The previous study by the authors showed that the class Ib antiarrhythmic drug lidocaine impairs but mexiletine augments vasorelaxation mediated by adenosine triphosphate-sensitive K+ channels. Lidocaine and mexiletine have different values of the negative logarithm of the drug-proton dissociation constant, indicating that the ion channel-blocking effects of these drugs under different pH levels may vary. However, the role of pH in the effects of lidocaine and mexiletine on vasodilation mediated by K+ channels has not been studied. Therefore, the current study was designed to examine whether the inhibition and augmentation of vasorelaxation in response to an adenosine triphosphate-sensitive K+ channel opener, levcromakalim, by the clinically relevant concentrations of lidocaine or mexiletine are modified by mild alkalinization or acidification in the isolated rat aorta.
Rings of the rat aorta without endothelium were suspended for isometric force recording. Three types of modified Krebs-Ringer solutions (pH 7.2, 7.4, and 7.6) were prepared by changing the composition of NaCl and NaHCO3. During contractions in response to phenylephrine (3 x 10(-7) M), relaxations in response to levcromakalim (10(-8) to 10(-5) M) were obtained. Lidocaine (10(-5) to 10(-4) M), mexiletine (10(-5) to 10(-4) M), or glibenclamide (10(-5) M) was applied 15 min before addition of phenylephrine.
Relaxations in response to levcromakalim, which are abolished by the selective adenosine triphosphate-sensitive K+ channel antagonist glibenclamide (10(-5) M), were not different among the three pH groups. In the normal Krebs-Ringer solution of pH 7.4, lidocaine significantly reduced these relaxations in a concentration-dependent fashion. Alkalinization of pH 7.6 augmented the inhibitory effect of lidocaine on these relaxations, whereas acidification of pH 7.2 substantially abolished this effect. In contrast, mexiletine pH independently augmented relaxations in response to levcromakalim. Glibenclamide (10(-5) M) abolished these relaxations in arteries treated with mexiletine (10(-4) M) in any pH group.
These results suggest that even under conditions of such mild alkalosis or acidosis, vasorelaxation via adenosine triphosphate-sensitive K+ channels is dependent on pH in the presence of clinically relevant concentrations of lidocaine but not mexiletine.
THE class Ib antiarrhythmic drugs lidocaine and mexiletine reportedly inhibit cardiac Na+channels, resulting in their antiarrhythmic action. 1,2Most antiarrhythmic drugs exist as both charged and uncharged forms of these drugs. The uncharged form seems to dissolve readily into the lipid phase of the cell membrane and reaches the binding sites of Na+channels. 3Because the ratio of the uncharged form compared with the charged form of antiarrhythmic drugs is determined by the negative logarithm of the drug-proton dissociation constant (pKa) of the drug and pH of the external solution, it is conceivable that extracellular pH plays an important role in the Na+channel–blocking effects of each antiarrhythmic drug. Lidocaine and mexiletine have different pKa values, indicating that the Na+channel–blocking effects of these antiarrhythmic drugs under different pH levels may vary. 1However, even in cardiac myocytes, the role of mild changes of pH in the effects of lidocaine and mexiletine on ion channels has not been demonstrated.
Increasing evidence suggests that adenosine triphosphate (ATP)-sensitive K+channels play an important role in physiologic and pathophysiologic vasodilation. 4Previous studies, including ours, showed the inhibitory or augmenting effects of lidocaine and mexiletine on the activity of ATP-sensitive K+channels. 5–9pH changes may be capable of modifying these effects of class Ib antiarrhythmic drugs on vasodilation mediated by ATP-sensitive K+channels because extracellular pH seems to contribute to the effects of lidocaine and mexiletine on Na+channels by changing the ratio of the uncharged to the charged form of the drugs. 1,3However, the role of pH changes in the vasodilation mediated by K+channels has not been well-studied. Therefore, the current study was designed to examine whether the inhibition and augmentation of vasorelaxation in response to an ATP-sensitive K+channel opener, levcromakalim, by the clinically relevant concentrations of lidocaine or mexiletine are modified by the mild alkalinization or acidification in the isolated rat aorta.
The study was approved by the institutional animal care and use committee (Wakayama Medical Collage, Wakayama, Japan). The experiments were performed on thoracic aortic rings obtained from male Wistar rats (300–400 g) that were anesthetized with inhalation of 3% halothane in 100% oxygen (3 l/min). Thoracic aortic rings 2 mm in length were studied in modified Krebs-Ringer bicarbonate solution (control solution pH 7.4) of the following composition (mm): NaCl, 119; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.17; KH2PO4, 1.18; NaHCO3, 25; and glucose, 11. Three types of modified Krebs-Ringer solutions (pH 7.2, 7.4, and 7.6) were prepared by changing the composition of NaCl and NaHCO3(131 or 101 mm NaCl and 13 or 43 mm NaHCO3for the acid [pH 7.2] or alkaline [pH 7.6] solutions, respectively). The pH of the bathing solution was continuously monitored with a pH meter (CyberScan pH 100; Eutech Instruments PTE Ltd., Ayer Rajah Crescent, Singapore) throughout the experiments. In all rings, the endothelium was removed mechanically because our previous study showed that in the rat aorta, vasorelaxation in response to levcromakalim is augmented in the presence of functional endothelium. 10Several rings cut from the same artery were studied in parallel. Each ring was connected to an isometric force transducer and suspended in an organ chamber filled with 10 ml control solution (37°C) bubbled with 95% O2–5% CO2gas mixture. The artery was gradually stretched to the optimal point of its length–tension curve as determined by the contraction in response to phenylephrine (3 × 10−7m). In most of the studied arteries, optimal tension was achieved at approximately 1.5 g. The endothelial removal was evaluated by the absence of relaxation induced by acetylcholine (10−5m). Preparations were equilibrated for 90 min. After equilibration, rings were assigned to one of three pH groups (pH 7.2, 7.4, or 7.6), and the normal solution of pH 7.4 was replaced with acid or alkaline solution in pH 7.2 and 7.6 groups, respectively. During submaximal contractions to phenylephrine (3 × 10−7m), concentration–response curves for levcromakalim (10−8to 10−5m) were obtained in the absence or in the presence of lidocaine, mexiletine, or glibenclamide. Concentration–response curves were obtained in a cumulative fashion. Only one concentration–response curve was made from each ring. Lidocaine (10−5to 10−4m), mexiletine (10−5to 10−4m), or glibenclamide (10−5m) was given 15 min before addition of phenylephrine (3 × 10−7m). The relaxations were expressed as a percentage of the maximal relaxations to papaverine (3 × 10−4m), which is added at the end of experiments to produce maximal relaxations (= 100%) of the arteries.
The following pharmacologic agents were used: dimethyl sulfoxide, glibenclamide, lidocaine hydrochloride, and phenylephrine (Sigma, St. Louis, MO). Mexiletine hydrochloride and levcromakalim were gifts from Boehringer Ingelheim Pharm. KG. (Ingelheim, Germany) and SmithKline Beecham Pharmaceutical Company (Betchworth, Surrey, Great Britain), respectively. Drugs were dissolved in distilled water such that volumes of less than 60 μl were added to the organ chambers. Stock solutions of levcromakalim (10−5m) and glibencla-mide (10−5m) were prepared in dimethyl sulfoxide (3 × 10−4m). The concentrations of drugs are expressed as final molar concentration.
The data are expressed as mean ± SD; n refers to the number of rats from which the aorta was taken. Statistical analysis was performed using repeated measures analysis of variance, followed by the Scheffé F test for multiple comparison. Differences were considered to be statistically significant when P was less than 0.05.
During submaximal contractions in response to phenylephrine (3 × 10−7m), a selective ATP-sensitive K+channel opener, levcromakalim (10−8to 10−5m) induced concentration-dependent relaxations in the rat thoracic aorta without endothelium (fig. 1). These relaxations, which are abolished by a selective ATP-sensitive K+channel antagonist, glibenclamide (10−5m), were not different among the three pH groups (fig. 1). At normal pH (pH 7.4), lidocaine (3 × 10−5, 10−4m) significantly reduced relaxations in response to levcromakalim in a concentration-dependent fashion (fig. 2). Alkalinization (pH 7.6) augmented the inhibitory effect of these concentrations of lidocaine (fig. 2and table 1). However, acidification (pH 7.2) substantially abolished this effect of lidocaine on vasodilator responses to levcromakalim, although it seemed to be a tendency of the shift in the concentration–response curve (fig. 2and table 1). In contrast to lidocaine, mexiletine (3 × 10−5, 10−4m) augmented relaxations in response to levcromakalim in the pH-independent fashion (fig. 3and table 2). Glibenclamide (10−5m) abolished these relaxations in arteries treated with mexiletine (10−4m) in any pH group (fig. 4). Neither lidocaine nor mexiletine produced any effects on contractions to phenylephrine in any pH group (data not shown).
This is the first study showing the differential role of pH changes in the effects of the class Ib antiarrhythmic drugs lidocaine and mexiletine on vasorelaxation mediated by K+channels. Our results suggest that even under conditions of such mild alkalosis or acidosis, vasorelaxation via ATP-sensitive K+channels is dependent on pH in the presence of clinically relevant concentrations of lidocaine but not mexiletine.
In the current study, glibenclamide, which has been shown to be a selective antagonist of ATP-sensitive K+channels, abolished relaxations in response to levcromakalim. 11,12These results are consistent with our recent study of the isolated rat aorta showing that vasorelaxation to levcromakalim is completely inhibited by glibenclamide. 10Our previous finding in the rat aorta that glibenclamide did not affect relaxations in response to nitric oxide donors also reinforces the selectivity of glibenclamide on ATP-sensitive K+channels in this preparation. 6
In the rat aorta, relaxations induced by levcromakalim were not different in any pH group. These results are consistent with the evidence that modulation of vasorelaxation in response to ATP-sensitive K+channel openers induced by alkalinization or acidification within such physiologically ranged changes of pH has not been demonstrated. Although in isolated coronary and cerebral arteries decreased extracellular pH, beyond the values in the current study, reportedly produces vasorelaxation mediated by ATP-sensitive K+channels, it seems that the mild degree of changes in the extracellular pH in our study is at least partly because of these differential results. 13,14
In the current study, mild pH changes affected the inhibitory effect of lidocaine on vasodilation in response to levcromakalim, whereas they did not alter the augmenting effect of mexiletine. It may be possible that the mechanism of observed differences in mild changes of pH effects between these two antiarrhythmic drugs is the result of alterations in the ionization of ATP-sensitive K+channel protein, which affects its ability to respond more to lidocaine than to mexiletine. Lidocaine and mexiletine have different pKa values of 7.85 or 9.3, respectively, suggesting that the differential proportionof uncharged form between these antiarrhythmic drugs in the same pH solution may be at least partly because of the differential pH-dependency of these drugs for vasodilation mediated by ATP-sensitive K+channels. 1However, when one considers the previous studies showing the ratio of the uncharged to the charged form of lidocaine and mexiletine in the different extracellular pH, alteration of only 0.2 or 0.4 pH units would be expected to alter the amount of the uncharged drug a very small degree. 1Therefore, the differential ratio of the uncharged to the charged form of the compound in such mild alkalosis and acidosis may not be responsible for our results regarding the differential modulator effects of lidocaine and mexiletine on vasodilation mediated by ATP-sensitive K+channels. pH-dependent effects of lidocaine and mexiletine on Na+channels have been demonstrated only in a relatively large extent of pH changes in cardiac myocytes. 1,15We cannot rule out the possibility that our results from lidocaine and mexiletine may be mediated by some components other than ATP-sensitive K+channels, including G-protein coupled receptors, because it has been demonstrated that the activity of G-protein is related to the inward rectifier K+channel modulation in cardiac myocytes. 12,16
The ATP-sensitive K+channel is a complex of two proteins: the sulfonylurea receptor, which is a member of the ATP-binding cassette transporter family, and a smaller protein, Kir6.1 or 6.2, which belongs to the inward rectifier K+channel family. 16Because recent direct functional and biochemical studies have shown that the sulfonylurea receptor of the ATP-sensitive K+channel is a primary target of the openers of this channel, it is likely that the differential pH-dependent effects of lidocaine and mexiletine on vasorelaxation to ATP-sensitive K+channel openers are caused by the effects of these class Ib antiarrhythmic drugs on the sulfonylurea receptor of ATP-sensitive K+channels in vascular smooth muscle cells. 17However, further biochemical studies are necessary to clarify the mechanisms responsible for the effects of lidocaine and mexiletine on ATP-sensitive K+channels.
The therapeutic ranges of plasma concentrations of lidocaine and mexiletine used as antiarrhythmic drugs have been reported as 8 × 10−6to 5 × 10−5and 8 × 10−7to 10−5m for lidocaine and mexiletine, respectively. 18,19Because approximately 50% of lidocaine and mexiletine is bound to plasma proteins, concentrations of lidocaine or mexiletine used in the current study are within and beyond the free plasma concentrations in the clinical situations, respectively. 20Therefore, our results suggest that in the clinical situations, lidocaine pH-dependently impairs vasodilation mediated by ATP-sensitive K+channels, whereas clinically relevant concentrations of mexiletine may not affect these vasodilator effects.
Class Ib antiarrhythmic drugs are usually administered to treat ventricular arrhythmias, including ventricular premature contractures, ventricular tachycardia, and ventricular fibrillation, which can be often seen in patients with ischemic heart disease or during cardiopulmonary resuscitation. 21,22In these situations, vital organs may be subject to hypoxia, leading to acidosis in systemic circulation and local circulation of the organs. During hypoxia, acidosis, and ischemia, ATP-sensitive K+channels are activated, resulting in arterial dilation and increased tolerance of tissues to ischemia. 13,23,24Therefore, when one uses lidocaine and mexiletine in these patients with acidosis, it may be speculated that lidocaine does not affect but mexiletine may augment beneficial vasodilator effects induced by activation of ATP-sensitive K+channels, which play an important role in regulation of circulation during hypoxia, acidemia, and ischemia. In contrast, several types of ATP-sensitive K+channel openers are now available to treat cardiovascular disorders, including hypertension and ischemic heart disease. 25Because the patients with these disease states often have ventricular arrhythmias, lidocaine and mexiletine can be coadministered with ATP-sensitive K+channel openers. If these patients are slightly hyperventilated or hypoventilated in combination with metabolic acidosis or alkalosis during anesthesia and intensive care, the effects of these openers will be easily pH-dependently modified by lidocaine but not mexiletine. However, it may be difficult to extrapolate the current in vitro study to the clinical setting because of the following reasons. First, in the current study, many of the reported force changes produced by lidocaine and mexiletine were relatively small. Second, the effects of these antiarrhythmic drugs seem to be limited to vasodilation mediated by ATP-sensitive K+channels and to be unrelated to other mechanisms affecting vasocontraction because lidocaine and mexiletine did not alter baseline tone as well as contractions to phenylephrine in this study. Therefore, in the clinical setting, when multiple factors interact to regulate vascular smooth muscle tone, the role of mild pH changes in inhibitory as well as augmenting effects of lidocaine and mexiletine on ATP-sensitive K+channels may be modest.
Even considering our results from conduit arteries, such as the aorta, it is still unclear whether our results have relevance to the smooth muscle function in resistance blood vessels, such as cerebral arterioles. However, because it is well-known that ATP-sensitive K+channels play a major role in vasodilation, especially that of smaller arteries, the current study also indicates the possibility that lidocaine and mexiletine may differently produce pH-dependent and -independent modulation of pathophysiologically and pharmacologically induced vasodilator responses via ATP-sensitive K+channels in resistance vascular beds.