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Cardiovascular Research 2005 65(2):505-512; doi:10.1016/j.cardiores.2004.10.038
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Copyright © 2004, European Society of Cardiology

Euhydric hypercapnia increases vasoreactivity of rat pulmonary arteries via HCO3– transport and depolarisation

Marie Vanková1, Vladimir A. Snetkov, Greg A. Knock, Philip I. Aaronson and Jeremy P.T. Ward*

Department of Asthma, Allergy and Respiratory Science, GKT School of Medicine, Guy's Hospital Campus, London SE1 1UL, UK

* Corresponding author. Cardiovascular Biology and Medicine, 2nd floor New Hunt's House, King's College London, Guy's Hospital Campus, London SE1 1UL, UK. Tel.: + 44 20 7848 6695; fax: +44 20 7403 8640. Email address: Jeremy.ward{at}kcl.ac.uk

Received 28 August 2004; revised 9 October 2004; accepted 26 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To examine whether altered PCO2 or HCO3– at normal pH potentiate agonist-induced vasoconstriction of small pulmonary arteries, and if so to determine the mechanism.

Methods: Small intrapulmonary arteries (IPA) from rats were mounted on a myograph and PGF2{alpha} (3 µM)-induced tension recorded before and 40 min after replacing normal bath solution (5% CO2, 24 mM [HCO3–], pH 7.4) with one containing either normal [HCO3–] (24 mM) gassed with 10% CO2 (pH 7.12; hypercapnic acidosis) or high [HCO3–] (48 mM) gassed with 10% CO2 (pH 7.4; euhydric hypercapnia).

Results: Hypercapnic acidosis had no significant effect on the response of IPA to PGF2{alpha}. Euhydric hypercapnia however caused a substantial ~5.5-fold potentiation of the response (n=17, p<0.001) in the majority of preparations, whilst 20% of IPA (11 of 58) developed a slow spontaneous vasoconstriction after ~20 min. No equivalent responses to euhydric hypercapnia were observed in either mesenteric or renal arteries. Both the potentiation of PGF2{alpha}-induced vasoconstriction and the spontaneous vasoconstriction in IPA were inhibited by the L-type channel blocker diltiazem (10 µM). The potentiation was also suppressed by DIDS, an inhibitor of anion transporters, removal of extracellular Na+, and anthracene-9-carboxylic acid (A9C; 200 µM), reported to inhibit Ca2+-activated Cl– channels. Inhibition of nitric oxide synthase with L-NAME (100 µM) did not prevent potentiation. Depolarisation with 20 mM [K+] mimicked the effect of euhydric hypercapnia in that it also potentiated the response to PGF2{alpha} (>sixfold, n=6).

Conclusions: Euhydric hypercapnia increases vasoreactivity of IPA, but not mesenteric or renal arteries, via a mechanism involving Na+-dependent HCO3– transport, activation of Ca2+-dependent Cl– channels, and subsequent depolarisation. These results may have consequences for patients with CO2-retaining chronic respiratory disease where plasma [HCO3–] is raised following renal compensation, and could explain the increased propensity to pulmonary hypertension and increased mortality in such patients.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Patients developing hypoxaemia as a result of respiratory disease or critical illness tend to have an increased pulmonary vascular resistance (PVR) due to vasoconstriction of small pulmonary arteries [hypoxic pulmonary vasoconstriction (HPV)]. Respiratory dysfunctions that lead to hypoxaemia are also likely to cause perturbations in CO2 exchange, leading to hypercapnia and respiratory acidosis. However, in chronic respiratory disease, the respiratory acidosis is corrected primarily by renal compensation and increased plasma [HCO3–]. These conditions are reported to promote development of pulmonary hypertension and increase mortality in COPD patients [1], suggesting that hypercapnia per se and/or the increase in [HCO3–] may affect pulmonary vascular reactivity.

For many years, there has been a general acceptance that hypercapnia increases PVR in perfused lungs and in vivo, and this is indeed consistent with a recent study in healthy humans [2]. However, hypercapnia has also been reported to reduce PVR and suppress agonist-induced vasoconstriction and HPV in perfused lungs [3,4], and Brimioulle et al. [5] reported that whereas metabolic acidosis enhanced HPV in intact dogs, an equivalent respiratory acidosis was without effect. Conversely, Gordon et al. [6] have shown in piglets that although an acute hypercapnic acidosis had no immediate effect on PVR, if maintained for 60 min then baseline PVR was increased and HPV was potentiated. This is perhaps consistent with the study of Balanos et al. [2] in humans, where the increase in PVR during hypercapnia developed slowly over several hours.

Other studies have also shown that hypercapnic acidosis suppresses the response to vasoconstrictors in isolated pulmonary arteries [7]. This suppression was attributed to the effects of acidosis, as euhydric hypercapnia (i.e., pH maintained at 7.4) did not cause any significant changes in agonist-induced tension [8,9]. However, in a subsequent paper, the same group reported that euhydric hypercapnia enhanced depolarisation-induced vasoconstriction in an endothelium- and nitric-oxide-dependent manner, and suggested that elevated CO2 suppressed nitric oxide production [10]. Although this might be consistent with reports of hypercapnia-induced vasoconstriction, it remains unclear why, if hypercapnia does suppress nitric oxide production, a similar potentiation of agonist-induced vasoconstriction was not observed in the same preparation [8,9].

We have therefore reevaluated the effects of hypercapnic acidosis and euhydric hypercapnia in small intrapulmonary arteries (IPA) of the rat, using a protocol designed to detect any delayed effects similar to those described by Gordon et al. [6]. In addition, we examined the role of Na+-dependent HCO3– transport, as it has been suggested that this may play a role in HPV in small pulmonary arteries [11].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Male Wistar rats (200–300 g) were anaesthetized with sodium pentobarbital (55 mg.kg–1 IP) and killed by cervical dislocation as approved by the local Home Office Inspector. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The lungs, intestines and kidneys were excised and placed in cold physiological salt solution (PSS, see below). Small (250–550 µm i.d.) IPA, mesenteric and renal arteries were dissected free of adventitia, mounted in a temperature-controlled myograph at 37 °C (Danish MyoTechnology, Aarhus, Denmark), and gassed continuously with 95% air/5% CO2 (pH 7.4). After 30 min, arteries were stretched to give an equivalent transmural pressure of 30 mm Hg for pulmonary and 100 mm Hg for systemic arteries as previously described [12]. Preparation viability was assessed by measuring the response to 80 mM K+ PSS (KPSS; isotonic replacement of NaCl by KCl), and was deemed suitable for use in experiments only when they produced at least 5 mN tension. Prior to beginning experiments, arteries were equilibrated with three 2-min exposures to KPSS.

2.1. Measurement of intracellular pH in isolated arteries
Following mounting, arteries were incubated with PSS containing 1 µM BCECF/AM for 30 min at room temperature, followed by a further 15 min at 37 °C to facilitate intracellular hydrolytic cleavage of the dye. The preparation was washed with PSS and stimulated repeatedly with high KPSS until a stable tension response was achieved. The myograph was mounted on an inverted microscope (Zeiss Axiovert 200) equipped with an imaging system (Universal Imaging, Downingtown, PA, USA) and illuminated alternately at 490 and 440 nm while measuring the intensities of emitted light via a 550-nm filter using a CCD camera. The ratio was calibrated with nigericin as described by Aalkjaer and Cragoe [13].

2.2. Protocol
Studies on the potentiation of PGF2{alpha}-induced vasoconstriction were performed as follows: After a stable baseline was obtained for >20 min, PGF2{alpha} was added directly to the bath to give a final concentration of 3 µM (~EC10; tension: 8.8 ± 0.5% of the response to KPSS, n=58; change in bath volume <0.1%). After developed tension reached a plateau, the preparation was washed repeatedly with fresh PSS and allowed to return to baseline. Following incubation with any inhibitors for 20 min, the process was repeated. The bath solution was then exchanged for prewarmed and appropriately gassed experimental solution containing inhibitors as required, the gassing of the bath changed if necessary, and the preparation allowed to stabilise for 40 min before addition of the next PGF2{alpha} challenge. In timed controls, there was no significant change in PGF2{alpha}-induced tension at 40 min (n=6).

2.3. Solutions
Control physiological salt solution (PSS; in mM): NaCl 118; NaHCO3 24; KCl 4; CaCl2 1.8; MgSO4 1; NaH2PO4 0.434; glucose 5.56. HEPES-buffered PSS (in mM): NaCl 130; KCl 4; CaCl2 1.8; MgCl2 1; glucose 5.56; HEPES 10, pH adjusted to 7.4 with NaOH. Low Na+ PSS: N-methyl-D-glucamine (NMDG) 118; choline bicarbonate 24; CaCl2 1.8; MgSO4 1; NaH2PO4 0.434; glucose 5.56; pH adjusted to 7.4 with HCl. High [HCO3–] PSS and 80 mM [K+] containing PSS (KPSS) were made by iso-osmotic substitution for NaCl. Unless otherwise stated, all experimental solutions were adjusted to pH 7.4, and were isotonic with normal PSS. All chemicals and drugs were obtained from Sigma-Aldrich (Poole, Dorset, UK) or Calbiochem (Notts., UK). 4,4'-Diisothiocyanato-stilbene-2,2'-di-sulfonic acid (DIDS) and acetazolamide were made up as stock solutions in DMSO; final concentration of DMSO was <0.1%, which had no effect on its own.

2.4. Calculations and statistics
Developed tension is presented as a percentage of the maximum tension obtained to the final 2-min exposure to KPSS during the equilibration procedure, and expressed as % KPSS. Results are shown as mean ± S.E.M., and means compared using paired or unpaired Student's t test as appropriate (SigmaStat, SPSS, Chicago, USA). A difference was deemed significant if P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effect of hypercapnic acidosis on IPA
Changing the aerating gas from 5% CO2 to 10% or 15% in air without adjusting [HCO3–] (hypercapnic acidosis) had no effect on baseline tension, and did not significantly alter the response to 3 µM PGF2{alpha} (Control: 11.3 ± 1.0% KPSS, n=19; 10% CO2, pH 7.12: 11.2 ± 1.4% KPSS, n=9; 15% CO2, pH 6.95: 10.3 ± 1.7% KPSS, n=10).

3.2. Effect of increased PCO2 and [HCO3–] (euhydric hypercapnia) on IPA
IPA were challenged with 3 µM PGF2{alpha} under control conditions, washed and exposed to euhydric hypercapnia by exchanging the PSS for one containing 48 mM [HCO3–] gassed with 10% CO2, pH 7.4. There was little or no immediate change in tension (0.9 ± 0.4% KPSS) following transition to euhydric hypercapnia, but thereafter we observed two patterns of response. In the majority of preparations, tension remained stable for at least 40 min, but the tension induced by 3 µM PGF2{alpha} at 40 min was greatly potentiated (Control: 10.3 ± 0.7% KPSS; euhydric hypercapnia: 56.5 ± 7.1% KPSS; n=17; p<0.001; Fig. 1A). This potentiation was reversed by 10 µM diltiazem (Fig. 1A), and was prevented by preincubation with diltiazem (Control, diltiazem: 9.7 ± 1.7% KPSS; euhydric hypercapnia, diltiazem: 9.6 ± 1.6% KPSS; n=5; NS; data summarised in Fig. 2). We examined whether euhydric hypocapnia (CO2 2%, [HCO3–] 10 mM, pH 7.4) might conversely suppress PGF2{alpha}-induced tension, but this was without effect (Control: 10.7 ± 1.0% KPSS; Euhydric hypocapnia: 10.8 ± 0.6% KPSS; n=4; NS).


Figure 1
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  		Fig. 1 Typical traces of the two types of response exhibited by IPA following transition to euhydric hypercapnia. The initial response to depolarisation with KPSS (80 mM [K+]) is shown for comparison. Panel A shows the potentiation of 3 µM PGF2{alpha}-induced tension after 40 min of euhydric hypercapnia, and its reversal back to the level of the control PGF2{alpha} challenge by 10 µM diltiazem. Panel B shows development of a spontaneous constriction after ~20 min of euhydric hypercapnia, and its reversal to baseline by diltiazem.

 

Figure 2
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  		Fig. 2 Summarised data of the effects of inhibitors and Na+ substitution on the potentiation of PGF2{alpha}-induced tension following 40 min of euhydric hypercapnia. The filled bars show the extent of 3 µM PGF2{alpha}-induced tension under control conditions but in the presence of the respective inhibitor. The open bars show tension following 40 min euhydric hypercapnia. *p<0.01 compared to the respective control; n=4–17.

 
In ~20% of all IPA exposed to euhydric hypercapnia (11 of 58), a relatively slow spontaneous vasoconstriction of variable size developed after a consistent delay of 18.8 ± 1.8 min (Fig. 1B). Application of the L-type Ca2+ channel blocker diltiazem (10 µM) caused immediate vasorelaxation by 90.6 ± 13.5% (Fig. 1B), suggesting that the vasoconstriction was due to a slowly developing depolarisation and activation of Ca2+ entry via L-type channels. To test whether the potentiation of PGF2{alpha}-induced tension observed above was also related to a slowly developing mechanism, we also examined the effects of euhydric hypercapnia after 20 min. PGF2{alpha}-induced tension was only potentiated by a factor of 1.9 ± 0.3 (n=3) at this time. All further experiments were carried out only on IPA that showed a stable baseline tension for 40 min after transition to euhydric hypercapnia.

In order to determine whether a raised PCO2 alone might affect the response to PGF2{alpha}, we examined whether 10% CO2 in the absence of HCO3– could induce potentiation. HEPES PSS was gassed with 10% CO2 and pH corrected to 7.4 with NaOH. No potentiation of PGF2{alpha}-induced tension was observed under these conditions (Fig. 3; Control: 9.9 ± 1.4% KPSS; High CO2, HEPES: 7.4 ± 2.2% KPSS; n=4; NS), suggesting that the potentiation is related to the raised [HCO3–] rather than raised pCO2. The response to PGF2{alpha} was therefore also examined in the absence of CO2 in the aerating gas, using HEPES PSS with and without 24 or 48 mM [HCO3–] (adjusted to pH 7.4). The addition of HCO3– did potentiate PGF2{alpha}-induced tension, but only by a factor of 2 (HEPES: 5.1 ± 0.6% KPSS; HEPES, 24 mM HCO3–: 10.2 ± 1.2% KPSS; n=5; p<0.01). Increasing [HCO3–] to 48 mM had no further effect (n=4; data not shown).


Figure 3
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  		Fig. 3 A trace typical of four experiments showing the lack of potentiation of PGF2{alpha}-induced tension in an IPA following 40 min of HEPES PSS gassed with 10% CO2, pH 7.4.

 
Changes in intracellular pH (pHi) during hypercapnic acidosis (10% CO2, 24 mM [HCO3–], pH 7.12), and euhydric hypercapnia were examined using BCECF. Fig. 4 shows a typical trace representative of three others, demonstrating that although hypercapnic acidosis caused the expected intracellular acidification of about 0.23 pH units, euhydric hypercapnia also caused acidification (~0.15 pH units), although extracellular pH was normal. There was some recovery in pHi towards normal over 40 min, but nevertheless, pHi was still acid at the end of this period. Following return to normal PSS, there was a small alkaline overshoot of similar size to the extent of recovery during the preceding 40 min, presumably reflecting increased intracellular [HCO3–].


Figure 4
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  		Fig. 4 A trace typical of three experiments in which pHi was estimated in IPA using BCECF. A standard NH4Cl challenge was initially performed, showing the typical response of an initial alkalisation followed by transient acidification on washout. Note that challenge with 3 µM PGF2{alpha} after 40 min of euhydric hypercapnia had no discernable effect on pHi. The changes in pHi in response to NH4Cl challenge and hypercapnic acidosis are similar to those previously reported in small IPA [11].

 
It has been suggested that depolarisation-induced vasoconstriction of rat IPA is potentiated under euhydric hypercapnia via a reduction in nitric oxide production from the endothelium [10], whereas agonist-induced vasoconstriction is not [9]. We examined whether the potentiation of PGF2{alpha}-induced vasoconstriction observed here was also due to inhibition of nitric oxide production. IPA were incubated in 100 µM L-NAME for 20 min. L-NAME itself potentiated the response to 3 µM PGF2{alpha} (22.5 ± 4.2% KPSS). Nevertheless, in the presence of L-NAME, euhydric hypercapnia still caused a significant further potentiation of the response to PGF2{alpha} (69.8 ± 9.3% KPSS; n=9; p<0.001; Fig. 2). We also examined the effects of removal of the endothelium by rubbing the IPA lumen with a human hair; adequate removal was ascertained by lack of relaxation to 1 µM acetylcholine following constriction with PGF2{alpha}. As with L-NAME, removal of the endothelium potentiated the response to PGF2{alpha} in normal PSS (31.6 ± 4.6% KPSS, n=6). Following transition to euhydric hypercapnia, endothelium-denuded IPA exhibited a stable baseline for 40 min. The subsequent response to PGF2{alpha} was further potentiated (57.8 ± 4.1% KPSS, n=6, p<0.005), and did not differ significantly from that of intact IPA in the presence euhydric hypercapnia and L-NAME.

The data suggest that both the potentiation of PGF2{alpha}-induced vasoconstriction and the slow spontaneous vasoconstriction are due to depolarisation. It would therefore be predicted that mild depolarisation with 20 mM [K+] should mimic the effect of euhydric hypercapnia and enhance the response to PGF2{alpha}. Depolarisation with 20 mM [K+] caused an increase in tension of 10.4 ± 1.9% KPSS (n=6). Under these conditions, the response to 3 µM PGF2{alpha} was significantly potentiated (65.1 ± 5.8% KPSS, n=6, p<0.001). Conversely, euhydric hypercapnia substantially enhanced vasoconstriction to 20 mM [K+] (Fig. 5, n=3). Both of these findings are consistent with the hypothesis that euhydric hypercapnia causes smooth muscle depolarisation in IPA.


Figure 5
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  		Fig. 5 A trace typical of three experiments showing potentiation of the response to 20 mM [K+] in euhydric hypercapnia.

 
3.3. Mechanism of high [HCO3–]-induced depolarisation in IPA
We examined the effect of DIDS, a nonspecific inhibitor of anion transport and Cl– channels. DIDS (100 µM) itself caused a small but significant reduction in 3 µM PGF2{alpha}-induced tension (Control: 8.0 ± 0.6% KPSS; DIDS: 5.3 ± 0.7% KPSS; n=7; p=0.04); however, it completely abolished potentiation in euhydric hypercapnia (4.9 ± 1.2% KPSS; n=7; Fig. 2). We investigated involvement of Na+-dependent HCO3– transport by removal of extracellular Na+, using as substitutes the impermeant cation NMDG and choline HCO3 for NaCl and NaHCO3, respectively (remaining [Na+]<0.5 mM). Na+ substitution itself caused a small increase in the response to PGF2{alpha}, but again completely abolished any further potentiation in euhydric hypercapnia (Low Na+, control: 19.8 ± 7.0% KPSS; Low Na+, euhydric hypercapnia: 17.6 ± 4.1% KPSS; n=4; p<0.01; Fig. 2).

The carbonic anhydrase inhibitor acetazolamide is reported to have an IC50 of ~1 µM, although doses between 100 µM and 1 mM are commonly used [14]. At 10 µM, acetazolamide had no effect on IPA reactivity (n=4; data not shown). At 100 µM, it caused a significant reduction in PGF2{alpha}-induced tension under control conditions (4.8 ± 0.8% KPSS, n=4), and also reduced the potentiation in euhydric hypercapnia such that this was no longer significant from control (15.0 ± 4.9% KPSS; NS; Fig. 2).

Increasing the outward electrochemical gradient for Cl– by substitution of extracellular Cl– with either methyl sulphonate or iodide elicited a slowly developing but powerful vasoconstriction in IPA (n=9; data not shown), similar to the spontaneous vasoconstriction elicited by euhydric hypercapnia. This implies that a significant Cl– conductance exists in IPA under basal conditions. The potential role of Cl– channels was examined by use of anthracene-9-carboxylic acid (A9C), which has been described as a blocker of Ca2+-activated Cl– channels [15]. A9C (200 µM) itself caused a small suppression of PGF2{alpha}-induced tension, but completely abolished its potentiation by euhydric hypercapnia (A9C, control: 6.0 ± 1.5% KPSS; A9C, euhydric hypercapnia: 6.4 ± 2.7% KPSS; n=4; NS; Fig. 2).

3.4. Effect of euhydric hypercapnia on systemic arteries
To establish whether the potentiation of PGF2{alpha}-induced tension by euhydric hypercapnia was specific to IPA, we performed experiments in small mesenteric and renal arteries. As shown in Fig. 6, no potentiation of PGF2{alpha}-induced tension was observed in mesenteric arteries (Control: 5.1 ± 1.1% KPSS; Euhydric hypercapnia: 4.4 ± 1.1% KPSS; n=10; NS); similar results were obtained for renal arteries (Control: 9.7 ± 1.6% KPSS; Euhydric hypercapnia: 7.3 ± 1.3% KPSS; n=5; NS). The lack of potentiation was not due to suppression of contraction by high CO2-induced intracellular acidification, because depolarisation of mesenteric arteries with 20 mM [K+] caused significant and similar degrees of potentiation of the response to PGF2{alpha} in both control conditions and euhydric hypercapnia (Fig. 7). This also indicates that it is the mechanism causing the depolarisation in response to euhydric hypercapnia that is specific to IPA, and not the effects of the depolarisation itself.


Figure 6
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  		Fig. 6 A trace typical of 10 experiments showing that euhydric hypercapnia does not cause potentiation of 3 µM PGF2{alpha}-induced constriction in small mesenteric arteries.

 

Figure 7
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  		Fig. 7 Summarised data for mesenteric and renal arteries. The filled bars show tension in response to 3 µM PGF2{alpha} (mesenteric, renal) or 20 mM [K+] (mesenteric, 20 mM KCl) under control conditions. The open bars show the response following 40 min euhydric hypercapnia. As in IPA, depolarisation with 20 mM [K+] potentiated the response to PGF2{alpha} in mesenteric arteries (hatched bar); however, this was not significantly depressed during euhydric hypercapnia (grey bar). *p<0.05, n=3–10.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The key novel observation from this study is that euhydric hypercapnia (10% CO2, 48 mM [HCO3–], pH 7.4) substantially increased the vasoreactivity of rat small IPA (Fig. 2), but did not affect vasoreactivity of small mesenteric or renal arteries (Fig. 7). The data strongly suggest that the mechanism involves smooth muscle depolarisation. In contrast, neither euhydric hypocapnia nor hypercapnic acidosis had any significant effect on the response of IPA to low concentrations of PGF2{alpha}. The latter is consistent with previous reports from McLoughlin et al. [7–9] showing that agonist-induced vasoconstriction of pulmonary arteries is resistant to acidosis, which they have suggested is due to the greater importance of Rho-kinase-mediated Ca2+-sensitisation mechanisms in this tissue [16]. PGF2{alpha} has been reported to induce vasoconstriction in pulmonary artery primarily via Ca2+-sensitisation rather than voltage-gated Ca2+ entry [8,17], and this is consistent with our data which show that the L-type channel blocker diltiazem had no effect on constriction induced by 3 µM PGF2{alpha} in normal PSS.

IPA exhibited two patterns of response after exposure to euhydric hypercapnia, with the majority showing a stable basal tension for 40 min, followed by potentiation of both PGF2{alpha}- and 20 mM [K+]-induced vasoconstriction. In ~20% of IPA, however, a slow spontaneous constriction developed after a consistent delay of ~20 min. Both potentiation and spontaneous constriction were reversed by diltiazem, suggesting that both responses were due to the same mechanism, namely, activation of voltage-dependent L-type Ca2+ channels by depolarisation. Moreover, depolarisation with 20 mM [K+] mimicked the effect of euhydric hypercapnia.

The reason why some IPA exhibit a spontaneous constriction during euhydric hypercapnia whilst the majority did not is not clear, as there were no obvious differences between groups. The inability to predict which IPA would spontaneously constrict, coupled with the variable nature of the response, made further experimentation difficult. However, the relationship between depolarisation and constriction is very steep in IPA and effectively has a threshold, so small differences in the extent of depolarisation have profound effects on constriction, or whether or not it occurs at all (e.g., Ref. [18]). Euhydric hypercapnia may depolarise IPA close to this threshold, but in some cases exceed it and so cause constriction. This does not easily explain the apparently consistent nature of the delay before the spontaneous constriction, which needs further study.

Our findings contrast with those reported by Sweeney et al. in rat conduit pulmonary artery [9] and IPA [8], where euhydric hypercapnia did not alter phenylephrine- or PGF2{alpha}-induced vasoconstriction, respectively. There are, however, differences in protocol that might explain these conflicting results. In both studies, Sweeney et al. examined the effects of euhydric hypercapnia after agonist-induced vasoconstriction was established, as opposed to the current study where preparations were preincubated with euhydric hypercapnia for 40 min before challenging with PGF2{alpha}. Moreover, the dose of agonist and size of vasoconstriction were greater, at the EC50 or EC70 [8,9] compared to ~EC10 in the present study. We have previously shown that vasoconstriction of IPA to higher concentrations of PGF2{alpha} (50 µM) has a significant component (~50%) that is verapamil-sensitive, indicating that PGF2{alpha} does induce depolarisation at higher concentrations [19]. This could potentially mask any depolarisation induced by euhydric hypercapnia. Finally, Sweeney et al. [8,9] measured the response 15–20 min after initiation of euhydric hypercapnia, rather than the 40 min used here. If euhydric hypercapnia causes a slowly developing response (see below), 20 min might not have been sufficient to show any significant effect under the above conditions.

The delay in onset and slow nature of the spontaneous constriction plus the reduced potentiation of PGF2{alpha}-induced tension after 20 min implies that the underlying mechanism is slowly developing. This is reminiscent of the phenomenon reported by Gordon et al. [6], whereby an acute (5–10 min) hypercapnic acidosis had no effect on PVR in intact piglets, but when sustained for up to an hour caused a significant rise in basal PVR and potentiated HPV. There could also be parallels with the slow increase in PVR that has been reported during hypercapnia in humans [2]. However, we did not show any alterations in either basal tension or the response to PGF2{alpha} of IPA for up to 40 min of hypercapnic acidosis. On the other hand, both of these studies were performed in intact organisms, and although renal compensation is unlikely to be effective within hours, some degree of extra-renal compensation may occur much faster, with a rise in plasma [HCO3–] due to titration of body nonbicarbonate buffers [20]. It is not possible to say whether plasma [HCO3–] did increase in these studies [2,6], as these data were not provided.

It could be argued that both the potentiating effect of euhydric hypercapnia that we report here and the hypercapnia-associated increases in PVR reported in the previous two studies are due to vasoconstriction in response to hypercapnia per se, and that the reason we do not see an effect of hypercapnic acidosis is that, in our preparation, this vasoconstriction is suppressed by extracellular or intracellular acidosis. This would seem unlikely on two grounds. The first is that PGF2{alpha}-induced vasoconstriction in rat IPA has been shown elsewhere to be unaltered by either hypercapnic acidosis, as also reported here, or an equivalent normocapnic acidosis [8]. The second is that the difference in degree of intracellular acidosis that we observed in IPA during hypercapnic acidosis and euhydric hypercapnia was relatively small (~0.08 pH units), and the amount of recovery of pHi after 40 min was also small (Fig. 4). It would seem implausible that these small differences in pHi could have such profound effects on PGF2{alpha}-induced tension, especially in the light of the study of Hyvelin et al. [16], where it was shown that agonist-induced Rho-kinase-mediated Ca2+ sensitisation in pulmonary artery was essentially unaffected by changes in cytosolic pH.

Nevertheless, we attempted to determine whether the observed effects of euhydric hypercapnia were related to the hypercapnia itself or the increase in [HCO3–], by using HEPES buffered PSS with the addition of either CO2 or HCO3– alone. HEPES PSS gassed with 10% CO2 (pH 7.4) did not affect PGF2{alpha}-induced tension, again suggesting that the potentiation of PGF2{alpha}-induced tension is not related to hypercapnia per se. Conversely, HCO3– containing HEPES gassed with air did show potentiation, albeit only by a factor of 2. Even so, these data might be construed as suggesting that it is primarily the increase in extracellular [HCO3–] that is responsible for observed effects of euhydric hypercapnia. However, these results using HEPES should perhaps be interpreted with some caution, as it is not clear how removal of extracellular CO2 or HCO3– would affect the relative intracellular concentrations of this buffer pair. HEPES is also known to suppress contraction in vascular smooth muscle [21].

Although Sweeney et al. [8,9] have reported that euhydric hypercapnia does not enhance agonist-induced vasoconstriction, the same group have also reported that it does enhance depolarisation-induced vasoconstriction, which they suggested was mediated via inhibition of endothelium-derived nitric oxide production by hypercapnia [10]. However, we found that although both inhibition of nitric oxide synthase with L-NAME and removal of the endothelium potentiated the response to PGF2{alpha}, this was considerably less than that induced by euhydric hypercapnia, and neither L-NAME nor removal of the endothelium prevented further potentiation during subsequent euhydric hypercapnia (see above and Fig. 2). This strongly suggests that neither nitric oxide nor the endothelium plays a role in the potentiation of PGF2{alpha}-induced tension observed here.

As the response to euhydric hypercapnia is slowly developing, the depolarisation is unlikely to be related to any direct effect on ion channels. A more plausible explanation is that it is directly or indirectly related to a change in the composition of intracellular fluid as a result of a transport process, and the implication that the raised [HCO3–] is critical suggests the Na+-dependent HCO3–-Cl– transporter, which has been shown to be important both for recovery from intracellular acidosis and HPV in cat small IPA [11]. Consistent with this, both DIDS, a nonspecific inhibitor of anion transport, and removal of extracellular Na+ abolished the potentiation of PGF2{alpha}-induced tension during euhydric hypercapnia. Our HEPES experiments could be interpreted as showing that both HCO3– and CO2 are required. It has been suggested that carbonic anhydrases associated with certain HCO3– transporters may regulate their action, and perhaps act as sensors for intracellular CO2/HCO3– balance [22]. We found that inhibition of carbonic anhydrase with acetazolamide suppressed the potentiation of PGF2{alpha}-induced tension (Fig. 2), perhaps consistent with a regulatory function on the transporter. Interestingly, like the Na+-dependent HCO3–-Cl– transporter, carbonic anhydrase has also been implicated in HPV [23].

It has been suggested that Cl– channels may play a significant role in the regulation of pulmonary artery tone [24,25]. DIDS also blocks Cl– channels, and it is possible that the depolarisation associated with euhydric hypercapnia could be ultimately due to activation of an outward Cl– current. Consistent with this hypothesis, blockade of Ca2+-activated Cl– channels with A9C completely abolished the potentiation of PGF2{alpha}-induced tension (Fig. 2). The mechanism by which increased HCO3– transport is linked to activation of these Cl– channels remains to be ascertained. We believe that it is unlikely to be due a rise in intracellular Ca2+ secondary to an initial depolarisation caused by increased transporter activity, as one would predict that in that case there would still be some potentiation of PGF2{alpha}-induced tension in the presence of A9C. Moreover, modulation of Na+-dependent HCO3–-Cl– transport in vascular smooth muscle has been shown to have little effect on membrane potential [26].

In summary, our results suggest that euhydric hypercapnia enhances the vasoreactivity of rat small IPA, but not mesenteric or renal arteries, by mechanisms involving enhanced HCO3– transport, activation of Ca2+-activated Cl– channels and subsequent depolarisation. The most logical initial stimulus is the effect of an increased transmembrane HCO3– gradient on the normal intracellular alkalisation mechanisms activated by the fall in pHi. The fact that this phenomenon appears to be specific to pulmonary arteries is of particular interest, especially considering that it has been suggested that both the Na+-dependent HCO3–-Cl– transporter and Ca2+-activated Cl– channels play an important role in the regulation of pulmonary vascular tone and HPV [11,24,25]. It is also of note that both A9C-inhibitable Cl– channels and HCO3–-Cl– transport have been implicated in the response of the carotid body chemosensor to hypercapnia and hypoxia [27]. From a functional and clinical standpoint, these findings may also at least partly explain why chronic hypercapnia with a compensated acidosis is associated with promotion of pulmonary hypertension and increased mortality in COPD patients [1].


   Acknowledgements
 
This work was supported by the Wellcome Trust (grant numbers 069759 [MV] and 062554).


   Notes
 
1 Current address: Department of Pathological Physiology 2. Lekarska Fakulta UK V Uvalu 84 Praha 5, 150 000 Czech Republic. Back

Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. MacNee W. Pathophysiology of cor pulmonale in chronic obstructive pulmonary disease. Part one. Am. J. Respir. Crit. Care Med. (1994) 150:833–852.[ISI][Medline]
  2. Balanos G.M., Talbot N.P., Dorrington K.L., Robbins P.A. Human pulmonary vascular response to 4 h of hypercapnia and hypocapnia measured using Doppler echocardiography. J. Appl. Physiol. (2003) 94:1543–1551.[Abstract/Free Full Text]
  3. Barer G.R., Shaw J.W. Pulmonary vasodilator and vasoconstrictor actions of carbon dioxide. J. Physiol. (1971) 213:633–645.[Abstract/Free Full Text]
  4. Baudouin S.V., Evans T.W. Action of carbon dioxide on hypoxic pulmonary vasoconstriction in the rat lung: evidence against specific endothelium-derived relaxing factor-mediated vasodilation. Crit. Care Med. (1993) 21:740–746.[ISI][Medline]
  5. Brimioulle S., Lejeune P., Vachiery J.L., Leeman M., Melot C., Naeije R. Effects of acidosis and alkalosis on hypoxic pulmonary vasoconstriction in dogs. Am. J. Physiol. (1990) 258:H347–H353.[ISI][Medline]
  6. Gordon J.B., Rehorst-Paea L.A., Hoffman G.M., Nelin L.D. Pulmonary vascular responses during acute and sustained respiratory alkalosis or acidosis in intact newborn piglets. Pediatr. Res. (1999) 46:735–741.[ISI][Medline]
  7. Sweeney M., O'Regan R.G., McLoughlin P. Effects of hypercapnia on steady state, phenylephrine-induced tension in isolated rings of rat pulmonary artery. Adv. Exp. Med. Biol. (1996) 410:463–469.[Medline]
  8. Sweeney M., O'Regan R.G., McLoughlin P. Effects of changes in pH and PCO2 on wall tension in isolated rat intrapulmonary arteries. Exp. Physiol. (1999) 84:529–539.[Abstract]
  9. Sweeney M., Beddy D., Honner V., Sinnott B., O'Regan R.G., McLoughlin P. Effects of changes in pH and CO2 on pulmonary arterial wall tension are not endothelium dependent. J. Appl. Physiol. (1998) 85:2040–2046.[Abstract/Free Full Text]
  10. Lynch F., Sweeney M., O'Regan R.G., McLoughlin P. Hypercapnia-induced contraction in isolated pulmonary arteries is endothelium-dependent. Respir. Physiol. (2000) 121:65–74.[CrossRef][ISI][Medline]
  11. Madden J.A., Ray D.E., Keller P.A., Kleinman J.G. Ion exchange activity in pulmonary artery smooth muscle cells: the response to hypoxia. Am. J. Physiol. Lung Cell. Mol. Physiol. (2001) 280:L264–L271.[Abstract/Free Full Text]
  12. Leach R.M., Robertson T.P., Twort C.H., Ward J.P.T. Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries. Am. J. Physiol. (1994) 266:L223–L231.[ISI][Medline]
  13. Aalkjaer C., Cragoe E.J. Jr. Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels. J. Physiol. (1988) 402:391–410.[Abstract/Free Full Text]
  14. Berg J.T., Ramanathan S., Gabrielli M.G., Swenson E.R. Carbonic anhydrase in mammalian vascular smooth muscle. J. Histochem. Cytochem. (2004) 52:1101–1106.[Abstract/Free Full Text]
  15. Piper A.S., Greenwood I.A. Anomalous effect of anthracene-9-carboxylic acid on calcium-activated chloride currents in rabbit pulmonary artery smooth muscle cells. Br. J. Pharmacol. (2003) 138:31–38.[CrossRef][ISI][Medline]
  16. Hyvelin J.M., O'Connor C., McLoughlin P. Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. (2004) 287:L673–L684.[Abstract/Free Full Text]
  17. Himpens B., Kitazawa T., Somlyo A.P. Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflugers Arch. (1990) 417:21–28.[CrossRef][ISI][Medline]
  18. Robertson T.P., Dipp M., Ward J.P.T., Aaronson P.I., Evans A.M. Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat. Br. J. Pharmacol. (2000) 131:5–9.[CrossRef][ISI][Medline]
  19. Priest R.M., Robertson T.P., Leach R.M., Ward J.P.T. Membrane potential-dependent and -independent vasodilation in small pulmonary arteries from chronically hypoxic rats. J. Pharmacol. Exp. Ther. (1998) 285:975–982.[Abstract/Free Full Text]
  20. Giebisch G., Berger L., Pitts R.F. The extra-renal response to acute acid–base disturbances of respiratory origin. J. Clin. Invest. (1955) 34:231–245.[ISI][Medline]
  21. Kane J., Macdonald R.L., Zhang J., Sima B. HEPES inhibits contractile responses of canine basilar artery. Neurol. Res. (1997) 19:527–533.[ISI][Medline]
  22. Sterling D., Reithmeier R.A., Casey J.R. A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers. J. Biol. Chem. (2001) 276:47886–47894.[Abstract/Free Full Text]
  23. Deem S., Hedges R.G., Kerr M.E., Swenson E.R. Acetazolamide reduces hypoxic pulmonary vasoconstriction in isolated perfused rabbit lungs. Respir. Physiol. (2000) 123:109–119.[CrossRef][ISI][Medline]
  24. Yuan X.J. Role of calcium-activated chloride current in regulating pulmonary vasomotor tone. Am. J. Physiol. (1997) 272:L959–L968.[ISI][Medline]
  25. Wang Q., Wang Y.X., Yu M., Kotlikoff M.I. Ca(2+)-activated Cl- currents are activated by metabolic inhibition in rat pulmonary artery smooth muscle cells. Am. J. Physiol. (1997) 273:C520–C530.[ISI][Medline]
  26. Aalkjaer C., Hughes A. Chloride and bicarbonate transport in rat resistance arteries. J. Physiol. (Lond.) (1991) 436:57–73.[Abstract/Free Full Text]
  27. Iturriaga R., Mokashi A., Lahiri S. Anion exchanger and chloride channel in cat carotid body chemotransduction. J. Auton. Nerv. Syst. (1998) 70:23–31.[CrossRef][ISI][Medline]

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