Developmental alterations in the biophysical properties of Cav1.3 Ca2+ channels in mouse inner hair cells

Prior to hearing onset, spontaneous action potentials activate voltage-gated Ca_v1.3 Ca²⁺ channels in mouse inner hair cells (IHCs), which triggers exocytosis of glutamate and excitation of afferent neurons. In mature IHCs, Ca_v1.3 channels open in response to evoked receptor potentials, causing graded changes in exocytosis required for accurate sound transmission. Developmental alterations in Ca_v1.3 properties may support distinct roles of Ca_v1.3 in IHCs in immature and mature IHCs, and have been reported in various species. It is not known whether such changes in Ca_v1.3 properties occur in mouse IHCs, but this knowledge is necessary for understanding the roles of Ca_v1.3 in developing and mature IHCs. Here, we describe age-dependent differences in the biophysical properties of Ca_v1.3 channels in mouse IHCs. In mature IHCs, Ca_v1.3 channels activate more rapidly and exhibit greater Ca²⁺-dependent inactivation (CDI) than in immature IHCs. Consistent with the properties of Ca_v1.3 channels in heterologous expression systems, CDI in mature IHCs is not affected by increasing intracellular Ca²⁺ buffering strength. However, CDI in immature IHCs is significantly reduced by strong intracellular Ca²⁺ buffering, which both slows the onset of, and accelerates recovery from, inactivation. These results signify a developmental decline in the sensitivity of CDI to global elevations in Ca²⁺, which restricts negative feedback regulation of Ca_v1.3 channels to incoming Ca²⁺ ions in mature IHCs. Together with faster Ca_v1.3 activation kinetics, increased reliance of Ca_v1.3 CDI on local Ca²⁺ may sharpen presynaptic Ca²⁺ signals and improve temporal aspects of sound coding in mature IHCs.     

Calcium channels play a pivotal role in the sequence of ionic changes involved in initiation of mouse sperm acrosomal exocytosis

The sequence of ionic changes involved in initiation of acrosomal exocytosis in capacitated mouse spermatozoa was investigated. Earlier studies demonstrated that a large influx of Na⁺ is required for exocytosis, this Na⁺ apparently being associated with an increase in intracellular pH (pH_i) via an Na⁺-H⁺ exchanger. This rise in pH_i may in turn activate calcium channels and permit the influx of extracellular Ca²⁺ needed to trigger acrosomal exocytosis. In the present study, the dihydropyridine voltage-dependent calcium channel antagonist nifedipine was able to inhibit significantly exocytosis in sperm cells treated in various ways capable of stimulating acrosomal loss. The monovalent cation ionophore monensin can promote Na⁺ entry required for both capacitation and acrosomal exocytosis, as demonstrated by using chlortetracycline to monitor changes in sperm functional potential. In the presence of 10 nM nifedipine, monensin treatment accelerated capacitation but was unable to trigger exocytosis. The requirement for internalization of a high concentration of Na⁺ can be bypassed by the addition of 25 mM NH₄CI to raise the pH_i of cells capacitated in 25NH₄CI to raise the pH_i of cells capacitated in 25 mM Na⁺ (insufficient Na⁺ to support exocytosis under usual conditions). Again, introduction of nifedipine was able to inhibit exocytosis. In a third experimental approach, amiloridestimulated exocytosis in capacitated cells was significantly inhibited by nifedipine. In contrast to these treatments directed at specific mechanisms, the ability of the Ca²⁺ inophore A23187 to promote more general entry of Ca²⁺ and thereby to accelerate capacitation and exocytosis was not inhibited by nifedipine. Finally, monensin-treated cells exhibited a rise and then a fall in ⁴⁵Ca²⁺ uptake, the time course of which paralleled stimulation of acrosomal exocytosis in similarly treated cells. Nifedipine significantly reduced this uptake. The fact that nifedipine can block exocytosis induced by a variety treatments strongly suggests that voltage-dependent calcium channels play a pivotal role in the response. These results are consistent with the following sequence of ionic changes in capacitated cells leading to acrosomal exocytosis: [Na⁺]_i ↑ → [H]_i↓ → pH_i ↑ → activation of calcium channels → [Ca²⁺]_i ↑ → exocytosis. Given that zona-induced exocytosis is reportedly an indirect response, mediated by voltage-dependent calcium channels, and that the Na⁺-H⁺ exchanger in somatic cells can be activated by receptor-mediated mechanisms, we suggest that sperm-zona inter action promotes an influx of Na⁺ by activating an Na⁺-H⁺ exchanger and thereby initiating the above sequence of changes. © 1993 Wiley-Liss, Inc.     

Calcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledons

In developing seeds of bean (Phaseolus vulgaris L.), phloem‐imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K⁺ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole‐cell currents across their plasma membranes were measured with the patch‐clamp technique. A weakly rectified cation current displaying a voltage‐dependent blockade by external Ca²⁺ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal‐cell pH and Calcium‐dependent Cation Conductance (Pv‐CD‐pHCaCC) was highly selective for K⁺ over Ca²⁺ and Cl^–. For K⁺ current through Pv‐CD‐pHCaCC a sigmoid shaped current–voltage (I–V) curve was observed with negative conductance at voltages between ?200 and ?140 mV. This negative K⁺ conductance was Ca²⁺ dependent. With other univalent cations (Na⁺, Rb⁺, NH₄⁺) the currents were smaller and were not Ca²⁺ dependent. Reversal potentials remained constant when external K⁺ was substituted with these cations, suggesting that Pv‐CD‐pHCaCC channels were non‐selective. The Pv‐CD‐pHCaCC would provide a pathway for K⁺ and other univalent cation influx into developing cotyledons. These cation influxes could be co‐ordinated with sucrose influx via pH and Ca²⁺dependence.     

H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.     

Fine Tuning of CaV1.3 Ca2+ Channel Properties in Adult Inner Hair Cells Positioned in the Most Sensitive Region of the Gerbil Cochlea

Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca²⁺ inflow through Ca_V1.3 (L-type) Ca²⁺ channels. We investigated the macroscopic (whole-cell) and elementary (cell-attached) properties of Ca²⁺ currents in IHCs positioned at the middle turn (frequency ∼2 kHz) of the adult gerbil cochlea, which is their most sensitive hearing region. Using near physiological recordings conditions (body temperature and a Na⁺ based extracellular solution), we found that the macroscopic Ca²⁺ current activates and deactivates very rapidly (time constant below 1 ms) and inactivates slowly and only partially. Single-channel recordings showed an elementary conductance of 15 pS, a sub-ms latency to first opening, and a very low steady-state open probability (P _o: 0.024 in response to 500-ms depolarizing steps at ∼−18 mV). The value of P _o was significantly larger (0.06) in the first 40 ms of membrane depolarization, which corresponds to the time when most Ca²⁺ channel openings occurred clustered in bursts (mean burst duration: 19 ms). Both the P _o and the mean burst duration were smaller than those previously reported in high-frequency basal IHCs. Finally, we found that middle turn IHCs are likely to express about 4 times more Ca²⁺ channels per ribbon than basal cells. We propose that middle-turn IHCs finely-tune Ca_V1.3 Ca²⁺ channel gating in order to provide reliable information upon timing and intensity of lower-frequency sounds.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Impact of gating modulation in CaV1.3 L-type calcium channels

Ca_V1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells Ca_V1.3 inward calcium current (I_Ca) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing Ca_V1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing Ca_V1.3 channels. Ca_V1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel’s negative activation range and slows calcium-dependent inactivation.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

High Calcium Permeability of Serotonin 5-HT3 Receptors on Presynaptic Nerve Terminals from Rat Striatum

Abstract: The serotonin 5-HT₃ receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT₃ receptors induce Ca²⁺ influx. Whereas postsynaptic 5-HT₃ receptors induce depolarization, being permeant to Na⁺ and K⁺, the basis of presynaptic 5-HT₃ receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT₃ receptor agonist, induced increases in internal free Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca²⁺]_i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT₃ receptor antagonist, but were insensitive to the removal of external free Na⁺ (substituted with N-methyl-d -glucamine), to prior depolarization induced on addition of 20 mM K⁺, or to voltage-gated Ca²⁺ channel blockade by 10 µM Co²⁺/Cd²⁺ or by 1 µMω-conotoxin MVIIC/1 µMω-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca²⁺ influx induced by 5-HT₃ receptor activation in NG108-15 cells by 1 µM mCPBG was substantially reduced by 10 µM Co²⁺/Cd²⁺ and was completely blocked by 1 µM nitrendipine, an L-type Ca²⁺ channel blocker. We conclude that in contrast to the perikaryal 5-HT₃ receptors, presynaptic 5-HT₃ receptors appear to be uniquely calcium-permeant.     

Optimizing synaptic architecture and efficiency for high-frequency transmission

Bursts of neuronal activity are transmitted more effectively as synapses mature. However, the mechanisms that control synaptic efficiency during development are poorly understood. Here, we study postnatal changes in synaptic ultrastructure and exocytosis in a calyx-type nerve terminal. Vesicle pool size, exocytotic efficiency (amount of exocytosis per Ca influx), Ca current facilitation, and the number of active zones (AZs) increased with age, whereas AZ area, number of docked vesicles per AZ, and release probability decreased with age. These changes led to AZs that are less prone to multivesicular release, resulting in reduced AMPA receptor saturation and desensitization. A greater multiplicity of small AZs with few docked vesicles, a larger pool of releasable vesicles, and a higher efficiency of release thus promote prolonged high-frequency firing in mature synapses.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately ?30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca²⁺ spikes with a maximum rate of rise of 7.5 Vs^?1 in microstome and 4.7 Vs^?1 in macrostome cells. Depolarizing voltage steps from a holding potential of ?40 mV induced an inward Ca²⁺ ‐current (I_ca) peaking at ?10 mV, reaching approximately the same value in microstome (?1.4 nA) and macrostome cells (?1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I_Ca in these morphs supports the notion that the voltage‐gated Ca²⁺ channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na⁺‐free solution and was only partially inhibited by extracellular Cs⁺. The inward rectification was completely blocked by replacing Ca²⁺ with Co²⁺ or Ba²⁺ in the recording solution, and is probably due to Ca²⁺ ‐activated inward K⁺ current secondary to Ca²⁺ influx through channels activated by hyperpolarization.     

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca²⁺ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca²⁺ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca²⁺ transport was specific to midtubule segments, where 97% of the Ca²⁺ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca²⁺ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca²⁺ influx is facilitated by voltage‐gated Ca²⁺ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca²⁺ had opposite effects on tubule Ca²⁺ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca²⁺ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca²⁺ sequestration through stimulatory (cAMP) and inhibitory (Ca²⁺) regulatory pathways.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

Hypoxia‐induced increase in intracellular calcium concentration in endothelial cells: Role of the Na+‐glucose cotransporter

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet‐activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca²⁺]_i) which then activates the phospholipase A₂ (PLA₂). The link between the decrease in ATP and the increase in [Ca²⁺]_i was not known and is investigated in this work. We first showed that the presence of extracellular Na⁺ was necessary to observe the hypoxia‐induced increase in [Ca²⁺]_i and the activation of PLA₂. This increase was not due to the release of Ca²⁺ from intracellular stores, since thapsigargin did not inhibit this process. The Na⁺/Ca²⁺ exchanger was involved since dichlorobenzamil inhibited the [Ca²⁺]_i and the PLA₂ activation. The glycolysis was activated, but the intracellular pH (pH_i) in hypoxic cells did not differ from control cells. Finally, the hypoxia‐induced increase in [Ca²⁺]_i and PLA₂ activation were inhibited by phlorizin, an inhibitor of the Na⁺‐glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na⁺ through the activated Na⁺‐glucose cotransport followed by the activation of the Na⁺/Ca²⁺ exchanger, resulting in a net influx of Ca²⁺. J. Cell. Biochem. 84: 115–131, 2002. © 2001 Wiley‐Liss, Inc.     

Plasma membrane Ca²+ transporters mediate virus-induced acquired resistance to oxidative stress

This paper reports the phenomenon of acquired cross‐tolerance to oxidative stress in plants and investigates the activity of specific Ca²⁺ transport systems mediating this phenomenon. Nicotiana benthamiana plants were infected with Potato virus X (PVX) and exposed to oxidative [either ultraviolet (UV‐C) or H₂O₂] stress. Plant adaptive responses were assessed by the combined application of a range of electrophysiological (non‐invasive microelectrode ion flux measurements), biochemical (Ca²⁺‐ and H⁺‐ATPase activity), imaging (fluorescence lifetime imaging measurements of changes in intracellular Ca²⁺ concentrations), pharmacological and cytological transmission electrone microscopy techniques. Virus‐infected plants had a better ability to control UV‐induced elevations in cytosolic‐free Ca²⁺ and prevent structural and functional damage of chloroplasts. Taken together, our results suggest a high degree of crosstalk between UV and pathogen‐induced oxidative stresses, and highlight the crucial role of Ca²⁺ efflux systems in acquired resistance to oxidative stress in plants.     

Spermidine oxidase‐derived H2O2 regulates pollen plasma membrane hyperpolarization‐activated Ca2+‐permeable channels and pollen tube growth

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca²⁺ concentration that accompanies pollen tube growth. Using the whole‐cell patch clamp and outside‐out single‐channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization‐activated Ca²⁺ current: the addition of Spd cannot induce the channel open probability increase in excised outside‐out patches, indicating that the effect of Spd in the induction of Ca²⁺ currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization‐activated Ca²⁺‐permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca²⁺‐permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal‐encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca²⁺‐permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca²⁺ influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd‐derived H₂O₂ signals Ca²⁺ influx, thereby regulating pollen tube growth.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.