clamp pulses was characterized quantitatively as the difference between the total Ca2+ entering the cell via L-type Ca2+ channels and [Ca2+]i, in experiments in which SR function had been abolished. As total calcium increased during the entry of Ca2+, total calcium was found to agree reasonably well with that predicted by assuming that Ca2+ could bind to endogenous intracellular Ca2+ ligands and to indo-1. 5. The results indicate that, in the absence of Na+, the major factors determining the removal of cytoplasmic free Ca2+ are the Ca(2+)-pumping ATPase of the SR and the binding of Ca2+ to endogenous and exogenous Ca2+ ligands. 6. Several hypothetical 'Ca2+ removal functions' were fitted to the declining phase of [Ca2+]i transients. The best fit was one in which the flux of Ca2+ through the SR Ca(2+)-pumping ATPase was described by a Michaelis-Menten-type equation. The decline of the [Ca2+]i transient was thus described by a linear, first-order differential equation having terms giving the rate of Ca2+ transport by the SR Ca(2+)-pumping ATPase (Vmax and KM), the rates of complexation of Ca2+ with the various Ca2+ ligands (L), and a leak of Ca2+ into the cytoplasm from the SR (FSR,leak).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Local control of excitation-contraction coupling in rat heart cells.

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Visualization of quantal synaptic transmission by dendritic calcium imaging.

As changes in synaptic strength are thought to be critical for learning and memory, it would be useful to monitor the activity of individual identified synapses on mammalian central neurons. Calcium imaging of cortical neurons grown in primary culture was used to visualize the activation of individual postsynaptic elements by miniature excitatory synaptic currents elicited by spontaneous quantal release. This approach revealed that the probability of spontaneous activity differed among synapses on the same dendrite. Furthermore, synapses that undergo changes in activity induced by glutamate or phorbol ester treatment were identified.

Animals↗
PubMed1994

Nitric oxide decreases [Ca2+]i in vascular smooth muscle by inhibition of the calcium current.

Endothelium derived relaxing factor (nitric oxide, or NO) activates cytoplasmic guanylate cyclase in vascular smooth muscle and decreases vascular tone through cGMP-dependent mechanisms that are not yet understood fully. In cultured vascular smooth muscle cells (A7r5 cell line) sodium nitroprusside (NP), a vasodilator that decomposes into nitric oxide, lowered [Ca2+]i in cells in which [Ca2+]i was elevated after depolarization. NP decreased current through voltage-gated calcium channels, but did not affect release of calcium from intracellular stores. Hemoglobin, a scavenger of NO, reversed the effect of NP on [Ca2+]i and 8-Br-cGMP, a membrane permeant form of cGMP, mimicked the effect of NP on [Ca2+]i and on calcium currents. Thus, the signal transduction mechanism of endothelium dependent relaxation of vascular smooth muscle involves a decrease in [Ca2+]i by inhibition of Ca2+ entry. Relaxation or vasodilation would then result from decreased activity of myosin light chain kinase, in addition to myosin light chain dephosphorylation.

Animals↗
PubMed1994

Differential regulation of calcium/calmodulin-dependent protein kinase II and p42 MAP kinase activity by synaptic transmission.

Calcium/calmodulin-dependent protein kinase II (CaMK) and p42 mitogen-activated protein kinase (MAPK) are enriched in neurons and possess the capacity to become persistently active, or autonomous, following removal of the activating stimulus. Since persistent kinase activation may be a mechanism for information storage, we have used primary cultures of cortical neurons to investigate whether kinase autonomy can be triggered by bursts of spontaneous synaptic activity. We and others have found that both these kinases respond to synaptic stimulation, but differ markedly in their kinetics of activation and inactivation, as well as in their sensitivity to NMDA receptor blockade. While 90% of maximal CaMK activation was observed after only 10 sec of synaptic bursting, MAPK activity was unaffected at this early time and rose to only 30% of maximal after 2 min of stimulation. Following blockade of synaptic stimulation, CaMK activity decreased by 50% in 10-30 sec, while MAPK activity decayed by 50% within 6-10 min. Although MAPK exhibited relatively slow activation, short periods of synaptic activity could trigger the MAPK activation process, which persisted in the absence of synaptic stimulation. Comparison of the effect of NMDA receptor blockade on synaptic activation of these kinases revealed that CaMK activity is preferentially suppressed. As previous immunocytochemical studies indicate that CaMK is concentrated in dendritic processes in the vicinity of synapses, we measured synaptic calcium transients in fine dendritic processes (approximately 1 microns diameter) to assess their sensitivity to NMDA receptor blockade. Calcium transients in these fine processes were reduced by up to 90% by NMDA receptor blockade, possibly accounting for the profound sensitivity of CaMK to this treatment. The sharp contrast between the regulation of CaMK and MAPK by synaptic activity indicates that they may mediate neuronal responses to different patterns of afferent stimulation. The relatively slow activation and inactivation of MAPK suggests that it may be able to integrate information from multiple, infrequent bursts of synaptic activity.

Amino Acid Sequence↗
PubMed1993

Rapid communication between neurons and astrocytes in primary cortical cultures.

The identification of neurotransmitter receptors and voltage-sensitive ion channels on astrocytes (reviewed by Barres, 1991) has renewed interest in how these cells respond to neuronal activity. To investigate the physiology of neuron astrocyte signaling, we have employed primary cortical cultures that contain both neuronal and glial cells. As the neurons in these cultures exhibit synchronous spontaneous synaptic activity, we have used both calcium imaging and whole-cell recording techniques to identify physiological activity in astrocytes related to neuronal activity. Whole-cell voltage-clamp records from astrocytes revealed rapid inward currents that coincide with bursts of electrical activity in neighboring neurons. Calcium imaging studies demonstrate that these currents in astrocytes are not always associated with slowly propagating calcium waves. Inclusion of the dye Lucifer yellow within patch pipettes confirmed that astrocytes are extensively coupled to each other but not to adjacent neurons, indicating that the currents observed are not due to gap junction connections between these cell types. These currents do not reflect widespread diffusion of glutamate or potassium released during neuronal activity since a population of small, round, multipolar presumed glial cells that are not dye coupled to adjacent cells did not display electrical currents coincident with neuronal firing, even though they respond to locally applied glutamate and potassium. These findings indicate that, in addition to the relatively slow signaling conveyed by calcium waves, astrocytes also display rapid electrical responses to neuronal activity.

Animals↗
PubMed1992

Modulation of L-type calcium channels by sodium ions.

It is universally believed that the removal of external sodium ions is without effect on calcium current. We now report that in enzymatically isolated guinea pig ventricular cells, the replacement of external sodium ions with certain other cations causes a 3- to 6-fold increase in peak L-type calcium current. The increase in current is reversibly blocked by L-type calcium-channel antagonists, not mediated by changes in internal calcium, and is inhibited by intracellular 5'-adenylyl imidodiphosphate, a nonhydrolyzable ATP analogue. The effects of sodium removal (and isoproterenol) were almost completely blocked by intracellular application of a specific (peptide) inhibitor of cAMP-dependent protein kinase. These experiments demonstrate a previously unknown effect of sodium ions to modulate calcium-channel phosphorylation via cAMP-dependent protein kinase.

Adenylyl Cyclases↗