Evoked phasic release in frog nerve terminals obtained after block of Ca2+ entry by Cd2+

Cutaneous pectoris muscles of frogs were isolated, mounted in a chamber and superfused with Ringer's solution. With a macro-patch-clamp electrode [5] placed on a section of a motor nerve terminal, quantal synaptic currents were elicited by depolarizing pulses and recorded. The electorde tip and the section of the terminal recorded from were perfused rapidly by Ringer's solution alone or containing 20–500 M Cd²⁺ to block Ca²⁺ inflow. Separate superfusion of the muscle and the rest of the terminal with normal or elevated Ca²⁺ Ringer's solution provided a sufficiently high resting Ca²⁺ concentration in the terminal even when Ca²⁺ inflow was blocked by Cd²⁺. The depolarization level of maximal Ca²⁺ inflow into the terminal was found by measuring maximal test pulse facilitation, F _c [6]. In control solution as well as in the case of Cd²⁺ block, the rate of phasic release after depolarizing pulses rose further when depolarization was increased past the level of F _c, and reached a saturation level which was maintained at estimated depolarizations up to +200 mV. Block of Ca²⁺ inflow by Cd²⁺ decreased release substantially, but did not suppress it. The depression of release was greater in the range of large Ca²⁺ inflow (around F _c) than for very large depolarizations. The time course of phasic release was unaltered by blockage of Ca²⁺inflow. It is concluded that Ca²⁺ inflow contributes to the promotion of evoked release only in the depolarization range in which Ca²⁺ inward current is large. When Ca²⁺ concentration in the terminal is sufficiently high, release can be evoked by depolarization in the absence of Ca²⁺ inflow, the voltage-dependent release factor, S, compensating to a great extent for the lack of Ca²⁺ inflow.     

Ca2+ dynamics at the frog motor nerve terminal

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.     

Contribution of Ca2+ inflow to quantal,phasic transmitter release from nerve terminals of frog muscle

Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca²⁺ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cd _e ²⁺ or with an intracellular, EGTA-buffered solution containing less than 0.1 M Ca _e ²⁺ . Pulse application and recording were by a perfused macro-patch-clamp electrode. The muscle outside the electrode (bath) was superfused with Ringer's solutions containing Cd _b ²⁺ to block Ca²⁺ inflow and normal (1.8mM) or elevated (10 mM) Ca _b ²⁺ . The depolarization level of the terminal during current pulses that generated maximal Ca²⁺ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca²⁺ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca²⁺ inflow is high. However, for large depolarizations (>1.5 relative units), the depression of release by block of Ca²⁺ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca²⁺ inflow. If the extra-electrode superfusion of Ca _b ²⁺ of the muscle was elevated to 10 mM and Cd _b ²⁺ was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1M Ca _e ²⁺ raised the average release paradoxically. With 0.5 mM Cd _b ²⁺ this paradoxical increase of release was, on average, 4-fold at 6 °C, and 19-fold at 16 °C. Quantal endplate currents recorded in less than 0.1 M Ca _e ²⁺ had slightly increased amplitudes, and decay time constants were prolonged by about 50%. The results are interpreted to support the Ca²⁺/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca²⁺ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca²⁺ concentration is sufficiently high, large depolarizations can elicit release independent of the presence or absence of Ca²⁺ inflow.     

Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.     

Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in a high K+ solution

The inhibitory effect of a high external Ca²⁺ ([Ca²⁺]_o) on spontaneous transmitter release in a high K⁺ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca²⁺ ([Ca²⁺]_i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca²⁺]_i; increasing [Ca²⁺]_o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K⁺ concentration (7–15 mM), their facilitatory effects on m.e.p.p. frequency decreased, disappeared and eventually reversed to depressant actions. Since a rise in the external K⁺ concentration would increase the steady state level of [Ca²⁺]_i, it is suggested that when the [Ca²⁺]_i is preset at a high level, manipulations so as to further increase [Ca²⁺]_i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca²⁺]_i.     

Ultrastructural correlates of experimentally altered transmitter release efficacy in frog motor nerve terminals

After experimentally inducing long term changes in transmitter release, a series of frog neuromuscular junctions were studied with intracellular recording and then semi-serially sectioned and examined in the electron microscope. Transmitter release per unit length of motor nerve terminal was well correlated with several measures of the length of individual presynaptic active zones and with the number of mitochondria per terminal. Total release from each terminal correlated with estimates of the total amount of active zone. This study of neuromuscular junctions in sartorius muscles of the frog Rana pipiens was undertaken to search for ultrastructural correlates of the increase in transmitter release efficacy that follows denervation of the contralateral sartorius. This treatment typically results in greatly enhanced release at some synapses while others appear unaffected. In the present study, nine identified junctions with known physiological properties were sectioned every 6 micron throughout much of their length to yield 40-105 cross-sectional profiles per junction. Overall, these 9 synapses showed a 33-fold range in quantal transmitter release and an 18-fold range in release per unit nerve terminal. Release correlated with estimates of active zone size. No correlations were found between release and the density of synaptic vesicles adjacent to active zones. Our results suggest that active zones in motor nerve terminals are plastic structures, and that changes in active zone size may be the structural basis of long term changes in transmitter release and synaptic efficacy.     

L-Type calcium channels unmasked by cell-permeant Ca2+ buffer at mouse motor nerve terminals

 The involvement of the different types of voltage-dependent calcium channels (VDCC) in both DM-BAPTA-AM-incubated and EGTA-AM-incubated mature mice levator auris neuromuscular junctions (NMJ) was studied. We evaluated the effects of ω-agatoxin IVA (ω-Aga IVA), nitrendipine and ω-conotoxin GVIA (ω-CgTX) (P/Q-, L- and N-type VDCC blockers, respectively) on perineurial calcium currents (I _Ca) and nerve-evoked transmitter release. The application of ω-Aga IVA (100 nM) drastically reduced perineurial I _Ca (>90%) and nerve-evoked transmitter release (>90% of reduction in quantal content, m) at both DM-BAPTA-AM-incubated and EGTA-AM-incubated NMJ. The L-type VDCC antagonist nitrendipine (10 μM) caused a significant reduction (23±9%, n=5) of perineurial I _Ca at DM-BAPTA-AM-incubated NMJ. In addition, after the block of P/Q-type VDCC with ω-Aga IVA (100 nM), nitrendipine reduced (>90%, n=2) the remaining perineurial I _Ca. Such reduction was not observed at EGTA-AM-incubated NMJ, before or after the total block of P/Q-type VDCC. Moreover, nitrendipine did not significantly reduce the quantal content of DM-BAPTA-AM-incubated NMJ. Finally, the application of ω-CgTX (5 μM) did not significantly affect perineurial I _Ca or nerve-evoked transmitter release at either DM-BAPTA-AM-incubated or EGTA-AM-incubated NMJ. These results show the existence of a nitrendipine-sensitive, L-type component of perineurial I _Ca in DM-BAPTA-AM-incubated NMJ of mature mice. Received: 2 September 1998 / Received after revision:13 October 1998 / Accepted:14 October 1998     

Inhibition of Ca2+ inflow at nerve terminals of frog muscle blocks facilitation while phasic transmitter release is still considerable

Action potentials were triggered in the motor nerve by a suction electrode and calcium currents (i_Ca) in the nerve terminals were measured by means of a perfused macro-patch-clamp electrode on the distal portion of the end-plates. Postsynaptic currents were blocked by adding d-tubocurarine, whereas presynaptic Na⁺ (i_Na) and K⁺ (i_K) currents were blocked by adding tetrodotoxin (TTX), tetraethylammonium and 3,4-diaminopyridine, respectively, to the perfusate of the electrode. The current components which could be suppressed by addition of Cd²⁺ to the perfusate were taken as presynaptic i_Ca. The observed effects on the presynaptic current components were very similar to those reported previously. If the electrode was perfused with Ringer's solution containing the blockers for i_Na and i_K, the same, obviously complete block of i_Ca was obtained by 50 and 100μM Cd²⁺, an average of 96% block by 20μM Cd²⁺ and 50% block by about 5μM Cd²⁺. Using the same type of electrode and similar locations on motor nerve terminals, postsynaptic quantal currents and twin-pulse facilitation (F_d) were elicited by variable-duration (0.5–3 ms) depolarizing pulses. When the electrode was perfused with Ringer's solution containing TTX, 20μM Cd²⁺ added to the perfusate reduced the rate of phasic release of quanta insignificantly for short depolarizing pulses and by a factor of about 10 for longer pulses. F_d was blocked almost completely. Addition of 50μM Cd²⁺ to the perfusate had a greater depressive effect on release after short depolarizing pulses and reduced release after longer pulses by a factor of about 100. The double-logarithmic slope of the dependence of the rate of release on pulse duration was reduced from values of about 8 in the controls to about 4 in 50μM Cd²⁺. F_d was completely blocked. The effects of Cd²⁺ on release and F_d were reversible. If the Ca²⁺ in the perfusate of the electrode was lowered, the block by Cd²⁺ became more effective. Comparison of the effects of Cd²⁺ on presynaptic i_Ca and on release and facilitation shows that there is considerable phasic release at Cd²⁺ concentrations which block i_Ca, while facilitation is reduced approximately in proportion to the extent of block of i_Ca. The latter observation is in line with the “residual Ca²⁺” concept of facilitation. Phasic release in spite of block of i_Ca by Cd²⁺ is assumed to be mediated by the non-Ca, depolarization controlled activator in concert with a considerable maintained Ca²⁺ concentration in the terminal.     

Ca(2+)-dependent Ca(2+) clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals

Ca(2+) clearance in frog motor nerve terminals was studied by fluorometry of Ca(2+) indicators. Rises in intracellular Ca(2+) ([Ca(2+)](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca(2+) transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca(2+) pumps at the cell membrane by external Li(+) and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li(+) alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca(2+) transients. Blocking Ca(2+) pumps at Ca(2+) stores by thapsigargin did not affect 100 stimuli-induced Ca(2+) transients but increased the initial decay time constant of 200 stimuli-induced Ca(2+) transients with no change in other parameters. Inhibiting mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca(2+) transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca(2+) transients against [Ca(2+)](i) yielded the supralinear [Ca(2+)](i) dependence of Ca(2+) efflux out of the cytosol. Blocking Ca(2+) extrusion or mitochondrial Ca(2+) uptake significantly reduced this [Ca(2+)](i)-dependent Ca(2+) efflux. Thus Ca(2+)-dependent mitochondrial Ca(2+) uptake and plasmalemmal Ca(2+) extrusion clear out a small Ca(2+) load in frog motor nerve terminals, while thapsigargin-sensitive Ca(2+) pump boosts the clearance of a heavy Ca(2+) load. Furthermore, the activity of plasmalemmal Ca(2+) pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.     

Ultrastructural correlates of naturally occurring differences in transmitter release efficacy in frog motor nerve terminals

Summary Motor nerve terminals in cutaneous pectoris muscles of the frogRana pipiens release more transmitter and form synapses with higher levels of effectiveness than do those in sartorius muscles. Neuromuscular junctions from these two muscles were compared in the electron microscope to search for ultrastructural correlates of differences in transmitter release and synaptic effectiveness. The following measurements were made from cross-sections of junctions with known levels of effectiveness: (a) the presence of active zones, the presumed sites of transmitter release, (b) active zone size, (c) the perimeter, cross-sectional area, height and width of nerve terminals, (d) number of mitochondria, (e) vesicle density, and (f) the extent to which Schwann cells wrap terminals. Nerve terminals in the two muscles did not differ in size, shape or vesicle density. The more strongly releasing cutaneous pectoris terminals did, however, have significantly larger active zones due to deeper invagination of the terminal into the postsynaptic gutter and lesser interposition of Schwann cell processes between presynaptic and postsynaptic membranes. Cutaneous pectoris terminals also contained more mitochondria, presumably to supply the greater energy demand imposed by high release levels.     

Inhibition of mitochondrial Ca2+ uptake affects phasic release from motor terminals differently depending on external [Ca2+

We investigated how inhibition of mitochondrial Ca2+ uptake affects stimulation-induced increases in cytosolic [Ca2+] and phasic and asynchronous transmitter release in lizard motor terminals in 2 and 0.5 mM bath [Ca2+]. Lowering bath [Ca2+] reduced the rate of rise, but not the final amplitude, of the increase in mitochondrial [Ca2+] during 50-Hz stimulation. The amplitude of the stimulation-induced increase in cytosolic [Ca2+] was reduced in low-bath [Ca2+] and increased when mitochondrial Ca2+ uptake was inhibited by depolarizing mitochondria. In 2 mM Ca2+, end-plate potentials (epps) depressed by 53% after 10 s of 50-Hz stimulation, and this depression increased to 80% after mitochondrial depolarization. In contrast, in 0.5 mM Ca2+ the same stimulation pattern increased epps by approximately 3.4-fold, and this increase was even greater (transiently) after mitochondrial depolarization. In both 2 and 0.5 mM [Ca2+], mitochondrial depolarization increased asynchronous release during the 50-Hz train and increased the total vesicular release (phasic and asynchronous) measured by destaining of the styryl dye FM2-10. These results suggest that by limiting the stimulation-induced increase in cytosolic [Ca2+], mitochondrial Ca2+ uptake maintains a high ratio of phasic to asynchronous release, thus helping to sustain neuromuscular transmission during repetitive stimulation. Interestingly, the quantal content of the epp reached during 50-Hz stimulation stabilized at a similar level ( approximately 20 quanta) in both 2 and 0.5 mM Ca2+. A similar convergence was measured in oligomycin, which inhibits mitochondrial ATP synthesis without depolarizing mitochondria, but quantal contents fell to <20 when mitochondria were depolarized in 2 mM Ca2+.     

Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

We studied inactivation of Ca(2+)-induced Ca(2+) release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca(2+)](i) due to CICR evoked by a depolarizing pulse decreased markedly within 10-20 ms to a much slower rate despite persistent Ca(2+) entry and little depletion of Ca(2+) stores. The Ca(2+) entry elicited by the subsequent pulse within 50 ms, during which the [Ca(2+)](i) level remained unchanged, did not generate a distinct [Ca(2+)](i) rise. This mode of [Ca(2+)](i) rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 microm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct >or= 50 ms after depolarization and depended on [Ca(2+)](i). The inactivation was prevented by BAPTA (>or= 100 microm) but not by EGTA (相似文献   

Free radical-dependent Ca2+ signaling: role of Ca2+-induced Ca2+ release

Previously we have shown that Fe3+/ascorbate-induced Ca2+ release from scallop sarcoplasmic reticulum (SR) is due to Ca2+-channel gating by free radicals. This study is aimed at demonstrating that Ca2+-induced Ca2+ release (CICR) plays a role in this kind of Ca2+ release. Scallop SR vesicles were incubated with fluo-3 and exposed to Fe3+/ascorbate. Fluorimetric recordings showed massive Ca2+ release, with maximum rate and 50% release occurring at 30 min after exposure. Conversely, the use of the probe for reactive oxygen species dihydrorhodamine or the assay of malondialdehyde allowed oxyradical production to be traced for approximately 5 min only. Hence, although Ca2+ release started just after exposure to Fe3+/ascorbate, most release occurred after free radical exhaustion. Ruthenium red addition after Fe3+/ascorbate slowed down the Ca2+ release, whereas cyclic adenosine 5'-diphosphoribose addition accelerated it, indicating that the free radical-induced Ca2+ release from SR vesicles triggers a mechanism of CICR that dramatically increases the initial effect.     

Calmodulin and cyclic ADP-ribose interaction in Ca2+ signaling related to cardiac sarcoplasmic reticulum: superoxide anion radical-triggered Ca2+ release

Reactive oxygen species (ROS) are often shown to damage cellular functions. The targets of oxidative damage depend on the nature of ROS produced and the site of generation. In contrast, ROS can also regulate signal transduction. In this case, ROS may either induce or enhance events, which lead to forward directions of cellular signaling. The consequences of regulation of signal transduction can be observed in physiological processes such as muscle contraction. Here, we discuss the concentration-dependent effects of superoxide anion radical (*O2-) on Ca2+ release from the cardiac sarcoplasmic reticulum (SR). Recent studies suggest that the ADP-ribosyl cyclase pathway, through its production of cyclic adenosine 5'-diphosphoribose (cADPR), may control Ca2+ mobilization in cardiac muscle cells. *O2- has dual effects that are concentration dependent. At low concentrations (nearly nanomolar levels), *O2- induces Ca2+ release by stimulating synthesis of cADPR, which requires calmodulin for sensitization of ryanodine-sensitive Ca2+-release channels (RyRC). At these low concentrations, *O2- is responsible for regulation of cellular signal transduction. At higher concentrations (micromolar levels), *O2- produces a loss in the function of calmodulin that is to inhibit RyRC. This results in an increase in Ca2+ release, which is linked to cell injury. The difference in the functions of low and high concentrations of *O2- may result in two distinct physiological roles in cardiac muscle Ca2+ signaling.     

钙诱导钙释放机制与慢性心力衰竭的关系

目的:探讨钙诱导的钙释放(CICR)过程与慢性心力衰竭之间的关系.方法:采用冠状动脉左前降支结扎手术制作大鼠慢性心衰模型,酶解法分离成年大鼠心室肌细胞,然后使用NiCl2(5mmol/L)和毒胡萝卜素(TG, 100nmol/L)分别阻断钠钙交换体和钙泵,采用膜片钳和激光扫描共聚焦显微镜同步实时系统记录心肌细胞的L-型钙电流(ICa.L)以及CICR过程中的钙瞬变.结果:心衰大鼠心肌细胞的ICa.L低于正常大鼠心肌细胞的ICa.L;心衰组心肌细胞内CICR过程中的钙火花发生率低于正常对照组.结论:心肌细胞CICR功能的异常与心力衰竭的发生机制有着十分密切的关系,可能为导致心力衰竭发生的重要原因之一.     

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.     

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+. The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid or release of Ca2+ from intracellular stores such as mitochondria. Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose, so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10(-4)M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10(-4)M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.     

Different effects of toosendanin on perineurially recorded Ca(2+) currents in mouse and frog motor nerve terminals.

By perineurial recording, the effects of toosendanin (TSN), a presynaptic blocker, on nerve terminal calcium currents (I(Ca)) were observed in innervated triangularis sterni of the mouse and cutaneous pectoris of the frog. It was found that TSN blocked the slow component of I(Ca) insensitive to nifedipine and omega-conotoxin-GVIA, and increasing the extracellular Ca(2+) concentration partially antagonized the inhibitory effect in mouse motor nerve terminals. However, in the frog, TSN increased the slow component of I(Ca) and this effect disappeared in the presence of nifedipine in perfusion solution. Based on previous data showing that the slow component of I(Ca) were mediated by different subtypes of calcium channels in mouse and frog motor nerve terminals, we presume that TSN could exercise different effects on various subtypes of calcium channels.