Cytosolic Free Calcium Mobilization Research Articles

Role of cyclic AMP and cyclic GMP as modulators of platelet cytosolic calcium

Studies from our laboratory at the University of Minnesota as well as that of others have demonstrated that agonist-mediated receptor stimulation leads to the activation of phospholipase C and formation of second messengers, diacyl glycerol and inositol trisphosphate, and mobilization of cytosolic free calcium. Elevation in intracellular calcium activates phospholipase A2 and release of arachidonic acid. This fatty acid is further converted to active metabolites, prostaglandin endoperoxides, and thromboxanes (TXA 2 ). TXA 2 promote fibrinogen binding, irreversible aggregation, and the release of granule contents, including serotonin and ADP. Agents that promote irreversible aggregation, facilitate fibrinogen binding, and those drugs that dissociate this process do so by lowering the cytosolic calcium levels. Endogenous antagonists such as PGE 1 , PGI 2 , and NO exert their inhibitory effect on platelet function by the action of cyclic nucleotides, cyclic AMP and cyclic GMP. Epinephrine-induced modulation of alpha-adrenergic receptor restores the action of agonists in drug-induced refractory platelets. As far as the role of cyclic nucleotides is considered, the observations are consistent with the concept that cyclic nucleotides participation in the biologic regulation may be modulated by cytoplasmic cations, and that a change in intracellular cyclic nucleotides and appropriate cations serves to promote the participation of both regulatory effectors. Anti-platelet drugs such as aspirin, indomethacin, and PGE 1 do not inhibit platelet interactions with the vessel wall. Based on this collective knowledge, novel antiplatelet drugs should be developed for the prevention of acute vascular events.

Lipopolysaccharide-mediated cell activation without rapid mobilization of cytosolic free calcium

The involvement of cytosolic free calcium (Ca 2+) as a second messenger in lipopolysaccharide (LPS)-activated cellular pathways remains controversial. Using human monocyte-like THP-1 cells stably transfected with glycosylphosphatidylinositol-anchored human CD14, we have monitored the concentration of intracellular Ca 2+ after sequential addition of LPS, ATP, and ionomycin. Whereas ATP and ionomycin induced rapid elevations of cytosolic Ca 2+, no such changes were observed after LPS addition. These data argue against the participation of cytosolic Ca 2+ in the CD14-dependent LPS activation pathway.

Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma.

Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLCgamma) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2. The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation-activated PLCgamma. The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase-mediated mechanism, as determined by the blockade of short-term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)-dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non-confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLCgamma with a concentration- and time-profile similar to that of [Ca(2+)](i) mobilization. The present study reveals that H(2)O(2) activates PLCgamma on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca(2+)](i) mobilization, shape-changing actions and damage by H(2)O(2) in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H(2)O(2) and may help to design pharmacologically protective strategies.

Effect of Bradykinin on Cultured Bovine Corneal Endothelial Cells

Purpose: To clarify the effect of bradykinin on cytosolic free calcium mobilization and cell proliferation in cultured bovine corneal endothelial cells (BCEC). Methods: The cytosolic free calcium concentration (Ca²⁺]_i) was measured with the InCa^TM Imaging System after the treatment of bradykinin (10^–11 to 10^–7 M) alone or with the pretreatments of EGTA, bradykinin receptor (Bk₁ and Bk₂) antagonists and an inhibition of phospholipase C (U-73122). Also, the effect of bradykinin on cell proliferation in BCEC was evaluated using cell counts. Results: In BCEC, [Ca²⁺]_i in the resting state was 87 ± 9 nM. Bradykinin induced an increment of [Ca²⁺]_i in a concentration-dependent manner and its 50% effective concentration was approximately 5 × 10^–11 M. A [Ca²⁺]_i increment at 10^–8 M bradykinin was inhibited with the pretreatment of EGTA, an extracellular calcium chelator. U-73122 (5 × 10^–6 M) attenuated the bradykinin-induced [Ca²⁺]_i increment. The pretreatment of HOE-140 (Bk₂ antagonist) almost attenuated the bradykinin (10^–8 M)-induced [Ca²⁺]_i increase, but des-Arg⁹-[Leu⁸]-bradykinin (Bk₁ antagonist) did not suppress it. To investigate the physiological effect of bradykinin, the effect of bradykinin on cell proliferation was studied. 10^–8 M of bradykinin produced a significant increase in cell numbers. This mitogenic effect of bradykinin was inhibited by the Bk₂ antagonist. Conclusions: Bradykinin-induced stimulation of the signal transduction pathway in BCEC is coupled with the Bk₂ type receptor. Furthermore, bradykinin produces the mitogenic effect in BCEC.

Involvement of erythropoietin-induced cytosolic free calcium mobilization in activation of mitogen-activated protein kinase and DNA synthesis in vascular smooth muscle cells.

Human recombinant erythropoietin (rHuEPO) induces cytosolic free calcium ([Ca2+]i) mobilization, an activation of mitogen-activated protein (MAP) kinase and DNA synthesis in several tissues. We explored the mechanism of rHuEPO-induced [Ca2+]i mobilization and its role in the activation of MAP kinase and DNA synthesis in vascular smooth muscle cells (VSMC). [Ca2+]i concentrations were measured by fura-2. MAP kinase activation was analyzed using an immunocomplex kinase assay and Western blotting. DNA synthesis was measured as an incorporation of 5-bromo-2'-deoxyuridine. Although addition of rHuEPO significantly increased [Ca2+]i, either in the presence or absence of extracellular Ca2+, the peak level and sustained elevation of [Ca2+]i were significantly reduced in the absence of extracellular Ca2+. Pretreatment with genistein completely blocked the elevation of [Ca2+]i in both conditions. Calphostin C and staurosporine did not completely block the elevation of [Ca2+]i. Staurosporine reduced its peak level in a dose-dependent manner, whereas calphostin C reduced its peak level at concentrations over 1 nmol/l in the presence of extracellular Ca2+. Similar results to those with staurosporine were observed with nifedipine. In the absence of extracellular Ca2+, their dose-dependent effects disappeared even though rHuEPO increased [Ca2+]i. rHuEPO activated MAP kinase and DNA synthesis, both of which were significantly suppressed by the chelation of intracellular Ca2+. These findings suggest that rHuEPO increases [Ca2+]i by both Ca2+ influx and Ca2+ release from intracellular stores. Tyrosine phosphorylation is critical in the regulation of [Ca2+]i, but protein kinase C activation is important only in the regulation of Ca2+ influx. Dihydropyridine-sensitive L-type Ca2+ channels seem to be involved in rHuEPO-induced Ca2+ influx. In addition, increase of [Ca2+]i by rHuEPO stimulates MAP kinase activation and DNA synthesis in VSMC.

Purinoceptor-mediated calcium mobilization and proliferation in HaCaT keratinocytes

To investigate the effect of nucleotides on cytosolic free calcium mobilization and proliferation activity in HaCaT keratinocytes, nucleotides-induced intracellular free calcium concentration ([Ca 2+] i) and cell proliferation observed. [Ca 2+] i to the extracellular nucleotides was determined using Ca 2+ sensitive indicator, Fura-2/AM with digital video fluorescence imaging microscopy, and cell proliferation was evaluated by counting of cell number. An adenosine 5′-triphosphate (ATP)-induced [Ca 2+] i increase was observed from the concentration of 10 −8 M and was more conspicuous at higher concentrations in a concentration-dependent manner. Additionally, other nucleotides such as ADP, UTP, and 2-me- S-ATP also induced a [Ca 2+] i increase in a concentration-dependent manner. However, adenosine induced a slight increase of [Ca 2+] i only at 10 −3 M. α,-methylene-ATP did not evoke any rise in [Ca 2+] i. The maximal response observed occurred with ATP and UTP at a concentration of 10 −4 M. The ATP-induced transient [Ca 2+] i increase was attenuated by the pretreatment with phospholipase C (PLC) inhibitor, U-73122 (10 μM) for 30 min. ATP-induced [Ca 2+] i increase and cell proliferation were inhibited by putative P2Y receptor antagonist, suramin (10 −4 M). When the HaCaT cells were stimulated with nucleotides on a concentration of 10 −4 M and cultured for 5 days, the order of effect on cell proliferation was observed to be ATP>UTP>ADP>2-me- S-ATP. Based on these results, we suggest that extracellular ATP stimulate HaCaT keratinocytes proliferation via purinoceptor-mediated [Ca 2+] i mobilization

Modulation of Endothelin-1-Induced Cytosolic Free Calcium Mobilization and Mitogen-Activated Protein Kinase Activation by Erythropoietin in Vascular Smooth Muscle Cells

Background: It has been reported that human recombinant erythropoietin (rHuEPO) modulates the sensitivity of the cardiovascular system to vasoconstrictors. We investigated whether rHuEPO has modulative effects on the endothelin-1 (ET-1)-induced elevation of cytosolic free calcium concentration ([Ca²⁺]_i) and mitogen-activated protein (MAP) kinase activation in vascular smooth muscle cells (VSMC). Methods: [Ca²⁺]_i was measured by fura-2/AM, and MAP kinase activation was analyzed by Western blotting. Results: Exposure of VSMC to rHuEPO prior to stimulation with ET-1 enhanced both basal and ET-1-induced elevation of [Ca²⁺]_i in a dose-dependent manner in the presence of extracellular Ca²⁺. The synergistic effect was also retained in the absence of extracellular Ca²⁺ after exposure to rHuEPO. However, the effect was diminished in the presence of extracellular Ca²⁺ combined with the intracellular Ca²⁺ release inhibitor TMB-8, PKC inhibitor, or PKC depletion. Exposure to rHuEPO also had a synergistic effect on the activation of MAP kinase induced by ET-1; however, this effect was diminished in the presence of the Ca²⁺ chelator BAPTA-AM. Conclusion: The results suggest that rHuEPO has synergistic effects on ET-1-induced [Ca²⁺]_i mobilization, particularly on intracellular Ca²⁺ release, and MAP kinase activation in VSMC.

Purinoceptor-Mediated Calcium Mobilization and Cellular Proliferation in Cultured Bovine Corneal Endothelial Cells

In the present study, we investigated the effect of adenosine triphosphate (ATP) on cytosolic free calcium mobilization and mitogenic activity in cultured bovine corneal endothelial cells (BCEC). The [Ca2+]i was determined using a Ca2+ sensitive indicator, Fura-2/AM, and cell proliferation was evaluated by counting the cell number. ATP, its metabolites and analogs caused transient increase in [Ca2+]i in a concentration-dependent manner (10-7M-10-3M) and the potency of agonists was ordered as follows: 2-methylthio-ATP > uridine triphosphate > ATP > adenosine diphosphate. Adenosine monophosphate and adenosine did not affect [Ca2+]i. ATP (10-4M) also promoted the accumulation of inositol trisphosphate (IP3). The ATP-induced transient [Ca2+]i increase and IP3 accumulation were attenuated by pretreatment with a phospholipase C inhibitor, U-73122 (5μM), for 30 min. ATP (10-5 M) significantly enhanced the proliferation of BCEC. ATP-induced [Ca2+]i increase and cell proliferation were inhibited by a purinoceptor antagonist, suramin (10-4 M). Thus, the present study indicates that BCEC contain P2 purinoceptors that regulate their proliferation.

Silica increases cytosolic calcium and causes cell injury in renal cell lines.

The purpose of this study was to clarify the effect of silica-induced cytosolic free calcium mobilization and cell injury in immortalized cell lines from transgenic mice kidney harboring SV40 T-antigen gene. The proximal convoluted tubule (S1)- and the inner medullary collecting tubule (IMCT)-originated cell lines were used. Cytosolic free calcium concentration ([Ca2+]i) was measured employing Fura-2 fluorescence and cell injury was evaluated by a vital dye exclusion procedure. Silica increased [Ca2+]i in a concentration-dependent manner in S1 (60 micrograms/ml-600 micrograms/ml) and IMCT (6 micrograms/ml-600 micrograms/ml). Silica caused a biphasic increase in [Ca2+]i which was composed of an initial rapid rise and following sustained phase. Ca2+ removal from the medium resulted in abolishment of initial and sustained phase of silica (600 micrograms/ml)-induced [Ca2+]i in both cell lines. Silica-induced cell injury was increased in a dose-dependent manner. This silica-induced cell injury was attenuated by the pretreatment with EGTA (100 microM) and nifedipine (1 microM). Cellular ATP content ([ATP]i) by silica also decreased in a concentration-dependent manner. The relationship between [Ca2+]i and [ATP]i showed that [ATP]i depletion caused [Ca2+]i to rise. This study suggests that 1) the elevation of [Ca2+]i caused by silica is due mainly to influx through plasma membrane Ca2+ channel and non specific membrane damage (at high concentration) and 2) nephrotoxicity of silica shows site-specificity within the kidney.

Improved cytosolic free calcium mobilization and superoxide production in bicarbonate-based peritoneal dialysis solution.

Intraperitoneal phagocytes play an important role in local host defence to prevent CAPD peritonitis. The intracellular calcium [Ca2+]i is thought to be involved in the regulation of various cell functions. This study therefore investigates the effect of lactate-based dialysis solution (LBDS) and bicarbonate-based dialysis solution (BBDS) on cytosolic free calcium mobilization and superoxide production (SP) as important steps in signal transduction and bacterial killing. We studied changes in [Ca2+]i and SP following stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) in polymorphonuclear neutrophils (PMNs) incubated in either LBDS-pH 5.2, LBDS adjusted to pH 7.4, 1:10 diluted spent and fresh LBDS or BBDS-pH 7.4 with different glucose concentrations, comparing the data with cells treated with Hanks buffer (HBSS) pH 7.4 as control. To elucidate the effect of glucose and lactate PMNs were additionally incubated in HBSS-pH 7.4, containing glucose (HBSS-Glu-pH 7.4) or lactate (HBSS-Lact-pH 7.4) in the same concentrations as contained in CAPD solutions and tested as above. PMNs were isolated from healthy blood donors and incubated with dialysis solution 10 min prior to stimulation with fMLP. [Ca2+]i mobilization and SP were completely inhibited in PMNs incubated in LBDS pH 5.2. pH adjustment of LBDS to 7.4 and 1:10 dilution of spent and fresh LBDS corrected some of the suppression of the calcium influx and superoxide production. BBDS pH 7.4, however, preserved physiological cell function significantly better at low (1.5 and 2.3%) glucose concentrations. In comparison to conventional lactate-based dialysis solution, pH adjusted and 1:10 diluted LBDS, bicarbonate-based dialysis solution is more biocompatible since it preserves significantly better neutrophil cell functions.

Regulation of monocyte integrin expression by beta-family chemokines.

In the present study we investigated the ability of three monocyte chemokines (MCP-1, MIP-1 alpha, and RANTES) to modulate monocyte adhesion molecules in an attempt to evaluate their potential to induce tissue infiltration of macrophages in vivo. All three chemokines tested induced increased expression of the alpha-chains of two members of beta 2 family of integrins, CD11b and CD11c, and their common beta-chain (CD18). They had no effect on CD11a expression. Enhancement of CD11b and CD11c was dose dependent and followed a distinct time course with peak levels at 4 h. Levels declined to reach basal levels by 24 h. In contrast, IL-1 induced enhancement remained high after 24 h of stimulation. However, the increases caused by chemokines were not mediated by IL-1 as indicated by lack of inhibition by the IL-1R antagonist. Studies on the mechanism of integrin up-regulation showed that mobilization of cytosolic free calcium is an important signaling event in this response and that up-regulation is associated with mobilization from intracellular pools mediated by microtubules. Enhanced CD11b and CD11c expression by chemokines was also found to result in enhancement of monocyte binding to endothelial cells. Further studies indicated that monocyte binding to endothelial cells follows similar dose-response kinetics as the up-regulation of integrins and can be partially blocked by Abs to CD11b and CD11c. These results suggest that modulation of the integrin expression by chemokines may facilitate the tissue trafficking of monocytes during inflammation.

Enhancement of intracellular sodium by vasopressin in spontaneously hypertensive rats.

The arginine vasopressin-induced increase in intracellular sodium concentration was augmented in cultured rat vascular smooth muscle cells derived from 12-week-old spontaneously hypertensive rats (SHR) compared with those from 12-week-old normotensive Wistar-Kyoto (WKY) rats. This difference was enhanced by treatment with a Na+,K(+)-ATPase inhibitor, ouabain. The calcium-free state did not affect the basal intracellular sodium concentration but completely blocked the arginine vasopressin-induced increase in intracellular sodium concentration in both cell groups. The arginine vasopressin-mobilized cytosolic free calcium was enhanced in SHR compared with WKY rats. This enhancement was diminished but not completely inhibited in the calcium-free state. Also, arginine vasopressin-produced intracellular alkalinization was augmented in SHR. Pretreatment of both cell groups with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, completely blocked arginine vasopressin-induced intracellular alkalinization and increased intracellular sodium concentration. Scatchard analysis showed that the V1 receptor number of either quiescent or proliferative cells of SHR was five to seven times greater than that of WKY rats, without any change in receptor affinity. These findings therefore indicate that the arginine vasopressin-induced increase in intracellular sodium concentration is augmented in vascular smooth muscle cells of SHR mediated through the enhancement of the mobilization of cytosolic free calcium and the activity of sodium-hydrogen exchange, which depends on an increase in V1 receptor number.

Cocaine suppresses proliferation of phytohemagglutinin-activated human peripheral blood T-cells.

Cocaine has been reported to modulate the immune system of experimental animals. Also, we observed that the drug suppresses the phytohemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells (PBMCs) but at concentrations extremely high relative to those seen in the blood of drug abusers. This suggested that cocaine had a relatively weak effect when tested under conditions of optimum proliferation. We, therefore, decided to examine the effect of lower concentrations of cocaine on suboptimum PHA-induced proliferation. At 0.09-12 microM, cocaine had no effect on the proliferation of PBMC in response to 0.2 micrograms/ml PHA. However, depleting the population of B-cells and monocytes resulted in a drug-induced suppression of the residual T-cells. Maximum suppression by cocaine was observed at 3 microM with both higher and lower concentrations of the drug causing less suppression. Suppression of proliferation was not influenced by either the age or sex of the peripheral blood lymphocytes (PBL) donors. The suppression of T-cell-enriched PBLs induced by cocaine could be eliminated by increasing the dose of the mitogen but was enhanced by preincubating the cells with the drug for 24 h. Also, the drug suppressed cytosolic free calcium mobilization in Fura 2/AM loaded enriched T-cell populations. These results show that cocaine moderately but consistently suppresses PHA-induced proliferation of T-cells from random blood donors at drug concentrations observed in drug abusers. The suppression is only evident under conditions of suboptimum proliferation and is accompanied by a corresponding decrease in the mobilization of cytosolic free calcium. It is suggested that the weak suppressive effect of cocaine is reversed by either helper factors produced by accessory cells or increasing concentrations of mitogen. These factors when present possibly overcome a drug-induced reduction in calcium mobilization and lymphocyte activation.

Calcium Distribution and Mobilization in Single Rat Parotid Acinar Cells Investigated by Digital Imaging Microscopy

The intracellular distribution and mobilization of cytosolic free calcium in single rat parotid acinar cells was analyzed by a digital imaging microscope equipped with a microspectrofluorometer, using calcium-sensitive dye fura-2. In the resting state, intracellular distribution of cytosolic free calcium concentration ([Ca2+]i) was heterogeneous: [Ca2+]i in the nuclear and perinuclear region was usually higher than that in the cytoplasm. By Ca(2+)-ionophore ionomycin and muscarinic agonist carbachol stimulation in the presence of 1 mM extracellular Ca2+, [Ca2+]i increased markedly and the gradient of [Ca2+]i between the nuclear region and the cytoplasm decreased. In ionomycin stimulation, [Ca2+]i increased homogeneously and this homogeneous increase was irreversible. In carbachol stimulation the gradient of [Ca2+]i between the nuclear region and the cytoplasm obviously reappeared within 2 min. By carbachol stimulation in the absence of extracellular Ca2+ (added 1 mM EGTA), [Ca2+]i returned to the prestimulation level after the initial transient increase. The distribution of [Ca2+]i also returned approximately to the prestimulation state. The gradient of [Ca2+]i between the nuclear region and the cytoplasm did not disappear even when [Ca2+]i elevated at the peak value.

Mitogenic response of near‐diploid mouse cell line m5S/1M induced by epidermal growth factor

A nonmalignant near-diploid cell line m5s/1M, established by Sasaki and Kodama (J. Cell. Res., 131:114-122, 1987), was shown to respond to the epidermal growth factor (EGF). The m5s/1M cells showed high sensitivity to post-confluence inhibition of cell division and formed a uniform monolayer after the cells had become confluent. The addition of EGF resulted in loss of contact-dependent inhibition of growth and caused a massive piling up of a multilayered array of cells after they had become confluent. When EGF was removed from the medium, the cell number decreased rapidly, and the cells formed a uniform monolayer at the density observed in the absence of EGF. m5S/1M cells have high- and low-affinity receptors for EGF (approximately 40,000 receptors per cell), and the apparent dissociation constants of the EGF-binding reactions were 3.3 nM and 0.15 nM, respectively. The effect of EGF on the intracellular mobilization of Ca2+ and the formation of inositol phosphates was studied by using the calcium-sensitive fluorescent indicator fura 2 and [3H]inositol. EGF had no effect either on the mobilization of cytosolic free calcium [( Ca2+]i) or on the formation of inositol phosphates in m5s/1M cells, whereas bradykinin induced a rapid increase in both [Ca2+]i and inositol phosphates. Analysis of the glycosphingolipid (GSL) composition of m5S/1M cells showed that globotriaosylceramide (Gb3Cer), which is known to be a Burkitt lymphoma-associated antigen, is specifically expressed in the EGF-treated cells. The expression of Gb3Cer is dependent on the presence of EGF, with a reversible shift in GSL composition being observed in the presence or absence of EGF.

Effects of Prostaglandin F2 on the Mobilization of Cytosolic Free Calcium in Vascular Smooth Muscle Cells and on the Tension of Aortic Strips from Rats

The possible mechanism of action of prostaglandin F2 alpha on vascular smooth muscle was investigated. Prostaglandin F2 alpha induced sustained tonic vasoconstriction of rat aortic strips, accompanied by rapid increase in the cytosolic free Ca2+ concentration [( Ca2+]i) in the vascular smooth muscle cells. Prostaglandin F2 alpha also evoked rapid increase in production of inositol 1,4,5,-trisphosphate (Ins-P3). Moreover, it increased release of Ca2+ from nonmitochondrial pools in digitonin-permeabilized vascular smooth muscle cells, although this effect was much weaker than that of Ins-P3. These results suggest that the sustained action of prostaglandin F2 alpha may involve not only a rise in [Ca2+]i mediated by Ins-P3, but also a direct stimulation of Ca2+ from intracellular stores.