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Effects of modulators of TASK potassium channels on rat pulmonary artery tone

Effects of modulators of TASK potassium channels on rat pulmonary artery tone Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn019 ......................................................................................................................................................................................................................................... Research article Effects of modulators of TASK potassium channels on rat pulmonary artery tone Sajni Dipak Shah* Faculty of Life Sciences, University of Manchester, Core Technology Facility, Manchester, UK. * Corresponding author: 15 Crundale Avenue, Kingsbury, London NW9 9PJ, UK. Tel: þ44 (0)7828 974984. Email: sajds@hotmail.co.uk Supervisor: Professor A. M. Gurney, Faculty of Life Sciences, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, UK. ........................................................................................................................................................................................................................................ TWIK-related acid-sensitive potassium (TASK) channels have been implicated as having a role in maintaining and mediating the tone of pulmonary arteries by influencing the membrane potential of the smooth muscle cells. Inhibition of these channels would be expected to promote depolarization, calcium influx and contraction. The purpose of this study was to investigate the effects of TASK modulators on rat intrapulmonary artery tone. The modulators included pH, an important physiological TASK modulator, and drugs that inhibit TASK channels, such as bupivacaine, methanandamide and zinc. Small vessel myography was used to measure the tone of both conduit and resistance pulmonary arteries. Cumulative bupivacaine and methanandamide dose–response curves were compared with phenyle- phrine (a sympathomimetic vasoconstrictor). The effects of pH were investigated on vessel tone and responses to bupivacaine, metha- nandamide or zinc chloride. Bupivacaine and methanandamide (.10 mM) resulted in increased artery tone, with similar effects seen in conduit and resistance vessels. Zinc had no effect, possibly reflecting an inhibitory action on calcium channels. In the presence of endo- thelial blockers, methanandamide (100 mM) still resulted in an increase in artery tone, implying an action on smooth muscle. The appli- cation of nifedipine resulted in the inhibition of the response seen with bupivacaine (100 mM), implying that voltage-gated calcium entry was involved. Changing the pH from 7.3 to 8.3 resulted in vasoconstriction, and a relaxation was seen in acidic conditions. This is oppo- site to the result expected for TASK channel modulation, but may reflect the multiple effects of pH on smooth muscle. The contractions seen with bupivacaine and methanandamide were increased at pH 8.3 but inhibited at pH 6.3, consistent with an effect on TASK chan- nels. Responses to bupivacaine and methanandamide were, however, very small, suggesting that currents produced by TASK channels may not be a major factor in contracting intrapulmonary arteries, but may still have a minor role. Key words: arterial tone, TASK channels, rat, bupivacaine, methanandamide, pH. ........................................................................................................................................................................................................................................ The a-subunit of the two-pore domain channels, which Introduction are mainly expressed as homodimers, comprised four trans- The TWIK-related acid-sensitive potassium (TASK) channels, membrane segments, two pore-forming domains and a a subtype of the two-pore domain family of potassium chan- short N-terminus and a long C-terminus, both of which 6, 7 8 9 nels, have been implicated as having a role in maintaining and are intracellular. Five TASK channels, TASK-1, 22, 10 11 12 mediating the tone of pulmonary arteries. Thus, the inhibition 23, 24 and 25, have been cloned thus far. Studies of TASK channels prevents potassium efflux and results in have found the presence of TASK-1 proteins and mRNA in depolarization, and activation leads to hyperpolarization of rat, rabbit and human PASMCs as well as in rat endothelial 1–6 2–5 the pulmonary artery smooth muscle cells (PASMCs). cells, suggesting a role for TASK channels, particularly Depolarization of the cell membrane is expected to enhance TASK-1, in setting resting membrane potential. A small the open probability of L-type calcium channels, allowing role has also been suggested for TASK-2 by the finding that calcium entry into the cell which leads to constriction of the small interfering RNA directed against the TASK-2 mRNA artery. Conversely, hyperpolarization would result in the reduced the smooth muscle resting membrane potential and closure of voltage-dependent calcium channels, thus decreas- the effects of pH on membrane potential in intact intrapul- ing calcium entry and subsequent vasodilation. monary arteries (IPAs). ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 114 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Three known modulators of TASK channels have been MgCl 1, glucose 5 and CaCl 1. The pH was adjusted to 2 2 investigated in rat, rabbit and human PASMCs: bupivacaine, 7.3 with NaOH, bubbled with air and maintained at 37ºC. a local anaesthetic, the endogenous cannabinoid anand- 2þ amide and Zn . In electrophysiological studies, a voltage- Myography insensitive potassium current was found to be inhibited by these three drugs, suggesting that they might cause an Chart software (J. Dempster, Strathclyde University) was 2–4 3 increase in IPA tone. Gardener et al. looked at the used to record the isometric tensions of the vessels. After effect of anandamide and bupivacaine on vessel tone and the vessels were mounted, resting tensions of 5 and 4 mN found a gradual increase, which was concentration- were applied to the large and small vessels, respectively, to dependent. Whether it was due to an effect on TASK and provide some initial tone, followed by an equilibration voltage-gated calcium channels was not investigated. period of 30 min. At the start of every experiment, vessels An important modulator of membrane potential in these were contracted three times using 50 mM KCl, with relax- 2–5 studies was pH, which also affects the TASK channels. ation back to baseline occurring between each contraction Increasing and decreasing the extracellular pH of PASMCs in response to being washed with PSS. The final potassium had the effect of activating a potassium current, leading to contraction was used as the reference against which all hyperpolarization, or inhibition of the current, leading to measurements were made. 2– 5,13 depolarization of the membrane, respectively. In theory, this suggests that an alkaline environment would Experimental procedures cause a relaxation in arterial tone, although this may only be seen in conditions that raise baseline tone, whereas an Individual phenylephrine, bupivacaine and methanandamide increase in baseline arterial tone would be seen with a dose–response curves were created by applying increasing 29 23 more acidic pH. Surprisingly, few studies have addressed and cumulative concentrations (10 M–10 M) of the 2–5 the effects of pH on isolated pulmonary arteries; there- drugs to the 5 ml bath of PSS surrounding the vessels. fore, it is not yet clear if this prediction holds true. The interval between each application was 5 min or after The aim of this study was to investigate the effects of the response was seen to reach its peak. TASK channel modulator drugs and pH on rat IPA tone. After creating the dose–response curve for methananda- Both large conduit and small resistance vessels were used, mide followed by washing, the endothelial inhibitors, because arteries from different levels of the pulmonary arter- N-nitro-L-arginine methyl ester (L-NAME) (100 mM) and ial tree are known to respond differently to a variety of phys- indomethacin (10 mM), were added to the myograph iological stimuli, and the size and source of arteries are chamber and left to equilibrate for 40 min. A second thought to be important factors in determining responsive- dose–response curve was then created in the same manner ness to pH. as the first in order to compare the two conditions. Nifedipine, a calcium channel blocker, was used to inves- tigate the mechanism of the bupivacaine contraction. The Materials and methods response to 100 mM bupivacaine was recorded. After washing off the bupivacaine with PSS, 1 mM nifedipine Preparation of vessels was administered for 10 min followed by 100 mM Sprague Dawley rats (250–300 g) were killed by cervical dis- bupivacaine. location in accordance with Schedule 1 of the UK Animals The effect of pH was examined by adjusting the PSS to pH (Scientific procedures) Act and the lungs and heart 6.3 (acid) or pH 8.3 (alkaline) using 1 M HCl or NaOH. removed. Both large (conduit) and small (resistance) IPAs After equilibration at each pH, either bupivacaine or metha- were required. Large vessels were taken from the middle of nandamide (10 and 100 mM) or zinc chloride (100 and the main conduit artery running the length of the lung lobe 200 mM) was applied individually and the responses with an approximate diameter of 500 mm. The small recorded. vessels, with an approximate diameter of 200 mm, were taken from the second- and third-order branches. Vessels Data analysis were cut into segments 1–2 mm long and suspended hori- zontally in 5 ml baths of a multi-channel myograph Cursor measurements of the amplitudes of responses were (Danish Myotechnology, Aarhus, Denmark). The larger entered into Excel where the drug responses were calculated vessels were mounted on pins attached to a force transducer; as a percentage of maximal 50 mM KCl-induced tension. the smaller vessels were gently threaded onto steel wires Data are displayed as a means+ standard error of the attached to the force transducer. The baths contained physio- mean. Data were analysed using the paired t-test or logical salt solution (PSS) of composition (in millimolar): one-way analysis of variance (ANOVA) where appropriate. NaCl 122, KCl 5, HEPES 10, KH PO 0.5, NaH PO 0.5, Significance was assumed if P, 0.05. 2 4 2 4 ......................................................................................................................................................................................................................................... 115 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Reagents used in small and large vessels were analysed by a paired t-test and not found to be significantly different. Stock solutions (10 mM) of phenylephrine (Sigma) and bupi- Methanandamide, another TASK channel inhibitor, also vacaine hydrochloride (Sigma) were prepared with distilled caused a concentration-dependent increase in vessel tension; water, methanandamide (TOCRIS) was dissolved in however, the response was very small (Fig. 1B and D). The ethanol and stored in aliquots in the freezer. A 100 mM methanandamide (n ¼ 5) response was much smaller than stock solution of zinc chloride (Fluka) was prepared and (, 20%) that induced by KCl or phenylephrine. As can be stored in the fridge. These solutions were serially diluted in seen in Fig. 1D, arterial tension did not increase until PSS as required. 100 mM methanandamide was administered. A paired t-test showed that the maximum tensions recorded in response to 100 mM methanandamide for the large and small IPAs were Results not significantly different. Vessels were used for experiments only if they contracted in response to the application of 50 mM KCl. In each case, The Influence of the endothelium on the effects of three reproducible responses, averaging 2–3 mN in ampli- methanandamide tude for both large and small IPAs, were obtained to 50 mM The influence of the endothelium was inhibited with the use KCl before the experiments began. of the endothelial blockers L-NAME and indomethacin, which inhibit the release of the vasodilators nitric oxide The effect of TASK channel inhibitors on vessel tone and prostacyclin, respectively. Physical removal of the endo- The effect of applying increasing concentrations of bupiva- thelium was not undertaken due to the difficulty in removing caine (n ¼ 5) on IPA tone can be seen in Fig. 1A and C. the endothelium from the small vessels. The response to Both large and small IPAs were noted to have increased 100 mM methanandamide seen following the addition of tone with increasing concentrations of bupivacaine; endothelial blockers (n ¼ 4) was similar to that seen with however, significant changes in tone were only seen at 10 methanandamide alone (Fig. 2). A paired t-test comparing and 100 mM for the large IPA and 100 mM for the small the tensions produced at 100 mM methanandamide in the IPA. The tensions produced were much lower than presence and absence of endothelial blockers for both types (,30%) those achieved by 50 mM KCl or phenylephrine. of vessels suggests that the responses to methanandamide The maximum tensions produced by 100 mM bupivacaine were not affected by the endothelial blockers. Figure 1. Effects of bupivacaine (BUP) and methanandamide (ANA) on pulmonary artery tone. Raw traces show the tension produced in a large IPA in response to increasing concentrations (1 nM–100 mM) of bupivacaine (A) or a single application of 100 mM methanandamide (B). Cumulative concen- tration–response curves for bupivacaine (C, n ¼ 3) and methanandamide (D, n ¼ 5) show the mean+ SEM. Increase in tone expressed as a percentage of that induced by 50 mM KCl in the same tissue. The concentration–effect curve for phenylephrine is shown for comparison. ......................................................................................................................................................................................................................................... 116 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. Influence of the endothelium on the contractile response to methanandamide. Responses to 100 mM methanandamide, expressed as a percentage of the response to 50 mM KCl, are shown in large and small IPAs, before and after the addition of the endothelial blockers, L-NAME (100 mM) and indomethacin (10 mM), to the myograph chamber. Bars show mean+ SEM. of four to five experiments. The effect of inhibitors of depolarization-induced calcium entry on the responsiveness of IPA to TASK channel inhibitors Nifedipine, a calcium channel antagonist, inhibits Figure 3. Effect of nifedipine on the contractile response to bupivacaine. depolarization-induced calcium entry, thus indicating that (A) Raw trace showing tension induced by 100 mM bupivacaine in a large activation of voltage-gated calcium channels has occurred. IPA before and after applying 1 mM nifedipine. (B) Histogram showing Such a step was proposed as part of the mechanism involving mean tension (+SEM n ¼ 3), measured as a percentage of the response 2, 3 TASK channel inhibition in pulmonary arteries. Thus, if induced by 50 mM KCl, in response to 100 mM bupivacaine, in the absence (con) and presence of 1 mM nifedipine (nif). Responses are the contraction caused by TASK channel inhibitors is due shown for large and small IPAs. **P, 0.01 versus control (paired t-test). to voltage-gated calcium entry, it should be blocked by nife- dipine. The addition of 100 mM bupivacaine produced an increase in IPA tone. However, when bupivacaine was The change in tension induced by pH 6.3 compared with added after the administration of 1 mM nifedipine, this that induced at pH 8.3 was found to be significantly different response was inhibited (Fig. 3). The raw trace of this using a paired t-test for both types of vessels (large IPA: response can be seen in Fig. 3A and the mean results from P, 0.005 and small IPA: P, 0.01). both large and small IPAs in Fig. 3B. This pattern of block was seen with both the large and small IPAs. A paired The effect of pH on the responsiveness of IPA t-test comparing the mean bupivacaine-induced tensions in to TASK channel inhibitors the absence and presence of nifedipine showed that the The concentrations of 10 and 100 mM bupivacaine (n ¼ 5) results for the large vessel were significantly different and 100 mM methanandamide (n ¼ 3) were chosen to be (Fig. 3B, P, 0.01). applied in the presence of PSS adjusted to pH 8.3 and 6.3, as it was shown by the dose–response curves in Fig. 1 that The effect of pH on artery tone these concentrations produced adequate increases in tone The effect of changing the pH of the bath solution was to to allow the effects of pH on their activity to be cause an increase or decrease in the vessel tone, although demonstrated. the effect was usually small and variable. Changing pH The effect of pH on bupivacaine and methanandamide from 7.3 to 8.3, thus making it more alkaline, produced an responses can be seen in Fig. 4. In the raw traces shown increase in the IPA tone, whereas changing pH from 7.3 to (Fig. 4A and B), changing pH had little effect by itself, but the more acidic pH 6.3 caused a decrease in tension. The it did influence the response to bupivacaine and methanand- mean percentage increases in tone (relative to the 50 mM amide. The bar charts (Fig. 4C and D) show that the KCl-induced contraction) on changing pH from 7.3 to 8.3 responses to these drugs were inhibited at pH 6.3, whereas (n ¼ 6) for the large and small vessels were 16+ 5% and the responses were enhanced at pH 8.3. This pattern is 56+ 19%, respectively. On changing from pH 7.3 to 6.3 clear at 100 mM bupivacaine and methanandamide, which (n ¼ 6), the percentage decreases in tone were 25+ 2% produced substantial contractions at pH 8.3, but had no and 211+ 6% for the large and small arteries, respectively. effect at pH 6.3. A similar response pattern was seen with ......................................................................................................................................................................................................................................... 117 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 4. Effect of pH on contractile responses to bupivacaine (BUP) and methanandamide (ANA). Raw traces show the effect on tension in a large IPA of changing external pH from 7.3 to 6.3 and then 8.3, and the effect of 10 mM and 100 mM bupivacaine (A)or100 mM methanandamide (B) applied at each pH. Histograms show mean tension (+SEM, n ¼ 3) induced by 10 or 100 mM bupivacaine (C) or 100 mM methanandamide (D) at pH 6.3, 7.3 and 8.3 in large and small IPAs. Responses are expressed as a percentage of the contractile response to 50 mM KCl. **P, 0.01 (ANOVA) comparing pH 6.3, 7.3 and 8.3. the lower concentration of 10 mM bupivacaine; however, this application of nifedipine indicates that bupivacaine probably concentration produced small effects when compared with does cause the opening of L-type calcium channels via TASK those recorded for 100 mM bupivacaine, which, in this channel inhibition. In the presence of nifedipine, calcium was experiment, were only measurable at pH 8.3. Using unable to enter the cell and cause vasoconstriction, despite ANOVA, it was found that the differences in tension seen bupivacaine causing depolarization. As pulmonary arteries with changing the pH were statistically significant for bupi- do not express sodium channels, a local anaesthetic action vacaine at 100 mM on the large artery (P, 0.01). on Na channels cannot contribute to the result, although Zinc chloride, at concentrations blocking TASK channels inhibition of another K channel cannot be excluded. in PASMCs (100 and 200 mM), was found to produce no The increase in the vessel tone at the higher concentrations changes in tension at pH 6.3, 7.3 or 8.3. of 10 and 100 mM bupivacaine in large and small vessels may reflect the point at which the numbers of inhibited TASK channels reach the level required to cause increased Discussion tension. This is possibly the concentrations at which suffi- cient numbers of TASK channels are closed to bring the Bupivacaine was found to increase vessel tone. Gardener membrane potential to the calcium channel activation et al. also found this when investigating the effect of bupi- threshold, after which calcium enters and results in contrac- vacaine on pulmonary arteries, with the increase in tone tion. Gardener et al. found similar concentration depen- being concentration-dependent. Bupivacaine is a local anaes- dence with an increase in the vessel tone occurring at a thetic drug that is believed to inhibit TASK channels. It has concentration slightly higher than 10 mM. been suggested that TASK channel inhibition leads to Methanandamide, another putative inhibitor of TASK-1 depolarization due to a decrease in the potassium efflux. channels, also increased arterial muscle tension at concen- The resulting depolarization enhances the open probability trations above 10 mM. Gardener et al. reported similar find- of L-type calcium channels. Subsequently, calcium enters ings. This contraction may have been due to depolarization the smooth muscle cells, resulting in muscle contraction as a result of TASK channel inhibition; however, this was and vasoconstriction. By using nifedipine to block these not studied further. Methanandamide is a cannabinoid, so calcium channels, it was possible to test whether this is an action on cannabinoid receptors cannot be ruled out. the mechanism by which bupivacaine exerts its effects. The Two cannabinoid receptors, CB1 and CB2, were found to fact that bupivacaine did not increase vessel tone after the ......................................................................................................................................................................................................................................... 118 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... be present in the endothelium upon which anandamide may opposite to what is found in the intact lung and is 17, 18 exert relaxant effects. This would counteract and limit hypothesized from the effects of pH on TASK channels. the amplitude of any direct contractile action of Findings from studies investigating TASK channel currents methanandamide on the smooth muscle. TASK channels and their influence on the membrane potential showed that have been found to be present in the endothelium as well an acidic pH causes depolarization and an alkaline pH 2–4 as in the smooth muscle cells of pulmonary arteries, causes hyperpolarization due to inhibition or activation of 2–5 and the inhibition of endothelial TASK channels could also TASK currents, respectively. Thus, it would be expected influence the constrictor effect of methanandamide. To that acidic conditions would result in vasoconstriction and investigate the possible influence of the endothelium on alkaline conditions would cause vasodilation. The present responses to methanandamide, the effect of blocking the finding is consistent with one other study, in which relax- release of endothelium-derived relaxing factors on its ation of pulmonary artery in the presence of extracellular effects was, therefore, investigated. The endothelial blockers acidosis was seen, as well as with the known characteristics used, L-NAME and indomethacin, are nitric oxide synthase of systemic vessels. and cyclooxygenase inhibitors, respectively. They inhibit The effects of pH that was observed on IPA tone cannot be the production of nitric oxide and prostacyclin, both of due to its effect on TASK channels. As pH can affect the which are relaxing factors produced by the endothelium. If activities of many pathways involved in regulating vascular the constrictor effect of methanandamide was limited by tone, it is possible that TASK channel inhibition does con- concurrent endothelium-dependent relaxation, it would be tribute, but is masked by opposing effects. Vascular tone is expected that the addition of endothelial blockers would mainly determined by the intracellular concentration of cause it to be increased. This was not observed. calcium ions, and the influence of extracellular and intra- Methanandamide produced a response in the presence of cellular acidosis and alkalosis on the calcium ion concen- the endothelial blockers that was not significantly different tration has been investigated with conflicting results. from that in their absence. However, it cannot be ruled out Wakabayashi and Groschner found that a decrease in the that TASK channel inhibition or activation of cannabinoid extracellular, but not intracellular, proton concentration receptors in the endothelium by methanandamide may (i.e. extracellular alkalosis) caused calcium entry into endo- result in the release of factors not blocked by L-NAME and thelial cells, which resulted in the release of vasoactive indomethacin. One such factor that may be involved is the mediators and thus a decrease in the vascular tone. It has endothelium-derived hyperpolarizing factor (EDHF), which also been suggested that the vasodilator effects seen in acid has been implicated in cannabinoid-induced mesenteric pH may be due to its action on the endothelium; however, 17 13,21 vasodilation in the rat. Thus, an EDHF-dependent relax- two studies, one in which the endothelium was ation may be masking the full methanandamide contraction. removed and one using endothelial blockers, found that the Although both bupivacaine and methanandamide caused relaxation was endothelium-independent. Klockner and constriction of IPA, the tensions produced were very small. Isenberg suggested that extracellular protons exert their Phenylephrine, an a-adrenergic agonist, is known to cause effects in the same way as intracellular acidification as they constriction of IPA and its effects showed the vessels used can permeate the membrane of vascular smooth muscle to be viable, as did the constriction to 50 mM KCl. The cells. Krampetz and Rhoades found that intracellular alka- responses to bupivacaine and methanandamide were far losis caused direct contraction of PASMCs through the smaller than those produced by phenylephrine, even at release of calcium from intracellular sites; however, they 100 mM, which is well above the concentrations producing also found that intracellular acidosis caused contraction via 16,19 50% block of TASK channels. This indicates that voltage-activated calcium channels. pH has been found to TASK channel block does not induce the maximal IPA affect the activity of L-type calcium channels. Intracellular tension and suggests that TASK currents are not of major acidification decreases, and intracellular alkalosis increases, 22, 23, 25, 26 importance in contracting IPA, but may still have a minor calcium influx through these channels. It must role. be noted, however, that the aforementioned effects of pH on vascular smooth muscle cells do not necessarily occur in The effect of pH on tone and responsiveness of IPA IPA. to TASK channel inhibitors Although pH did not affect basal tone in the way expected In the intact rat lung, acidosis causes vasoconstriction and of TASK channel modulation, it did influence the responses alkalosis causes vasodilation in the presence of elevated to bupivacaine and methanandamide. There was a very basal tone. Surprisingly, however, there is very little in slight or no response from both the drugs at pH 6.3. This the literature about the effects of extracellular pH on tone may be due to the acid conditions inhibiting TASK channels in isolated IPA. In this study, changing pH to create an so that no further modulation by bupivacaine and methanan- acidic environment had the effect of relaxing IPA, whereas damide can take place. The inhibitory effect of an acid pH is in more alkaline conditions, the IPA constricted. This is believed to occur due to protonation of the histidine residue ......................................................................................................................................................................................................................................... 119 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... at position 98 on the TASK subunit, which leads to channel research into the development of TASK modulator drugs 10,26 block, thus preventing the efflux of potassium ions. With will help, in the future, to clarify the role of TASK channels the channel already blocked, further interaction with in regulating pulmonary artery tone. modulator drugs would have no effect. Alkaline conditions caused an enhanced response to both Acknowledgements drugs. Patch clamp studies have shown that an alkaline pH results in hyperpolarization, which is reversed by both drugs Obtaining rats and microdissection of the pulmonary vessels and to a membrane potential that is close to that reached were carried out by Professor A. Gurney (Supervisor), Boris 2–4, 8 when the drugs are applied at pH 7.3. From these Manoury (Research Fellow), Shreena Joshi (PhD Student) effects of pH, it might be expected that, in terms of vessel and Sarah Etheridge (Technician). All other work in this tone, the alkaline pH would have little effect on responsive- project was carried out by the student: Sajni Dipak Shah. ness to bupivacaine and methanandamide. The enhanced I would like to thank Professor A. M. Gurney for guidance effect of the drugs at pH 8.3 can be explained, however, if throughout. we consider the initial contraction seen with the change of pH from 7.3 to 8.3. This caused the tension to be slightly Funding higher than normal, so that the subsequent administration of TASK channel blockers may have more easily caused vasocon- Biotechnology and Biological Sciences Research Council striction as the efflux of potassium ions was inhibited. The (grant BBS/B/11761/2). initially higher baseline tone at pH 8.3, compared with 7.3 or 6.3, may therefore have had a synergistic effect with the References actions of bupivacaine and methanandamide. Although zinc chloride is also an inhibitor of TASK chan- 1. Gurney AM, Osipenko ON, Macmillan D et al. (2002). 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Exp Physiol 84: and role of TASK channels in IPA, it is hoped that further 529–539. ......................................................................................................................................................................................................................................... 120 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 14. Chootip K, Ness KF, Wang Y et al. (2002) Regional variation in P2 receptor 21. Sweeney M, Beddy D, Honner V et al. (1998) Effects of changes in pH and expression in the rat pulmonary arterial circulation. Br J Pharmacol 137: 637–646. CO on pulmonary arterial wall tension are not endothelium dependent. J Appl Physiol 85: 2040–2046. 15. 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Ja´rai Z, Wagner JA, Varga K et al. (1999) Cannabinoid-induced mesenteric 25. Iino S, Hayashi H, Saito H et al. (1994) Effects of intracellular pH on calcium vasodilation through an endothelial site distinct from CB1 or CB2 receptors. currents and intracellular calcium ions in the smooth muscle of rabbit portal PNAS 96: 14136–14141. vein. Exp Physiol 79: 669–680. 19. Zoratti C, Kipmen-Korgun D, Osibow K et al. (2003) Anandamide initiates 26. Schuhmann K, Voelker C, Hofer GF et al. (1997) Essential role of the beta 2þ Ca signaling via CB receptor linked to phospholipase C in calf pulmonary subunit in modulation of C-class L-type Ca2þ channels by intracellular endothelial cells. Br J Clin Pharm 140: 1351–1362. pH. FEBS Lett 408: 75–80. 2þ 20. Gao Y, Tassiopoulos AK, McGraw DJ et al. (1999) Segmental pulmonary vas- 27. Busselberg D, Michael D, Evans ML et al. (1992) Zinc (Zn ) blocks voltage cular responses to changes in pH in rat lungs: role of nitric oxide. Acta gated calcium channels in cultured rat dorsal root ganglion cells. Brain Res Anaesthesiol Scand 43: 64–70. 593: 77–81. ........................................................................................................................................................................................................................................ Submitted on 26 September 2007; accepted on 28 January 2008; advance access publication 17 April 2008 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Effects of modulators of TASK potassium channels on rat pulmonary artery tone

Bioscience Horizons , Volume 1 (2) – Jun 17, 2008

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Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn019 ......................................................................................................................................................................................................................................... Research article Effects of modulators of TASK potassium channels on rat pulmonary artery tone Sajni Dipak Shah* Faculty of Life Sciences, University of Manchester, Core Technology Facility, Manchester, UK. * Corresponding author: 15 Crundale Avenue, Kingsbury, London NW9 9PJ, UK. Tel: þ44 (0)7828 974984. Email: sajds@hotmail.co.uk Supervisor: Professor A. M. Gurney, Faculty of Life Sciences, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, UK. ........................................................................................................................................................................................................................................ TWIK-related acid-sensitive potassium (TASK) channels have been implicated as having a role in maintaining and mediating the tone of pulmonary arteries by influencing the membrane potential of the smooth muscle cells. Inhibition of these channels would be expected to promote depolarization, calcium influx and contraction. The purpose of this study was to investigate the effects of TASK modulators on rat intrapulmonary artery tone. The modulators included pH, an important physiological TASK modulator, and drugs that inhibit TASK channels, such as bupivacaine, methanandamide and zinc. Small vessel myography was used to measure the tone of both conduit and resistance pulmonary arteries. Cumulative bupivacaine and methanandamide dose–response curves were compared with phenyle- phrine (a sympathomimetic vasoconstrictor). The effects of pH were investigated on vessel tone and responses to bupivacaine, metha- nandamide or zinc chloride. Bupivacaine and methanandamide (.10 mM) resulted in increased artery tone, with similar effects seen in conduit and resistance vessels. Zinc had no effect, possibly reflecting an inhibitory action on calcium channels. In the presence of endo- thelial blockers, methanandamide (100 mM) still resulted in an increase in artery tone, implying an action on smooth muscle. The appli- cation of nifedipine resulted in the inhibition of the response seen with bupivacaine (100 mM), implying that voltage-gated calcium entry was involved. Changing the pH from 7.3 to 8.3 resulted in vasoconstriction, and a relaxation was seen in acidic conditions. This is oppo- site to the result expected for TASK channel modulation, but may reflect the multiple effects of pH on smooth muscle. The contractions seen with bupivacaine and methanandamide were increased at pH 8.3 but inhibited at pH 6.3, consistent with an effect on TASK chan- nels. Responses to bupivacaine and methanandamide were, however, very small, suggesting that currents produced by TASK channels may not be a major factor in contracting intrapulmonary arteries, but may still have a minor role. Key words: arterial tone, TASK channels, rat, bupivacaine, methanandamide, pH. ........................................................................................................................................................................................................................................ The a-subunit of the two-pore domain channels, which Introduction are mainly expressed as homodimers, comprised four trans- The TWIK-related acid-sensitive potassium (TASK) channels, membrane segments, two pore-forming domains and a a subtype of the two-pore domain family of potassium chan- short N-terminus and a long C-terminus, both of which 6, 7 8 9 nels, have been implicated as having a role in maintaining and are intracellular. Five TASK channels, TASK-1, 22, 10 11 12 mediating the tone of pulmonary arteries. Thus, the inhibition 23, 24 and 25, have been cloned thus far. Studies of TASK channels prevents potassium efflux and results in have found the presence of TASK-1 proteins and mRNA in depolarization, and activation leads to hyperpolarization of rat, rabbit and human PASMCs as well as in rat endothelial 1–6 2–5 the pulmonary artery smooth muscle cells (PASMCs). cells, suggesting a role for TASK channels, particularly Depolarization of the cell membrane is expected to enhance TASK-1, in setting resting membrane potential. A small the open probability of L-type calcium channels, allowing role has also been suggested for TASK-2 by the finding that calcium entry into the cell which leads to constriction of the small interfering RNA directed against the TASK-2 mRNA artery. Conversely, hyperpolarization would result in the reduced the smooth muscle resting membrane potential and closure of voltage-dependent calcium channels, thus decreas- the effects of pH on membrane potential in intact intrapul- ing calcium entry and subsequent vasodilation. monary arteries (IPAs). ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 114 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Three known modulators of TASK channels have been MgCl 1, glucose 5 and CaCl 1. The pH was adjusted to 2 2 investigated in rat, rabbit and human PASMCs: bupivacaine, 7.3 with NaOH, bubbled with air and maintained at 37ºC. a local anaesthetic, the endogenous cannabinoid anand- 2þ amide and Zn . In electrophysiological studies, a voltage- Myography insensitive potassium current was found to be inhibited by these three drugs, suggesting that they might cause an Chart software (J. Dempster, Strathclyde University) was 2–4 3 increase in IPA tone. Gardener et al. looked at the used to record the isometric tensions of the vessels. After effect of anandamide and bupivacaine on vessel tone and the vessels were mounted, resting tensions of 5 and 4 mN found a gradual increase, which was concentration- were applied to the large and small vessels, respectively, to dependent. Whether it was due to an effect on TASK and provide some initial tone, followed by an equilibration voltage-gated calcium channels was not investigated. period of 30 min. At the start of every experiment, vessels An important modulator of membrane potential in these were contracted three times using 50 mM KCl, with relax- 2–5 studies was pH, which also affects the TASK channels. ation back to baseline occurring between each contraction Increasing and decreasing the extracellular pH of PASMCs in response to being washed with PSS. The final potassium had the effect of activating a potassium current, leading to contraction was used as the reference against which all hyperpolarization, or inhibition of the current, leading to measurements were made. 2– 5,13 depolarization of the membrane, respectively. In theory, this suggests that an alkaline environment would Experimental procedures cause a relaxation in arterial tone, although this may only be seen in conditions that raise baseline tone, whereas an Individual phenylephrine, bupivacaine and methanandamide increase in baseline arterial tone would be seen with a dose–response curves were created by applying increasing 29 23 more acidic pH. Surprisingly, few studies have addressed and cumulative concentrations (10 M–10 M) of the 2–5 the effects of pH on isolated pulmonary arteries; there- drugs to the 5 ml bath of PSS surrounding the vessels. fore, it is not yet clear if this prediction holds true. The interval between each application was 5 min or after The aim of this study was to investigate the effects of the response was seen to reach its peak. TASK channel modulator drugs and pH on rat IPA tone. After creating the dose–response curve for methananda- Both large conduit and small resistance vessels were used, mide followed by washing, the endothelial inhibitors, because arteries from different levels of the pulmonary arter- N-nitro-L-arginine methyl ester (L-NAME) (100 mM) and ial tree are known to respond differently to a variety of phys- indomethacin (10 mM), were added to the myograph iological stimuli, and the size and source of arteries are chamber and left to equilibrate for 40 min. A second thought to be important factors in determining responsive- dose–response curve was then created in the same manner ness to pH. as the first in order to compare the two conditions. Nifedipine, a calcium channel blocker, was used to inves- tigate the mechanism of the bupivacaine contraction. The Materials and methods response to 100 mM bupivacaine was recorded. After washing off the bupivacaine with PSS, 1 mM nifedipine Preparation of vessels was administered for 10 min followed by 100 mM Sprague Dawley rats (250–300 g) were killed by cervical dis- bupivacaine. location in accordance with Schedule 1 of the UK Animals The effect of pH was examined by adjusting the PSS to pH (Scientific procedures) Act and the lungs and heart 6.3 (acid) or pH 8.3 (alkaline) using 1 M HCl or NaOH. removed. Both large (conduit) and small (resistance) IPAs After equilibration at each pH, either bupivacaine or metha- were required. Large vessels were taken from the middle of nandamide (10 and 100 mM) or zinc chloride (100 and the main conduit artery running the length of the lung lobe 200 mM) was applied individually and the responses with an approximate diameter of 500 mm. The small recorded. vessels, with an approximate diameter of 200 mm, were taken from the second- and third-order branches. Vessels Data analysis were cut into segments 1–2 mm long and suspended hori- zontally in 5 ml baths of a multi-channel myograph Cursor measurements of the amplitudes of responses were (Danish Myotechnology, Aarhus, Denmark). The larger entered into Excel where the drug responses were calculated vessels were mounted on pins attached to a force transducer; as a percentage of maximal 50 mM KCl-induced tension. the smaller vessels were gently threaded onto steel wires Data are displayed as a means+ standard error of the attached to the force transducer. The baths contained physio- mean. Data were analysed using the paired t-test or logical salt solution (PSS) of composition (in millimolar): one-way analysis of variance (ANOVA) where appropriate. NaCl 122, KCl 5, HEPES 10, KH PO 0.5, NaH PO 0.5, Significance was assumed if P, 0.05. 2 4 2 4 ......................................................................................................................................................................................................................................... 115 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Reagents used in small and large vessels were analysed by a paired t-test and not found to be significantly different. Stock solutions (10 mM) of phenylephrine (Sigma) and bupi- Methanandamide, another TASK channel inhibitor, also vacaine hydrochloride (Sigma) were prepared with distilled caused a concentration-dependent increase in vessel tension; water, methanandamide (TOCRIS) was dissolved in however, the response was very small (Fig. 1B and D). The ethanol and stored in aliquots in the freezer. A 100 mM methanandamide (n ¼ 5) response was much smaller than stock solution of zinc chloride (Fluka) was prepared and (, 20%) that induced by KCl or phenylephrine. As can be stored in the fridge. These solutions were serially diluted in seen in Fig. 1D, arterial tension did not increase until PSS as required. 100 mM methanandamide was administered. A paired t-test showed that the maximum tensions recorded in response to 100 mM methanandamide for the large and small IPAs were Results not significantly different. Vessels were used for experiments only if they contracted in response to the application of 50 mM KCl. In each case, The Influence of the endothelium on the effects of three reproducible responses, averaging 2–3 mN in ampli- methanandamide tude for both large and small IPAs, were obtained to 50 mM The influence of the endothelium was inhibited with the use KCl before the experiments began. of the endothelial blockers L-NAME and indomethacin, which inhibit the release of the vasodilators nitric oxide The effect of TASK channel inhibitors on vessel tone and prostacyclin, respectively. Physical removal of the endo- The effect of applying increasing concentrations of bupiva- thelium was not undertaken due to the difficulty in removing caine (n ¼ 5) on IPA tone can be seen in Fig. 1A and C. the endothelium from the small vessels. The response to Both large and small IPAs were noted to have increased 100 mM methanandamide seen following the addition of tone with increasing concentrations of bupivacaine; endothelial blockers (n ¼ 4) was similar to that seen with however, significant changes in tone were only seen at 10 methanandamide alone (Fig. 2). A paired t-test comparing and 100 mM for the large IPA and 100 mM for the small the tensions produced at 100 mM methanandamide in the IPA. The tensions produced were much lower than presence and absence of endothelial blockers for both types (,30%) those achieved by 50 mM KCl or phenylephrine. of vessels suggests that the responses to methanandamide The maximum tensions produced by 100 mM bupivacaine were not affected by the endothelial blockers. Figure 1. Effects of bupivacaine (BUP) and methanandamide (ANA) on pulmonary artery tone. Raw traces show the tension produced in a large IPA in response to increasing concentrations (1 nM–100 mM) of bupivacaine (A) or a single application of 100 mM methanandamide (B). Cumulative concen- tration–response curves for bupivacaine (C, n ¼ 3) and methanandamide (D, n ¼ 5) show the mean+ SEM. Increase in tone expressed as a percentage of that induced by 50 mM KCl in the same tissue. The concentration–effect curve for phenylephrine is shown for comparison. ......................................................................................................................................................................................................................................... 116 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. Influence of the endothelium on the contractile response to methanandamide. Responses to 100 mM methanandamide, expressed as a percentage of the response to 50 mM KCl, are shown in large and small IPAs, before and after the addition of the endothelial blockers, L-NAME (100 mM) and indomethacin (10 mM), to the myograph chamber. Bars show mean+ SEM. of four to five experiments. The effect of inhibitors of depolarization-induced calcium entry on the responsiveness of IPA to TASK channel inhibitors Nifedipine, a calcium channel antagonist, inhibits Figure 3. Effect of nifedipine on the contractile response to bupivacaine. depolarization-induced calcium entry, thus indicating that (A) Raw trace showing tension induced by 100 mM bupivacaine in a large activation of voltage-gated calcium channels has occurred. IPA before and after applying 1 mM nifedipine. (B) Histogram showing Such a step was proposed as part of the mechanism involving mean tension (+SEM n ¼ 3), measured as a percentage of the response 2, 3 TASK channel inhibition in pulmonary arteries. Thus, if induced by 50 mM KCl, in response to 100 mM bupivacaine, in the absence (con) and presence of 1 mM nifedipine (nif). Responses are the contraction caused by TASK channel inhibitors is due shown for large and small IPAs. **P, 0.01 versus control (paired t-test). to voltage-gated calcium entry, it should be blocked by nife- dipine. The addition of 100 mM bupivacaine produced an increase in IPA tone. However, when bupivacaine was The change in tension induced by pH 6.3 compared with added after the administration of 1 mM nifedipine, this that induced at pH 8.3 was found to be significantly different response was inhibited (Fig. 3). The raw trace of this using a paired t-test for both types of vessels (large IPA: response can be seen in Fig. 3A and the mean results from P, 0.005 and small IPA: P, 0.01). both large and small IPAs in Fig. 3B. This pattern of block was seen with both the large and small IPAs. A paired The effect of pH on the responsiveness of IPA t-test comparing the mean bupivacaine-induced tensions in to TASK channel inhibitors the absence and presence of nifedipine showed that the The concentrations of 10 and 100 mM bupivacaine (n ¼ 5) results for the large vessel were significantly different and 100 mM methanandamide (n ¼ 3) were chosen to be (Fig. 3B, P, 0.01). applied in the presence of PSS adjusted to pH 8.3 and 6.3, as it was shown by the dose–response curves in Fig. 1 that The effect of pH on artery tone these concentrations produced adequate increases in tone The effect of changing the pH of the bath solution was to to allow the effects of pH on their activity to be cause an increase or decrease in the vessel tone, although demonstrated. the effect was usually small and variable. Changing pH The effect of pH on bupivacaine and methanandamide from 7.3 to 8.3, thus making it more alkaline, produced an responses can be seen in Fig. 4. In the raw traces shown increase in the IPA tone, whereas changing pH from 7.3 to (Fig. 4A and B), changing pH had little effect by itself, but the more acidic pH 6.3 caused a decrease in tension. The it did influence the response to bupivacaine and methanand- mean percentage increases in tone (relative to the 50 mM amide. The bar charts (Fig. 4C and D) show that the KCl-induced contraction) on changing pH from 7.3 to 8.3 responses to these drugs were inhibited at pH 6.3, whereas (n ¼ 6) for the large and small vessels were 16+ 5% and the responses were enhanced at pH 8.3. This pattern is 56+ 19%, respectively. On changing from pH 7.3 to 6.3 clear at 100 mM bupivacaine and methanandamide, which (n ¼ 6), the percentage decreases in tone were 25+ 2% produced substantial contractions at pH 8.3, but had no and 211+ 6% for the large and small arteries, respectively. effect at pH 6.3. A similar response pattern was seen with ......................................................................................................................................................................................................................................... 117 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 4. Effect of pH on contractile responses to bupivacaine (BUP) and methanandamide (ANA). Raw traces show the effect on tension in a large IPA of changing external pH from 7.3 to 6.3 and then 8.3, and the effect of 10 mM and 100 mM bupivacaine (A)or100 mM methanandamide (B) applied at each pH. Histograms show mean tension (+SEM, n ¼ 3) induced by 10 or 100 mM bupivacaine (C) or 100 mM methanandamide (D) at pH 6.3, 7.3 and 8.3 in large and small IPAs. Responses are expressed as a percentage of the contractile response to 50 mM KCl. **P, 0.01 (ANOVA) comparing pH 6.3, 7.3 and 8.3. the lower concentration of 10 mM bupivacaine; however, this application of nifedipine indicates that bupivacaine probably concentration produced small effects when compared with does cause the opening of L-type calcium channels via TASK those recorded for 100 mM bupivacaine, which, in this channel inhibition. In the presence of nifedipine, calcium was experiment, were only measurable at pH 8.3. Using unable to enter the cell and cause vasoconstriction, despite ANOVA, it was found that the differences in tension seen bupivacaine causing depolarization. As pulmonary arteries with changing the pH were statistically significant for bupi- do not express sodium channels, a local anaesthetic action vacaine at 100 mM on the large artery (P, 0.01). on Na channels cannot contribute to the result, although Zinc chloride, at concentrations blocking TASK channels inhibition of another K channel cannot be excluded. in PASMCs (100 and 200 mM), was found to produce no The increase in the vessel tone at the higher concentrations changes in tension at pH 6.3, 7.3 or 8.3. of 10 and 100 mM bupivacaine in large and small vessels may reflect the point at which the numbers of inhibited TASK channels reach the level required to cause increased Discussion tension. This is possibly the concentrations at which suffi- cient numbers of TASK channels are closed to bring the Bupivacaine was found to increase vessel tone. Gardener membrane potential to the calcium channel activation et al. also found this when investigating the effect of bupi- threshold, after which calcium enters and results in contrac- vacaine on pulmonary arteries, with the increase in tone tion. Gardener et al. found similar concentration depen- being concentration-dependent. Bupivacaine is a local anaes- dence with an increase in the vessel tone occurring at a thetic drug that is believed to inhibit TASK channels. It has concentration slightly higher than 10 mM. been suggested that TASK channel inhibition leads to Methanandamide, another putative inhibitor of TASK-1 depolarization due to a decrease in the potassium efflux. channels, also increased arterial muscle tension at concen- The resulting depolarization enhances the open probability trations above 10 mM. Gardener et al. reported similar find- of L-type calcium channels. Subsequently, calcium enters ings. This contraction may have been due to depolarization the smooth muscle cells, resulting in muscle contraction as a result of TASK channel inhibition; however, this was and vasoconstriction. By using nifedipine to block these not studied further. Methanandamide is a cannabinoid, so calcium channels, it was possible to test whether this is an action on cannabinoid receptors cannot be ruled out. the mechanism by which bupivacaine exerts its effects. The Two cannabinoid receptors, CB1 and CB2, were found to fact that bupivacaine did not increase vessel tone after the ......................................................................................................................................................................................................................................... 118 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... be present in the endothelium upon which anandamide may opposite to what is found in the intact lung and is 17, 18 exert relaxant effects. This would counteract and limit hypothesized from the effects of pH on TASK channels. the amplitude of any direct contractile action of Findings from studies investigating TASK channel currents methanandamide on the smooth muscle. TASK channels and their influence on the membrane potential showed that have been found to be present in the endothelium as well an acidic pH causes depolarization and an alkaline pH 2–4 as in the smooth muscle cells of pulmonary arteries, causes hyperpolarization due to inhibition or activation of 2–5 and the inhibition of endothelial TASK channels could also TASK currents, respectively. Thus, it would be expected influence the constrictor effect of methanandamide. To that acidic conditions would result in vasoconstriction and investigate the possible influence of the endothelium on alkaline conditions would cause vasodilation. The present responses to methanandamide, the effect of blocking the finding is consistent with one other study, in which relax- release of endothelium-derived relaxing factors on its ation of pulmonary artery in the presence of extracellular effects was, therefore, investigated. The endothelial blockers acidosis was seen, as well as with the known characteristics used, L-NAME and indomethacin, are nitric oxide synthase of systemic vessels. and cyclooxygenase inhibitors, respectively. They inhibit The effects of pH that was observed on IPA tone cannot be the production of nitric oxide and prostacyclin, both of due to its effect on TASK channels. As pH can affect the which are relaxing factors produced by the endothelium. If activities of many pathways involved in regulating vascular the constrictor effect of methanandamide was limited by tone, it is possible that TASK channel inhibition does con- concurrent endothelium-dependent relaxation, it would be tribute, but is masked by opposing effects. Vascular tone is expected that the addition of endothelial blockers would mainly determined by the intracellular concentration of cause it to be increased. This was not observed. calcium ions, and the influence of extracellular and intra- Methanandamide produced a response in the presence of cellular acidosis and alkalosis on the calcium ion concen- the endothelial blockers that was not significantly different tration has been investigated with conflicting results. from that in their absence. However, it cannot be ruled out Wakabayashi and Groschner found that a decrease in the that TASK channel inhibition or activation of cannabinoid extracellular, but not intracellular, proton concentration receptors in the endothelium by methanandamide may (i.e. extracellular alkalosis) caused calcium entry into endo- result in the release of factors not blocked by L-NAME and thelial cells, which resulted in the release of vasoactive indomethacin. One such factor that may be involved is the mediators and thus a decrease in the vascular tone. It has endothelium-derived hyperpolarizing factor (EDHF), which also been suggested that the vasodilator effects seen in acid has been implicated in cannabinoid-induced mesenteric pH may be due to its action on the endothelium; however, 17 13,21 vasodilation in the rat. Thus, an EDHF-dependent relax- two studies, one in which the endothelium was ation may be masking the full methanandamide contraction. removed and one using endothelial blockers, found that the Although both bupivacaine and methanandamide caused relaxation was endothelium-independent. Klockner and constriction of IPA, the tensions produced were very small. Isenberg suggested that extracellular protons exert their Phenylephrine, an a-adrenergic agonist, is known to cause effects in the same way as intracellular acidification as they constriction of IPA and its effects showed the vessels used can permeate the membrane of vascular smooth muscle to be viable, as did the constriction to 50 mM KCl. The cells. Krampetz and Rhoades found that intracellular alka- responses to bupivacaine and methanandamide were far losis caused direct contraction of PASMCs through the smaller than those produced by phenylephrine, even at release of calcium from intracellular sites; however, they 100 mM, which is well above the concentrations producing also found that intracellular acidosis caused contraction via 16,19 50% block of TASK channels. This indicates that voltage-activated calcium channels. pH has been found to TASK channel block does not induce the maximal IPA affect the activity of L-type calcium channels. Intracellular tension and suggests that TASK currents are not of major acidification decreases, and intracellular alkalosis increases, 22, 23, 25, 26 importance in contracting IPA, but may still have a minor calcium influx through these channels. It must role. be noted, however, that the aforementioned effects of pH on vascular smooth muscle cells do not necessarily occur in The effect of pH on tone and responsiveness of IPA IPA. to TASK channel inhibitors Although pH did not affect basal tone in the way expected In the intact rat lung, acidosis causes vasoconstriction and of TASK channel modulation, it did influence the responses alkalosis causes vasodilation in the presence of elevated to bupivacaine and methanandamide. There was a very basal tone. Surprisingly, however, there is very little in slight or no response from both the drugs at pH 6.3. This the literature about the effects of extracellular pH on tone may be due to the acid conditions inhibiting TASK channels in isolated IPA. In this study, changing pH to create an so that no further modulation by bupivacaine and methanan- acidic environment had the effect of relaxing IPA, whereas damide can take place. The inhibitory effect of an acid pH is in more alkaline conditions, the IPA constricted. This is believed to occur due to protonation of the histidine residue ......................................................................................................................................................................................................................................... 119 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... at position 98 on the TASK subunit, which leads to channel research into the development of TASK modulator drugs 10,26 block, thus preventing the efflux of potassium ions. With will help, in the future, to clarify the role of TASK channels the channel already blocked, further interaction with in regulating pulmonary artery tone. modulator drugs would have no effect. Alkaline conditions caused an enhanced response to both Acknowledgements drugs. Patch clamp studies have shown that an alkaline pH results in hyperpolarization, which is reversed by both drugs Obtaining rats and microdissection of the pulmonary vessels and to a membrane potential that is close to that reached were carried out by Professor A. Gurney (Supervisor), Boris 2–4, 8 when the drugs are applied at pH 7.3. From these Manoury (Research Fellow), Shreena Joshi (PhD Student) effects of pH, it might be expected that, in terms of vessel and Sarah Etheridge (Technician). All other work in this tone, the alkaline pH would have little effect on responsive- project was carried out by the student: Sajni Dipak Shah. ness to bupivacaine and methanandamide. The enhanced I would like to thank Professor A. M. Gurney for guidance effect of the drugs at pH 8.3 can be explained, however, if throughout. we consider the initial contraction seen with the change of pH from 7.3 to 8.3. This caused the tension to be slightly Funding higher than normal, so that the subsequent administration of TASK channel blockers may have more easily caused vasocon- Biotechnology and Biological Sciences Research Council striction as the efflux of potassium ions was inhibited. The (grant BBS/B/11761/2). initially higher baseline tone at pH 8.3, compared with 7.3 or 6.3, may therefore have had a synergistic effect with the References actions of bupivacaine and methanandamide. Although zinc chloride is also an inhibitor of TASK chan- 1. Gurney AM, Osipenko ON, Macmillan D et al. (2002). Potassium channels underlying the resting potential of pulmonary artery smooth muscle cells. nels and blocks TASK-like currents in pulmonary artery 2 Clin Exp Pharmacol Physiol 29: 330–333. myocytes, it had no effect on vessel tone at any pH. One 2. Gurney AM, Osipenko ON, Macmillan D et al. (2003) Two-pore domain pot- explanation for the lack of effect could be the finding by assium channel, TASK-1, in pulmonary artery smooth muscle cells. Circ Res Busselberg et al. that zinc can block voltage-gated 93: 957. calcium channels. As activation of L-type calcium channels 3. Gardener MJ, Johnson IT, Burnham MP et al. (2004) Functional evidence of a is essential for contraction mediated by TASK channel inhi- role for two-pore domain potassium channels in rat mesenteric and pul- bition, block of these channels by zinc would inhibit any monary arteries. Brit J Pharmacol 142: 192–202. contraction that could potentially occur. 4. Olschewski A, Yingji L, Tang B et al. (2006) Impact of TASK-1 in human pul- monary artery smooth muscle cells. Circ Res 98: 1072–1080. Comparison of the large and small IPA? 5. Go¨ nczi M, Szentandrassy N, Johnson IT et al. (2006) Investigation of the role of TASK-2 channels in rat pulmonary arteries; pharmacological and func- Responses generally seemed smaller in the small IPA, but no tional studies following RNA interference procedures. Brit J Pharm 147: significant differences were measured. This may reflect the 496–505. small number of experiments performed, so more studies 6. O’ Connell AD, Morton MJ, Hunter M (2002) Two-pore domain potassium are required for clarification. channels—molecular sensors. Biochim Biophys Acta Biomembr 1566: 152–161. 7. Lesage F, Lazdunski M (2000) Molecular and functional properties of Conclusions two-pore domain potassium channels. Am J Physiol Renal Physiol 279: F793–F801. The TASK channel inhibitor drugs, bupivacaine and metha- 8. Duprat F, Lesage F, Fink M et al. (1997) TASK, a human background Kþ nandamide, increase IPA tone; however, zinc had no effect. channel to sense external pH variations near physiological pH. EMBO J 16: The hypothesis that vasoconstriction would occur in acid 5464–5471. conditions and vasodilation in alkaline conditions was dis- 9. Reyes R, Duprat F, Lesage F et al. (1998) Cloning and expression of a novel proved; thus TASK channels do not appear to be involved pH-sensitive two pore domain K channel from human kidney. J Biol in the tissue response to pH. The bupivacaine- and Chem 273: 30863–30869. methanandamide-induced contractions were enhanced at 10. Kim Y, Bang H, Kim D (2000) TASK-3, a new member of the tandem pore K channel family. J Biol Chem 275: 9340–9347. alkaline pH and inhibited in acidosis, supporting the pre- 11. Decher N, Maier M, Dittrich W et al. (2001) Characterization of TASK-4, a sence and function of TASK channels in rat IPA. It must be novel member of the pH sensitive, two-pore domain potassium channel stressed, however, that the effects of TASK inhibitors were family. FEBS Lett 492: 84–89. small, meaning that either TASK channels have a small 12. Kim D, Gnatenco C (2001) TASK-5, a new member of the tandem-pore Kþ role in mediating IPA tone or the drugs used are non-selective channel family. Biochem Biophys Res Commun 284: 923–930. and have additional actions that mask the effect of TASK 13. Sweeney M, O’Regan RG, McLoughlin P (1999) Effects of changes in pH and inhibition. With the already strong evidence for the presence PCO2 on wall tension in isolated rat intrapulmonary arteries. 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Ja´rai Z, Wagner JA, Varga K et al. (1999) Cannabinoid-induced mesenteric 25. Iino S, Hayashi H, Saito H et al. (1994) Effects of intracellular pH on calcium vasodilation through an endothelial site distinct from CB1 or CB2 receptors. currents and intracellular calcium ions in the smooth muscle of rabbit portal PNAS 96: 14136–14141. vein. Exp Physiol 79: 669–680. 19. Zoratti C, Kipmen-Korgun D, Osibow K et al. (2003) Anandamide initiates 26. Schuhmann K, Voelker C, Hofer GF et al. (1997) Essential role of the beta 2þ Ca signaling via CB receptor linked to phospholipase C in calf pulmonary subunit in modulation of C-class L-type Ca2þ channels by intracellular endothelial cells. Br J Clin Pharm 140: 1351–1362. pH. FEBS Lett 408: 75–80. 2þ 20. Gao Y, Tassiopoulos AK, McGraw DJ et al. (1999) Segmental pulmonary vas- 27. Busselberg D, Michael D, Evans ML et al. (1992) Zinc (Zn ) blocks voltage cular responses to changes in pH in rat lungs: role of nitric oxide. Acta gated calcium channels in cultured rat dorsal root ganglion cells. Brain Res Anaesthesiol Scand 43: 64–70. 593: 77–81. ........................................................................................................................................................................................................................................ Submitted on 26 September 2007; accepted on 28 January 2008; advance access publication 17 April 2008 .........................................................................................................................................................................................................................................

Journal

Bioscience HorizonsOxford University Press

Published: Jun 17, 2008

Keywords: Key words arterial tone TASK channels rat bupivacaine methanandamide pH

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