We found that 3,4-diaminopyridine (3,4-DAP), a voltage-gated potassium channel (KV) inhibitor, elicits pH-sensitive periodic contractions (PCs) of coronary smooth muscles. Underlying mechanisms of PCs, however, remained to be elucidated. The present study was performed to examine the roles of ion channels in the genesis of PCs. To determine the electromechanical changes of smooth muscles, isolated coronary arterial rings from beagles were suspended in organ chambers filled with Krebs-Henseleit solution, and 10−2 M 3,4-DAP was added to elicit PCs. 3,4-DAP caused periodic spike-and-plateau depolarization accompanied by contraction. PCs were not produced when the CaCl2 concentration in the chamber was ≤0.3 × 10−3 or ≥10−2 M. PCs were eliminated by a CaCl2 concentration ≥5 × 10−3 M or by lowering pH below 7.20 with HCl and recovered by the addition of iberiotoxin or charybdotoxin, which inhibit large-conductance calcium-activated potassium channels (KCa), or by elevating pH above 7.35 with NaOH. PCs, as well as the spike-and-plateau depolarization, were eliminated by nifedipine, which inhibits L-type voltage-gated calcium channels (CaV). Influx of Ca2+ through L-type CaV, which was opened because closing of KCa, secondary to 3,4-DAP-induced closing of KV, resulted in contraction; the intracellular Ca2+ increased by this influx opened KCa, leading to closure of CaV and consequent cessation of Ca2+ influx with resultant relaxation. These processes were repeated spontaneously to cause PCs. H+ and OH− were considered to act as the opener and closer of KCa, respectively.
Periodic anginal attacks associated with elevation of the ST segment on the electrocardiogram, which are related to coronary spasm, are common in patients with vasospastic angina (Prinzmetal et al., 1959; Murao et al., 1975; Maseri et al., 1978). The cycle length of the ST elevation ranges from 5 to 30 min (Murao et al., 1975), but the underlying mechanisms of coronary spasm and its periodic occurrence are not well known.
Coronary arteries are very sensitive to pH changes in this category of coronary artery disease; anginal attacks caused by coronary spasm are evoked by the elevation of blood pH by hyperventilation and Tris buffer infusion, and they are suppressed by lowering blood pH by hypoventilation (Yasue et al., 1978).
Coronary spasm can be provoked by intracoronary administration of ergonovine (Siegel, 2010) or acetylcholine (Yasue, et al., 1978) in patients. In animals, coronary spasm can be provoked by histamine (Yamamoto et al., 1987) or serotonin (Fukai et al., 1993; Mitsuoka et al., 1995). However, none of them can provoke pH-sensitive periodic coronary spasm.
During a search for an experimental model for this type of periodic coronary spasm, we discovered that 3,4-diaminopyridine (3,4-DAP), which inhibits (closes) voltage-gated potassium channels (KV) (Kirsch and Narahashi, 1978; Robertson and Nelson, 1994; Flet et al., 2010) and is used clinically to treat neuromuscular diseases (Flet et al., 2010), causes spontaneous and periodic contractions (PCs) of isolated in vitro animal and human coronary arteries that are sensitive to the changes in pH and continue for more than 12 h with a cycle length similar to that of vasospastic anginal attacks (Uchida and Sugimoto, 1984). These PCs are associated with perinuclear vacuolization of the coronary smooth muscle cells, a typical morphological change observed with spasm (Joris and Majno, 1981; Uchida, 1985; Uchida et al., 2011). However, the underlying mechanisms for the periodic nature and pH-sensitive nature of PCs remained obscure.
The present in vitro study aimed to clarify the periodic nature and pH-sensitive nature of the PCs of coronary smooth muscles.
Materials and Methods
Preparation of Coronary Arterial Rings.
We conducted the experiments at the Jikei University School of Medicine Institute for Animal Experiments, and the protocol was approved by the University Administrative Panel on Laboratory Animal Care.
To prepare the coronary arterial rings, 41 male beagles (8–15 kg) were anesthetized with sodium pentobarbital (30 mg/kg i.v.), and each beating heart was removed and immersed in cold Krebs-Henseleit solution (KHS) that contained 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM NaHCO3, and 10 mM glucose.
The proximal segments (2–3 cm in length) of the left anterior descending coronary artery and circumflex artery were carefully dissected free of the surrounding tissues by using two pairs of microscopic dissection forceps and then removed from the myocardium by transecting both ends. The isolated arteries were cut into rings that were 2 mm long.
Recording of Mechanical Activity.
Each coronary arterial ring was suspended by two tungsten hooks under a resting tension of 2 g in an organ chamber filled with 20 ml of KHS at 37°C and gassed with 95% O2 and 5% CO2. The pH of the solution was 7.40 to 7.45.
Ten to 14 rings obtained from a beagle were suspended in 10 to 14 chambers simultaneously, and each ring was used for a different examination. Two grams of resting tension were applied to prevent contraction-induced detachment of the electrode used for measurement of electrical activity and because force developed maximally at this resting tension in a preliminary study. Force was measured with an isometric force displacement transducer (T-7-30; Tokyo Boldwin, Tokyo, Japan) and recorded on an ink writing oscillograph (Type 8s; Sanei Instrument, Tokyo, Japan).
Recording Both Transmembrane Potential and Mechanical Activity.
Each ring was suspended in an organ chamber (2 ml volume) and perfused with KHS at the rate of 5 ml/min. Force was recorded as described above.
Transmembrane potentials (TMPs) were recorded with glass microelectrodes filled with 3 M KCl and having a tip resistance of 30 to 80 MΩ. The microelectrodes were mounted on a hydraulic micro-manipulator (Narushige Scientific Instrument Lab, Tokyo, Japan) and inserted from the outer surface of the rings. The criteria for successful penetration included a sharp drop from baseline on entry of the electrode into a cell and a sharp return to zero on withdrawal of the electrode (Harder, 1980).
After a 1-h equilibration period, the viability of each ring was tested by raising the potassium (K+) concentration by 2.5 × 102 M. At 15 min after repeated washing, 3,4-DAP (10−2 M; Nakarai Chemical Co, Tokyo, Japan) was added to the chamber, and 3 to 30 min later, PCs developed in all rings.
When the cycle length and developed tension stabilized, drugs that might have affected the cell membrane or intracellular contraction systems were dissolved in distilled water and added to the chamber.
Evaluation of Drug Effects.
The level of tension achieved after administration of 10−6 M papaverine was called 0 g, and the tension of the contraction phase, duration of contraction at 20% of tension development, and the tension of the relaxation phase were compared with the average values of the three control cycles. The time to the reappearance of PCs after drug administration was called the reappearance time; when it was significantly longer than the average value of the control three control cycle length, the PCs were considered as having been eliminated by the particular drug.
Likewise, the changes in TMP before and after administration of each drug were examined.
The drugs that were used in the present study were: 1) inhibitors of calcium-activated-potassium channels (KCa); large-conductance KCa inhibitors, iberiotoxin (IbTX; Sigma-Aldrich Japan, Tokyo, Japan) (Latorre et al., 1989; Marchenko and Sage, 1996); charybdotoxin (ChTX; Sigma-Aldrich Japan) (Miller et al., 1985); small-conductance KCa inhibitor, apamine (Muraki et al., 1997); 2) CaCl2 (Wako Pure Chemicals, Osaka, Japan); 3) inhibitors of L-type voltage-gated-calcium channels (CaV), nifedipine (Wako Pure Chemicals) (Medina et al., 2010), verapamil (Wako Pure Chemicals) (Ko et al., 2010); 4) agonist of L-type CaV, Bay K8644 [S-(−)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester] (Sigma-Aldrich Japan) (Amobi et al., 2010); 5) acidic and alkaline substances, HCl, lactic acid, NaOH, NaHCO3 (Wako Pure Chemicals); 6) inhibitors of ion pumps: Na-pump (K+-Na+-ATPase) inhibitor, ouabain (Sigma-Aldrich Japan) (Briggs et al., 1996); 7) hybrid of ATP-sensitive potassium channel (KATP) opener and nitrate: nicorandil (Chugai Pharmaceutical Co, Tokyo, Japan) (Uchida et al., 1978; Yanagisawa et al., 1979); 8) KATP opener, pinacidil (Wako Pure Chemicals) (Davies et al., 2010); 9) KATP inhibitor, glibencramide (Wako Pure Chemicals) (Lindauer et al., 2003; Medina et al., 2010); 10) drugs that affect the sarcoplasmic reticulum (SR), caffeine (Wako Pure Chemicals) (Somlyo and Somlyo, 1976), dantrolene (Wako Pure Chemicals) (Kuba, 1980); 11) drugs that affect the mitochondria, oligomycin (Sigma-Aldrich Japan) (Visneskii et al., 1980); 12) β-adrenergic receptor agonist, isoproterenol (Sigma-Aldrich Japan) (Petkov and Nelson, 2005); 13) β-adrenergic receptor blocker, propranolol (Wako Pure Chemicals) (Romana-Rouza et al., 2009); and 14) α-and β-adrenergic receptor stimulant, phenylephrine (Wako Pure Chemicals) (Romana-Rouza et al., 2009).
Because PCs of coronary artery rings obtained from the same beagle tend to provide a uniform response to a given stimulus (Uchida, 1985), each study group was composed of rings obtained from different beagles; therefore, the number of rings also indicated the number of beagles in the present study.
To compare the potency of drug effects on PCs, it is necessary to compare their effects, for example, on developed tension, duration of contraction, the area under tension curve, or cycle length of PCs as reported previously (Uchida, 1985) or to compare maximum percentage of inhibition or oscillatory response (Watts et al., 1993). In contrast, because the present study was aimed to examine the effects on PCs of individual drug and not to compare potency among drugs whether or not a given drug eliminated or recovered with a given dosage was presented.
The data were expressed as mean ± S.D. and were tested by Student's t test. A p < 0.05 was considered significant.
PCs Induced by 3,4-DAP
At 3 to 30 (21 ± 6) min after administration of 10−2 M 3,4-DAP, the isolated coronary arterial rings from the beagles began to contract abruptly and contracted periodically and spontaneously thereafter in all preparations for more than 12 h (Fig. 1 and Table 1).
PCs were elicited by 3,4-DAP only in the presence of CaCl2 in concentrations between 0.3 × 10−3 and 5 × 10−3 M. They did not appear below or above this range (Table 2).
The contraction time, cycle length, and developed tension were almost constant for 12 h (Table 3).
Changes in TMP Induced by 3.4-DAP
The TMP of the smooth muscles of the isolated coronary rings was −48 mV on average before (resting potential) the administration of 10−2 M 3,4-DAP and immediately after it increased to −37 mV, but it was not followed by obvious tension development (Table 4). However, 3 to 30 min later, an abrupt spike-and-plateau depolarization accompanied by tension development occurred (Fig. 2A). High-speed recording of TMP revealed that a slight depolarization preceded this spike-and-plateau depolarization (a and b in Fig. 2B).
The spike was approximately 0 mV, but the initial portion of the plateau was −22.5 mV on average. The plateau decreased gradually (slow repolarization) and changed into fine oscillation (possibly Ca2+ oscillation; arrow O in Fig. 2A). When TMP reached −27.5 mV, an abrupt repolarization occurred and TMP returned to the baseline level (Fig. 2A and Table 5).
In three of the rings, small and periodic waves were superimposed on the partial depolarization wave that was caused by 3,4-DAP. The cycle length of these waves ranged from 5 s to 5 min, and the waves were not accompanied by a spike-and-plateau depolarization and contraction (Fig. 2C), nor were they inhibited by nifedipine, which inhibits CaV (Medina et al., 2010).
Effects on PCs of Drugs that May Affect Ion Channels
PCs were lengthened in the contraction phase by lower concentrations and were replaced by tonic contractions (TCs) at higher concentrations of IbTX or ChTX (Latorre et al., 1989; Marchenko and Sage, 1996), indicating that the relaxation phase (i.e., opening of KCa) was inhibited by these agents (Fig. 3A and Table 6). However, PCs were not influenced by apamine, which inhibits small-conductance KCa (Marchenko and Sage, 1996) (Table 6).
Lowering pH to 7.30 or below with HCl or lactic acid always resulted in the elimination of PCs. In contrast, elevation of pH to 7.33 or more with NaOH or NaHCO3 resulted in the reappearance of PCs (Fig. 5 and Table 8). However, PCs were replaced by TCs when pH exceeded 7.8 with NaOH (Table 8). Lowering pH with lactic acid or increasing percentage of CO2 in the gas and elevating pH with NaHCO3 or increasing percentage of O2 in the gas had the same effects. NaCl had no obvious effects on PCs, indicating that H+, OH−, and HCO3− affected PCs (Tables 7 and 8). Inhibitory effect of HCl on PCs was eliminated by IbTX or ChTX (Table 7).
Inhibitors of Ion Pumps.
The PCs were changed into TCs by the administration of ouabain (Briggs et al., 1996) (Fig. 3B and Table 6). However, the TCs thus induced were eliminated by nifedipine and CaCl2 (Fig. 3, C and D and Table 9).
Drugs that Affect the SR.
Ca2+ released from the SR regulates smooth muscle contraction (Quarrie et al., 2011), but PCs were not influenced by caffeine, which inhibits the uptake and accelerates the release of Ca2+ from the SR (Somlyo and Somlyo AP, 1976), and by dantrolene, which inhibits Ca2+ release from the SR (Kuba, 1980) (Table 6).
Drug that Affects the Mitochondria.
Ca+ uptake and release by the mitochondria influence contraction. However, PCs were not influenced by oligomycin (Table 6).
Nicorandil is a hybrid of KATP opener and nitrate (Uchida et al., 1978; Yanagisawa et al., 1979), and PCs were eliminated by administration of 10−4 M nicorandil (Table 6). The inhibitory effect of nicorandil on PCs was inhibited not by KCa inhibitor but by a KATP inhibitor glibencramide (Lindauer et al., 2003) (Table 7). Likewise, the inhibitory effect of pinacidil on PCs was inhibited by glibencramide (Table 7).
After administration of 10−4 M nicorandil, TMP was lowered (hyperpolarization) and the spike-and-plateau depolarization did not appear (Table 10).
β-Adrenergic Receptor Agonist.
β-Adrenergic receptors cause smooth muscle relaxation through cAMP. A β-adrenergic receptor agonist isoproterenol inhibited PCs (Table 6). The PCs inhibited by isoproterenol (10−6 M) were recovered by propranolol (10−6 M) in six preparations.
A number of studies have been performed on the mechanisms of vascular smooth muscle contraction, and the roles of endothelium, ion channels, pumps, exchanges, receptors, and intracellular factors in vascular smooth muscle contraction have been clarified considerably (Vanhoutte et al., 2005; Uhrenholt et al., 2007).
In addition, many studies have been performed on the mechanisms of vascular spasm, suggesting participation of endothelial dysfunction (Félétou, 2010), increase of vasoconstricting substances (Vanhoutte et al., 2005), decrease of vasorelaxing substances (McNeish et al., 2010), increased calcium influx, hypersensitivity of vascular smooth muscles, and genomic changes (Murakami et al., 2010; Takefuji et al., 2010). However, the exact ion mechanisms of coronary smooth muscle spasm, especially of periodic spasm and its pH-sensitive nature, have remained obscure.
In the present in vitro study, 3,4-DAP, a KV inhibitor, elicited PCs of coronary smooth muscles. The cycle length of PCs and pH-sensitive nature were similar to those of coronary spasm in patients with vasospastic angina (Murao et al., 1975; Uchida, 1985).
Based on the results of present study, the following ion mechanisms are considered to participate in 3,4-DAP-induced PCs.
Participation of KCa in PCs.
Administration of 3,4-DAP elevated TMP, indicating partial depolarization to some extent. The depolarization consisted of a rapid component, which occurred immediately after drug administration, and then a very slow component, indicating that the KV was partially closed (inhibited) abruptly and then slowly closed thereafter. When TMP reached a certain level, a small depolarization occurred (b in Fig. 7). Although the exact interactions between KV and KCa were not examined, this relatively slow depolarization that preceded the spike-and-plateau depolarization might be related to closing (inhibition) of the KCa, and it triggered the opening (activation) of CaV, and consequent Ca2+ influx, leading to the spike-and-plateau depolarization and contraction (Figs. 7 and 8).
The small and periodic depolarization waves that were not accompanied by a spike-and-plateau depolarization were observed in a few preparations, It was considered to be the result of a coupling failure between the KCa and CaV.
In high concentrations, Ca2+ eliminated PCs, and the PCs were restored by IbTX and ChTX, which inhibit large-conductance KCa (Miller et al., 1985). Furthermore, PCs were replaced by TCs by the administration of IbTX and ChTX. However, apamine, which inhibits small-conductance KCa, did not influence PCs (Muraki et al., 1997). Taken together, it is conceivable that large-conductance KCa plays an important role in the genesis of PCs.
PCs were elicited by 3,4-DAP within a limited range of CaCl2 concentrations, which indicates that Ca2+ in lower concentrations caused contraction and in high contractions it eliminated contractions by opening (activating) KCa.
The spike-and-plateau depolarization mimics those of the mesenteric arterial smooth muscles of rat (Chen and Khalil, 2008) and pregnant uterine muscles that are contracted by ergonovine, which is used clinically to provoke coronary spasm (Osa and Ogasawara, 1984).
Role of L-Type CaV.
The spike-and-plateau depolarization, as well as PCs, were eliminated by nifedipine. We therefore considered that the spike-and-plateau depolarization was caused by calcium influx through the L-type CaV (Figs. 7 and 8). In this respect, PCs in the present study differ from Ca2+-insensitive vascular contraction (McNair et al., 2004).
Role of pH.
In the present study, the PCs were extremely sensitive to changes in pH; PCs were eliminated by lowering the pH with H+ or CO2, and the PCs reappeared by elevating the pH with OH−, HCO3−, or O2. Furthermore, PCs eliminated by H+ were restored by IbTX and ChTX which inhibit large-conductance KCa, indicating that H+ acted as an opener of large-conductance KCa (Figs. 7 and 8).
Smooth muscle contraction results in the production of H+ by mitochondria, and H+ is released into the cytoplasm (Somlyo and Somlyo, 1976). In addition to the influx of extracellular H+ into the cell, the H+ released from mitochondria might have contributed to the relaxation phase of PCs by the opening of KCa.
Because NaOH and NaHCO3 also restored the PCs that were suppressed by H+, and because OH−- and HCO3−-induced TCs were eliminated by Ca2+, which activates KCa and nifedipine, OH− and HCO3− might have acted as closers (inhibitors) of KCa (Fig. 8).
Roles of Ion Exchangers and Pumps.
In addition to the Ca2+ pump, the Na+-Ca2+ exchanger plays a role in the exclusion of intracellular Ca2+. In the present study, PCs were replaced by TCs after the administration of ouabain, which inhibits the Na+-K+ pump through inhibition of the Na+-K+-ATPase, which is coupled to the Na+-Ca2+ exchanger (Kim et al., 2005; Prichard et al., 2010). It is noteworthy, however, that the TCs induced by ouabain were eliminated by CaCl2 and nifedipine, suggesting that ouabain elicited TCs not by acting on its target ion pump but by closing KCa (Fig. 8).
Role of the SR.
The SR is the Ca2+ storage site. The PCs were not inhibited by caffeine, which inhibits uptake and accelerates release of Ca2+ from the SR (Somlyo and Somlyo, 1976), and dantrolene, which inhibits Ca2+ release from SR (Kuba, 1980), indicating that intracellular Ca2+ not from the SR but from other sites such as the extracellular space contributed mainly to eliciting PCs, as in the case of nitric-oxide production by vascular endothelial cells (Rusko et al., 1992).
Role of the Mitochondria.
Because oligomycin, which inhibits Ca2+ movement from the mitochondria (Visneskii et al., 1980), did not affect PC, it is unlikely that Ca2+ supplied from the mitochondria participated in eliciting PCs.
Role of KATP.
Nicoranidil, a hybrid of KATP opener and nitrate, also inhibited PCs, and its inhibitory effect was not blocked by KCa inhibitors but was blocked by glibenclamide, a KATP inhibiter (Lindauer et al., 2003). Because nicorandil caused hyperpolarization of the cell membrane in the present and previous studies (Yanagisawa et al., 1979), the rise in TMP necessary for KCa closing or opening of CaV was prevented by this agent, with resultant inhibition of PCs.
Role of β-adrenergic Receptors.
Isoproterenol, a β-adrenoceptor stimulant, eliminated PCs, probably through activation of cAMP because propranolol blocked the inhibitory action of isoproterenol on PCs, although activation of KCa can not be denied (Petkov and Nelson, 2005).
Possible Underlying Mechanisms of PCs.
Pulling all of the results together, the following is the mechanism that we considered to be involved in the induction of PCs by 3,4-DAP.
3,4-DAP caused a partial depolarization of TMP by closing (inhibiting) KV. When TMP reached to a threshold potential, the KCa closed, resulting in a slight depolarization. (arrow b in Fig. 7). When TMP attained the threshold level by this depolarization, coupled by KCa, the L-type CaV opened and an abrupt Ca2+ influx occurred, with resultant spike-and-plateau depolarization and contraction. When the intracellular Ca2+ concentration ([Ca2+]i), reached to a certain level by this influx, Ca2+ itself opened the large-conductance KCa with resultant closing of CaV and consequent repolarization and relaxation.
Stimulated by this process, Ca2+ efflux was induced through the Na+-Ca2+ exchanger and/or Ca2+ pump, and H+ efflux was induced through the Na+-H+ exchanger. The K+-Na+ pump might also participated in this process by regulating the K+-Na+ exchanger or H+-Ca2+ exchanger (Prichard et al., 2010). When Ca2+ was considerably excluded, KCa closed again and the processes were repeated, resulting in PCs (Figs. 7 and 8).
It is conceivable that intracellular Ca2+ played a key role in PCs, although channels, exchangers, and receptors other than those mentioned above might have affected PCs through, or not through, altering TMP (Figs. 7 and 8).
Possible Mechanism of Periodic Nature of Coronary Artery.
The periodic nature of 3,4-DAP-induced contraction of a single smooth muscle cell can be explained by the above-mentioned mechanism. PCs observed in the present study indicated simultaneous contraction of the smooth muscle cells that constituted the ring. However, why multiple smooth muscles contracted simultaneously remains to be elucidated.
Oscillatory contractions can be induced in rat tail or mesenteric artery by sympathetic denervation or tetraethylammonium chloride. An increased cell-to-cell communication by increased gap expression has been proposed as the underlying mechanism of the oscillatory contractions (Watts et al., 1994; Slovut, 2004). Similar mechanism may underlay the PCs observed in the present study.
Similarity and Dissimilarity to PCs in the Other Arteries.
Periodic or rhythmic contractions are produced in the human vas deferens by Bay K8644,which is blocked by nifedipine and verapamil (Amobi et al., 2010). Burke et al. (2010) also observed PCs of the rat pulmonary artery induced by phenylephrine and Bay K8644. In the present study, however, PCs was not induced by Bay K8644, whereas they were suppressed by phenylephrine. Gianchini et al. (2009) observed rhythmic contractions of mouse mesenteric artery induced by phenylephrine in the presence but not in the absence of the endothelium. The 3,4-DAP-induced PCs occur irrespective of the presence or absence of the endothelium (Uchida, 1985), indicating that the PCs in the present study are different from the rhythmic contractions observed by Burke et al. and Gianchini et al.
Clinically, in patients with vasospastic angina the attacks caused by coronary spasm often occur periodically with a cycle length ranging in minutes. The attacks are evoked by elevating blood pH and inhibited by lowering blood pH. Based on the results presented in this study, 3,4-DAP-induced PCs in isolated canine coronary artery rings seem to share some characteristics in common with human coronary vasospasm with respect to periodic nature and pH-sensitive nature.
Analyzing electrophysiological properties of KCa by patch clump method is necessary to obtain definite evidence of periodic closing and opening of KCa, which were considered to underlay the PCs.
PCs of beagle coronary smooth muscles were elicited by 3,4-DAP, which inhibits KV. The cycle length and pH-sensitive nature of PCs were similar to those of coronary spasm in patients with vasospastic angina.
We considered that the closing of KV induced by 3,4-DAP resulted in the closing of KCa and consequent opening of L-type CaV, leading to Ca2+ influx and contraction, and the increased intracellular Ca2+ by this influx opened KCa and consequent Cav closure and relaxation, and these processes were repeated spontaneously, resulting in PCs, and H+ that was produced by mitochondria acted as a KCa opener.
Participated in research design: Yasumi Uchida.
Conducted experiments: Yasumi Uchida, Yuko Maezawa, Yoshiro Maezawa, Yasuto Uchida, and Nakamura.
Performed data analysis: Yuko Maezawa and Yoshiro Maezawa.
Wrote or contributed to the writing of the manuscript: Yasumi Uchida.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- periodic contraction
- tonic contraction
- transmembrane potential
- voltage-gated potassium channel
- calcium-activated potassium channel
- ATP-sensitive potassium channel
- voltage-gated calcium channel
- Krebs-Henseleit solution
- Bay K8644
- S-(−)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester
- sarcoplasmic reticulum
- intracellular Ca2+ concentration
- intracellular H+ concentration.
- Received February 17, 2011.
- Accepted June 14, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics