Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.     

Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.     

Differences in intracellular calcium mobilization by interferon-beta and interferon-gamma in RPMI-4788 cells

Human interferon (IFN) stimulates a 1.5- to 1.7-fold transient increase in the concentration of cytoplasmic-free calcium ion ([Ca2+]i) within 10-20 s upon exposure of RPMI-4788 cells to IFN. This early event of IFN-induced [Ca2+]i mobilization was measurable by loading the cells with Fura-2AM, a fluorescent Ca2+ indicator. The mobilization induced by IFN-beta or IFN-gamma was dependent on the concentration of each IFN. The increased [Ca2+]i gradually returned to its resting level within 60 s. The addition of EGTA (0.5-10 mM) to medium induced a marked decrease in the amount of [Ca2+]i mobilized by IFN-beta and a partial decrease by IFN-gamma. This finding suggests that the mechanisms of [Ca2+]i mobilization by IFN-beta and IFN-gamma might be different. While IFN-beta-induced mobilization may be mainly from an influx of the extracellular calcium ion ([Ca2+]o), IFN-gamma-induced mobilization may be a summation of an influx of [Ca2+]o and a release from intracellular Ca2+ stores.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca²⁺-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (?log EC₅₀ = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca²⁺-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G_q-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, α₁(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca²⁺ mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.     

Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells

C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.     

The relationship between alpha 1-adrenergic receptor occupation and the mobilization of intracellular calcium

We have simultaneously quantitated alpha 1-adrenergic receptor occupation and agonist-elicited Ca2+ mobilization monitored as unidirectional 45Ca2+ efflux from intact BC3H-1 muscle cells in order to examine the relationship between the number of surface receptors occupied and the functional response. [3H]Prazosin has been used to measure receptor number as well as the binding kinetics with surface receptors, and the observed equilibrium and kinetic constants are in close accord with values obtained previously in cellular homogenates. Since alpha 1-agonist-elicited 45Ca2+ efflux can be monitored over intervals of 3 min or less and prazosin dissociation from its receptor has a t 1/2 of 44 min, prazosin can be employed to produce a pseudoirreversible inactivation of receptors. A comparison of the remaining receptors and residual response reveals an inverse linear relationship between receptors inactivated by prazosin and 45Ca2+ efflux. A similar result is obtained following fractional receptor inactivation with the irreversible alkylating agent phenoxybenzamine. Parameters of receptor occupation and response also correlate well for the agonist phenylephrine and for the competitive antagonist phentolamine. The unitary relationship between sites available for occupation and response indicates that the alpha 1 receptor does not function as an oligomer where fewer bound antagonist molecules are required to block the receptor than sites of agonist occupation necessary for activation. Moreover, substantial evidence has accrued in intact smooth muscle for a receptor reserve or nonlinear coupling between alpha 1 receptor occupation and contraction in smooth muscle. Our findings demonstrate that such behavior does not exist for alpha 1 receptor-elicited mobilization of Ca2+ in the BC3H-1 muscle cell.     

HVEM signaling in monocytes is mediated by intracellular calcium mobilization

Herpes virus entry mediator (HVEM) is a member of the TNF receptor (TNFR) superfamily and is expressed on many immune cells, including T and B cells, NK cells, monocytes, and neutrophils. Interaction of HVEM with its ligand, LIGHT, costimulates T cells and increases the bactericidal activity of monocytes and neutrophils. The interaction recruits cytoplasmic TNFR-associated factor adaptor proteins to the intracellular domain of HVEM. This leads to NFkappaB activation as a result of IkappaBalpha degradation and/or JNK/AP-1 activation, and ultimately results in the expression of genes required for cell survival, cytokine production, or cell proliferation. In this study, we show that treatment of human monocytes with recombinant human LIGHT (rhLIGHT) induces rapid elevation of intracellular calcium concentration ([Ca(2+)](i)) in a HVEM-specific manner in parallel with TNF-alpha production, and enhances the bactericidal activities of monocytes. Immunoprecipitation and Western blotting analyses revealed phosphorylation of phospholipase Cgamma1 (PLCgamma1) but not PLCgamma2. rhLIGHT-induced Ca(2+)response was completely abolished by silencing PLCgamma1, or preincubating monocytes with PLC inhibitors, antagonists of the inositol-1,4,5-triphosphate receptor, or [Ca(2+)](i) chelators. Furthermore, these PLC/Ca(2+) inhibitors also blocked rhLIGHT-mediated IkappaBalpha degradation, generation of reactive oxygen species, TNF-alpha production and the bactericidal activities of monocytes. Our results indicate that Ca(2+)is a downstream mediator of the LIGHT/HVEM interaction in monocytes.     

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.     

Transient inositol (1,4,5) trisphosphate accumulation under vasopressin stimulation in WRK1 cells: correlation with intracellular calcium mobilization

In the rat mammary tumoral cell line (WRK1 cells), vasopressin was previously described to stimulate a phospholipase C. In this study, we have analysed the effect of vasopressin both on intracellular calcium mobilization and on the accumulation of inositol phosphates. Maximal concentration of vasopressin simultaneously induces an accumulation of Ins(1,4,5)P3 and a rise of intracellular calcium concentration. Both these two phenomena are transient and exhibit similar kinetics. A sustained accumulation of InsP2, Ins(1,3,4)P3 and InsP are observed later. Yet no stimulation of InsP4 can be objectified. These results indicate that Ins(1,4,5)P3 is the major inositol phosphate involved in intracellular calcium mobilization.     

P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells

We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca(2+)(i) mobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca(2+)(i) mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca(2+)](i) in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5' nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca(2+)](i) in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca(2+)](i), suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca(2+)](i). Furthermore, adenosine, ADP, UDP, or adenosine 5'-O-(3-thiotriphosphate) did not induce Ca(2+)(i) mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4'-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca(2+)(i) response, whereas pertussis toxin, a G(i/o)-protein inhibitor, inhibited fluid flow-induced increases in [Ca(2+)](i) in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca(2+)(i) response to fluid flow. Finally, a decreased percentage of MC3T3-E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca(2+)](i), and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization in osteoblastic cells.     

Direct relationship between intracellular calcium mobilization and phospholipase D activation in prostaglandin E-stimulated human erythroleukemia cells.

The relationship between calcium mobilization and phospholipase D (PLD) activation in response to E-series prostaglandins (PGEs) was investigated in human erythroleukemia cells. Intracellular free Ca2+ concentration ([Ca2+]i) was increased by PGE1 and PGE2 over the same concentration range at which PLD activation was seen. Pretreatment of cells with pertussis toxin greatly inhibited the PGE-stimulated increase in [Ca2+]i, implying that a G protein participates in the PGE receptor signaling process. The peak level and also the plateau level of Ca2+ mobilization stimulated by these prostaglandins were markedly decreased in Ca(2+)-depleted medium, indicating that both extracellular and intracellular Ca2+ stores contribute to the changes in [Ca2+]i. Likewise, activation of PLD by PGE1 and PGE2 was abolished by pertussis toxin pretreatment or incubation in Ca(2+)-depleted medium. U73122, a putative phospholipase C inhibitor, blocked both Ca2+ mobilization and PLD activation in PGE-stimulated cells. Furthermore, the intracellular loading of BAPTA, a Ca2+ chelator, inhibited both Ca2+ mobilization and PLD activation by PGE1 and PGE2 in a similar dose-dependent manner. Simultaneous measurement of [Ca2+]i and PLD activity in the same cell samples indicated that PLD activity increases as a function of [Ca2+]i in a similar fashion in cells stimulated either by PGEs or by the calcium ionophore ionomycin. Taken together, these findings suggest that a rise in [Ca2+]i is necessary for PGE-stimulated PLD activity in human erythroleukemia cells.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium.

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.     

Is there crosstalk between extracellular and intracellular calcium mobilization in jasmonic acid signaling

The changes in cytosolic Ca²⁺ levels play an important role in the jasmonic acid (JA) signal transduction pathway. We demonstrate that an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis leaf cells was affected by pretreatment with heparin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). With pretreatment of heparin, an antagonist of inositol 1,4,5-trisphosphate (IP₃) sensitive channels, the basal and JA induced fluorescence of [Ca²⁺]_cyt were both decreased. Furthermore, heparin and TMB-8, another antagonist of IP₃ sensitive channels, enhanced the JA-induced gene expression of JR1. These data suggest that there may be a fine tune control system between extracellular and intracellular Ca²⁺ mobilization in JA signaling pathway.