Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Angiotensin II type I receptor modulates intracellular free Mg2+ in renally derived cells via Na+-dependent Ca2+-independent mechanisms

Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) with a concomitant decrease in cytosolic free Mg(2+) concentration ([Mg(2+)](i)). In the present study we demonstrate that this hormone-induced decrease in [Mg(2+)](i) is independent of [Ca(2+)](i) but dependent on extracellular Na(+). [Mg(2+)](i), [Ca(2+)](i), and [Na(+)](i) were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg(2+)](i) and increased [Na(+)](i) in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT(1) receptor blocker) but not by PD123319 (selective AT(2) receptor blocker). Imipramine and quinidine (putative inhibitors of the Na(+)/Mg(2+) exchanger) and removal of extracellular Na(+) abrogated Ang II-mediated [Mg(2+)](i) effects. In cells pretreated with thapsigargin (reticular Ca(2+)-ATPase inhibitor), Ang II-stimulated [Ca(2+)](i) transients were attenuated (p < 0.01), whereas agonist-induced [Mg(2+)](i) responses were unchanged. Clamping the [Ca(2+)](i) near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca(2+)](i) increases but failed to alter Ang II-induced [Mg(2+)](i) responses. Benzamil, a selective blocker of the Na(+)/Ca(2+) exchanger, inhibited [Na(+)](i) but not [Mg(2+)](i) responses. Our data demonstrate that in MDCK cells, AT(1) receptors modulate [Mg(2+)](i) via a Na(+)-dependent Mg(2+) transporter that is not directly related to [Ca(2+)](i). These data support the notion that rapid modulation of [Mg(2+)](i) is not simply a result of Mg(2+) redistribution from intracellular buffering sites by Ca(2+) and provide evidence for the existence of a Na(+)-dependent, hormonally regulated transporter for Mg(2+) in renally derived cells.     

Calcium microdomains in mitochondria and nucleus

Endomembranes modify the progression of the cytosolic Ca(2+) wave and contribute to generate Ca(2+) microdomains, both in the cytosol and inside the own organella. The concentration of Ca(2+) in the cytosol ([Ca(2+)](C)), the mitochondria ([Ca(2+)](M)) and the nucleus ([Ca(2+)](N)) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca(2+) from the high [Ca(2+)](C) microdomains generated during cell activation near Ca(2+) channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca(2+) signal to the bulk cytosol. This shaping of [Ca(2+)](C) signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca(2+)](M) signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca(2+)](C) signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca(2+)](N) signals could be generated by direct release of stored Ca(2+) into the nucleoplasm. Ca(2+) release could even be restricted to subnuclear domains. Putative Ca(2+) stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca(2+) into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain.     

Voltage-dependent changes of TRPV6-mediated Ca2+ currents

The physiological role and activation mechanism for most proteins of the transient receptor potential (TRP) family are unknown. This is also the case for the highly Ca(2+) selective transient receptor potential vanilloid type 6 (TRPV6) channel. Patch clamp experiments were performed on transiently transfected human embryonic kidney (HEK) cells to address this issue. Currents were recorded under various conditions of intracellular Ca(2+) buffering and monitored at the same voltage throughout. No TRPV6-mediated Ca(2+) entry was detected under in vivo Ca(2+) buffering conditions at a slightly negative holding potential; however, moderate depolarization resulted in current activation. Very similar results were obtained with different Ca(2+) chelators, either EGTA or BAPTA dialyzing the cell. TRPV6 channel activity showed a negative correlation with the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and was modulated by the membrane potential: Hyperpolarization decreases and depolarization increases TRPV6-mediated currents. Monovalent ions permeated TRPV6 channels in the absence of extracellular divalent cations. These currents were resistant to changes in the holding potential while the negative correlation to the [Ca(2+)](i) was conserved, indicating that the voltage-dependent current changes depend on blocking and unblocking the charge carrier Ca(2+) within the pore. In summary, these results suggest that the voltage dependence of TRPV6-mediated Ca(2+) influx is of physiological importance since it occurs at cytosolic Ca(2+) buffering and takes place within a physiologically relevant membrane potential range.     

Patterns of [Ca2+](i) mobilization and cell response in human spermatozoa exposed to progesterone

Human spermatozoa stimulated with progesterone (a product of the cumulus and thus encountered by sperm prior to fertilization in vivo) apparently mobilize Ca(2+) and respond very differently according to the way in which the steroid is presented. A progesterone concentration ramp (0-3 microM) induces [Ca(2+)](i) oscillations (repetitive store mobilization) which modify flagellar beating, whereas bolus application of micromolar progesterone causes a single large transient (causing acrosome reaction) which is apparently dependent upon Ca(2+) influx. We have investigated Ca(2+)-mobilization and functional responses in human sperm exposed to 3 muM progesterone. The [Ca(2+)](i) response to progesterone was abolished by 4 min incubation in 0 Ca(2+) medium (2 mM EGTA) but in nominally Ca(2+)-free medium (no added Ca(2+); 0 EGTA) a smaller, slow response occurred. Single cell imaging showed a similar effect of nominally Ca(2+)-free medium and approximately 5% of cells generated a small transient even in the presence of EGTA. When cells were exposed to EGTA-containing saline (5 min) and then returned to nominally Ca(2+)-free medium before stimulation, the [Ca(2+)](i) transient was greatly delayed (approximately 50 s) and rise time was doubled in comparison to cells not subjected to EGTA pre-treatment. We conclude that mobilization of stored Ca(2+) contributes a 'slow' component to the progesterone-induced [Ca(2+)](i) transient and that incubation in EGTA-buffered saline is able rapidly to deplete this store. Analysis of flagellar activity induced by 3 muM progesterone showed an effect (modified beating) associated with the [Ca(2+)](i) transient, in >80% of cells bathed in nominally Ca(2+)-free medium. This was reduced greatly in cells subjected to 5 min EGTA pre-treatment. The store-mediated transient showed a pharmacological sensitivity similar to that of progesterone-induced [Ca(2+)](i) oscillations (consistent with filling of the store by an SPCA) suggesting that the transient induced by micromolar progesterone is a 'single shot' activation of the same store that generates Ca(2+) oscillations.     

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.     

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.     

Growth plate chondrocyte maturation is regulated by basal intracellular calcium

Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca(2+) concentration ([Ca(2+)](i)) from 50 to 100 nM. To determine if this rise in [Ca(2+)](i) functionally participates in the maturational process of growth plate chondrocytes (GPCs), we examined its effect on several markers of hypertrophy, including annexin V, bone morphogenetic protein-6, type X collagen, and indian hedgehog. Expression of these genes was determined under conditions either where the Ca(2+) chelator EGTA was used to deplete extracellular Ca(2+) and lower [Ca(2+)](i) to < 50 nM or where the extracellular addition of 5 mM CaCl(2) was used to elevate [Ca(2+)](i) to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl(2), 4 mM EGTA significantly inhibited their expression. This effect was recapitulated in sternal chondrocytes and was reversed following withdrawal of EGTA. Based on these findings, we hypothesized that the EGTA-induced suppression of these genes was mediated by a factor whose expression is responsive to changes in basal [Ca(2+)](i). Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca(2+)](i) on PTHrP expression. Suggesting that low [Ca(2+)](i) suppression of hypertrophy was PTHrP-dependent in GPCs, (a) treatment with 4 mM EGTA increased PTHrP expression, (b) the EGTA effect was rescued by blocking PTHrP binding to its receptor with the competitive antagonist TIP(7-39), and (c) EGTA could mimic the PTHrP stimulation of AP-1 binding to DNA. Additionally, PTHrP promoter analysis identified a domain (-1498 to -862, relative to the start codon) involved with conferring Ca(2+) sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca(2+) in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca(2+)](i).     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

Calcium dynamics in catecholamine-containing secretory vesicles

We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.     

Role of calcium in phototoxicity of 2-butylamino-2-demethoxy-hypocrellin A to human gastric cancer MGC-803 cells

After incubation with 2-butylamino-2-demethoxy-hypocrellin A (2-BA-2-DMHA), photodynamically induced change in the cytoplasmic free calcium concentration ([Ca(2+)](i)) and its effect on cell damage were investigated in human gastric cancer (MGC-803). Fluorescence spectrophotometry measurement indicated that the photosensitization of MGC-803 by 2-BA-2-DMHA caused an increase in intracellular calcium [Ca(2+)](i), and this increase in [Ca(2+)](i) showed a dependence on the concentration of 2-BA-2-DMHA, light dose and extracellular [Ca(2+)](e). This phenomenon of intracellular calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). Furthermore, the results from MTT assay and flow cytometry analysis suggested that chelation of extracellular calcium by EGTA or intracellular calcium by BAPTA could inhibit photodynamically induced cell killing, while increase of [Ca(2+)](i) by thapsigargin (TG), a highly specific inhibitor of the Ca(2+)-ATPase, or by A23187, a calcium ionophore could enhance this action. Meanwhile, the nucleus morphology was also investigated by fluorescence microscopy. The results indicated that the increase in intracellular Ca(2+) concentration was responsible for 2-BA-2-DMHA photodynamically induced damage to MGC-803.     

Calreticulin modulates capacitative Ca2+ influx by controlling the extent of inositol 1,4,5-trisphosphate-induced Ca2+ store depletion

Calreticulin (CRT) is a highly conserved Ca(2+)-binding protein that resides in the lumen of the endoplasmic reticulum (ER). We overexpressed CRT in Xenopus oocytes to determine how it could modulate inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) influx. Under conditions where it did not affect the spatially complex elevations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) due to InsP(3)-induced Ca(2+) release, overexpressed CRT decreased by 46% the Ca(2+)-gated Cl(-) current due to Ca(2+) influx. Deletion mutants revealed that CRT requires its high capacity Ca(2+)-binding domain to reduce the elevations of [Ca(2+)](i) due to Ca(2+) influx. This functional domain was also required for CRT to attenuate the InsP(3)-induced decline in the free Ca(2+) concentration within the ER lumen ([Ca(2+)](ER)), as monitored with a "chameleon" indicator. Our data suggest that by buffering [Ca(2+)](ER) near resting levels, CRT may prevent InsP(3) from depleting the intracellular stores sufficiently to activate Ca(2+) influx.     

An osmotically induced cytosolic Ca2+ transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance of Saccharomyces cerevisiae

Hyperosmotic stress caused by NaCl, LiCl, or sorbitol induces an immediate and short duration ( approximately 1 min) transient cytosolic Ca(2+) ([Ca(2+)](cyt)) increase (Ca(2+)-dependent aequorin luminescence) in Saccharomyces cerevisiae cells. The amplitude of the osmotically induced [Ca(2+)](cyt) transient was attenuated by the addition of chelating agents EGTA or BAPTA, cation channel pore blockers, competitive inhibitors of Ca(2+) transport, or mutations (cch1Delta or mid1Delta) that reduce Ca(2+) influx, indicating that Ca(ext)(2+) is a source for the transient. An osmotic pretreatment (30 min) administered by inoculating cells into media supplemented with either NaCl (0.4 or 0.5 m) or sorbitol (0.8 or 1.0 m) enhanced the subsequent growth of these cells in media containing 1 m NaCl or 2 m sorbitol. Inclusion of EGTA in the osmotic pretreatment media or the cch1Delta mutation reduced cellular capacity for NaCl but not hyperosmotic adaptation. The stress-adaptive effect of hyperosmotic pretreatment was mimicked by exposing cells briefly to 20 mm CaCl(2). Thus, NaCl- or sorbitol-induced hyperosmotic shock causes a [Ca(2+)](cyt) transient that is facilitated by Ca(2+) influx, which enhances ionic but not osmotic stress adaptation. NaCl-induced ENA1 expression was inhibited by EGTA, cch1Delta mutation, and FK506, indicating that the [Ca(2+)](cyt) transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance.     

Gonadotropin-releasing hormone-stimulated sperm binding to the human zona is mediated by a calcium influx

The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca(2+)-free medium and in the presence of Ca(2+) channel antagonists. We also examined the GnRH effect on the intracellular free Ca(2+) concentration ([Ca(2+)](i)). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca(2+) was present in the medium. In Ca(2+)-free medium or in the presence of 400 nM nifedipine, 80 microM diltiazem, or 50 microM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca(2+)](i) in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca(2+)](i) was fast and transient, from a basal [Ca(2+)](i) of 413 +/- 22 nM to a peak value of 797 +/- 24 nM. The GnRH-induced increase in [Ca(2+)](i) was entirely due to a Ca(2+) influx from the extracellular medium because the increase in [Ca(2+)](i) was blocked by the Ca(2+) chelator EGTA and by the Ca(2+) channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca(2+) influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca(2+) influx but inhibited the progesterone-activated Ca(2+) influx. Finally, the GnRH-induced Ca(2+) influx was blocked by two specific GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH and Ac-(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2), D-Trp(3,6)-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca(2+)](i) through T-type, voltage-operated calcium channels.     

Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA

Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.     

Calcium signal transmission between ryanodine receptors and mitochondria

Control of energy metabolism by increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) may represent a fundamental mechanism to meet the ATP demand imposed by heart contractions, but the machinery underlying propagation of [Ca(2+)] signals from ryanodine receptor Ca(2+) release channels (RyR) to the mitochondria remains elusive. Using permeabilized cardiac (H9c2) cells we investigated the cytosolic [Ca(2+)] ([Ca(2+)](c)) and [Ca(2+)](m) signals elicited by activation of RyR. Caffeine, Ca(2+), and ryanodine evoked [Ca(2+)](c) spikes that often appeared as frequency-modulated [Ca(2+)](c) oscillations in these permeabilized cells. Rapid increases in [Ca(2+)](m) and activation of the Ca(2+)-sensitive mitochondrial dehydrogenases were synchronized to the rising phase of the [Ca(2+)](c) spikes. The RyR-mediated elevations of global [Ca(2+)](c) were in the submicromolar range, but the rate of [Ca(2+)](m) increases was as large as it was in the presence of 30 microm global [Ca(2+)](c). Furthermore, RyR-dependent increases of [Ca(2+)](m) were relatively insensitive to buffering of [Ca(2+)](c) by EGTA. Therefore, RyR-driven rises of [Ca(2+)](m) appear to result from large and rapid increases of perimitochondrial [Ca(2+)]. The falling phase of [Ca(2+)](c) spikes was followed by a rapid decay of [Ca(2+)](m). CGP37157 slowed down relaxation of [Ca(2+)](m) spikes, whereas cyclosporin A had no effect, suggesting that activation of the mitochondrial Ca(2+) exchangers accounts for rapid reversal of the [Ca(2+)](m) response with little contribution from the permeability transition pore. Thus, rapid activation of Ca(2+) uptake sites and Ca(2+) exchangers evoked by RyR-mediated local [Ca(2+)](c) signals allow mitochondria to respond rapidly to single [Ca(2+)](c) spikes in cardiac cells.     

Low [Mg(2+)](o) induces contraction and [Ca(2+)](i) rises in cerebral arteries: roles of ca(2+), PKC, and PI3

Removal of extracellular Ca(2+) concentration ([Ca(2+)](o)) and pretreatment of canine basilar arterial rings with either an antagonist of voltage-gated Ca(2+) channels (verapamil), a selective antagonist of the sarcoplasmic reticulum Ca(2+) pump [thapsigargin (TSG)], caffeine plus a specific antagonist of ryanodine-sensitive Ca(2+) release (ryanodine), or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]- mediated Ca(2+) release antagonist (heparin) markedly attenuates low extracellular Mg(2+) concentration ([Mg(2+)](o))-induced contractions. Low [Mg(2+)](o)-induced contractions are significantly inhibited by pretreatment of the vessels with G?-6976 [a protein kinase C-alpha (PKC-alpha)- and PKC-betaI-selective antagonist], bisindolylmaleimide I (Bis, a specific antagonist of PKC), and wortmannin or LY-294002 [selective antagonists of phosphatidylinositol-3 kinases (PI3Ks)]. These antagonists were also found to relax arterial contractions induced by low [Mg(2+)](o) in a concentration-dependent manner. The absence of [Ca(2+)](o) and preincubation of the cells with verapamil, TSG, heparin, or caffeine plus ryanodine markedly attenuates the transient and sustained elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by low-[Mg(2+)](o) medium. Low [Mg(2+)](o)-produced increases in [Ca(2+)](i) are also suppressed markedly in the presence of G?-6976, Bis, wortmannin, or LY-294002. The present study suggests that both Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from intracellular stores [both Ins(1,4,5)P(3) sensitive and ryanodine sensitive] play important roles in low-[Mg(2+)](o) medium-induced contractions of isolated canine basilar arteries. Such contractions are clearly associated with activation of PKC isoforms and PI3Ks.     

Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.     

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein a subunit (Galpha) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca(2+)](cyt)) and Ca(2+) channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Galpha null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Galpha overexpression lines (wGalpha and cGalpha) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca(2+)](cyt) was lower in gpa1-1 and gpa1-2 and higher in wGalpha and cGalpha. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca(2+)-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba(2+) > Ca(2+) > Mg(2+) > Sr(2+) > Mn(2+). The activity of the Ca(2+)-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGalpha and cGalpha. The results suggest that Galpha may participate in pollen germination through modulation of the hyperpolarization-activated Ca(2+) channel in the PM of pollen cells.