Generation Of Intracellular Ca2 Research Articles

Impact of TRP Channels on Extracellular Matrix Remodeling: Focus on TRPV4 and Collagen.

Disturbed remodeling of the extracellular matrix (ECM) is frequently observed in several high-prevalence pathologies that include fibrotic diseases of organs such as the heart, lung, periodontium, liver, and the stiffening of the ECM surrounding invasive cancers. In many of these lesions, matrix remodeling mediated by fibroblasts is dysregulated, in part by alterations to the regulatory and effector systems that synthesize and degrade collagen, and by alterations to the functions of the integrin-based adhesions that normally mediate mechanical remodeling of collagen fibrils. Cell-matrix adhesions containing collagen-binding integrins are enriched with regulatory and effector systems that initiate localized remodeling of pericellular collagen fibrils to maintain ECM homeostasis. A large cadre of regulatory molecules is enriched in cell-matrix adhesions that affect ECM remodeling through synthesis, degradation, and contraction of collagen fibrils. One of these regulatory molecules is Transient Receptor Potential Vanilloid-type 4 (TRPV4), a mechanically sensitive, Ca2+-permeable plasma membrane channel that regulates collagen remodeling. The gating of Ca2+ across the plasma membrane by TRPV4 and the consequent generation of intracellular Ca2+ signals affect several processes that determine the structural and mechanical properties of collagen-rich ECM. These processes include the synthesis of new collagen fibrils, tractional remodeling by contractile forces, and collagenolysis. While the specific mechanisms by which TRPV4 contributes to matrix remodeling are not well-defined, it is known that TRPV4 is activated by mechanical forces transmitted through collagen adhesion receptors. Here, we consider how TRPV4 expression and function contribute to physiological and pathological collagen remodeling and are associated with collagen adhesions. Over the long-term, an improved understanding of how TRPV4 regulates collagen remodeling could pave the way for new approaches to manage fibrotic lesions.

Purinergic signaling in tanycytes and its contribution to nutritional sensing.

Tanycytes are hypothalamic radial glial-like cells with an important role in the regulation of neuroendocrine axes and energy homeostasis. These cells have been implicated in glucose, amino acids, and fatty acid sensing in the hypothalamus of rodents, where they are strategically positioned. While their cell bodies contact the cerebrospinal fluid, their extensive processes contact neurons of the arcuate and ventromedial nuclei, protagonists in the regulation of food intake. A growing body of evidence has shown that purinergic signaling plays a relevant role in this homeostatic role of tanycytes, likely regulating the release of gliotransmitters that will modify the activity of satiety-controlling hypothalamic neurons. Connexin hemichannels have proven to be particularly relevant in these mechanisms since they are responsible for the release of ATP from tanycytes in response to nutritional signals. On the other hand, either ionotropic or metabotropic ATP receptors are involved in the generation of intracellular Ca2+ waves in response to hypothalamic nutrients, which can spread between glial cells and towards neighboring neurons. This review will summarize recent evidence that supports a nutrient sensor role for tanycytes, highlighting the participation of purinergic signaling in this process.

Introduction to ion channels and calcium signaling in the microcirculation.

The microcirculation is the network of feed arteries, arterioles, capillaries and venules that supply and drain blood from every tissue and organ in the body. It is here that exchange of heat, oxygen, carbon dioxide, nutrients, hormones, water, cytokines, and immune cells takes place; essential functions necessary to maintenance of homeostasis throughout the life span. This chapter will outline the structure and function of each microvascular segment highlighting the critical roles played by ion channels in the microcirculation. Feed arteries upstream from the true microcirculation and arterioles within the microcirculation contribute to systemic vascular resistance and blood pressure control. They also control total blood flow to the downstream microcirculation with arterioles being responsible for distribution of blood flow within a tissue or organ dependent on the metabolic needs of the tissue. Terminal arterioles control blood flow and blood pressure to capillary units, the primary site of diffusional exchange between blood and tissues due to their large surface area. Venules collect blood from capillaries and are important sites for fluid exchange and immune cell trafficking. Ion channels in microvascular smooth muscle cells, endothelial cells and pericytes importantly contribute to all of these functions through generation of intracellular Ca2+ and membrane potential signals in these cells.

Purinergic receptor stimulation induces calcium oscillations and smooth muscle contraction in small pulmonary veins.

We investigated the excitation-contraction coupling mechanisms in small pulmonary veins (SPVs) in rat precision-cut lung slices. We found that SPVs contract strongly and reversibly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced vasoconstriction in SPVs was associated with the stimulation of purinergic P2Y2 receptors in vascular smooth muscle cell, activation of phospholipase C-β and the generation of intracellular Ca2+ oscillations mediated by cyclic Ca2+ release events via the inositol 1,4,5-trisphosphate receptor. Active constriction of SPVs may play an important role in the development of pulmonary hypertension and pulmonary oedema. The small pulmonary veins (SPVs) may play a role in the development of pulmonary hypertension and pulmonary oedema via active changes in SPV diameter, mediated by vascular smooth muscle cell (VSMC) contraction. However, the excitation-contraction coupling mechanisms during vasoconstrictor stimulation remain poorly understood in these veins. We used rat precision-cut lung slices and phase-contrast and confocal microscopy to investigate dynamic changes in SPV cross-sectional luminal area and intracellular Ca2+ signalling in their VSMCs. We found that the SPV (∼150μm in diameter) contract strongly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced SPV contraction was fast, concentration-dependent, completely reversible upon ATP washout, and inhibited by purinergic receptor antagonists suramin and AR-C118925 but not by MRS2179. Immunofluorescence showed purinergic P2Y2 receptors expressed in SPV VSMCs. ATP-induced SPV contraction was inhibited by phospholipase Cβ inhibitor U73122 and accompanied by intracellular Ca2+ oscillations in the VSMCs. These Ca2+ oscillations and SPV contraction were inhibited by the inositol 1,4,5-trisphosphate receptor inhibitor 2-APB but not by ryanodine. The results of the present study suggest that ATP-induced vasoconstriction in SPVs is associated with the activation of purinergic P2Y2 receptors in VSMCs and the generation of Ca2+ oscillations.

Chimeras of sperm PLC reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

Mechanisms of Phospholipase C-Regulated Calcium Entry

In a variety of cell types, activation of phospholipase C-linked receptors results in the generation of intracellular Ca2+ signals comprised of components of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. This entry of Ca2+ occurs by either of two general mechanisms: the release of stored Ca2+ can activate, by an unknown mechanism, store-operated channels in the plasma membrane, a process known as capacitative calcium entry. Alternatively, second messengers generated at the plasma membrane can activate Ca2+ channels more directly, a non-capacitative calcium entry process. This review summarizes current knowledge of the underlying signaling mechanisms and the nature of the channel molecules responsible for these two general categories of regulated Ca2+ entry.

IL-1 induced release of Ca2+ from internal stores is dependent on cell-matrix interactions and regulates ERK activation.

The cellular mechanisms that modulate interleukin-1 (IL-1) signaling are not defined. In fibroblasts, IL-1 signaling is affected by the nature of cell-matrix adhesions including focal adhesions, adhesive domains that sequester IL-1 receptors. We conducted studies to elucidate which steps of cellular Ca2+ handling are affected by focal adhesions and by which mechanisms focal adhesions modulate IL-1-induced Ca2+ signals and ERK activation in human gingival fibroblasts. Cells were plated on poly-l-lysine or fibronectin and treated with tenascin, Hep-I, or SPARC peptides to inhibit focal adhesion formation. These treatments blocked IL-1 and thapsigargin-induced Ca2+ release from the endoplasmic reticulum, indicating that the ER-release pathway is focal adhesion dependent. Focal adhesions were also required for Ca2+ entry through store-operated channels and for IL-1-induced ERK activation. Thus interactions with the extracellular matrix and focal adhesion formation regulate IL-1-induced generation of intracellular Ca2+ signals that in turn are required for ERK activation.

RyR1 and RyR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells.

Ryanodine receptors (RyRs) are expressed on the endoplasmic reticulum of many cells, where they form intracellular Ca2+-release channels that participate in the generation of intracellular Ca2+ signals. Here we report studies on the intracellular localisation and functional properties of transfected RyR1 or RyR3 channels in HEK 293 cells. Immunofluorescence studies indicated that both RyR1 and RyR3 did not form clusters but were homogeneously distributed throughout the endoplasmic reticulum. Ca2+ release experiments showed that transfected RyR1 and RyR3 channels responded to caffeine, although with different sensitivity, generating a global release of Ca2+ from the entire endoplasmic reticulum. However, video imaging and confocal microscopy analysis revealed that, in RyR3-expressing cells, local spontaneous Ca2+ release events were observed. No such spontaneous activity was observed in RyR1-expressing cells or in control cells. Interestingly, the spontaneous release events observed in RyR3-expressing cells were restricted to one or two regions of the endoplasmic reticulum, suggesting the formation of a further subcellular organisation of RyR3 in Ca2+ release units. These results demonstrate that different RyR isoforms can engage in the generation of distinct intracellular Ca2+ signals in HEK 293 cells.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

Structure and function of inositol 1,4,5-trisphosphate receptor.

Generation of intracellular Ca2+ signals in response to Ca(2+)-mobilizing stimuli is a critical event in the control of many cellular processes. Inositol 1,4,5-trisphosphate (IP3) represents a dominant second messenger subserving the release of Ca2+ from intracellular store sites. The protein on the surface of which the IP3 receptor is located comprises an IP3-gated Ca2+ channel, and binding of IP3 to this receptor triggers the release of Ca2+ through this channel. The receptor for IP3 displays a close resemblance to the ryanodine receptor, another intracellular Ca2+ channel, in many molecular and physiological properties. Many lines of evidence strongly suggest the central role that the IP3 receptor plays in the conversion of numerous external stimuli to intracellular Ca2+ signals characterized by complex spatiotemporal patterns such as Ca2+ waves and oscillations. In this review, we shall summarize our current knowledge of the structure and function of the IP3 receptor in order to understand the way how the activity of this important receptor is regulated to accomodate itself to the generation of diverse intracellular Ca2+ signals.

Structure and Function of Inositol 1,4,5-Trisphosphate Receptor

Generation of intracellular Ca2+ signals in response to Ca2+-mobilizing stimuli is a critical event in the control of many cellular processes. Inositol 1,4,5-trisphosphate (IP3) represents a dominant second messenger subserving the release of Ca2+ from intracellular store sites. The protein on the surface of which the IP3 receptor is located comprises an IP3-gated Ca2+ channel, and binding of IP3 to this receptor triggers the release of Ca2+ through this channel. The receptor for IP3 displays a close resemblance to the ryanodine receptor, another intracellular Ca2+ channel, in many molecular and physiological properties. Many lines of evidence strongly suggest the central role that the IP3 receptor plays in the conversion of numerous external stimuli to intracellular Ca2+ signals characterized by complex spatiotemporal patterns such as Ca2+ waves and oscillations. In this review, we shall summarize our current knowledge of the structure and function of the IP3 receptor in order to understand the way how the activity of this important receptor is regulated to accomodate itself to the generation of diverse intracellular Ca2+ signals.

Nuclei from fertilized mouse embryos have calcium-releasing activity

During mammalian fertilization, the sperm triggers a series of intracellular Ca2+ oscillations which initiate oocyte activation and the formation of pronuclei. Oocyte activation can be induced artificially by a variety of chemical and physical stimuli which elevate intracellular calcium. We show that the transfer of nuclei from 1- and 2-cell-stage fertilized mouse embryos to unfertilized oocytes stimulates the completion of meiosis and the formation of pronuclei. Nuclei from embryos that had developed to the 4-cell stage did not stimulate meiotic resumption. The ability to cause oocyte activation was specific to nuclei transferred from fertilized embryos as nuclei from parthenogenetic embryos or cytoplasts from fertilized or parthenogenetic embryos did not induce activation. Nucleus-induced oocyte activation was associated with the generation of intracellular Ca2+ transients, which were seen after nuclear envelope breakdown of the transferred nuclei. Treatment of the oocyte with the intracellular Ca2+ chelator, BAPTA, prior to nuclear transfer inhibited intracellular Ca2+ transients and oocyte activation. The specific Ca(2+)-releasing activity of the nucleus was not caused by sperm-induced protein synthesis since similar activity was present in nuclei originating from embryos exposed to cycloheximide throughout fertilization. The specific ability of nuclei from fertilized embryos to stimulate Ca2+ transients and oocyte activation was also found in nuclei from embryos parthenogenetically activated by the injection of a partially purified cytosolic sperm factor. The results suggest that the fertilizing sperm introduces Ca(2+)-releasing activity which becomes associated with the nucleus of early mammalian embryos.

Uptake of Metabolites by Postcapillary Venules: Mechanism for the Control of Arteriolar Diameter

The microcirculation is the network of feed arteries, arterioles, capillaries and venules that supply and drain blood from every tissue and organ in the body. It is here that exchange of heat, oxygen, carbon dioxide, nutrients, hormones, water, cytokines, and immune cells takes place; essential functions necessary to maintenance of homeostasis throughout the life span. This chapter will outline the structure and function of each microvascular segment highlighting the critical roles played by ion channels in the microcirculation. Feed arteries upstream from the true microcirculation and arterioles within the microcirculation contribute to systemic vascular resistance and blood pressure control. They also control total blood flow to the downstream microcirculation with arterioles being responsible for distribution of blood flow within a tissue or organ dependent on the metabolic needs of the tissue. Terminal arterioles control blood flow and blood pressure to capillary units, the primary site of diffusional exchange between blood and tissues due to their large surface area. Venules collect blood from capillaries and are important sites for fluid exchange and immune cell trafficking. Ion channels in microvascular smooth muscle cells, endothelial cells and pericytes importantly contribute to all of these functions through generation of intracellular Ca2+ and membrane potential signals in these cells.

Cholera toxin inhibits resting human T cell activation via a cAMP-independent pathway.

The catalytic subunit of cholera toxin (CT) can chemically modify the alpha polypeptides of certain G-binding proteins and thus alter their function. In order to study the involvement of CT-sensitive G proteins in T cell activation, we have utilized CT in an in vitro system in which purified, resting human peripheral T cells are activated by anti-CD3 antibodies and rIL-2. Perturbation of the TCR/CD3 molecular complex by anti-CD3 antibodies causes changes in membrane phospholipids and induces a rise in cytoplasmic Ca2+. These events, however, are insufficient to allow progression into cellular proliferation and addition of IL-2 is required. Under these conditions, treatment of cells with a low concentration of CT (2 ng/ml) causes a significant inhibition of the anti-CD3-induced calcium event as well as the anti-CD3 plus IL-2-stimulated proliferation. Under our experimental conditions, inhibition of both proliferation and intracellular Ca2+ elevation by CT requires the involvement of the TCR/CD3 complex. This is supported by the observation that the toxin does not inhibit either the proliferation triggered by ionomycin and PMA or the Ca2+ influx induced by the ionophore. These data suggest that in TCR/CD3-mediated T cell activation CT acts at a point between TCR/CD3 perturbation and the generation of intracellular Ca2+. In view of the ability of CT to activate the alpha subunit of the G protein that stimulates adenyl cyclase (G alpha s), it is possible that the effect of CT on T cells is secondary to intracellular elevation of cAMP. However, measurement of cAMP levels both early after CT addition and at later time points, when proliferation is maximal, reveals lack of cyclic nucleotide accumulation. The presented data are consistent with the interpretation that the CT-mediated inhibition is caused by the modification of a G-binding protein that is either directly or indirectly associated with triggering of T cells via the TCR/CD3 molecular complex. The data also suggest that this protein is not G alpha s and it probably represents an as yet unidentified moiety or one of the several G proteins that have been recently described as regulators of phospholipase C activation.