Effects of tetanus toxin on spontaneous and evoked transmitter release at inhibitory and excitatory synapses in the rat SDCN neurons

We observed the effects of tetanus toxin (TeNT) on spontaneous miniature and evoked postsynaptic currents at inhibitory (glycinergic) and excitatory (glutamatergic) synapses in SDCN of rat spinal cord, by use of ‘synaptic bouton’ preparations, under voltage clamp condition. TeNT (>10 pM) dose-dependently decreased the frequency without affecting amplitude of glycinergic spontaneous miniature IPSCs. However, TeNT (100 pM) had no effect on frequency or amplitude of glutamatergic spontaneous EPSCs. Focal paired electrical stimulation of ‘synaptic boutons’ elicited two consecutive glycinergic eIPSCs or glutamatergic eEPSCs with large amplitude and low failure rate (Rf). TeNT (100 pM) reduced the amplitude and increased the failure rate of the first glycinergic eIPSCs and greatly enhanced the ratio of the second to first (P2/P1) eIPSCs. Application of 4-AP restored glycinergic eIPSCs suppressed by TeNT (100 pM). However, TeNT (100 pM) had no effect on the amplitude, Rf or P2/P1 ratio of glutamatergic eEPSCs. These results show that TeNT pre-synaptically affects spontaneous and evoked, and inhibitory and excitatory neurotransmitter release differentially, thereby suggesting that molecular events underlying spontaneous and evoked, inhibitory and excitatory neurotransmitter release may be different in CNS, and that the release machinery becomes less sensitive to Ca²⁺ in TeNT poisoned ‘synaptic boutons’.     

Effects of scorpion toxin on excitatory and inhibitory presynaptic terminals

The effects of scorpion toxin (STX) on both spontaneous and evoked glycinergic and glutamatergic postsynaptic currents were studied by using both the mechanically dissociated single SDCN neuron (synaptic bouton preparation) and the ‘focal electrical stimulation technique’. In the experimental condition where Na⁺ channels on postsynaptic soma membrane were blocked by intracellular perfusion of QX-314, STX increased dose-dependently the frequency of spontaneous glycinergic and glutamatergic postsynaptic currents (sIPSC and sEPSC, respectively) without affecting the amplitude, suggesting STX acts on inhibitory and excitatory presynaptic nerve terminal. Such a facilitatory effect of STX on sIPSC was stronger than that on sEPSC. On the other hand, STX significantly enhanced the averaged current amplitude and decreased the failure rate (Rf) of both evoked inhibitory and excitatory postsynaptic currents (eIPSC and eEPSC, respectively), indicating that STX increases not only the release frequency of glycine and glutamate but also the amount of their release from the both presynaptic nerve endings. These effects of STX were completely removed by adding Na⁺ or Ca²⁺ channel blockers, indicating that STX increases Ca²⁺ influx through Ca²⁺ channels triggered by activating voltage-dependent Na⁺ channels on the nerve terminals. In addition, the difference of STX actions on the amplitude of spontaneous and evoked currents was discussed.     

P- and R-type Ca channels regulating spinal glycinergic nerve terminals

The contributions of P- and R-type Ca²⁺ channels on glycinergic nerve endings (boutons) projecting to the rat spinal sacral commissural nucleus (SDCN) neurons are not understood. Thus, we investigated the functional role of P- and R-type Ca²⁺ channels by measuring the inhibitory postsynaptic currents (eIPSCs) evoked from individual nerve endings (boutons) by focal electrical stimulation. The current amplitude and failure rate (Rf) of glycinergic eIPSCs varied directly with changes in [Ca²⁺]_o. Low concentration of ω-Aga IVA (P-type selective antagonist) suppressed eIPSCs as much as high concentration (both P- and Q-type selective) indicating little contribution of Q-type Ca²⁺ channels. Antagonism of R-type Ca²⁺ channels with SNX-482 and Ni²⁺ greatly decreased the current amplitude and increased failure rate (Rf) of glycinergic eIPSCs. Overall, our results suggest that the dominant control of glycine release depends on Ca²⁺ entry through P- and R-type Ca²⁺ channels that ubiquitously populate spinal glycine release sites.     

Neurotoxin A2NTX blocks fast inhibitory and excitatory transmitter release from presynaptic terminals

Our recent study showed a possibility that newly developed A2 type botulinum toxin (A2NTX) inhibits both spontaneous and evoked transmitter release from inhibitory (glycinergic or GABAergic) and excitatory (glutamatergic) nerve terminals using rat spinal sacral dorsal commissural nucleus neurons. In the present study, to determine the modulatory effect of A2NTX on glycinergic and glutamatergic release probabilities, we tested the effects of A2NTX on a single inhibitory or excitatory nerve ending adherent to a dissociated neuron that was activated by paired-pulse stimuli by using the focal electrical stimulation technique. The results of the present paired-pulse experiments showed clearly that A2NTX enhanced paired-pulse facilitation of evoked glycinergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents and increased the failure rate (Rf) of the first postsynaptic currents (P(1)) and both the responses. These effects of A2NTX on the amplitude and Rf of the P(1) and the second postsynaptic currents (P(2)) and paired-pulse ratio were rescued by application of 4-aminophthalimide. In summary, the present results showed that A2NTX acts purely presynaptically and inhibits the release machinery of transmitters such as glycine and glutamate, and the transmitter release machinery became less sensitive to intracellular free-Ca(2+) in A2NTX poisoned nerve terminals.     

Dual effect of 2,4-dinitrophenol on the spontaneous release of transmitter at the frog neuromuscular junction.

The uncoupling agent 2,4-Dinitrophenol (DNP) has a dual effect on the spontaneous release of transmitter at the frog neuromuscular junction, causing an initial fall in miniature endplate potential (MEPP) frequency followed by a dramatic rise. The latter effect is probably associated with the release of Ca²⁺ from the mitochondria. The initial fall in MEPP rate is independent of [Ca²⁺]₀ but is largely suppressed by pretreatment with theophylline; it is suggested that DNP also combines with another intracellular Ca²⁺-store, so reducing Ca²⁺ leakage and causing a fall in the steady-state level of [Ca²⁺]_i. The results confirm that MEPP frequency is largely determined by [Ca²⁺]_i at the presynaptic terminals.     

Dantrolene and neutromuscular junction: Evidence for intracellular calcium stores

Dantrolene sodium (DaNa) markedly depresses the frequency of spontaneous miniature potentials at the frog neuromuscular junction. Its action is still observed at low [Ca²⁺]_o and is little affected by temperature; its effect is not readily reversible. Theophylline antagonises the action of DaNa. DaNa prevents the stimulatory effect of the ionophore A23187 at 25°C, but is ineffective in antagonising the action of X537A. DaNa and theophylline do not affect the functioning and integrity of mitochondria in vitro and it is suggested that DaNa acts at intracellular Ca²⁺ stores (other than the mitochondria) in the presynaptic terminals.     

Idebenone inhibition of glutamate release from rat cerebral cortex nerve endings by suppression of voltage-dependent calcium influx and protein kinase A

The present study was aimed at investigating the effect and the possible mechanism of idebenone on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Idebenone inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration dependent. Inhibition of glutamate release by idebenone was prevented by chelating extracellular Ca²⁺, or by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to DL-threo-beta-benzyl-oxyaspartate, a glutamate transporter inhibitor. Idebenone decreased the depolarization-induced increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_C),whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The inhibitory effect of idebenone on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. Furthermore, the idebenone effect on 4-AP-evoked Ca²⁺ influx and glutamate release was completely abolished by the protein kinase A (PKA) inhibitors, H89 and KT5720. On the basis of these results, it was concluded that idebenone inhibits glutamate release from rat cortical synaptosomes and this effect is linked to a decrease in [Ca²⁺]_C contributed by Ca²⁺ entry through presynaptic voltage-dependent Ca²⁺ channels and to the suppression of PKA signaling cascade.     

Pharmacological changes in cellular Ca2+ homeostasis parallel initiation of atrial arrhythmogenesis in murine langendorff-perfused hearts

• 1 Intracellular Ca²⁺ overload has been associated with established atrial arrhythmogenesis. The present experiments went on to correlate acute initiation of atrial arrhythmogenesis in Langendorff‐perfused mouse hearts with changes in Ca²⁺ homeostasis in isolated atrial myocytes following pharmacological procedures that modified the storage or release of sarcoplasmic reticular (SR) Ca²⁺ or inhibited entry of extracellular Ca²⁺.
• 2 Caffeine (1 mmol/L) elicited diastolic Ca²⁺ waves in regularly stimulated atrial myocytes immediately following addition. This was followed by a decline in the amplitude of the evoked transients and the disappearance of such diastolic events, suggesting partial SR Ca²⁺ depletion.
• 3 Cyclopiazonic acid (CPA; 0.15 µmol/L) produced more gradual reductions in evoked Ca²⁺ transients and abolished diastolic Ca²⁺ events produced by the further addition of caffeine.
• 4 Nifedipine (0.5 µmol/L) produced immediate reductions in evoked Ca²⁺ transients. Further addition of caffeine produced an immediate increase followed by a decline in the amplitude of the evoked Ca²⁺ transients, without eliciting diastolic Ca²⁺ events.
• 5 These findings correlated with changes in spontaneous and provoked atrial arrhythmogenecity in mouse isolated Langendorf‐perfused hearts. Thus, caffeine was pro‐arrhythmogenic immediately following but not > 5 min after application and both CPA and nifedipine pretreatment inhibited such arrhythmogenesis.
• 6 Together, these findings relate acute atrial arrhythmogenesis in intact hearts to diastolic Ca²⁺ events in atrial myocytes that, in turn, depend upon a finite SR Ca²⁺ store and diastolic Ca²⁺ release following Ca²⁺‐induced Ca²⁺ release initiated by the entry of extracellular Ca²⁺.

     

The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

Background and purpose:

The sarcoplasmic reticulum (SR) releases Ca²⁺ via inositol 1,4,5-trisphosphate receptors (IP₃R) in response to IP₃-generating agonists. Ca²⁺ release subsequently propagates as Ca²⁺ waves. To clarify the role of IP₃ production in wave generation, the contribution of a key enzyme in the production of IP₃ was examined using a phosphoinositide-specific phospholipase C (PI-PLC) inhibitor, U-73122.

Experimental approach:

Single colonic myocytes were voltage-clamped in whole-cell configuration and cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) measured using fluo-3. SR Ca²⁺ release was evoked either by activation of IP₃Rs (by carbachol or photolysis of caged IP₃) or ryanodine receptors (RyRs; by caffeine).

Key results:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto transients. The drug also inhibited [Ca²⁺]_cyto increases, evoked by direct IP₃R activation (by photolysis of caged IP₃) and RyR activation (by caffeine), which do not require PI-PLC activation. U-73122 also increased steady-state [Ca²⁺]_cyto and slowed the rate of Ca²⁺ removal from the cytoplasm. An inactive analogue of U-73122, U-73343, was without effect on either IP₃R- or RyR-mediated Ca²⁺ release.

Conclusions and implications:

U-73122 inhibited carbachol-evoked [Ca²⁺]_cyto increases. However, the drug also reduced Ca²⁺ release when evoked by direct activation of IP₃R or RyR, slowed Ca²⁺ removal and increased steady-state [Ca²⁺]_cyto. These results suggest U-73122 reduces IP₃-evoked Ca²⁺ transients by inhibiting the SR Ca²⁺ pump to deplete the SR of Ca²⁺ rather than by inhibiting PI-PLC.     

Reduction in extracellular Ca^2＋ attenuates endothelium-dependent relaxation more than nitroprussideinduced relaxation

Aim:

To quantitatively assess the effect of lowering external Ca²⁺ ([Ca²⁺]_o) on both endothelium-dependent and -independent relaxations in rabbit aorta.

Methods:

Isometric contractions and relaxations of isolated aortae were recorded. When assessing the effect of reduced [Ca²⁺]_o on relaxations, the normal [Ca²⁺]_o solution was substituted with one of the reduced [Ca²⁺]_o solutions for one aorta, while a paired aorta was replenished with normal [Ca²⁺]_o solution.

Results:

The extent of acetylcholine (ACh)-induced relaxation, which is dependent on an intact endothelium, is time-dependent, and inversely related to [Ca²⁺]_o in a range of 0.02–2 mmol/L. ACh-induced relaxations were not significantly altered by the magnitude of the precontraction induced by PGF_2α. Nitroprusside-induced relaxations, which are independent of the endothelium, are also attenuated by reduced [Ca²⁺]_o. Relaxant responses to ACh were significantly more susceptible to reduced [Ca²⁺]_o than nitroprusside-induced relaxations. A maximally effective relaxing concentration of D600, an L-type Ca channel blocker methoxyverapamil, (10⁻⁵ mol/L) attenuated ACh-induced relaxations, whereas nitroprusside-induced relaxations were unaffected by D600.

Conclusion:

Thus, endothelium-dependent relaxation is more dependent on [Ca²⁺]_o than endothelium-independent relaxation, and it seems likely that [Ca²⁺]_o plays an important role not only in contractile processes, but also in relaxant processes as well.     

Exposure of piglet coronary arterial muscle cells to low concentrations of Mg2+ found in blood of ischemic heart disease patients result in rapid elevation of cytosolic Ca2+: Relevance to sudden infant death syndrome

Exposure of cultured piglet primary neonatal coronary arterial smooth muscle cells to concentrations of ionized Mg²⁺ ([Mg²⁺]_o (i.e., 0.48, 0.3, 0.15 mM) found in blood of patients presenting with ischemic heart disease and in hypoxic neonates resulted in concentration-dependent elevation in intracellular free Ca²⁺ ions ([Ca²⁺]_i; the lower the [Mg²⁺]_o, the higher the [Ca²⁺]_i rise. The lowest concentration of [Mg²⁺]_o tested, i.e., 0.15 mM, resulted in a clear rounding-up (i.e., contraction) of many of the coronary smooth muscle cells; reintroduction of normal 1.2 mM [Mg²⁺]_o failed to restore either normal [Ca²⁺]_i or cell shape.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Regulation by FK506 and rapamycin of Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle: the role of FK506 binding proteins and mTOR

Background and purpose:

The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP₃R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR).

Experimental approach:

Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca²⁺]_cyto measured using fluo-3. IP₃Rs were activated by photolysis of caged IP₃ and RyRs activated by hydrostatic application of caffeine.

Key results:

FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca²⁺]_cyto rise evoked by activation of either RyR or IP₃R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP₃-evoked [Ca²⁺]_cyto rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP₃-evoked [Ca²⁺]_cyto release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP₃R-mediated Ca²⁺ release. The mTOR inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, like rapamycin, decreased IP₃-evoked Ca²⁺ release.

Conclusions and implications:

Ca²⁺ release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP₃R-mediated Ca²⁺ release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP₃-mediated Ca²⁺ release may be explained by mTOR inhibition.     

L‐type Ca2+ channel opener BayK 8644‐induced Ca2+ influx and Ca2+ release in human oral cancer cells (OC2)

The effect of BayK 8644, a chemical widely used to activate L‐type Ca²⁺ channels, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) has not been explored to date. The present study examined whether BayK 8644 altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura‐2. BayK 8644 (10 pM–10 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. BayK 8644‐induced Ca²⁺ influx was blocked by nifedipine, but was not altered by the store‐operated Ca²⁺ entry inhibitors, econazole and SKF96365; protein kinase C modulators phorbol 12‐myristate 13‐acetate (PMA) and GF109203X; the protein kinase A inhibitor H89; and the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺‐free medium, after pretreatment with 1 µM BayK 8644, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pretreatment abolished BayK 8644‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change BayK 8644‐induced [Ca²⁺]_i rises. Collectively, in OC2 cells, BayK 8644 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via L‐type Ca²⁺ channels. Drug Dev Res 69: 2008. © 2008 Wiley‐Liss, Inc.     

Mechanisms of 5-hydroxytryptamine-induced inhibition in the porcine myometrium

1 The present experiments were designed to clarify the mechanisms of the inhibitory response of 5-hydroxytryptamine (5-HT) in the porcine uterine circular muscle. 2 Inhibitory responses induced by 5-HT (1 n m –1 μm ) were not affected by apamin (1 μm ), charybdotoxin (100 n m ) or glibenclamide (20 μm ) but were significantly attenuated by 4-aminopyridine (3 m m ) and tetraethylammonium (3 m m ). 3 Imidazole (100 μm ) decreased but 3-isobutyl-1-methylxanthine (30 μm ), milrinone (30 μm ) and Ro 20–1724 (10 and 30 μm ) potentiated the 5-HT-induced inhibition. On the other hand, zaprinast (3–30 μm ) had no significant effect on the inhibitory response of 5-HT. 4 5-HT caused a time (0–5 min)-and concentration (1 n m –1 μm )-dependent increase in the tissue cyclic AMP level, but had no effect on the tissue cyclic GMP level. A significant correlation (P < 0.05) was observed between the inhibition of contraction and tissue cyclic AMP level. 5 The effect of 5-HT on contractile force and cytosolic Ca²⁺ level ([Ca²⁺]_i) was investigated using fura-PE3-loaded myometrial strips. A low concentration of 5-HT (≤ 10 n m ) inhibited the spontaneous contraction without changing the amplitude of the spontaneous [Ca²⁺]_i increase, but a higher concentration of 5-HT (≥ 100 n m ) decreased the resting [Ca²⁺]_i and inhibited both the spontaneous [Ca²⁺]_i increase and spontaneous contraction. 6 High-K⁺ (50 m m ) caused increases in muscle contractile force and [Ca²⁺]_i. 5-HT concentration-dependently inhibited the high-K⁺-induced contraction (EC₅₀, 45 n m ) with only a small decrease in [Ca²⁺]_i increase. 7 Carbachol also caused increases in muscle contractile force and [Ca²⁺]_i. 5-HT significantly decreased both the carbachol-induced contraction and [Ca²⁺]_i increase, but was more potent at inhibition of contractile force than [Ca²⁺]_i. 8 In Ca²⁺ -loaded myometrial strips, carbachol, but not caffeine, caused a transient increase in [Ca²⁺]_i and contraction in the absence of external Ca²⁺ (EGTA, 1 m m ). 5-HT inhibited both the carbachol-induced increases in [Ca²⁺]_i release and contractile force. 9 In the β-escin permeabilized myometrium, 5-HT significantly inhibited the Ca²⁺-induced contraction. 10 The present results indicate that 5-HT stimulates tissue cyclic AMP production, and inhibits the porcine uterine muscle contractility by a reduction in [Ca²⁺]_i and in Ca²⁺ sensitivity of the contractile elements. Activation of K⁺ channels might be partially involved in 5-HT-induced inhibition of the myometrial contractility.     

Cardiac alternans and intracellular calcium cycling

Cardiac alternans refers to a condition in which there is a periodic beat‐to‐beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat‐to‐beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca²⁺ transient amplitude (Ca²⁺ alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (V_m) and intracellular calcium ([Ca²⁺]_i). Bidirectional coupling refers to the fact that, in cardiac cells, V_m depolarization and the generation of action potentials cause the elevation of [Ca²⁺]_i that is required for contraction (a process referred to as excitation–contraction coupling); conversely, changes of [Ca²⁺]_i control V_m because important membrane currents are Ca²⁺ dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca²⁺ signalling. Herein we review how two key factors of cardiac cellular Ca²⁺ cycling, namely the release of Ca²⁺ from internal stores and the capability of clearing the cytosol from Ca²⁺ after each beat, determine the conditions under which alternans occurs. The contributions from key Ca²⁺‐handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca²⁺ release channels) are discussed.     

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

Background and Purpose

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved.

Experimental Approach

End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques.

Key Results

Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A₃ receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A₃ receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd²⁺, the removal of extracellular Ca²⁺ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the G_i/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62.

Conclusion and Implications

Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A₃ receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A₃ receptors appear to be coupled to G_i/o protein. PKC and calmodulin may be involved in these effects of inosine.     

The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells

Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca²⁺ levels ([Ca²⁺]_i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca²⁺]_i rises which were reduced by removing extracellular Ca²⁺. Eugenol-induced [Ca²⁺]_i rises were not altered by store-operated Ca²⁺ channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca²⁺]_i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca²⁺]_i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca²⁺]_i rises by inducing PLC-dependent release of Ca²⁺ from the endoplasmic reticulum and caused Ca²⁺ influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway.     

Effect of melamine on [Ca2+]i and viability in PC3 human prostate cancer cells

Melamine is thought to be an endocrine disrupter that affects physiology in cells. This study examined the effect of melamine on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Melamine evoked [Ca²⁺]_i rises concentration-dependently. Melamine-evoked Ca²⁺ entry was inhibited by nifedipine, econazole, SKF96365, GF109203X and phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin inhibited melamine-evoked [Ca²⁺]_i rise. Conversely, treatment with melamine abolished thapsigargin-evoked [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter melamine-evoked [Ca²⁺]_i rise. Melamine at 500–800 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, our data suggest that in PC3 cells, melamine induced [Ca²⁺]_i rises by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ entry. Melamine also caused Ca²⁺-independent cell death.     

3,3'-Diindolylmethane alters Ca2+ homeostasis and viability in MG63 human osteosarcoma cells

Abstract: The effect of the natural product 3,3′‐diindolylmethane (DIM) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺‐sensitive fluorescent dye fura‐2 was applied to measure [Ca²⁺]_i. DIM at concentrations of 40–80 μM induced a [Ca²⁺]_i rise in a concentration‐dependent manner. The response was reduced partly by removing Ca²⁺. DIM‐evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited or abolished DIM‐induced [Ca²⁺]_i rise. Incubation with DIM also inhibited thapsigargin or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished DIM‐induced [Ca²⁺]_i rise. At concentrations of 10–50 μM, DIM killed cells in a concentration‐dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration‐dependent manner. In sum, in MG63 cells, DIM induced a [Ca²⁺]_i rise by evoking phospholipase C‐dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C‐sensitive store‐operated Ca²⁺ channels. DIM caused cell death that may involve apoptosis.