Readdition Of Ca2 Research Articles

Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly.

Intestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca2+ concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca2+ into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this principle, the Ca2+-switch assay was established to investigate tight junction assembly in fully differentiated intestinal epithelial cells. This chapter provides a stepwise protocol for culture of intestinal epithelial cell monolayers using T84 cell line as an in vitro model and the Ca2+-switch assay for evaluating tight junction assembly.

Self-Assembly of Switchable Protein Nanocages via Allosteric Effect

Protein cages have promising applications in highly efficient gene transportation. Many methods of engineering protein–interface interactions have been developed to build such viral mimics. Missing...

Zn2+ ions inhibit gene transcription following stimulation of the Ca2+ channels Cav1.2 and TRPM3.

Zinc, a trace element, is necessary for the correct structure and function of many proteins. Therefore, Zn2+ has to be taken up by the cells, using specific Zn2+ transporters or Ca2+ channels. In this study, we have focused on two Ca2+ channels, the L-type voltage-gated Cav1.2 channel and the transient receptor potential channel TRPM3. Stimulation of either channel induces an intracellular signaling cascade leading to the activation of the transcription factor AP-1. The influx of Ca2+ ions into the cytoplasm is essential for this activity. We asked whether extracellular Zn2+ ions affect Cav1.2 or TRPM3-induced gene transcription following stimulation of the channels. The results show that extracellular Zn2+ ions reduced the activation of AP-1 by more than 80% following stimulation of either voltage-gated Cav1.2 channels or TRPM3 channels. Experiments performed with cells maintained in Ca2+-free medium revealed that Zn2+ ions cannot replace Ca2+ ions in inducing gene transcription via stimulation of Cav1.2 and TRPM3 channels. Re-addition of Ca2+ ions to the cell culture medium, however, restored the ability of these Ca2+ channels to induce a signaling cascade leading to the activation of AP-1. Secretory cells, including neurons and pancreatic β-cells, release Zn2+ ions during exocytosis. We propose that the released Zn2+ ions function as a negative feedback loop for stimulus-induced exocytosis by inhibiting Ca2+ channel signaling.

Up-regulation of Orai1 expression and store operated Ca(2+) entry following activation of membrane androgen receptors in MCF-7 breast tumor cells.

BackgroundMembrane androgen receptors (mAR) are functionally expressed in a variety of tumor-cells including the breast tumor-cell line MCF-7. They are specifically activated by testosterone albumin conjugates (TAC). The mAR sensitive signaling includes activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and reorganization of the actin filament network. Signaling of tumor-cells may further involve up-regulation of pore forming Ca2+ channel protein Orai1, which accomplishes store operated Ca2+ entry (SOCE). This study explored the regulation of Orai1 abundance and SOCE by mAR.MethodsActin filaments were visualized utilizing confocal microscopy, Rac1 activity using GST-GBD assay, Orai1 transcript levels by RT-PCR and total protein abundance by western blotting, Orai1 abundance at the cell surface by confocal microscopy and FACS-analysis, cytosolic Ca2+ activity ([Ca2+]i) utilizing Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following readdition of Ca2+ after store depletion with thapsigargin (1 μM).ResultsTAC treatment of MCF-7 cells was followed by Rac1 activation, actin polymerization, transient increase of Orai1transcript levels and protein abundance, and transient increase of SOCE. The transient increase of Orai1 protein abundance was abrogated by Rac1 inhibitor NSC23766 (50 μM) and by prevention of actin reorganization with cytochalasin B (1 μM).ConclusionsmAR sensitive Rac1 activation and actin reorganization contribute to the regulation of Orai1 protein abundance and SOCE.

Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ∼1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 μm Ca(2+) showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mm Ca(2+), > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mm EDTA showed large (5.6- to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mm Ca(2+) showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.

Force augmentation and stimulated actin polymerization in swine carotid artery

The phenomenon of posttetanic potentiation, in which a single submaximal contraction or series of submaximal contractions strengthens a subsequent contraction, has been observed in both skeletal and cardiac muscle. In this study, we describe a similar phenomenon in swine carotid arterial smooth muscle. We find that a submaximal K(+) depolarization increases the force generation of a subsequent maximal K(+) depolarization; we term this "force augmentation." Force augmentation was not associated with a significant increase in crossbridge phosphorylation or shortening velocity during the maximal K(+) depolarization, suggesting that the augmented force was not caused by higher crossbridge phosphorylation or crossbridge cycling rates. We found that the characteristics of the tissue before the maximal K(+) depolarization predicted the degree of force augmentation. Specifically, measures of stimulated actin polymerization (higher prior Y118 paxillin phosphorylation, higher prior F-actin, and transition to a more solid rheology evidenced by lower noise temperature, hysteresivity, and phase angle) predicted the subsequent force augmentation. Increased prior contraction alone did not induce force augmentation since readdition of Ca(2+) to Ca(2+)-depleted tissues induced a partial contraction that was not associated with changes in noise temperature or with subsequent force augmentation. These data suggest that stimulated actin polymerization may produce a substrate for increased crossbridge mediated force, a process we observe as force augmentation.

Role of symmetric dimethylarginine in vascular damage by increasing ROS via store-operated calcium influx in monocytes

The guanidines asymmetric dimethylarginine (ADMA), a marker of endothelial dysfunction, and its counterpart symmetric dimethylarginine (SDMA), considered inert, are accumulated in chronic kidney disease (CKD). The present study evaluates their effect on monocyte function, since previous data demonstrated leukocyte activation by other guanidino compounds. The effect of ADMA and SDMA on reactive oxygen species (ROS) production in human whole blood at baseline and after N-formyl-methionine-leucine-phenylalanine (fMLP) stimulation was evaluated. By using the fluorescent probe Fluo3-AM, the role of changes in monocytic cytoplasmic calcium ([Ca2+]i) was studied. Thapsigargin, and removal followed by addition of extracellular Ca2+ (Ca2+(ex)), was used to investigate the contribution of store-operated Ca2+-channels (SOCs). SKF96365 was used as a selective inhibitor of the SOCs. A pharmacologic intervention with captopril, known to affect Ca2+ influx, was tested. SDMA enhanced ROS production in fMLP-stimulated monocytes using heparinized blood, and this effect was abolished in EDTA-anticoagulated blood. In the presence of SDMA, an increased Ca2+ entry from the extracellular milieu resulted in an elevated amplitude of the peak [Ca2+]i change triggered by fMLP. None of these effects were seen with ADMA. Depletion of the intracellular stores with thapsigargin in the absence of Ca2+(ex), followed by re-addition of Ca2+(ex) triggered a significantly larger Ca2+ entry after SDMA treatment versus saline. This effect was prevented with SKF96365, as was the SDMA-enhanced oxidative burst after fMLP. Pre-incubation with captopril also reduced the increased ROS production seen with SDMA. SDMA, a uraemic retention solute considered inert, stimulates ROS production of monocytes by acting on Ca2+ entry via SOCs. This pro-inflammatory effect may trigger vascular pathology and may be involved in altering the prevalence of cardiovascular disease in CKD.

Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE)

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca(2+) entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca(2+) store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1(D76A), which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1(D76A) puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.

STIM1 Knockdown Reveals That Store-operated Ca2+ Channels Located Close to Sarco/Endoplasmic Ca2+ ATPases (SERCA) Pumps Silently Refill the Endoplasmic Reticulum

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.

A Rich1/Amot Complex Regulates the Cdc42 GTPase and Apical-Polarity Proteins in Epithelial Cells

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Nuclear Factor-κB Activated by Capacitative Ca2+ Entry Enhances Muscarinic Receptor-mediated Soluble Amyloid Precursor Protein (sAPPα) Release in SH-SY5Y Cells

G(q/11) protein-coupled muscarinic receptors are known to regulate the release of soluble amyloid precursor protein (sAPPalpha) produced by alpha-secretase processing; however, their signaling mechanisms remain to be elucidated. It has been reported that a muscarinic agonist activates nuclear factor (NF)-kappaB, a transcription factor that has been shown to play an important role in the Alzheimer disease brain, and that NF-kappaB activation is regulated by intracellular Ca2+ level. In the present study, we investigated whether NF-kappaB activation plays a role in muscarinic receptor-mediated sAPPalpha release enhancement and contributes to a changed capacitative Ca2+ entry (CCE), which was suggested to be involved in the muscarinic receptor-mediated stimulation of sAPPalpha release. Muscarinic receptor-mediated NF-kappaB activation was confirmed by observing the translocation of the active subunit (p65) of NF-kappaB to the nucleus by the muscarinic agonist, oxotremorine M (oxoM), in SH-SY5Y neuroblastoma cells expressing muscarinic receptors that are predominantly of the M3 subtype. NF-kappaB activation and sAPPalpha release enhancement induced by oxoM were inhibited by NF-kappaB inhibitors, such as an NF-kappaB peptide inhibitor (SN50), an IkappaB alpha kinase inhibitor (BAY11-7085), a proteasome inhibitor (MG132), the inhibitor of proteasome activity and IkappaB phosphorylation, pyrrolidine dithiocarbamate, the novel NF-kappaB activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino) quinazoline), and by an intracellular Ca2+ chelator (TMB-8). Furthermore, both oxoM-induced NF-kappaB activation and sAPPalpha release were antagonized by CCE inhibitors (gadolinium or SKF96365) but not by voltage-gated Ca2+-channel blockers. On the other hand, treatment of cells with NF-kappaB inhibitors (SN50, BAY11-7085, MG132, or pyrrolidine dithiocarbamate) did not inhibit muscarinic receptor-mediated CCE. These findings provide evidence for the involvement of NF-kappaB regulated by CCE in muscarinic receptor-mediated sAPPalpha release enhancement.

Calcium feedback mechanisms regulate oscillatory activity of a TRP‐like Ca2+ conductance in C. elegans intestinal cells

Inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in Caenorhabditis elegans intestinal epithelial cells regulate the nematode defecation cycle. The role of plasma membrane ion channels in intestinal cell oscillatory Ca2+ signalling is unknown. We have shown previously that cultured intestinal cells express a Ca2+-selective conductance, I(ORCa), that is biophysically similar to TRPM7 currents. I(ORCa) activates slowly and stabilizes when cells are patch clamped with pipette solutions containing 10 mm BAPTA and free Ca2+ concentrations of approximately 17 nm. However, when BAPTA concentration is lowered to 1 mm, I(ORCa) oscillates. Oscillations in channel activity induced simultaneous oscillations in cytoplasmic Ca2+ levels. Removal of extracellular Ca2+ inhibited I(ORCa) oscillations, whereas readdition of Ca2+ to the bath caused a rapid and transient reactivation of the current. Experimental manoeuvres that elevated intracellular Ca2+ blocked current oscillations. Elevation of intracellular Ca2+ in the presence of 10 mm BAPTA to block I(ORCa) oscillations led to a dose-dependent increase in the rate of current activation. At intracellular Ca2+ concentrations of 250 nm, current activation was transient. Patch pipette solutions buffered with 1-4 mm of either BAPTA or EGTA gave rise to similar patterns of I(ORCa) oscillations. We conclude that changes in Ca2+ concentration close to the intracellular opening of the channel pore regulate channel activity. Low concentrations of Ca2+ activate the channel. As Ca2+ enters and accumulates near the pore mouth, channel activity is inhibited. Oscillating plasma membrane Ca2+ entry may play a role in generating intracellular Ca2+ oscillations that regulate the C. elegans defecation rhythm.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.

Zinc ions are endogenous modulators of neurotransmitter‐stimulated capacitative Ca2+ entry in both cultured and in situ mouse astrocytes

Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.

Capacitative calcium entry in testosterone-induced intracellular calcium oscillations in myotubes

Ca2+ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca2+ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca2+ entry (CCE) on intracellular Ca2+ oscillations induced by testosterone at the single-cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca2+ rise, accompanied by Ca2+ oscillations in a majority of myotubes. Spectral analysis of the Ca2+ oscillations revealed a periodicity of 20.3 +/- 1.8 s (frequency of 49.3 +/- 4.4 mHz). In Ca(2+)-free medium, an increase in intracellular Ca2+ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca2+ response. This intracellular Ca2+ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP3). Intracellular Ca2+ store depletion in Ca(2+)-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-pump inhibitor, followed by re-addition of extracellular Ca2+, gave a fast rise in intracellular Ca2+, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca2+ release and led to CCE after re-addition of Ca2+ to Ca(2+)-free extracellular medium. The CCE blockers 2-aminoethyl diphenylborate and La3+, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca2+ oscillations and CCE. The steady increase in Ca2+ induced by testosterone was not, however, affected by either La3+ or cytochalasin D. These results demonstrate testosterone-induced Ca2+ oscillations in myotubes, mediated by the interplay of IP3-sensitive Ca2+ stores and Ca2+ influx through CCE.

Effect of dexamethasone on voltage-gated K + channels in Jurkat T-lymphocytes

The voltage-gated K+ channel Kv1.3 is an important regulator of lymphocyte function. Activation of lymphocytes is accompanied by stimulation, whereas CD95-induced apoptosis by inhibition, of Kv1.3. The channel serves to maintain cell membrane potential, a prerequisite for signalling through the Ca2+ release-activated Ca2+ channel I(CRAC). As glucocorticoids are known to regulate lymphocyte function, the present study addressed the effect of dexamethasone on voltage-gated K+ channels in Jurkat T-lymphocytes. In whole-cell patch-clamp experiments current families evoked by 200-ms potential steps every 15 s from -70 mV to values from -120 to +100 mV revealed the functional expression of voltage-gated K+ channels. Pre-treatment of Jurkat T-lymphocytes for 2-3 h with 1 microM dexamethasone led to a significant decrease of voltage-gated K+ currents. Fura-2-fluorescence measurements showed that the readdition of Ca2+ to Ca(2+)-depleted cells led to a rapid increase of cytosolic Ca2+ activity. This increase of Ca2+ activity was blunted by both the K+ channel blocker margatoxin (10 nM) and 24 h pre-treatment with dexamethasone (1 microM). In conclusion, dexamethasone inhibits voltage-gated K+ channels in Jurkat T-lymphocytes, an effect impeding Ca2+ entry through I(CRAC).

Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential.

We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca2+ ATPase of the sarco-endoplasmic reticulum (SERCA-ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, [Ca2+](i), plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca2+ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA-ATPase inhibitors induced a rise in [Ca2+](i) both in Ca2+ and Ca2+-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca2+-stimulated a rise in [Ca2+](i). This demonstrated that internal Ca2+ store depletion can evoke the opening of Ca2+-channels on sperm plasma membrane, thus showing the existence of "capacitative" Ca2+ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca2+ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca2+ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca2+-dependent K+ channels. When K+-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in [Ca2+](i) plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca2+ stores and that the capacitative Ca2+ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.

The supramolecular organisation of fibrillin-rich microfibrils determines the mechanical properties of bovine zonular filaments.

The zonular filaments from the eyes of cows are rich in microfibrils containing fibrillin. Tensile tests, stress-relaxation tests and X-ray diffraction studies were used to study the relationship between the mechanical behaviour of zonular filaments and the molecular packing and structure of the fibrillin-rich microfibrils. Zonular filaments show a non-linear (J-shaped) stress-strain curve and appreciable stress-relaxation. It is proposed that the non-linear properties are due to local variations in waviness in the microfibrils or assemblies of microfibrils, which straighten out and become more regularly aligned with strain. Previous and current X-ray diffraction results consistently show a partial ordering of microfibrils in zonular filaments into staggered aggregates which become more ordered and laterally aligned on stretching. Although the removal and re-addition of Ca(2+) is known to change the molecular structure of fibrillin, no effect was observed on the tensile properties of the zonular filaments. It is hypothesised that strain-induced deformation in the supramolecular aggregate packing may not be Ca(2+)-sensitive but could dominate the mechanical behaviour of microfibrillar arrays in zonular filaments.

Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation.

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca2+ influx through L-type voltage-dependent Ca2+ channels as defined by a sensitivity to 1 microM nifedipine. This Ca2+ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca2+ followed by the readdition of Ca2+ allows the coupling of a class of L-type Ca2+ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [3H]-D-aspartate. KCl stimulation in the absence of external Ca2+ ('predepolarization') enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca2+ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca2+ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca2+ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca2+ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells.

We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.