High conductance potassium channels activation by acid exposure in rat aorta is endothelium-dependent

Background We investigated, previously, the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. These studies suggested that extracellular acidosis promotes vasodilation mediated by NO, KATP and SKCa, and maybe other K+ channels in isolated rat thoracic aorta. This study was carried out to investigate the paxilline-mediated hyperpolarization induced by acid exposure. Results The relaxation response to HCl-induced extracellular acidification (7.4–6.5) was measured in rat aortic rings pre-contracted with phenylephrine (PE, 10−6 M). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence of paxilline (10−6 M), which is an inhibitor of high calcium conductance potassium BKCa channels. In rings with endothelium, paxilline inhibits relaxation, triggered by acidification at all pH values lower than 7.2 and had no effect on rings without endothelium, showing that the activation of BKCa is endothelium-dependent. Conclusion High conductance potassium channel activation induced by acid exposure is endothelium-dependent.


Background
We investigated, previously, the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The extracellular acidosis failed in inducing any changes in the vascular tone of rings pre-contracted with potassium chloride (KCl). However, it caused endothelium-dependent, and endothelium-independent, relaxations in rings pre-contracted with PE. These relaxations were inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. These results suggested that extracellular acidosis promotes vasodilation mediated by NO, K ATP and SK Ca , and maybe other K + channels in the isolated rat thoracic aorta [1]. In rings with endothelium, paxilline inhibits relaxation induced by acidification at all pH values lower than 7.2. Otherwise, paxilline had no effect on rings without endothelium, showing that the activation of BK Ca is endotheliumdependent. After an extensive literature search, we assumed this data as original. Therefore, the rationale of the present study was to test a hypothesis that the relaxation induced by extracellular acidification includes the activation of high conductance BK Ca potassium channels.

Methods
The experimental procedures and animal handling were reviewed and approved by the Institutional Animal Care Review Board (CETEA-Ethics Committees of Animal Experiments of the Faculty of Medicine of Ribeirão Preto-University of São Paulo). This 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). Before the experiments, the rats were housed under standard laboratory conditions (12 h light/dark cycle at 21 °C),
The thoracic aorta was cut into rings (4-5 mm in length). The endothelium was removed from some rings by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker's forceps. This method removes the endothelium, but it does not affect the ability of the vascular smooth muscle to contract or relax. Then, these rings were placed in isolated organ baths (10 mL) filled with Krebs solution, maintained at 37 °C, and bubbled with 95 % O 2 /5 % CO 2 (pH 7.4). Each arterial ring was suspended by two stainless steel clips placed through the lumen. One clip was anchored to the bottom of the organ bath, while the other was connected to a strain gauge for measurement of the isometric force with the aid of the Grass FT03 equipment (Grass Instrument Company, Quincy, MA, USA). Each ring was stretched to a resting tension of 1.5 g and allowed to equilibrate for 60 min. During this period, tissues were washed every 15 min. The efficacy of the procedure for endothelium removal was confirmed by the lack of relaxant effects induced by acetylcholine (10 −6 M) in rings precontracted with PE (10 −6 M-EC 80 ). Endothelium was considered to be present when the Ach-induced relaxation was at least 80 %. Then, each ring was washed and re-equilibrated for 30 min. Aortic rings were then precontracted with PE (10 −6 M), and pH-response curves were obtained after a stable plateau was reached. The pH-response curves allowed assessment of the relaxation response to HCl-induced (1N) extracellular acidification (7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5). For this purpose, an electrode (Analion G2134, Ribeirão Preto, SP, Brazil) connected to a pH-meter (MS tecnopon MPA 210, Piracicaba, SP, Brazil) was immersed in the organ bath, enabling real-time analysis of the Krebs solution pH, each point of the curve was obtained by adding a sufficient volume (approximately 2-3 μL) of HCl to the preparation, in order to decrease the pH in 0.1 units. The time elapsed between consecutive additions of acid was 1-2 min, which was necessary for observation of the effect of pH-changes and achievement of the plateau. Considering that the pH curve ranged from 7.4 to 6.5, the total final volume of HCl added to the organ bath was about 30 μL. The paxilline added to the bath was 10 −6 M. Preparations were kept in contact with the drugs for an incubation period of 30 min. Phenylephrine (PE) and paxilline were purchased from Sigma Chemical Company (St. Louis, MO, USA). All the drugs were prepared with distilled water.
The results are shown as mean ± SEM. In the relaxation study, the changes in vascular tension are expressed as a percent of relaxation about the maximal contraction achieved by EC 80 PE-stimulated contraction, a convention that corrects interanimal variability. Statistical analysis was performed using two-way repeated-measures ANOVA and Bonferroni post-test (Prism 4.0, GraphPad Software Inc, San Diego, CA, USA). Values were considered to be statistically significant at p values less than 0.05.

Discussion
Several types of K + channels have been identified in the vascular smooth muscle. These channels had been shown to be involved in the vasodilation seen in cerebral circulation under acidosis condition. K ATP and K Ca are the most important potassium channels engaged in the relaxant response [2][3][4][5][6] in the presence of decreased extracellular pH. It is known that high potassium concentrations can inhibit the activity of all potassium channels [4,7].
To confirm the involvement of this two potassium channels in acidosis-induced vasodilation, we previously tested the specific channels K ATP and K Ca under acidosis condition concluding that their vascular beds actions were endothelium-independent. Vasodilation in response to acidosis was completely abolished when only one of these channels was blocked. The inhibitory effect of glibenclamide or apamin on vasodilation HClinduced, was potenciated by endothelium removal. The association between L-NAME and specific potassium channels blockers, inhibited the HCl-relaxation almost a hundred percent. [1]. This data is suggestive that hyperpolarization and NO/cGMP has some "cross-talked" mechanism. It has been shown that the vasodilator response to NO in rat middle cerebral artery is mediated by activation of K Ca channels via a cGMP-independent pathway [8], a finding that is in accordance with our results about the important role of NO and K Ca channel activity in acidosis.
To evaluate the involvement of BK Ca , this study used paxilline [9], because although some studies have used charybdotoxin as a blocker of BK Ca [2,10], it is known that this drug is not unique to this channel [11][12][13][14]. The present studies, using the blocker paxilline, showed an interesting data: the effect of acidification mediated by potassium channel type BK Ca is dependent on the endothelium. However, it is prudent to consider the fact that paxilline inhibit the relaxation only in rings with endothelium, suggesting that the opening of these channels should be dependent on some endothelium-derived factor. From this point of view, the paxilline BK Ca channel blocker effect, by itself, should not participate in the relaxation induced by acidification. In addition, it is possible to speculate that the endothelium role is dependent on calcium conductance, since the acidosis-induced relaxation blocked by apamin (small conductance Ca 2+ -activated K + channel blocker-SK Ca ) was endothelium-independent, and paxilline (a high calcium conductance BK Ca ) was endothelium-dependent.
The present study demonstrated the contribution of BK Ca for dilation evoked by decreased pHo and paxilline proved to be useful as a pharmacological tool. For example, recent study about the vasorelaxant effect of cilostazol a selective inhibitor of type III phosphodiesterase (PDE3), was not affected by removal of the endothelium and the results were quite similar to the decreased pHo effect. Application of a nitric oxide synthase inhibitor and a small conductance Ca 2+ -activated K + (SK Ca ) channel inhibitor did not affect cilostazol-induced vasorelaxation, which was endothelium-independent and only blocked by paxilline [15]. In conclusion, high conductance potassium channel (BK Ca ) activation induced by acid exposure is endothelium-dependent (Fig. 3).