Quantitative measurement of Ca(2+)-dependent calmodulin-target binding by Fura-2 and CFP and YFP FRET imaging in living cells

Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.     

Trypanosoma evansi: a convenient model for studying intracellular Ca(2+) homeostasis using fluorometric ratio imaging from single parasites

The aim of this work was to measure, for the first time, the basal cytosolic Ca(2+) levels of Trypanosoma evansi and to explore the possibility of observing changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) using fluorescence ratio imaging techniques in single isolated parasites of this species. Under appropriate loading conditions, the high intracellular levels of the Ca(2+) fluorescence probe Fura-2 permits resolution, in real time, of single parasite [Ca(2+)](i) signals. Measurements of the basal [Ca(2+)](i) indicate that homeostatic mechanisms maintain [Ca(2+)](i) at 106 +/- 38 (n = 32) nM in the presence of 2 mM extracellular calcium. The resting [Ca(2+)](i) was unaffected by changes in extracellular Ca(2+) in the range from 0 to 10 mM. The Ca(2+) ionophore A23187 induced a large increase in [Ca(2+)](i) which (i) reached a steady state value even in the simultaneous presence of both external calcium and ionophore and (ii) returned to base line upon removal of extracellular Ca(2+). A dose-response curve of the protonophore nigericin shows that T. evansi contains an important pH-sensitive intracellular pool which may be released by this drug with a K(1/2) of 8 microM. These data demonstrate that this parasite contains highly efficient systems to control [Ca(2+)](i). Finally, our results, with the use of sera as source of an antibody-complement to induce Ca(2+) entry, demonstrate that it is possible to resolve fast [Ca(2+)](i) signals in single parasites from T. evansi.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].     

Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot [Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot [Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude [Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced [Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot [Ca(2+)](i) signals. Neuronally evoked increases in astrocytic [Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.     

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.     

Cytosolic Ca2+ buffers

"Ca(2+) buffers," a class of cytosolic Ca(2+)-binding proteins, act as modulators of short-lived intracellular Ca(2+) signals; they affect both the temporal and spatial aspects of these transient increases in [Ca(2+)](i). Examples of Ca(2+) buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Besides their proven Ca(2+) buffer function, some might additionally have Ca(2+) sensor functions. Ca(2+) buffers have to be viewed as one of the components implicated in the precise regulation of Ca(2+) signaling and Ca(2+) homeostasis. Each cell is equipped with proteins, including Ca(2+) channels, transporters, and pumps that, together with the Ca(2+) buffers, shape the intracellular Ca(2+) signals. All of these molecules are not only functionally coupled, but their expression is likely to be regulated in a Ca(2+)-dependent manner to maintain normal Ca(2+) signaling, even in the absence or malfunctioning of one of the components.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria

Cytosolic Ca(2+) ([Ca(2+)](i)) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP(3)R) driven through cycles of activation/inactivation by local Ca(2+) feedback. Consequently, modulation of the local Ca(2+) gradients influences IP(3)R excitability as well as the duration and amplitude of the [Ca(2+)](i) oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP(3)-dependent [Ca(2+)](i) oscillations in intact hepatocytes, apparently by altering the local Ca(2+) gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP(3) sensitivity for Ca(2+) release from intracellular stores. These effects on IP(3)-dependent [Ca(2+)](i) signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca(2+) fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca(2+) by sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), lowering basal and inter-spike [Ca(2+)](i). In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (DeltaPsi(m)), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca(2+) uptake and retention by the mitochondria during a rise in [Ca(2+)](i). We suggest that CSA suppresses local Ca(2+) feedback by enhancing mitochondrial and endoplasmic reticulum Ca(2+) uptake, these actions of CSA underlie the lower IP(3) sensitivity found in permeabilized cells and the impaired IP(3)-dependent [Ca(2+)](i) signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca(2+)](i) signals, which, in turn, may adjust the output of downstream Ca(2+)-sensitive pathways.     

Lack of evidence for presenilins as endoplasmic reticulum Ca2+ leak channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.     

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.     

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca(2+)](i) response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH(2)O (hypotonic) evoked transient increase in [Ca(2+)](i) of principal cells of rat CCDs. The [Ca(2+)](i) increase was markedly attenuated by the removal of extracellular Ca(2+)(.) The application of a P(2) purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca(2+)](i) increase. The [Ca(2+)](i) increase in response to extracellular hypotonicity was not influenced by application of Gd(3+) and ruthenium red. On the other hand, a voltage-gated Ca(2+) channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca(2+)](i) increase in the principal cells. In order to assess Ca(2+) entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn(2+). The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn(2+), indicating that a Ca(2+)-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn(2+) quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca(2+) entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca(2+)](i) elevation of principal cells in rat CCD.     

Spatial organization of Ca(2+) entry and exocytosis in mouse pancreatic beta-cells

Secretion from single pancreatic beta-cells was imaged using a novel technique in which Zn(2+), costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta-cells was limited to an active region that comprised approximately 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca(2+)](i) at single cells, colocalization of exocytotic release sites and Ca(2+) entry was observed when cells were stimulated by glucose or K(+). Treatment of cells with the Ca(2+) ionophore 4-Br-A23187 induced large Ca(2+) influx around the entire cell circumference. Despite the nonlocalized increase in [Ca(2+)](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca(2+) channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca(2+)](i), but instead involves spatial localization of other components of the exocytotic machinery.     

Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells

Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.     

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.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.