Acute effect of beta amyloid on synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks

The effects of beta amyloid (Abeta) on cytoplasmic Ca(2+) ([Ca(2+)](c)) have been studied extensively, but the current literature on this aspect is confusing. We reported that 20 microM Abeta(25-35) significantly inhibited the synchronized spontaneous cytoplasmic Ca(2+) transients immediately after application, whereas it had little effect on the baseline [Ca(2+)](c) concentration in neurons. Abeta(1-42) had a similar effect on the Ca(2+) transients as Abeta(25-35), while it increased baseline [Ca(2+)](c) concentration gradually. However, Abeta(1-40) had little effect on either Ca(2+) transients or baseline [Ca(2+)](c). Such differential effects of Abeta on Ca(2+) signals might explain, at least partially, the confusing observations from the previous studies and provide important therapeutic implications for preventing or reversing early neuron damage in Alzheimer's disease.     

Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs

Dysregulation of intracellular Ca(2+) homeostasis may underlie amyloid beta peptide (Abeta) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Abeta(1-42) oligomers, the assembly state correlating best with cognitive decline in AD, but not Abeta fibrils, induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Abeta oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca(2+) overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca(2+) overload, cytochrome c release and cell death induced by Abeta oligomers. Our results indicate that i) mitochondrial Ca(2+) overload underlies the neurotoxicity induced by Abeta oligomers and ii) inhibition of mitochondrial Ca(2+) overload provides a novel mechanism of neuroprotection by NSAIDs against Abeta oligomers and AD.     

Cellular redox state protects acetaldehyde-induced alteration in cardiomyocyte function by modifying Ca2+ release from sarcoplasmic reticulum

Recent studies indicate that low concentrations of acetaldehyde may function as the primary factor in alcoholic cardiomyopathy by disrupting Ca(2+) handling or disturbing cardiac excitation-contraction coupling. By producing reactive oxygen species, acetaldehyde shifts the intracellular redox potential from a reduced state to an oxidized state. We examined whether the redox state modulates acetaldehyde-induced Ca(2+) handling by measuring Ca(2+) transient using a confocal imaging system and single ryanodine receptor type 2 (RyR2) channel activity using the planar lipid bilayer method. Ca(2+) transient was recorded in isolated rat ventricular myocytes with incorporated fluo 3. Intracellular reduced glutathione level was estimated using the monochlorobimane fluorometric method. Acetaldehyde at 1 and 10 microM increased Ca(2+) transient amplitude and its relative area in intact myocytes, but acetaldehyde at 100 microM decreased Ca(2+) transient area significantly. Acetaldehyde showed a minor effect on Ca(2+) transient in myocytes in which intracellular reduced glutathione content had been decreased against challenge of diethylmaleate to a level comparable to that induced by exposure to approximately 50 microM acetaldehyde. Channel activity of the RyR2 with slightly reduced cytoplasmic redox potential from near resting state (-213 mV) or without redox fixation was augmented by all concentrations of acetaldehyde (1-100 microM) used here. However, acetaldehyde failed to activate the RyR2 channel, when the cytoplasmic redox potential was kept with a reduced (-230 mV) or markedly oxidized (-180 mV) state. This result was similar to effects of acetaldehyde on Ca(2+) transient in diethylmaleate-treated myocytes, probably being in oxidized redox potential. The present results suggest that acetaldehyde acts as an RyR2 activator to disturb cardiac muscle function, and redox potential protects the heart from acetaldehyde-induced alterations in myocytes.     

The role of Ca2+ channel modulation in the neuroprotective actions of estrogen in beta-amyloid protein and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) cytotoxic models

Physiologically relevant concentrations of 17beta-estradiol (E2) are neuroprotective in both beta-amyloid protein 25-35 (Abeta) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cytotoxicity in SK-N-SH cells. MPTP, but not Abeta, induces apoptosis in this cell line. The L-type calcium channel blocker nifedipine or decreased extracellular Ca(2+) concentration blocked Abeta-induced cell death, but not MPTP-induced cell death. Other blockers selective for different Ca(2+) channel subtypes had no effects on either Abeta or MPTP induced death. Western blot analysis for L-type Ca(2+) channel alpha(1)-subunits demonstrated that Abeta increases the expression of the neuronal alpha(1C) and alpha(1D) subunits of L-type channels. Both E2 and nifedipine inhibit the increase in expression of these by Abeta. MPTP also increases expression of alpha(1C) and alpha(1D), but the increases were not influenced by E2 or nifedipine. These observations suggested that Abeta cytotoxicity in SK-N-SH cells may involve increased availability of calcium to cells, whereas MPTP induced cytotoxicity does not require extracellular Ca(2+). Both cytotoxic models were associated with increased expression of Ca(2+) channel alpha(1) subunits, and neuroprotection associated with inhibition of that increase. These studies reveal that nifedipine, in addition to its direct action of nifedipine on Ca(2+) channels, may also protect neurons from Abeta toxicity through the suppression of the channel protein overexpression. A new action of dihydropyridines (DHPs) may be considered in the regulation of calcium homeostasis.     

Carboxyl-terminal peptide of beta-amyloid precursor protein blocks inositol 1,4,5-trisphosphate-sensitive Ca2+ release in Xenopus laevis oocytes

The effects of Alzheimer's disease-related amyloidogenic peptides on inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) mobilization were examined in Xenopus laevis oocytes. Intracellular Ca(2+) was monitored by electrophysiological measurement of the endogenous Ca(2+)-activated Cl(-) current. Application of a hyperpolarizing pulse released intracellular Ca(2+) in oocytes primed by pre-injection of a non-metabolizable inositol 1,4,5-trisphosphate analogue. The carboxyl terminus of the amyloid precursor protein inhibited inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca(2+) release in a dose-dependent manner. Equimolar beta-amyloid peptides Abeta(1-40) or Abeta(1-42) had no effect, and whereas a truncated carboxyl terminus lacking the Abeta domain was equipotent to the full-length one, a carboxyl terminus fragment lacking the NPTY sequence was less effective than the full-length fragment. The inhibition induced by the carboxyl terminus was not associated with the block of the Ca(2+)-dependent Cl(-) channel itself or compromised Ca(2+) influx. We conclude that the carboxyl terminus of the amyloid precursor protein inhibits inositol 1,4,5-trisphosphate-sensitive Ca(2+) release and could thus disrupt Ca(2+) homeostasis and that the carboxyl terminus is much more effective than the beta-amyloid fragments used. By perturbing the coupling of inositol 1,4,5-trisphosphate and Ca(2+) release, the carboxyl terminus of the amyloid precursor protein can potentially be involved in inducing the neural toxicity characteristic of Alzheimer's disease.     

Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes.

Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist.     

Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses including neurotransmitter release, muscle contraction, and cell growth and proliferation [1]. During intracellular Ca(2+) signaling, mitochondria rapidly take up significant amounts of Ca(2+) from the cytosol, and this stimulates energy production, alters the spatial and temporal profile of the intracellular Ca(2+) signal, and triggers cell death [2-10]. Mitochondrial Ca(2+) uptake occurs via a ruthenium-red-sensitive uniporter channel found in the inner membrane [11]. In spite of its critical importance, little is known about how the uniporter is regulated. Here, we report that the mitochondrial Ca(2+) uniporter is gated by cytosolic Ca(2+). Ca(2+) uptake into mitochondria is a Ca(2+)-activated process with a requirement for functional calmodulin. However, cytosolic Ca(2+) subsequently inactivates the uniporter, preventing further Ca(2+) uptake. The uptake pathway and the inactivation process have relatively low Ca(2+) affinities of approximately 10-20 microM. However, numerous mitochondria are within 20-100 nm of the endoplasmic reticulum, thereby enabling rapid and efficient transmission of Ca(2+) release into adjacent mitochondria by InsP(3) receptors on the endoplasmic reticulum. Hence, biphasic control of mitochondrial Ca(2+) uptake by Ca(2+) provides a novel basis for complex physiological patterns of intracellular Ca(2+) signaling.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.     

Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Selectivity signatures of three isoforms of recombinant T-type Ca2+ channels

Voltage-gated Ca(2+) channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca(2+) over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca(2+) permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca(2+)-channel alpha1 subunits, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca(v)3 channels select Ca(2+) over Na(+) by affinity. Ca(v)3.1 and Ca(v)3.2 discriminate Ca(2+), Sr(2+) and Ba(2+) based on the ion's effects on the open channel probability, whilst Ca(v)3.3 discriminates based on the ion's intrapore binding affinity. All Ca(v)3s were characterized by much smaller difference in the K(D) values for Na(+) current blockade by Ca(2+) (K(D1) approximately 6 microM) and for Ca(2+) current saturation (K(D2) approximately 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na(+)/Ca(2+) current at close to physiological Ca(2+) concentrations, which was the strongest for Ca(v)3.3, smaller for Ca(v)3.2 and the smallest for Ca(v)3.1. In addition to intrapore Ca(2+) binding site(s) Ca(v)3.2, but not Ca(v)3.1 and Ca(v)3.3, is likely to possess an extracellular Ca(2+) binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca(2+) current in native cells.     

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.     

Contribution of both Ca2+ entry and Ca2+ sensitization to the alpha1-adrenergic vasoconstriction of rat penile small arteries

Sympathetic adrenergic nerves maintain the flaccid state of the penis through the tonic release of norepinephrine that contracts trabecular and arterial smooth muscle. Simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension and experiments with alpha-toxin-permeabilized arteries were performed in branches of the rat dorsal penile artery to investigate the intracellular Ca(2+) signaling pathways underlying alpha(1)-adrenergic vasoconstriction. Phenylephrine increased both [Ca(2+)](i) and tension, these increases being abolished by extracellular Ca(2+) removal and reduced by about 50% by the L-type Ca(2+) channel blocker nifedipine (0.3 microM). Non-L-type Ca(2+) entry through store-operated channels was studied by inhibiting the sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA (30 microM) induced variable phasic contractions that were abolished by extracellular Ca(2+) removal and by the store-operated channels antagonist 2-aminoethoxydiphenyl borate (2-APB, 50 microM) and largely inhibited by nifedipine (0.3 microM). CPA induced a sustained increase in [Ca(2+)](i) that was reduced in a Ca(2+)-free medium. Under conditions of L-type channels blockade, Ca(2+) readmission after store depletion with CPA evoked a sustained and marked elevation in [Ca(2+)](i) not coupled to contraction. 2-APB (50 microM) inhibited the rise in [Ca(2+)](i) evoked by CPA and the nifedipine-insensitive increases in both [Ca(2+)](i) and contraction elicited by phenylephrine. In alpha-toxin-permeabilized penile arteries, activation of G proteins with guanosine 5'-O-(3-thiotriphosphate) and of the alpha(1)-adrenoceptor with phenylephrine both enhanced the myofilament sensitivity to Ca(2+). This Ca(2+) sensitization was reduced by selective inhibitors of PKC, tyrosine kinase (TK), and Rho kinase (RhoK) by 43%, 67%, and 82%, respectively. As a whole, the present data suggest the alpha(1)-adrenergic vasoconstriction in penile small arteries involves Ca(2+) entry through both L-type and 2-APB-sensitive receptor-operated channels, as well as Ca(2+) sensitization mechanisms mediated by PKC, TK, and RhoK. A capacitative Ca(2+) entry coupled to noncontractile functions of the smooth muscle cell is also demonstrated.     

Effects of (-)-epigallocatechin-3-gallate in Ca2+ -permeable non-selective cation channels and voltage-operated Ca2+ channels in vascular smooth muscle cells

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.     

Amyloid induced suicidal erythrocyte death.

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl(-) by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Abeta (1-42) could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca(2+) activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Abeta (1-42) (> or = 0.5 microM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Abeta (1-40) (1 microM). Abeta (1-42) (> or = 1.0 microM) further significantly decreased forward scatter of erythrocytes. The effect of Abeta (1-42) (> or = 0.5 microM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca(2+) activity. The presence of Abeta (1-42) further significantly enhanced the eryptosis following Cl(-) depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Abeta (1-42), which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer's disease.     

A helix-breaking mutation in the epithelial Ca(2+) channel TRPV5 leads to reduced Ca(2+)-dependent inactivation

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.     

Effects of redox potential and Ca2+ on the inositol 1,4,5-trisphosphate receptor L3-1 loop region: implications for receptor regulation

Inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major intracellular Ca(2+) channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP(3)R and the regulators, and whether it involves conformational changes of the lumenal regions of IP(3)R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca(2+), on the L3-1 lumenal loop region of IP(3)R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca(2+) showed little effect on the L3-1 conformation, suggesting that the regulation of Ca(2+) is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys(34) and Cys(42). A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys(34)-Cys(42) region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys(34) and Cys(42) may act together as a reduction sensor, and that Cys(65) may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca(2+) can regulate IP(3)R through totally different mechanisms: Ca(2+) by the indirect effect and the redox potential by direct action causing conformational changes.     

Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.