Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Role of L- and N-type calcium channels in the pathophysiology of traumatic spinal cord white matter injury

Recent work has suggested a potential role for voltage-gated Ca(2+) channels in the pathophysiology of anoxic central nervous system white matter injury. To examine the relevance of these findings to neurotrauma, we conducted electrophysiological studies with inorganic Ca(2+) channels blockers and L- and N-subtype-specific calcium channel antagonists in an in vitro model of spinal cord injury. Confocal immunohistochemistry was used to examine for localization of L- and N-type calcium channels in spinal cord white matter tracts. A 30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber and injured with a modified clip (2g closing force) for 15s. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential at two points with glass microelectrodes. The compound action potential decreased to 71.4+/-2.0% of control (P<0. 05) after spinal cord injury. Removal of extracellular Ca(2+) promoted significantly greater recovery of compound action potential amplitude (86.3+/-7.6% of control; P< 0.05) after injury. Partial blockade of voltage-gated Ca(2+) channels with cobalt (20 microM) or cadmium (200 microM) conferred improvement in compound action potential amplitude. Application of the L-type Ca(2+) channel blockers diltiazem (50 microM) or verapamil (90 microM), and the N-type antagonist omega-conotoxin GVIA (1 microM), significantly enhanced the recovery of compound action potential amplitude postinjury. Co-application of the L-type antagonist diltiazem with the N-type blocker omega-conotoxin GVIA showed significantly greater (P<0.05) improvement in compound action potential amplitude than application of either drug alone. Confocal immunohistochemistry with double labelling for glial fibrillary acidic protein, GalC and NF200 demonstrated L- and N-type Ca(2+) channels on astrocytes and oligodendrocytes, but not axons, in spinal cord white matter.In conclusion, the injurious effects of Ca(2+) in traumatic central nervous system white matter injury appear to be partially mediated by voltage-gated Ca(2+) channels. The presence of L- and N-type Ca(2+) channels on periaxonal astrocytes and oligodendrocytes suggests a role for these cells in post-traumatic axonal conduction failure.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons posses both “big” and “small” types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM ω-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 μM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM ω-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 μM ω-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM ω-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly.The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker.

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 microM D600 or 10 microM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.     

Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin

The biophysical properties of T-type voltage-gated calcium channels are well suited to pacemaking and to supporting calcium flux near the resting membrane potential in both excitable and non-excitable cells. We have identified a new scorpion toxin (kurtoxin) that binds to the alpha 1G T-type calcium channel with high affinity and inhibits the channel by modifying voltage-dependent gating. This toxin distinguishes between alpha 1G T-type calcium channels and other types of voltage-gated calcium channels, including alpha 1A, alpha 1B, alpha 1C and alpha 1E. Like the other alpha-scorpion toxins to which it is related, kurtoxin also interacts with voltage-gated sodium channels and slows their inactivation. Kurtoxin will facilitate characterization of the subunit composition of T-type calcium channels and help determine their involvement in electrical and biochemical signaling.     

Reciprocal synaptic interactions between rod bipolar cells and amacrine cells in the rat retina.

Reciprocal synaptic transmission between rod bipolar cells and presumed A17 amacrine cells was studied by whole cell voltage-clamp recording of rod bipolar cells in a rat retinal slice preparation. Depolarization of a rod bipolar cell evoked two identifiable types of Ca2+ current, a T-type current that activated at about -70 mV and a current with L-type pharmacology that activated at about -50 mV. Depolarization to greater than or equal to -50 mV also evoked an increase in the frequency of postsynaptic currents (PSCs). The PSCs reversed at approximately ECl (the chloride equilibrium potential), followed changes in ECl, and were blocked by gamma-aminobutyric acidA (GABAA) and GABAC receptor antagonists and thus were identified as GABAergic inhibitory PSCs (IPSCs). Bipolar cells with cut axons displayed the T-type current but lacked an L-type current and depolarization-evoked IPSCs. Thus L-type Ca2+ channels are placed strategically at the axon terminals to mediate transmitter release from rod bipolar cells. The IPSCs were blocked by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that non-NMDA receptors mediate the feed-forward bipolar-to-amacrine excitation. The NMDA receptor antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid had no consistent effect on the depolarization-evoked IPSCs, indicating that activation of NMDA receptors is not essential for the feedforward excitation. Tetrodotoxin (a blocker of voltage-gated Na+ channels) reversibly suppressed the reciprocal response in some cells but not in others, indicating that graded potentials are sufficient for transmitter release from A17 amacrine cells, but suggesting that voltage-gated Na+ channels, under some conditions, can contribute to transmitter release.     

L-type calcium channels but not N-methyl-D-aspartate receptor channels mediate rhythmic activity induced by cholinergic agonist in motoneurons from turtle spinal cord slices

Rhythmic activity induced by different combinations of N-methyl-D-aspartate (NMDA), serotonin (5-HT), muscarine and D-tubocurarine was monitored intracellularly in lumbar motoneurons in a slice preparation from adult turtles. Low concentration of NMDA (7.5-15 microM) combined with 5-HT (10-80 microM) induced rhythmic motoneuron discharge which was underlied by intrinsic voltage oscillations resistant to tetrodotoxin. This oscillatory activity was abolished by 2-amino-5-phosphonopentanoic acid (AP5), a competitive blocker of NMDA receptors and by nifedipine a selective blocker of L-type calcium channels. In contrast, rhythmicity induced by the cholinergic agents muscarine and d-tubocurarine was abolished by nifedipine but not by AP5 nor by high [Mg2+]o. These results show that different receptor agonists induce intrinsic oscillations in mature motoneurons by independent routes. Each oscillatory mechanism depends on L-type calcium channels but only NMDA/5-HT-induced oscillations depend on voltage-sensitive NMDA-activated ionophores.     

Hypoxic increase in nitric oxide generation of rat sensory neurons requires activation of mitochondrial complex II and voltage-gated calcium channels

Recently, we have demonstrated that sensory neurons of rat lumbar dorsal root ganglia (DRG) respond to hypoxia with an activation of endothelial nitric oxide (NO) synthase (eNOS) resulting in enhanced NO production associated with mitochondria which contributes to resistance against hypoxia. Extracellular calcium is essential to this effect. In the present study on rat DRG slices, we set out to determine what types of calcium channels operate under hypoxia, and which upstream events contribute to their activation, thereby focusing upon mitochondrial complex II. Both the metallic ions Cd2+ and Ni2+, known to inhibit voltage-gated calcium channels and T-type channels, respectively, and verapamil and nifedipine, typical blocker of L-type calcium channels completely prevented the hypoxic neuronal NO generation. Inhibition of complex II by thenoyltrifluoroacetone at the ubiquinon binding site or by 3-nitropropionic acid at the substrate binding site largely diminished hypoxic-induced NO production while having an opposite effect under normoxia. An additional blockade of voltage-gated calcium channels entirely abolished the hypoxic response. The complex II inhibitor malonate inhibited both normoxic and hypoxic NO generation. These data show that complex II activity is required for increased hypoxic NO production. Since succinate dehydrogenase activity of complex II decreased at hypoxia, as measured by histochemistry and densitometry, we propose a hypoxia-induced functional switch of complex II from succinate dehydrogenase to fumarate reductase, which subsequently leads to activation of voltage-gated calcium channels resulting in increased NO production by eNOS.     

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.     

Voltage-gated calcium channels are not involved in generation and propagation of spreading depression (SD) in the brainstem of immature rats

Spreading depression (SD) can be elicited in the brainstem of rats younger than 13 days when excitability is enhanced by acetate superfusion [F. Richter, S. Rupprecht, A. Lehmenkühler, H.-G. Schaible, Spreading depression can be elicited in brain stem in immature but not adult rats, J. Neurophysiol. 90 (2003) 2163--2170]. To investigate whether voltage-gated calcium channels (VGCCs) modify initiation and propagation of SD in this type of tissue, we applied specific blockers to L-, T-, P/Q-, and N-type VGCCs locally or systemically. SD-related d.c. potentials and concomitant increases in extracellular potassium concentration ([K(+)](e)) were unaffected by the L- and T-type VGCC blocker flunarizine that was applied either systemically (up to 2mg/kg body weight) or by superfusion onto the brainstem (40 microM). In addition, local application of the P/Q-type VGCC blocker omega-agatoxin (1 microM) or of the N-type VGCC blocker omega-conotoxin (1 microM) to the brainstem surface did not influence SD. The results indicate that VGCCs do not modify the generation or propagation of SDs in the brainstem of the immature rat. Blockade of N-type VGCCs disturbed the normal breathing rhythm. Application of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (250-1000 microM) that elicited SD in the immature cortex, failed to elicit SD in the immature brainstem. In summary, it is likely that K(+) initiates and propagates brainstem SDs.     

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic-clonic seizures. IC neurons are known to have Ca(2+) channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca(2+) channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca(2+) channels were activated by depolarizing pulses from a holding potential of -90 mV in 10 mV increments using 5 mM barium (Ba(2+)) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca(2+) channel currents that could be blocked by 50 microM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 microM, L-type channel blocker), omega-conotoxin GVIA (1 microM, N-type channel blocker), and omega-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) omega-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca(2+) channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 microM nickel and inactivated with tau of 15-50 ms, was considered as R-type Ca(2+) channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca(2+) channel antibodies showed that IC neurons express Ca(2+) channel alpha(1A), alpha(1B), alpha(1C), alpha(1D), and alpha(1E) subunits. We conclude that IC neurons express functionally all members of HVA Ca(2+) channels, but only a subset of these neurons appear to have developed functional LVA channels.     

Localization of amiloride-sensitive sodium current and voltage-gated calcium currents in rat fungiform taste cells

Recent studies have shown that taste cells transducing bitter, sweet, and umami stimuli do not possess high-threshold voltage-gated calcium channels required for synaptic transmission at conventional synapses, suggesting some sort of signal processing inside taste buds prior to the activation of nerve endings. To evaluate whether this is a general paradigm for the physiology of taste reception, we studied the transduction pathway underlying the detection of sodium ions (salty stimulus). In laboratory rodents, Na(+) is thought to be transduced, at least in part, through amiloride-sensitive sodium channels (ASSCs). Therefore we used the patch-clamp techniques to analyze the occurrence pattern of amiloride-sensitive sodium currents and voltage-gated calcium currents (both low-voltage-activated T-type current and high-voltage-activated L-type current) among taste cells in rat fungiform papillae. Because taste cells turnover, we focused our attention on cells possessing large voltage-gated sodium currents, a sign of "mature" cells. We found that cells expressing functional ASSCs either did not possess any calcium currents or exhibited only T-type calcium currents, which is believed to play a role in repetitive firing. On the contrary, cells lacking functional ASSCs were endowed with L-type calcium currents, which are thought to mediate calcium influx required for neurotransmitter exocytosis. Therefore our data suggest that sodium-detecting cells are unlikely to use conventional synaptic communication to transfer taste information to nerve endings. Our findings on sodium taste detection support the recent model of taste transduction, involving separate groups of taste cells: chemosensitive cells and cells forming conventional synapses.     

Roles of Ca2+ influx through ATP-activated channels in catecholamine release from pheochromocytoma PC12 cells.

1. Extracellular ATP evokes catecholamine release concomitant with depolarization in pheochromocytoma PC12 cells. Roles of Ca2+ influx through ATP-activated channels during the catecholamine release were investigated. 2. Norepinephrine or dopamine release induced by > or = 100-microM concentrations of ATP was insensitive to 300 microM Cd2+, whereas the release induced by increasing extracellular KCl (50-150 mM) was completely blocked by this concentration of Cd2+. 3. ATP (100 microM) increased the intracellular free Ca2+ concentration measured with fura-2. The increase was not affected by 300 microM Cd2+ or 100 microM nicardipine, suggesting that Ca2+ influx through ATP-activated channels but not through voltage-gated Ca2+ channels contributes to the ATP-evoked catecholamine release. 4. Inward currents permeating through voltage-gated Ca2+ channels were measured using the whole-cell voltage clamp. In the presence of 10 microM ATP, a concentration that induces an ATP-activated channel-mediated current equivalent to that induced by 100 microM ATP during the depolarization in "non-voltage clamped" cells, the Ca2+ current activated by a voltage step to +10 mV was reduced. The reduction in the Ca2+ channel-mediated current was not observed when the extracellular Ca2+ was replaced with Ba2+. 5. The ATP (100 microM)-evoked dopamine release was inhibited by 300 microM Cd2+ when measured with extracellular Ba2+ instead of Ca2+. This effect of Ba2+ may not be related to K+ channel-blocking activity, because the ATP-evoked dopamine release obtained with 5 mM tetraethylammonium (TEA) was not inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium channels coupled to neurotransmitter release at dually innervated neuromuscular junctions in the newborn rat

We studied the effect of several calcium channel blockers (omega-Conotoxin-GVIA, 1 and 3microM; omega-Agatoxin-IVA, 100nM; Nitrendipine, 1 and 10microM) on evoked transmitter release at singly and dually innervated endplates of the levator auris longus muscle from three- to six-day-old rats. In dually innervated fibers, a second endplate potential may appear after the first one when we increase the stimulation intensity. The lowest and highest endplate potential amplitudes are designated "small endplate potential" and "large endplate potential", respectively. The percentage of doubly innervated junctions remains almost constant throughout the age range examined. Nevertheless, the percentage of junctions innervated by three or more terminal axons drops, whereas the singly innervated junctions increase. Therefore, between postnatal days 3 and 6, roughly half the neuromuscular junctions may experience the final process of axonal elimination. The synaptic efficacy of the large endplate potential in dual junctions, measured as the mean amplitude of the synaptic potential and mean quantal content, was the same as in the junctions that had become recently mono-innervated in the same postnatal period. In singly innervated fibers, the endplate potential size was strongly reduced by both the P/Q-type voltage-dependent calcium channel blocker omega-Agatoxin-IVA (79.17+/-4.02%; P < 0.05) and the N-type voltage-dependent calcium channel blocker omega-Conotoxin-GVIA (56.31+/-7.80%; P < 0.05), whereas endplate potential amplitude was not significantly changed by the L-type voltage-dependent calcium channel blocker Nitrendipine. In dually innervated fibers, the P/Q-type voltage-dependent calcium channel blocker omega-Agatoxin-IVA and L-type voltage-dependent calcium channel blocker Nitrendipine increased the size of the small endplate potential (161.29+/-47.87% and 109.32+/-11.03%, respectively; P < 0.05 in both cases) and reduced the large endplate potential (74.42+/-15.32% and 70.91+/-10.04%, respectively; P < 0.05 in both cases). The N-type voltage-dependent calcium channel blocker omega-Conotoxin-GVIA significantly increased the small endplate potential in the first few minutes after toxin application (at 10min: 90.23+/-17.38%; P < 0.05). This increase was not maintained, while the large endplate potential was strongly inhibited (69.25+/-7.5%; P < 0.05). In conclusion, in the dually innervated endplates of the newborn rat, presynaptic calcium channel types can have different roles in transmitter release from each of the two inputs, which suggests that nerve terminal voltage-dependent calcium channels are involved in neonatal synaptic maturation.     

Toosendanin, a triterpenoid derivative, increases Ca2+ current in NG108-15 cells via L-type channels

Toosendanin, a triterpenoid derivative extracted from Melia toosendan Sieb et Zucc, was demonstrated to be a selective presynaptic blocker and an effective antibotulismic agent in previous studies. Here, we observed its effects on Ca(2+) channels in NG108-15 cells by whole-cell patch-clamp recording. Obtained data showed that toosendanin concentration dependently increased the high-voltage-activated (HVA) Ca(2+) current with an EC(50) of 5.13 microM in differentiated NG108-15 cells. The enhancement effect was still observed when the cells were pretreated with 5 microM omega-conotoxin MVIIC. However, when the cells were preincubated with 5 microM nifedipine or 10 microM verapamil-containing solution, the effect was absent. In undifferentiated NG108-15 cells, which only express T-type Ca(2+) channels, toosendanin did not affect Ca(2+) currents. These results show that toosendanin increases Ca(2+) influx in NG108-15 cells via L-type Ca(2+) channels.     

Agatoxin-IVA-sensitive calcium channels mediate the presynaptic and postsynaptic nicotinic activation of cardiac vagal neurons

Whole cell currents and miniature glutamatergic synaptic events (minis) were recorded in vitro from cardiac vagal neurons in the nucleus ambiguus using the patch-clamp technique. We examined whether voltage-dependent calcium channels were involved in the nicotinic excitation of cardiac vagal neurons. Nicotine evoked an inward current, increase in mini amplitude, and increase in mini frequency in cardiac vagal neurons. These responses were inhibited by the nonselective voltage-dependent calcium channel blocker Cd (100 microM). The P-type voltage-dependent calcium channel blocker agatoxin IVA (100 nM) abolished the nicotine-evoked responses. Nimodipine (2 microM), an antagonist of L-type calcium channels, inhibited the increase in mini amplitude and frequency but did not block the ligand gated inward current. The N- and Q-type voltage-dependent calcium channel antagonists conotoxin GVIA (1 microM) and conotoxin MVIIC (5 microM) had no effect. We conclude that the presynaptic and postsynaptic facilitation of glutamatergic neurotransmission to cardiac vagal neurons by nicotine involves activation of agatoxin-IVA-sensitive and possibly L-type voltage-dependent calcium channels. The postsynaptic inward current elicited by nicotine is dependent on activation of agatoxin-IVA-sensitive voltage-dependent calcium channels.     

Mechanisms of unmodified CdSe quantum dot-induced elevation of cytoplasmic calcium levels in primary cultures of rat hippocampal neurons

Quantum dots (QDs) have shown great promise for applications in biology and medicine, which is being challenged by their potential nanotoxicity. Reactive oxygen species (ROS) produced by QDs are believed to be partially responsible for QD cytotoxicity. Cytoplasmic Ca(2+) plays an important role in the development of ROS injury. Here we found unmodified cadmium selenium (CdSe) QDs could elevate cytoplasmic calcium levels ([Ca(2+)](i)) in primary cultures of hippocampal neurons, involved both extracellular Ca(2+) influx and internal Ca(2+) release. More specifically, verapamil and mibefradil (L-type and T-type calcium channels antagonists, respectively) failed to prevent extracellular Ca(2+) influx under QD insult, while omega-conotoxin (N-type antagonist) could partially block this Ca(2+) influx. Surprisingly, this Ca(2+) influx could be well blocked by voltage-gated sodium channels (VGSCs) antagonist, tetrodotoxin (TTX). QD-induced internal Ca(2+) release could be avoided by clonazepam, a specific inhibitor of mitochondrial sodium-calcium exchangers (MNCX), and also by TTX. Furthermore, dantrolene, an antagonist of ryanodine (Ry) receptors in endoplasmic reticulum (ER), almost abolished internal Ca(2+) release, while 2-APB [inositol triphosphate (IP(3)) receptors antagonist] failed to block this Ca(2+) release, indicating that released Ca(2+) from mitochondria, which was induced by extracellular Na(+) influx, further triggered much more Ca(2+) release from ER. Our results imply that more research on the biocompatibility and biosafety of QD is both warranted and necessary.     

Hydrogen peroxide modulates whole cell Ca2+ currents through L-type channels in cultured rat dentate granule cells

Modification of voltage-gated Ca(2+) channels by hydrogen peroxide, a membrane-permeable form of reactive oxygen species, in cultured dentate granule cells was examined using the whole cell patch clamp technique. Pretreatment with hydrogen peroxide (1 and 10 microM) for 2 h enhanced the Ca(2+) current without affecting its voltage dependence. The enhancement was completely cancelled by 1 mM glutathione, an antioxidant, and 2 microM nifedipine, an L-type Ca(2+) channel blocker. In contrast, the enhancement of the Ca(2+) current was not mimicked by pretreatment with 10 microg/ml tunicamycin, an endoplasmic reticulum stressor. These results suggest that oxidative stress induced by hydrogen peroxide selectively regulates the activity of L-type Ca(2+) channels.     

Effects of anandamide and noxious heat on intracellular calcium concentration in nociceptive drg neurons of rats

As an endogenous agonist at the cannabinoid receptor CB1 and the capsaicin-receptor TRPV1, anandamide may exert both anti- and pronociceptive actions. Therefore we studied the effects of anandamide and other activators of both receptors on changes in free cytosolic calcium ([Ca(2+)](i)) in acutely dissociated small dorsal root ganglion neurons (diameter: < or =30 microm). Anandamide (10 microM) increased [Ca(2+)](i) in 76% of the neurons. The EC(50) was 7.41 microM, the Hill slope was 2.15 +/- 0.43 (mean +/- SE). This increase was blocked by the competitive TRPV1-antagonist capsazepine (10 microM) and in Ca(2+)-free extracellular solution. Neither exclusion of voltage-gated sodium channels nor additional blockade of voltage-gated calcium channels of the L-, N-, and/or T-type, significantly reduced the anandamide-induced [Ca(2+)](i) increase or capsaicin-induced [Ca(2+)](i) transients (0.2 microM). The CB1-agonist HU210 (10 microM) inhibited the anandamide-induced rise in [Ca(2+)](i). Conversely, the CB1-antagonist AM251 (3 microM) induced a leftward shift of the concentration-response relationship by approximately 4 microM (P < 0.001; Hill slope, 2.17 +/- 0.75). Intracellular calcium transients in response to noxious heat (47 degrees C for 10 s) were highly correlated with the anandamide-induced [Ca(2+)](i) increases (r = 0.84, P < 0.001). Heat-induced [Ca(2+)](i) transients were facilitated by preincubation with subthreshold concentrations of anandamide (3 microM), an effect that was further enhanced by 3 microM AM251. Although anandamide acts on both TRPV1 and CB1 receptors in the same nociceptive DRG neurons, its pronociceptive effects dominate. Anandamide triggers an influx of calcium through TRPV1 but no intracellular store depletion. It facilitates the heat responsiveness of TRPV1 in a calcium-independent manner. These effects of anandamide differ from those of the classical exogenous TRPV1-agonist capsaicin and suggest a primarily modulatory mode of action of anandamide.