An oxidized metabolite of linoleic acid increases intracellular calcium in rat adrenal glomerulosa cells

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.     

Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries

We developed an in situ assay system to simultaneously monitor intracellular Ca(2+) concentration ([Ca(2+)](i), fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 microM) stimulated a rapid increase in [Ca(2+)](i) followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 microM) induced a gradual, small increase in [Ca(2+)](i) and a slow increase in intracellular NO levels. Removal of extracellular Ca(2+) and depletion of Ca(2+) stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca(2+)](i) by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca(2+)](i) and intracellular NO production in the intact endothelium is a powerful tool to study Ca(2+)-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca(2+) in tyrosine phosphorylation-induced NO production.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca(2+) concentration ([Ca(2+)](o)) regulates the functions of many cell types through a G protein-coupled [Ca(2+)](o)-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca(2+)](o), neomycin, and gadolinium) failed to increase intracellular Ca(2+) concentration ([Ca(2+)](i)), the CaR agonist spermine stimulated an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) and was abolished after depletion of an intracellular Ca(2+) pool with thapsigargin or after blocking IP(3)- and ryanodine receptor-mediated Ca(2+) release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca(2+)](i) and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca(2+)](i), primarily due to release of IP(3)- and ryanodine-sensitive intracellular Ca(2+) stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.     

The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca(2+) ([Ca(2+)](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca(2+)](i), and sustained Ca(2+) increase disappeared in the absence of extracellular Ca(2+). However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca(2+)](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H(2)O(2), with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H(2)O(2) scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca(2+) response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca(2+) response to extracellular H(2)O(2) and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca(2+)](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca(2+) response to EGF, but not vice versa. These results suggested that intracellular H(2)O(2) regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca(2+)](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca(2+)](i) by producing intracellular H(2)O(2).     

Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.     

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.     

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.     

Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells

The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca(2+) plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca(2+)](i)) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca(2+) dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca(2+)](i) in U937-derived macrophages in both acute and chronic treatments (P<0.01). In particular, the increases of the induced [Ca(2+)](i) were different in the presence or absence of extracellular Ca(2+) under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca(2+)](i) induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

Sperm factor initiates capacitance and conductance changes in mouse eggs that are more similar to fertilization than IP(3)- or Ca(2+)-induced changes

We used patch clamp electrophysiology and concurrent imaging with the Ca(2+)-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca(2+) concentration ([Ca(2+)](i)) during mouse egg fertilization. We found an approximately 2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca(2+)](i) at sperm fusion. This was followed approximately 1 min later by a rise in [Ca(2+)](i) that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-microM peak [Ca(2+)](i) favored capacitance decrease, while higher [Ca(2+)](i) favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca(2+)](i). The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca(2+)](i) dependence. Raising [Ca(2+)](i) by the intracellular introduction of IP(3) initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca(2+)](i) was increased modestly by activation of an endogenous Ca(2+) current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca(2+)](i) and that sperm and the active component of sperm factor may be doing more than initiating the IP(3)-mediated release of intracellular Ca(2+).     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Effect of hydrogen peroxide and superoxide anions on cytosolic Ca2+: comparison of endothelial cells from large-sized and small-sized arteries

We compared the Ca(2+) responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H(2)O(2)) caused an increase in cytosolic Ca(2+) levels ([Ca(2+)](i)) in both cell types. The [Ca(2+)](i) rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP(3)) receptors. Removal of Ca(2+) from the bath also decreased the [Ca(2+)](i) rises in response to H(2)O(2). In addition, treatment of endothelial cells with H(2)O(2) reduced the [Ca(2+)](i) responses to subsequent challenge of ATP. The decreased [Ca(2+)](i) responses to ATP were resulted from a pre-depletion of intracellular Ca(2+) stores by H(2)O(2). Interestingly, we also found that Ca(2+) store depletion was more sensitive to H(2)O(2) treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca(2+)](i) rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H(2)O(2) but not by hydroxyl radical. H(2)O(2) contribution in HX-XO-induced [Ca(2+)](i) rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H(2)O(2) could induce store Ca(2+) release via phospholipase C-IP(3) pathway in endothelial cells. Resultant emptying of intracellular Ca(2+) stores contributed to the reduced [Ca(2+)](i) responses to subsequent ATP challenge. The [Ca(2+)](i) responses were more sensitive to H(2)O(2) in endothelial cells of small-sized arteries than those of large-sized arteries.     

Role of calcium and calmodulin in ciliary stimulation induced by acetylcholine

The goal of this work was to elucidate the molecular events underlying stimulation of ciliary beat frequency (CBF) induced by acetylcholine (ACh) in frog esophagus epithelium. ACh induces a profound increase in CBF and in intracellular Ca(2+) concentration ([Ca(2+)](i)) through M(1) and M(3) muscarinic receptors. The [Ca(2+)](i) slowly decays to the basal level, while CBF stabilizes at an elevated level. These results suggest that ACh triggers Ca(2+)-correlated and -uncorrelated modes of ciliary stimulation. ACh response is abolished by the phospholipase C (PLC) inhibitor U-73122 and by depletion of intracellular Ca(2+) stores but is unaffected by reduction of extracellular Ca(2+) concentration and by blockers of Ca(2+) influx. Therefore, ACh activates PLC and mobilizes Ca(2+) solely from intracellular stores. The calmodulin inhibitors W-7 and calmidazolium attenuate the ACh-induced increase in [Ca(2+)](i) but completely abolish the elevation in CBF. Therefore, elevation of [Ca(2+)](i) is necessary for CBF enhancement but does not lead directly to it. The combined effect of Ca(2+) elevation and of additional factors, presumably mobilized by Ca(2+)-calmodulin, results in a robust CBF enhancement.     

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.