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Regulation of noncapacitative calcium entry by arachidonic acid and nitric oxide in endothelial cells

Department of Animal and Human Biology, University of Torino, Italy, and Nanostructured Interfaces and Surfaces Centre of Excellence (NIS)

¹Correspondence: Department of Animal and Human Biology, University of Torino, Via Accademia Albertina 13, 10123 Torino, Italy. E-mail: luca.munaron{at}unito.it

SPECIFIC AIMS

Several peptides, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), activate the release of arachidonic acid (AA) and nitric oxide (NO) in endothelial cells (ECs). Both messengers are involved in EC activation, proliferation, and motility, key events in angiogenesis and inflammatory processes, and control calcium homeostasis in different ways. It was recently suggested that NO acts as a downstream mediator of AA-induced calcium entry in smooth muscle cells and isolated mouse parotid cells.

We investigated the relationships among intracellular calcium, AA and NO in bovine aortic endothelial cells (BAEC). Using mainly simultaneous Ca²⁺ and NO fluorimetric confocal imaging, we provide evidence for a complex pathway leading to noncapacitative calcium entry (NCCE). AA is able to induce NCCE through two different pathways: one dependent on eNOS recruitment and NO release, the other NO-independent.

PRINCIPAL FINDINGS

Ca²⁺ and NO elevation activated by AA stimulation
NO has been suggested to mediate AA-induced NCCE in smooth muscle cells and isolated mouse parotid cells. To verify this hypothesis in our cells, intracellular Ca²⁺ and NO levels were measured simultaneously in BAECs stimulated with 5 µM AA after incorporation of Calcium Green 1 AM and DAR-4M AM, a cell-permeant fluorescent probe sensitive to NO and insensitive to pH.

Application of 5 µM AA induced a prolonged and simultaneous elevation of Ca²⁺ and NO ((F/F₀)_Ca=0.35±0.15, (F/F₀)_NO=0.67±0.22, 70 cells; Fig. 1 ).

View larger version (10K): [in this window] [in a new window]   Figure 1. Arachidonic acid activates simultaneous [Ca]i and NO increase. A representative experiment showing the increase of [NO]i and [Ca]i induced by 5 µM AA in a single cell.

To clarify the causal relationship connecting NO and Ca²⁺ signals, the same experiment was performed in a calcium-free extracellular solution (0 Ca-3 mM EGTA) to abolish calcium influx, the only pathway of calcium increase in these cells induced by AA. Under these conditions, AA was still able to promote NO increment, unmasking a calcium-independent pathway for NO release ((F/F₀)_NO=0.65±0.13, 49 cells).

The ability of AA to induce calcium increase in cells pretreated with thapsigargin (1 µM for 10 min), an inhibitor of SERCA that completely and irreversibly depletes intracellular calcium stores, confirmed that the response was entirely due to noncapacitative calcium entry ((F/F₀)_Ca=0.66±0.30, 32 cells).

To verify a potential direct effect of AA on eNOS, we used ETYA (5,8,11,14-eicosatetraynoic acid), an acetylenic AA analog that blocks all AA metabolizing enzymes by acting as a false substrate. Application of 5 µM ETYA triggered NO increase in both the presence and absence of calcium in the extracellular medium (respectively (F/F₀)_NO=0.69±0.22 and 0.78±0.14, 28 and 20 cells).

cGMP assay was performed to confirm this evidence. Stimulation with either 5 µM AA, 5 µM ETYA, 10 µM SNAP, or 2 mM L-Arg significantly increased cGMP levels (n=4); consistent with the existence of a mechanism of NO release induced by AA in a calcium-independent way, cGMP levels were augmented when AA was added to a medium containing 3 mM EGTA to prevent calcium influx.

These data suggest the ability of AA to activate NO release in a direct way even if an additional effect of AA metabolites on eNOS cannot be excluded.

Bovine aortic endothelial cells express eNOS, as demonstrated by RT-PCR experiments (n=3).

Calcium signals activated by NO in single BAECs
Recent data from different cell types, including ECs, suggest that NO is able to induce calcium signals, but NO-dependent calcium entry inhibition has also been described. For these reasons we analyzed at the single cell level the effects of NO on calcium signaling in BAECs. They were assessed by the use of either L-Arg (a widely used eNOS activator that enters the cell via a specific membrane carrier) or NO donors (NOC 18 and SNAP).

Application of 2 mM L-Arg induced increases of [Ca]_i and [NO]_i ((F/F₀)_Ca=0.31±0.07, (F/F₀)_NO=0.67±0.25, 55 cells); the latter was reversible and strictly dependent on L-Arg presence (34 cells). Moreover it was completely due to calcium entry, as suggested by its complete inhibition in the presence of 3 mM external EGTA; subsequent calcium addition to the external bath completely restored the response to L-Arg (4 cells).

Ratiometric calcium measurements using the indo-1 probe were performed to quantitatively test CCE and NCCE contribution to the response to L-Arg. L-Arg (2 mM) was still able to induce calcium increase in cells pretreated with thapsigargin (TG, 1 µM for 10 min), confirming, as in the case of AA, that the response was entirely due NCCE (R(400/480)=0.15±0.1 for controls, 9 cells, and 0.17±0.05 with TG, 8 cells).

To exclude the possibility that NO-induced calcium entry was due to an effect on cPLA2 and indirectly to AA, cells were stimulated with L-Arg in the presence of a widely used cPLA2 inhibitor, AACOCF3 (preincubation with 5 µM AACOCF3 for 10 min): calcium entry could still be observed (18 cells).

Stimulation with either 10 µM SNAP or 1 mM NOC18, two independent NO donors, triggered calcium increases very similar to those described for 2 mM L-Arg application ((F/F₀)_Ca=0.38±0.11 and 0.29±0.04; respectively 28 and 24 cells).

Electrophysiological measurements performed in whole cell configuration, voltage clamp mode, confirmed the ability of SNAP to activate a cationic current with a V_rev near 0 mV (n=4). Cl^– involvement could be excluded because V_h= –50 mV corresponds to V_Cl in the ionic conditions used.

The next set of experiments was performed to investigate the possible involvement of guanylyl cyclase and cGMP as mediators of NO-induced calcium entry. We tested the effects of 8-Br-cGMP, a membrane-permeable cGMP analog, and ODQ, a widely used guanylyl cyclase inhibitor.

Direct application of either 1 mM 8-Br-cGMP or 10 µM ODQ on resting cells failed to activate any calcium signal; subsequent perfusion with 2 mM L-Arg induced a calcium entry indistinguishable from control experiments (respectively 70 and 12 cells).

In cell pretreated with 10 µM ODQ for 10 h, stimulation with 2 mM L-Arg or 10 µM SNAP was still able to increase calcium levels (respectively (F/F₀)_Ca=0.41±0.18 and 0.31±0.12, 21 and 9 cells).

Finally, acute application of 1 mM 8-Br-cGMP or 10 µM ODQ on the calcium increase induced by 2 mM L-Arg exerted no detectable effect (respectively 6 and 8 cells; Fig. 2 ).

View larger version (9K): [in this window] [in a new window]   Figure 2. Direct application of 10 µM ODQ on the calcium entry activated by 2 mM L-Arg failed to exert any effect.

AA induces calcium signals independently from NO
To evaluate whether AA-induced calcium signals were totally dependent on NO release, BAECs were pretreated with L-NAME (5 mM for 30 min) or L-NMMA (1 mM for 30 min), two unrelated and widely used eNOS inhibitors. Under these conditions, 5 µM AA was still able to increase calcium levels, showing the existence of a NO-independent calcium entry component (respectively (F/F₀)_Ca=0.36±0.19 and (F/F₀)_Ca=0.33±0.08; respectively 36 and 20 cells).

Ratiometric calcium measurements using indo-1 probe were performed in cells to quantitatively confirm this evidence: the amplitude of the calcium increase, expressed as R(400/480), in control cells stimulated with 5 µM AA was 0.33±0.25 (8 cells); in cells pretreated with L-NMMA, 0.35±0.24 (8 cells).

NO and cell proliferation
BAECs were plated in DMEM+10%FCS for 18 h, then stimulated in DMEM+1%FCS for 24 h, with or without (control) different agonists able to induce an increase of NO levels, such as L-Arg (2 mM) or the NO donors sp/NO and SNAP, at different concentrations.

Stimulation with either L-Arg or low doses of NO donors induced a significant increase in cell number. Considering the experiments (n=10) as a whole and expressing the data as percentages of growth referred to the control condition (cells in DMEM+1%FCS, 100% of growth), values obtained under different conditions were the following: 2 mM L-Arg, 141.2±20.8%; 1 µM sp/NO, 145.3±17.1%; 1 µM SNAP, 159.0±29.4%; 10 µM SNAP, 152.2±26.3%.

Higher doses of sp/NO (5, 10, 50 µM) caused a significant loss of cells, accordingly with the proapoptotic effects described by others; high doses of SNAP (50 µM) inhibited proliferation.

CONCLUSIONS AND SIGNIFICANCE

Recent papers support the idea that NO mediates AA-induced NCCE in smooth muscle cells and isolated mouse parotid cells.

ECs represent an interesting model to test the general validity of this hypothesis for several reasons: AA and NO are two critical intracellular messengers in these cells, and both are involved in the control of EC activation, proliferation and motility, key events in angiogenesis and inflammatory processes. Their ability to control calcium homeostasis in different cell types, including ECs, has been well documented by several authors.

The major aims of this work were to study calcium signals activated by AA and NO in BAECs at the single cell level and to clarify the functional relationships among the related signaling pathways. The conclusions are summarized in Fig. 3 .

View larger version (11K): [in this window] [in a new window]   Figure 3. Schematic picture showing the intracellular pathways leading to NCCE in BAECs.

In BAECs the release of AA at low concentrations gives rise to a complex intracellular signaling network, including two distinct pathways for NCCE activation: one "shorter" and "directly" mediated by AA itself and another "longer" and mediated by NOS/NO recruitment (Fig. 3) . Due to the high heterogeneity of EC types, these two modes could act in parallel in the same cell or alternatively in physiological or pathological conditions: they may play different and specific roles in each endothelial cell type depending on the tissue-dependent expression of intracellular factors (including NOS types and calcium channels).

Some relevant points remain to be clarified. The molecular identity of calcium channels modulated by AA and/or NO is still elusive, even if some members of TRPC channels family could be involved. The development of specific blockers, potentially available also in the pharmacological intervention of the vascular pathologies in which AA and NO are involved, could help to address this topic.

Another aim should be to investigate in more detail the molecular mechanisms responsible for modulation of calcium channels by AA and NO: they could be direct or mediated by downstream intracellular factors.

Finally, a detailed analysis of elementary (local or spatially restricted) calcium and NO intracellular events will unmask spatiotemporal patterns potentially responsible for the specificity of the response.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4110fje;

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