Pflfigers Arch (1992) 421:409-415

Journal of Physiology 9 Springer-Verlag1992

Aromatic aldehydes and aromatic ketones open ATP-sensitive K + channels in guinea-pig ventricular myocytes Zheng Fan* Keiko Nakayama, Tohru Sawanobori, and Masayasu Hiraoka Department of Cardiovascular Diseases, Medical Research Institute, TokyoMedical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan Received January 20, 1992/Receivedafter revision April 24, 1992/AcceptedMay 8, 1992

Abstract. Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxyacetophenone, on the adenosine-5'-triphosphate(ATP)sensitive K + channel current (/K.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5-1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of /K.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K + concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATE Thus we identified this channel as IK.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on IK.ATP was not reduced by addition of cysteine (10mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of IK.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP. These results suggest that these compounds: (a) modulate IK.ATP perhaps through an intracellular mechanism, (b) bind covalently to proteins to form a Schiff base which may by responsible for the effects, and (c)may require an ADP-dependent process.

* P r e s e n t address: Department of Medicine, Cardiac Electrophysiology Laboratories, 5841 South Maryland Avenue, Hospital Box 440, Chicago, IL 60637, USA C o r r e s p o n d e n c e to: M. Hiraoka

Key words: 3,4-Dihydroxybenzaldehyde -

2,3-Dihydroxybenzaldehyde - 2.4-Dihydroxyacetophenone ATP-sensitive K + channel - guinea-pig ventricular myocytes

Introduction Recently, increasing numbers of reports have appeared to demonstrate that so-called "K + channel openers", such as cromakalim [4, 17], nicorandil [10], and pinacidil [1, 6, 7, 13], can activate ATP-sensitive K + channels (IK.ATP) in cardiac cells and pancreatic fl-cells [12], and also in certain smooth muscle cells [14, 19]. Although it became obvious that the molecular mechanisms of the action of these compounds may be different because of their chemical diversity, understanding of these mechanisms would be important for both drug development and clarification of the modulation and functional structure of the IK.ATP channel. However, so far the molecular mechanisms of action of these drugs on the [K.ATP channel have not been proposed [3]. Chemical modification of channel proteins is one of the approaches to identify the specific reaction processes and binding sites of the target channels. We have previously shown that 3,4-dihydroxybenzaldehyde, and aldehyde compound, could reversibly shorten the action potential duration in ventricular muscles of the guinea-pig [5]. This effect was quite similar to those produced by "K + channel openers" [1, 4, 10, 13]. Therefore, we questioned whether this effect is caused by an opening of IK.ATP channels or not. We further questioned whether other aldehyde and ketone compounds can have a similar effect. This is interesting because aldehyde or ketone compounds are known to have two major modification effects on proteins: to form a reversible Schiff base with flee amino groups of proteins [24] and to react reversibly with thiol groups on proteins [16, 18]. Since the above two reactions can be distinguished, it is possible to determine a key step in the mechanism of how these com-

410 A "50% threshold" criterion was used to detect events with the help of manual confirmation. The open probability is expressed by the openstate probability times the number of channels activated in the patch

pounds open IK.ATP channels. On the other hand, it is also interesting since many drugs and biologically useful reagents are aldehydes and ketones and are sometimes used as vehicles for other agents thought to be active on

(nx Po). Data were expressed as means_+SE. Statistical comparison was made using a paired t-test. A p value of less than 0.05 was considered significant.

[K.ATP c h a n n e l s .

In this study, we report for the first time that some aldehydes and ketones including 3,4-dihydroxybenzaldehyde open IK.ATP channels in ventricular myocytes of guinea pigs. A short abstract has been published elsewhere [11].

Results

Effects of 3,4-dihydroxybenzaldehyde on membrane potentials and currents

Materials and methods

3,4-Dihydroxybenzaldehyde (0.5-2raM) shortened the action-potential duration (APD), measured both at 20~ (ADP20) and 90O7o (ADP90) repolarization, without changing the resting membrane potential. This effect was reversed or "blocked" by further application of glibenclamide (0.3 IxM), a specific I~:.ATPblocker [8]. Figure 1A illustrates typical recordings of action potentials in the control, after 3,4-dihydroxybenzaldehyde (1 raM) and after 3,4-dihydroxybenzaldehyde (1 raM) with glibenclamide (0.3 ~tM), from a single experiment. The shortening effect appeared within 2 - 3 min upon application of 3,4-dihydroxybenzaldehyde, and was quickly reversible (< 1 rain) after 10 rain application. Similar results were obtained in three other cells. APD20 and APD90 were 417+62ms and 581+84ms in the control, 121+42ms and 185 + 65 ms in 3,4-dihydroxybenzaldehyde ( P < 0.001 in comparison with the control), and 468+91 ms and 649_+101 ms in 3,4-dihydroxybenzaldehyde with glibenclamide (P

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes.

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihyd...
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