Xenopus Myocytes Research Articles

An (un)paralleled process?

exercise is challenged because classical biochemical models are unable to explain the increased rate of oxidative ATP production on the basis of altered concentrations of respiratory substrates alone. The paper by Zoladz et al. (2013) exploresparallelactivationtheoryinhuman skeletal muscle by applying innovative reasoning,asfollows:(i) adaptationtofaster oxygenuptakekineticsfollowingendurance exercise training, observed in as few as two training sessions, occurs more rapidly than theexpansionofthemitochondrialvolume, suggesting enhancements of allosteric control in mitochondrial energy supply; and (ii) following training, a more tightly controlled, metabolically stable, parallel system would be accompanied by a reduction in the energy demands of exercise. The data show convincing support for these hypotheses, in that moderateintensity exercise training speeded the kinetics aerobic adjustment during exercise by almost 25% and reduced the oxygen cost of the exercise by nearly 5%; these effects were observed in the absence of changes in markers of mitochondrial respiratory capacity or biogenesis. These human data are consistent with features of parallel control recently demonstrated in single Xenopus myocytes (Gandra et al. 2012) andinthegastrocnemius‐superficialdigital flexorcomplexofthedog(W¨ ustetal.2011). Considering the wide interest in the control of oxidative metabolism during exercise, the molecular mechanisms contributing to kinetic control of oxidative phosphorylation in vivo have received relatively little attention. As such, the training-induced reduction in sarcoplasmic

Visualization of Ca2+ Signaling During Embryonic Skeletal Muscle Formation in Vertebrates

Dynamic changes in cytosolic and nuclear Ca(2+) concentration are reported to play a critical regulatory role in different aspects of skeletal muscle development and differentiation. Here we review our current knowledge of the spatial dynamics of Ca(2+) signals generated during muscle development in mouse, rat, and Xenopus myocytes in culture, in the exposed myotome of dissected Xenopus embryos, and in intact normally developing zebrafish. It is becoming clear that subcellular domains, either membrane-bound or otherwise, may have their own Ca(2+) signaling signatures. Thus, to understand the roles played by myogenic Ca(2+) signaling, we must consider: (1) the triggers and targets within these signaling domains; (2) interdomain signaling, and (3) how these Ca(2+) signals integrate with other signaling networks involved in myogenesis. Imaging techniques that are currently available to provide direct visualization of these Ca(2+) signals are also described.

Effect of Calcium on Spontaneous Quantal Transmitter Secretion from ACh-Loaded Myocytes

Spontaneous secretions occur in both neurons and non-neuronal cells, and calcium is important for these secretion processes. However, the detailed roles of calcium on the secretions have not yet been identified. In the present study, cultured Xenopus myocytes loaded with exogenous acetylcholine (ACh) into the cytoplasm in the absence of extracellular Ca 2+ undergo spontaneous quantal ACh secretion as detected by the appearance of pulsatile miniature endplate currents. Analysis of the frequencies, amplitudes, and time courses of these currents suggests that similar cellular mechanisms are involved in the secretions of ACh in normal medium and Ca 2+-free solution. Various doses of ryanodine were used to regulate the intracellular Ca 2+ to different levels. The spontaneous ACh secretion from myocytes in Ca 2+-free medium was decreased by reducing intracellular Ca 2+ levels and enhanced by increasing cytosolic Ca 2+ levels. These observations demonstrate that the spontaneous secretion from isolated myocytes and the effect of ryanodine on ACh-loaded cells are both independent of extracellular Ca 2+ while Ca 2+ in the sarcoplasmic reticulum plays a crucial role in the secretions.

Quantal transmitter release by glioma cells: quantification of intramembrane particle changes

Glial cells in situ are able to release neurotransmitters such as glutamate or acetylcholine (ACh). Glioma C6BU-1 cells were used to determine whether the mechanisms of ACh release by a glial cell line are similar or not to quantal release from neurones. Individual C6BU-1 cells, pre-filled with ACh, were moved into contact with a Xenopus myocyte that was used as a real-time ACh detector. Upon electrical stimulation, C6BU-1 cells generated evoked ACh impulses which were Ca 2+-dependent and quantal (quantal steps of ca. 100 pA). Changes in plasma membrane ultrastructure were investigated by using a freeze-fracture technique designed for obtaining large and flat replicas from monolayer cell cultures. A transient increase in the density of medium and large size intramembrane particles – and a corresponding decrease of small particles – occurred in the plasma membrane of C6BU-1 cells stimulated for ACh release. Changes in interaction forces between adjacent medium and large particles were investigated by computing the radial distribution function and the interaction potential. In resting cells, the radial distribution function revealed a significant increase in the probability to find two particles separated by an interval of 24 nm; the interaction potential suggested repulsive forces for intervals shorter than 24 nm and attractive forces between 24 and 26 nm. In stimulated cells, this interaction was displaced to 21 nm and made weaker, despite of the fact that the overall particle density increased. The nature of this transient change in intramembrane particles is discussed, particularly with regard to the mediatophore proteolipid which is abundant in the membranes C6-BU-1 like in those of cholinergic neurones. In conclusion, evoked ACh release from pre-filled C6-BU-1 glioma cells is quantal and Ca 2+-dependent. It is accompanied by a transient changes in the size distribution and the organisation of intramembrane particles in the plasma membrane. Thus, for the release characteristics, glioma cells do not differ fundamentally from neurones.

Neurotransmitter release through the V0 sector of V-ATPase.

Neurotransmitter release through the V0 sector of V-ATPase.

Evoked acetylcholine release by immortalized brain endothelial cells genetically modified to express choline acetyltransferase and/or the vesicular acetylcholine transporter.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Calcium Signaling in the Developing Xenopus Myotome

Embryonic Xenopus myocytes generate spontaneous calcium (Ca2+) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca2+ dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca2+ transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP3 and ryanodine receptors in the intact myotome by eliciting Ca2+ elevations in response to photorelease of caged IP3 and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca2+ release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation.

Acetylcholine synthesis and quantal release reconstituted by transfection of mediatophore and choline acetyltranferase cDNAs.

Neuroblastoma N18TG-2 cells cannot synthesize or release acetylcholine (ACh), and do not express proteins involved in transmitter storage and vesicle fusion. We restored some of these functions by transfecting N18TG-2 cells with cDNAs of either rat choline acetyltransferase (ChAT), or Torpedo mediatophore 16-kDa subunit, or both. Cells transfected only with ChAT synthesized but did not release ACh. Cells transfected only with mediatophore expressed Ca2+-dependent ACh release provided they were previously filled with the transmitter. Cell lines produced after cotransfection of ChAT and mediatophore cDNAs released the ACh that was endogenously synthesized. Synaptic-like vesicles were found neither in native N18TG-2 cells nor in ChAT-mediatophore cotransfected clones, where all the ACh content was apparently cytosolic. Furthermore, restoration of release did not result from enhanced ACh accumulation in intracellular organelles consecutive to enhanced acidification by V-ATPase, as Torpedo 16 kDa transfection did not increase, but decreased the V-ATPase-driven proton transport. Using ACh-sensitive Xenopus myocytes for real-time recording of evoked release, we found that cotransfected cells released ACh in a quantal manner. We compared the quanta produced by ChAT-mediatophore cotransfected clones to those produced by clones transfected with mediatophore alone (artificially filled with ACh). The time characteristics and quantal size of currents generated in the myocyte were the same in both conditions. However, cotransfected cells released a larger proportion of their initial ACh store. Hence, expression of mediatophore at the plasma membrane seems to be necessary for quantal ACh release; the process works more efficiently when ChAT is operating as well, suggesting a functional coupling between ACh synthesis and release.

Constitutive Secretion of Exogenous Neurotransmitter by Nonneuronal Cells: Implications for Neuronal Secretion

Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.

Characterization of single inward rectifier potassium channels from embryonic Xenopus laevis myocytes.

Single inward rectifier K+ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K+ channels (Kir(0.3), Kir(0.5) and Kir(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K+]o respectively. Extracellular Ba2+ blocked the normally high channel activity in a concentration-dependent manner (KA = 7.8 microM, -90 mV). In whole-cell recordings of inward rectifier K+ current, marked voltage dependence of Ba2+ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to EK, outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg(2+)-free solution at pH 9.1. This was probably also true for Kir(0.3), Kir(0.5) and Kir(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-gamma S had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K+ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states.

Enhancement of quantal transmitter release and mediatophore expression by cyclic AMP in fibroblasts loaded with acetylcholine

Neuronal properties such as neurotransmitter uptake and release can be expressed in non-neuronal cells. We show here that fibroblasts—mouse cell line L-M(TK−)—are able to take up acetylcholine from the external medium and to release it in response to a calcium influx. Release was assessed biochemically by a luminescence method, but it was also elicited from individual fibroblasts and recorded in real-time using a Xenopus myocyte as an acetylcholine detector. After treatment for three to six days with dibutyryl-cyclic AMP, the cells changed their shape and acetylcholine release was greatly enhanced. Surprisingly, in differentiated fibroblasts the time-course transmitter release exhibited a high degree of variability even for the successive responses evoked from the same cell; many currents recorded in myocytes on electrical stimulation of fibroblasts had an extremely long duration (up to 1s or more). This suggested that the release sites were kept open for a very long time. Cyclic AMP treatment also caused a marked increase in the expression of mediatophore 16,000 mol. wt proteolipid in fibroblast membranes. Mediatophore is an acetylcholine-translocating protein which is abundant in cholinergic presynaptic plasma membranes.It is concluded that cyclic AMP differentiation of fibroblasts prolongs the duration of acetylcholine release at individual sites and enhances the expression of the 16,000 mol. wt proteolipid forming mediatophore.

Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca(2+)-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed, discrete amplitude levels, like in naturally occurring synapses.

Microfilament-membrane interactions in Xenopus myocytes.

We used quick-freeze, deep-etch, rotary-replication transmission electron microscopy to determine at molecular resolution the organization of microfilaments at the cytoplasmic surface of the sarcolemma of Xenopus myocytes. We demonstrate that actin microfilaments interact with the sarcolemma in two distinct ways. In one, which resembled focal contacts in Xenopus fibroblasts [Samuelsson et al., 1993: J. Cell Biol. 122:485-496], bundles of microfilaments approached the sarcolemma at sites containing aggregates of membrane-associated particles. Immunogold cytochemistry showed that these particle aggregates contained vinculin, talin and beta 1-integrin. In the second, which covered most of the cytoplasmic surface of the sarcolemma, individual actin microfilaments formed an extensive, lattice-like array. Particle aggregates associated with this array of actin microfilaments also labeled with antibodies to vinculin, talin and beta 1-integrin. The unique, lattice-like association of actin microfilaments with the membrane in Xenopus myocytes suggests that the organization of actin filaments over most of the sarcolemma is distinct from focal contacts, mediating widespread associations of the actin cytoskeleton with the cytoplasmic membrane face.

Erratic Deposition of Agrin during the Formation of Xenopus Neuromuscular Junctions in Culture

In order to disclose the mechanisms that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses in vivo, agrin deposition usually lagged well behind the other synaptic markers during development in culture, and was not detectable at many differentiated junctions. Agrin deposition at nerve-muscle contacts in culture also appeared to require the presence of other synaptic components. A similarly variable deposition occurred on noninnervated myocytes, where agrin again collected near sites of HSPG and AChR accumulation on some cells. Profuse agrin accretion occurred consistently, however, within the extracellular matrices of surrounding epithelial cells derived from both myotomes and neural tubes. In cocultures of Ambystoma neurons and Xenopus myocytes Ambystoma agrin collected at some chimeric neuromuscular junctions, but also accumulated on noninnervated myocytes and in the extracellular matrices of salamander neuroendothelial cells. Based upon these observations we conclude that (a) focal agrin deposition is not required for synaptic differentiation on Xenopus myocytes and (b) agrin may be one of several muscle basal lamina components that stem mainly from the secreted products of nearby epithelial cells.

Electrical activity and calcium influx regulate ion channel development in embryonic Xenopus skeletal muscle

The development of electrical excitability involves complex coordinated changes in ion channel activity. Part of this coordination appears to be due to the fact that the expression of some channels is dependent on electrical activity mediated by other channel types. For example, we have previously shown that normal potassium current development in embryonic skeletal muscle cells of the frog Xenopus laevis is dependent on sodium channel activity. To examine the interrelationships between the development of different ionic currents, we have made a detailed study of electrical development in cultured Xenopus myocytes using whole-cell patch-clamp recording. The initial expression of potassium, sodium, and calcium currents is followed by a brief period during which the densities of potassium currents decrease, while at the same time sodium and calcium current densities continue to increase, which may increase electrical excitability during this time. The normal developmental increase in both potassium and sodium currents is inhibited by the sodium channel blocker tetrodotoxin, suggesting that electrical activity normally stimulates the expression of both these currents. These effects of electrical activity appear to be mediated via activation of voltage-gated calcium channels. We suggest that the developmental acquisition of sodium and calcium channels by these cells, possibly coupled with a transient decrease in potassium current density, lead to an increase in electrical excitability and calcium entry, and that this calcium entry provides a critical developmental cue controlling the subsequent development of mature electrical properties.

Convertible modes of inactivation of potassium channels in Xenopus myocytes differentiating in vitro.

1. Voltage-dependent inactivating single-channel potassium currents were recorded in cell-attached and inside-out patches from embryonic Xenopus myocytes differentiating in culture. 2. Channels with rapid inactivation (time constants < 25 ms) and with slow inactivation (time constants > 80 ms) recorded after one day in vitro appear to belong to two functionally different classes. Rapidly and slowly inactivating channels show steady-state inactivation with potentials of half-inactivation of -74 +/- 7 and -44 +/- 9 mV. They exhibit voltage-dependent activation, with times to half-maximal activation of 0.79 +/- 0.09 and 1.17 +/- 0.22 ms when stepped from -120 to +40 mV. Rapidly inactivating channels also have a lower open probability than slowly inactivating ones. The channels have similar conductances of 23 +/- 6 and 17 +/- 4 pS and extrapolated reversal potentials close to the potassium equilibrium potential. 3. In cell-attached patches, inactivation behaviours of channels with rapid or slow inactivation do not change during recording. After patch excision, rapidly inactivating channels usually switch to a slow inactivation mode. Slowly inactivating channels derived from rapidly inactivating channels after patch excision retain their conductance and extrapolated reversal potential, but are not distinguishable from native slowly inactivating channels with respect to steady-state inactivation, activation and inactivation times, as well as open probabilities. 4. The change in inactivation behaviour of rapidly inactivating channels after patch excision is reversed by application of reduced dithiothreitol (DTT). In contrast, channels with slow inactivation in the cell-attached mode do not change in to rapidly inactivating channels after application of DTT in the excised configuration, suggesting that these channels belong to a structurally different class. 5. Frequent observation of superposing channel openings indicates clustering of inactivating potassium channels in the myocyte membrane, since many patches lack channel activity. Clustering does not depend on the presence of differentiating neurones. 6. Channels with rapid inactivation increase 6-fold in density during the first day in culture in the presence of neurones; channel density decreases in their absence. Channels with slow inactivation increase 2-fold in density in the presence or absence of differentiating neurones during this period. 7. Channels with rapid or slow inactivation in cell-attached membrane belong to functionally distinct classes that are developmentally regulated differently. Reversible changes from rapid to slow inactivation mode after patch excision suggest that the channels may be structurally related.

Spontaneous quantal transmitter secretion from myocytes and fibroblasts: comparison with neuronal secretion

When exogenous ACh is loaded into the cytoplasm of cultured amphibian myocytes and fibroblasts, the cells undergo spontaneous quantal ACh secretion, as detected by the appearance of pulsatile membrane currents in Xenopus myocytes which are manipulated into contact with the cells. These currents resemble in many ways the miniature endplate currents (MEPCs) observed at developing neuromuscular synapses formed on these Xenopus myocytes. Analyses of the frequency, amplitude, and time course of these currents suggests similarity in the cellular mechanisms involved in the packaging and secretion of ACh quanta in fibroblasts, myocytes, and developing neurons. The size of the ACh packets released by the non-neuronal cells were found to be very similar to the size of the neuronal ACh quanta, which are thought to result from the exocytotic release of synaptic vesicles. Moreover, the kinetics with which the ACh packets are discharged from all three cell types are comparable, although the speed of secretion in non-neuronal cells is somewhat slower and more irregular. The spontaneous quantal ACh secretion from neurons and myocytes was decreased by reducing cytosolic Ca2+ level and enhanced by activation of protein kinase C with phorbol ester, but secretion from fibroblasts was unaffected by both treatments. The spontaneous secretion from fibroblasts did show some sensitivity to a rise in cytosolic Ca2+ after treatment with a Ca2+ ionophore. These observations support the hypothesis that the basic machinery for transmitter secretion operating in neurons derive from a more ubiquitous mechanism used for constitutive secretion and membrane trafficking in non-neuronal cells, and neuronal differentiation involves expression of additional unique components for the regulation of the spontaneous quantal secretion.

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.

Pharmacological evidence for a lack of role for protein kinase C in staurosporine-induced morphological changes in embryonic Xenopus myocytes

Staurosporine, an inhibitor of protein kinases, induced outgrowth of cultured embryonic Xenopus myocytes. The outgrowing membrane elicited by staurosporine was stained uniformly with fluorescein isothiocyanate-phalloidin. Pretreatment with microfilament-disrupting agents but not microtubule inhibitors inhibited staurosporine-induced membrane outgrowth. Microfilament assembly is thus required for the action of staurosporine. Protein kinase C activators did not antagonize the membrane outgrowing effect of staurosporine. Furthermore, none of H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), H-8 N[2-(methylamino)ethyl]-5isoquinoline sulfonamide), sphingosine, phloretin, genistein or calmidazolium induced any significant morphological changes of embryonic myocytes, indicating that tyrosine kinases protein kinase C, protein kinase A or calmodulin-dependent protein kinases may not invloved in the membrane outgrowing action of staurosporine. Total pretein content of myocytes was not altered by staurosporine and protein or RNA synthesis inhibitors did not inhibit the membrane outgrowth induced by staurosporine. Furthermore, membrane outgrowth induced by staurosporine was less pronounced in older cultured myocytes or myocytes acutely isolated at later stages of tadpoles, indicating that there is different developmental susceptibility to the action of staurosporine.

Chapter 6 Application of Patch Clamp Methods to the Study of Calcium Currents and Calcium Channels

Chapter 6 Application of Patch Clamp Methods to the Study of Calcium Currents and Calcium Channels