Thapsigargin-sensitive Stores Research Articles

Calcium signaling in prostate cancer cells of increasing malignancy

Calcium signaling controls a large variety of cell functions, including proliferation and apoptosis, and plays a major role in neoplastic transformation. Prostate cancer (PCa) is one of the most common malignancies in men. The transition to castration-resistant prostate cancer (CRPC), a lethal form that is still lacking an effective cure, could be influenced by fine tuning intracellular calcium ([Ca2+]i) homeostasis. This study investigates [Ca2+]i dynamics in metastatic PCa cell lines that mimic the progression of PCa to CRPC: (i) well differentiated LNCaP cells that require androgen for survival, and (ii) poorly differentiated, highly aggressive androgen-insensitive prostate cancer (AIPC) PC3 and DU145 cells. In AIPC cells, ATP induces a fast rise in [Ca2+]i, due to release from intracellular stores and sensitive to phospholipase C inhibitors, while LNCaP cells do not respond to ATP challenge. Moreover, AIPC cells showed a reduced capacity to store Ca2+ in thapsigargin-sensitive stores and limited store-operated calcium entry, with respect to androgen-dependent LNCaP cells. Finally, green tea extract causes [Ca2+]i elevation and inhibits proliferation in PC3 and DU145 cells, but is ineffective in LNCaP cells. The consequences of these differences are discussed and interpreted in this study with reference to previously proposed models for Ca2+ dependence of prostate carcinogenesis.

The WD40 repeat protein, WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by Gq-coupled receptors

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-β and the thromboxane A2 receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gαq and PLCβ. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.

Na +/H + exchanger inhibitor induces vasorelaxation through nitric oxide production in endothelial cells via intracellular acidification-associated Ca2 + mobilization

The objective of this study was to determine the mechanism by which Na+/H+ exchanger (NHE) inhibitors induce vasodilatation. The NHE inhibitors, 5-(N,N-dimethyl)-amiloride (DMA), cariporide, and amiloride, evoked endothelium-dependent relaxation in rat aortas with ED50 values of 16, 89, and 148μM, respectively, and these effects were abolished by treatment with NG-nitro-l-arginine methyl ester (L-NAME). The relaxation effects induced by DMA and cariporide were strongly attenuated in aortas of the endothelial NO synthase (eNOS)-deficient mice, as compared to the effects in wild-type mice. The DMA-induced relaxation in rat aorta was attenuated by a calmodulin (CaM) inhibitor, calmidazolium, and a soluble guanylyl cyclase inhibitor, [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but was not affected by a phosphoinositide 3-kinase inhibitor, wortmannin. Immunoblots for endothelial eNOS on immunoprecipitated CaM complexes showed that DMA enhanced the association of eNOS with CaM in rat aortas. Both DMA and cariporide induced the reduction of intracellular pH (pHi) in bovine aortic endothelial cells (BAECs), which was accompanied by a sustained elevation of cytosolic Ca2+ ([Ca2+]i). This DMA-induced rise of [Ca2+]i was not affected by removing external Ca2+ from the buffer, but was abolished in thapsigargin-pretreated BAECs. These results suggest that lowering of pHi by NHE inhibitors in endothelial cells induces the mobilization of Ca2+ from the thapsigargin-sensitive stores of endoplasmic reticulum, which in turn stimulates NO production via the CaM-dependent activation of eNOS.

Neuropeptide TLQP-21, a VGF Internal Fragment, Modulates Hormonal Gene Expression and Secretion in GH3 Cell Line

In the present study we demonstrated that TLQP-21, a biologically active peptide derived from the processing of the larger pro-VGF granin, plays a role in mammotrophic cell differentiation. We used an established in vitro model, the GH3 cell line, which upon treatment with epidermal growth factor develops a mammotrophic phenotype consisting of induction of prolactin expression and secretion, and inhibition of growth hormone. Here we determined for the first time that during mammotrophic differentiation, epidermal growth factor also induces Vgf gene expression and increases VGF protein precursor processing and peptide secretion. After this initial observation we set out to determine the specific role of the VGF encoded TLQP-21 peptide on this model. TLQP-21 induced a trophic effect on GH3 cells and increased prolactin expression and its own gene transcription without affecting growth hormone expression. TLQP-21 was also able to induce a significant rise of cytoplasmic calcium, as measured by Fura2AM, due to the release from a thapsigargin-sensitive store. TLQP-21-dependent rise in cytoplasmic calcium was, at least in part, dependent on the activation of phospholipase followed by phosphorylation of PKC and ERK. Taken together, the present results demonstrate that TLQP-21 contributes to differentiation of the GH3 cell line toward a mammotrophic phenotype and suggest that it may exert a neuroendocrine role in vivo on lactotroph cells in the pituitary gland.

Functional Expression of TRPV4 Channels in Human Collecting Duct Cells: Implications for Secondary Hypertension in Diabetic Nephropathy

Background. The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct. Methods. Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca2+-dependent mechanical stimulation within a novel system of the human collecting duct (HCD). Results. Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca2+]i that was dependent upon thapsigargin-sensitive store release and Ca2+ influx. At 48 hrs, high glucose and mannitol (25 mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown. Conclusion. Our data confirm expression of Ca2+-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy.

Mammalian cell invasion by closely related Trypanosoma species T. dionisii and T. cruzi

Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within this genus, Trypanosoma dionisii from bats and Trypanosoma cruzi that causes Chagas’ disease, belonging to the subgenus Schizotrypanum, can invade mammalian cells. The mechanisms of cell invasion by T. dionisii are poorly understood. To address that question, metacyclic trypomastigotes (MT) and human epithelial HeLa cells were used. Similarly to genetically divergent T. cruzi strains G (TcI) and CL (TcVI), associated, respectively with marsupial and human infections, T. dionisii infectivity increased under nutritional stress, a condition that induces host cell lysosome exocytosis required for parasite internalization. For efficient internalization, T. dionisii depended on MT protein tyrosine kinase (PTK) and Ca2+ mobilization from acidocalcisomes, whereas T. cruzi strains also relied on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) and Ca2+ released from thapsigargin-sensitive compartments. T. dionisii-induced signaling in host cells implicated PKC and Ca2+ mobilized from thapsigargin-sensitive stores, like T. cruzi, but without PI3K involvement. Unlike T. cruzi, T. dionisii metacyclic forms did not use l-proline as source of energy required for internalization. Molecules related to T. cruzi surface glycoproteins involved in MT-host cell interaction were undetectable in T. dionisii. The difference in the surface profile of the two species was also inferred from the susceptibility of T. dionisii metacyclic forms to complement-mediated lysis, as opposed to complete resistance of T. cruzi. In summary, the two Trypanosoma species display distinct surface profiles but invade host cells through a common mechanism involving lysosome mobilization to the site of parasite entry.

Oleic Acid Versus Linoleic and α-Linolenic Acid. Different Effects on Ca2+ Signaling in Rat Thymocytes

Background: Oleic acid is the principal fatty acid of olive oil composition and is reported to play a crucial role in its healthy aspects. However, the detailed mechanism of action is poorly understood. Aims: This study aims to elucidate the role of oleic acid in calcium signaling in rat thymocytes, in comparison to linoleic and linolenic acid. Methods: Fatty acids were applied to thymocytes isolated from wistar rats and loaded with Fura-2 to measure calcium signals. Results: The main results showed a concentration-dependent increase in [Ca²⁺]_i induced by the 3 fatty acids. Raising the number of unsaturations resulted in greater increases. Two different pathways contributed to the increase induced by the polyunsaturated fatty acids: an IP₃- independent release from the thapsigargin-sensitive stores and an extracellular calcium entry by econazole and nifedipine-insensitive channels. However, the OA-induced increases in [Ca²⁺]_i seemed to be due mostly to the Ca²⁺ recruited from the intracellular stores. Conclusion: This study demonstrates that the fatty acids tested induce increases in [Ca²⁺]_i in rat thymocytes, with differences in close relation to the degree of unsaturation. Such differences could be responsible for their different physiological action.

Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor, ZnR, to Trigger Signaling Leading to Epithelial Repair

A role for Zn(2+) in accelerating wound healing is established, yet, the signaling pathways linking Zn(2+) to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn(2+) induces a metabotropic Ca(2+) response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn(2+)-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn(2+) and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn(2+) was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn(2+) probe, ZnAF-2. Chelation of Zn(2+) and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca(2+) signaling. The signaling triggered by Zn(2+), via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn(2+), which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.

Diversity of Lysophosphatidic Acid Receptor-Mediated Intracellular Calcium Signaling in Early Cortical Neurogenesis

Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that can induce pleomorphic effects in neural progenitor cells (NPCs) from the cerebral cortex, including alterations in ionic conductance. LPA-induced, calcium-mediated conductance changes have been reported; however, the underlying molecular mechanisms have not been determined. We show here that activation of specific cognate receptors accounts for nearly all intracellular calcium responses evoked by LPA in acutely cultured nestin-positive NPCs from the developing mouse cerebral cortex. Fast-onset changes in intracellular calcium levels required release from thapsigargin-sensitive stores by a pertussis toxin-insensitive mechanism. The influx of extracellular calcium through Cd(2+)/Ni(2+)-insensitive influx pathways, approximately one-half of which were Gd(3+) sensitive, contributed to the temporal diversity of responses. Quantitative reverse transcription-PCR revealed the presence of all five known LPA receptors in primary NPCs, with prominent expression of LPA(1), LPA(2), and LPA(4). Combined genetic and pharmacological studies indicated that NPC responses were mediated by LPA(1) (approximately 30% of the cells), LPA(2) (approximately 30%), a combination of receptors on single cells (approximately 30%), and non-LPA(1,2,3) pathways (approximately 10%). LPA responsivity was significantly reduced in more differentiated TuJ1(+) cells within cultures. Calcium transients in a large proportion of LPA-responsive NPCs were also initiated by the closely related signaling lipid S1P (sphingosine-1-phosphate). These data demonstrate for the first time the involvement of LPA receptors in mediating surprisingly diverse NPC calcium responses involving multiple receptor subtypes that function within a single cell. Compared with other known factors, lysophospholipids represent the major activator of calcium signaling identified within NPCs at this early stage in corticogenesis.

Role of calcium signalling and phosphorylations in disruption of the epithelial junctions by Pseudomonas aeruginosa quorum sensing molecule

In Pseudomonas aeruginosa, cell–cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We demonstrate here that 3O-C 12-HSL can induce changes in calcium signalling through influx and release of calcium from thapsigargin-sensitive stores and delocalization of inositol 1,4,5-trisphosphate receptors (IP 3R), but not of ryanodine receptors (RyR). In parallel, P. aeruginosa 3O-C 12-HSL disrupts junctions in human Caco-2 cells as evidenced by a reduction of the expression and distribution of ZO-3 and JAM-A. Using co-immunoprecipitation we also found an alteration in the binding of ZO-3 to JAM-A in protein complexes. Moreover, 3O-C 12-HSL-treatment resulted in tyrosine hyperphosphorylation of ZO-3 and JAM-A. On the contrary, serine and threonine residues of ZO-1 and JAM-A became less phosphorylated after exposition of 3O-C 12-HSL. The 3O-C 12-HSL-induced intracellular calcium signalling and alteration in the phosphorylation status of junction proteins furthermore correlated with changes in the association between JAM-A–ZO-3. The calcium inhibitors thapsigargin, xestospongin C, and dantrolene partly prevented the 3O-C 12-HSL-induced decreases in TER and increases in the paracellular flux of 10 kDa dextran. These findings clearly suggest that P. aeruginosa 3O-C 12-HSL can cause the loss of epithelial barrier function via calcium signalling and further alteration in the phosphorylation status of junction proteins; and that bacterial quorum sensing signals represent inter-kingdom signalling.

The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER

Bestrophin-1, mainly analyzed in overexpression experiments, functions as Ca(2+)-dependent Cl(-) channel. Analysis of endogenously expressed bestrophin-1 suggested an influence on intracellular Ca(2+). The aim of the study is to analyze the influence of endogenously expressed bestrophin-1 on Ca(2+) homeostasis. Primary cultures of retinal pigment epithelial (RPE) cells were established from wild-type and bestrophin-1-deficient mice. Intracellular free Ca(2+) ([Ca(2+)](i)) was recorded by Ca(2+) imaging; through immunocytochemistry and differential centrifugation, subcellular localization of bestrophin-1 was analyzed. RPE cells of bestrophin-1-deficient mice showed higher levels of resting [Ca(2+)](i) than cells from wild-type mice. In cells from knockout mice and wild-type mice, ATP led to increases in [Ca(2+)](i) subsequent to phospholipase C activation. ATP-induced Ca(2+) in bestrophin-1-deficient mice rose faster and decayed slower. In cells from wild-type mice, ATP led to [Ca(2+)](i) increase via depletion of Ca(2+) from thapsigargin-sensitive stores. In cells from bestrophin-1-deficient mice, ATP-dependent increase in [Ca(2+)](i) resulted in 40% of cells from depletion of bafilomycin-sensitive and in 60% from thapsigargin-sensitive Ca(2+) stores. After differential centrifugation, bestrophin-1 was found in fractions enriched of ClC-3 Cl channel and myosin-7A. Co-localization analysis of bestrophin-1, with beta-catenin or pan-cadherin, in fresh sections of porcine retina, revealed bestrophin-1 in the basolateral membrane. A portion of endogenously expressed bestrophin-1,localized in the endoplasmic reticulum, influenced uptake of Ca(2+) into Ca(2+) stores. Therefore, bestrophin-1 possibly conducts Cl(-) as counter ion for Ca(2+) uptake into cytosolic Ca(2+) stores.

Therapeutic (High) Doses of Rituximab Activate Calcium Mobilization and Inhibit B-cell Growth via an Unusual Mechanism Triggered Independently of Both CD20 and Fcγ Receptors

Rituximab is a CD20-specific monoclonal antibody that effectively targets and depletes B lymphocytes in vivo, primarily via indirect cytotoxic mechanisms. Direct effects on B cells may also contribute to B-cell depletion but are less clearly defined. In this report, we demonstrate that monomeric rituximab, at the high concentrations found in plasma following infusion of therapeutic doses, induces prolonged low-amplitude release of calcium from thapsigargin-sensitive intracellular stores and reduces the growth of Ramos B cells in culture. Intracellular calcium release was triggered via a signaling pathway distinct from the lipid raft-dependent and src family kinase-dependent pathway that is activated by CD20 hypercrosslinking or B-cell receptor association. The response was independent of both CD20 and Fc receptor binding, and was also triggered by some, but not all, irrelevant monoclonal IgG1 antibodies. The data indicate that unique regions within IgG may contribute to direct effects of therapeutic monoclonal antibodies delivered at suprasaturating concentrations.

Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca2+ from thapsigargin-sensitive stores. Here, we have studied Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca2+]i was elevated and agonist-induced [Ca2+]i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca2+. Importantly, [Ca2+]i increases and Ca2+ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca2+ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca2+ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca2+]i increase and the other with a slower and prolonged [Ca2+]i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca2+]i increases, reflecting overall Ca2+ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca2+ storage and elevated basal [Ca2+]i. S1P metabolism thus plays a role not only in acute Ca2+ mobilization but also in long-term regulation of Ca2+ homeostasis.

Lysophospholipid Receptor-Mediated Calcium Signaling in Human Keratinocytes

The lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), stimulate chemotaxis and induce differentiation of human keratinocytes. As Ca(2+) plays an important role in keratinocyte differentiation, we studied Ca(2+) signaling by S1P and LPA in these cells, known to express mRNA transcripts of the S1P(1-5) and LPA(1-3) receptors, and the receptor subtypes involved in this process. S1P and LPA caused transient increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)), with pEC(50) values of 8.5+/-0.11 and 7.5+/-0.23, respectively. The [Ca(2+)](i) increases are apparently mediated by stimulation of phospholipase C and involve Ca(2+) mobilization from thapsigargin-sensitive stores and subsequent Ca(2+) influx. The LPA-induced [Ca(2+)](i) increases were not inhibited by the LPA(1/3) receptor antagonist, dioctanoylglycerol pyrophosphate. The S1P-induced [Ca(2+)](i) increases were largely inhibited by the putative S1P(3) antagonist, BML-241, and the S1P(1/3) antagonist, VPC23019. The S1P(1)-specific agonist, SEW2871, did not increase [Ca(2+)](i) but stimulated chemotaxis of keratinocytes, which was fully blocked by S1P(1) antisense oligonucleotides. The data indicate that LPA and S1P potently increase [Ca(2+)](i) in human keratinocytes and that the effect of LPA is mediated by LPA(2), whereas that of S1P is mediated at least to a large part by S1P(3). The S1P(1) receptor, without stimulating [Ca(2+)](i) increases, mediates chemotaxis of keratinocytes.

α 1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway

Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both α- and β-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the α-adrenoceptor initiates the venom production cycle. Our results show that the α 1-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of α 1-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca 2+ from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, α 1-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake α 1-adrenoceptor (260 bp) with higher identity with α 1D and α 1B-adrenoceptors from different species. These results suggest that α 1-adrenoceptors in the venom secreting cells are probably coupled to a G q protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.

Extracellular Acidification Elicits Spatially and Temporally Distinct Ca2+ Signals

Extracellular acidification accompanies neoplastic transformation of tissues and increases with tumor aggressiveness [1, 2]. The intracellular signaling cascade triggered by this process remains poorly understood and may be linked to recently discovered proton-activated G protein-coupled receptors such as OGR1 and G2A [3, 4]. Here, we report that OGR1 and G2A are expressed in human medulloblastoma tissue and its corresponding neuronal cell line. We show that extracellular acidification activates phospholipase C, IP(3) formation, and subsequent Ca2+ release from thapsigargin-sensitive stores in neurons. The number of responsive cells and the amount of Ca2+ released from stores correlated positively with the extent of extracellular acidification. Ca2+ release recruited the MEK/ERK pathway, providing a mechanistic explanation for how acidification stimulates cell growth. In addition, acidification activated Ca2+-permeable ion channels through a mechanism dependent on phospholipase C but independent of store depletion or a cytoplasmic Ca2+ rise. Hence, extracellular acidification, to levels seen in tumor tissue, activates temporally and spatially distinct pathways that elevate Ca2+ and may be directly relevant for tumor cell biology.

Identification and localisation of SERCA 2 isoforms in mammalian sperm

Upon binding to the egg's zona pellucida, capacitated spermatozoa will undergo a calcium-dependent exocytotic event called acrosome reaction. During this process, Ca2+ depletion from internal stores is followed by an important rise in [Ca2+]i due to a massive Ca2+ influx. Previous reports have shown that the acrosome can act as a Ca2+ store and that depletion of thapsigargin-sensitive stores induces acrosome exocytosis in capacitated spermatozoa from different mammalian species. The effect of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs), suggests the presence and implication of SERCA in the active Ca2+ uptake during mammalian sperm capacitation. Although the presence of a thapsigargin-sensitive Ca2+-ATPase has been debated, the aim of this study was to clearly determine whether SERCAs are present in mammalian spermatozoa. Using three different anti-SERCA 2 antibodies, mono- and polyclonal, which recognised the same protein, we successfully identified and localised SERCA 2 in human, mouse and bovine sperm. Western blot analysis suggests that more than one SERCA 2 splice variant are present, one detected in the fraction containing the outer acrosomal membranes and another one present in the subcellular fraction containing the sperm midpiece. These results were confirmed by indirect immunofluorescence where SERCA 2 was observed in the acrosome and midpiece regions of human sperm. SERCA 2 immunohistochemical studies on human testis and PCR-amplification of mRNA encoding for each SERCA 2 splice variant in spermatogenic cells support the presence of this Ca2+-ATPase family in mature spermatozoa. In this paper, we clearly demonstrate, for the first time, the presence of SERCA 2 in mammalian sperm.

The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.

The extracellular zinc-sensing receptor mediates intercellular communication by inducing ATP release

Taste and salivary secretion disorders have been linked to zinc deficiency, indeed zinc is found in secretory granules in the salivary gland. The signaling role for the zinc release in this tissue, however, is poorly understood. Here, we address the signaling pathways and physiological role of the zinc-sensing receptor, ZnR, in the ductal salivary gland cell line, HSY. Exposure of these cells to zinc triggered intracellular Ca 2+ release from thapsigargin-sensitive stores. The Gαq inhibitor, YM-254890 (1 μM), eliminated the Zn 2+-dependent Ca 2+ response, demonstrating that ZnR is a Gαq-coupled receptor. Dose–response curves yielded an apparent K 0.5 of 36 μM and a Hill coefficient of 7 in the absence of extracellular Ca 2+, and K 0.5 of 55 μM with a Hill coefficient of 3 in its presence. This indicates that although Zn 2+ is essential for ZnR activation, Ca 2+ may affect the receptor co-operativity. The homologous desensitization pattern of ZnR was characterized by pre-exposure of cells to Zn 2+ at concentrations found to activate the receptor. Re-exposure of cells to Zn 2+ elicited an attenuated Zn 2+-dependent Ca 2+ response for at least 3 h, indicating that the ZnR is strongly desensitized by Zn 2+. Finally, we studied the paracrine affects of ZnR using a co-culture consisting of the HSY cells and vascular smooth muscle cells (VSMCs). While no Zn 2+-dependent Ca 2+ release was observed in VSMC alone, application of Zn 2+ to the co-culture induced a Ca 2+ rise in both HSY cells and VSMC. This Ca 2+ rise was inhibited by the ATP scavenger, apyrase. Taken together, our results demonstrate that ZnR activity is monitored in salivary cells and is modulated by extracellular Ca 2+. We further show that ZnR enhances secretion of ATP, thereby linking zinc to key signaling pathways involved in modification of salivary secretions by the ductal cells.

Arachidonic acid mediates non‐capacitative calcium entry evoked by CB1‐cannabinoid receptor activation in DDT1 MF‐2 smooth muscle cells

Cannabinoid CB1-receptor stimulation in DDT1 MF-2 smooth muscle cells induces a rise in [Ca2+]i, which is dependent on extracellular Ca2+ and modulated by thapsigargin-sensitive stores, suggesting capacitative Ca2+ entry (CCE), and by MAP kinase. Non-capacitative Ca2+ entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine H1-receptor-evoked increases in [Ca2+]i in DDT1 MF-2 cells. In the current study, both Ca2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+]i were investigated. In the whole-cell patch clamp configuration, the CB-receptor agonist CP 55, 940 evoked a transient, Ca2+-dependent K+ current, which was not blocked by the inhibitors of CCE, 2-APB, and SKF 96365. AA, but not its metabolites, evoked a transient outward current and inhibited the response to CP 55,940 in a concentration-dependent manner. CP 55,940 induced a concentration-dependent release of AA, which was inhibited by the CB1 antagonist SR 141716. The non-selective Ca2+ channel blockers La3+ and Gd3+ inhibited the CP 55,940-induced current at concentrations that had no effect on thapsigargin-evoked CCE. La3+ also inhibited the AA-induced current. CP 55,940-induced AA release was abolished by Gd3+ and by phospholipase A2 inhibition using quinacrine; this compound also inhibited the outward current. The CP 55,940-induced AA release was strongly reduced by the MAP kinase inhibitor PD 98059. The data suggest that in DDT1 MF-2 cells, AA is an integral component of the CB1 receptor signaling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase.