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Meeting Abstracts  |   August 2005
In Vitro  Electrophysiologic Effects of Morphine in Rabbit Ventricular Myocytes
Author Affiliations & Notes
  • Guo-Sheng Xiao, M.D.
    *
  • Jing-Jun Zhou, Ph.D.
    *
  • Guan-Ying Wang, Ph.D.
    *
  • Chun-Mei Cao, Ph.D.
    *
  • Gui-Rong Li, Ph.D.
  • Tak-Ming Wong, Ph.D.
  • * Research Fellow, Department of Physiology, † Assistant Professor, Departments of Medicine and Physiology, ‡ Professor and Head, Department of Physiology, Faculty of Medicine, The University of Hong Kong.
Article Information
Meeting Abstracts   |   August 2005
In Vitro  Electrophysiologic Effects of Morphine in Rabbit Ventricular Myocytes
Anesthesiology 8 2005, Vol.103, 280-286. doi:
Anesthesiology 8 2005, Vol.103, 280-286. doi:
MORPHINE is still widely used in postoperative and preoperative care1,2 and sedation in critically ill patients.3 The drug is also effective in relieving the chest pain associated with acute myocardial infarction.4–6 Morphine has been shown to mediate cardioprotection of preconditioning,7,8 and the effect of morphine results from direct action at the level of the cardiac myocytes via  δ- and κ-opioid receptors.9 In addition, morphine showed antiarrhythmic activity in ischemia-reperfusion–induced arrhythmias.10 Therefore, morphine is used in prognostic myocardial damage in cardiac surgery.11 
Electrophysiologic effects of morphine on cardiac myocytes are not well understood. In multicellular preparations, morphine at higher therapeutic concentrations decreases the maximal upstroke velocity of action potential depolarization and increases the action potential duration (APD).12,13 At the cellular level, the compound inhibits Na+current in rat and human cardiac myocytes and has no effect on L-type Ca2+current (ICa.L), transient outward K+current, and inwardly rectifying K+current (IK1).14 A recent study in ischemia-reperfused guinea pig myocardium showed that morphine at clinically relevant concentrations decreases ischemia-induced conduction blocks and reperfusion-induced ventricular arrhythmias, which is believed to be mainly due to partial reverse of ischemia-induced membrane depolarization and decrease in action potential amplitude and the maximal upstroke velocity of action potential depolarization.15 However, the ionic mechanisms involved in the effects of morphine remain to be clarified. The current study was therefore designed to determine the effects of morphine at clinically relevant concentrations on the action potential and ionic currents in isolated rabbit ventricular myocytes using a whole cell patch clamp technique.
Materials and Methods
The New Zealand white rabbits used in the current study were cared for in accordance with the Guide for the Care and Use of Laboratory Animals  ,16 and the experimental protocol was approved by the Ethics Committee on the Use of Animals in Teaching and Research, The University of Hong Kong (Pokfulam, Hong Kong SAR, China).
Ventricular Myocytes Preparation
Ventricular myocytes were isolated from the hearts of rabbits with a procedure previously described.17 Briefly, New Zealand white rabbits of either sex (1.5–2.5 kg) were anesthetized with pentobarbital (30 mg/kg, intravenous injection), and their hearts were quickly removed and placed in oxygenated Tyrode solution. Hearts were mounted on a Langendorff system and perfused for approximately 5 min with normal Tyrode solution and for an additional 10–12 min with Ca2+-free Tyrode solution. Then, perfusion solution was switched to the Ca2+-free Tyrode solution containing 0.5 mg/ml collagenase (CLS II; Worthington Biochemical, Freehold, NJ) and 1 mg/ml bovine serum albumin. The myocytes isolated from the softened heart were stored in high-K+storage solution. Myocytes were placed in the recording chamber (0.3 ml) mounted on the stage of an inverted microscope and superfused at approximately 2 ml/min with external solution. Only quiescent, rod-shaped cells showing clear striations were selected for experiments. The experiments were performed at 36°C for recording action potentials and delayed rectifier K+current (IK, i.e.  , IKrand IKs) or room temperature (21°–22°C) for recording ICa.Land IK1.
Solutions and Drugs
The high-K+storage solution contained 10 mm KCl, 10 mm KH2PO4, 20 mm glucose, 120 mm K-glutamate, 10 mm taurine, 0.5 mm EGTA, 10 mm HEPES, and 1.8 mm MgSO4(pH adjusted to 7.2 with KOH). The Tyrode solution contained 140 mm NaCl, 5.4 mm KCl, 1.0 mm MgCl2, 1.8 mm CaCl2, 0.33 mm NaH2PO4, 10 mm glucose, and 10 mm HEPES (pH adjusted to 7.4 with NaOH). When Tyrode solution was used to record K+currents, 200 μm Cd2+and 3 mm 4-aminopyridine were added to respectively block ICa.Land transient outward K+current (Ito). The pipette solution for K+current recordings contained 20 mm KCl, 110 mm potassium aspartate, 1.0 mm MgCl2, 10 mm HEPES, 5 mm EGTA, 0.1 mm GTP, 5 mm Mg2ATP, and 5 mm sodium phosphocreatine (pH adjusted to 7.2 with KOH). An N  -methyl-d-glucamine Tyrode solution, containing 140 mm N  -methyl-d  -glucamine, 5.4 mm CsCl, 1.0 mm MgCl2, 1.8 mm CaCl2, 0.33 mm KH2PO4, 10 mm glucose, and mm 10 HEPES, was used when ICa.Lwas measured (pH adjusted to 7.4 with CsOH). The pipette solution for ICa.Lrecording contained 20 mm CsCl, 110 mm cesium aspartate, 1.0 mm MgCl2, 10 mm HEPES, 5 mm EGTA, 0.1 mm GTP, 5 mm Mg2ATP, and 5 mm sodium phosphocreatine (pH adjusted to 7.2 with CsOH). Morphine (sulfate salt) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and dissolved in distilled water. All other chemicals were obtained from Sigma-Aldrich.
Based on the pharmacokinetics of morphine in humans, i.e.  , a volume of distribution of 3.2 l/kg and 35% protein binding, the equilibrate plasma concentration is approximately 0.06 μm after intravenous injection of 0.2 mg/kg morphine.18 We therefore used clinically relevant concentrations (0.01–1 μm) of morphine. To study the interaction of morphine with opioid receptors, effects of morphine on ICa.Land IK1were determined in the presence of 5 μm naltrindole (NTD; a selective δ-opioid receptor antagonist), 5 μm nor-binaltorphimine (nor-BNI; a selective κ-opioid receptor antagonist), or 5 μm CTOP (a selective μ-opioid receptor antagonist).
Data Acquisition and Analysis
The whole cell patch clamp technique was used17,19 to record membrane current. Borosilicate glass electrodes (1.2 mm OD) were pulled with a Brown-Flaming puller (model P-97; Sutter Instrument Co., Novato, CA) and had a tip resistance of 2–3 MΩ when filled with pipette solution. The tip potentials were compensated before the pipette touched the cell. After a gigaseal was obtained, the cell membrane was ruptured by gentle suction to establish the whole cell configuration. Data were acquired with the use of EPC-9 (Heka Electronik, Lambrecht, Germany). Command pulses were generated by a 12-bit digital-to-analog converter controlled by Pulse software (Heka Electronik). The recording of ionic currents were low-pass filtered at 5 kHz and stored on the hard disk of a computer. The series resistance (Rs) was electrically compensated to minimize the capacitive surge on the current recording and voltage drop across the clamped membrane and was kept at a constant value during the current recording. Membrane capacitance was 116.5 ± 12.8 pF (n = 45). To account for differences in cell size, all mean data are expressed as current density (i.e.  , normalized to cell membrane capacitance). For recording action potential, a perforated patch was applied using a procedure of back-filling amphotericin-B (160 μg/ml; Sigma-Aldrich) in the K+pipette solution.20 Action potentials were recorded in current clamp mode. Recorded membrane potentials were corrected by 10 mV for the liquid junction potential between the pipette and external solutions.
Data Analysis and Statistics
Nonlinear curve fitting was performed using Sigmaplot (SPSS, Chicago, IL). Concentration–response effects of morphine were fit to the Hill equation: E = Emax/[1 + (EC50/C)b], where E is the effect at concentration C, Emaxis the maximal effect, EC50is the concentration for half-maximal effect, and b  is the Hill coefficient. Curves for steady state activation (d  ) and inactivation (f  ) of ICa.Lwere fit to Boltzmann relations for activation and inactivation as follows: d  (or f  ) = 1/{1 + exp[(V0.5− V)/K]}, where V is the membrane potential, V0.5is a midpoint of potential for activation or inactivation, and K is a slope factor.
Comparison of two means was performed using the paired and unpaired Student t  tests, and comparison of several means was performed using analysis of variance, then with a Dunnett post hoc  test for the comparison with control. Statistical differences were considered significant if the P  value was less than 0.05. Results are presented as mean ± sd.
Results
Effects of Morphine on Action Potentials in Isolated Rabbit Ventricular Myocytes
Figure 1shows representative action potentials recorded at 1 Hz in current clamp mode from a rabbit ventricular myocyte in the presence of 0.01, 0.1, and 1 μm morphine and after washout of the drug. The APD was substantially increased, and the resting membrane potential was slightly hyperpolarized by application of morphine. The effects were partially reversed by washout of the drug for 10 min. The effects on action potential variables in each experiment exposed to three concentrations of morphine are summarized in table 1.
Fig. 1. Effect of morphine on action potentials. Action potentials were recorded from a representative rabbit ventricular myocyte, showing that action potential duration is prolonged and resting membrane potential is slightly hyperpolarized by the application of morphine at 0.01, 0.1, and 1 μm (6 min for each concentration). The effect was only partially reversed upon drug washout (10 min). 
Fig. 1. Effect of morphine on action potentials. Action potentials were recorded from a representative rabbit ventricular myocyte, showing that action potential duration is prolonged and resting membrane potential is slightly hyperpolarized by the application of morphine at 0.01, 0.1, and 1 μm (6 min for each concentration). The effect was only partially reversed upon drug washout (10 min). 
Fig. 1. Effect of morphine on action potentials. Action potentials were recorded from a representative rabbit ventricular myocyte, showing that action potential duration is prolonged and resting membrane potential is slightly hyperpolarized by the application of morphine at 0.01, 0.1, and 1 μm (6 min for each concentration). The effect was only partially reversed upon drug washout (10 min). 
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Table 1. Effects of Morphine on Action Potential Variables in Rabbit Ventricular Myocytes 
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Table 1. Effects of Morphine on Action Potential Variables in Rabbit Ventricular Myocytes 
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Effect of Morphine on ICa.L
Because ICa.Lis mainly responsible for the plateau of action potentials of the cardiac muscle, we determined the effect of morphine on the current. Figure 2Adisplays ICa.Ltracings recorded from a representative cell with 300-ms voltage steps to between −40 and +60 mV from −50 mV as shown in the inset. Morphine at 0.1 μm substantially increased ICa.Lafter administration for 6 min. The effect was partially reversed upon washout for 10 min. Figure 2Bshows current–voltage relations of mean values of ICa.Ldensity (n = 12) in the absence and presence of 0.1 μm morphine. The density of ICa.Lat +10 mV corresponding to peak current was significantly increased by 23 ± 8% (from 5.9 ± 1.9 to 7.3 ± 1.7 pA/pF; P  < 0.05).
Fig. 2. Effect of morphine on L-type Ca2+current (ICa.L). (  A  ) ICa.Ltraces recorded in a representative myocyte using 300-ms steps to between −40 and +60 mV from −50 mV as shown in the  inset  during control, after the application of 0.1 μm morphine for 6 min, and after washout of the drug for 10 min. ICa.Lwas increased by the application of 0.1 μm morphine and was partially reversed by drug washout. (  B  ) Current–voltage relations of ICa.Ldensity (n = 12) in the absence (control;  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). Data are expressed as mean ± sd. *  P  < 0.05  versus  control. (  C  ) Concentration–response relation of morphine effect on ICa.L.  Numbers within parentheses  are number of experiments. 
Fig. 2. Effect of morphine on L-type Ca2+current (ICa.L). (  A  ) ICa.Ltraces recorded in a representative myocyte using 300-ms steps to between −40 and +60 mV from −50 mV as shown in the  inset  during control, after the application of 0.1 μm morphine for 6 min, and after washout of the drug for 10 min. ICa.Lwas increased by the application of 0.1 μm morphine and was partially reversed by drug washout. (  B  ) Current–voltage relations of ICa.Ldensity (n = 12) in the absence (control;  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). Data are expressed as mean ± sd. *  P  < 0.05  versus  control. (  C  ) Concentration–response relation of morphine effect on ICa.L.  Numbers within parentheses  are number of experiments. 
Fig. 2. Effect of morphine on L-type Ca2+current (ICa.L). (  A  ) ICa.Ltraces recorded in a representative myocyte using 300-ms steps to between −40 and +60 mV from −50 mV as shown in the  inset  during control, after the application of 0.1 μm morphine for 6 min, and after washout of the drug for 10 min. ICa.Lwas increased by the application of 0.1 μm morphine and was partially reversed by drug washout. (  B  ) Current–voltage relations of ICa.Ldensity (n = 12) in the absence (control;  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). Data are expressed as mean ± sd. *  P  < 0.05  versus  control. (  C  ) Concentration–response relation of morphine effect on ICa.L.  Numbers within parentheses  are number of experiments. 
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Figure 2Cillustrates the concentration–response relation for the augmentation of ICa.Lby morphine. The mean data were fit to the Hill equation, and the EC50(at +10 mV) was 0.47 μm with a Hill coefficient of 0.31, and the Emaxwas 47%.
Effects of morphine on steady state voltage dependence of ICa.Lactivation and inactivation were evaluated in rabbit ventricular cells. Voltage-dependent activation of ICa.Lwas determined, as described previously,21 from the current–voltage relation of ICa.Lin figure 2Bon the basis of the formulation: dt= It/[gx(Vt− Vr)], where dtis the activation variable and Itis ICa.Lat a test potential Vt, gxis the maximum conductance, and Vris the reversal potential. Calculated values were normalized to the maximum value in each cell to obtain the activation variable d  . Figure 3Aillustrates the voltage protocol and representative recordings used to assess ICa.Linactivation. The inactivation variable f  ) was determined as ICa.Lat a given prepulse potential divided by the maximum ICa.Lin the absence of a prepulse. Figure 3Bshows the results obtained from analysis of voltage-dependent activation and inactivation before and after 0.1 μm morphine treatment. Mean data are shown by the symbols, and the curves shown are best-fit Boltzmann distributions. The midpoint of voltage (V0.5) and slope factor for activation averaged −5.7 ± 1.2 mV and 6.4 ± 1.1 for control, and those for morphine (0.1 μm) were −6.7 ± 1.3 mV and 6.1 ± 1.0 (n = 9), respectively. The V0.5and slope factor for inactivation averaged −20.1 ± 1.1 mV and −4.6 ± 0.9 for control and −20.4 ± 0.8 mV and −4.5 ± 0.7 for morphine (0.1 μm) treatment (n = 10), respectively. The steady state activation and inactivation of ICa.Lwere not affected by morphine. In addition, recovery of ICa.Lfrom inactivation, determined with the procedure as previously described,21 was not influenced by 0.1 μm morphine, either (n = 7).
Fig. 3. Absence of effect of morphine (0.1 μm) on voltage-dependent activation and inactivation of L-type Ca2+current (ICa.L). (  A  ) Representative current recordings used to determine voltage dependence of ICa.Linactivation. Cell was conditioned with 500-ms prepulses from holding potential of −80 mV to between −100 and 0 mV and back to −80 mV for 5 ms, then subjected to 300-ms test pulse to +10 mV. (  B  ) Mean values of voltage-dependent activation and inactivation relations for ICa.L. No significant differences of V0.5and slope factor were observed for activation and inactivation of ICa.Lbefore (control;  open circles  ) and after application of 0.1 μm morphine (  filled circles  ). 
Fig. 3. Absence of effect of morphine (0.1 μm) on voltage-dependent activation and inactivation of L-type Ca2+current (ICa.L). (  A  ) Representative current recordings used to determine voltage dependence of ICa.Linactivation. Cell was conditioned with 500-ms prepulses from holding potential of −80 mV to between −100 and 0 mV and back to −80 mV for 5 ms, then subjected to 300-ms test pulse to +10 mV. (  B  ) Mean values of voltage-dependent activation and inactivation relations for ICa.L. No significant differences of V0.5and slope factor were observed for activation and inactivation of ICa.Lbefore (control;  open circles  ) and after application of 0.1 μm morphine (  filled circles  ). 
Fig. 3. Absence of effect of morphine (0.1 μm) on voltage-dependent activation and inactivation of L-type Ca2+current (ICa.L). (  A  ) Representative current recordings used to determine voltage dependence of ICa.Linactivation. Cell was conditioned with 500-ms prepulses from holding potential of −80 mV to between −100 and 0 mV and back to −80 mV for 5 ms, then subjected to 300-ms test pulse to +10 mV. (  B  ) Mean values of voltage-dependent activation and inactivation relations for ICa.L. No significant differences of V0.5and slope factor were observed for activation and inactivation of ICa.Lbefore (control;  open circles  ) and after application of 0.1 μm morphine (  filled circles  ). 
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Figure 4shows the effects of 0.1 μm morphine on ICa.Lat +10 mV in the absence and presence of opioid receptor antagonists. These three types of opioid receptor antagonists themselves had no effect on ICa.L. NTD or nor-BNI, but not CTOP, abolished the action of morphine on the current, suggesting that the enhancement of ICa.Lby morphine is mediated by both δ- and κ-opioid receptors.
Fig. 4. Opioid receptor antagonists and morphine effects on L-type Ca2+current (ICa.L). ICa.Lwas increased by the administration of 0.1 μm morphine, and the increase was partially reversed upon drug washout for 10 min. Pretreatment (5 min) with nor-binaltorphimine (nor-BNI, 5 μm, n = 7) and naltrindole (NTD, 5 μm, n = 8), but not CTOP (5 μm, n = 6), prevented morphine-induced increase of ICa.L. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 4. Opioid receptor antagonists and morphine effects on L-type Ca2+current (ICa.L). ICa.Lwas increased by the administration of 0.1 μm morphine, and the increase was partially reversed upon drug washout for 10 min. Pretreatment (5 min) with nor-binaltorphimine (nor-BNI, 5 μm, n = 7) and naltrindole (NTD, 5 μm, n = 8), but not CTOP (5 μm, n = 6), prevented morphine-induced increase of ICa.L. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 4. Opioid receptor antagonists and morphine effects on L-type Ca2+current (ICa.L). ICa.Lwas increased by the administration of 0.1 μm morphine, and the increase was partially reversed upon drug washout for 10 min. Pretreatment (5 min) with nor-binaltorphimine (nor-BNI, 5 μm, n = 7) and naltrindole (NTD, 5 μm, n = 8), but not CTOP (5 μm, n = 6), prevented morphine-induced increase of ICa.L. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
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In addition, the prolongation of cardiac APD by morphine was prevented by NTD or nor-BNI, but not by CTOP. In cells with pretreatment of 5 μm NTD and 5 μm nor-BNI, APD50(at 1 Hz) was 124.2 ± 31.6 and 121.7 ± 36.5 ms, respectively, and 122.9 ± 39.1 and 125.7 ± 35.3 ms after application of 0.1 nm morphine (n = 6; not significant). Nevertheless, in cells pretreated with 5 μm CTOP, APD50was 126.8 ± 34.9 and 142.3 ± 41.3 ms in the absence and presence of 0.1 μm morphine, respectively (n = 8; P  < 0.05). These results indicate that the prolongation of cardiac APD by morphine is related to the increase of ICa.Land mediated by δ- and κ-opioid receptors.
Effect of Morphine on IK
Figure 5displays the effect of morphine on IK(IKrand IKs) in a typical experiment. Morphine at 0.1 μm had no effect on IK. In a total of six ventricular myocytes, the tail current (measured at −30 mV from the peak tail to the completed deactivation level) of IKwas 2.2 ± 0.8 pA/pF in control and 2.2 ± 0.9 after application of 0.1 μm morphine (n = 6; not significant). No change in IKtail current was observed even when the concentration of morphine was increased to 1 μm (2.3 ± 0.7 pA/pF, n = 6; not significant).
Fig. 5. Effect of morphine on delayed rectifier K+current (IK). (  A  ) IK(including IKrand IKs) was recorded during control in a representative cell in the presence of 200 μm Cd2+(to block L-type Ca2+current) and 3 mm 4-aminopyridine (to block Ito). The current was elicited by 2-s voltage steps to between −40 and +60 mV (20-mV increment) from −50 mV, then to −30 mV (to record IKcurrent;  arrow  ). (  B  ) Current traces recorded after application of 100 nm morphine in the same cell as in  A  . No change in IK(assessed by tail current) was observed with morphine administration. The  dashed lines  indicate the zero current. 
Fig. 5. Effect of morphine on delayed rectifier K+current (IK). (  A  ) IK(including IKrand IKs) was recorded during control in a representative cell in the presence of 200 μm Cd2+(to block L-type Ca2+current) and 3 mm 4-aminopyridine (to block Ito). The current was elicited by 2-s voltage steps to between −40 and +60 mV (20-mV increment) from −50 mV, then to −30 mV (to record IKcurrent;  arrow  ). (  B  ) Current traces recorded after application of 100 nm morphine in the same cell as in  A  . No change in IK(assessed by tail current) was observed with morphine administration. The  dashed lines  indicate the zero current. 
Fig. 5. Effect of morphine on delayed rectifier K+current (IK). (  A  ) IK(including IKrand IKs) was recorded during control in a representative cell in the presence of 200 μm Cd2+(to block L-type Ca2+current) and 3 mm 4-aminopyridine (to block Ito). The current was elicited by 2-s voltage steps to between −40 and +60 mV (20-mV increment) from −50 mV, then to −30 mV (to record IKcurrent;  arrow  ). (  B  ) Current traces recorded after application of 100 nm morphine in the same cell as in  A  . No change in IK(assessed by tail current) was observed with morphine administration. The  dashed lines  indicate the zero current. 
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Effect of Morphine on IK1
Figure 6Aillustrates representative IK1tracings elicited by 300-ms voltage steps to between −120 and 0 mV from −40 mV during control, after the application of 0.1 μm morphine, and after washout of the drug. IK1was increased after the application of 0.1 μm morphine for approximately 5 min. The effect was reversed only partially after washout of the drug for 10 min. Figure 6Bis the current–voltage relation of IK1density (n = 12) in the absence and presence of 0.1 μm morphine. IK1was significantly increased by morphine in both inward (−120 and −110 mV) and outward (−70 to −40 mV; P  < 0.05) components. At −60 mV, IK1was 2.8 ± 1.0 pA/pF during control. After the application of 0.01, 0.1, and 1 μm morphine, IK1was increased to 3.1 ± 1.1, 3.5 ± 0.9, and 3.7 ± 1.0 pA/pF, respectively (i.e.  , increased by 11 ± 7, 25 ± 9, and 32 ± 11%; P  < 0.05 for 0.1 and 1 μm morphine).
Fig. 6. Effect of morphine on inward rectifier K+current (IK1). (  A  ) IK1traces recorded using 300-ms voltage steps to between −120 and 0 mV from a holding potential of −40 mV in a representative ventricular myocyte during control, after the application of 0.1 μm morphine (6 min), and after washout of the drug (10 min). (  B  ) (  a  ) Current–voltage relation of IK1density determined with the same protocol as  A  in the absence (  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). (  b  ) Expanded plots for the outward components of IK1from  B  (  a  ) to show the difference before and after the application of 0.1 μm morphine. (  C  ) Mean values of relative IK1at −60 mV (n = 12) in the absence and presence of 0.1 μm morphine, and opioid receptor antagonists,  i.e.  , 5 μm nor-binaltorphimine (nor-BNI), 5 μm naltrindole (NTD), or 5 μm CTOP. The augmentation of IK1by morphine was not prevented by the pretreatment (5 min) with any opioid receptor antagonist. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 6. Effect of morphine on inward rectifier K+current (IK1). (  A  ) IK1traces recorded using 300-ms voltage steps to between −120 and 0 mV from a holding potential of −40 mV in a representative ventricular myocyte during control, after the application of 0.1 μm morphine (6 min), and after washout of the drug (10 min). (  B  ) (  a  ) Current–voltage relation of IK1density determined with the same protocol as  A  in the absence (  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). (  b  ) Expanded plots for the outward components of IK1from  B  (  a  ) to show the difference before and after the application of 0.1 μm morphine. (  C  ) Mean values of relative IK1at −60 mV (n = 12) in the absence and presence of 0.1 μm morphine, and opioid receptor antagonists,  i.e.  , 5 μm nor-binaltorphimine (nor-BNI), 5 μm naltrindole (NTD), or 5 μm CTOP. The augmentation of IK1by morphine was not prevented by the pretreatment (5 min) with any opioid receptor antagonist. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 6. Effect of morphine on inward rectifier K+current (IK1). (  A  ) IK1traces recorded using 300-ms voltage steps to between −120 and 0 mV from a holding potential of −40 mV in a representative ventricular myocyte during control, after the application of 0.1 μm morphine (6 min), and after washout of the drug (10 min). (  B  ) (  a  ) Current–voltage relation of IK1density determined with the same protocol as  A  in the absence (  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). (  b  ) Expanded plots for the outward components of IK1from  B  (  a  ) to show the difference before and after the application of 0.1 μm morphine. (  C  ) Mean values of relative IK1at −60 mV (n = 12) in the absence and presence of 0.1 μm morphine, and opioid receptor antagonists,  i.e.  , 5 μm nor-binaltorphimine (nor-BNI), 5 μm naltrindole (NTD), or 5 μm CTOP. The augmentation of IK1by morphine was not prevented by the pretreatment (5 min) with any opioid receptor antagonist. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
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Figure 6Csummarizes the mean data of morphine effect on IK1at −60 mV in the absence and presence of opioid receptor antagonists. Pretreatment with 5 μm NTD, 5 μm nor-BNI, or 5 μm CTOP did not affect the action of morphine on the current, indicating that the increase of IK1by morphine is not mediated by opioid receptors.
Discussion
In the current study, we demonstrated that morphine at 0.01–0.1 μm prolonged APD and increased ICa.Lin isolated rabbit ventricular myocytes. The effects were prevented by blockade of δ- or κ-opioid receptors. At 0.1–1 μm, morphine also hyperpolarizes the resting membrane potential and enhances IK1, but this effect was not mediated by opioid receptors. Based on the pharmacokinetics of morphine in humans, i.e.  , a volume of distribution of 3.2 l/kg and 35% protein binding, the equilibrate plasma concentration is approximately 0.06 μm after intravenous injection of 0.2 mg/kg morphine.18 Therefore, the concentrations at 0.01–0.1 μm used in the current study are within therapeutic concentrations.
It is well known that prolongation of action potential is dependent on increase of inward current (e.g.  , ICa.L) or decrease of outward K currents (IKor IK1). A previous report demonstrated that morphine prolonged cardiac APD in cardiac tissue,13 but the ionic mechanisms are not understood. In the current study, using a whole cell patch clamp technique, we provided evidence that morphine prolonged cardiac APD by increasing ICa.L, not by decreasing IKor IK1. The increase of ICa.Lby morphine had an EC50of 0.5 μm with a Hill coefficient of 0.31. The small Hill coefficient suggested a wide effective concentration range of morphine.
The current observation on the increase of cardiac ICa.Lby morphine was supported by previous studies using 45Ca2+or spectrofluorometry, in which morphine was found to induce transmembrane Ca2+influx in cardiac myocytes.9,22 In contrast to the current finding, stimulation of opioid receptor with 0.01 μm leucine-enkephalin, a δ-opioid receptor agonist, was shown to reduce ICa.Lin rat ventricular myocytes.23 This discrepancy may be due to the use of different opioid receptor agonists in the two studies: morphine in the current study and leucine-enkephalin in the previous study.23 Actually, the opposing effects of opioids on voltage-dependent Ca2+channels (ICa) were documented in nerve cells. Most of the studies indicated inhibitory effects of opioids on ICa.L,24–26 but stimulation was also observed.27 
It was reported that morphine at 30 μm did not affect ICa.Lin rat ventricular myocytes.14 The discrepancies between these findings might be related to drug concentration, species, experimental procedures, opioid receptor subtypes involved, or other unknown factors. In the heart, L-type calcium channels play an important role in generating electrical activity and excitation–contraction coupling. It is well established that increases in ICa.Lproduce positive inotropic effects and induce triggered arrhythmias, which is highly undesirable in patients with acute myocardial infarction. However, increases in ICa.Lby Bay Y5959, a Ca2+promoter, have recently been shown to prevent the initiation of reentrant ventricular tachycardia in the epicardial border of healing infarcted canine heart,28 indicating antiarrhythmic actions of an increase in ICa.L.
A previous study reported that activation of opioid receptors would hyperpolarize cell membrane in neurons.29 In the current study, we showed that morphine at the therapeutic concentration of 0.1 μm increased IK1in cardiac myocytes via  an opioid receptor–independent pathway. There is evidence that a reduction in IK1is associated with arrhythmias. Patients with Andersen syndrome, a rare inherited disease with a mutation in a major contributor of the IK1channel, Kir2.1, which causes a reduction in IK1, exhibit complex ventricular ectopy and polymorphic ventricular tachycardia.30,31 Therefore, it is likely that an increase in IK1hyperpolarizes the resting membrane potential, thus producing a membrane stabilizing effect.
It should be noted that the concentrations of morphine used in the previous study29 were much higher than those used in clinical practice. Recently, it was found that morphine at low concentrations produced preconditioning in brain Purkinje cells.32 Yvon et al.  15 reported that morphine at clinically relevant concentrations did not modify action potential variables in guinea pig ventricle in normoxic conditions but significantly attenuated the ischemia-induced depolarization and increase in amplitude of action potential and Vmax, and therefore reduced the incidence of conduction block during ischemia and reperfusion-induced arrhythmias. Nevertheless, we observed that morphine at 0.1 and 1 μm hyperpolarized the resting membrane potential significantly and prolonged APD in single ventricular myocytes from rabbit hearts. Analyses of ionic currents showed that morphine increases both ICa.Land IK1. The increase in ICa.Land IK1would prolong and shorten the cardiac APD, respectively, thus resulting in a moderate prolongation of the APD. The effects of morphine on these two types of currents would be responsible for decreased ischemia-reperfusion–induced arrhythmias as observed by Yvon et al.  15 Together with its analgesic and cardioprotective action, morphine at the therapeutic concentrations may be very useful in the management of acute myocardial infarction.
The following limitations must be considered in the assessment of the relevance of our study. The species differences, in vitro  study, healthy myocardium versus  diseased myocardium, normal physiologic conditions versus  abnormal conditions (e.g.  , hypoxia, ischemia),33 and interaction with other anesthetic agents34 should all be taken into consideration. In addition, no clear explanation was available for the observed slow reversibility of morphine effect (by washout). Possible explanations for this phenomenon would be the tight binding of morphine with cell membrane protein and the participation of intracellular events. It has been found that several signal pathways (e.g.  , protein kinase A, protein kinase C, protein kinase G) are involved in physiologic actions mediated by opioid receptors.35–37 The slow reversibility is likely related to the fact that the effects from these pathways may require a longer time to disappear after washout of the receptor ligand.
It is interesting to note that blockade of either δ- or κ-opioid receptors abolished the effect of morphine. A similar phenomenon has been reported previously by us. The infarct-sparing effect of preconditioning of remifentanil, an ultrarapid-acting opioid receptor agonist, is abolished by blockade of κ- or δ-opioid receptors.38 Further study is required to determine whether this is related to interaction of κ-opioid receptors with δ-opioid receptors.
In conclusion, the current study has demonstrated that morphine at a clinically relevant concentration of 0.1 μm increases ICa.Land IK1in isolated rabbit ventricular myocytes. Its action on ICa.Lis mediated by δ- and κ-opioid receptors, whereas its effect on IK1is not mediated by opioid receptors.
The authors thank Haiying Sun, B.Sc. (Research Assistant, Department of Medicine, The University of Hong Kong, Hong Kong, China), and Chi-Pui Mok (Technician, Department of Physiology, The University of Hong Kong, Hong Kong) for excellent technical support.
References
Aubrun F, Bunge D, Langeron O, Saillant G, Coriat P, Riou B: Postoperative morphine consumption in the elderly patient. Anesthesiology 2003; 99:160–5Aubrun, F Bunge, D Langeron, O Saillant, G Coriat, P Riou, B
Matot I, Oppenheim-Eden A, Ratrot R, Baranova J, Davidson E, Eylon S, Peyser A, Liebergall M: Preoperative cardiac events in elderly patients with hip fracture randomized to epidural or conventional analgesia. Anesthesiology 2003; 98:156–63Matot, I Oppenheim-Eden, A Ratrot, R Baranova, J Davidson, E Eylon, S Peyser, A Liebergall, M
Dahaba AA, Grabner T, Rehak PH, List WF, Metzler H: Remifentanil versus  morphine analgesia and sedation for mechanically ventilated critically ill patients: A randomized double-blind study. Anesthesiology 2004; 101:640–6Dahaba, AA Grabner, T Rehak, PH List, WF Metzler, H
Everts B, Karlson B, Abdon NJ, Herlitz J, Hedner T: A comparison of metoprolol and morphine in the treatment of chest pain in patients with suspected acute myocardial infarction: The MEMO study. J Intern Med 1999; 245:133–41Everts, B Karlson, B Abdon, NJ Herlitz, J Hedner, T
Shannon AW, Harrigan RA: General pharmacologic treatment of acute myocardial infarction. Emerg Med Clin North Am 2001; 19:417–31Shannon, AW Harrigan, RA
Antman EM, Braunwald E: Acute myocardial infarction, Harrison's Principles of Internal Medicine. Edited by Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL. New York, McGraw-Hill, 2001, pp 953–71Antman, EM Braunwald, E Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL New York McGraw-Hill
Williams JT, Christie MJ, Manzoni O: Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev 2001; 81:299–343Williams, JT Christie, MJ Manzoni, O
Miki T, Cohen MV, Downey JM: Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits. Mol Cell Biochem 1998; 186:3–12Miki, T Cohen, MV Downey, JM
Ela C, Barg J, Vogel Z, Hasin Y, Eilam Y: Distinct components of morphine effects on cardiac myocytes are mediated by the kappa and delta opioid receptors. J Mol Cell Cardiol 1997; 29:711–20Ela, C Barg, J Vogel, Z Hasin, Y Eilam, Y
Sarne Y, Flitstein A, Oppenheimer E: Anti-arrhythmic activities of opioid agonists and antagonists and their stereoisomers. Br J Pharmacol 1991; 102:696–8Sarne, Y Flitstein, A Oppenheimer, E
Fellahi JL, Gue X, Richomme X, Monier E, Guillou L, Riou B: Short- and long-term prognostic value of postoperative cardiac troponin I concentration in patients undergoing coronary artery bypass grafting. Anesthesiology 2003; 99:270–4Fellahi, JL Gue, X Richomme, X Monier, E Guillou, L Riou, B
Brasch H: Influence of the optical isomers (+)- and (–)-naloxone on beating frequency, contractile force and action potentials of guinea-pig isolated cardiac preparations. Br J Pharmacol 1986; 88:733–40Brasch, H
Alarcon S, Hernandez J, Laorden ML: Effects of morphine: An electrophysiological study on guinea-pig papillary muscle. J Pharm Pharmacol 1992; 44:275–7Alarcon, S Hernandez, J Laorden, ML
Hung CF, Tsai CH, Su MJ: Opioid receptor independent effects of morphine on membrane currents in single cardiac myocytes. Br J Anaesth 1998; 81:925–31Hung, CF Tsai, CH Su, MJ
Yvon A, Hanouz JL, Terrien X, Ducouret P, Rouet R, Bricard H, Gerard JL: Electrophysiological effects of morphine in an in vitro model of the “border zone” between normal and ischaemic-reperfused guinea-pig myocardium. Br J Anaesth 2002; 89:888–95Yvon, A Hanouz, JL Terrien, X Ducouret, P Rouet, R Bricard, H Gerard, JL
United States Department of Health and Human Services: Guide for the Care and Use of Laboratory Animals. National Institutes of Health Publication No. 85-23, revised 1985.
Li GR, Baumgarten CM: Modulation of cardiac Na+current by gadolinium, a blocker of stretch-induced arrhythmias. Am J Physiol 2001; 280:H272–9Li, GR Baumgarten, CM
Stanski DR, Greenblatt DJ, Lowenstein E: Kinetics of intravenous and intramuscular morphine. Clin Pharmacol Ther 1978; 24:52–9Stanski, DR Greenblatt, DJ Lowenstein, E
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981; 391:85–100Hamill, OP Marty, A Neher, E Sakmann, B Sigworth, FJ
Li GR, Zhang M, Satin LS, Baumgarten CM: Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes. Am J Physiol 2002; 282:H1270–7Li, GR Zhang, M Satin, LS Baumgarten, CM
Li GR, Yang B, Feng J, Bosch RF, Carrier M, Nattel S: Transmembrane ICacontributes to rate-dependent changes of action potentials in human ventricular myocytes. Am J Physiol 1999; 276:H98–106Li, GR Yang, B Feng, J Bosch, RF Carrier, M Nattel, S
Ventura C, Spurgeon H, Lakatta EG, Guarnieri C, Capogrossi MC: Kappa and delta opioid receptor stimulation affects cardiac myocytes function and Ca2+release from an intracellular pool in myocytes and neurons. Circ Res 1992; 70:66–81Ventura, C Spurgeon, H Lakatta, EG Guarnieri, C Capogrossi, MC
Xiao RP, Spurgeon HA, Capogrossi MC, Lakatta EG: Stimulation of opioid receptors on cardiac ventricular myocytes reduces L-type Ca2+channel current. J Mol Cell Cardiol 1993; 25:661–6Xiao, RP Spurgeon, HA Capogrossi, MC Lakatta, EG
Bourinet E, Soong TW, Stea A, Snutch TP: Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc Natl Acad Sci U S A 1996; 93:1486–91Bourinet, E Soong, TW Stea, A Snutch, TP
Acosta CG, Lopez HS: Delta opioid receptor modulation of several voltage-dependent Ca2+currents in rat sensory neurons. J Neurosci 1999; 19:8337–48Acosta, CG Lopez, HS
Jin W, Lee NM, Loh HH, Thayer SA: Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma glioma hybrid NG108-15 cells. Mol Pharmacol 1992; 42:1083–9Jin, W Lee, NM Loh, HH Thayer, SA
Eriksson PS, Nilsson M, Wagberg M, Hansson E, Ronnback L: Kappa-opioid receptors on astrocytes stimulate L-type Ca2+channels. Neuroscience 1993; 54:401–7Eriksson, PS Nilsson, M Wagberg, M Hansson, E Ronnback, L
Cabo C, Schmitt H, Wit AL: New mechanism of antiarrhythmic drug action: Increasing L-type calcium current prevents reentrant ventricular tachycardia in the infracted canine heart. Circulation 2000; 102:2417–25Cabo, C Schmitt, H Wit, AL
Grudt TJ, Williams JT: Kappa-opioid receptors also increase potassium conductance. Proc Natl Acad Sci USA 1993; 90:11429–32Grudt, TJ Williams, JT
Jongsma HJ, Wilders R: Channelopathies: Kir2.1 mutations jeopardize many cell functions. Curr Biol 2001; 11:R747–50Jongsma, HJ Wilders, R
Tristani-Firouzi M, Jensen JL, Donaldson MR, Sansone V, Meola G, Hahn A, Bendahhou S, Kwiecinski H, Fidzianska A, Plaster N, Fu YH, Ptacek LJ, Tawil R: Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Anderson syndrome). J Clin Invest 2002; 110:381–8Tristani-Firouzi, M Jensen, JL Donaldson, MR Sansone, V Meola, G Hahn, A Bendahhou, S Kwiecinski, H Fidzianska, A Plaster, N Fu, YH Ptacek, LJ Tawil, R
Lim YJ, Zheng S, Zuo Z: Morphine preconditions Purkinje cells against cell death under in vitro  simulated ischemia–reperfusion conditions. Anesthesiology 2004; 100:562–8Lim, YJ Zheng, S Zuo, Z
Blaise G, Langleben D, Hubert B: Pulmonary arterial hypertension: Pathophysiology and anesthetic approach. Anesthesiology 2003; 98:1415–21Blaise, G Langleben, D Hubert, B
Ludwig LM, Patel HH, Gross GJ, Kersten JR, Pagel PS, Warltier DC: Morphine enhances pharmacological preconditioning by isoflurane: Role of mitochondrial KATPchannels and opioid receptors. Anesthesiology 2003; 98:705–11Ludwig, LM Patel, HH Gross, GJ Kersten, JR Pagel, PS Warltier, DC
Smith FL, Javed RR, Elzey MJ, Dewey WL: The expression of a high level of morphine antinociceptive tolerance in mice involves both PKC and PKA. Brain Res 2003; 985:78–88Smith, FL Javed, RR Elzey, MJ Dewey, WL
Wang GY, Zhou JJ, Shan J, Wong TM: Protein kinase C-epsilon is a trigger of delayed cardioprotection against myocardial ischemia of kappa-opioid receptor stimulation in rat ventricular myocytes. J Pharmacol Exp Ther 2001; 299:603–10Wang, GY Zhou, JJ Shan, J Wong, TM
Tso PH, Wong YH: Molecular basis of opioid dependence: Role of signal regulation by G-proteins. Clin Exp Pharmacol Physiol 2003; 30:307–16Tso, PH Wong, YH
Zhang Y, Irwin MG, Wong TM: Remifentanil preconditioning protects against ischemic injury in the intact rat heart. Anesthesiology 2004; 101:918–23Zhang, Y Irwin, MG Wong, TM
Fig. 1. Effect of morphine on action potentials. Action potentials were recorded from a representative rabbit ventricular myocyte, showing that action potential duration is prolonged and resting membrane potential is slightly hyperpolarized by the application of morphine at 0.01, 0.1, and 1 μm (6 min for each concentration). The effect was only partially reversed upon drug washout (10 min). 
Fig. 1. Effect of morphine on action potentials. Action potentials were recorded from a representative rabbit ventricular myocyte, showing that action potential duration is prolonged and resting membrane potential is slightly hyperpolarized by the application of morphine at 0.01, 0.1, and 1 μm (6 min for each concentration). The effect was only partially reversed upon drug washout (10 min). 
Fig. 1. Effect of morphine on action potentials. Action potentials were recorded from a representative rabbit ventricular myocyte, showing that action potential duration is prolonged and resting membrane potential is slightly hyperpolarized by the application of morphine at 0.01, 0.1, and 1 μm (6 min for each concentration). The effect was only partially reversed upon drug washout (10 min). 
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Fig. 2. Effect of morphine on L-type Ca2+current (ICa.L). (  A  ) ICa.Ltraces recorded in a representative myocyte using 300-ms steps to between −40 and +60 mV from −50 mV as shown in the  inset  during control, after the application of 0.1 μm morphine for 6 min, and after washout of the drug for 10 min. ICa.Lwas increased by the application of 0.1 μm morphine and was partially reversed by drug washout. (  B  ) Current–voltage relations of ICa.Ldensity (n = 12) in the absence (control;  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). Data are expressed as mean ± sd. *  P  < 0.05  versus  control. (  C  ) Concentration–response relation of morphine effect on ICa.L.  Numbers within parentheses  are number of experiments. 
Fig. 2. Effect of morphine on L-type Ca2+current (ICa.L). (  A  ) ICa.Ltraces recorded in a representative myocyte using 300-ms steps to between −40 and +60 mV from −50 mV as shown in the  inset  during control, after the application of 0.1 μm morphine for 6 min, and after washout of the drug for 10 min. ICa.Lwas increased by the application of 0.1 μm morphine and was partially reversed by drug washout. (  B  ) Current–voltage relations of ICa.Ldensity (n = 12) in the absence (control;  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). Data are expressed as mean ± sd. *  P  < 0.05  versus  control. (  C  ) Concentration–response relation of morphine effect on ICa.L.  Numbers within parentheses  are number of experiments. 
Fig. 2. Effect of morphine on L-type Ca2+current (ICa.L). (  A  ) ICa.Ltraces recorded in a representative myocyte using 300-ms steps to between −40 and +60 mV from −50 mV as shown in the  inset  during control, after the application of 0.1 μm morphine for 6 min, and after washout of the drug for 10 min. ICa.Lwas increased by the application of 0.1 μm morphine and was partially reversed by drug washout. (  B  ) Current–voltage relations of ICa.Ldensity (n = 12) in the absence (control;  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). Data are expressed as mean ± sd. *  P  < 0.05  versus  control. (  C  ) Concentration–response relation of morphine effect on ICa.L.  Numbers within parentheses  are number of experiments. 
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Fig. 3. Absence of effect of morphine (0.1 μm) on voltage-dependent activation and inactivation of L-type Ca2+current (ICa.L). (  A  ) Representative current recordings used to determine voltage dependence of ICa.Linactivation. Cell was conditioned with 500-ms prepulses from holding potential of −80 mV to between −100 and 0 mV and back to −80 mV for 5 ms, then subjected to 300-ms test pulse to +10 mV. (  B  ) Mean values of voltage-dependent activation and inactivation relations for ICa.L. No significant differences of V0.5and slope factor were observed for activation and inactivation of ICa.Lbefore (control;  open circles  ) and after application of 0.1 μm morphine (  filled circles  ). 
Fig. 3. Absence of effect of morphine (0.1 μm) on voltage-dependent activation and inactivation of L-type Ca2+current (ICa.L). (  A  ) Representative current recordings used to determine voltage dependence of ICa.Linactivation. Cell was conditioned with 500-ms prepulses from holding potential of −80 mV to between −100 and 0 mV and back to −80 mV for 5 ms, then subjected to 300-ms test pulse to +10 mV. (  B  ) Mean values of voltage-dependent activation and inactivation relations for ICa.L. No significant differences of V0.5and slope factor were observed for activation and inactivation of ICa.Lbefore (control;  open circles  ) and after application of 0.1 μm morphine (  filled circles  ). 
Fig. 3. Absence of effect of morphine (0.1 μm) on voltage-dependent activation and inactivation of L-type Ca2+current (ICa.L). (  A  ) Representative current recordings used to determine voltage dependence of ICa.Linactivation. Cell was conditioned with 500-ms prepulses from holding potential of −80 mV to between −100 and 0 mV and back to −80 mV for 5 ms, then subjected to 300-ms test pulse to +10 mV. (  B  ) Mean values of voltage-dependent activation and inactivation relations for ICa.L. No significant differences of V0.5and slope factor were observed for activation and inactivation of ICa.Lbefore (control;  open circles  ) and after application of 0.1 μm morphine (  filled circles  ). 
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Fig. 4. Opioid receptor antagonists and morphine effects on L-type Ca2+current (ICa.L). ICa.Lwas increased by the administration of 0.1 μm morphine, and the increase was partially reversed upon drug washout for 10 min. Pretreatment (5 min) with nor-binaltorphimine (nor-BNI, 5 μm, n = 7) and naltrindole (NTD, 5 μm, n = 8), but not CTOP (5 μm, n = 6), prevented morphine-induced increase of ICa.L. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 4. Opioid receptor antagonists and morphine effects on L-type Ca2+current (ICa.L). ICa.Lwas increased by the administration of 0.1 μm morphine, and the increase was partially reversed upon drug washout for 10 min. Pretreatment (5 min) with nor-binaltorphimine (nor-BNI, 5 μm, n = 7) and naltrindole (NTD, 5 μm, n = 8), but not CTOP (5 μm, n = 6), prevented morphine-induced increase of ICa.L. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 4. Opioid receptor antagonists and morphine effects on L-type Ca2+current (ICa.L). ICa.Lwas increased by the administration of 0.1 μm morphine, and the increase was partially reversed upon drug washout for 10 min. Pretreatment (5 min) with nor-binaltorphimine (nor-BNI, 5 μm, n = 7) and naltrindole (NTD, 5 μm, n = 8), but not CTOP (5 μm, n = 6), prevented morphine-induced increase of ICa.L. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
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Fig. 5. Effect of morphine on delayed rectifier K+current (IK). (  A  ) IK(including IKrand IKs) was recorded during control in a representative cell in the presence of 200 μm Cd2+(to block L-type Ca2+current) and 3 mm 4-aminopyridine (to block Ito). The current was elicited by 2-s voltage steps to between −40 and +60 mV (20-mV increment) from −50 mV, then to −30 mV (to record IKcurrent;  arrow  ). (  B  ) Current traces recorded after application of 100 nm morphine in the same cell as in  A  . No change in IK(assessed by tail current) was observed with morphine administration. The  dashed lines  indicate the zero current. 
Fig. 5. Effect of morphine on delayed rectifier K+current (IK). (  A  ) IK(including IKrand IKs) was recorded during control in a representative cell in the presence of 200 μm Cd2+(to block L-type Ca2+current) and 3 mm 4-aminopyridine (to block Ito). The current was elicited by 2-s voltage steps to between −40 and +60 mV (20-mV increment) from −50 mV, then to −30 mV (to record IKcurrent;  arrow  ). (  B  ) Current traces recorded after application of 100 nm morphine in the same cell as in  A  . No change in IK(assessed by tail current) was observed with morphine administration. The  dashed lines  indicate the zero current. 
Fig. 5. Effect of morphine on delayed rectifier K+current (IK). (  A  ) IK(including IKrand IKs) was recorded during control in a representative cell in the presence of 200 μm Cd2+(to block L-type Ca2+current) and 3 mm 4-aminopyridine (to block Ito). The current was elicited by 2-s voltage steps to between −40 and +60 mV (20-mV increment) from −50 mV, then to −30 mV (to record IKcurrent;  arrow  ). (  B  ) Current traces recorded after application of 100 nm morphine in the same cell as in  A  . No change in IK(assessed by tail current) was observed with morphine administration. The  dashed lines  indicate the zero current. 
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Fig. 6. Effect of morphine on inward rectifier K+current (IK1). (  A  ) IK1traces recorded using 300-ms voltage steps to between −120 and 0 mV from a holding potential of −40 mV in a representative ventricular myocyte during control, after the application of 0.1 μm morphine (6 min), and after washout of the drug (10 min). (  B  ) (  a  ) Current–voltage relation of IK1density determined with the same protocol as  A  in the absence (  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). (  b  ) Expanded plots for the outward components of IK1from  B  (  a  ) to show the difference before and after the application of 0.1 μm morphine. (  C  ) Mean values of relative IK1at −60 mV (n = 12) in the absence and presence of 0.1 μm morphine, and opioid receptor antagonists,  i.e.  , 5 μm nor-binaltorphimine (nor-BNI), 5 μm naltrindole (NTD), or 5 μm CTOP. The augmentation of IK1by morphine was not prevented by the pretreatment (5 min) with any opioid receptor antagonist. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 6. Effect of morphine on inward rectifier K+current (IK1). (  A  ) IK1traces recorded using 300-ms voltage steps to between −120 and 0 mV from a holding potential of −40 mV in a representative ventricular myocyte during control, after the application of 0.1 μm morphine (6 min), and after washout of the drug (10 min). (  B  ) (  a  ) Current–voltage relation of IK1density determined with the same protocol as  A  in the absence (  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). (  b  ) Expanded plots for the outward components of IK1from  B  (  a  ) to show the difference before and after the application of 0.1 μm morphine. (  C  ) Mean values of relative IK1at −60 mV (n = 12) in the absence and presence of 0.1 μm morphine, and opioid receptor antagonists,  i.e.  , 5 μm nor-binaltorphimine (nor-BNI), 5 μm naltrindole (NTD), or 5 μm CTOP. The augmentation of IK1by morphine was not prevented by the pretreatment (5 min) with any opioid receptor antagonist. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
Fig. 6. Effect of morphine on inward rectifier K+current (IK1). (  A  ) IK1traces recorded using 300-ms voltage steps to between −120 and 0 mV from a holding potential of −40 mV in a representative ventricular myocyte during control, after the application of 0.1 μm morphine (6 min), and after washout of the drug (10 min). (  B  ) (  a  ) Current–voltage relation of IK1density determined with the same protocol as  A  in the absence (  open circles  ) and presence of 0.1 μm morphine (  filled circles  ). (  b  ) Expanded plots for the outward components of IK1from  B  (  a  ) to show the difference before and after the application of 0.1 μm morphine. (  C  ) Mean values of relative IK1at −60 mV (n = 12) in the absence and presence of 0.1 μm morphine, and opioid receptor antagonists,  i.e.  , 5 μm nor-binaltorphimine (nor-BNI), 5 μm naltrindole (NTD), or 5 μm CTOP. The augmentation of IK1by morphine was not prevented by the pretreatment (5 min) with any opioid receptor antagonist. Data are expressed as mean ± sd. *  P  < 0.05  versus  control. 
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Table 1. Effects of Morphine on Action Potential Variables in Rabbit Ventricular Myocytes 
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Table 1. Effects of Morphine on Action Potential Variables in Rabbit Ventricular Myocytes 
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