Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Ceramide increase cytoplasmic Ca2+ concentration in Jurkat T cells by liberation of calcium from intracellular stores and activation of a store-operated calcium channel

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.     

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

Involvement of STIM1 and Orai1 in EGF-mediated cell growth in retinal pigment epithelial cells

Background

In non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca²⁺ store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line).

Results

EGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation.

Conclusions

Our results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders.     

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells

Background and aims

Steroid hormones target K⁺ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3.

Results

Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K⁺ conductance, which is abolished by clotrimazole (30 μM), an inhibitor of the intermediate conductance calcium activated K⁺ channel (IK_Ca). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K⁺ conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IK_Ca channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IK_Ca channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation.

Conclusions

Estradiol rapidly regulates a K⁺ conductance through the IK_Ca channel in an estrogen receptor independent manner. E2 stimulates the translocation of IK_Ca to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.     

Selenium potentiates the anticancer effect of cisplatin against oxidative stress and calcium ion signaling-induced intracellular toxicity in MCF-7 breast cancer cells: involvement of the TRPV1 channel

Background: In breast cancers, calcium signaling is a main cause of proliferation and apoptosis of breast cancer cells. Although previous studies have implicated the transient receptor potential vanilloid 1 (TRPV1) cation channel, the synergistic inhibition effects of selenium (Se) and cisplatin in cancer and the suppression of ongoing apoptosis have not yet been investigated in MCF-7 breast cancer cells. This study investigates the anticancer properties of Se through TRPV1 channel activity in MCF-7 breast cancer cell line cultures when given alone or in combination with cisplatin. Materials: The MCF-7 cells were divided into four groups: the control group, the Se-treated group (200?nM), the cisplatin-treated group (40?μM) and the Se?+?cisplatin-treated group. Results: The intracellular free calcium ion concentration and current densities increased with TRPV1 channel activator capsaicin (0.01?mM), but they decreased with the TRPV1 blocker capsazepine (0.1?mM), Se, cisplatin, and Se?+?cisplatin incubations. However, mitochondrial membrane depolarization, apoptosis, and the caspase 3, and caspase 9 values increased in the Se-treated group and the cisplatin-treated group, although Western blot (procaspase 3 and 9) results and the cell viability levels decreased with the Se and Se?+?cisplatin treatments. Apoptosis and caspase-3 were further increased with the Se?+?cisplatin treatment. Intracellular reactive oxygen species production increased with the cisplatin treatment, but not with the Se treatment. Conclusion: This study’s results report, for the first time, that at a cellular level, Se and cisplatin interact on the same intracellular toxic cascade, and the combination of these two drugs can result in a remarkable anticancer effect through modulation of the TRPV1.     

Ceramide-1-P induces Ca2+ mobilization in Jurkat T-cells by elevation of Ins(1,4,5)-P3 and activation of a store-operated calcium channel

Sphingolipids comprise a very important class of second messengers involved in cell growth, differentiation, and apoptosis, among other different functions. Recently, these lipids have been implicated in calcium mobilization in different cell lines, including Jurkat T-lymphocytes. However, the effect of each particular sphingolipid appears to be cell-line specific. Among them, the least studied is ceramide-1-P (Cer-1-P). Here, we show that Cer-1-P increased the intracellular Ca(2+) concentration in Jurkat T-cells. Furthermore, laser-scanning confocal microscopy indicated that Ca(2+) is released from the endoplasmic reticulum. An effect on store-operated Ca(2+) channels was evidenced by whole-cell "patch clamp" measurements after Cer-1-P induced Ca(2+) store depletion. The mechanism of action of Cer-1-P resembles that of the Jurkat anti-TCR antibody, but differs from that of ceramide, since Cer-1-P induced an increase in Ins(1,4,5)-P(3).     

Lysophosphatidic acid-LPA1 receptor-Rho-Rho kinase-induced up-regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.