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.     

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.     

Enhancement of Ca2+-induced Ca2+ release by cyclic ADP-ribose in frog motor nerve terminals

Ca2+-induced Ca2+ release (CICR) occurs via activation of ryanodine receptors (RyRs) in frog motor nerve terminals after RyRs are primed for activation by repetitive Ca2+ entries, thereby contributing to synaptic plasticity. To clarify how the mechanism of CICR becomes activable by repetitive Ca2+ entries, we studied effects of a RyR modulator, cyclic ADP-ribose (cADPr), on CICR by Ca2+ imaging techniques. Use-dependent binding of fluorescent ryanodine and its blockade by ryanodine revealed the existence of RyRs in the terminals. Repetition of tetani applied to the nerve produced repetitive rises in intracellular Ca2+ ([Ca2+]i) in the terminals. The amplitude of each rise slowly waxed and waned during the course of the stimulation. These slow rises and decays were blocked by ryanodine, indicating the priming, activation and inactivation of CICR. Uncaging of caged-cADPr loaded in the terminals increased the amplitude of short tetanus-induced rises in [Ca2+]i and the amplitude, time to peak and half decay time of the slow waxing and waning rises in [Ca2+]i evoked by repetitive tetani. A cADPr blocker, 8-amino-cADPr, loaded in the terminals decreased the slow waxing and waning component of rises and blocked all the actions of exogenous cADPr. It is concluded that cADPr enhances the priming and activation of CICR. The four-state model for RyRs suggests that cADPr inhibits the inactivation of CICR and increases the activation efficacy of RyR.     

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.     

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.     

Inhibition of mitochondrial function affects cellular Ca2+ handling in pancreatic B-cells

The mitochondrial inhibitors NaN(3) and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) were used to study the role of mitochondria in pancreatic B-cell Ca2+ homeostasis. In glucose-stimulated B-cells NaN(3) and FCCP both increased the K(ATP) current and thus hyperpolarized the cell membrane potential, as expected for agents depleting cellular ATP. NaN(3) and FCCP stopped the glucose-induced oscillations in the cytosolic free Ca2+ concentration ([Ca2+](c)) and elicited a biphasic response. After a first rapid and transient increase, [Ca2+](c) rose in a second slow phase to a sustained level. In cells pretreated with thapsigargin the first inhibitor-induced rise in [Ca2+](c) was absent, suggesting that it may be due to Ca2+ mobilization from intracellular stores. The glucose-induced oscillations were terminated again by NaN(3) and FCCP, respectively, but the slow increase in [Ca2+](c)of the second phase was still present. A minute increase in [Ca2+](c)elicited by NaN(3) or FCCP was even visible after the removal of extracellular Ca2+, suggesting that the inhibitors also mobilize Ca2+ from mitochondria. NaN(3) and FCCP induced Ca2+ influx into B-cells treated with low glucose concentrations whose voltage-dependent Ca2+ channels are closed. Experiments with thapsigargin-preincubated cells indicate that disturbance of mitochondrial function stimulates Ca2+ influx through voltage-independent Ca2+ pathways. During the NaN(3)-induced increase in [Ca2+](c), K+-elicited depolarizations of the cells did not further augment [Ca2+](c). Evidently, this is due to a direct inhibitory effect of azide on L-type Ca2+ channels. The data demonstrate that disturbing the mitochondrial function affects cellular Ca2+ homeostasis in B-cells at several sites. Thus, it is concluded that intact mitochondrial function is a prerequisite for regular Ca2+ handling in B-cells.     

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.     

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.     

Caffeine affects Ca uptake and Ca release from intracellular stores: fura-2 measurements in isolated snail neurones

Using the fluorescent probe fura-2, the average cytoplasmic concentration of free Ca2+ [( Ca]i) was measured in isolated voltage-clamped neurons of the snail Helix pomatia. In normal Ringer solution [Ca]i transients elicited by membrane depolarizations lasting 30-100 s have a voltage dependence similar to that of the calcium current. In the presence of caffeine [Ca]i transients did not depend on the testing voltage, indicating Ca release from intracellular stores. In both cases [Ca]i decayed after the transient increase. The rate of [Ca]i decline was monoexponential and independent of the membrane potential. In caffeine-containing solution the decline was 3 times faster. Steady membrane depolarization in the presence of caffeine induced periodic changes in [Ca]i. A simple model to describe these oscillations on the basis of Ca release from and Ca uptake into intracellular stores predicted that the oscillations could be initiated and modulated by Ca influx into the cytoplasm, which is in line with experimental data.     

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.     

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     

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Ovalbumin-induced sensitization affects non-quantal acetylcholine release from motor nerve terminals and alters contractility of skeletal muscles in mice

Skeletal muscles play key roles in the development of various pathologies, including bronchial asthma and several types of auto-immune disorders, e.g. polymyositis. Since most of these maladies have an immunological/allergic element, this paper is devoted to assessing the impact of immunobiological reorganization on the functional properties of isolated skeletal muscles in mice. A combination of two methods (myography and electrophysiology) was used to evaluate extensor digitorum longus (EDL) and diaphragmatic muscle (DM) in this regard. Conventional myographic technique showed that ovalbumin-induced sensitization (OS) produced different changes in the contractile properties of EDL and DM. The amplitudes of carbachol (CCh)-induced contractions increased in DM but decreased in EDL. Those changes were inversely related to OS-mediated changes of non-quantal acetylcholine (ACh) release intensity within the muscle endplate, as shown by the electrophysiologically measured H-effect. These results clearly show that OS-mediated changes of non-quantal ACh release alter the functional properties of postjunctional ACh receptors and therefore contribute to the disturbance of CCh-induced contractility of skeletal muscles. Other mechanisms of OS-mediated changes of skeletal muscle contractility are also proposed and discussed.     

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.     

Ca2+-induced Ca2+ release in Aplysia bag cell neurons requires interaction between mitochondrial and endoplasmic reticulum stores

Intracellular Ca2+ is influenced by both Ca2+ influx and release. We examined intracellular Ca2+ following action potential firing in the bag cell neurons of Aplysia californica. Following brief synaptic input, these neuroendocrine cells undergo an afterdischarge, resulting in elevated Ca2+ and the secretion of neuropeptides to initiate reproduction. Cultured bag cell neurons were injected with the Ca2+ indicator, fura-PE3, and subjected to simultaneous imaging and electrophysiology. Delivery of a 5-Hz, 1-min train of action potentials (mimicking the fast phase of the afterdischarge) produced a Ca2+ rise that markedly outlasted the initial influx, consistent with Ca2+-induced Ca2+ release (CICR). This response was attenuated by about half with ryanodine or depletion of the endoplasmic reticulum (ER) by cyclopiazonic acid. However, depletion of the mitochondria, with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, essentially eliminated CICR. Dual depletion of the ER and mitochondria did not reduce CICR further than depletion of the mitochondria alone. Moreover, tetraphenylphosphonium, a blocker of mitochondrial Ca2+ release, largely prevented CICR. The Ca2+ elevation during and subsequent to a stimulus mimicking the full afterdischarge was prominent and enhanced by protein kinase C activation. Traditionally, the ER is seen as the primary Ca2+ source for CICR. However, bag cell neuron CICR represents a departure from this view in that it relies on store interaction, where Ca2+ released from the mitochondria may in turn liberate Ca2+ from the ER. This unique form of CICR may be used by both bag cell neurons, and other neurons, to initiate secretion, activate channels, or induce gene expression.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

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