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Cardiovascular Research 2003 60(2):326-336; doi:10.1016/S0008-6363(03)00527-3
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Copyright © 2003, European Society of Cardiology

Role of Ca2+-sensitive K+ channels in the remission phase of pulmonary hypertension in chronic obstructive pulmonary diseases

Sébastien Bonnet*,a, Jean-Pierre Savineaua, Wilfrid Barillotb, Eric Dubuisc, Christophe Vandierc and Pierre Bonnet*,c

aINSERM (EMI 0356), Université Bordeaux 2, 146 rue Léo-Saignat, 33076 Bordeaux, France
bINSERM U441, avenue du haut-Lévêque, 33600 Pessac, France
cLaboratoire de physiopathologie de la paroi artérielle (LABPART), Dept. de Physiologie, Faculté de Médecine, 2 bis Boulevard Tonnellé, 37032 Tours, Indre et Loire, France

*Corresponding authors. Tel.: +33-2-47-36-60-91; fax: +33-2-47-36-60-64. Email address: sebastien.bonnet{at}lpcr.u-bordeaux2.fr bonnet{at}med.univ-tours.fr

Received 6 February 2003; revised 4 July 2003; accepted 7 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: Clinically, the effect of chronic hypoxia (CH) in the pulmonary circulation alternates between phases of pulmonary artery hypertension (CH-PAHT) and normoxic normotensive remission (N-RE). Little information is available on the role of calcium-sensitive potassium channels (BKCa) in both CH-PAHT and N-RE phases. In the present study, we investigated the effects of both CH and N-RE on BKCa channels activity and their consequences on hypoxic pulmonary vasoconstriction (HPV). Methods: Using isolated ring preparation, the patch-clamp technique, RT-PCR and Western immunoblotting, we examined the role of the BKCa channel in normoxic, CH-PAHT and N-RE rat pulmonary artery smooth muscle cells (PASMCs). Results: In intrapulmonary arterial rings, acute hypoxia induced contraction in control vessels, relaxation in the N-RE rats, and had no effect in CH-PAHT. The hypoxia-induced relaxation in the N-RE rat pulmonary arteries was abolished by iberiotoxin (IbTx), a specific BKCa blocker. The IbTx-sensitive whole-cell KCa channel current was reduced in CH-PAHT and increased in N-RE rat PASMCs. The BKCa channel conductance and voltage sensitivity were not altered in CH and N-RE rat PASMCs, whereas its calcium sensitivity was decreased and increased in CH and N-RE rat PASMCs, respectively. Results of RT-PCR and Western blot analysis revealed a decrease in the mRNA and protein of the BKCa {alpha}-subunit in CH, whereas no change at protein level was observed in the N-RE. Conclusion: In rat PASMCs, CH and N-RE are associated with a down- and up-regulation of BKCa activity, respectively, mainly due to modifications of its Ca2+ sensitivity. This could explain the acute hypoxic pulmonary constriction and relaxation observed in CH and N-RE rats, respectively.

KEYWORDS Ion channels; K-channel; Pulmonary circulation; Smooth muscle; Single channel currents; Pulmonary artery; Chronic hypoxia; COPD; Hypertension; Ca2+-sensitive K+ channels

Abbreviations: CH-PAHT, chronic hypoxic pulmonary artery hypertension • N-RE, normoxic remission • CH, chronic hypoxia • PA, pulmonary artery • PASMCs, pulmonary artery smooth muscle cells • HPV, hypoxic pulmonary vasoconstriction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Patients suffering from chronic obstructive pulmonary diseases (COPD) are affected by successive hypoxic episodes leading to pulmonary artery hypertension [1] accompanied by pulmonary artery (PA) remodelling [2,3]. In an early stage of COPD, chronic hypoxic pulmonary artery hypertension (CH-PAHT) alternates with phases of clinical remission during which the regression of CH-PAHT is usually observed [1]. The cellular mechanisms underlying this cyclic pathophysiology of the pulmonary circulation during COPD are not fully elucidated. Recently, we have observed that chronic hypoxia (CH) in rats following 3 weeks of normoxic-remission (N-RE) show neither CH-PAHT nor membrane depolarization [4,5]. It is thus of great importance to ensure that during N-RE periods, the pulmonary artery (PA) retains its capability to adapt to further hypoxic stress while still developing the hypoxic pulmonary vasoconstriction (HPV). It is now well recognized that the pulmonary artery smooth muscle cells (PASMCs) play an important role in the hypoxic response. Hypoxia induces membrane depolarization which in turn increases intracellular calcium concentration ([Ca2+]i) in PASMCs thus promoting vasomotor tone in the pulmonary vasculature [6,7]. PASMCs membrane potential (Em) is controlled by numerous potassium channels including Kv and KCa channels [3,4]. Previous studies have revealed that CH down-regulates voltage-gated potassium currents (IKv) in PASMCs from both animals and humans [8,9].

However, no detailed studies have been performed on the electrophysiological properties and changes in the expression of BKCa at the protein and transcript level in CH and N-RE rats PASMCs. The BKCa channels conduct ionic currents that mediate membrane hyperpolarization and vascular relaxation [10]. BKCa channel activity is regulated by a number of factors including Em, cytosolic calcium concentration ([Ca2+]i) and channel phosphorylation [10]. The open probability of BKCa in PASMCs is relatively low at resting Em but increases with membrane depolarization during exposure to acute hypoxia. In this regard, activation of BKCa channel appears to act as a contractile negative feedback mechanism functioning to adjust pulmonary vascular tone [10–13].

We designed experiments to investigate if the electrophysiological properties and expression of BKCa channels are altered during CH and after N-RE, and tested the hypothesis that after CH-PAHT, the activity of BKCa channel is increased, thus promoting the occurrence of the HPV and accounting for the clinical remission phase.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1. Chronic hypoxia
Male Wistar rats (220 g body weight) were separated into three groups. The first group (control) was housed in ambient room air, whereas both second and third groups were exposed to CH for 3 weeks in a hypobaric chamber (50 kPa). Then the second group was tested immediately (CH group), whereas the third group was left under normoxic conditions for a further 3 weeks (N-RE group) [5].

CH-PAHT was assessed by measuring pulmonary arterial pressure (PAP) and the ratio of right ventricle (RV) to left ventricle plus septum (LV+S) weight. PAP was measured by means of a polyethylene catheter inserted into the right jugular vein and pushed through the RV into the pulmonary artery as detailed previously [5].

The experimental investigations were carried out in agreement with the Guideline for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publications No. 85-23, revised 1996) and European Directives (86/609/CEE).

2.2. Tissue and cell preparation
Rats were anaesthetized by an intraperitoneal injection of ethyl carbamate (40 mg/kg). The right and left branches of the conductive pulmonary arteries (internal diameter≥400 µm) were carefully dissected. For mechanical measurements, PA rings (3 mm in length) were prepared. For the study of membrane currents, enzymatic isolation of PASMCs was performed according to a method described previously [5]. Only elongated, smooth and optically refractive cells were used for patch-clamp measurements within 6 h of isolation.

2.3. Resting tension recording
Isometric contraction was determined in endothelium-denuded PA rings (mechanically removed by gently rubbing the intimal surface with the tip of small forceps) using a computerized isolated organ bath system filled with PSS solution, gassed either with air or N2 to induce hypoxia. Rings were set at optimal length by applying a passive load of 20 mN. At the beginning of each experiment, the ring preparations were treated with high K+ solution (80 mM) to obtain two consecutive contractions. The mean amplitude of these reference contractions was not significantly different in three groups of rats (data not shown). These high K+ induced contractions were used to normalize the contractions induced by acute hypoxia. Acute hypoxia (PO2~10–20 mm Hg) was applied for 20–30 min and changes in basal tone were continuously recorded. The involvement of BKCa in the acute hypoxia response was tested by the application of a selective blocker of BKCa channels, iberiotoxin (IbTx) 100 nmol/l.

2.4. Patch-clamp recording
In isolated cells, electrophysiological recordings were obtained using the patch-clamp technique [5]. Currents were normalized to cell capacitance and were expressed as picoamperes per picofarad (pA/pF).

Net macroscopic K+ currents were generated by stepwise 10 mV depolarizing pulses (400 ms duration; 5-s intervals) from a constant holding potential of –80 to +60 mV. The BKCa current was defined as the difference between the outward current recorded with and without IbTx (100 nM). Unitary BKCa currents were obtained in inside-out patches of PASMCs bathed in symmetrical 145 mmol/l KCl and subjected to membrane potentials of –60 to +60 mV (10-mV increments). Currents were filtered at 5 kHz and sampled at 50 kHz. Averaged current amplitudes were obtained for the calculation of single-channel conductance [14]. The open state was defined as 50% of the predominant single-channel current amplitude, and NPo was calculated to obtain voltage-activation relationships at different [Ca2+]i levels. In some experiments, pipette tips were briefly loaded with drug-free pipette solution and then back-filled with pipette solution containing 100 nmol/l IbTx. Unitary currents were recorded immediately during 1 to 2 min in inside-out patches and measurements were repeated at the same membrane potential after 5 min to allow drug diffusion to the outside patch surface.

2.5 Evaluation of BKCa expression by Western immunoblotting
Pulmonary arteries isolated from different rats were pooled (N = 5 to 10 rats per groups). All samples were frozen in liquid N2 and homogenized in RIPA buffer (1% NP40, 0.5% deoxycholate sodium, 0,1% SDS, aprotinin 10µg/ml, leupeptin 100 µg/ml, PEFA 1 mM in PBS) with polytron (bioblock scientific) and then centrifuged at 2000 x g. The extract was checked by Coomassie staining. Each sample was loaded at 10 µg in 7.5% resolving acrylamide gel and electrotransferred to nylon membrane. Immunoblot analyses used rabbit monoclonal antibody against rat BKCa (Alomone Labs no. APC-021 at 1/500) and mouse monoclonal anti-β-actin (Santa Cruz Biotechnology clone C-2 at 1/500). The secondary antibody coupled to horseradish peroxidase was used at 1/5000 dilution. The immunoreactive bands were revealed with an ECL kit (Amersham Pharmacia Biotechnology). For quantification, the photodensity of BKCa bands was normalized to β-actin using Scion Image program from the National Institutes of Health. Each experiment was repeated twice with three different protein isolations.

2.6 Evaluation of BKCa transcripts
BKCa transcripts were evaluated using reverse-transcriptase polymerase chain reaction (RT-PCR) according to a method previously described [15]. The following sense and antisense primers were designed from coding regions of the BKCa {alpha}-subunit [GenBank accession number RNU93052; 5'-CGCCATTAAGTCGGGCTGAT-3' and 5'-GACGGCAAATGCTGTCCC-3'] and of smooth muscle specific {alpha}-actin [GenBank accession number BTU-35433; 5'-ATCCATCGTGGGACGTCCCAGACA-3' and 5'-GTCAGGCAGTTCGTAGCTCTTCT-3'] (Operon Technologies, Alameda, CA). Samples were initially incubated for 5 min at 95 °C followed by 36 cycles of 1 min at 95 °C, 1 min at 59 °C, and 2 min at 72 °C, with a final extension step of 7 min at 72 °C. Amplified cDNA products were analyzed on a 1% agarose gel containing ethidium bromide using a 100-bp DNA ladder as a molecular weight marker. Photodensity of BKCa bands was normalized to that of á-actin (Scion Image program). Each experiment was repeated twice with three different RNA isolations.

2.7. Solutions and chemicals
The PSS contained (in mM): NaCl, 138.6; KCl, 5.4; CaCl2, 1.8; MgCl2, 1.2; NaH2PO4, 0.33; HEPES, 10 and glucose, 11; pH was adjusted to 7.4 using NaOH. The dissociation solution contained (in mM): NaCl, 145; KCl, 4; MgCl2, 1; HEPES, 10 and glucose, 10; pH was adjusted to 7.3 using NaOH. For whole cell patch-clamp recordings, the pipette solution contained (in mM): glutamic acid, 125; KCl, 20, Na2ATP, 1; CaCl2, 0.37; MgCl2, 1; HEPES, 10; EGTA, 1; pH was adjusted to 7.2 using KOH. For single channel recordings, the external solution contained (in mM): 140 KCl, 10 HEPES, 5 EGTA, and CaCl2 (0.3; 2; 4.3), MgCl2 (1.3; 1.2; 1.1); pH was adjusted to 7.2 using KOH. Different [Ca2+] was obtained using a computer program developed by Godt and Lindley [16]. The internal solution contained (in mM) 140 KCl, 10 HEPES, 5 EGTA, 0 CaCl2, 1 MgCl2 and pH was adjusted to 7.2 using KOH.

BSA (fraction V), collagenase (type H), dithioerythritol, (type IV), IbTx, papain, trypsin inhibitor (type 1-S) were from Sigma (St. Quentin Fallavier, France). Stock solutions of IbTx were prepared in distilled water and then diluted to the appropriate concentration in PSS.

2.8. Analysis of data and statistics
Values are expressed as mean±S.E.M. Intergroup differences were assessed by repeated-measures ANOVA or factorial ANOVA, as appropriate. Post hoc analysis used Dunnett and Fisher test (NCSS 2001, Kaysville, UT, USA). The lower case n refers to the number of cells or artery rings and upper case N to the number of animals. Differences were considered significant when p<0.05. For unitary BKCa currents, NPo values from five to six different inside-out patches were fitted by a Boltzmann function of the following form: NPo=NPmax/[1+exp(V–V0.5)/K], where N is the number of channels in the patch, Po is the single channel open-state probability, and V1/2 is the voltage for half-maximal activation. K represents the slope factor, which is an indicator of single-channel voltage sensitivity [17].


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1. Reversal of CH-induced pulmonary hypertension
In control group, the mean PAP was 11.3±1.5 mm Hg (N = 8). It was significantly increased after 3 weeks of hypoxia (29±1.4 mm Hg, N = 8, p<0.05). However, after 3 weeks of N-RE, the mean PAP returned to a value similar to that under control conditions, 14.1±2.1 mm Hg (N = 7, p>0.05, Fig. 1). CH-PAHT caused an increase in the weight ratio of RV/LV+S from 0.28±0.2 (N = 10) to 0.64±0.25 (N = 8) (p<0.05). This increase was also fully reversed after 3 weeks of N-RE (0.28±0.2, N = 10, vs. 0.31±0.22, N = 8; p>0.05).


Figure 1
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  		Fig. 1 Reversal of chronic hypoxia-induced pulmonary hypertension. Original pulmonary artery pressure traces recording in vivo in control (A), CH (B) and N-RE rats (C). CH increased the mean pulmonary artery pressure (B). After 3 weeks of N-RE the values were not different from those of the control rats (C).

 
3.2. Effect of acute hypoxia on arterial basal tone
In resting conditions, acute hypoxia (10 mm Hg) induced a significant contraction of 8±1% (n = 12; N = 4) of the maximal KCl contraction in PA rings from control rats. In CH rats, the acute hypoxic contraction was significantly lowered to 0.8±0.01% (n = 12; N = 4; p<0.05) (Fig. 2A,Ba). Whereas in N-RE acute hypoxia significantly relaxed PA rings to –18±2% (n = 17; N = 6) of the basal tone (Fig. 2Ca).


Figure 2
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  		Fig. 2 Effect of acute hypoxia on resting tension in the pulmonary arterial rings. Original traces of resting tension in the absence (left panel) and in presence (right panel) of IbTx (100 nmol/l) in PA rings from control (A), CH (B) and N-RE (C) rats. Acute hypoxia induced a significant contraction in rings from control rats and a relaxation in rings from N-RE rats; this relaxation was entirely abolished in IbTx pretreated rings.

 
Exposure of PA rings to IbTx (100 nmol/l) had no significant effect on the basal tone in all three preparations (p>0.05). Effects of acute hypoxia were then tested in IbTx (100 nmol/l) pre-incubated PA rings for 10–20 min. In control and CH PA rings, IbTx did not significantly change (p>0.05) the acute hypoxia responses previously observed (Fig. 2Ab,Bb). In N-RE PA rings, IbTx significantly inhibited the acute hypoxia-induced relaxation and restored acute hypoxia-induced contraction that averaged 6±2% (n = 6; N = 4) of the maximal KCl contraction (Fig. 2Cb).

3.3 Effects of N-RE on BKCa current density
Fig. 3 shows original traces of macroscopic outward currents of PASMCs from the three groups of rats. In PASMCs isolated from CH rats, the amplitude of the BKCa current, recorded at +60 mV, was significantly decreased (49.6±9%, n = 7; N = 4, p<0.05) when compared to control PASMCs. In contrast, in PASMCs from N-RE rats, the currents were increased by +62.5±8%, (n = 8; N = 4, p<0.05).


Figure 3
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  		Fig. 3 Effect of CH and N-RE on the amplitude of whole-cell BKCa currents in pulmonary arterial smooth muscle cells (PASMCs). (A, B) Outward currents with and without IbTx (100 nmol/l). Currents were elicited by incremental 10 mV depolarizing steps from –80 to 60 mV. (C), Current–voltage relationships of outward currents with and without IbTx. Current–voltage relationships of outward Kca currents were calculated as the difference between outward current recorded with and without IbTx. Current amplitude was normalized to cell capacitance to obtain current density. Data points are mean±S.E.M. n = 6, 4 and 8 in PASMCs from control, CH and N-RE rats, respectively. *p<0.05.

 
In all groups, IbTx reduced the outward current at +60 mV by 36% (n = 8; N = 5) in control, 24% in CH (n = 7; N = 4), and 51% (n = 8; N = 4) in N-RE groups (Fig. 3B).

Fig. 3C shows that KCa current density was 4.6 and 4.1 times lower in CH rat PASMCs at 0 and +60 mV, respectively (p<0.05), when compared to control rat PASMCs. After 3 weeks of N-RE, KCa current density was 2.35- and 1.8-fold higher in N-RE rat PASMCs at 0 and +60 mV, respectively (p<0.05), compared with control rat PASMCs. It should be noted that no significant difference was observed between mean values of cell capacitance in PASMCs obtained from control (14.3±2 pF; n = 10; N = 4); CH (15.2±3.1 pF; n = 10; N = 4) and N-RE (13.8±2 pF; n = 10; N = 4) rats (p>0.05).

3.4 Effects of N-RE on KCa channels single-channel properties
Subsequent experiments were performed to test the possibility that modified single-channel properties could account for the observed alterations in BKCa current density. The opening probability of single channels (NPo) was recorded in inside-out patches of PASMCs isolated from the different groups of rats clamped at +60 mV and exposed to 100 nmol/l Ca2+. Under control condition, NPo was 0.48±0.07 (n = 6; N = 4, Fig. 4A). The opening frequency was significantly (p<0.05) decreased to 0.27±0.02 (n = 6; N = 4) in PASMCs isolated from CH and increased to 0.72±0.05; (n = 6; N = 4) in N-RE rats. Exposure of the outside patch surface to IbTx (100 nmol/l) significantly reduced NPo under each experimental condition, i.e. control, CH and N-RE (n = 3, each, p<0.05). In the range of tested potentials (–40 and+60 mV), the amplitude of single-channel current was similar in patches of control, CH and N-RE and identical to those observed before IbTx exposure which proves that only IbTx-sensitive potassium current were studied. The resulting current–voltage relationship (Fig. 4B) indicated a single-channel conductance of 228, 234 and 238 pS in control, CH and N-RE rats PASMCs, respectively.


Figure 4
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  		Fig. 4 Effect of CH and N-RE on single-channel BKCa currents from inside-out PASMCs patches. (A) Unitary KCa currents in PASMCs patches from control (a), CH (b), N-RE (c) and N-RE in the presence of IbTx (100 nmol/l) (d). Membrane potential was maintained at +60 mV with a [Ca2+]i of 100 nmol/l. (B) Current–voltage relationship for the unitary currents were obtained in PASMCs patches from control, CH and N-RE rats. Linear fit revealed mean unitary conductance of 228 ± pS (control), 234 ± pS (CH) and 238 ± pS (N-RE). Data points are means±S.E.M. n = 6 for each curve.

 
Fig. 5 illustrates the relationship between normalized NPo and membrane potential for BKCa of PASMCs patched membranes in control, CH and N-RE rats. NPo was calculated from unitary current recordings obtained at membrane potentials (20-mV steps) between –60 and +60 mV and at three different [Ca2+]i: 10, 100 and 1000 nM. NPo values obtained from five to six different inside-out patches were fitted by a Boltzmann function (see Materials and methods). Values for K were similar for all activation curves at different [Ca2+]i and between preparations averaging 27.6±2.3 (n = 5; N = 4); 28.8±2.5 (n = 6; N = 4) and 28.7±1.9 (n = 5; N = 4) for a [Ca2+]i of 100 nmol/l, in PASMCs patch membranes from control, CH and remission rats, respectively (p>0.05). V0.5 were significantly (p<0.05) different in CH and N-RE rats PASMCs compared to controls for all [Ca2+]i (Table 1). Calcium sensitivity of the BKCa channels was evaluated by the linear relation between V1/2 and log[Ca2+]i (Fig. 5D). We observed a significant decrease (p<0.05) of the slope coefficient in CH groups compared to control groups (–20.1±1, n = 6; N = 4 vs. –28.6±2.2, n = 5; N = 4) and a significant increase (p<0.05) in N-RE compared to control (–39.3±1.2, n = 5; N = 4 vs. –28.6±2.2 n = 5; N = 4).


Figure 5
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  		Fig. 5 Effect of CH and N-RE on the NPo–voltage relationship at different pCa for BKCa in PASMCs inside-out patches. pCa=–log[Ca2+]. (A) pCa 8; (B) pCa 7; (C) pCa 6. Normalized NPo was plotted as a function of membrane potential and fitted to the Boltzmann equation. Each fit includes NPo values obtained from five to six patches at 10 mV interval between –60 and +60 mV. (D) Linear relationship between V1/2 and log of [Ca2+]. The slope coefficient represents the calcium sensitivity. Data points are means±S.E.M. n = 5 to 6; N = 4 for each curve.

 

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  		Table 1 Effect of CH and N-RE on V1/2 values at different calcium concentration [Ca2+]

 
3.5 Effects of N-RE on BKCa channel gene expression
Fig. 6Aa shows cycle-dependant amplification products of 637 and 407 bp corresponding to transcripts for smooth muscle {alpha}-actin and the BKCa {alpha}-subunit, respectively. Compared to control, the photodensity of the 407 bp bands corresponding to BKCa {alpha}-subunit mRNA was significantly (p<0.05) lower in CH rats PA and higher in N-RE rats PA (Fig. 6Ad). At the same cycle number, {alpha}-actin (637 bp) was equally amplified, providing evidence of experimental consistency between reactions (Fig. 6Ad). Performing amplification without the RT reaction or in the absence of specific primers produced no detectable products (data not shown).


Figure 6
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  		Fig. 6 Effect of CH and N-RE on the expression of the BKCa {alpha}-subunit. (A) Multiplex RT-PCR amplification products correlating to the BKCa {alpha}-subunit gene (407 bp) and the {alpha}-actin gene (637 bp) from PASMCs of control (a), CH (b) and N-RE (c) rats. RT-PCR show lower and higher expression BKCa {alpha}-subunit in CH and N-RE compared to control rats, respectively. (B) Histograms of the BKCa {alpha}-subunit/{alpha}-actin density band ratio at 29 and 32 cycles. We observed a significant increase of the BKCa {alpha}-subunit mRNA in N-RE groups, whereas at 32 cycles we observed a significant decrease in CH. Data points are mean density±S.E.M. (p<0.05). (C) Immunoblot of the BKCa {alpha}-subunit. BKCa {alpha}-subunit was recognized by the antibody as a 125-kD immunoreactive band. BKCa {alpha}-subunit is down-regulated in CH vs. control groups, whereas its expression was similar between control and N-RE groups. (D) Ratio of the BKCa {alpha}-subunit/β-actin density. Data points are means density±S.E.M. (p<0.05).

 
3.6 Effects of N-RE on BKCa {alpha}-subunit expression
To assess whether the alteration in mRNA levels was accompanied by an alteration in the protein expression level, we determined the protein expression level of BKCa channel by Western blotting. Fig. 6Ba shows three adjacent lanes loaded with PASMC membrane protein from control (left), CH (middle) and N-RE (right) rats and containing the 125 kD band corresponding to the BKCa {alpha}-subunit. Fig. 6B displays a subsequent Western blot showing down-regulation of the BKCa in membranes from CH compared to control rats (p<0.05). This down-regulation was fully reversed in N-RE rats and no significant difference (p>0.05) was observed between control and N-RE rats (Fig. 6Bb).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
In the present study, we observed that despite normalization of the pulmonary artery (PA) pressure and right ventricular hypertrophy (RVH) after the N-RE period, the behavior of the PA rings to a further hypoxic challenge is profoundly altered. In N-RE PA rings, acute hypoxia induces significant relaxation. We demonstrated that BKCa channels do not modulate the HPV in control and CH conditions, whereas following N-RE the BKCa channels are responsible for hypoxia-induced relaxation due to the resulting increased Ca2+ sensitivity. These data suggest that after CH, BKCa might play a major role in regulating the pulmonary arterial tone especially in response to hypoxia.

In control isolated PA rings, exposure to acute hypoxia induced vasoconstriction, whereas after 3 weeks of CH a further acute challenge had no effect. In N-RE isolated PA rings, acute hypoxia induced vasorelaxation. To the best of our knowledge this is the first time that such a hypoxic response has been observed.

In the three tested conditions, IbTx had no effect on the basal tone, suggesting that BKCa channels are not directly involved in the regulation of the PA resting tone. Such lack of effect by IbTx was also evident in HPV suggesting that BKCa channels do not contribute to this condition, in contrast, in N-RE rats IbTx blocked acute hypoxia-induced relaxation, suggesting that BKCa play a major role in this phenomenon. However, although the reason for this differential response of the BKCa channel to various levels of molecular oxygen is not yet fully known, it might reflect changes at the transcriptional and expression level of the channel.

There is now convincing evidence that acute hypoxia inhibits Kv channels in PASMCs [9,18], inducing membrane depolarization and a rise in the [Ca2+]i. There is also accumulated evidence that acute hypoxia directly induces Ca2+ release from intracellular stores, which contribute to the rise in [Ca2+]i and to vasoconstriction [19,20]. We hypothesized that an acute hypoxia-induced [Ca2+]i increase is not sufficient for a large activation of the BKCa channels. In contrast, in the N-RE rats, where BKCa channels are much more calcium sensitive, the hypoxia-induced [Ca2+]i increase would activate many more BKCa channels, which, would mask the hypoxic vasoconstriction and thus lead to the hypoxia-induced vasorelaxation.

The mechanism for the decrease or increase in total whole cell current might be, at least in part, a change in one of the different parameters given in the equation I = NiPo, where N is the channel number, i is the unitary current amplitude, and Po is the channel open-state probability. In CH rat PASMCs, we found a decrease in both Po and BKCa mRNA and protein expression of the BKCa pore-forming {alpha}-subunit, suggesting a decrease in N. In contrast, in N-RE rat PASMCs, Po and mRNA expression were strikingly increased. Nevertheless, no significant change was observed in the protein level in N-RE rats PASMCs compared to control rats. The fact that arterial PO2 is the main altered factor during the CH and N-RE periods suggests that chronic change in cellular oxygenation could modulate both activity and gene expression of BKCa in the PASMCs. Abnormal K+ channels expression has previously been reported in PAs under chronic reduction of oxygen concentration [8,21]. Several molecular mechanisms are known to modify the activity of BKCa including its voltage or Ca2+ sensitivity, the coupling of {alpha}- to β-subunits, and the generation of {alpha}-subunit isoforms. In this respect, in PASMCs, a decrease in both voltage and Ca2+ sensitivity has been suggested as potential mechanisms for the observed decrease in Po of BKCa channels exposed to hypoxia [9]. In the present study, BKCa single-channel conductance was similar under all three conditions. Furthermore, K, the index of the voltage sensitivity of the channel, has similar values in the three groups. Therefore, these data appear to indicate that neither the conductance nor the voltage sensitivity of the channel can explain the increase in channel activity found in PASMCs patch membranes from N-RE rats. On the other hand, in the same preparations, the value of slope coefficient of the relation V1/2 plotted as a function of log calcium concentration is directly correlated to the Ca2+ sensitivity of the channel [17]. The significant increase in the slope coefficient demonstrates that after 3 weeks of N-RE, BKCa channels are much more calcium sensitive. Thus, the change in calcium sensitivity of the BKCa appears to be the mechanism that determines the up-regulation of BKCa activity after N-RE as protein expression is not modified in N-RE compared to control rats.

Our findings also indicated that CH alone down-regulated the BKCa activity with similar but opposite mechanisms (decrease in genomic and protein expression of the channel and less calcium sensitivity). Previous studies have shown that a change in PO2 could alter gene expression, as hypoxia decreases Kv channel expression in pulmonary arteries [8].

A key transcriptional response to a decrease in O2 partial pressure is the induction of hypoxia-inducible factor 1 (HIF-1) [22,23]. HIF-1 activates the transcription of genes involved in the development of PAHT. In this regard, Shimoda et al. [22] have shown that a partial deficiency of HIF-1{alpha} in mice prevents K+ currents inhibition induced by CH. Therefore, it is plausible that the prevalence of HIF-1 could be a triggering mechanism responsible for changes in the expression of BKCa channels under CH and reversal to normoxic conditions. Furthermore, the fact that mRNA but not the protein levels of the {alpha}-subunit in PA from the N-RE group is altered might suggest the involvement of a disturbance in the protein synthesis or rapid turn-over that requires further investigation at the cellular and molecular level.

The co-existence of the β-subunit with the {alpha}-subunit is critical for the voltage and Ca2+ sensitivity of the BKCa channel. Several mechanisms, including the coupling between {alpha}- and β-subunits and the generation of alternatively spliced {alpha}-subunit isoforms, have been suggested to account for the changes in Ca2+ sensitivity of BKCa observed in membranes from CH and N-RE groups [24,25]. Given that the β-subunit is obligatory for the sensitivity of the KCa channel, this view appears to further suggest that the β-subunit could be one of the target molecular components for oxygen sensing capability of the KCa channel in the pulmonary vasculature. This idea awaits thorough investigation.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
The increase in calcium sensitivity of the BKCa channels is responsible for the acute hypoxic relaxation of the pulmonary artery observed in remission rats. These long lasting modifications could be the first step of more important alterations of the pulmonary artery vasoreactivity observed in pathological situations whereby successive and repetitive hypoxic stress are observed.


   Acknowledgements
 
We thank Pr Nancy Rush for her helpful comments in the elaboration of the manuscript.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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