Chonnam Med J.  2009 Apr;45(1):38-46. 10.4068/cmj.2009.45.1.38.

Effects of micron-Opioid Agonist on ATP-sensitive Potassium Channel Activity in Isolated Ventricular Cardiomyocytes

• Piao LH
• Wu M
• Kim JH

• KMID: 2172282
• DOI: http://doi.org/10.4068/cmj.2009.45.1.38

Abstract

It is well known that opioid agonists are released from the myocardium during hypoxia or ischemia, and that ATP-sensitive potassium channels (K(ATP) channels) exist in the cardiac cell membrane and function as a cardioprotector preventing the myocardium from ischemic damage. In the present study, therefore, to determine whether opioid agonists are involved in the regulation of ATP-sensitive potassium channel activities, effects of the micron-opioid agonist DAMGO were examined on K(ATP) channel activities by using excised inside-out and cell-attached patch clamp techniques in enzymatically (collagenase and protease) isolated mouse ventricular cardiac myocytes. In the excised inside-out patches, DAMGO (1~300 micron mol/L) inhibited K(ATP) channel activities in a dose-dependent manner. K(ATP) channel activity, which had been attenuated by the addition of ATP (100 micron mol/L) to the internal solution, was not reactivated by DAMGO. The fashion of the single-channel inhibition by DAMGO was that both channel opening frequency and mean open-time were decreased, but the amplitudes of single channel currents and channel conductances were not altered. The half-maximal inhibition concentration (IC50) for DAMGO was 18 micron mol/L. In the cell- attached patch configuration, however, DAMGO (1~300 micron mol/L) increased dinitrophenol (50 micron mol/L)- induced K(ATP) channel activities. It was inferred that the micron-opioid agonist is involved in the regulation of ATP-sensitive potassium channel activity in cardiac myocardium, agonizing (through internal target) or antagonizing (through external target) the inhibitory action of ATP in a competitive manner, thereby attenuating or enhancing the channel openings.

Keyword

micron-Opioid agonist; K(ATP) channels; Myocytes, Cardiac

MeSH Terms

Adenosine Triphosphate
Animals
Anoxia
Cell Membrane
Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
Felodipine
Ischemia
KATP Channels
Mice
Myocardium
Myocytes, Cardiac
Patch-Clamp Techniques
Potassium
Potassium Channels
Adenosine Triphosphate
Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
Felodipine
KATP Channels
Potassium
Potassium Channels

Figure

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  Fig. 1 Patch clamp configurations used in the present study. Excised inside-out (left) and cell-attached (right) configurations respectively.

• 

  Fig. 2 Representative recording of the typical KATP channel activity in the excised inside-out patch at -60 mV holding potential. The channel activity was appeared immediately after making inside-out patch, and 1 mM ATP almost completely inhibited the channel activity. The channel activity reappeared when the ATP was washed out from the bath solution, and then 50 µM glibenclamide inhibited the channel activity.

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  Fig. 3 I-V properties of the ATP-sensitive K+ channel (KATP) currents. Recordings were obtained in excised inside-out patch membrane of isolated rat ventricular myocytes at different clamp potentials ranging from -100 to +100 mV (right panel). The current-voltage relationship (left panel) was plotted with the single channel currents of the right panel.

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  Fig. 4 Effect of linoleic acid on the KATP channel activities. Recordings show that DAMGO (1~100 µmol/L) inhibited the channel activities at -60 mV holding potential in the excised inside-out patch.

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  Fig. 5 Dose-response curve of KATP channel inhibition by the DAMGO in the excised inside-out patch. Each points denote the mean of 3~4 experiments and the vertical bar is SEM.

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  Fig. 6 Influence of DAMGO (100 µM) on the current-voltage curves of the ATP-sensitive K+ channel (KATP). The current-voltage relationship was plotted with the single channel currents in the excised inside-out patch at different clamp potentials, and the channel conductance was not changed by the DAMGO.

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  Fig. 7 Effect of DAMGO (100 µM) on the attenuated KATP channel activity in the presence of internal 100 µM ATP in the inside-out patch at -60 mV holding potential. DAMGO did not increase the channel activity.

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  Fig. 8 A trace showing typical KATP channel activities in a cell-attached patch of an isolated mouse cardiac myocyte at -60 mV holding potential (HP). Dinitrophenol (DNP) induced channel activities and these activities were inhibited by glibenclamide.

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  Fig. 9 Effects of DAMGO (10 and 100 µmol/L) on the dinitrophenol (DNP)-induced KATP channel activities in the cell-attached patch at -60 mV holding potential (HP). DAMGO increased the DNP-induced channel activities in a dose-dependent manner.

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  Fig. 10 Effects of DAMGO on the dinitrophenol (DNP)-induced channel activities in the cell-attached patches. Relative activity at 1.0 represents the channel activities for 30 sec of DNP superfusion. Each columns and vertical bars represent mean ±SEM of 4 to 5 experiments. * and † indicate significant differences at p<0.05 (*) and p<0.01 (†) compared with the DNP-induced control activities, respectively.

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