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Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation
Nitric Oxide 13 (2005) 145–151 www.elsevier.com/locate/yniox
Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation Renata S.A. Costa, Jamil Assreuy ¤ Department of Pharmacology, UFSC, Campus Universitário, Trindade, Bloco D/CCB, P.O. Box 476, Florianópolis, SC 88049-900, Brazil Received 8 April 2005; revised 21 May 2005 Available online 1 July 2005
Abstract Several nitric oxide (NO) eVects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important eVectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-D,L-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1–1000 M) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 M) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 M) prevented NO eVects, conWrming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 M), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 M) prevented SNAP inhibitory eVects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 M) was ineVective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative eVect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1–100 M), inhibited cell proliferation. In addition, sub-eVective concentrations of ODQ (100 nM) and TEA (10 M) synergized in blocking SNAP antiproliferative eVects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation. 2005 Elsevier Inc. All rights reserved. Keywords: Guanylate cyclase; Nitric oxide; Potassium channel; Proliferation; Smooth muscle cell
Excessive vascular smooth muscle cell (VSMC) proliferation is considered to be a hallmark of artherosclerosis [1]. In the cardiovascular system, nitric oxide (NO) formed by the endothelium diVuses across VSMC membranes and plays an important role in the vasculature, including regulation of contractile activity, prevention of platelet and monocyte adhesion, and inhibition of cell proliferation [2,3]. Additionally, it has been shown that NO decreases VSMC proliferation in vitro and in vivo
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Corresponding author. Fax: +55 48 337 5479. E-mail address: [email protected] (J. Assreuy).
1089-8603/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.05.010
[4–7]. It has been accepted that soluble guanylate cyclase (sGC) activation with the consequent increase in cyclic guanosine-3⬘-5⬘-monophosphate (cGMP) levels is the principal intracellular event for NO inhibitory eVect [4,5]. In contrast, the events that occur downstream from cGMP formation are still much debated. It has been well known that cGMP activates cGMP-dependent protein kinase (PKG) in smooth muscle cells and the activation, in turn, elicits inhibition of VSMC proliferation [8]. Otherwise, the NO antiproliferative eVect on VSMC can be independent of cGMP [9] or at least independent of PKG activation [10]. Potassium channels are a diverse and ubiquitous family of membrane proteins
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transducing external signals across the cell membrane to the cell interior. More recently, potassium channel activation has emerged as an important mediator of NO eVects in smooth muscle cell contractility and vascular tone [11]. However, little is known concerning the role of potassium channels in NO inhibitory eVect on VSMC proliferation. Thus, in this report, we investigated the role of the potassium channels on VSMC proliferation and its relation to NO/cGMP/PKG pathway, using the rat aorta vascular smooth muscle cell line A7r5.
a humidiWed incubator at 37 °C with a 5% CO2 atmosphere. Every 4–5 days, cultures were conXuent and were passaged using 0.25% trypsin/0.03% EDTA and a split ratio of 1:4. For experimental protocols, conXuent A7r5 cells were harvested as mentioned above, resuspended in culture medium, and cell viability was determined by trypan blue exclusion. A total of 3.5 £ 103 viable cells/well were seeded in 200 l medium in 96-well plates. After 24 h, test compounds were added in a maximum volume of 20 l. Cell counting
Experimental procedures Drugs Tissue culture media, serum, and antibiotics were from Gibco (São Paulo, SP, Brazil). S-Nitroso-N-acetylD,L-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) were synthesized in our laboratory according to published methods [12,13]. Glyceryl trinitrate (GTN; 1,2,3-propanotriol trinitrate) was kindly donated by Cristália Laboratories (São Paulo, SP, Brazil). 8-Bromoguanosine-3⬘-5⬘-cyclic monophosphate (8-Br-cGMP; a cell-permeable analogue of cGMP), ODQ (oxadiazoloquinoxalinone; a selective inhibitor of the soluble guanylate cyclase), KT5823 (a selective inhibitor of cGMPdependent protein kinase), NS1619 (an activator of high conductance calcium-activated potassium channels), TEA (tetraethylammonium; a non-selective blocker of potassium channels), 4-AP (4-aminopyridine; a selective blocker of voltage-dependent potassium channels), and glibenclamide (a selective blocker of ATP-dependent potassium channels) were purchased from Sigma Chemical (St. Louis, MO, USA). Charybdotoxin (a blocker of the high and intermediate conductance calcium-activated potassium channels), iberiotoxin (a selective blocker of high conductance calcium-activated potassium channels), and apamin (a selective blocker of the small conductance calcium-activated potassium channels) were purchased from Alomone Laboratories (Jerusalem, Israel). All solutions were prepared in culture medium except glibenclamide and KT5823 that were dissolved in DMSO, GTN that was dissolved in 1 mM HCl, and toxins, which were dissolved in PBS containing 1 mg/ml protease-free bovine serum albumin. Cell culture The rat aortic vascular smooth muscle cell line A7r5 was obtained from Rio de Janeiro Cell Bank (UFRJ, RJ, Brazil) and was used at passages 2–15. Cells were grown and passaged in plastic culture Xasks in Dulbecco’s modiWed Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, 10 mM Hepes, pH 7.4 (referred to as DMEM), in
At the end of the experimental periods, each well was washed twice with Ca2+/Mg2+-free PBS. Cells were then detached using 100 l of 0.25% trypsin/0.03% EDTA solution, incubated at 37 °C for 30 min. Trypsin was then inactivated with 10 l of fetal calf serum, cells were quantiWed in Neubauer chambers, and the results are expressed as cell number/mm3. Analysis of data Data are expressed as means § SD of triplicate wells. Each experiment was repeated at least twice with similar results. Statistical signiWcance was determined by oneway analysis of variance (ANOVA) followed by Bonferroni’s post hoc t test. A P value of less than 0.05 was considered signiWcant.
Results NO donors and cell proliferation After seeding 3.5 £ 103 cells in each well of a 96-well plate, A7r5 cell line reached conXuence after 96 h (around 60 £ 103 § 6 cells/mm3, n D 10). After this time, cell number remained constant for 1–2 days when cells started to die and detach from the substrate. Thus, the 96 h time point was chosen as the end-point to evaluate cell proliferation. The NO donor SNAP (Fig. 1A) caused a concentration-dependent inhibition of A7r5 cell proliferation with an IC50 value of around 90 M (calculated using GraphPad Prism 3 software). A similar eVect was seen with GTN or GSNO, with IC50 values of »70 and 540 M, respectively. In addition, neither NAP nor GSH (the non-nitrosylated parent compounds of NO donors) had eVect on cell proliferation. Besides inhibiting VSMC proliferation [4], NO can induce apoptosis [14]. To conWrm that NO inhibitory eVect was not due to its toxicity to cells, Trypan blue exclusion tests were carried out in cells treated with large range of SNAP concentrations. Cell death became apparent when SNAP 300 M or higher was employed (53 § 18% viability, n D 3). Therefore, the maximal SNAP concentration used throughout
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age of their antiproliferative eVects by KT5823 (vehicle 47 § 5; SNAP 100 M 20 § 0.3; SNAP + KT5823 1 M 49 § 1 £ 103 cells/mm3, n D 3; 8-Br-cGMP 100 M 18 § 1; 8-Br-cGMP + KT5823 38 § 5 £ 103 cells/mm3, n D 3). KT5823 did not aVect cell proliferation by itself (51 § 9). Potassium channel blockers and NO antiproliferative eVect TEA (a non-selective blocker of potassium channels; Fig. 2A) and 4-AP (a blocker of voltage-dependent potassium channels; Fig. 2B) completely blocked the antiproliferative eVects of the lower concentration of
Fig. 1. EVects of NO donors on the proliferation of A7r5 rat vascular smooth muscle cell line and the dependence on soluble guanylate cyclase. Cells were seeded (3.5 £ 103 cells/well) and after 24 h, compounds were added to cultures. (A) 1–100 M for SNAP, GTN, and NAP and 1–1000 M for GSNO and GSH. (B) 1–100 M for 8-BrcGMP and SNAP and 1 M for ODQ. Ninety-six hours after seeding, cell number was obtained by counting, as detailed in Experimental procedures. Each point is the means § SD of triplicate wells. Similar results were obtained in at least two independent experiments. * P < 0.05 when compared to the control group (A) (no compound) or the correspondent SNAP or 8-Br-cGMP groups (B) ANOVA followed by Bonferroni’s post hoc t test).
this report was 100 M, which always yielded cell viability greater than 95%. NO/cGMP/PKG pathway and inhibition of A7r5 cell proliferation As shown in Fig. 1B, 8-Br-cGMP (a cell-permeable analogue of cGMP) inhibited A7r5 cell proliferation in a dose-dependent fashion with an IC50 value of »70 M, yielding a dose–response curve very similar to that of SNAP. Pretreatment of cells with ODQ prevented the onset of SNAP antiproliferative eVect. Cell proliferation was unchanged by ODQ alone (39 § 7 vs 43 § 5 £ 103 cells/mm3, vehicle and ODQ, respectively, n D 3). However, SNAP and 8-Br-cGMP eVects were mediated through PKG as demonstrated by the block-
Fig. 2. EVects of potassium channel blockers on NO-induced inhibition of A7r5 rat vascular smooth muscle cell line proliferation. Cells (3.5 £ 103 cells/well) were seeded and after 24 h, compounds (SNAP, 1–100 M; tetraethylammonium, TEA 300 M; 4-aminopyridine, 4AP 100 M; glibenclamide, GBN 1 M; iberiotoxin, IbTx 100 nM; apamin 100 nM and charybdotoxin, ChTx 100 nM) were added. Vehicle was DMSO 0.05% for glibenclamide, saline with 1 mg/ml BSA for toxins or saline for the other compounds. Ninety-six hours after seeding, the cell number was obtained by counting, as detailed in Experimental procedures. Bars are means § SD of triplicate wells. Similar results were obtained in two independent experiments. *P < 0.05 compared to control (no SNAP) and #P < 0.05, compared to the equivalent SNAP group (ANOVA followed by Bonferroni’s post hoc t test).
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SNAP (10 M). When the NO donor was used at concentrations of 100 M, the inhibitory eVect of TEA and 4-AP became partial. On the other hand, NO donor eVect was unaVected by glibenclamide (a blocker of ATP-dependent potassium channels; Fig. 2C). Iberiotoxin (Fig. 2D) blocked SNAP eVects at lower concentrations of the NO donor and caused a partial inhibition at the higher concentrations of the NO donor. On the other hand, apamin (Fig. 2E) and charybdotoxin (Fig. 2F) blocked the antiproliferative eVect induced by 10 M SNAP, but failed to aVect the antiproliferative eVect induced by higher SNAP concentrations (100 M). None of the potassium channel blockers caused cytotoxicity or aVected cell proliferation by themselves (Fig. 2), at least within the range of concentrations employed. Potassium channel blockers and 8-Br-cGMP-induced inhibition of A7r5 cell proliferation TEA and 4-AP completely reversed 8-Br-cGMP antiproliferative eVect as shown in Figs 3A and B, respectively. Iberiotoxin and apamin also eYciently blocked 8-Br-cGMP antiproliferative eVects, as shown in Figs. 3C and D, respectively.
EVect of simultaneous inhibition of guanylate cyclase and potassium channel on NO-induced inhibition of A7r5 cell proliferation The combination of sub-eVective concentrations of ODQ (100 nM) and TEA (10 M) completely prevented the onset of SNAP antiproliferative eVect (Fig. 4). Potassium channel opener- and NO-induced inhibition of A7r5 cell proliferation To conWrm whether calcium-activated potassium channels are involved in mediating the inhibitory eVects of NO, an activator of high conductance calcium-activated potassium channel, NS1619, was tested on cell proliferation. NS1619 antiproliferative eVect was concentration-dependent with an IC50 value of »70 M. For comparison, SNAP was present in matched experiments (Fig. 5).
Discussion Our results showed that NO donors inhibited the proliferation of the rat embryonic thoracic aorta smooth muscle cell line (A7r5). Besides inhibiting VSMC prolifer-
Fig. 3. EVects of potassium channel blockers on 8-Br-cGMP-induced inhibition of A7r5 rat vascular smooth muscle cell line proliferation. Cells (3.5 £ 103 cells/well) were seeded and after 24 h, compounds (8-Br-cGMP, 1–100 M; tetraethylammonium, TEA 300 M; 4-aminopyridine, 4-AP 100 M; iberiotoxin, IbTx 100 nM and apamin 100 nM) were added. Vehicle was saline with 1 mg/ml BSA for toxins or saline for the other compounds. Ninety-six hours after seeding, the cell number was obtained by counting, as detailed in Experimental procedures. Bars are means § SD of triplicate wells. Similar results were obtained in two independent experiments. *P < 0.05 compared to control (no 8-Br-cGMP; ANOVA followed by Bonferroni’s post hoc t test).
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Fig. 4. EVect of the simultaneous blockage of soluble guanylate cyclase and potassium channels on NO-induced inhibition of A7r5 cell line proliferation. Cells were seeded (3.5 £ 103 cells/well) and after 24 h, incubated with vehicle or with SNAP in the presence or absence of sub-eVective concentrations of ODQ (1 nM) or TEA (10 M), or with the two blockers together. Ninety-six hours after seeding, cell number was obtained by counting, as detailed in Experimental procedures. Bars are means § SD of triplicate wells. Similar results were obtained in at least two independent experiments. *P < 0.05 compared to control (no SNAP; ANOVA followed by Bonferroni’s post hoc t test).
Fig. 5. EVects of NS1619 and SNAP on A7r5 cell line proliferation. Cells (3.5 £ 103 cells/well) were seeded and after 24 h, compounds (1– 100 M for both NS1619 and SNAP) were added to diVerent cultures. Vehicle was DMSO 0.05% for NS1619 or saline for SNAP. Ninety-six hours after seeding, the cell number was obtained by counting, as detailed in Experimental procedures. Points are means § SD of triplicate wells. Similar results were obtained in two independent experiments. *P < 0.05 compared to control group (absence of added compound; ANOVA followed by Bonferroni’s post hoc t test).
ation, our results also showed that NO can induce cell death, although the latter eVect occurred only at concentrations higher than those inhibiting proliferation. The NO inhibitory eVect was concentration-dependent and seems to be attributable to NO only since NAP and GSH, the non-nitrosylated parent compounds of nitrosothiols SNAP, and GSNO, respectively, were devoid of any eVect. GSNO displayed essentially the same antiproliferative eVect as SNAP, although it was less potent, probably due to its slower kinetics of NO release [15]. In addition, GTN (an organic nitrate and not a nitrosothiol) used in the clinical setting as a vasodilator also inhibited VSMC prolifer-
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ation. Therefore, this NO eVect cannot be attributable to non-selective eVect of nitrosothiols. Several NO eVects are mediated through soluble guanylate cyclase activation, with the correspondent increase in cyclic guanosine-3⬘-5⬘-monophosphate (cGMP) levels. Cyclic GMP eVects, in turn, are mediated through cGMP-dependent protein kinase (PKG), cGMP-regulated phosphodiesterases [16] or cyclic nucleotide-gated cation channels [17]. The relevance of NO and cGMP pathway in inhibiting SMC proliferation, initially observed by Garg and Hassid [4], is well established today forming the basis of potential clinical applications [18]. The dependence on guanylate cyclase/ cGMP pathway in mediating NO antiproliferative eVects on A7r5 cell line was demonstrated by showing the inhibitory action of ODQ, a potent and selective inhibitor of soluble guanylate cyclase and of KT5823, a speciWc inhibitor of PKG. In addition, 8-Br-cGMP, a membrane-permeant analogue of cGMP, eYciently mimicked NO eVect and was also blocked by KT5823. Therefore, our data conWrm and extend the literature concerning NO antiproliferative eVect on smooth muscle cells and show that the A7r5 smooth muscle cell line seems to be identical to a primary vascular smooth muscle cell, at least as far as the guanylate cyclase/cGMP pathway is concerned. Potassium channels are essential to several cell functions, including proliferation [19,20], they are relevant targets for NO [21–25] and SMC proliferation is central to processes such as atherosclerosis [1]. These three lines of evidence provide the basis of the most important Wnding of this report, the relationship among NO, SMC proliferation, and potassium channels. This point has not been examined in the literature and we took advantage of a previous publication from our laboratory, showing that NO antiproliferative eVects on a tumor cell line could be inhibited by potassium channel blockers [26]. When examined together, our results are clearly indicative that a signiWcant portion of NO antiproliferative eVect on vascular smooth muscle cells is mediated through potassium channel activation. However, some sub-types seem to be more relevant than others in this regard. For instance, voltage-dependent, as well members of the calcium-dependent sub-families (notably IKCa and SKCa) channels are important for NO eVects. Although our arguments are based on the use of blockers of potassium channels, at least concerning the calcium-activated sub-types, the availability of an opener of these channels (NS1619) allowed us to directly show the inhibitory role of this potassium channel family on vascular smooth muscle cells proliferation. On the other hand, the ATP-dependent sub-type does not seem to be involved in the NO antiproliferative eVect in spite of its involvement in several other NO actions [27,28].
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As for the mechanism by which NO may be recruiting to activate potassium channels, some points are worth discussing. First, the results showing that the eVects of 8-Br-cGMP (which mimicked NO) could be blocked by potassium channel inhibitors are suggestive that cGMP (either as a product of NO-dependent guanylate cyclase activation or exogenously added) may be causing phosphorylation of potassium channels via protein kinase G. A similar Wnding is reported in the literature [24]. Second, the Wnding that sub-eVective concentrations of ODQ and TEA when used simultaneously blocked NO antiproliferative eVect suggests that at least part of potassium channel activation could be an event situated downstream to guanylate cyclase activation. Third, it is important to note that when higher (and more eVective in inhibiting cell proliferation) NO donor concentrations were employed, the inhibition caused by potassium channel blockers was not complete, as it was in the lower SNAP concentrations. This was especially true for the voltage-dependent and iberiotoxin-sensitive channels (BKCa). Taking into account that all potassium channel blockers completely prevented 8-Br-cGMP antiproliferative eVects, our results are suggestive that, at least for voltage- and high conductance calcium-dependent potassium channels, some other mechanism induced by NO may be operative. As discussed above, NO can nitrosylate cysteine sulfydryls [29,30] of several proteins, among them potassium channels [25]. Thus, it is temptating to speculate that S-nitrosylation may represent an alternative pathway for potassium channel modulation and inhibition of SMC proliferation, mainly when higher NO concentrations are concerned. The experiment showing that sub-eVective concentrations of ODQ + TEA abolished NO antiproliferative eVect would favor the suggestion that phosphorylation seems to be a more relevant mechanism for NO-dependent potassium channel activation. However, since we do not know the consequences of S-nitrosylation on potassium channel activation/phosphorylation, we cannot exclude the possibility that NO may be activating potassium channels via S-nitrosylation. In summary, we show that the mechanism by which NO inhibits VSMC proliferation involves cGMP and, for the Wrst time, that some sub-types of potassium channels seem to have a key role in this eVect.
Acknowledgments The skillful technical assistance of Mrs. Adriane Madeira is gratefully acknowledged. We also thank Dr. J.B. Calixto for allowing us the use of his tissue culture facilities. This work was supported by Conselho Nacional de Desenvolvimento CientíWco e Tecnológico (CNPq, Brazil) and by PRONEX/MCT/Brazil.
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Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation Renata S.A. Costa, Jamil Assreuy ¤ Department of Pharmacology, UFSC, Campus Universitário, Trindade, Bloco D/CCB, P.O. Box 476, Florianópolis, SC 88049-900, Brazil Received 8 April 2005; revised 21 May 2005 Available online 1 July 2005
Abstract Several nitric oxide (NO) eVects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important eVectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-D,L-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1–1000 M) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 M) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 M) prevented NO eVects, conWrming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 M), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 M) prevented SNAP inhibitory eVects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 M) was ineVective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative eVect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1–100 M), inhibited cell proliferation. In addition, sub-eVective concentrations of ODQ (100 nM) and TEA (10 M) synergized in blocking SNAP antiproliferative eVects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation. 2005 Elsevier Inc. All rights reserved. Keywords: Guanylate cyclase; Nitric oxide; Potassium channel; Proliferation; Smooth muscle cell
Excessive vascular smooth muscle cell (VSMC) proliferation is considered to be a hallmark of artherosclerosis [1]. In the cardiovascular system, nitric oxide (NO) formed by the endothelium diVuses across VSMC membranes and plays an important role in the vasculature, including regulation of contractile activity, prevention of platelet and monocyte adhesion, and inhibition of cell proliferation [2,3]. Additionally, it has been shown that NO decreases VSMC proliferation in vitro and in vivo
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Corresponding author. Fax: +55 48 337 5479. E-mail address: [email protected] (J. Assreuy).
1089-8603/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.05.010
[4–7]. It has been accepted that soluble guanylate cyclase (sGC) activation with the consequent increase in cyclic guanosine-3⬘-5⬘-monophosphate (cGMP) levels is the principal intracellular event for NO inhibitory eVect [4,5]. In contrast, the events that occur downstream from cGMP formation are still much debated. It has been well known that cGMP activates cGMP-dependent protein kinase (PKG) in smooth muscle cells and the activation, in turn, elicits inhibition of VSMC proliferation [8]. Otherwise, the NO antiproliferative eVect on VSMC can be independent of cGMP [9] or at least independent of PKG activation [10]. Potassium channels are a diverse and ubiquitous family of membrane proteins
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transducing external signals across the cell membrane to the cell interior. More recently, potassium channel activation has emerged as an important mediator of NO eVects in smooth muscle cell contractility and vascular tone [11]. However, little is known concerning the role of potassium channels in NO inhibitory eVect on VSMC proliferation. Thus, in this report, we investigated the role of the potassium channels on VSMC proliferation and its relation to NO/cGMP/PKG pathway, using the rat aorta vascular smooth muscle cell line A7r5.
a humidiWed incubator at 37 °C with a 5% CO2 atmosphere. Every 4–5 days, cultures were conXuent and were passaged using 0.25% trypsin/0.03% EDTA and a split ratio of 1:4. For experimental protocols, conXuent A7r5 cells were harvested as mentioned above, resuspended in culture medium, and cell viability was determined by trypan blue exclusion. A total of 3.5 £ 103 viable cells/well were seeded in 200 l medium in 96-well plates. After 24 h, test compounds were added in a maximum volume of 20 l. Cell counting
Experimental procedures Drugs Tissue culture media, serum, and antibiotics were from Gibco (São Paulo, SP, Brazil). S-Nitroso-N-acetylD,L-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) were synthesized in our laboratory according to published methods [12,13]. Glyceryl trinitrate (GTN; 1,2,3-propanotriol trinitrate) was kindly donated by Cristália Laboratories (São Paulo, SP, Brazil). 8-Bromoguanosine-3⬘-5⬘-cyclic monophosphate (8-Br-cGMP; a cell-permeable analogue of cGMP), ODQ (oxadiazoloquinoxalinone; a selective inhibitor of the soluble guanylate cyclase), KT5823 (a selective inhibitor of cGMPdependent protein kinase), NS1619 (an activator of high conductance calcium-activated potassium channels), TEA (tetraethylammonium; a non-selective blocker of potassium channels), 4-AP (4-aminopyridine; a selective blocker of voltage-dependent potassium channels), and glibenclamide (a selective blocker of ATP-dependent potassium channels) were purchased from Sigma Chemical (St. Louis, MO, USA). Charybdotoxin (a blocker of the high and intermediate conductance calcium-activated potassium channels), iberiotoxin (a selective blocker of high conductance calcium-activated potassium channels), and apamin (a selective blocker of the small conductance calcium-activated potassium channels) were purchased from Alomone Laboratories (Jerusalem, Israel). All solutions were prepared in culture medium except glibenclamide and KT5823 that were dissolved in DMSO, GTN that was dissolved in 1 mM HCl, and toxins, which were dissolved in PBS containing 1 mg/ml protease-free bovine serum albumin. Cell culture The rat aortic vascular smooth muscle cell line A7r5 was obtained from Rio de Janeiro Cell Bank (UFRJ, RJ, Brazil) and was used at passages 2–15. Cells were grown and passaged in plastic culture Xasks in Dulbecco’s modiWed Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, 10 mM Hepes, pH 7.4 (referred to as DMEM), in
At the end of the experimental periods, each well was washed twice with Ca2+/Mg2+-free PBS. Cells were then detached using 100 l of 0.25% trypsin/0.03% EDTA solution, incubated at 37 °C for 30 min. Trypsin was then inactivated with 10 l of fetal calf serum, cells were quantiWed in Neubauer chambers, and the results are expressed as cell number/mm3. Analysis of data Data are expressed as means § SD of triplicate wells. Each experiment was repeated at least twice with similar results. Statistical signiWcance was determined by oneway analysis of variance (ANOVA) followed by Bonferroni’s post hoc t test. A P value of less than 0.05 was considered signiWcant.
Results NO donors and cell proliferation After seeding 3.5 £ 103 cells in each well of a 96-well plate, A7r5 cell line reached conXuence after 96 h (around 60 £ 103 § 6 cells/mm3, n D 10). After this time, cell number remained constant for 1–2 days when cells started to die and detach from the substrate. Thus, the 96 h time point was chosen as the end-point to evaluate cell proliferation. The NO donor SNAP (Fig. 1A) caused a concentration-dependent inhibition of A7r5 cell proliferation with an IC50 value of around 90 M (calculated using GraphPad Prism 3 software). A similar eVect was seen with GTN or GSNO, with IC50 values of »70 and 540 M, respectively. In addition, neither NAP nor GSH (the non-nitrosylated parent compounds of NO donors) had eVect on cell proliferation. Besides inhibiting VSMC proliferation [4], NO can induce apoptosis [14]. To conWrm that NO inhibitory eVect was not due to its toxicity to cells, Trypan blue exclusion tests were carried out in cells treated with large range of SNAP concentrations. Cell death became apparent when SNAP 300 M or higher was employed (53 § 18% viability, n D 3). Therefore, the maximal SNAP concentration used throughout
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age of their antiproliferative eVects by KT5823 (vehicle 47 § 5; SNAP 100 M 20 § 0.3; SNAP + KT5823 1 M 49 § 1 £ 103 cells/mm3, n D 3; 8-Br-cGMP 100 M 18 § 1; 8-Br-cGMP + KT5823 38 § 5 £ 103 cells/mm3, n D 3). KT5823 did not aVect cell proliferation by itself (51 § 9). Potassium channel blockers and NO antiproliferative eVect TEA (a non-selective blocker of potassium channels; Fig. 2A) and 4-AP (a blocker of voltage-dependent potassium channels; Fig. 2B) completely blocked the antiproliferative eVects of the lower concentration of
Fig. 1. EVects of NO donors on the proliferation of A7r5 rat vascular smooth muscle cell line and the dependence on soluble guanylate cyclase. Cells were seeded (3.5 £ 103 cells/well) and after 24 h, compounds were added to cultures. (A) 1–100 M for SNAP, GTN, and NAP and 1–1000 M for GSNO and GSH. (B) 1–100 M for 8-BrcGMP and SNAP and 1 M for ODQ. Ninety-six hours after seeding, cell number was obtained by counting, as detailed in Experimental procedures. Each point is the means § SD of triplicate wells. Similar results were obtained in at least two independent experiments. * P < 0.05 when compared to the control group (A) (no compound) or the correspondent SNAP or 8-Br-cGMP groups (B) ANOVA followed by Bonferroni’s post hoc t test).
this report was 100 M, which always yielded cell viability greater than 95%. NO/cGMP/PKG pathway and inhibition of A7r5 cell proliferation As shown in Fig. 1B, 8-Br-cGMP (a cell-permeable analogue of cGMP) inhibited A7r5 cell proliferation in a dose-dependent fashion with an IC50 value of »70 M, yielding a dose–response curve very similar to that of SNAP. Pretreatment of cells with ODQ prevented the onset of SNAP antiproliferative eVect. Cell proliferation was unchanged by ODQ alone (39 § 7 vs 43 § 5 £ 103 cells/mm3, vehicle and ODQ, respectively, n D 3). However, SNAP and 8-Br-cGMP eVects were mediated through PKG as demonstrated by the block-
Fig. 2. EVects of potassium channel blockers on NO-induced inhibition of A7r5 rat vascular smooth muscle cell line proliferation. Cells (3.5 £ 103 cells/well) were seeded and after 24 h, compounds (SNAP, 1–100 M; tetraethylammonium, TEA 300 M; 4-aminopyridine, 4AP 100 M; glibenclamide, GBN 1 M; iberiotoxin, IbTx 100 nM; apamin 100 nM and charybdotoxin, ChTx 100 nM) were added. Vehicle was DMSO 0.05% for glibenclamide, saline with 1 mg/ml BSA for toxins or saline for the other compounds. Ninety-six hours after seeding, the cell number was obtained by counting, as detailed in Experimental procedures. Bars are means § SD of triplicate wells. Similar results were obtained in two independent experiments. *P < 0.05 compared to control (no SNAP) and #P < 0.05, compared to the equivalent SNAP group (ANOVA followed by Bonferroni’s post hoc t test).
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SNAP (10 M). When the NO donor was used at concentrations of 100 M, the inhibitory eVect of TEA and 4-AP became partial. On the other hand, NO donor eVect was unaVected by glibenclamide (a blocker of ATP-dependent potassium channels; Fig. 2C). Iberiotoxin (Fig. 2D) blocked SNAP eVects at lower concentrations of the NO donor and caused a partial inhibition at the higher concentrations of the NO donor. On the other hand, apamin (Fig. 2E) and charybdotoxin (Fig. 2F) blocked the antiproliferative eVect induced by 10 M SNAP, but failed to aVect the antiproliferative eVect induced by higher SNAP concentrations (100 M). None of the potassium channel blockers caused cytotoxicity or aVected cell proliferation by themselves (Fig. 2), at least within the range of concentrations employed. Potassium channel blockers and 8-Br-cGMP-induced inhibition of A7r5 cell proliferation TEA and 4-AP completely reversed 8-Br-cGMP antiproliferative eVect as shown in Figs 3A and B, respectively. Iberiotoxin and apamin also eYciently blocked 8-Br-cGMP antiproliferative eVects, as shown in Figs. 3C and D, respectively.
EVect of simultaneous inhibition of guanylate cyclase and potassium channel on NO-induced inhibition of A7r5 cell proliferation The combination of sub-eVective concentrations of ODQ (100 nM) and TEA (10 M) completely prevented the onset of SNAP antiproliferative eVect (Fig. 4). Potassium channel opener- and NO-induced inhibition of A7r5 cell proliferation To conWrm whether calcium-activated potassium channels are involved in mediating the inhibitory eVects of NO, an activator of high conductance calcium-activated potassium channel, NS1619, was tested on cell proliferation. NS1619 antiproliferative eVect was concentration-dependent with an IC50 value of »70 M. For comparison, SNAP was present in matched experiments (Fig. 5).
Discussion Our results showed that NO donors inhibited the proliferation of the rat embryonic thoracic aorta smooth muscle cell line (A7r5). Besides inhibiting VSMC prolifer-
Fig. 3. EVects of potassium channel blockers on 8-Br-cGMP-induced inhibition of A7r5 rat vascular smooth muscle cell line proliferation. Cells (3.5 £ 103 cells/well) were seeded and after 24 h, compounds (8-Br-cGMP, 1–100 M; tetraethylammonium, TEA 300 M; 4-aminopyridine, 4-AP 100 M; iberiotoxin, IbTx 100 nM and apamin 100 nM) were added. Vehicle was saline with 1 mg/ml BSA for toxins or saline for the other compounds. Ninety-six hours after seeding, the cell number was obtained by counting, as detailed in Experimental procedures. Bars are means § SD of triplicate wells. Similar results were obtained in two independent experiments. *P < 0.05 compared to control (no 8-Br-cGMP; ANOVA followed by Bonferroni’s post hoc t test).
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Fig. 4. EVect of the simultaneous blockage of soluble guanylate cyclase and potassium channels on NO-induced inhibition of A7r5 cell line proliferation. Cells were seeded (3.5 £ 103 cells/well) and after 24 h, incubated with vehicle or with SNAP in the presence or absence of sub-eVective concentrations of ODQ (1 nM) or TEA (10 M), or with the two blockers together. Ninety-six hours after seeding, cell number was obtained by counting, as detailed in Experimental procedures. Bars are means § SD of triplicate wells. Similar results were obtained in at least two independent experiments. *P < 0.05 compared to control (no SNAP; ANOVA followed by Bonferroni’s post hoc t test).
Fig. 5. EVects of NS1619 and SNAP on A7r5 cell line proliferation. Cells (3.5 £ 103 cells/well) were seeded and after 24 h, compounds (1– 100 M for both NS1619 and SNAP) were added to diVerent cultures. Vehicle was DMSO 0.05% for NS1619 or saline for SNAP. Ninety-six hours after seeding, the cell number was obtained by counting, as detailed in Experimental procedures. Points are means § SD of triplicate wells. Similar results were obtained in two independent experiments. *P < 0.05 compared to control group (absence of added compound; ANOVA followed by Bonferroni’s post hoc t test).
ation, our results also showed that NO can induce cell death, although the latter eVect occurred only at concentrations higher than those inhibiting proliferation. The NO inhibitory eVect was concentration-dependent and seems to be attributable to NO only since NAP and GSH, the non-nitrosylated parent compounds of nitrosothiols SNAP, and GSNO, respectively, were devoid of any eVect. GSNO displayed essentially the same antiproliferative eVect as SNAP, although it was less potent, probably due to its slower kinetics of NO release [15]. In addition, GTN (an organic nitrate and not a nitrosothiol) used in the clinical setting as a vasodilator also inhibited VSMC prolifer-
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ation. Therefore, this NO eVect cannot be attributable to non-selective eVect of nitrosothiols. Several NO eVects are mediated through soluble guanylate cyclase activation, with the correspondent increase in cyclic guanosine-3⬘-5⬘-monophosphate (cGMP) levels. Cyclic GMP eVects, in turn, are mediated through cGMP-dependent protein kinase (PKG), cGMP-regulated phosphodiesterases [16] or cyclic nucleotide-gated cation channels [17]. The relevance of NO and cGMP pathway in inhibiting SMC proliferation, initially observed by Garg and Hassid [4], is well established today forming the basis of potential clinical applications [18]. The dependence on guanylate cyclase/ cGMP pathway in mediating NO antiproliferative eVects on A7r5 cell line was demonstrated by showing the inhibitory action of ODQ, a potent and selective inhibitor of soluble guanylate cyclase and of KT5823, a speciWc inhibitor of PKG. In addition, 8-Br-cGMP, a membrane-permeant analogue of cGMP, eYciently mimicked NO eVect and was also blocked by KT5823. Therefore, our data conWrm and extend the literature concerning NO antiproliferative eVect on smooth muscle cells and show that the A7r5 smooth muscle cell line seems to be identical to a primary vascular smooth muscle cell, at least as far as the guanylate cyclase/cGMP pathway is concerned. Potassium channels are essential to several cell functions, including proliferation [19,20], they are relevant targets for NO [21–25] and SMC proliferation is central to processes such as atherosclerosis [1]. These three lines of evidence provide the basis of the most important Wnding of this report, the relationship among NO, SMC proliferation, and potassium channels. This point has not been examined in the literature and we took advantage of a previous publication from our laboratory, showing that NO antiproliferative eVects on a tumor cell line could be inhibited by potassium channel blockers [26]. When examined together, our results are clearly indicative that a signiWcant portion of NO antiproliferative eVect on vascular smooth muscle cells is mediated through potassium channel activation. However, some sub-types seem to be more relevant than others in this regard. For instance, voltage-dependent, as well members of the calcium-dependent sub-families (notably IKCa and SKCa) channels are important for NO eVects. Although our arguments are based on the use of blockers of potassium channels, at least concerning the calcium-activated sub-types, the availability of an opener of these channels (NS1619) allowed us to directly show the inhibitory role of this potassium channel family on vascular smooth muscle cells proliferation. On the other hand, the ATP-dependent sub-type does not seem to be involved in the NO antiproliferative eVect in spite of its involvement in several other NO actions [27,28].
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As for the mechanism by which NO may be recruiting to activate potassium channels, some points are worth discussing. First, the results showing that the eVects of 8-Br-cGMP (which mimicked NO) could be blocked by potassium channel inhibitors are suggestive that cGMP (either as a product of NO-dependent guanylate cyclase activation or exogenously added) may be causing phosphorylation of potassium channels via protein kinase G. A similar Wnding is reported in the literature [24]. Second, the Wnding that sub-eVective concentrations of ODQ and TEA when used simultaneously blocked NO antiproliferative eVect suggests that at least part of potassium channel activation could be an event situated downstream to guanylate cyclase activation. Third, it is important to note that when higher (and more eVective in inhibiting cell proliferation) NO donor concentrations were employed, the inhibition caused by potassium channel blockers was not complete, as it was in the lower SNAP concentrations. This was especially true for the voltage-dependent and iberiotoxin-sensitive channels (BKCa). Taking into account that all potassium channel blockers completely prevented 8-Br-cGMP antiproliferative eVects, our results are suggestive that, at least for voltage- and high conductance calcium-dependent potassium channels, some other mechanism induced by NO may be operative. As discussed above, NO can nitrosylate cysteine sulfydryls [29,30] of several proteins, among them potassium channels [25]. Thus, it is temptating to speculate that S-nitrosylation may represent an alternative pathway for potassium channel modulation and inhibition of SMC proliferation, mainly when higher NO concentrations are concerned. The experiment showing that sub-eVective concentrations of ODQ + TEA abolished NO antiproliferative eVect would favor the suggestion that phosphorylation seems to be a more relevant mechanism for NO-dependent potassium channel activation. However, since we do not know the consequences of S-nitrosylation on potassium channel activation/phosphorylation, we cannot exclude the possibility that NO may be activating potassium channels via S-nitrosylation. In summary, we show that the mechanism by which NO inhibits VSMC proliferation involves cGMP and, for the Wrst time, that some sub-types of potassium channels seem to have a key role in this eVect.
Acknowledgments The skillful technical assistance of Mrs. Adriane Madeira is gratefully acknowledged. We also thank Dr. J.B. Calixto for allowing us the use of his tissue culture facilities. This work was supported by Conselho Nacional de Desenvolvimento CientíWco e Tecnológico (CNPq, Brazil) and by PRONEX/MCT/Brazil.
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