Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μM) and capsaicin (1 μM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 μM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 μM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.     

Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons

Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2–based ratiometric Ca²⁺-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 µM, 100 µM and 300 µM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca²⁺ imaging protocols revealed that HiK⁺ (30 mM)-induced intracellular free Ca²⁺ ([Ca²⁺]_i) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 µM, 100 µM and 300 µM LEV (respectively), without any significant effect on basal levels of [Ca²⁺]_i. This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca²⁺, providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.     

Inhibition of spontaneous activity of rabbit atrioventricular node cells by KB-R7943 and inhibitors of sarcoplasmic reticulum Ca(2+) ATPase

The atrioventricular node (AVN) can act as a subsidiary cardiac pacemaker if the sinoatrial node fails. In this study, we investigated the effects of the Na-Ca exchange (NCX) inhibitor KB-R7943, and inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA), using thapsigargin or cyclopiazonic acid (CPA), on spontaneous action potentials (APs) and [Ca(2+)](i) transients from cells isolated from the rabbit AVN. Spontaneous [Ca(2+)](i) transients were monitored from undialysed AVN cells at 37°C using Fluo-4. In separate experiments, spontaneous APs and ionic currents were recorded using the whole-cell patch clamp technique. Rapid application of 5 μM KB-R7943 slowed or stopped spontaneous APs and [Ca(2+)](i) transients. However, in voltage clamp experiments in addition to blocking NCX current (I(NCX)) KB-R7943 partially inhibited L-type calcium current (I(Ca,L)). Rapid reduction of external [Na(+)] also abolished spontaneous activity. Inhibition of SERCA (using 2.5 μM thapsigargin or 30 μM CPA) also slowed or stopped spontaneous APs and [Ca(2+)](i) transients. Our findings are consistent with the hypothesis that sarcoplasmic reticulum (SR) Ca(2+) release influences spontaneous activity in AVN cells, and that this occurs via [Ca(2+)](i)-activated I(NCX); however, the inhibitory action of KB-R7943 on I(Ca,L) means that care is required in the interpretation of data obtained using this compound.     

Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) augments late sodium current (I(Na.L)) in cardiomyocytes. This study tests the hypothesis that both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) mediate the effect of increased [Ca(2+)](i) to increase I(Na.L). Whole cell and open cell-attached patch clamp techniques were used to record I(Na.L) in rabbit ventricular myocytes dialyzed with solutions containing various concentrations of [Ca(2+)](i). Dialysis of cells with [Ca(2+)](i) from 0.1 to 0.3, 0.6, and 1.0 μM increased I(Na.L) in a concentration-dependent manner from 0.221 ± 0.038 to 0.554 ± 0.045 pA/pF (n = 10, P < 0.01) and was associated with an increase in mean Na(+) channel open probability and prolongation of channel mean open-time (n = 7, P < 0.01). In the presence of 0.6 μM [Ca(2+)](i), KN-93 (10 μM) and bisindolylmaleimide (BIM, 2 μM) decreased I(Na.L) by 45.2 and 54.8%, respectively. The effects of KN-93 and autocamtide-2-related inhibitory peptide II (2 μM) were not different. A combination of KN-93 and BIM completely reversed the increase in I(Na.L) as well as the Ca(2+)-induced changes in Na(+) channel mean open probability and mean open-time induced by 0.6 μM [Ca(2+)](i). Phorbol myristoyl acetate increased I(Na.L) in myocytes dialyzed with 0.1 μM [Ca(2+)](i); the effect was abolished by G?-6976. In summary, both CaMKII and PKC are involved in [Ca(2+)](i)-mediated augmentation of I(Na.L) in ventricular myocytes. Inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial dysfunction and cardiac arrhythmias caused by calcium overload.     

Dicoumarol activates Ca2+-permeable cation channels triggering erythrocyte cell membrane scrambling

Dicoumarol, a widely used anticoagulant, may cause anemia, which may result from enhanced erythrocyte loss due to bleeding or due to accelerated erythrocyte death. Erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether dicoumarol induces eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cation channel activity utilizing whole cell patch clamp, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding, and hemolysis from haemoglobin release. Exposure of erythrocytes for 48 hours to dicoumarol (=10 μM) significantly increased [Ca(2+)](i), enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 μM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of Ca(2+). In conclusion, dicoumarol stimulates suicidal death of erythrocytes by stimulating Ca(2+) entry and subsequent triggering of Ca(2+) dependent cell membrane scrambling.     

Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

Bovine adrenocortical cells express bTREK-1 K(+) (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca(2+) and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca(2+)-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca(2+) ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 μM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP](i). BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca(2+) concentration ([Ca(2+)](i)) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% (n=11) and 59.3 ± 8.4% (n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP(2)) analog DiC(8)PI(4,5)P(2) in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability (P(o)) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca(2+) (10-35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K(+) channels by a Ca(2+)-dependent mechanism that does not require the depletion of membrane-associated PIP(2). They further indicate that the Ca(2+) source is located in close proximity within a "Ca(2+) nanodomain" of bTREK-1 channels, where [Ca(2+)](i) may reach concentrations of >10 μM. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.     

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.     

Extracellular calcium controls background current and neuronal excitability via an UNC79-UNC80-NALCN cation channel complex

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)'s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCN's intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion.     

Reductions in external divalent cations evoke novel voltage-gated currents in sensory neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg(2+) and Ca(2+) from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na(+) influx that then causes depolarization-mediated activation of voltage-gated Ca(2+) channels (Ca(V)), which allows Ca(2+) influx upon divalent re-introduction. Inhibition of Ca(V) (ω-conotoxin, nifedipine) or Na(V) (tetrodotoxin, lidocaine) fails to reduce the Na(+) influx. The Ca(2+) influx is inhibited by Ca(V) inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg(2+) or Ca(2+) alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg(2+) and Ca(2+) from external solutions evokes a large slowly-inactivating voltage-gated current (I(DF)) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca(2+) alone fails to evoke I(DF). Evidence suggests I(DF) is a non-selective cation current. The I(DF) is not reduced by inhibition of Na(V) (lidocaine, riluzole), Ca(V) (cilnidipine, nifedipine), K(V) (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca(2+) (IC(50)～0.5 μM) or Mg(2+) (IC(50)～3 μM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.     

Functional triads consisting of ryanodine receptors, Ca(2+) channels, and Ca(2+)-activated K(+) channels in bullfrog sympathetic neurons. Plastic modulation of action potential

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca(2+) ([Ca(2+)](i)) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 microm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 microM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1-2 microM), a Ca(2+)-pump blocker, or omega-conotoxin GVIA (omega-CgTx, 1 microM), a N-type Ca(2+) channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca(2+) entry thus triggered the regenerative activation of Ca(2+)-induced Ca(2+) release (CICR) in the submembrane region, followed by buffered Ca(2+) diffusion in the deeper cytoplasm. Computer simulation assuming Ca(2+) release in the submembrane region reproduced the Ca(2+) dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca(2+)-activated K(+) channel blocker, or omega-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca(2+)](i) gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca(2+)](i) rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca(2+)-activated K(+) current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca(2+)](i), and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca(2+) channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.     

Is Ca2+ release from internal stores involved in membrane excitation in characean cells?

An action potential in characean cells is accompanied by an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) which subsequently causes cessation of cytoplasmic streaming. Two Ca(2+ )origins are postulated for the increase in [Ca(2+)](c), extracellular and intracellular ones. For the extracellular origin, a Ca(2+) influx through voltage-dependent Ca(2+)-permeable channels is postulated. For the intracellular origin, a chain of reactions is assumed to occur, involving phosphoinositide-specific phospholipase C (PI-PLC) activation, production of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-dependent Ca(2+) release from internal stores [Biskup et al. (1999) FEBS Lett. 453: 72]. The hypothesis of the intracellular Ca(2+) origin was tested in three ways: injection of IP(3) into the streaming endoplasm, application of inhibitors of PI-PLC (U73122 and neomycin) and application of an inhibitor of IP(3)-receptor (2-aminoethoxydiphenyl borate; 2APB). Injection of 1 mM IP(3) into Chara cells did not change the rate of cytoplasmic streaming. Both U73122 (20 micro M) and neomycin (200 micro M) did not affect the generation of the action potential, cessation of cytoplasmic streaming and the increase in [Ca(2+)](c) caused by electric stimulus even 20-30 min after application. 2APB depolarized the membrane and inhibited the excitability of the plasma membrane. The results are not consistent with the data obtained by Biskup et al. (1999) who found inhibition of the excitatory inward current by neomycin and U73122. The hypotheses of internal and external Ca(2+) origins are discussed in the light of the present results.     

cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.     

Muscarinic receptor activation determines the effects of store-operated Ca(2+)-entry on excitability and energy metabolism in pyramidal neurons

In various cell types, depletion of intracellular Ca(2+)-stores results in store-operated Ca(2+)-entry (SOCE) across the cellular membrane. However, the effects of SOCE on neuronal membrane excitability and mitochondrial functions in central neurons are not well defined. We investigated such cellular downstream effects in pyramidal neurons of rat organotypic hippocampal slice cultures by applying electrophysiological and fluorescence imaging techniques. We report that SOCE is associated with (i) elevations of Ca(2+)-concentration in individual neuronal mitochondria ([Ca(2+)](m)). In addition, SOCE can result in (ii) hyperpolarizing neuronal membrane currents, (iii) increase in extracellular K(+)-concentration ([K(+)](o)), (iv) mitochondrial membrane depolarization, and (v) changes in intracellular redox state (NAD(P)H and FAD fluorescence), the latter reflecting responses of energy metabolism. These additional downstream effects of SOCE required concomitant muscarinic receptor activation by carbachol or acetylcholine, and were suppressed by agonist washout or application of antagonist, atropine. We conclude that muscarinic receptor activation determines the downstream effects of SOCE on neuronal membrane excitability and energy metabolism. This mechanism might have significant impact on information processing and neurometabolic coupling in central neurons.     

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.     

Spike-dependent calcium influx in dendrites of the cricket giant interneuron

Identified wind-sensitive giant interneurons in the cricket's cercal sensory system integrate cercal afferent signals and release an avoidance behavior. A calcium-imaging technique was applied to the giant interneurons to examine the presence of the voltage-dependent Ca(2+) channels (VDCCs) in their dendrites. We found that presynaptic stimuli to the cercal sensory nerve cords elevated the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the dendrites of the giant interneurons. The dendritic Ca(2+) rise coincided with the spike burst of the giant interneurons, and the rate of Ca(2+) rise depended on the frequency of the action potentials. These results suggest that the action potentials directly caused [Ca(2+)](i) increase. Observation of the [Ca(2+)](i) elevation induced by depolarizing current injection demonstrates the presence of the VDCCs in the dendrites. Although hyperpolarizing current injection into the giant interneuron suppressed action potential generation, EPSPs could induce no [Ca(2+)](i) increase. This result means that ligand-gated channels do not contribute to the synaptically stimulated Ca(2+) elevation. On the other hand, antidromically stimulated spikes also increased [Ca(2+)](i) in all cellular regions including the dendrites. And bath application of a mixture of Ni(2+), Co(2+), and Cd(2+) or tetrodotoxin inhibited the [Ca(2+)](i) elevation induced by the antidromic stimulation. From these findings, we suppose that the axonal spikes antidromically propagate and induce the Ca(2+) influx via VDCCs in the dendrites. The spike-dependent Ca(2+) elevation may regulate the sensory signals processing via second-messenger cascades in the giant interneurons.     

Revisiting the ionic mechanisms of early afterdepolarizations in cardiomyocytes: predominant by Ca waves or Ca currents?

Early afterdepolarizations (EADs) have been implicated in severe cardiac arrhythmias and sudden cardiac deaths. However, the mechanism(s) for EAD genesis, especially regarding the relative contribution of Ca(2+) wave (CaW) vs. L-type Ca current (I(Ca,L)), still remains controversial. In the present study, we simultaneously recorded action potentials (APs) and intracellular Ca(2+) images in isolated rabbit ventricular myocytes and systematically compared the properties of EADs in the following two pharmacological models: 1) hydrogen peroxide (H(2)O(2); 200 μM); and 2) isoproterenol (100 nM) and BayK 8644 (50 nM) (Iso + BayK). We assessed the rate dependency of EADs, the temporal relationship between EADs and corresponding CaWs, the distribution of EADs over voltage, and the effects of blockers of I(Ca,L), Na/Ca exchangers, and ryanodine receptors. The most convincing evidence came from the AP-clamp experiment, in which the cell membrane clamp was switched from current clamp to voltage clamp using a normal AP waveform without EAD; CaWs disappeared in the H(2)O(2) model, but persisted in the Iso + BayK model. We postulate that, although CaWs and reactivation of I(Ca,L) may act synergistically in either case, reactivation of I(Ca,L) plays a predominant role in EAD genesis under oxidative stress (H(2)O(2) model), while spontaneous CaWs are a predominant cause for EADs under Ca(2+) overload condition (Iso + BayK model).     

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Ca(2+) signaling in porcine duodenal glands by muscarinic receptor activation

The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular Ca(2+) concentration ([Ca(2+)](i)) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [Ca(2+)](i), and the increase was markedly inhibited by atropine or 4-diphenylacetoxy-N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M(3) subtype far exceeded that for either M(1) or M(2) as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca(2+)](i) evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca(2+) deprivation. Caffeine had no effect on [Ca(2+)](i). ACh elicited Ca(2+)-dependent K(+) currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of M(3) receptors linked to Ca(2+) release in porcine duodenal glands.